Dermatol Clin 25 (2007) xiiixiv

Preface

Batya B. Davidovici, MD Ronni Wolf, MD Guest Editors In chemistry, corpora non agunt nisi liquida (substances are active only in solution). In chemotherapy, corpora non agunt nisi xate (substances do not act unless bound). Paul Ehrlich (who introduced the concept of the receptor), in an address to the International Medical Congress (London), 1913.

Receptors are molecular structures within a cell or on its surface to which substances such as hormones, drugs, and cellular or immunologic components selectively bind, causing a change in cell activity. Receptors lie at the very heart of every biological system. They have a pivotal role in the regulation of all cell functions, development, proliferation, and dierentiation in health and disease. Receptors are the foundations of pharmacology. The past decade has witnessed the emergence of the so-called new biology, which identied and cloned almost all receptors and ion channels. The eld of dermatology has joined other disciplines in being transformed from an art and a mainly morphologic discipline to a science. Molecular biology has moved from bench to bedside. Twenty years ago, we would have been incredulous that the knowledge of molecular biology would no longer be the exclusive domain of the basic scientist, but that it would become an essential part of mainstream medical practice. We would have found it even more farfetched to think that an issue of the Dermatologic Clinics, a journal

that focuses on topics relevant to ordinary dermatology practice, would be devoted to Cutaneous Receptors: Clinical Implications and Therapeutic Relevance. There is nothing for it but to face the reality that the dermatologist of today must be familiar with the basic molecular defects underlying various skin diseases. They must also be familiar with the new drugs and biological agents that target specic cell surface molecules of immunocytes, keratinocytes, and cytokines, as well as with older drugs, such as steroids, retinoids, and vitamin D analogs. Understanding all aspects of receptors and their ligands will allow clinicians to have a better grasp of the normal physiology of the skin and its interaction with the environment, as well as of the pathophysiology of diseases. They will be better equipped to diagnose some diseases (eg, cutaneous lymphomas, melanoma, and so forth) that cannot be dierentiated without those sophisticated methods, and to maximize the therapeutic benet of many new, but also old, drugs, while minimizing their adverse eects. We hope that the present issue of the Dermatologic Clinics will help clinicians incorporate the new information and approaches into the fabric of their daily practice, and that the content will provide practitioners with a useful and interesting reference. This issue is the nal product of the eorts and cooperation of an extraordinary team. We oer our sincere thanks to the contributors, all rst-rate
derm.theclinics.com

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.07.002

xiv

PREFACE

and extremely busy scientists and dermatologists who have been more than generous in giving of their valuable time to ensure the success of this project. Their talent, experience, and knowledge enables us to oer our colleagues a comprehensive, intelligent, and interesting collection of articles that will impact many thousands of patients. Ms. Ali Gavenda, Editor at Elsevier Science, did a masterful job in coordinating, organizing, and assuring the completion of this issue of the Dermatologic Clinics, and for this we extend our thanks and gratitude. Last, but by no means least, we have nothing but praise for the Consulting Editor, Bruce H. Thiers, MD, for conferring upon us the honor and unique privilege of being Guest Editors of this important issue of the Dermatologic Clinics, and for helping

us from the earliest steps of preparation to the completion of this project. Ronni Wolf, MD Dermatology Unit Kaplan Medical Center Rechovot 76100 Israel E-mail address: wolf_r@netvision.net.il Batya B. Davidovici, MD Dermatology Unit Kaplan Medical Center Rechovot 76100 Israel E-mail address: bdavidovici@yahoo.com

Dermatol Clin 25 (2007) 467476

Keratinocyte Cytokine and Chemokine Receptors

Yalc n Tu zu n, MDa,*, Meltem Antonov, MDa, Neslihan Dolar, MDa, Ronni Wolf, MDb
a

Department of Dermatology, Cerrahpasa Medical Faculty, Istanbul University, 34098 Istanbul, Turkey b Dermatology Unit, Kaplan Medical Center, Rechovot 76100, Israel

Cytokines, which include the large family of chemokines, are soluble polypeptide mediators that play pivotal roles in communication between cells of the hematopoietic system and other cells in the body [1]. Chemokines represent small, secreted proteins that mediate directional migration in vitro and are shown to critically regulate leukocyte trafcking in vivo. Chemokines bind pertussis toxinsensitive, 7-transmembranespanning G-protein coupled receptors (GPCR). Notably, the majority of small molecule antagonist therapeutics prescribed today target GPCR, making chemokine receptors a prime target for large-scale small molecule antagonist screening. Chemokine receptor signaling involves dierent pathways sustaining cell survival, including gene expression and, most importantly, those enabling directional cell migration [2]. Keratinocytes are the major cell population of the epidermis and can be activated to produce various chemokines, including the CCL chemokines, regulated on activation of normal T-cell expressed and secreted (RANTES)/CCL5 and monocyte chemoattractant protein 1 (MCP-1)/ CCL2, and the CXCL chemokines, interferon (IFN)-ginduced protein of 10 kd (inducible protein [IP-10])/CXCL10 and interleukin [IL]-8/ CXCL8). Cytokines released by inltrating T cells are among the strongest signals for chemokine expression in keratinocytes [3]. There is increasing evidence that keratinocytes participate directly in cutaneous immunologic diseases. Activated keratinocytes produce inammatory cytokines and
* Corresponding author. E-mail address: yalcintuzun@yahoo.com (Y. Tu zu n).

express MHC class II molecules and CD54. They also express costimulatory molecules, such as CD80 and CD40 [4]. Tumor necrosis factor a and tumor necrosis factor R1 Tumor necrosis factor a (TNF-a) plays an important role not only in immunity and inammation but also in the control of cell proliferation, dierentiation, and apoptosis. Its inuence on dierentiation is divergent and it is shown that dierent cell types are aected in dierent ways. It is believed that TNF-a induces skin keratinocyte dierentiation, but a study of how important TNF-a signaling is to cellular dierentiation shows that it clearly is involved in dierentiation of macrophages and dendritic cells. No clear regulation, however, is seen in keratinocytes in this study [5]. The biologic eects of TNF-a are mediated through either of the membrane-bound TNF receptors, TNFR1 (p-55) or TNFR2 (p-75). Human keratinocytes are shown to lack TNFR2, indicating that TNF-a responsiveness of these cells depends critically on regulation of TNFR1 expression. UV (ultraviolet) B radiation is found to regulate TNFR1 expression in keratinocytes by stimulating TNF-a release, which then acts in an intracrine, autocrine, and paracrine manner [6]. UVB irradiation has multiple eects on mammalian skin, including the induction of cytokines, such as TNF-a and other pathways, which are implicated in the formation of sunburn cells, which are apoptotic keratinocytes. UVB radiation has been noted to induce keratinocytes to synthesize
derm.theclinics.com

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.003

468

ZU N TU

et al

and secrete a variety of cytokines (IL-1, IL6, TNF-a, and IL-10) [7]. Irradiation with highdose UVB is known to induce apoptosis of keratinocytes in vivo and in vitro. This is shown mediated by various cellular events, such as direct cross-linking of Fas and the 55-kd TNF receptor (TNFR1) and p53-mediated signals. UVB mediates intermediate and delayed apoptosis. Intermediate apoptosis is mediated by the activation of receptors on the cellular membrane. A study shows that a combination of low-dose UVB and TNF-a signicantly induces TNFR1 aggregation and keratinocyte apoptosis. Addition of antiTNFR1 antibody signicantly inhibits the induction of apoptosis by these two stimuli. It was concluded that increased clustering of TNFR1 was involved in apoptosis [8]. Thalidomide is an eective agent in the treatment of photosensitivity diseases, such as actinic prurigo, polymorphous light eruption, hydroa vacciniforme, and chronic cutaneous lupus erythematosus (CCLE), and photosensitivity in AIDS. The anti-inammatory properties of thalidomide are, in part, related to its ability to inhibit TNF-a production by mononuclear cells. There is no information, however, about the eects of thalidomide on epithelial cell TNF-a production. TNFR1 is a possible target of thalidomide [7]. UV-induced apoptosis of keratinocytes is postulated to play a role in the pathophysiology of cutaneous lupus erythematosus. The self-antigens exposed during the apoptotic process may trigger the production of autoantibodies. Thus, thalidomide may confer photoprotection in the skin by decreasing the release of self-antigens in apoptotic bodies. An experiment demonstrated that thalidomide inhibits the formation of apoptotic bodies (ie, sunburn cells) after a single, acute exposure to UVB. Mice treated with thalidomide before being irradiated with UVB showed almost no changes related to UVB histologically, whereas untreated mice showed the typical histologic appearance of acute UVB irradiation. It also was shown that thalidomide caused a dose-dependent inhibition of the increase in epidermal sunburn cell counts induced by UVB irradiation. Despite this, thalidomide did not inhibit the increase in the number of cyclobutane pyrimidine dimers induced by increasing doses of UVB. Another nding is that thalidomide did not inhibit UVB-induced upregulation of IL-6 steady-state messenger RNA (mRNA). This suggests that there is specicity to the anti-inammatory eect of thalidomide for UVB-induced cytokine gene expression. This

study suggests that thalidomide may confer resistance to the death pathway of keratinocytes [7]. Another study, however, demonstrates no change in the presence of sunburn cells before and after being treated with thalidomide in patients who had CCLE. Thalidomide causes destabilization of TNF-a mRNA and may bind to a death receptor mediating apoptosis. The investigators examined, in three patients who had CCLE, the eects of thalidomide on minimal erythema doses (MED) and presence of sunburn cells. All three patients who previously had failed rst- and second-line therapies were treated successfully with thalidomide and a signicant decrease in MED values was observed. When the frequency of sunburn cells was assessed before and during thalidomide treatment, however, no signicant dierence was seen. This study suggests that thalidomide does not decrease UV-induced apoptosis signicantly in nonlesional skin of patients who have cutaneous lupus erythematosus. This nding may be explained by the fact that multiple mechanisms of apoptosis are activated by UVB. Three pathways independently induce apoptosis secondary to UV, including activation of death receptors, such as TNFR1, direct photochemical DNA damage, and formation of reactive oxygen species. Blocking only the TNF-a pathway may not be sucient to alter the overall apoptotic activity. It is unlikely that thalidomide inhibits DNA damage and the p53-mediated apoptotic pathway, which is believed to be the most potent stimulus for UV-induced keratinocyte apoptosis [9]. UVB radiation induces the conversion of 7dehydrocholesterol to calcitriol and the release of TNF-a in human keratinocytes. Calcitriol is a hormonally active metabolite of vitamin D3, which regulates keratinocyte proliferation and dierentiation via vitamin D receptormediated processes. It has been described that there is an autonomous metabolic vitamin D3 pathway in human epidermis, which results in synthesis of calcitriol independent of hydroxylases located in the liver and kidney. Another study investigated the inuence of TNF-a on the autonomous epidermal vitamin D3 pathway in cultured keratinocytes. It was shown that the generation of calcitriol from vitamin D3 in keratinocytes was dose dependently upregulated in the presence of TNF-a. The generation of calcitriol, vitamin D3, and TNF-a all depended on the UVB wavelength and showed maximum levels at 300 nm [6]. Antimalarial agents also are known to suppress sunlight-induced cytokines, including

KERATINOCYTE CYTOKINE AND CHEMOKINE RECEPTORS

469

TNF-a, IL-1, and IL-6, and inhibit UV-induced phospholipase activity [9].

Interleukin 20 and interleukin 19 IL-20 is identied as a member of the IL-10 family, which includes IL-10, -19, -20, -22, -24, and -26. Despite a partial homology in their amino acid sequences, they have diverse biologic functions. IL-19 and IL-20 are each others closest relatives within the IL-10 family when comparing sequence homology. IL-19, -20, and -24 share receptor complexes; all three are capable of signaling through the IL-20Ra/IL-20Rb heterodimer, and both IL-20 and IL-24 also can use IL22R and IL-20R2. The main biologic eects of these three cytokines seem, however, distinct: immune activity with IL-19, skin biology with IL-20, and tumor apoptosis with IL-24 [1012]. A heterodimer consisting of an IL-20Ra subunit wand an IL-20Rb subunit was identied as the IL-20 receptor complex and is shown to bind IL-19. Another heterodimeric receptor complex for IL-20, consisting of IL-22Ra and IL-20Rb, also is described and this receptor complex is not activated by IL-19. At mRNA level, IL-20Ra is the receptor subunit expressed most widely. It is expressed in many tissues, with the highest level of expression in heart, lung, and skin. Only lung tissue and skin are known to have a strong expression of all three receptor subunits [11,12]. Recent studies suggest pathogenic roles of IL-19 and IL-20 in psoriasis. Under normal conditions, IL-20 mRNA is present at low levels in skin. Overexpression of IL-20 in transgenic mice acuses neonatal lethality with psoriasis-like skin abnormalities, including hyperkeratosis, thickened epidermis, and aberrant epidermal differentiation. Moreover, IL-20 receptor mRNA is markedly upregulated in human psoriatic skin compared with normal skin. Overexpression of IL-19 mRNA in psoriatic skin recently was demonstrated but the skin of mice overexpressing IL-19 seems normal. The biologic functions of IL20 in keratinocyte proliferation remain unclear but it has been ascribed a key role in the pathogenesis of psoriasis [10,12]. In a study, immunostaining for IL-20, IL20Ra, and IL-20Rb was performed in skin sections from patients who had psoriasis. Specimens from healthy skin and spongiotic dermatitis were stained as controls. The results demonstrated an increased expression of IL-19 and IL-20 in the

epidermis of psoriasis lesions and spongiotic dermatitis lesions compared with healthy controls. Similar patterns and expression levels were seen in psoriasis and spongiotic dermatitis. IL-20 and IL19, however, showed dierent expression patterns. IL-20R1 and IL-20R2 expression also was higher in both types of dermatitis than in healthy skin. The distribution pattern of IL-20R subunit, however, was dierent between psoriasis and spongiotic dermatitis. IL-20R2 was expressed on the basal and suprabasal keratinocytes in spongiotic dermatitis. The investigators hypothesized that increased expression of an activated IL-20 receptor alters the interactions between endothelial cells, immune cells, and keratinocytes, which leads to dysregulation of keratinocyte proliferation and dierentiation. The investigators also treated CD8-positive T cells with IL-20 to examine whether or not there was any altered expression level of keratinocyte growth factor (KGF) and found that IL-20 upregulated KGF expression in CD8-positive T cells and not CD4. It was speculated that IL-20 may trigger CD8-positive T cells locally and mediate proliferative reactions to induce hyperplasia of keratinocytes in psoriasis through production of KGF [10]. Serum levels of IL-20 were signicantly lower in patients who had psoriasis than in controls. This may have been because of increased binding to the increased receptors. There was no correlation between serum IL-20 levels and psoriasis area and severity index scores in patients who have psoriasis. IL-20 serum level did not correlate with the severity of the disease state [10]. In another study, IL-19 and IL-20 mRNA were found elevated in lesional and nonlesional psoriatic skin. Expression of IL-20Ra and IL20Rb mRNA, however, was decreased signicantly in lesional skin. This is the opposite of studies using in situ hybridization. IL-22Ra mRNA expression was similar in lesional and nonlesional skin. Their inverse relationship of IL20 mRNA expression is compatible with the idea that an increased amount of IL-20 is accompanied by a compensatory downregulation in its receptors. This is supported by a weak, but statistically insignicant, increase in receptor expression during treatment with cyclosporin. There was a strong correlation between IL-19 and IL-20 mRNA levels. A similar correlation between IL-20Ra and IL-20Rb mRNA expression was found. The mRNA expression of IL-20, but not of IL-19, exhibited a statistically signicant inverse correlation to IL-20Ra and IL-20Rb mRNA

470

ZU N TU

et al

expression. There was no signicant dierence in gene expression levels for any of the target genes when comparing the dierent types of psoriatic lesions (guttate and chronic plaque types). During short-term (28 days) treatment with calcipotriol or cyclosporine, IL-19 and IL-20 mRNA expression in lesional psoriatic skin decreased in parallel to or even preceding the improvement in histopathologic score. Neither IL-19, IL-20, nor receptor gene expression returned to normal, however, during a treatment period of 28 days. This nding is in accordance with the presence of ongoing disease activity seen at histologic evaluation. At present, IL-20 and possibly IL-19 seem attractive as potential targets in the treatment of psoriasis [11]. Another study observed that IL-19 and IL-20 mRNA were expressed exclusively in foci of keratinocytes located at the top of the dermal papillae. They were absent in monocytes/macrophages, endothelial cells, melanocytes, dendritic Langerhans cells, and T lymphocytes. IL-19 mRNA was detected in 16 of 20 patients and IL-20 mRNA was detected in 17 of 20 patients. The overall IL-19 mRNA expression was stronger than that of IL-20. In most cases, the foci with IL20 expression also were positive for IL-19 mRNA. Virtually no expression of IL-19 and IL-20 mRNA was detected in uninvolved psoriatic skin. The distinct localization of IL-19 and IL20 mRNA at the top of the papillae indicate that the keratinocyte is a source of mRNA in the psoriatic lesions. The absence of immunostaining with the proliferation marker MiB1 (Ki-67 antigen) showed that the foci of IL-19 and IL-20 expressing keratinocytes were not proliferating. Expression of mRNA for the receptor chains, IL20R1 and IL-20R2, was found throughout the psoriatic epidermis. IL-20Ra mRNA was expressed predominantly above the basal layer of keratinocytes, whereas IL-20Rb mRNA was distributed evenly in all layers of epidermis. IL-20Ra mRNA was expressed dierentially in the most supercial part of the psoriatic epidermis just beneath the stratum corneum. No expression of these receptors was seen in endothelial cells or leukocytes in dermis. Treatment with cyclosporine A and calcipotriol resulted in disappearance of the focal expression of IL-19 and IL-20 mRNA in keratinocytes at the top of the dermal papillae. In the calcipotriol-treated patients IL-19 and IL-20, mRNA expression did not decrease as rapidly as in the cyclosporine A group. Disappearance of IL-19 and IL-20 mRNA expressions correlated with the changes of the clinical and

histopathologic scores. Expression levels of mRNA for the three receptor chains were not aected by cyclosporine A or calcipotriol treatment [12].

Interleukin 18 IL-18 has been described to be produced by keratinocytes. In several inammatory skin diseasesdsuch as psoriasis and ADdIL-8 overexpression in the skin is demonstrated. The receptor of IL-18 is composed of an a-chain (IL-18Ra) and a b-chain (IL-18Rb or accessory-like protein). Although IL-18Ra represents the ligand-binding chain, IL-18Rb seems to assist in the formation of a high-anity complex and mediates intracellular signals. In a study, keratinocytes were found to express IL-18Ra. IL-18Ra was upregulated moderately by IFN-g alone. Expression was increased more than threefold by IFN-g in combination with TNF-a. IL-12Rb1 was found to be expressed on keratinocytes and slightly but reproducibly upregulated by IFN-g. IL-4 did not exert any signicant eect on receptor expression, but IL-4 given before incubation with the pairs of cytokines that had the strongest eects on IL-18R expression on keratinocytes led to a receptor downregulation. Keratinocytes are good responders to IL-4, whereas the spectrum of the biologic eects of this cytokine to keratinocytes is broad and includes proinammatory and anti-inammatory eects. Keratinocytes isolated from psoriasis and AD lesions showed, respectively, vefold and twofold higher expression of the receptor compared with normal skin. It was shown that IL-18 induced CXCL10 production in keratinocytes. In eczematous skin diseases, the development of skin lesions from acute to chronic is accompanied by a switch from a more Th2 cytokine pattern to a Th1-like cytokine pattern expressed in the cutaneous lesions. The process of the shift from Th2 to Th1 dominance in the lesions largely is unknown but it is speculated that IL-12 together with IL-18 leads to the activation of Th1 cells in this process. Not only may IL18 act directly on inltrating T cells to produce IFN-g but also keratinocytes may be involved directly in perpetuating a Th1-like reaction pattern. At sites of acute inammation, the IL-18R is upregulated on keratinocytes, rendering them highly responsive toward IL-18. By production of CXCL10, CXCR3 expressing IFN-gproducing T cells are attracted to the site of inammation. Here, they encounter MHC class II expressing keratinocytes,

KERATINOCYTE CYTOKINE AND CHEMOKINE RECEPTORS

471

which, in combination with superantigens, are shown to be present in AD epidermis and psoriasis, induce a T-cell stimulation resulting in high IFN-g production. IL-18 also induced MHC class I and II expression on the keratinocytes. It is known that IL-18 plays a part in the clearance of viruses. Downmodulation of IL-18induced immune responses by human papillomavirus (HPV) oncoproteins may contribute to viral pathogenesis or carcinogenesis. This may arise via HPV binding to the IL-18R, thus preventing IL-18 induction of IFN-g. In epidermal viral infections, such as herpes simplex virus, HPV, and varicella-zoster virus, in addition to inducing IFN-g and activating CD8 T cells, IL-18 produced by keratinocytes may act in an autocrine fashion. Although it is shown that the IL-18R complex can be upregulated on na ve T cells, Th1, natural killer (NK) cells, and B cells by IL-12, in this study, IL-12 did not upregulate the expression of IL-18Ra on keratinocytes. The study demonstrated, however, that keratinocytes express the Il-12Rb1 but not the high-anity receptor, IL-12Rb2 [4]. Interleukin 13 IL-13 is an immunoregulatory cytokine secreted predominantly by activated Th2 cells and mast cells. It shares many functional properties with IL-4, including the induction of IgE synthesis, CD23 expression by B cells, the upregulation of MHC class II on monocytes, and the induction of vascular cell adhesion molecule-1 on endothelial cells. IL-13 exerts its actions via binding to the complex receptor consisting of IL-4Ra and at least two other cell surface proteins, IL-13Ra1 and IL13Ra2. IL-13Ra1 itself binds IL-13 with low anity but when paired with IL-4Ra it binds Il-13 with high anity and forms a functional receptor. IL13Ra2 that can bind IL-13 with high anity has been postulated to be a decoy receptor because of its short cytoplasmic tail without any signaling motifs. IL-13Ra1 is expressed on the keratinocytes in healthy skin. The regulation of IL-13Ra1 and IL13Ra2 expression is not understood completely. It is possible that each cell has specic regulatory mechanisms of expression of these receptors [13]. One study shows that the immunohistochemical staining patterns of IL-13Ra1 are dierent when normal skin and skin of patients who have atopic dermatitis (AD) and psoriasis are

compared. Staining was seen mostly in lower layers of keratinocytes in normal skin whereas it was seen in upper layers in the skin of patients who had AD. Psoriatic skin showed a striking increase in positive staining of IL-13Ra1 and in all layers of keratinocytes. There also was an increase in expression in AD when compared with normal skin but it was less than in psoriatic skin. IL-4Ra was increased signicantly in AD and psoriasis compared with normal skin. It may be hypothesized that dierent staining patterns of IL-13Ra1 may reect the dierent mechanism of hyperplasia between these diseases [13]. IL-13 or IFN-g alone signicantly enhanced IL13Ra mRNA expression. In contrast, the same concentration of IL-4 had no eect on the IL-13Ra expression. Furthermore, simultaneous addition of IL-4 and IL-13 abolished this eect of IL-13. Combined stimulation of IL-4 with IFN-g suppressed, but not signicantly, the IL-13Ra1 mRNA expression which was upregulated by IFN-g alone. The observation that IFN-g seems to be a major cytokine for the induction of IL-13Ra1 is consistent with the nding that IL-13Ra1 is expressed more abundantly in psoriasis, which is a disease state with predominance of IFN-g, than in AD [13]. Even though IL-4 or IL-13 alone could not induce secretion of IL-6 and RANTES from keratinocytes 48 hours after stimulation, the presence of IL-13 or IL-4 signicantly enhanced the IFN-g-stimulated secretion of these cytokines. The IFN-ginduced secretion of IL-8 and IP-10 tended to increase (but not signicantly) in the presence of IL-4 or IL-13. So far, it is not known how IL-4 and IL-13 have dierent biologic eects on keratinocytes through the same receptor [13]. Interleukin 4, interleukin 13, and macrophage-derived chemokine in atopic dermatitis It is shown that the serum levels of macrophagederived chemokine (MDC), a ligand for CCR4 and chemoattractant for CCR4 expressing cells, such as Th2-type cells and IL-2 activated NK cells, are higher in patients who have AD (considered a Th2dominant disease) than healthy controls. Serum MDC levels signicantly correlate with the disease activity of AD. IL-4 and IL-13 share some homology with each other and have similar biologic functions. They are reported to play a key pathogenetic role in the Th2-dominant diseases AD, bronchial asthma, and bullous pemgoid.

472

ZU N TU

et al

Supernatant MDC levels in a human keratinocyte cell line were measured in response to stimulation with TNF-a, IFN-g, IL-4, and IL-13 and the eects of dexamethasone and tacrolimus on MDC production in these cells were investigated. The results showed that MDC expression in keratinocytes was upregulated strongly by TNF-a and IFN-g and slightly downregulated by IL-4 and IL-13. TNF-a and IFN-g, when combined, showed synergistic increase in MDC. IL-4 and IL-13 both inhibited TNF-a and IFN-g enhanced MDC production in a dose-dependent manner but not synergistically when combined. Tacrolimus, but not dexamethasone, dose dependently inhibited TNFa and IFN-g-enhanced MDC production. In leukocytes, other studies have shown that IL-4 and IL-13 upregulate production of MDC. These results suggest that MDC production is regulated dierently in keratinocytes and leukocytes. This study also suggests that tacrolimus is eective in treating AD, possibly in part because of the inhibition of MDC production by lesional keratinocytes [14]. CCR3 CCR3 is a chemokine receptor expressed on eosinophils, basophils, dendritic cells, and T cells that responds to RANTES, MCP-3, or eotaxin under inammatory conditions. Under inammatory skin conditions, it is suggested that these chemokines aect the function of skin constructing cells, such as keratinocytes, broblasts, and endothelial cells. The nding that CCR3 was expressed on keratinocytes suggests that these chemokines may participate in proliferation or dierentiation of keratinocytes under inammatory skin conditions. One study found that normal keratinocytes have weak but detectable CCR3 expression and that its expression is upregulated signicantly when keratinocytes are cultured with RANTES but not with eotaxin, IL-4 or, IFN-g. There also was enhanced immunoreactivity of CCR3 in the epidermis of inammatory lesions, especially of AD. It can be speculated that RANTES regulates CCR3 expression through another ligand, such as CCR1, CCR4, or CCR5, on keratinocytes because eotaxin did not aect CCR3 expression on keratinocytes. The expression of these receptors on keratinocytes, however, is not yet claried. Further study is needed to clarify the role of RANTES and CCR3 in inammatory lesions [15]. It is intriguing that IFN-g and TNF-a enhance RANTES production by keratinocytes and that RANTES induces CCR3 expression on

keratinocytes, because these molecules would aect inammatory process in skin diseases. In AD, IFNg and TNF-a are detectable and these cytokines have a pathogenetic role in the skin lesions of AD, and, in fact, the lesional epidermis expresses CCR3 strongly, as revealed in this study [15]. CCR3 is the major receptor on eosinophils and is expressed on basophils, suggesting an important role for allergic diseases, such as AD and bronchial asthma. The CC chemokines, eotaxin, eotaxin-2 and eotaxin-3, are identied as ligands that activate the CCR3 exclusively and seem to be monospecic. CCR3 also is activated by other ligands, such as RANTES, MCP-2, MCP-3, and MCP-4. A study conducted to investigate the presence of CCR3 on keratinocytes found that CCR3 mRNA is expressed in cultured human keratinocytes. CCR3 mRNA expression was concentrated to the basal layers of the epidermis and found in normal individuals and in disease states, such as bullous pemphigoid, AD, and psoriasis. The staining patterns in all these conditions were reported as similar. Compared to eosinophils, however, keratinocytes showed only a weak CCR3 expression [16]. In a previous study, expression of eotaxin and CCR3 was increased signicantly in the dermis of AD lesions but no signicant staining was observed in keratinocytes [17]. The expression of CCR3 on keratinocytes in the former study also was donor dependent and diered from healthy volunteers. Preincubation of cultured keratinocytes for 30 minutes with Th2-type cytokines (IL-4 and IL-5) did not alter expression of CCR3. It also was found that eotaxin induced a dose-dependent proliferation of keratinocytes, which was as eective as IL-8. Proliferation also was induced by other CCR3 ligands, such as RANTES and MCP-4. Eotaxin was more eective than these ligands but less effective than epidermal growth factor (EGF). Preincubation of keratinocytes with the anti-CCR3 MoAb 7B11 inhibited the proliferation of keratinocytes induced by eotaxin. This demonstrates that the proliferation of keratinocytes after stimulation with eotaxin can be inhibited by antagonizing CCR3-mediated eects [16].

Other cytokine, chemokine, and keratinocyte interactions in atopic dermatitis and psoriasis Initiation of AD lesions is believed mediated by means of early skin inltration of Th2 lymphocytes, releasing high levels of IL-4, IL-5, and IL-13.

KERATINOCYTE CYTOKINE AND CHEMOKINE RECEPTORS

473

Subsequently, the accumulation of activated monocytes, mature dendritic cells, and eosinophils determines a rise in IL-12 expression and the appearance of a mixed Th2/Th1 cytokine pattern, with reduced IL-4 and IL-13 and the presence of IFN-g. By contrast, psoriasis is characterized by many activated Th1 cells, focal intraepidermal collections of neutrophils, and dermal accumulation of monocytes and dendritic cells. IFN-g producing Th1 clones dominate psoriatic lesions and primarily are responsible for the epidermal changes. Moreover, keratinocytes, mast cells, monocytes, and dendritic cells may be active sources of TNF-a in both diseases [3]. One study observed that basal keratinocytes actively are committed to neosynthesizing RANTES in AD and psoriasis. RANTES is chemotactic for monocytes, dendritic cells, and Th1 lymphocytes. It also was shown that basal keratinocytes strongly express MCP-1 in patients who have AD and at higher levels in patients who have psoriasis. MCP-1 potently attracts monocytes and dendritic cells in vivo but it can induce migration of Th1 and Th2 cells. In the case of AD, RANTES and MCP-1 can contribute relevantly to the mixed Th1/Th2 inltrate that characterizes the chronic phase. IP-10 was expressed markedly in the epidermis of patients who have psoriasis but only weakly and limited to some areas in AD lesions. The higher expression of MCP-1 and especially IP-10 in epidermis of patients who had psoriasis compared with that of patients who had AD likely reects presence of more numerous Th1 cells in the former because IFN-g is the most potent inducer of these chemokines. The investigators also conrmed keratinocyte-associated IL-8 production in psoriasis but not in skin of patients who had AD or healthy skin. TNF-a was the most potent inducer of RANTES and IL-8. By contrast, IFN-g was by far the most eective stimulus for MCP-1 and IP-10 production. IL-4 also induced some RANTES, IP-10, and IL-8 expression but not MCP-1 expression, which is consistent with the notion that keratinocytes express functional IL-4 receptor. This observation, together with the capacity of IL-4 to reinforce the activity of TNF-a and IFN-g in promoting keratinocyte expression of CXCR3 agonistic chemokines, indicates that IL-4 might be implicated in the recruitment of Th1 cells in chronic AD [3]. An interesting observation of this study was that keratinocytes from patients who had AD produced increased amounts of RANTES but

reduced levels of IP-10 in response to IFN-g or TNF-a when compared with keratinocytes from normal control subjects or patients who had psoriasis. By contrast, keratinocytes from patients who had psoriasis responded to the same stimuli with an exaggerated expression of IL-8, MCP-1, and IP-10 production. These results may indicate the existence of genetically determined defects in the constitutive and induced chemokine production by keratinocytes of patientswho had AD and psoriasis. Moreover, this abnormal production appeared under a complex chemokine-associated, rather than stimulus-specic, control. The investigators concluded that the data support the hypothesis that contribution of keratinocytes to the pathogenesis of AD and psoriasis is linked to the presence of distinct, intrinsic alterations in their capacity to respond to proinammatory stimuli and that these abnormalities can modulate amplication and persistence of skin inammation [3]. RANTES is one of the C-C type chemokines, which specically attracts eosinophils, T lymphocytes of memory phenotype, and monocytes in vitro [18]. Chemokines produced by epidermal keratinocytes play an important part in the outcome and modulation of psoriasis. Psoriatic scales contain neutrophil-attracting and -activating factors, one of which has been identied as IL-8, a member of CXC chemokine family specic for neutrophils and T cells. Aberrant expression of IL-8 by lesional keratinocytes was observed by in situ hybridization and immunostaining. One study observed that the combination of TNF-a and IFN-g displayed a marked synergism in inducing RANTES production, although they did not induce it when given separately. TNF-a could induce IL-8 production by itself but was augmented when IFN-g was added. Cytokine-induced RANTES and IL-8 production were inhibited by tacalcitol. The mechanism of the therapeutic ecacy of active vitamin D3 still is not understood fully. These ndings contribute to the understanding of psoriasis and its treatment [18].

Cytokines and chemokines in ulcers The recruitment of leukocytes at the site of skin injury is mediated by chemokines and growth factors released by immune cells, keratinocytes, broblasts, and endothelial cells. CXC chemokine IL-8 preferentially attracts neutrophils, whereas

474

ZU N TU

et al

CC chemokine MCP-1 attracts monocytes. The specic eects of these chemokines on their target cells are mediated by receptors CXCR1 for IL-8 and CCR2A for MCP-1 [19]. Keratinocytes and endothelial cells are sources of MCP-1 and IL-8 chemokines upregulated in normal wound repair, which trigger leukocyte inltration in a direct manner. Keratinocytes and endothelial cells express CCR2 and CXCR1 chemokine receptors. Transforming growth factor (TGF)-b1 is chemotactic for inammatory cells and broblasts and when applied exogenously, stimulates formation of granulation tissue in incisional wounds. Granulocyte macrophage colony-stimulating factor (GM-CSF) seems to be a multipotent factor stimulating keratinocyte growth and leg ulcer healing. IGF-1, basic broblast growth factor, EGF, and IL-15 also stimulate keratinocyte proliferation and reepithelialization [19]. In a study of chemokines, cytokines, and growth factors in the margin of diabetic foot ulcers, it was seen that there was a signicantly increased expression of factors responsible for keratinocyte proliferation and migration in the epidermis (TGF-b1, GM-CSF, and EGF). The expression of MCP-1, IL-8, IL-10 and their receptors, CCR2A, CXCR1, and IL-10R, and of IL-15 in keratinocytes in the margin of diabetic foot ulcer remained unchanged compared with normal skin. The expression of TGF-b1 was strong in diabetic and normal basal keratinocytes, whereas there was signicantly increased expression of TGF-b1 and its receptor TGF-b1R in the suprabasal layer in the diabetic epidermis. In contrast to the normal epidermis, the diabetic foot ulcer keratinocytes expressed GM-CSF. The diabetic foot ulcer keratinocytes revealed signicantly increased expression of EGF compared with normal skin. No dierences in expression of platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-Ra and PDGF-Rb receptors, GM-CSFR, EGFR, vascular endothelial growth factor (VEGF) and its receptor VEGFRII, IGF-1, and NOS2 were observed. There was a lack of upregulation of IL-8, CCR2A, IL10R, GM-CSFR, PDGF, and their receptors, VEGF and VEGFRII, EGFR, IGF-1, and NOS2 in keratinocytes and endothelial cells in the diabetic foot ulcer. Lastly, there was a lack of upregulation of IL-10 and IL-15 in keratinocytes [19]. In this study, there was only signicant upregulation of CXCR in the endothelium. Data concerning expression of TGF-b1 and its

receptors in chronic leg ulcers are conicting. Both reduced TGF-b1 but not TGF-bRI expression and enhanced TGF-b1 staining in the suprabasal layers of the epidermis were observed in chronic venous leg ulcers. The observation of overexpression of EGF, GM-CSF, and TGF-b1 in diabetic foot ulcer epidermis could be responsible for keratinocyte proliferation, lack of keratinocyte apoptosis, and migration. Altogether, the proinammatory factors were expressed at higher levels in diabetic ulcer keratinocytes and endothelial cells compared with normal foot skin. The main deciency in dermal diabetic foot inamed tissues was related to the low expression of angiogenic factors. In contrast, there was strong expression of factors responsible for mobilization and extravasation of leukocytes. Alternatively, enhanced expression of factors responsible for keratinocyte proliferation and migration suggests an undisturbed capacity of these cells present in the diabetic foot ulcer margin to function [19]. In normal acute wounds, studies reveal the importance of the TGF-bs to stimulate the synthesis of multiple extracellular matrix components, including collagens, bronectin, vitronectin, tenascin, and proteoglycans. Many studies have shown that exogenously applied TGF-bs enhance and accelerate acute wound healing, especially in situations where the normal healing process is naturally or articially impaired. In humans, there are three isoforms: TGF-b1, -b2, and -b3. The action of the TGF-bs is mediated by a complex formed through binding to two specic high-anity transmembrane serine-threonine kinases. The type I receptor requires the type II receptor to bind ligand, whereas the type II receptor requires the type I receptor for signaling. Expressions of the type I and type II TGF-b receptors are increased in response to acute wounding [20]. In a study of the expression of TGF-b and its receptors in chronic wounds and normal unwounded skin, it was found that the levels of all the TGF-b ligands were reduced in nonhealing venous leg ulcers and that there is dierential expression of the TGF-b receptors within these chronic wounds compared with normal, unwounded tissue. TGF-b1 was present throughout the epidermis at the margins of the ulcers but staining was reduced markedly within the ulcers. TGF-b2 was observed in suprabasal epidermal cells in normal unwounded skin and no staining was observed in the epidermal margin of the ulcers. TGF-b3 immunostaining was observed

KERATINOCYTE CYTOKINE AND CHEMOKINE RECEPTORS

475

throughout the epidermis in normal unwounded skin. Reduced staining was observed at the margins of the ulcers. TGF-b type I receptor immunostaining was restricted to the basal epidermal cells of normal skin. In contrast, intense expression was seen throughout the epidermal margin of the ulcers, including the suprabasal layers. The pattern of the type II receptor expression was markedly dierent. Type II TGF-b receptor immunostaining was observed throughout the epidermis of unwounded skin. Diminished staining was observed in the epidermal margins of the ulcers. There was absence of TGF-bs and their receptor expression in nonhealing ulcers and the expression of these factors in healing ulcers. This nding was consistent between patients. Because the mechanism involved in TGF-b signaling involves the binding of the ligand to the type II receptor, which then recruits the type I receptor into the complex, thereby forming a heteromeric complex of two TGF-bRIIs and two TGF-bRIs, if one member of the complex is absent, as the TGF-bRII in these nonhealing venous ulcers, it might be expected that receptor signaling is compromised in these ulcers and that neither endogenous nor exogenous TGF-b ligand will have its expected eects on wound repair. The addition of exogenous TGF-bs to chronic wounds is suggested as a potential therapeutic modality. Given the results obtained in this study, the level of TGF-b receptor expression is an important factor to be considered in chronic dermal wounds. After the implementation of good wound management, the prole of the TGF-bs and their receptors resembles more closely that observed in the acute wounds. Once an ulcer has entered a healing phase, expression of TGF-b and its receptors is upregulated and at this stage exogenous addition of growth factors may be more eective [20]. In another study, TGF-b expression in human diabetic wounds was investigated and compared with diabetic foot ulcers. There was no signicant increase in TGF-b1 in diabetic chronic foot ulcers and chronic venous ulcers. There was an increase in TGF-b2 and TGF-b3 staining in both ulcer groups, however, when compared with normal skin and diabetic skin. There was no increase in TGF-bR types I and II in diabetic foot ulcers. The lack of an upregulation of receptors in diabetic foot ulcers would mean a limited response to any elevation of TGF-b1, if this were to occur. The high levels of TGF-b3 found in diabetic foot ulcers could compound the problem by inhibiting a response to any TGF-b1 present by saturating

the limited number of receptors expressed. Alternatively, the elevated expression of TGF-b3 may reduce the number of receptors present. Does failure to upregulate TGF-b1 and its receptors on wounding contribute to the initial failure of diabetic foot wounds to heal and to leave a chronic ulcer? Do the chronically elevated levels of TGF-b3 and depressed levels of TGF-b1 perpetuate nonhealing in a diabetic ulcer and in chronic wounds in general? These interesting questions raised by this study need to be addressed in future investigations. In conclusion, this study clearly demonstrates that the normal elevation of TGF-b1 expression in acute wound healing consistently is absent in diabetic foot ulcers and chronic venous ulcers, whereas TGF-b3 expression is enhanced in these ulcers. This may explain, in part, the chronicity of such wounds. Such a deciency of TGF-b1 may have a specic role in the pathogenesis/perpetuation of chronic ulcers in general [21].

Summary Chemokines are a superfamily of small, secreted proteins that regulate cell trac in homeostatic and inammatory conditions. Keratinocytes synthesize many chemokines, including members of the CC and CXC subfamilies, such as RANTES, gamma-interferon inducible protein-10, monokine induced by gamma-interferon, and thymus- and activation-regulated chemokine. They also express some chemokine receptors that mediate the inammatory or immune response by attracting various kinds of leukocytes.

References
[1] Williams IR, Rich BE, Kupper TS. Cytokines. In: Freedberg IM, Esen AZ, Wol K, et al, editors. Fitzpatricks dermatology in general medicine. 6th edition. New York: Mc Graw Hill; 2004. p. 28596. [2] Homey BM. Chemokines and inammatory skin diseases. Adv Dermatol 2005;21:25177. [3] Giustizieri ML, Mascia F, Frezzolini A, et al. Keratinocytes from patients with atopic dermatitis and psoriasis show a distinct chemokine production prole in response to T cell-derived cytokines. J Allergy Clin Immunol 2001;107:8717. [4] Witmann M, Purwar R, Hartman C, et al. Human keratinocytes respond to interleukin-18: implication for the course of chronic inammatory skin diseases. J Invest Dermatol 2005;124:122533.

476

ZU N TU

et al keratinocytes in the skin of psoriasis and atopic dermatitis. J Dermatol Sci 2003;33:3140. Xiao T, Kagami S, Saeki H, et al. Both IL-4 and IL-13 inhibit the TNF- a and IFN-l enhance MDC production in a human keratinocyte cell line, HaCaT clls. J Dermatol Sci 2003;31:1117. Wakugawa M, Nakamura K, Akatsuka M, et al. Expression oc CC chemokine receptor 3 on human keratinocytes in vivo and in vitro-upregulation by RANTES. J Dermatol Sci 2001;25:22935. Ptering H, Kluthe C, Dulkys Y, et al. Characterization of the CC chemokine receptor 3 on human keratinocytes. J Invest Dermatol 2001;116: 54955. Yawalkar N, Uguccioni M, Scharer J, et al. Enhanced expression of eotaxin and CCR3 in atopic dermatitis. J Invest Dermatol 1999;113:438. Fukuoka M, Ogino Y, Sato H, et al. RANTES expression in psoriatic skin and regulation of RANTES and IL-8 production in cultured epidermal keratinocytes by active vitamin D3 (tacalcitol). Br J Dermatol 1998;138:6370. Galkowska H, Wojewodska U, Olszewski WL. Chemokines, cytokines and growth factors in keratinocytes and dermal endothelial cells in the margin of chronic diabetic foot ulcers. Wound Repair Regen 2006;14:55865. Cowin AJ, Hatzirodos N, Holding CA, et al. Eect of healing on the expression of transforming growth factor s and their receptors in chronic venous leg ulcers. J Invest Dermatol 2001;117:12829. Jude EB, Blakytny R, Bulmert J, et al. Transforming growth factor- beta 1,2,3 and receptor type I and II in diabetic foot ulcers. Diabet Med 2002; 19:4407.

[5] Schling P, Rudolph C, Heimerl S, et al. Expression of tumor necrosis factor alpha and its receptors during cellular dierantion. Cytokine 2006;33:23945. [6] Lehmann B, Abraham S, Meurer M. Role for tumor necrosis factor- a in UVB- induced conversion of 7 dehydrocholesterol to 1 a, 25-dihydroxivitamin D3 in cultured keratinocytes. J Steroid Biochem Mol Biol 2004;90:5615. [7] Lu KQ, Brenneman S, Burns R, et al. Thalidomide inhibits UVB induced mouse keratinocyte apoptosis by both TNF a independent pathways. Photodermatol Photoimmunol Photomed 2003;19:27280. [8] Tsuru K, Horikawa T, Budiyanto A, et al. Low dose ultraviolet B radiation synergizes TNF-a to induce apoptosis of keratinocytes. J Dermatol Sci 2001;26: 20916. [9] Cummins D, Gaspari A. Photoprotection by thalidomide in patients with chronic cutaneous and systemic lupus erythematosus: discordant eects on minimal erythema dose and sunburn cell formation. Br J Dermatol 2004;151:45864. [10] Wei CC, Chen WY, Wang YC, et al. Detection of IL20 and its receptors on psoriatic skin. Clin Immunol 2005;17:6572. [11] Otkjaer K, Kragballe K, Fundig AT, et al. The dynamics of gene expression of interleukin-19 and interleukin-20 and their receptors in psoriasis. Br J Dermatol 2005;153:9118. [12] Romer J, Hasselager E, Lisby P, et al. Epidermal overexpression of interleukin-19 and -20 mRNA in psoriatic skin disappears after short term treatment with cyclosporine A or calcipotriol. J Invest Dermatol 2003;121:130611. [13] Wongpiyabovorn J, Suto H, Ushio H, et al. Up regulation of interleukin 13 receptor a1 on human

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

Dermatol Clin 25 (2007) 477485

Keratinocyte Growth Factor Receptors

Vincenzo de Giorgi, MDa,*, Serena Sestini, MDa, Daniela Massi, MDb, Ilaria Ghersetich, MDa, Torello Lotti, MDa
a b

Department of Dermatology, University of Florence, Via della Pergola 60, 50100, Florence, Italy Department of Human Pathology and Oncology, University of Florence, Viale GB Morgagni 85, 50134, Florence, Italy

A key strategy of cellular physiology to regulate the proliferation rate and dierentiation process is modulation of the number of functional growth factor receptors on the epithelial cell surface that are exposed to the actions of cognate ligands. The mechanisms responsible for such modulation may operate at dierent transcriptional, translational, and posttranslational levels, including ligand-induced down-regulation by endocytosis and subsequent intracellular receptor degradation. The keratinocyte growth factor receptor (KGFR) and the epidermal growth factor receptor (EGFR), among the growth factor receptors expressed on keratinocytes, are believed to play a unique, crucial role in controlling epithelial proliferation and dierentiation. Modulation of previous receptors has been observed in vivo and in vitro [1]. KGFR and EGFR play a crucial role in the proliferation rate and dierentiation process. Moreover, there are some important dierences among these receptors [1]. KGFR and EGFR have dierent distributions in normal human skin. This is because KGFRs are present mainly in the suprabasal spinous layers, whereas EGFRs are distributed predominantly in the basal layer. The increase in KGFRs is conned to cells committed to the dierentiation process, whereas

KGFRs are distributed mostly on suprabasal layers and have low expression on basal cells. In addition, in human primary cultured keratinocytes, KGFR expression is up-modulated during dierentiation induced by high Ca2 concentration in the culture medium, which suggests that receptor expression may regulate the proliferative/differentiative balance during dierentiation from basal to suprabasal cells. Keratinocyte growth factor (KGF) and epidermal growth factor (EGF) have dierent effects in response to the Ca2 dierentiative signal. This is because KGF promotes the expression of dierentiation markers in vitro, whereas EGF blocks such expression. KGF and EGF have dierent mitogenic action on conuent cells, because KGF exerts its mitogenic eect mostly after keratinocytes reach conuence, whereas the EGF eect decreases with conuence. Taken together, all of these ndings demonstrate that KGF and EGF play dierent roles in the control of keratinocyte proliferation and dierentiation and that the expression of the receptors for these growth factors may be crucial for such control [1]. Even if they play dierent roles, their actions are not independent. It has been demonstrated that there is induction of transforming growth factor a (TGF-a) in KGFtreated keratinocytes, hence the activation and down-modulation of the EGFR [2]. Thus, EGFR signaling pathway may act through the KGFR stimulation for at least certain aspects of epidermal growth and dierentiation.
derm.theclinics.com

* Corresponding author. E-mail address: vincenzo.degiorgi@uni.it; vdegi@ tin.it (V. de Giorgi).

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.017

478

DE GIORGI

et al

Keratinocyte growth factor receptor Architecture KGF (FGF7) is a member of the broblast growth factor (FGF) family, which asserts its biologic activity on epithelial cells of dierent tissues. KGF is produced by cells of mesenchymal origin and seems to participate in controlling epithelial dierentiation, wound healing, survival against apoptotic induction, and protection from DNA damage. KGF binds with high anity exclusively to the tyrosine kinase KGFR; however, similarly to the other members of the FGF family, KGF binds with low anity to heparan sulfate proteoglycans, molecules associated with the cell surface, and components of the extracellular matrix [3,4]. KGFR is a tyrosine kinase receptor; it is a splicing transcript variant of FGF receptor 2 (FGFR2) (KGFR FGFR2-IIIb) [1,5]. Four well-characterized FGFRs contain a single transmembrane domain, consisting of 21 amino acids, considered unimportant for signal transduction; an intracellular tyrosine kinase domain, with a typical short insert of 13 amino acids; and an extracellular FGF-binding domain composed of two or three immunoglobulin (Ig)-like domains. The transcripts encoding three FGFRs (FGFR1, -2, and -3) are spliced alternatively to produce isoforms that contain one of two dierent Ig-III domains. Alternative splicing of FGFR2 transcripts results in the production of two receptors that dier in the carboxy-terminal half of the Ig-III domain. This hemidomain is determined by the tissue-specic inclusion of exon IIIb or exon IIIc, which ultimately controls ligandbinding specicity. FGFR2(IIIb) primarily binds KGF, and FGF10 and is the isoform of choice in epithelial cells [6,7]. Ligand binding Binding of the specic ligands to KGFR results in receptor dimerization, with subsequent autophosphorylation on tyrosine residues within the intracellular domain and recruitment and phosphorylation of substrate proteins, such as phospholipase C-g (PLCg) and FGFR substrate 2 (FRS2). FRS2 is a lipid-anchored docking protein that becomes tyrosine phosphorylated and binds to Grb/Sos in response to KGF stimulation [1,4,8]. In the structure of KGFR, the tyrosine 769 (in the C-terminal region of the receptor) is a key residue for the regulation of KGFR-mediated

signal transduction. It is required for binding and tyrosine phosphorylation of PLCg protein. The tyrosine 769 in KGFR also is able to control the full activation of the mitogen-activated protein kinase (MAPK) pathway and the mitogenic response to KGF, by controlling the level of tyrosine phosphorylation of FRS2 protein, which, in turn, regulates the activation of the ras/MAPK pathway. The key MAPK cascade is extracellular signal-related kinase (ERK), also known as the classic Ras/Raf/MAPK kinase/MAPK pathway. The activation of ERK-1 and -2 (ERK1/2) occurs by way of phosphorylation by cytoplasmic dualspecicity MAPKs, mitogen-activated protein kinase (MEK)-1 and -2 (MEK1/2), and often is associated with the stimulation of cell proliferation. In the nucleus, ERK1/2 is supposed to phosphorylate and activate some transcription factors, eg, Elk-1, c-myc, and c-fos [9]. Heparan sulfate modulates kinetic parameters of KGFKGFR interactions; it assists the complexes in assuming the best conguration for ecient receptor activation. Interaction of the b4-b5 loop with the receptor is critical for ecient activity. Proper orientation is necessary to bring together the intracellular domains of the receptor dimers for transphosphorylation [4]. Chlatrin-mediated receptor endocytosis, following receptor activation, is believed to represent a key process for receptor down-modulation and attenuation of mitogenic signaling [1], because endocytosis and sorting in multivesicular bodies play a crucial role in attenuating survival signals and regulating apoptosis of the damaged cells [10]. Receptor sorting, as occurs in early endosomes for recycling to the cell surface or lysosomal degradation, may control important cellular responses leading to proliferation, dierentiation, and transformation. The intracellular endocytic tracking of KGF and KGFR involves entry by clathrincoated pits, transport to early endosomes, and sorting to late endosomes and lysosomes. Therefore, KGFKGFR complexes seem to be internalized dierently from other FGF-FGFRs, whereas their intracellular route seems identical to the pathway followed by several tyrosine kinase receptors, whose prototype is EGFR and its ligand is EGF. Dierently from EGFR, however, KGFRs are degraded slowly; the KGF ligand still interacts with the receptor in late endosomes, determining its targeting to lysosomal degradation. The persistence of receptor activity and, therefore, signaling in a late perinuclear endosomal compartment may account for the slow kinetics of degradation, presumably

KERATINOCYTE GROWTH FACTOR RECEPTORS

479

because receptor transfer to lysosomes occurs only after receptor dephosphorylation [3,11]. KGFR endocytosis is an important mechanism to regulate the death of damaged cells; it has been observed that UV-B exposure induces the activation and the internalization of the receptor. The ligand-independent receptor internalization is mediated by the production of reactive oxygen species. It is novel, but not surprising, that KGFR, being expressed mainly on the suprabasal keratinocytes, may stimulate mechanisms of protection to UV-B radiation [10]. Eects on keratinocytes Proliferation and dierentiation KGFR regulates the proliferation rate and the dierentiation process. It is conned to cells committed to the dierentiation process, is found mostly on suprabasal layers, and has low expression on the basal cells [1]. The activation of KGFR induces the production of the dierentiation-specic keratins, K1 and K10, in suprabasal layers [2,12]. Recent research showed that the abnormally thin epidermis seen in the absence of functional KGFR signaling is caused by a reduction in keratinocyte proliferation. Despite the loss of proliferative potential, the keratinocytes that form a one- to two-cell layer above the basal layer are able to express the proteins of early (K10) and late (loricrin) terminal dierentiation. In the same research, the analysis of hair follicles showed defects in their number and distribution, as well as thin hairs [13]. The role of KGFR in the induction of proliferation and dierentiation also has been studied in psoriasis, a chronic dermatosis characterized by the marked hyperplasia of epidermal keratinocytes and by incomplete epidermal dierentiation. In this disease, there is an increased expression of KGFR on suprabasal layers and on the basal cells, coincident with the expanded proliferative keratinocyte pool. The down-regulation of KGFR synthesis by UV-B suggests a possible mechanism for the antiproliferative action of this agent in the treatment of psoriasis. Taken together, these results suggest that increased KGFR-mediated signaling in keratinocytes in the lesional epidermis might account, in part, for the epidermal hyperplasia in psoriasis [14]. The oncogenic activation mechanism of receptor tyrosine kinases has been studied extensively in EGFR: gene amplication and

rearrangement, point mutation, deletion, and overexpression. The activation mode of KGFR in carcinogenesis has not been examined, however. In squamous cell carcinoma, there is an increased expression of KGFR in well-dierentiated forms, whereas its loss is associated with a shift to more virulent behavior. In particular settings, signaling through KGFR might serve to maintain epithelial dierentiation, inhibit tumor growth, and constrain tumor invasion [15]. Motility Consistent with its function in promoting wound healing, KGFR enhances the migration of normal human keratinocytes. In neoplastic cells, the overexpressed KGFR results in overexpression of matrix metalloproteinase and urokinase plasminogen activator and the acquisition of invasive properties [13]. Angiogenesis In endothelial cells, KGFR regulates motility, but it does not regulate the other angiogenic responses, such as proliferation and tube formation [16]. Epidermal growth factor receptor Architecture EGFR is a member of the structurally related erbB family of receptor tyrosine kinases, so named for its rst identication as an oncogene encoded by the avian erythroblastosis virus. Four erbB members have been identied: EGFR (ErbB1), human epidermal growth factor receptor (HER)2/Neu (ErbB2), HER3 (ErbB3), and HER4 (ErbB4) [17,18]. The sequence identity of the EGFR family varies from 37% (53% similarity) for EGFR and ErbB3 to 49% (64% similarity) for EGFR and ErbB2 [18,19]. EGFR is a 170-kd glycoprotein, and it is composed of three regions, corresponding with three functional layers. The extracellular region constitutes the input layer, consisting of glycosylated domains (amino-terminal domain). This region, located on the external side of the cell surface, is where ligand binding takes place and signals are received; it is called ectodomain and consists of four domains. The transmembrane region bridges the cellular membrane; here, a single transmembrane domain contains a single hydrophobic anchor sequence. Finally, the output layer lies on the intracellular region that contains the catalytic tyrosine kinase domain and

480

DE GIORGI

et al

a carboxyl-terminal region containing several tyrosine residues that become phosphorylated after receptor activation [1721]. When TGF-a or EGF binds to EGFR, this receptor dimerizes with itself (homodimerization) or other members of the family, such as c-erb B2 (heterodimerization). The dimerization (homodimerization or heterodimerization) occurs across the transmembrane region, forming the signalprocessing layer. The receptor is inactive when it is in a monomer state [1721]. Ligand binding On binding ligands, the tyrosine kinase activity increases and the receptor phosphorylates tyrosine residues on itself (autophosphorylation). Phosphorylated EGFR, like other activated receptor tyrosine kinases, phosphorylates and activates several signal transduction pathways downstream of EGFR, including phosphoinositide 3-kinaseAKT, ERK1/2, and signal transducer and activator of transcription (STAT)-3 [18,22]. EGFR is expressed constitutively in normal skin and hair follicles, and it controls various cell activities, including cell proliferation, apoptosis, cell mobility, and angiogenesis [18]. The protein tyrosine kinase activities of the EGFR family of receptor tyrosine kinase normally are subject to multiple regulatory mechanisms before and after growth factor stimulation. It was shown that deregulation of the protein kinase activity or constitutive tyrosine phosphorylation of the EGFR family plays an important role in a variety of human cancers [23]. EGFR can have two distinct conformations of the extracellular receptor domain that have dierent anity states: a tethered monomeric autoinhibited conguration and an extended dimeric conguration. The monomeric, unoccupied EGFR is maintained in an autoinhibited lowanity state by intramolecular interactions between a specic loop from the cysteine-rich domain II and a binding site on domain IV (designated the tethered conguration) [24]. It is believed that this tethered monomeric conguration is in equilibrium with an extended conguration that exhibits higher ligand-binding anity and is competent for dimer formation and resulting tyrosine kinase activation [19,23]. EGF binding promotes dimerization of EGFR that is driven by receptorreceptor interactions involving the same loop in domain II (the dimerization arm) that is responsible for forming

the intramolecular tether in the monomer [19, 2325]. Localization of EGFR to the cell membrane increases the eective concentration of the receptor, thus enhancing receptor dimerization. More than half of the unstimulated EGFRs on the cell surface are concentrated in caveolae, which account for approximately 5% to 10% of the membrane, thus facilitating dimerization further. The transmembrane and kinase domains have active roles in stabilizing the dimer. This aspect was demonstrated in an experiment in which a recombinant form of EGFR, consisting of only the transmembrane and kinase domains, is capable of self-association. The presence of the transmembrane domain enhances ligand-induced dimer formation in solution [19]. Ligand binding to the EGFR is required to elevate the receptors tyrosine kinase activity. The position-dependent eects of adding a cysteine residue in the membrane-proximal part of the EGFRs extracellular region suggests that a ligandassociated orientation of the EGF dimer is required for the activation of the tyrosine kinase domains. Clearly, dimerization of the EGFR, although necessary, is not sucient to activate the intracellular kinase [26,27]. The ligand-binding induces the predimerized EGFR to twist about a pivot point near or in the transmembrane domain and reorients the intracellular domain to form an active kinase conguration [27,28]. The monomeric EGFR has much reduced kinase activity compared with the dimerized receptor; it is assumed that in the absence of dimerization, the kinase is in an inactive conformation. The ligand binding increases the proportion of dimerized EGFR and the reorientation of the kinase domains in a way that increases the anity for ATP bindingdprobably as a result of conformational changedthereby enhancing the kinase activity [19]. Thus, tyrosine phosphorylation of cellular substrates is the rst and crucial step in transducing EGFR-mediated signals. The EGF stimulation of a cell results in the simultaneous activation of multiple pathways. Often, these pathways are functionally interlinked and, ideally, should not be considered in isolation [19]. Signaling pathways The cascade of biochemical events that leads from EGFR to the activation of the protooncogene Ras and, eventually, the serine/threonine kinase MAPK has been analyzed. The key player in EGF-dependent Ras activation is the

KERATINOCYTE GROWTH FACTOR RECEPTORS

481

adaptor protein Grb2. Grb2 is constitutively bound to the Ras exchange factor Sos and normally is localized in the cytosol. Following activation of the EGFR kinase and autophosphorylation, the SH2 domain of Grb2 can bind to EGFR. Relocation of the Grb2/Sos complex to the receptor at the plasma membrane facilitates the interaction of membrane-associated Ras with Sos, resulting in the exchange of Ras-bound GDP for GTP, and, hence, Ras activation. Activated Ras, in turn, activates the serine/threonine kinase Raf-1. Raf-1 activation, through a series of intermediate kinases, leads to the phosphorylation, activation, and nuclear translocation of Erk-1 and Erk-2, which catalyze the phosphorylation of nuclear transcription factors. Activation of the MAPKs also provides a negative feedback loop for this pathway because the Grb2-Sos complex is dissociated following MAPK phosphorylation of Sos [28,29]. Grb2 and Shc play important roles in the activation of other EGFR-dependent pathways because of their modular construction. Grb2 contains two SH2 domains and one SH3 domain, which enables it to interact with tyrosine-phosphorylated motifs and proline-rich regions of other proteins. Shc can be associated with specic tyrosine-phosphorylated sequences by way of its SH2 and polypyrimidine tract binding protein domain and because it is phosphorylated on tyrosine by activated receptors and cytosolic tyrosine kinases, it serves, in turn, as a binding partner for SH2-containing proteins. SH2 and SH3 domains recognize specic sequences preferentially, but not exclusively. Thus, they can bind to many proteins with dierent anity. For example, Grb2 was shown to complex with proteins involved in cytoskeletal reorganization (eg, focal adhesion kinase and dynamin), with negative regulators of growth factor action (eg, Cb1, Dab-2, and suppressor of cytokine signaling (SOCS)-1, and with the inositol phosphatase SH2 domaincontaining inositol phosphatase. Shc also was detected in complexes with many other proteins, including mitogen-activated protein/ERK kinase-1, which links it to c-jun N-terminal kinase pathway activation, and cadherin, implying a role for Shc in cellcell adhesion [19]. SH2- and SH3-mediated protein interactions are dependent on the anity and the relative concentration of the binding partner; high-anity interactions will be favored at low concentrations of the target molecule, but could be displaced by low-anity interactions driven by high

concentrations of alternative partners. It also is important to recognize that the local abundance of a protein may determine its availability for binding; colocalization of binding partners (eg, at the plasma membrane) will favor interactions, even when the anity is low [19]. In the case of EGFR signaling, the evidence for involvement by members of the Src family of kinases is overwhelming. Overexpression of Src proteins strongly enhances EGF-mediated proliferation and transformation in broblasts and epithelial cells. Conversely, inhibition of Src activity can block EGF-dependent DNA synthesis and reverse the transformed phenotype of EGFRor ErbB2-overexpressing cells. Therefore, Src is a signal transducer downstream of EGFR, but it is possible that the phosphorylation by Src may contribute signicantly to the activation of EGFR [19]. Activated c-Src (cytosolic tyrosine kinase) also is linked intimately to the activation of phosphatidylinositol-3-kinase (PI3-K). The Src-dependent phosphorylation of the EGFR molecule can provide a docking site for p85, presumably facilitating its phosphorylation by EGFR and the consequent activation of PI3-K. Src also directly phosphorylates and activates PI3-K, once again pointing to the large overlap in signal activation by Src and EGFR [19,30]. STAT proteins, in particular STAT-1, -3, and -5, also have been implicated in EGFR signaling. STAT proteins are inactive transcription factors that are activated and translocated to the nucleus upon specic receptor stimulation [19,31]. The mode of activation of STATs by EGFR seems to be signicantly dierent from that used by cytokine receptors. First, the ligand-dependent phosphorylation of STATs by EGFR does not require JAK kinases [19]. Second, STATs do not bind to the C-terminal phosphotyrosines of EGFR; it appears that STATs are constitutively associated with EGFR [32]. As in JAK kinase signaling, however, activation of STAT transcriptional activity is strictly dependent upon the EGFR tyrosine kinase activity [19]. Recent reports have implicated the Src kinase in EGF-dependent STAT activation, but it is unclear whether Src acts upstream or downstream of EGFR activation [19,32]. EGF stimulation of a cell has marked eects on its phospholipid metabolism, including phosphatidylinositol turnover and production of phosphatidic acid (PA) and arachidonic acid. Of the enzymes involved in these pathways, at least three can be activated directly by EGFR (ie, PLCg,

482

DE GIORGI

et al

PI3-K, and phospholipase D [PLD]), whereas others (eg, phospholipase A2) are regulated indirectly by EGF-mediated activation of other pathways [19]. PLD hydrolyzes phosphatidylcholine to generate choline and the second messenger PA. The mechanism of activation of PLD seems to require the physical association of PLD with EGFR, but not necessarily tyrosine phosphorylation. Activation of PLD may require a conformational change that is stabilized, but not induced, by tyrosine phosphorylation; a similar mode of activation (dependent on complex formation, but independent of tyrosine phosphorylation) has been proposed for PLCg, suggesting a common mechanism of activation for this class of molecules [19]. PLCg binds directly to the autophosphorylated EGFR and is phosphorylated by the EGFR kinase; however, for the activation of PLCg, direct association with the receptor is required, but not necessarily tyrosine phosphorylation. Once activated, PLCg catalyzes the hydrolysis of phosphatidylinositols (PtdIns) (4,5)-P2 to yield the important second messengers 1,2-diacylglycerol (DAG) and inositol 1,3,5-trisphosphate (IP3). IP3 mediates calcium release from intracellular stores, aecting a host of Ca2-dependent enzymes, whereas DAG is a cofactor for the activation of the serine/threonine kinase PKC. Thus, through IP3, EGFR can activate Ca2-dependent pathways, such as RaI and nuclear factor-kappa B (NF-kB), and, through PKC, can activate multiple signaling components, including the MAPK and JNK pathways and possibly the Na/H exchange [19]. PI3-Ks are major players in cellular functions, where they contribute to a variety of cellular processes, including proliferation, survival, adhesion, and migration [33]. PI3-Ks catalyze phosphorylation on the 30 position of PtdIns and are assigned to three classes according to their subunit structure and their preferred lipid substrate [34]. Of the three classes of typical PI3-Ks, only class Ia is activated by tyrosine kinase receptors. Interaction between PI3-K and the ErbB receptors is required for activation and is mediated by association of the phosphorylated receptors with the p85 subunit of PI3-K by way of the latters SH2 domain. The major binding partner of p85 is not EGFR, but ErbB3; however, activation of PI3-K is observed in response to EGFR ligands through formation of ErbB1/ErbB3 heterodimers, as well as potentially by Src

phosphorylation of EGFR, so it is relevant to this discussion of EGF-mediated signaling pathways [19]. PI3-K Ia generates phosphatidylinositol-3,4, 5-trisphosphate (PIP3). One of the best-characterized targets of this second messenger is Ser/Thr kinase Akt (PKB), which binds to the lipid and is translocated to the plasma membrane where it is phosphorylated and activated by phosphoinositide-dependent kinase-1 and possibly other kinases [35]. PKB/Akt is a major mediator of PI3-K action in survival and proliferation, and may well be the major mediator of the antiapoptotic eects of EGFR activation [19]. It was observed that the structure of the catalytic subunit of PI3-Kg shows a change in its conformation upon binding to Ras, consistent with a Ras-mediated activation model. Because activated Ras is one of the major downstream eectors of EGFR signaling, this mechanism may represent another way in which activated EGFR regulates PI3-K activity [19]. After activation, the EGFR is internalized, and by this mechanism, it undergoes degradation or recycling that results in transient down-regulation of signaling pathway [31]. Eects on keratinocytes Proliferation and dierentiation EGF/EGFRs have a role in cell proliferation; evidence is accumulating that overexpression of the ligands or receptors, as well as ligandindependent receptor activation, occurs in many epithelial cancers. The overfunction of EGFR is capable of altering the balance between cellular proliferation and death. Continuous EGFR stimulation increases the mitotic index and reduces the rate of apoptosis [36]. Generally, in transgene models, the latency period of tumor tends to be long and the incidence tends to be low, suggesting that the EGFR system provides only one of the steps in multistage carcinogenesis, and neoplastic transformation occurs when other genes within the target tissue also are mutated. Stimulation of the EGFR pathways may override the DNA damage check points, resulting in decreased apoptosis and the accumulation of secondary mutations [19]. Recent research demonstrates that EGFR is a major regulator of the UV response of the skin. UV exposure results in the rapid activation of EGFR by a reactive oxygen intermediate-mediated mechanism. UV triggers receptor

KERATINOCYTE GROWTH FACTOR RECEPTORS

483

phosphorylation by damaging receptor-associated phosphatases, thus blocking deactivation, altering receptor internalization and degradation, and increasing EGFR ligand expression at later time points. Thus, UV exposure results in a short-lived and immediate, as well as a more delayed and prolonged, activation of EGFR. Much of the UV response is due to the activation of MAPK family members and the NF-kB pathway. Among other eects, the EGFR-activated MAPKs phosphorylate p53 and prevent its activation. The activation of EGFR following UV exposure increases skin tumor growth by suppressing UV-induced cell cycle arrest and increasing UV-induced cell proliferation. Therefore, EGFR increases keratinocyte proliferation and epidermal hyperplasia following UV exposure, associated with the induction of cyclin D expression and the suppression of p21 expression. Therefore, chronic EGFR activation upon UV exposure may contribute to the deregulation of cell cycle progression during UV-induced skin carcinogenesis [37]. EGFR also is involved during keratinocyte dierentiation; it was shown that the activated PI3-K/Akt pathway promotes growth arrest and dierentiation [38]. The involvement of EGFR activation also seems to be present in psoriasis-like lesions and hyperkeratosis, eventually accompanied by alopecia or stunted hair growth [19]. Motility The involvement of EGFR signaling in various normal physiologic processes requiring cell movement and deregulation of the motility response in pathologic conditions, such as tumor invasion, is well documented [39]. Although EGFR stimulation can lead to cell proliferation and migration, these responses are separable and mediated by way of dierent signaling pathways. It was demonstrated that the motility elicited by EGFR stimulation requires kinase activity of the receptor and the presence of at least one autophosphorylation site, tyrosine992. The ability of EGF to induce cell movement correlates with the activation of PLCg, and movement can be inhibited by blocking the function of this enzyme. The observation that EGF stimulates MAPK activation in motogenic and nonmotogenic EGFR-expressing cells is consistent with the current view that the motility and mitogenic responses elicited by EGFR diverge at the immediate postreceptor level and that MAPK activation alone is not

sucient to induce a motility response. EGFinduced activation of PLCg stimulates cell motility by releasing PIP2 and gelsolin (and possibly other actin-modifying proteins, prolin, colin, and CapG) from the membrane, thereby restoring its ability to bind, sever, and cap polymerized actin laments, a process required for lopodia/lamellipodia extension and retraction in motile cells. Thus, EGFR-mediated activation of PLCg is believed to be critical for the reorganization of the actin cytoskeleton and contributes to the initiation of the asymmetric motile phenotype [19]. Although not sucient to stimulate motility by itself, MAPK may, in part, regulate cell motility by modulating integrin adhesive functions. The suppression of integrin activity correlates with MAPK activation and seems to result in the loss of cell spreading. The downstream elements of this pathway have yet to be identied and it is still unclear how MAPK activation relates specically to EGFR-mediated cell motility. One possibility is that MAPK stimulates the disassembly of focal adhesions. Of course, disassembly of the focal adhesions results in decreased cell adhesion to the substratum and is associated with initial motility shape change [19]. The family of intracellular calcium-dependent proteases, calpains, also is important for the focal de-adhesion that is necessary during retraction of the trailing edge of migrating cells. Inhibition of calpains results in an elongated, but ultimately immobile, cell. Calpain activation in EGF-stimulated cells coincides with cell compaction, detachment, and enhanced motility. The events leading to activation of calpain are not understood completely and may dier between isoforms. At least two isoforms of calpain, including m-calpain and M-calpain, have been implicated in cell motility through their ability to cleave several proteins found in adhesion complexes [40]. The former may be critical for integrin-mediated motility and seems to be more sensitive to activation by Ca2 uxes, possibly triggered through stretch-activated calcium channels [19]. Another model suggests that basal and EGFdirected haptotactic responses involve direct phosphorylation of myosin light chain kinase (MLCK) by MAPK. This phosphorylation event enhances MLCKs ability to phosphorylate the myosin regulatory light chain, thereby promoting myosin ATPase activity and polymerization of actin cables, a well-established role for myosin-II in the

484

DE GIORGI

et al [2] Dlugosz AA, Cheng C, Denning MF, et al. Keratinocyte growth factor receptor ligands induce transforming growth factor alpha expression and activate the epidermal growth factor receptor signaling pathway in cultured epidermal keratinocytes. Cell Growth Dier 1994;5:128392. [3] Belleudi F, Ceridono M, Capone A, et al. The endocytic pathway followed by the keratinocyte growth factor receptor. Histochem Cell Biol 2002;118:110. [4] Ostrovsky O, Berman B, Gallagher J, et al. Dierential eects of heparin saccharides on the formation of specic broblast growth factor (FGF) and FGF receptor complexes. J Biol Chem 2002;277: 244453. [5] Nagy N, Bata-Csorgo Z, Kopasz N, et al. The expression of keratinocyte growth factor receptor (FGFR2-IIIb) correlates with the high proliferative rate of HaCaT keratinocytes. Exp Dermatol 2006; 15:596605. [6] Baraniak AP, Chen JR, Garcia-Blanco MA. Fox-2 mediates epithelial cell-specic broblast growth factor receptor 2 exon choice. Mol Cell Biol 2006; 26:120922. [7] Visco V, Belleudi F, Marchese C, et al. Dierential response to keratinocyte growth factor receptor and epidermal growth factor receptor ligands of proliferating and dierentiating intestinal epithelial cells. J Cell Physiol 2004;200:3144. [8] Marchese C, Maresca V, Cardinali G, et al. UVB-induced activation and internalization of keratinocyte growth factor receptor. Oncogene 2003;22:242231. [9] Ceridono M, Belleudi F, Ceccarelli S, et al. Tyrosine 769 of the keratinocyte growth factor receptor is required for receptor signaling but not endocytosis. Biochem Biophys Res Commun 2005;327:52332. [10] Belleudi F, Leone L, Aimati L, et al. Endocytic pathways and biological eects induced by UVBdependent or ligand-dependent activation of the keratinocyte growth factor receptor. FASEB J 2006;20:3957. [11] Belleudi F, Visco V, Ceridono M, et al. Ligandinduced clathrin-mediated endocytosis of the keratinocyte growth factor receptor occurs independently of either phosphorylation or recruitment of eps 15. FEBS Lett 2003;553:26270. [12] Marchese C, Felici A, Visco V, et al. Fibroblast growth factor 10 induces proliferation and dierentiation of human primary cultured keratinocytes. J Invest Dermatol 2001;116:6238. [13] Petiot A, Conti FJA, Grose R, et al. A crucial role of Fgfr2-IIIb signalling in epidermal development and hair follicle patterning. Development 2003;130: 5493501. [14] Finch PW, Murphy F, Cardinale I, et al. Altered expression of keratinocyte growth factor and its receptor in psoriasis. Am J Pathos 1997;151:161928. [15] Finch PW, Rubin JS. Keratinocyte growth factor expression and activity in cancer: implications for use

modulation cell contraction. The primary role of the EGF-induced MAPKMLCK pathway in cell locomotion would be in the translocation of the cell body or retraction (rather than de-adhesion) of the trailing edge of migrating cells [19]. The role of PI3-K in EGF-mediated cell movement is less clear. It was reported that EGF-mediated activation of PI3-K leads to translocation of PLCg1 to the leading edge of migrating cells in a wound-healing assay. Migration could be inhibited by expression of the pleckstrin homology domain of PLCg1, providing evidence that this eect was mediated by direct interaction between the PLCg1 PH domain and PtdIns(3,4,5)-P3 [41]. EGF/PI3-K pathways are likely to be critical for the establishment of cell polarity during EGFR-mediated chemotaxis [42]. The directional migration of keratinocytes requires kinase activity and redistribution of the EGF receptor at the leading edge, resulting in asymmetric actin polymerization in migrating cells [43]. Angiogenesis The EGF-mediated activation of PI3-K and Akt induce sprouting of new vessels and enlargement of preexisting vessels [44]. EGFR-mediated pathways are involved in tumor angiogenesis through up-regulation of vascular endothelial growth factor (VEGF) and other mediators of angiogenesis [17]. Activated ras-triggered EGFR signaling modulates the balance of angiogenic factor expression (up-regulation of VEGF and inhibition of Angiotensin-1), leading to the establishment of active tumor angiogenesis [45]. Summary This article reviews the major features of KGFRs and EGFRs structure and function. KGFR and EGFR play dierent roles in the control of keratinocyte proliferation and dierentiation, and their expression may be crucial for such control. Improved knowledge on the molecular signaling mechanisms activated by these two receptors may favor the discovery and development of antagonists for cancer treatment. References
[1] Capone A, Visco V, Belleudi F, et al. Up-modulation of the expression of functional keratinocyte growth factor receptors induced by high cell density in the human keratinocyte HaCaT cell line. Cell Growth Dier 2000;11:60714.

KERATINOCYTE GROWTH FACTOR RECEPTORS

485

[16]

[17]

[18]

[19]

[20]

[21] [22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

in patients with solid tumors. J Natl Cancer Inst 2006;98:81224. Nakamura T, Mochizuki Y, Kanetake H, et al. Signals via FGF receptor 2 regulate migration of endothelial cells. Biochem Biophys Res Commun 2001; 289:8016. Ramsay Camp E, Summy J, Bauer TW, et al. Molecular mechanism of resistance to therapies targeting the epidermal growth factor receptor. Clin Cancer Res 2005;11:397405. Yarden Y, Sliwkowski MX. Untangling the erbB signalling network. Nat Rev Mol Cell Biol 2001;2: 12737. Jorissen RN, Walker F, Pouliot N, et al. Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res 2003;284:3153. Herbst RS, Shin DM. Monoclonal antibodies to target epidermal growth factor receptor-positive tumors: a new paradigm for cancer therapy. Cancer 2002;94:1593611. Wujcik D. EGFR as a target: rationale for therapy. Semin Oncol Nurs 2006;22:59. Nunes M, Shi C, Greenberger LM. Phosphorylation of extracellular signal-regulated kinase 1 and 2, protein kinase B, and signal transducer and activator of transcription 3 are dierently inhibited by an epidermal growth factor receptor inhibitor, EKB-569, in tumor cells and normal human keratinocytes. Mol Cancer Ther 2004;3:217. zcan F, Klein P, Lemmon MA, et al. On the nature O of low- and high-anity EGF receptors on living cells. PNAS 2006;103:573540. Garrett T, McKerm N, Lou M, et al. Crystal structure of a truncated epidermal growth factor receptor extracellular domain bound to transforming growth factor a. Cell 2002;110:76373. Ogiso H, Ishitani R, Nureki O, et al. Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains. Cell 2002; 110:77587. Yu X, Sharma KD, Takahashi T, et al. Ligand-independent dimer formation of epidermal growth factor receptor (EGFR) is a step separable from ligandinduced EGFR signaling. Mol Biol Cell 2002;13: 254757. Moriki T, Maruyama H, Maruyama IN. Activation of preformed EGF receptor dimers by ligandinduced rotation of the transmembrane domain. J Mol Biol 2001;311:101126. Liebmann C. Regulation of MAP kinase activity by peptide receptor signalling pathway: paradigms of multiplicity. Cell Signal 2001;13:77785. Pouyssegur J, Volmat V, Lenormand P. Fidelity and spatio-temporal control in MAP kinase (ERKs) signalling. Biochem Pharmacol 2002;64:75563. Kloth MT, Laughlin KK, Biscardi JS, et al. STAT 5b, a mediator of synergism between C-Src and the EGF receptor. J Biol Chem 2003;278:16719.

[31] Leonard WJ. Role of Jak kinases and STATs in cytokine signal transduction. Int J Hematol 2001;73: 2717. [32] Xia L, Wang L, Chung AS, et al. Identication of both positive and negative domains within the epidermal growth factor receptor COOH-terminal region for signal transducer and activator of transcription (STAT) activation. J Biol Chem 2002; 277:3071623. [33] Cantley LC. The phosphoinositide 3-kinase pathway. Science 2002;296:16557. [34] Djordjevic S, Driscoll PC. Structural insight into substrate specicity and regulatory mechanisms of phosphoinositide 3-kinases. Trends Biochem Sci 2002;27:42632. [35] Nicholson KM, Anderson NG. The protein kinase B/Akt signalling pathway in human malignancy. Cell Signal 2002;14:38195. [36] Ferby I, Reschke M, Kudlacek O, et al. Mig 6 is a negative regulator of EGF receptor-mediated skin morphogenesis and tumor formation. Nat Med 2006;12:56873. [37] El-Abaseri TB, Putta S, Hansen LA. Ultraviolet irradiation induces keratinocyte proliferation and epidermal hyperplasia through the activation of the epidermal growth factor receptor. Carcinogenesis 2006;27:22531. [38] Calautti E, Li J, Saoncella S, et al. Phosphoinositide 3-kinase signalling to Akt promotes keratinocyte dierentiation versus death. J Biol Chem 2005;280: 3285665. [39] Kassis J, Lauenburger DA, Turner T, et al. Tumor invasion as dysregulated cell motility. Semin Cancer Biol 2001;11:10517. [40] Glading A, Lauenburger DA, Wells A. Cutting to the chase: calpain proteases in cell motility. Trends Cell Biol 2002;12:4654. [41] Piccolo E, Innominato PF, Mariggio MA, et al. The mechanism involved in the regulation of phospholipase Cgamma1 activity in cell migration. Oncogene 2002;21:65209. [42] Weiner OD. Rac activation: P-Rex1- a convergence point for PIP(3) and Gbetagamma? Curr Biol 2002; 12:R42931. [43] Zhao M, Pu J, Forrester JV, et al. Membrane lipids, EGF receptors, and intracellular signals colocalize and are polarized in epithelial cells moving directionally in a physiological electric eld. FASEB J 2002; 16:8579. [44] Jiang BH, Zheng JZ, Aoki M, et al. Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells. Proc Natl Acad Sci U S A 2000; 97:174953. [45] Casanova ML, Larcher F, Casanova B, et al. A critical role for ras-mediated, epidermal growth factor receptor-dependent angiogenesis in mouse skin carcinogenesis. Cancer Res 2002;62:34027.

Dermatol Clin 25 (2007) 487501

Death Receptors and Apoptosis

Emmanuel Contassot, PhDa, Olivier Gaide, MD, PhDb, Lars E. French, MDa,*
a

Department of Dermatology, Zurich University Hospital, Gloriastrasse 31, 8091 Zurich, Switzerland b Departments of PathologyImmunology and Dermatology, University Medical Center and University Hospital, Geneva, Switzerland

Cell death by apoptosis is a tightly regulated physiologic process essential for embryonic development and the maintenance of tissue homeostasis, which relies on the elimination of old, superuous, damaged, or infected cells. Deregulation of the apoptotic process, leading to either increased or decreased cell death, can contribute to the pathogenesis of various diseases including autoimmune diseases or cancer. Apoptosis is characterized by morphologic and biochemical changes caused by activation of a family of proteases called caspases, which are pre-existing as inactive zymogen precursors in the cell. Activation of caspases is the central event in apoptosis, leading to the cleavage of numerous proteins involved in the cell structure, cell cycle control, and DNA synthesis and repair. A subfamily of the tumor necrosis factor receptor (TNF-R) superfamily can recruit and activate caspases in response to specic extracellular signals. Because the outcome of caspase recruitment generally is cell death, these receptors are known as death receptors. However, they may alternatively drive survival or dierentiation signals as well. During the last decade, major advances have been made in the understanding of the function of death receptorinduced apoptosis in both physiologic and pathological conditions. In this review, we focus on the expression and function of these death receptors in normal skin and in pathological situations involving the skin.

Death receptors and apoptosis signaling TNF and TNF-R harbor a conserved genetic arrangement and protein structure. Although the TNF family of genes are scattered on many chromosomes, clustering of some subfamilies to precise regions of the genome may indicate that TNFTNF-R super family members have evolved from a common ancestor protogene. One example is the direct TNF subfamily (TNF, LTa, LTab2) genes, which are all conned to the same locus (6p21.3). The same holds true for TNF-Rs and, strikingly, both the TNF-R1 and LTb-R (the receptors for TNF, LTa, LTab2) are also located on the same chromosomal region (12q13). Most of the receptor genes also show a similar arrangement of 10-11 exons, of which, one codes for the entire transmembrane domain and another one for an entire cytoplasmic signaling domain (such as the death domain [DD]), when present [13]. However, classication in the TNFTNF-R families is based on their unique structural properties, and not gene structure. The TNF family ligands are type II (C-terminal out) transmembrane proteins characterized by a conserved extracellular domain called the TNF homology domain. The TNF homology domain is a 150amino acid long sequence forming two stacked b-pleated sheets each formed by 5 antiparallel b-strands. One of the b-pleated sheets is involved in trimerization of the ligand, whereas the other is exposed at the surface and is involved in receptor binding [4,5]. Likewise, TNF-R family of receptors are either type I or type III transmembrane proteins characterized by cysteine-rich sequences in their extracellular domain, arranged in a precise pattern
derm.theclinics.com

* Corresponding author. E-mail address: lars.french@usz.ch (L.E. French).

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.010

488

CONTASSOT

et al

yielding a tertiary structure of b-strands and loops maintained by disulde bridges, which are responsible for ligand recognition. A subset of the TNF-R family carry an intracellular proteinprotein interaction motif, which consists of 6 a helixes tightly packed in a globular core with conserved structure. Because mutations in this domain prevented the induction of apoptosis by TNF-R, it was called a death domain (DD), and the receptors carrying it, death receptors (DR). To date, eight DR, namely Fas (CD95, Apo-1), TRAIL-receptor 1 (TRAIL-R1) (DR4) and 2 (DR5, Apo-2), TNF-R1, TRAMP (WSL-1, Apo-3), EDAR, p75 neurotrophin receptor (p75NTR), and DR6 have been identied (Fig. 1). Despite their name, not all of these death receptors induce apoptotic cell death. Indeed, each receptor may trigger specic signaling pathways, thus resulting in a variety of cellular outcomes. The prototypical DR signaling pathway is the pathway triggered by Fas. Upon exposure to its ligand, the receptor multimerizes, thus recruiting the adaptor protein FADD (Fas-

associated death domain) through binding to homotypic interaction of their death domain (DD DD) [6]. FADD, in turn, binds caspase-8 via homotypic interaction involving death eector domains (DED); a proteinprotein interaction motif that shares the same overall structure (ie, 6 similarly arranged alpha helixes) as the DD (Fig. 2). The resulting complex formed is called a death-inducing signaling complex (DISC). The recruitment of caspase-8 through FADD within the DISC leads to autoprocessing of caspase-8, an event responsible for its activation [7]. Active caspase-8, in turn, activates eector caspases such as caspase-3 and -7 causing cells to undergo apoptosis as a consequence of the cleavage of various vital cellular proteins [8]. In the death receptor subfamily, EDAR and p75NTR have dierent properties than the other DR in that they are unable to recruit FADD. Not surprisingly, these receptors are also unable to induce cell death. This is because DDDD interactions are relatively specic. For example, the DD of EDAR interacts with the DD of

Fig. 1. Death and decoy receptors and their respective ligands. All death receptors are type I proteins containing cysteine-rich domains. Death receptors contain a cytoplasmic sequence named death domain (DD). Decoy receptors (DcR) lack the DD.

DEATH RECEPTORS AND APOPTOSIS

489

Fig. 2. Death receptors can mediate pro-apoptotic or survival signals. Ligation of membrane-bound FasL to its receptor Fas. Fas recruits the Fas-associated death domain (FADD) through direct homotypic binding of their respective death domains (DD). FADD binds caspase-8 via homotypic interaction involving death eector domains (DED). The resulting complex formed is the death-inducing signaling complex (DISC). The recruitment of caspase-8 to the DISC leads to its activation. Active caspase-8, in turn, activates downstream eector caspases such as caspase-3 and -7 causing cells to undergo apoptosis. A cellular inhibitor of caspase-8 is FLIP. Downstream caspases can be inhibited by the inhibitory apoptosis proteins (IAPs) such as baculoviral p35, Crm-A, or SERP-2. Soluble EDA binds to its receptor EDAR, which recruits the cytoplasmic adaptor EDARADD. EDARADD, in turn, binds Traf, which leads to the activation of the IKK complex. This complex then phosphorylates IB, which is subsequently degraded, thus allowing the release of NFkB.

EDARADD (EDAR-associated protein with a DD) but not with the DD of FADD. Furthermore, TNF-R1 can recruit and activate caspase-8 through interactions with FADD, yet it does not always induce cell death. This is because TNF engagement of TNF-R1 also leads to the rapid recruitment of TRADD, TRAF2, and RIP-1, which in turns activates IKKg (by a still unknown mechanism) and results in NF-kB activation. This rapid generation of a pro-survival signal may block the function of a second, slower-forming complex recruited by TNF-R, consisting of TRADD, FADD, and caspase-8 [9]. Death receptor signaling is a tightly regulated process, achieved by controlling: (1) the expression of the ligand; (2) the proteolytic cleavage of the ligand from the cell membrane; (3) the

expression level of the receptor; (4) the expression of decoy receptors; (5) the recruitment to the death-signaling complex of intracellular inhibitors such as FLIP or (6) the control of caspase activation. Each of the above DR signaling control systems is detailed further below. Control of ligand and receptor activity is achieved by regulated expression, both in time and location. In the immune system, for example, constitutive expression of the cytotoxic FasL is restricted to immunoprivileged tissues [10]. Cellular levels of the adaptor proteins and their transduction partners allow further regulation of the cell response. As mentioned above, TNF ligands are expressed as membrane-bound proteins. The biologic activity of TNF ligands is aected very

490

CONTASSOT

et al

dierently by their solubilization. Whereas TNF is fully active in a soluble trimeric form, FasL is 1000 times less active than its membrane bound precursor, despite an ecient binding to the receptor [11]. However, upon cross-linking with an antibody, the soluble form of FasL becomes fully active. In vivo, FasL aggregation is probably mediated by its multimerization within the cell membrane of the activated T cell. The shedding of the ligand may thus represent a way to inactivate the ligand. Hence, solubilization of TNF ligandsddepending on the nature of the ligandd can either have little eect on activity (TNF) or totally abrogate it (FasL). Interestingly, this shedding is an absolute prerequisite for the activity of EDA, the ligand for EDAR (see below). In addition to receptors able to signal apoptosis, decoy receptors to FasL, namely DcR3 [12], and to TRAIL, namely TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2), have been identied (see Fig. 1) [13,14]. When overexpressed, these decoy receptors can compete with DR thus allowing the cell to escape death ligandinduced killing. Finally, cellular inhibitors of DR signaling such as FLIP also play an important role in the control of DR activity. FLIP is a homolog of viral FLIP [15], which has a high homology to procaspase-8 and -10 but lacks an active site [16]. When expressed at high levels, FLIP binds Fas or TRAIL-DISC via its DED and prevents the recruitment and activation of caspase-8 (by competing for binding sites). The activity of caspases is further regulated by the inhibitory apoptosis proteins (IAPs), a family of proteins that blocks caspase activation [17]. To date, six homologs of the baculoviral IAP p35 have been identied [18]. The role of death receptors in normal skin Fundamental research in the TNF eld has generated a considerable knowledge with respect to their physiologic function and their involvement in the pathogenesis of disease. Most TNF-R family members play a role in the development and function of the immune system [19]. The TNFTNF-R superfamily and death receptors are also involved in skin biology and disease. Of the ve currently known diseases that are linked directly to genetic mutations of TNF-R family members, three are caused by death receptor mutations [4]. The Fas pathway is defective in an autoimmune lymphoproliferative syndrome (ALPS; OMIM 601,859), the TNF receptor is aected in a familial periodic fever syndrome

(TRAPS; OMIM 142,680), and hydrotic ectodermal dysplasia (HED; OMIM 305,100) results from alterations in the EDAR signaling pathway. The expression and function of DRs and their ligands is reviewed below and summarized in Table 1. Expression and function of Fas and Fas-ligand in the skin To date, Fas and FasL are the best-studied death-receptor ligand pair in the epidermis. In normal epidermis, membrane Fas is expressed by basal and suprabasal keratinocytes [2023]. In vitro, Fas expression is found on primary keratinocytes, and keratinocyte apoptosis can be induced by agonist antibodies or soluble ligands [22,24,25]. In basal conditions, FasL is also weakly expressed in the epidermis [25,26]. The reasons for which the coexistence of Fas and FasL in the epidermis is not associated with spontaneous apoptosis in a normal skin is still somewhat controversial. Electron microscopy studies suggested that FasL can be detected at the cell surface of resting keratinocytes [26], but results of more recent FACS, confocal, and immunoelectron microscopy studies suggest that FasL is largely intracellular and therefore not accessible to Fas expressed at the surface of keratinocytes under physiologic conditions [27]. Apoptosis in the epidermis: a rampart against tumorigenesis? A key physiologic function of the death receptor-ligand pair resides in the protection against mutagenic eects of UV radiation. The skin is a major target of UV radiation and its damaging eects. One of the hallmarks of acutely UVexposed skin is the presence of sunburn cells (SC) [28,29], which have been identied as apoptotic keratinocytes [28]. A signicant biological response to acute UV light exposure is DNA damage, p53 up-regulation, and the generation of SC [30,31]. Chronic exposure to UV radiation results in mutations of p53, thus decreasing its capacity to trigger cell death, and this is associated with subsequently increased proliferation and tumorigenesis of keratinocytes [3234]. Although it has been shown that p53 is an important regulator of apoptosis in the skin, there is evidence that Fas and FasL also play a role in the balance between life and death after UV radiation. Murine models have revealed a transient up-regulation of Fas and FasL in UVexposed keratinocytes, whose interaction was shown to lead to SC formation [35]. Consequently,

DEATH RECEPTORS AND APOPTOSIS

491

the Fas-FasL pathway is thought to play a role in preventing the accumulation of cells with mutated p53. Interestingly, UVB also has the capacity to directly activate Fas without the need for FasL [36,37]. In contrast to short-term UV exposure, chronic, long-term exposure of keratinocytes to UV light leads to a down-regulation of Fas expression. This decreased expression is correlated with a decreased sensitivity to FasL-induced cell death, thus potentially contributing to an inappropriate cell death response to UV and subsequently to tumorigenesis. Direct proof of a Fas-FasL defect contributing to keratinocyte tumorigenesis is lacking, however. In humans, progression from the precancerous state of actinic keratosis to the invasive skin tumor that is squamous carcinoma, is accompanied by an up-regulation of FasL and a downregulation of Fas and of the TRAIL receptors [38]. Increased expression of FasL, TRAIL, and the death receptor signaling inhibitor FLIP in squamous cell carcinoma may then contribute to the immune escape of the tumor, whereas the lack of expression of Fas and TRAIL receptors has been suggested to prevent autolysis of the tumor [38]. Similarly, in human basal cell carcinoma, tumor cells have low to undetectable levels of Fas expression, whereas FasL expression is quite strong [39,40]. The molecular mechanism of loss of Fas expression in these cells is unknown but could be an indirect consequence of the oncogenic process [41,42]. Finally, FasL has been shown to be a significant eector molecule in UV-induced immunosuppression, a process believed to be critical in the

outgrowth and metastatization of skin cancers [43,44]. Apoptosis and keratinocyte dierentiation The epidermis is constantly subjected to environmental insults, such as chemical and physical injuries, and is a continually renewing tissue. The integrity of the skin is regulated by a balance between proliferation, dierentiation, and cell death. In normal skin, apoptosis is thought to play a major role in the maintenance of this equilibrium. Programmed cell death occurs during the terminal dierentiation of keratinocytes, a phenomenon known as cornication. Although sharing common features including caspase-3 activation, cornication and apoptosis are thought to be two distinct mechanisms in the epidermis [4548], and there is no evidence for a role of DRs in the formation of the stratum corneum. Moreover, there exists indirect evidence that DRs do not play a major role in keratinocyte dierentiation because: (1) mice decient for Fas (lpr/lpr), FasL (gld/gld), or TRAIL (TRAIL /) exhibit normal skin and (2) in humans, no sign of impaired formation of the stratum corneum has been observed in patients with lymphoproliferative syndromes caused by Fas [4951] or FasL mutations [52]. Expression and function of TRAIL receptors and their ligands in the skin Implication of the TRAIL/TRAIL receptor in skin biology has been the subject of fewer

Table 1 Expression and regulation of death receptors and ligands in the skin Molecule I. FasL Fas Cells Keratinocytes, bone marrowderived cells Basal keratinocytes Inducing factor UV, IFN-g, TNF, IL-1, .Doxorubicin, methotrexate UV (low dose) IFN- a/g, TNF Cisplatin Doxorubicin, methotrexate, cisplatin Doxorubicin, methotrexate, cisplatin, curcumin UV Danger signals Repressing factor

UV (high dose)

II. TRAIL TRAIL-R

Keratinocytes R1: suprabasal keratinocytes R2: granular layer keratinocytes R3, R4: keratinocytes Not expressed in normal conditions R1: all skin cells R2: bone marrowderived cells, DC and eccrine sweat ducts Keratinocytes Keratinocytes

UV (high dose) UV (high dose) UV (high dose)

III. TNF TNF-R

IV. EDA EDAR

Lef-1/b-Catenin? EDAR/NFkB

EDAR/NFkB

Abbreviation: IFN, interferon.

492

CONTASSOT

et al

investigations. Immunohistochemical analyses show that TRAIL and its receptors are constitutively expressed in normal skin. Although TRAIL-R1 is expressed mostly in the suprabasal layer, TRAIL-R2 is mainly found in the granular layer, TRAIL-R3 in the basal layer, and TRAILR4 is weakly expressed in the suprabasal layer [38,53]. TRAIL was detected in the cytoplasm of keratinocytes in the epidermis and the infundibulum and outer root sheath of hair follicles. Interestingly, TRAIL expression was not dierent in skin with acute sunburn or after photoprovocation testing, indicating that acute UV exposure does not affect its expression. However, TRAIL expression was found to be signicantly reduced in both chronically UV-exposed skin of elderly individuals and in UV-induced skin tumors such as basal and squamous cell carcinoma. In contrast, TRAIL expression was not altered in keratoacanthomas [53]. TRAIL induces apoptosis selectively in malignant cells and has therefore been proposed to act as a natural guardian eliminating transformed cell [5355]. It is possible that the down-regulation of TRAIL in elderly patients with chronic UV exposure may contribute to the increased risk of skin cancer observed in this population. In vitro, high doses of TRAIL are not able to trigger signicant levels of apoptosis in proliferating primary keratinocytes. However, low-dose irradiation could sensitize cultured primary keratinocytes by selectively down-regulating the expression of TRAIL decoy receptors [56]. At higher doses of UV radiation, TRAIL and TRAIL- receptor expression were decreased, thus blocking TRAIL-mediated elimination of damaged cells [38]. Whether the TRAIL/ TRAIL-receptor pair plays a role in the elimination of damaged cells after acute UV exposure in vivo remains to be determined. In contrast to primary keratinocytes, transformed keratinocyte cell lines were found to be very sensitive to TRAIL-induced cell death [54]. Interestingly, activation of the NF-kB pathway by the cytokine IL-1b could block TRAIL but not UV-induced cell death, suggesting that TRAIL plays only a minor role in UV-induced apoptosis of transformed cell death in vitro [57]. Expression and function of TNF-R1 and TNF-R2 and its ligand in the skin Although the expression of TNF-R1 is constitutive in most cell types, the expression of TNF-R2 is much more important in hematopoietic and

endothelial cells. In normal skin, TNF-R1 is expressed by keratinocytes and by a network of upper dermal dendritic cells [58]. TNF-R2 expression is restricted to eccrine sweat ducts and dermal dendritic cells and is absent within the epidermis [58]. TNF is not signicantly expressed in noninamed skin. TNF binding to its receptor can simultaneously activate several signaling pathways, including those that promote or inhibit apoptosis. The selection of the dominant pathway isdas previously indicatedddetermined by the intracellular concentration of TRAF2 and RIP-1. The biological eects of TNF are numerous and beyond the scope of this review. TNF is involved in the pathogenesis of several diseases including skin diseases such as psoriasis, granulomatous skin disease, and contact hypersensitivity. These topics are well reviewed elsewhere and will therefore not be addressed here [59,60].

Expression and function of EDAR and its ligand in the skin HED was clinically described long before the discovery of the TNF family. Indeed, the specic morphologic features of the disease, which include absent or abnormal teeth, sparse thin hairs, and the inability to sweat, were reported as early as 1875 by Charles Darwin [61] who described an Indian family suering from HED. His description of the inheritance pattern of the disease clearly suggested a recessive X-linked transmission, only 10 years after the publication of Gregor Mendels laws of genetics. Positional cloning of the defective genes in both humans and spontaneous mouse mutants led to the identication of the TNF family ligand EDA [62], its receptor EDAR, a TNF receptor homolog [63], and of EDARADD, an adaptor protein linking EDAR to a more general downstream signaling pathway [64]. EDAR triggering does not lead to cellular death, but rather leads to the activation of components of the NF-kB pathway, such as TRAF-6, IKKg (NEMO) and I-kB, which have been shown to act downstream of EDAR [65,66]. However, other TNFTNFRSF pathways require these signaling molecules as well, and HED symptoms are combined with immunodeciency, osteopetrosis, and lymphoedema when these proteins are defective (HEDID, OMIM 300,291 and OLEDAID, OMIM 300,301) [66].

DEATH RECEPTORS AND APOPTOSIS

493

The EDA molecule contains an extracellular protease cleavage site followed by a collagen domain and a C-terminal TNF homology domain. Extensive analysis of coding mutations from HED patients revealed a clear clustering of the mutations in the protease cleavage site as well as in the collagen and TNF homology domains [67]. The analysis of the expression pattern of EDA and EDAR and of the eect of mutations in vitro and in vivo is compatible with a scenario in which EDA is rst released in a soluble form by proteolysis, enabling the ligand to reach the spatially separate receptor-expressing cells. EDA then engages three EDAR molecules via its homotrimeric TNF homology domain. This alone appears insucient to trigger signaling and suggests that EDA, like the TNF family ligand FasL, requires further multimerization to gain biological activity [11]. In vitro experiments conrmed that the collagen domain of EDA, mimicking its homolog in the rst component of complement C1q, could play this aggregating role. Treatment of HED is still limited to the use of prosthetics for teeth and hair and adaptive behavior for body temperature regulation. Taking into account our knowledge of the essential functional features of EDA, we have been able to produce a recombinant form of the ligand that can stably revert the murine form of HED [68]. These results raise the possibility that HED may become one of the few human genetic diseases that can be cured by treatment with a recombinant protein.

Death-receptormediated apoptosis in cutaneous diseases Dysregulation of death receptor and/or ligand expression and function is known to occur in certain cutaneous diseases, such as toxic epidermal necrolysis, graft-versus-host disease (GVHD), and skin cancers. Although the evidence is not always direct, below we review the current knowledge in the eld as summarized in Table 2. Toxic epidermal necrolysis Toxic epidermal necrolysis (TEN; Lyells syndrome) is a life-threatening cutaneous adverse drug reaction in which apoptotic epidermal cell death results in the separation of large areas of skin at the dermo-epidermal junction, producing the appearance of scalded skin [69]. TEN occurs at an estimated incidence of 0.4 to 1.2 cases per million, most frequently as a result of sulfonamide,

anticonvulsvant, or nonsteroidal anti-inammatory drug use, and is associated with a mortality rate of about 30%. Eective therapeutic approaches or preventive measures are badly needed and are likely to result from an improved understanding of the molecular pathogenesis of the disease. To date, the precise sequence of molecular and cellular events that lead to the development of TEN after selected drug intake is only partially understood. The skin is the rst site of tissue damage at the onset of TEN [69], and keratinocyte apoptosis is a hallmark of early stages of TEN, and thus the rst clear morphologic sign of specic tissue damage in this disease. We have shown that keratinocyte apoptosis in lesional skin of patients with TEN is associated with highly increased keratinocyte FasL expression, together with conserved levels of keratinocyte Fas expression in vivo [25]. We have also shown that keratinocyte FasL is cytolytically active in TEN. This cytolytic activity can be blocked with monoclonal antibodies that interfere with the interaction of Fas and FasL, thus supporting the hypothesis that increased keratinocyte FasL expression is responsible for the keratinocyte apoptosis that characterizes TEN. The model we currently favor is therefore that in normal skin, low levels of FasL are expressed by keratinocytes and localized intracellularly. In lesional skin during TEN, high levels of FasL and Fas are expressed by keratinocytes such that the conditions are present for interaction between keratinocyte Fas and FasL on adjacent keratinocytes. Because Fas and FasL are coexpressed on a very large number of keratinocytes in lesional skin during TEN, keratinocyte apoptosis can potentially be very abundant and may explain the observed destruction of large areas of epidermis. It is now well accepted that various cutaneous drug reactions are associated with drug-specic T cell inltration of the skin [70,71]. There is good evidence for CD8 involvement in TEN, because these cells are consistently reported to be the most common inltrating cell in TEN histology, although usually only present in small numbers within the epidermis [72]. They are also the predominant cell type in TEN blister uid [73]. High expression of perforin, granzyme, and FasL in mononuclear cells in TEN blisters suggests moreover that the CD8 cells are activated [74]. Recently the concept of specic drug-induced cytotoxicity has been taken further by the direct demonstration, in TEN-derived CD8 lymphocytes, of drug-specic MHC class I cytotoxicity,

494

CONTASSOT

et al

Table 2 Therapeutic targets of death receptor signaling pathway in the skin DR pathway Fas/FasL Disease Toxic epidermal necrolysis (TEN) Modulation Intravenous immunoglobulins (IVIG) contain anti-Fas antibodies Anti-Fas blocking antibody (n.a.i.c) Recombinant soluble Fas:Fc IVIG contain anti-Fas antibodies Anti-Fas blocking antibody (n.a.i.c) Recombinant soluble Fas:Fc Recombinant soluble FasL to selectively kill tumor cells Interferon-a (intralesional) induces Fas-expression and promote tumor involution UV light induces Fas-mediate apoptosis of skin inltrating lymphocytes (amongst other eects) UV light and methotrexate induce Fas-mediate apoptosis of skin inltrating lymphocytes (amongst other eects) Recombinant soluble TRAIL to selectively kill tumor cells Recombinant soluble TRAIL to selectively kill tumor cells Anti-TNF blocking antibody Anti-TNF blocking antibody Anti-TNF blocking antibody Isolated limb perfusion with recombinant TNF and melphalan Recombinant Fc:EDA fusion molecule Anti-EDAR activating antibody

GVHD

Melanoma BCC Contact hypersensitivity Psoriasis

TRAIL/TRAIL-R

Melanoma BCC

TNF/TNF-R

Psoriasis Granulomatous disease (sarcoidosis, foreign body granuloma) Hidradenitis suppurativa Melanoma Hypohidrotic ectodermal dysplasia

EDA/EDAR

Abbreviation: n.a.i.c, not available in clinic.

mediated by granzyme but not by Fas or TRAIL. However, this cytotoxicity was shown in vitro against lymphocytes and not keratinocytes. Because Fas is expressed in keratinocytes, the role of FasL-mediated killing by activated CD8 T cells in TEN physiopathology remains to be demonstrated. Acute cutaneous graft-versus-host disease The therapeutic potential of allogeneic hematopoietic stem cell transplantation relies on the graft-versus-leukemia (GVL) eect in which donor T cells eradicate tumor cells expressing host or tumor associated antigens. Unfortunately, GVL eect is often associated with GVHD. GVHD is a major complication of hematopoietic stem cell transplantation, leading to signicant morbidity and mortality [75]. The rst phase of acute GVHD occurs before the donor cells are even infused. Tissue damage caused by factors such as underlying disease, treatment of underlying disease, infection,

and transplantation preconditioning can lead to cellular activation and release of inammatory cytokines including TNF, interleukin (IL)-1, and IL-6. These cytokines induce the up-regulation of host antigens and adhesion molecules allowing donor T cells to respond to host antigens. Damage to the gastrointestinal mucosa induced by preconditioning enables bacteria and bacterial endotoxins to enter the systemic circulation from the gastrointestinal tract thus increasing the secretion of inammatory cytokines from macrophages [76]. Regardless of the source of hematopoietic stem cells and the donor or the host characteristics, TNF plays a crucial role in the pathophysiology of GVHD [7779]. Infusion of anti-TNF antibodies has been shown to be of benet for transplanted hosts [80,81]. TNF is involved in a multistep process during acute GVHD. First, TNF activates dendritic cells and enhances antigen presentation. Second, TNF recruits eector cells, neutrophils, and monocytes into target tissues. Third, TNF causes direct tissue damage by

DEATH RECEPTORS AND APOPTOSIS

495

inducing apoptosis and necrosis [82,83]. Acute GVHD results from the attack of transplanted donor lymphocytes against the host tissues. In the eector phase of acute GVHD, target tissues including skin, liver, and gut are attacked by donor cytotoxic T lymphocytes [84]. As a result of target cell killing by donor T cells, increased apoptosis within target tissues is a key element of the pathogenesis of the immune-mediated skin, gut, and liver damage in acute GVHD [78,85]. Several reports have suggested a pivotal role of Fas/FasL interactions in the destruction of target tissues in the transplanted hosts. Using cytotoxic T cells from mice decient in both functional Fas ligand and perforin [86,87], it has been shown that development of lethal acute graft-versus-host disease in appropriate transplantation models can be virtually completely prevented [88,89]. Delayed mortality was also observed in animals grafted with cells decient in only one of the major cytolytic pathways (perforin or FasL). Recipients grafted with allogeneic FasL-defective donor T cells only exhibited minimal signs of hepatic and cutaneous GVHD pathology compared with controls [88]. Interestingly, Tsukada and colleagues [90] reported recently that selective inactivation of Fas, perforin, or TNF all signicantly inhibit murine acute GVHD. Whereas blockade of TNF results in the inhibition of both GVHD and GVL, blockade of Fas inhibits GVHD but not GVL reaction, suggesting that FasL and Fas may be an important therapeutic target. Similarly, inhibition of FasL with antibodies has been shown to have a therapeutic eect on murine acute GVHD [91,92]. Surprisingly, recent work has also found that CD8 T cells decient in both FasL and perforin were still able to induce acute GVHD in mice [93]. In addition to its cytolytic action, FasL was shown in this study to be required for the contraction of alloreactive T cells. Taken together, the results of these studies of acute GVHD show that lymphocyte FasL, which is induced during acute GVHD, is involved in the immune response after hematopoietic stem cell transplantation. Melanoma Fas/FasL signaling Changes in Fas expression and function have been reported in melanoma [9496]. In hematopoietic and solid tumors, absence of Fas expression often is associated with resistance to chemotherapeutic drugs [51,97]. In melanoma,

absence of Fas expression by melanoma cells is associated with a poor prognosis [98]. Whereas Fas signaling is functional in the early phases of melanoma development, it is frequently impaired in the metastatic phase [96,99,100]. Several mechanisms leading to nonfunctional Fas signaling in advanced melanomas could be involved. Mutations of N-ras and H-ras play a role in melanoma promotion [101103]. Because activated H-ras and N-ras can cause resistance to FasL-mediated apoptosis [41,42,104], it is likely that Fas downregulation may contribute to the pathogenesis of melanoma. Several studies have reported alterations of the p53 gene occurring in 1% to 5% of primary melanomas and 11% to 25% of metastatic melanomas [105]. Because p53 is known to trigger the expression of DRs such as Fas, alterations of p53 in advanced melanomas could also contribute to nonfunctional Fas signaling [106108]. In addition to a loss of expression of Fas, mutations in its DD have been reported in 7% of metastatic melanomas [95]. Overexpression of FLIP has also been suggested to be responsible for resistance to FasL in melanomas [109111]. In vitro data provide evidence that down-regulation of FLIP with the use of cisplatin [112], siRNA [113], or UVB [114] can restore the sensitivity of melanoma cells to FasL and TRAIL. In addition to FLIP, other downstream molecules, such as the members of the Bcl-2 family, can regulate apoptosis induced by death receptors. Indeed, high levels of the anti-apoptotic molecule Bcl-2 have been found to contribute to the resistance to FasL in melanoma [115], and the silencing of Bcl-2 and Bcl-xL, an other anti-apoptotic factor, induces apoptosis in melanomas at dierent stages. Similarly, decreased expression of the proapoptotic Bcl-2related proteins Bax and Bak are associated with poor prognosis in melanoma patients [116,117]. Tumor progression often occurs in spite of CD8 T cell inltration within the tumor in melanoma patients. Tumor cells, by expressing FasL, have, in principle, the ability to kill activated Fas-expressing CD8 cells and therefore escape elimination by the immune system [118,119], although this remains controversial [120]. TRAIL/TRAIL-receptor signaling The role and functions of TRAIL and its receptors have been less studied. TRAIL is a promising anticancer molecule because binding to TRAIL-R1 or TRAIL-R2 triggers apoptosis in most cancer cells without aecting normal cells

496

CONTASSOT

et al

[55]. Most tumors exhibit a predominant expression of TRAIL-R1, but sensitivity to TRAIL in melanoma has long been believed to be mediated by TRAIL-R2 [110,121,122]. Kurbanov and colleagues [123], recently showed that both TRAIL DRs are strongly expressed in melanomas in vivo and that TRAIL-R1 expressing melanoma cell lines are also highly sensitive to TRAIL. The expression of TRAIL-R1 and TRAIL-R2 is higher in melanomas than in nevi and is decreased in metastasis [124,125]. The latter raises the possibility that advanced melanomas may not respond to TRAIL as a single agent. TNF/TNF-R signaling Although melanoma cell lines and fresh melanoma biopsies express both TNF-R1 and TNF-R2 [126], melanomas are generally resistant to TNFmediated apoptosis. This observation led to the idea that TNF resistance could result from the functional signicance of survival molecules such as NF-kB [109]. The increased expression of TRAF2 observed during melanoma progression may help to protect cells from TNF-induced apoptosis. In support of this, expression of the dominant negative form of TRAF2, lacking the ring nger domain, is sucient to sensitize melanoma cells to apoptosis [127]. Furthermore, the abrogation of NF-kB activity sensitizes TNFresistant melanoma cells to TNF-induced apoptosis [128]. Indeed, TNF-R1-mediated activation of NF-kB results in the activation of FLIP and IAPs, which inhibit caspase-8 activation [129]. Resistance to DR-mediated apoptosis is likely to contribute to the pathogenesis of melanoma. Further insight into the regulation of these signaling pathways and the clinical development of recombinant death ligands such as TRAIL, may open new perspectives for the therapy of advanced melanoma. Nonmelanoma skin cancers Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) represent the majority of nonmelanoma skin cancers (NMSC), and BCC is the most common malignant neoplasm among whites. Long-term exposure of the epidermis to UV radiation is associated with an increased risk of both melanoma and NMSC. As discussed above, by preventing the accumulation of cells with p53 mutations, Fas and TRAIL receptors contribute to the defense of the skin against UV radiation insults. Progression of NMSC is associated with

a loss of expression of Fas and TRAIL-R2 and an increased expression of FasL by transformed cells [3840]. It is likely that, like melanomas, NMSC gain the ability to partially escape antitumor immune responses because of reduced sensitivity to FasL and thus possibly cytotoxic T lymphocytemediated cell death. In addition to the loss of functional DRs, FLIP has also been shown to be overexpressed in SSC [38]. Taken together, the alterations of the DRmediated apoptosis pathways in NMSC, could play an important role in the survival of transformed cells. TNF-Rassociated periodic syndrome Tumor necrosis factor receptorassociated periodic syndrome (TRAPS) is characterized by recurrent inammatory bouts in which the systemic symptoms are fever, abdominal pain, arthralgia, myalgia, lymphadenopathy. Skin symptoms frequently include a generalized eruption consisting of large erythematous migratory patches or plaques or ecchymotic lesions and/or amyloidosis [130]. The susceptibility gene for TRAPS is the gene encoding TNF-R1. Mutations aecting the extracellular domain of TNF-R1 have been identied in TRAPS patients. Mutations in cysteine-rich domains, especially cysteine mutations, have been shown to dramatically destabilize the protein structure and impair cell surface expression and TNF binding [131]. TNF-R1 signaling functions are quite complex, and the mechanisms by which the symptoms are triggered have not been clearly identied. Summary Interactions between death receptors from the TNF superfamily and their ligands play a crucial role in the development and the integrity of the epidermis. The major consequence resulting from DR targeting is apoptosis. The deregulation of DR signaling, including lack or overexpression of receptors and/or ligands, or intracellular events interfering with apoptosis signaling, is involved in the pathogenesis of certain cutaneous diseases. In the skin, apoptosis is a key event in the elimination of cells such as keratinocytes and melanocytes, that are damaged by environmental insults such as UV radiation. The loss of the ability of damaged cells to undergo apoptosis contributes to tumor pathogenesis and progression, and evidence suggests that DRs are implicated in this process to some extent. On the other hand, excessive

DEATH RECEPTORS AND APOPTOSIS

497

apoptosis can lead to tissue destruction as observed in cutaneous GVHD or TEN, and the FasFasL pair play a central role herein. The signicant advances that have been made during the last two decades in the understanding of death receptor biology and its role in disease, have already helped conceptualize therapeutic tools targeting their signaling pathways, some of which are already in clinical trials.

References
[1] Fuchs P, Strehl S, Dworzak M, et al. Structure of the human TNF receptor 1 (p60) gene (TNFR1) and localization to chromosome 12p13. Genomics 1992;13:21924. [2] Cheng J, Liu C, Koopman WJ, et al. Characterization of human Fas gene. Exon/intron organization and promoter region. J Immunol 1995;154: 123945. [3] Beltinger CP, White PS, Maris JM, et al. Physical mapping and genomic structure of the human TNFR2 gene. Genomics 1996;35:94100. [4] Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 2001;104:487501. [5] Bodmer JL, Schneider P, Tschopp J. The molecular architecture of the TNF superfamily. Trends Biochem Sci 2002;27:1926. [6] Schulze-Ostho K, Ferrari D, Los M, et al. Apoptosis signaling by death receptors. Eur J Biochem 1998;254:43959. [7] Salvesen GS, Dixit VM. Caspase activation: the induced-proximity model. Proc Natl Acad Sci U S A 1999;96:109647. [8] Fischer U, Janicke RU, Schulze-Ostho K. Many cuts to ruin: a comprehensive update of caspase substrates. Cell Death Dier 2003;10:76100. [9] Micheau O, Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 2003;114:18190. [10] Grith TS, Brunner T, Fletcher SM, et al. Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 1995;270:118992. [11] Schneider P, Holler N, Bodmer JL, et al. Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J Exp Med 1998;187:120513. [12] Pitti RM, Marsters SA, Lawrence DA, et al. Genomic amplication of a decoy receptor for Fas ligand in lung and colon cancer. Nature 1998;396: 699703. [13] Pan G, Ni J, Wei YF, et al. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 1997;277:8158.

[14] Sheridan JP, Marsters SA, Pitti RM, et al. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 1997;277:81821. [15] Thome M, Schneider P, Hofmann K, et al. Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 1997; 386:51721. [16] Irmler M, Thome M, Hahne M, et al. Inhibition of death receptor signals by cellular FLIP. Nature 1997;388:1905. [17] Roy N, Deveraux QL, Takahashi R, et al. The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specic caspases. EMBO J 1997;16:691425. [18] Deveraux QL, Reed JC. IAP family proteins suppressors of apoptosis. Genes Dev 1999;13: 23952. [19] Gravestein LA, Borst J. Tumor necrosis factor receptor family members in the immune system. Semin Immunol 1998;10:42334. [20] Leithauser F, Dhein J, Mechtersheimer G, et al. Constitutive and induced expression of APO-1, a new member of the nerve growth factor/tumor necrosis factor receptor superfamily, in normal and neoplastic cells. Lab Invest 1993;69:41529. [21] Oishi M, Maeda K, Sugiyama S. Distribution of apoptosis-mediating Fas antigen in human skin and eects of anti-Fas monoclonal antibody on human epidermal keratinocyte and squamous cell carcinoma cell lines. Arch Dermatol Res 1994;286: 396407. [22] Sayama K, Yonehara S, Watanabe Y, et al. Expression of Fas antigen on keratinocytes in vivo and induction of apoptosis in cultured keratinocytes. J Invest Dermatol 1994;103:3304. [23] Matsue H, Kobayashi H, Hosokawa T, et al. Keratinocytes constitutively express the Fas antigen that mediates apoptosis in IFN gamma-treated cultured keratinocytes. Arch Dermatol Res 1995;287: 31520. [24] Leverkus M, Yaar M, Gilchrest BA. Fas/Fas ligand interaction contributes to UV-induced apoptosis in human keratinocytes. Exp Cell Res 1997; 232:25562. [25] Viard I, Wehrli P, Bullani R, et al. Inhibition of toxic epidermal necrolysis by blockade of CD95 with human intravenous immunoglobulin. Science 1998;282:4903. [26] de Panlis G, Semenza D, Lavazza A, et al. Keratinocytes constitutively express the CD95 ligand molecule on the plasma membrane: an in situ immunoelectron microscopy study on ultracryosections of normal human skin. Br J Dermatol 2002;147:712. [27] Viard-Leveugle I, Bullani RR, Meda P, et al. Intracellular localization of keratinocyte Fas ligand explains lack of cytolytic activity under physiological conditions. J Biol Chem 2003;278:161838. [28] Young AR. The sunburn cell. Photodermatol 1987; 4:12734.

498

CONTASSOT

et al [44] Byrne SN, Halliday GM. High levels of Fas ligand and MHC class II in the absence of CD80 or CD86 expression and a decreased CD4 T cell Inltration, enables murine skin tumours to progress. Cancer Immunol Immunother 2003;52:396402. [45] Mitra RS, Wrone-Smith T, Simonian P, et al. Apoptosis in keratinocytes is not dependent on induction of dierentiation. Lab Invest 1997;76: 99107. [46] Gandarillas A, Goldsmith LA, Gschmeissner S, et al. Evidence that apoptosis and terminal dierentiation of epidermal keratinocytes are distinct processes. Exp Dermatol 1999;8:719. [47] Weil M, Ra MC, Braga VM. Caspase activation in the terminal dierentiation of human epidermal keratinocytes. Curr Biol 1999;9:3614. [48] Candi E, Schmidt R, Melino G. The cornied envelope: a model of cell death in the skin. Nat Rev Mol Cell Biol 2005;6:32840. [49] Rieux-Laucat F, Le Deist F, Hivroz C, et al. Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. Science 1995;268:13479. [50] Vaishnaw AK, Orlinick JR, Chu JL, et al. The molecular basis for apoptotic defects in patients with CD95 (Fas/Apo-1) mutations. J Clin Invest 1999; 103:35563. [51] Straus SE, Jae ES, Puck JM, et al. The development of lymphomas in families with autoimmune lymphoproliferative syndrome with germline Fas mutations and defective lymphocyte apoptosis. Blood 2001;98:194200. [52] Wu J, Wilson J, He J, et al. Fas ligand mutation in a patient with systemic lupus erythematosus and lymphoproliferative disease. J Clin Invest 1996;98: 110713. [53] Stander S, Schwarz T. Tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) is expressed in normal skin and cutaneous inammatory diseases, but not in chronically UV-exposed skin and non-melanoma skin cancer. Am J Dermatopathol 2005;27:11621. [54] Kothny-Wilkes G, Kulms D, Poppelmann B, et al. Interleukin-1 protects transformed keratinocytes from tumor necrosis factor-related apoptosis-inducing ligand. J Biol Chem 1998;273:2924753. [55] Yagita H, Takeda K, Hayakawa Y, et al. TRAIL and its receptors as targets for cancer therapy. Cancer Sci 2004;95:77783. [56] Qin JZ, Bacon P, Panella J, et al. Low-dose UV-radiation sensitizes keratinocytes to TRAIL-induced apoptosis. J Cell Physiol 2004;200:15566. [57] Kothny-Wilkes G, Kulms D, Luger TA, et al. Interleukin-1 protects transformed keratinocytes from tumor necrosis factor-related apoptosisinducing ligand- and CD95-induced apoptosis but not from ultraviolet radiation-induced apoptosis. J Biol Chem 1999;274:2891621.

[29] Daniels F Jr, Brophy D, Lobitz WC Jr. Histochemical responses of human skin following ultraviolet irradiation. J Invest Dermatol 1961;37:3517. [30] Henseleit U, Zhang J, Wanner R, et al. Role of p53 in UVB-induced apoptosis in human HaCaT keratinocytes. J Invest Dermatol 1997;109:7227. [31] Hall PA, McKee PH, Menage HD, et al. High levels of p53 protein in UV-irradiated normal human skin. Oncogene 1993;8:2037. [32] Brash DE, Rudolph JA, Simon JA, et al. A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proc Natl Acad Sci U S A 1991;88:101248. [33] Ananthaswamy HN, Loughlin SM, Cox P, et al. Sunlight and skin cancer: inhibition of p53 mutations in UV-irradiated mouse skin by sunscreens. Nat Med 1997;3:5104. [34] Ouhtit A, Gorny A, Muller HK, et al. Loss of Fas-ligand expression in mouse keratinocytes during UV carcinogenesis. Am J Pathol 2000;157: 197581. [35] Hill LL, Ouhtit A, Loughlin SM, et al. Fas ligand: a sensor for DNA damage critical in skin cancer etiology. Science 1999;285:898900. [36] Rehemtulla A, Hamilton CA, Chinnaiyan AM, et al. Ultraviolet radiation-induced apoptosis is mediated by activation of CD-95 (Fas/APO-1). J Biol Chem 1997;272:257836. [37] Aragane Y, Kulms D, Metze D, et al. Ultraviolet light induces apoptosis via direct activation of CD95 (Fas/APO-1) independently of its ligand CD95L. J Cell Biol 1998;140:17182. [38] Bachmann F, Buechner SA, Wernli M, et al. Ultraviolet light downregulates CD95 ligand and TRAIL receptor expression facilitating actinic keratosis and squamous cell carcinoma formation. J Invest Dermatol 2001;117:5966. [39] Buechner SA, Wernli M, Harr T, et al. Regression of basal cell carcinoma by intralesional interferonalpha treatment is mediated by CD95 (Apo-1/Fas)CD95 ligand-induced suicide. J Clin Invest 1997; 100:26916. [40] Gutierrez-Steil C, Wrone-Smith T, Sun X, et al. Sunlight-induced basal cell carcinoma tumor cells and ultraviolet-B-irradiated psoriatic plaques express Fas ligand (CD95L). J Clin Invest 1998;101: 339. [41] Fenton RG, Hixon JA, Wright PW, et al. Inhibition of Fas (CD95) expression and Fas-mediated apoptosis by oncogenic Ras. Cancer Res 1998;58: 3391400. [42] Peli J, Schroter M, Rudaz C, et al. Oncogenic Ras inhibits Fas ligand-mediated apoptosis by downregulating the expression of Fas. EMBO J 1999;18: 182431. [43] Hill LL, Shreedhar VK, Kripke ML, et al. A critical role for Fas ligand in the active suppression of systemic immune responses by ultraviolet radiation. J Exp Med 1999;189:128594.

DEATH RECEPTORS AND APOPTOSIS

499

[58] Kristensen M, Chu CQ, Eedy DJ, et al. Localization of tumour necrosis factor-alpha (TNF-alpha) and its receptors in normal and psoriatic skin: epidermal cells express the 55-kD but not the 75-kD TNF receptor. Clin Exp Immunol 1993;94:35462. [59] Schottelius AJ, Moldawer LL, Dinarello CA, et al. Biology of tumor necrosis factor-alpha- implications for psoriasis. Exp Dermatol 2004;13:193222. [60] Corsini E, Galli CL. Cytokines and irritant contact dermatitis. Toxicol Lett 1998;1023:27782. [61] Darwin C. The variation of animal and plants under domestication. 2nd edition. London: John Murray; 1875. [62] Kere J, Srivastava AK, Montonen O, et al. X-linked anhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a novel transmembrane protein. Nat Genet 1996;13:40916. [63] Monreal AW, Ferguson BM, Headon DJ, et al. Mutations in the human homologue of mouse dl cause autosomal recessive and dominant hypohidrotic ectodermal dysplasia. Nat Genet 1999;22:3669. [64] Headon DJ, Emmal SA, Ferguson BM, et al. Gene defect in ectodermal dysplasia implicates a death domain adapter in development. Nature 2001; 414:9136. [65] Thesle I, Mikkola ML. Death receptor signaling giving life to ectodermal organs. Sci STKE 2002; 2002(131):22. [66] Smahi A, Courtois G, Rabia SH, et al. The NF-kappaB signalling pathway in human diseases: from incontinentia pigmenti to ectodermal dysplasias and immune-deciency syndromes. Hum Mol Genet 2002;11:23715. [67] Schneider P, Street SL, Gaide O, et al. Mutations leading to X-linked hypohidrotic ectodermal dysplasia aect three major functional domains in the tumor necrosis factor family member ectodysplasin-A. J Biol Chem 2001;276:1881927. [68] Gaide O, Schneider P. Permanent correction of an inherited ectodermal dysplasia with recombinant EDA. Nat Med 2003;9:6148. [69] Roujeau JC, Stern RS. Severe adverse cutaneous reactions to drugs. N Engl J Med 1994;331: 127285. [70] Yawalkar N, Egli F, Hari Y, et al. Inltration of cytotoxic T cells in drug-induced cutaneous eruptions. Clin Exp Allergy 2000;30:84755. [71] Pichler WJ. T cells in drug allergy. T cells in drug allergy. Curr Allergy Asthma Rep 2002;2:915. [72] Miyauchi H, Hosokawa H, Akaeda T, et al. T-cell subsets in drug-induced toxic epidermal necrolysis. Possible pathogenic mechanism induced by CD8positive T cells. Arch Dermatol 1991;127:8515. [73] Correia O, Delgado L, Ramos JP, et al. Cutaneous T-cell recruitment in toxic epidermal necrolysis. Further evidence of CD8+ lymphocyte involvement. Arch Dermatol 1993;129:4668. [74] Posadas SJ, et al. Delayed reactions to drugs show levels of perforin, granzyme B, and Fas-L to be

[75] [76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85] [86]

[87]

[88]

[89]

related to disease severity. J Allergy Clin Immunol 2002;109:15561. Ferrara JL, Deeg HJ. Graft-versus-host disease. N Engl J Med 1991;324:66774. Hill GR, Crawford JM, Cooke KR, et al. Total body irradiation and acute graft-versus-host disease: the role of gastrointestinal damage and inammatory cytokines. Blood 1997;90:320413. Nestel FP, Price KS, Seemayer TA, et al. Macrophage priming and lipopolysaccharide-triggered release of tumor necrosis factor alpha during graftversus-host disease. J Exp Med 1992;175:40513. Gilliam AC, Whitaker-Menezes D, Korngold R, et al. Apoptosis is the predominant form of epithelial target cell injury in acute experimental graftversus-host disease. J Invest Dermatol 1996;107: 37783. Schmaltz C, Alpdogan O, Muriglan SJ, et al. Donor T cell-derived TNF is required for graftversus-host disease and graft-versus-tumor activity after bone marrow transplantation. Blood 2003; 101:24405. Herve P, Flesch M, Tiberghien P, et al. Phase I-II trial of a monoclonal anti-tumor necrosis factor alpha antibody for the treatment of refractory severe acute graft-versus-host disease. Blood 1992;79:33628. Cooke KR, Hill GR, Gerbitz A, et al. Tumor necrosis factor-alpha neutralization reduces lung injury after experimental allogeneic bone marrow transplantation. Transplantation 2000;70:2729. Laster SM, Wood JG, Gooding LR. Tumor necrosis factor can induce both apoptic and necrotic forms of cell lysis. J Immunol 1988;141:262934. Hill GR, Ferrara JL. The primacy of the gastrointestinal tract as a target organ of acute graft-versushost disease: rationale for the use of cytokine shields in allogeneic bone marrow transplantation. Blood 2000;95:27549. Ferrara JL, Levy R, Chao NJ. Pathophysiologic mechanisms of acute graft-vs.-host disease. Biol Blood Marrow Transplant 1999;5:34756. Sale GE. Does graft-versus-host disease attack epithelial stem cells? Mol Med Today 1996;2:1149. Lowin B, Hahne M, Mattmann C, et al. Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways. Nature 1994;370:6502. Kagi D, Vignaux F, Ledermann B, et al. Fas and perforin pathways as major mechanisms of T cellmediated cytotoxicity. Science 1994;265:52830. Baker MB, Altman NH, Podack ER, et al. The role of cell-mediated cytotoxicity in acute GVHD after MHC-matched allogeneic bone marrow transplantation in mice. J Exp Med 1996;183:264556. Braun MY, Lowin B, French L, et al. Cytotoxic T cells decient in both functional fas ligand and perforin show residual cytolytic activity yet lose their capacity to induce lethal acute graft-versus-host disease. J Exp Med 1996;183:65761.

500

CONTASSOT

et al [107] Chen YR, Tan TH. Lack of correlation in JNK activation and p53-dependent Fas expression induced by apoptotic stimuli. Biochem Biophys Res Commun 1999;256:5959. [108] Petak I, Tillman DM, Houghton JA. p53 dependence of Fas induction and acute apoptosis in response to 5-uorouracil-leucovorin in human colon carcinoma cell lines. Clin Cancer Res 2000; 6:443241. [109] Grith TS, Chin WA, Jackson GC, et al. Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells. J Immunol 1998;161:283340. [110] Zhang XD, Franco A, Myers K, et al. Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma. Cancer Res 1999;59:274753. [111] Bullani RR, Huard B, Viard-Leveugle I, et al. Selective expression of FLIP in malignant melanocytic skin lesions. J Invest Dermatol 2001;117:3604. [112] Song JH, Song DK, Herlyn M, et al. Cisplatin down-regulation of cellular Fas-associated death domain-like interleukin-1beta-converting enzymelike inhibitory proteins to restore tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human melanoma cells. Clin Cancer Res 2003;9:425566. [113] Chawla-Sarkar M, Bae SI, Reu FJ, et al. Down regulation of Bcl-2, FLIP or IAPs (XIAP and survivin) by siRNAs sensitizes resistant melanoma cells to Apo2L/TRAIL-induced apoptosis. Cell Death Differ 2004;11:91523. [114] Zeise E, Weichenthal M, Schwarz T, et al. Resistance of human melanoma cells against the death ligand TRAIL is reversed by ultraviolet-B radiation via downregulation of FLIP. J Invest Dermatol 2004;123:74654. [115] Raisova M, Hossini AM, Eberle J, et al. The Bax/ Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis. J Invest Dermatol 2001;117:33340. [116] Fecker LF, Geilen CC, Tchernev G, et al. Loss of proapoptotic Bcl-2-related multidomain proteins in primary melanomas is associated with poor prognosis. J Invest Dermatol 2006;126:136671. [117] Leverkus M, Gollnick H. Bak (and Bax) to the futureof primary melanoma prognosis? J Invest Dermatol 2006;126:12124. [118] Hahne M, Rimoldi D, Schroter M, et al. Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape. Science 1996; 274:13636. [119] Walker PR, Saas P, Dietrich PY. Role of Fas ligand (CD95L) in immune escape: the tumor cell strikes back. J Immunol 1997;158:45214. [120] Zaks TZ, Chappell DB, Rosenberg SA, et al. Fasmediated suicide of tumor-reactive T cells following activation by specic tumor: selective rescue by caspase inhibition. J Immunol 1999;162:32739.

[90] Tsukada N, Kobata T, Aizawa Y, et al. Graftversus-leukemia eect and graft-versus-host disease can be dierentiated by cytotoxic mechanisms in a murine model of allogeneic bone marrow transplantation. Blood 1999;93:273847. [91] Hattori K, Hirano T, Miyajima H, et al. Dierential eects of anti-Fas ligand and anti-tumor necrosis factor alpha antibodies on acute graft-versus-host disease pathologies. Blood 1998;91:40515. [92] Miwa K, Hashimoto H, Yatomi T, et al. Therapeutic eect of an anti-Fas ligand mAb on lethal graftversus-host disease. Int Immunol 1999;11:92531. [93] Maeda Y, Levy RB, Reddy P, et al. Both perforin and Fas ligand are required for the regulation of alloreactive CD8 T cells during acute graftversus-host disease. Blood 2005;105:20237. [94] Hug H. Fas-mediated apoptosis in tumor formation and defense. Biol Chem 1997;378:140512. [95] Shin MS, Park WS, Kim SY, et al. Alterations of Fas (Apo-1/CD95) gene in cutaneous malignant melanoma. Am J Pathol 1999;154:178591. [96] Bullani RR, Wehrli P, Viard-Leveugle I, et al. Frequent downregulation of Fas (CD95) expression and function in melanoma. Melanoma Res 2002; 12:26370. [97] Muschen M, Warskulat U, Beckmann MW. Dening CD95 as a tumor suppressor gene. J Mol Med 2000;78:31225. [98] Helmbach H, Rossmann E, Kern MA, et al. Drugresistance in human melanoma. Int J Cancer 2001; 93:61722. [99] Owen-Schaub LB, van Golen KL, Hill LL, et al. Fas and Fas ligand interactions suppress melanoma lung metastasis. J Exp Med 1998;188:171723. [100] Chan H, Bartos DP, Owen-Schaub LB. Activationdependent transcriptional regulation of the human Fas promoter requires NF-kappaB p50-p65 recruitment. Mol Cell Biol 1999;19:2098108. [101] Ball NJ, Yohn JJ, Morelli JG, et al. Ras mutations in human melanoma: a marker of malignant progression. J Invest Dermatol 1994;102:28590. [102] Jafari M, Papp T, Kirchner S, et al. Analysis of ras mutations in human melanocytic lesions: activation of the ras gene seems to be associated with the nodular type of human malignant melanoma. J Cancer Res Clin Oncol 1995;121:2330. [103] Wagner SN, Ockenfels HM, Wagner C, et al. Ras gene mutations: a rare event in nonmetastatic primary malignant melanoma. J Invest Dermatol 1995;104:86871. [104] Urquhart JL, Meech SJ, Marr DG, et al. Regulation of Fas-mediated apoptosis by N-ras in melanoma. J Invest Dermatol 2002;119:55661. [105] Hussein MR, Haemel AK, Wood GS. Apoptosis and melanoma: molecular mechanisms. J Pathol 2003;199:27588. [106] Bennett M, Macdonald K, Chan SW, et al. Cell surface tracking of Fas: a rapid mechanism of p53mediated apoptosis. Science 1998;282:2903.

DEATH RECEPTORS AND APOPTOSIS

501

[121] Hersey P, Zhang XD. How melanoma cells evade trail-induced apoptosis. Nat Rev Cancer 2001;1: 14250. [122] Zhang XY, Zhang XD, Borrow JM, et al. Translational control of tumor necrosis factor-related apoptosis-inducing ligand death receptor expression in melanoma cells. J Biol Chem 2004;279: 1060614. [123] Kurbanov BM, Geilen CC, Fecker LF, et al. Ecient TRAIL-R1/DR4-mediated apoptosis in melanoma cells by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). J Invest Dermatol 2005;125:10109. [124] Zhuang L, Lee CS, Scolyer RA, et al. Progression in melanoma is associated with decreased expression of death receptors for tumor necrosis factorrelated apoptosis-inducing ligand. Hum Pathol 2006;37:128694. [125] McCarthy MM, DiVito KA, Sznol M, et al. Expression of tumor necrosis factorrelated apoptosis-inducing ligand receptors 1 and 2 in melanoma. Clin Cancer Res 2006;12:385663. [126] Carrel S, Hartmann F, Salvi S, et al. Expression of type A and B tumor necrosis factor (TNF) receptors on melanoma cells can be regulated by dbc-

[127]

[128]

[129]

[130]

[131]

AMP and IFN gamma. Int J Cancer 1995;62: 7683. Ivanov VN, Fodstad O, Ronai Z. Expression of ring nger-deleted TRAF2 sensitizes metastatic melanoma cells to apoptosis via up-regulation of p38, TNFalpha and suppression of NF-kappaB activities. Oncogene 2001;20:224353. Bakker TR, Reed D, Renno T, et al. Ecient adenoviral transfer of NF-kappaB inhibitor sensitizes melanoma to tumor necrosis factor-mediated apoptosis. Int J Cancer 1999;80:3203. Baud V, Karin M. Signal transduction by tumor necrosis factor and its relatives. Trends Cell Biol 2001;11:3727. Toro JR, Aksentijevich I, Hull K, et al. Tumor necrosis factor receptor-associated periodic syndrome: a novel syndrome with cutaneous manifestations. Arch Dermatol 2000;136:148794. Rebelo SL, Bainbridge SE, Amel-Kashipaz MR, et al. Modeling of tumor necrosis factor receptor superfamily 1A mutants associated with tumor necrosis factor receptor-associated periodic syndrome indicates misfolding consistent with abnormal function. Arthritis Rheum 2006;54: 267487.

Dermatol Clin 25 (2007) 503513

Glucocorticoid and Sex Hormone Receptors: Clinical Implications and Therapeutic Relevance
Christina Stefanaki, MDa, George P. Chrousos, PhDb, Andreas Katsambas, MDa,*
a

Department of Dermatology, Andreas Sygros Hospital for Skin Diseases, University of Athens, 5 Ionos Dragoumi Street, Kaisariani, Athens 16121, Greece b First Department of Pediatrics, Agia Soa Childrens Hospital, University of Athens, Micras Asias and Thivon, Goudi, Athens, Greece

The skin is both a target and a source of various hormones [1]. Several skin diseases are characterized by disturbed hormone actions at the level of the skin, therefore the recognition of the precise underlying molecular mechanisms are of outmost importance for the development of more specic and eective drugs with fewer side eects. Glucocorticoids are the most widely used drugs in dermatology, although their long-term application is accompanied by severe side eects. On the other hand, sex steroids are also involved in the regulation of several cutaneous functions and their roles in hair growth and sebaceous gland activity have long been recognized [2]. Reviewing the molecular mechanisms of action of the steroid hormones, particularly their interaction with nuclear receptors is a major challenge. Glucocorticoids Glucocorticoids, secreted by the adrenal glands, aect almost every organ in the body and are involved in diverse physiologic processes that maintain homeostasis, behavior, and resting and stress-related metabolism. The immune and inammatory reactions participate in these processes [3]. Synthetic glycocorticoids are by far the

* Corresponding author. 5 Ionos Dragoumi Street, Kaisariani, Athens 16121, Greece. E-mail address: katsambas@internet.gr (A. Katsambas).

most widely used drugs for numerous diseases, including inammatory skin conditions. The eects of glycocorticoids are mediated through two distinct nuclear receptors the glucocorticoid (GR) and the mineralocorticoid receptor (MR). GR and MR belong to the family of steroid hormone receptors that also includes the androgen (AR), estrogen (ER), and progesterone (PR) receptors, all of which are members of the nuclear receptor superfamily [4]. Other classes of nuclear receptors (NRs) include the thyroid, retinoid, and orphan receptors [4]. While GRs are widely expressed in most cells types, only epithelial cells in the kidney, colon, salivary glands, and nonepithelial cells in the brain and heart express MRs [5]. Activation of epithelial MRs leads to Na retention and subsequently to an increase in blood pressure, while brain MRs are involved in glycocorticoid feedback [6]. Synthetic glucocorticoids that are in use today in clinical practice are GR-selective, therefore any undesirable side eects from Na retention are avoided. The classic GR receptor has three major functional domains. The N-terminal domain discriminates between the dierent steroid response elements and therefore is important for the selectivity of glucocorticoid eects and interactions with co-activators [7]. The central, highly conserved, DNA-binding domain contains two loops, which are stabilized by four cysteine residues each complexed to zinc ions [7]. The ligand steroid-binding domain, which was recently crystallized, favors dimerization and transactivation
derm.theclinics.com

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.011

504

STEFANAKI

et al

but also interactions with co-activators [7,8]. Several subdomains also exist with specic roles [9]. Glucocorticoids readily penetrate the cell membrane and interact with GRs, located in the cytoplasm in an inactive hetero-oligomeric complex with heat shock proteins (hsp) and other proteins, such as the immunophilin FKB51 (a tacrolimus binding protein) [4,10]. Upon ligand binding the receptor undergoes conformational changes, dissociates from the heat shock proteins, partly homodimerizes, and translocates into the nucleus through a nuclear pore by means of an active adenosine triphosphate-dependent process [9,10]. Tyrosine nitration has been described as a mechanism that enhances GR hsp90 dissociation and anti-inammatory activity [7]. The observation that a GR ligand, carrying a nitration domain, induced nitration, opens new avenues to be explored for more specic drugs [11]. In the nucleus, the GR-ligand complex can positively regulate (transactivate) target genes by specic binding to the glucocorticoid response elements (GRE) in the promoter or enhancer region of those genes, followed by induction of gene transcription [12]. The reverse is also possible; the activated GR-complex can bind to negative GREs leading to negative regulation of gene transcription (transrepression), possibly due to interference with the binding of essential transcription factors [12]. The number of genes per cell directly regulated by glucocorticoids was estimated to be between 10 and 100 [13]. It was demonstrated however, that about 20% of the expressed human leukocyte genome is positively or negatively regulated by glucocorticoids [14]. In epidermal cells, the synthesis of basal cell specic (K5, K14) and disease-associated keratins (K6, K16, K17) is under the control of four negative GREs [15]. This may contribute to the thinning of the epidermis (decreased K5-K14) and poor wound healing (decreased K6-K16) observed with glucocorticoid treatment [16]. Transcriptional eects by activated GRs may also be the result of direct protein-protein interactions with other transcription factors modifying their action on their target genes, eg, activator protein-1 (AP-1), nuclear factor-kB (NF-kB), Smad3, p-53, and CRE-binding protein [10]. NF-kB and AP-1 are key transcription factors involved in the regulation of cytokines and adhesion molecules, consequently DNA-independent GR eects are sucient for a large part of their anti-inammatory actions [13]. These interactions are not simple, as multiple cofactors that

specically modulate interactions of GR with NF-kB and AP-1 remain to be further elucidated [4]. Topical glucocorticoids are widely used in dermatology for their anti-inammatory eects, with skin atrophy being their most feared side eect. This is caused not only by changes in keratin expression, but also because of decreased proliferative activity of keratinocytes and skin broblasts and decreased collagen synthesis [17,18]. Other glucocorticoid-mediated cutaneous side eects are delayed wound healing, acne, telangiectasia, and hypertrichosis [7]. Systemic glucocorticoids have also been associated with numerous side eects (eg, osteoporosis, cataracts, diabetes mellitus, psychiatric disorders, adrenal insuciency, and systemic immunosuppresion) [12]. A leading hypothesis is that the benecial anti-inammatory eects of glucocorticoids are mediated primarily by mechanisms mediated in part by GR interaction with other proteins, whereas the side eects might result mainly from the actions of the GR via GREs [5,8,19]. However, some side eects are exerted via both interaction of the GR with other proteins and through direct interaction with GREs in the regulatory region of responsive genes [2022]. The identication of novel selective GR steroidal or nonsteroidal ligands causing a receptor conformation that prefers interaction of the GR to other regulatory proteins to GR-DNA binding is the goal of several pharmaceutical companies. Such compounds have demonstrated dissociated eects in vitro but did not show similar actions in vivo [21]. Nonsteroidal compounds have also been developed that bind selectively and with high anity to the GR receptor and demonstrate strong anti-inammatory action in vitro with a better side-eect prole [23,24]. No such compounds are commercially available to date. Apart from GR-selective agonists, GR antagonists have also been developed, such as RU-486 (mifepristone) [7]. By inhibiting dexamethasoneinduced transactivation, this compound prevents hypercortisolisms eects on target tissues including pituitary inhibition and skin blanching [7]. With respect to NF-kB and AP-1 suppression, it acts as a weak partial agonist [2527]. A more selective glucocorticoid antagonist ZK98299 does not modulate NF-kB activity [25,26]. The human GR gene, located on chromosome 5, encodes two GR variants: GR-a and GR-b [28,29]. The GR-a variant is translated into multiple GR-a

GLUCOCORTICOID AND SEX HORMONE RECEPTORS

505

isoforms and most probably GR-b is similarly translated into an equal number of isoforms [9,30]. GR-b does not bind glucocorticoids, but does bind to DNA and may interfere with the action of glucocorticoids by blocking CRE binding or by competition with GR-a for transcriptional nuclear receptor co-activators [10,29,3133]. Interestingly, GR-b had no eect on GR-a binding capacity or specicity in vitro in skin cells [34]. The dierent GR-a isoforms share the same DNA and ligand-binding domains but have dierent N-terminal domains [30]. Moreover, the dierent GRa mRNA isoforms display dierent potency in translational activity, while the protein isoforms have distinct transactivation or transrepression patterns and thus may transduce dierent hormone signals, depending on their inherent activities and relative expression in dierent tissues [9,30]. Given that human cells express 16 distinct GR-a and GR-b isoforms, which may form 256 homo- or heterodimers, one can realize the complexity of glucocorticoid action. Dierent GR polymorphisms or mutations may result in reduced sensitivity to glucocorticoids, such as in autoimmune or allergic disorders (systemic lupus erythematosus, rheumatoid arthritis, glucocorticoid-resistant asthma) or in decreased sensitivity (diabetes mellitus type 2, essential hypertension) [9]. Elevated tissue levels of GR-b have been associated with glucocorticoidresistant asthma and ulcerative colitis [3537]. Whether increased expression of GR-b, after prolonged topical glucocorticoid administration, is the cause of diminished response to treatment (tachyphylaxis) remains to be explored [7]. The GR contains two transactivation domains, AF-1 and AF-2, in its N-terminal and ligandbinding domain, respectively. Certain CR-a isoforms contain only a part of AF-1, whereas others completely lack AF-1 [30]. The GR N-terminal domain interacts with the b subunit of the G protein complex, located in the inner surface of the plasma membrane and this may partly explain some nongenomic eects of GR [38]. Consequently, the dierent length of the N-terminal domain in the various GR isoforms may contribute to dierent nongenomic eects of the variants. Glucocorticoids vary greatly in their anity of binding to the GR and in their bioavailability. They are divided into three functional subgroups, A, B, and C, on the basis of the mechanisms by which they activate the formation of precursors of prostanoids, showing dierent eects on the release of arachinoic acid via activation of cPLA2 and COX2 and not on the basis of their

GR-binding anity, which poorly predicts their potency [39,40]. Steroids of group C (dexamethasone, hydrocortisone, triamcinolone acetonide, and beclomethasone) inhibit both cPLA2 activity and COX2 expression and induce both genomic and nongenomic eects. Methylprednisolone, of group B, which only inhibits cPLA2 without any eect on COX2, exhibits only nongenomic eects. The new generation of glycocorticoids (mometasone, uticasone, and budesonide) belong to group A, which includes prednisolone and beclomethasone dipropionate. They have no signicant eects on arachidonic acid release and cPLA2 activity (nongenomic eect) but they inhibit COX2 expression (genomic eect) [7,40]. Clobetasol propionate only slightly diered with respect to NF-kB transrepression to hydrocortisone, implying that the NF-kB transrepression does not reect the potential for skin diseases [28]. Interestingly, transrepression is more potently induced than transactivation with low glucocorticoid concentrations [41]. Recently, a new steroid was developed, prednicarbate, with an improved benet/risk ratio, combining signicant antiinammatory activity with minor reduction in skin thickness [4244].

Sex hormones Androgens The function of the skin and particularly of its appendages (sebaceous glands and hair follicles) strongly depends on biologically active sex hormones, which exert their eects through binding to specic nuclear receptors. Androgen (AR), estrogen (ER), and progesterone (PR) receptors, members of the nuclear receptor superfamily, are present in healthy human skin [45]. In the classic steroidogenic organs, the gonads and the adrenal gland, testosterone is derived mainly via two biosynthetic pathways: (1) progesterone, 17a-hydroxyprogesterone, D4-androstenedione or (2) 17a-hydroxypregnenolone, DHEA, D5-androstenediol [2]. In the skin, where all the necessary enzymes exist, testosterone is derived from enzymatic conversion of the weaker and more abundant androgen DHEA-sulfate and DHEA [46]. Testosterone can be further metabolized to dihydro-testosterone (DHT), a potent tissue androgen, through the action of 5a-reductase. Furthermore, testosterone can be inactivated to estradiol by aromatase, converted to weaker

506

STEFANAKI

et al

androgens, or can be cleared from the circulation by glucuronidation [47]. Androgens exert their biologic eects by binding to the AR. The anity of DHT for the AR is approximately vefold higher than that of testosterone, whereas DHEA has a relatively low anity [47]. The number of ARs is believed to dene an increased sensitivity of a target to androgens [47]. The reverse also is true, elevated levels of DHT can increase both AR and 5a-reductase levels in the skin thus generating a cycle of enhanced androgen activity [48]. ARs are ligand-activated transcriptional factors that encode three major functional domains: a modulatory N-terminal, a DNA-binding, and a C-terminal androgen-binding domain [49]. The most variable of the three is the N-terminal domain, which is involved in transcriptional activation [50]. The ARs rst recognize and then bind to testosterone or DHT within the cytoplasm of androgen target cells [51]. Upon androgen binding the ARs dimerize and migrate to the nucleus, where they bind with cofactors to the promoter region of androgen-sensitive target genes (androgen-responsive elements [ARE]), thus initiating the appropriate cell response [52,53]. The AREs can be divided into two main categories: the inverted repeat AREs, which bind both to AR and GR, and the direct repeat AREs, which are specic for the AR [51,54,55]. In this way, androgens are able to activate or to suppress the transcription of various genes. The activity of the AR can be modulated by coregulators: co-activators and corepressors. Co-activators are proteins that enhance liganddependent transcriptional activity, whereas corepressors repress or silence basal transcriptional activity [51]. The list of AR co-activators and corepressors is growing rapidly [56]. Many signaling pathways inuence androgen action via coregulators and crosstalk with AR [56]. Further characterization of these interactions may lead to the development of new more eective therapies for androgen-dependent diseases. However, the mechanism of action and the in vivo importance of coregulators have not been elucidated as yet. Molecular chaperones are involved in protein folding, tracking, and degradation. It has become apparent that in the case of NRs, including the AR, chaperones have an eect on the transcriptional activity of the receptors, making them potential targets of future therapies [51]. The AR gene is located in the proximal long arm of chromosome X at Xq11-12 [49]. Mutations

in the AR gene are the cause of the androgen insensitivity syndrome, where male sexual development is deterred or absent and the action of androgens on skin appendages is prevented [57]. Sequence variations occur more frequently in the AR gene than in other steroid receptor genes and possibly these polymorphisms aect androgen sensitivity and phenotypic expression [58,59]. An example of a common genetic variation is the number of tricluteotide repeats in exon 1 of the androgen receptor, which vary widely among humans [60]. Expansion of this region of the androgen receptor has been implicated recently in the development of androgen-related skin disorders in men and women alike, including androgenetic alopecia and hirsutism [61,62]. In the skin, ARs have been identied in the epidermis. The sebaceous glands, the secretory cells of eccrine glands [45], the apocrine glands [63], and sweat and sebaceous glands are responsible for the vast majority of androgen metabolism in skin [64]. Regarding the hair follicle, ARs have been detected in the beard, axillary, and frontal dermal papilla cells (DPCs), but not in the DPCs of the occipital scalp hair follicles [65]. In contrast, follicular epithelial cells (FECs) do not have the characteristics of androgen target cells [65]. These ndings suggest that the primary target cells of androgens in human hair follicles are DPCs, which mediate the signals to FECs in a paracrine fashion and that there is marked regional variation in hair concerning androgen sensitivity. Androgens stimulate hair growth from the beard and axillary skin and suppress hair growth from the frontal hair but have no eect on occipital hair. Systemic or local overproduction of active androgens results in acne and androgenetic alopecia in both males and females and in hirsutism in females. The involvement of AR in these conditions indicates that their blockade would be a logical approach to treatment. Several agents with anti-androgenic properties have been tried over the years with variable success rates. The clinical ecacy of orally administered cyproterone acetate in the treatment of acne and hirsutism in females has long been established. Cyproterone acetate may be given either in a high dose during the rst 10 days of a 21-day ethinylestradiol cycle or 2 mg of cyproterone acetate can be combined with 35 mg ethinylestradiol in a marketed oral contraceptive [66]. Chlormadinone acetate and dienogest are newer progestogens with antiandrogenic activity. The antiandrogenic activity of

GLUCOCORTICOID AND SEX HORMONE RECEPTORS

507

dienogest is similar to chlormadinone acetate and approximately one third of that of cyproterone acetate [66]. Combined oral contraceptives containing these agents are usually well tolerated with the most common side eects being headache, breast tenderness, and nausea. Flutamide is a potent selective nonsteroidal antiandrogen. It may increase the transaminase levels in 32% of patients, however it rarely causes fatal liver toxicity [67]. Spironolactone is an aldosterone antagonist, which also competes with androgens at the level of the androgen receptor. It is less eective than the other antiandrogens, and the diuretic eects, the menstrual disturbances, and other adverse eects make its use troublesome. All antiandrogens have a possible feminizing eect on a male fetus, therefore when they are administered, adequate contraception is mandatory [67]. The compounds mentioned above have already been tested topically but the results were rather disappointing [68]. However, a novel nonsteroidal antiandrogen compound, RU 58,841 has demonstrated a promising activity prole [6870], and is currently in phase II trials for the treatment of androgen-dependent acne and alopecia. The absence of systemic androgen eects in animals at doses up to 100 times the local active dose makes RU 58,841 an ideal topical antiandrogen [68]. Recently, a RU 58,841-myristate prodrug was developed, loaded on solid lipid nanoparticles, specically targeting the hair follicle [71]. Androgens and ARs have also been implicated in wound healing [72]. Indeed, the reduction in androgen levels may accelerate healing via eects on mesenchymal and/or inammatory cells and androgens, although the AR may inhibit tissue repair [72]. Local AR blockade may be a novel therapeutic strategy for elderly males with impaired wound healing.

Estrogens Estrogens are derived from the conversion of cholesterol to androstenedione and nally to estradiol. Androgens may also be converted by the enzyme aromatase to estrogens in peripheral tissue and particularly in adipose tissue [73]. Estradiol is primarily secreted by the ovary in adult women, whereas in men, estrogens are formed from extraglandular aromatization. Estradiol is the most potent estrogen. Estrone a product of peripheral conversion, androstenedione, 16-a

dehydroepiandrostenone, and estriol are very weak estrogens [74]. Estrogens exert their actions in the skin through their specic receptors, the ER-a and ER-b, which also belong to the superfamily of steroid nuclear receptors [75]. The gene for ER-a is located on chromosome 6, whereas the one for ER-b is on chromosome 14 [74]. Recent reports suggest that ER polymorphisms may inuence the susceptibility to certain diseases and individual responses to endogenous or exogenous estrogens [76,77]. Upon estrogen binding, the ERs undergo conformational changes, leading to the formation of either ER-a/ER-b heterodimers or homodimers depending on the cells expression repertoire [78]. Activated receptors act as transcription factors that bind estrogen response elements (ERE) in the regulatory regions of target genes, thus promoting or repressing their transcriptional activity [79]. Interestingly the genes that express c-fos protein that form part of the growth-promoting AP-1 transcription factor contain EREs and respond to estrogen [74,80]. Many genes induced by estrogens lack EREs yet respond to estrogens possibly aecting transcription via protein-protein interactions with other transcription factors. Estrogens, acting through ER-a or ER-b or possibly other ER subtypes, can participate in extranuclear signaling events in many tissues including the cardiovascular, digestive, and neural systems [78,81,82]. Some of these responses to estrogens occur in the cytoplasm or at the cell membranes rather than through altering gene expression and these processes may mediate rapid adaptive actions through activation of extranuclear signaling cascades [78]. Cross-talk between estrogens and growth factors, such as the insulin-like growth factor 1, transforming growth factor-alpha (TGFa), and epidermal growth factor (EGF) have been reported in several tissues [83,84]. These nongenotropic eects were found primarily to aect cell growth and survival and to protect cells from apoptosis. Estrogen action is characterized by staggering complexity. Both ER-a and ER-b may activate transcription, but in the presence of estradiol, ER-a is an activator, whereas ER-b is an inhibitor or silencer at activating protein-1 sites [85]. Moreover, the transcriptional potency of ER-a is down-regulated by ER-b in an estradiol dose-dependent manner [86,87]. A variant of ER-b, called ER-b ins may act as a dominant negative regulator of ER-a, independent of the estradiol

508

STEFANAKI

et al

concentration [88,89]. Therefore, precise information about the exact expression patterns of ER (a or b) is crucial to elucidate the role of estrogens in skin and hair biology. Estrogen receptor agonists include estrogens contained in contraceptives and hormone replacement regimens. A new class of agonists has been developed, the selective estrogen receptor modulators (SERMS), whose relative agonist/antagonist activities dier among cell types. Tamoxifen, a rst-generation SERM, functions as an estrogen antagonist in the breast and agonist in bone and uterus. Raloxifen, a second-generation SERM, manifests estrogenic activity in bone and the cardiovascular system, while opposing estrogen action in the uterus and breast [90]. Third-generation SERMS, like lasofoxifen, particularly in combination with a classic estrogen, may have an improved risk/benet prole in postmenopausal women [91,92]. The development of pure subtype-specic ER (a or b) modulators is ongoing. Phytoestrogens are plant compounds that have SERM activity [93]. Isoavones and lignans comprise the two main groups of phytoestrogens. They are weaker than endogenous estrogens, are not stored within the tissues, possess both agonist and antagonist properties, and interact with both ER-a and ER-b [94,95]. Phytoestrogens have shown in vitro the capacity to improve epithelial cell proliferation, collagen synthesis, and also to have a protective eect on collagen and elastic bers against enzymatic degeneration [96,97]. Estradiol rescues keratinocytes from apoptosis induced by UV-oxidative stress [98]. Many phytoestrogens are known antioxidants and it has been postulated that their antioxidant activity may be mediated by ER-b [93]. Orally administered isoavones inhibited UV-induced matrix metalloproteinases and collagen degradation [99]. Genistein, an isoavone, may protect from UV-induced skin damage and have antiphotocarcinogenesis, antioxidant, and antiphotoaging properties [100,101]. ER immunolocalization studies have shown that ER-b is widely expressed in human skin and its appendages [102104]. ER-a immunoreactivity is expressed in the sebaceous and sweat glands [45,102,103]. Both basal cells and sebocytes express both ER-a and ER-b [45,102]. Hormone replacement therapy (HRT) in postmenopausal women increases epidermal surface lipids and, thus, moisture and skin barrier function [105]. However, the role of estrogens, under normal conditions, remains uncertain.

In the past it was suggested that only ER-b is expressed in keratinocytes [102]; however, epidermal ER-a immunolabeling was recently demonstrated [45]. Estradiol binds keratinocytes with high anity, up-regulates ER-b receptors, and induces keratinocyte proliferation [106]. The positive eects of estrogens on epidermal thickness is primarily mediated by ER-a rather than ER-b, therefore the use of a specic ER-a agonist might be useful in increasing epidermal thickness in conditions associated with epidermal atrophy, and vice versa [107]. ER-a and ER-b expression has been documented in human skin broblasts, with ER-a predominating [108,109]. A direct hormonal action mediated by ER in broblasts may be stimulatory on collagen, reversing the declining collagen synthesis that is observed in menopause [108,110]. Up-regulation of estrogen and insulin growth factor-I (IGF-1) receptors, increase of prolidase activity for collagen synthesis, and inhibition of tissue-degrading metalloproteinases have been observed with administration of estrogens and the SERM raloxifen [108]. Increased skin collagen and thickness has been demonstrated by HRT [74,109]. Topically applied estradiol improves cutaneous function of aged skin by increasing the amount and improving the organization of connective tissue (up-regulation of procollagen type I and tropoelastin, down-regulation of MMP-1) and, therefore, might be of clinical benet [111]. In addition to their eects on connective tissue, estrogens also decrease neutrophil chemotaxis [110], and this is of great importance in wound healing. DHEA by conversion to estrogens and acting through ER has a direct eect on proinammatory cytokine production by macrophages and accelerates wound repair when administered locally [112]. The delays in wound healing caused by an inappropriately excessive inammatory response, in both elderly males and females, can be diminished by a topical natural estrogen or estrogen agonist [110]. The reports regarding ER-a and ER-b expression in cells of the derma papilla and the outer root sheath of the hair follicles have been rather conicting. Pelletier and colleagues [102] have observed only ER-b expression in the derma papilla cells and the cells of the outer root sheath, whereas Kariya and colleagues [45] demonstrated only ER-a labeling in a proportion of cells of the outer root sheath. Despite these discrepancies, ER-a and ER-b have about 60% homology and

GLUCOCORTICOID AND SEX HORMONE RECEPTORS

509

nearly equal binding anity for a large number of ligands [74], and, in general, estrogens appear to be able to inuence hair growth. However, the control of hair growth by estrogens is complex, as it is dependent on species, gender, and anatomical location. In mice, rats, guinea pigs, and dogs, estrogens exhibit inhibitory properties on hair growth [86]. In humans, estradiol prolongs the anagen phase of scalp hair follicles in vivo [86]. In males it has a stimulatory eect on hair shaft elongation in the frontotemporal area, whereas in females it has an inhibitory eect in the frontotemporal and occipital scalp skin [112,113]. This may help explain the clinical observation that topical estradiol or high systemic estradiol levels during estrogen-based contraception and pregnancy increase the telogen/anagen ratio, improving a preexisting telogen euvium and causing a telogen euvium postpartum, due to estradiol withdrawal [112]. Topical estradiol has long been used for the treatment of androgenetic alopecia in women [112]. In mice, ER-a expression is hair cycle dependent, whereas ER-b expression is more constant throughout the hair cycle, suggesting that stimulation of ER-a and not ER-b functions as a molecular cycle brake in this species [86,107]. Whether this applies to humans remains to be answered. Therefore, topically administered specic ER modulators may be explored in the future to treat conditions like hirsutism, androgenetic alopecia, and telogen euvium. However, before such an intervention is attempted several questions should be answered: the sex- and locationdependent dierences with respect to estrogen receptor subtype expression (a or b) and the inuence of vehicles in ER expression and estrogen metabolism. An understanding of the estrogen-signaling system of follicular stem cells could also have important implications for cutaneous neoplasia, as the follicular stem cell may represent the target cell for chemical carcinogens and the precursor cell of skin cancer [114]. However, the immunoreaction of ER-a was signicantly lower in sweat and sebaceous gland neoplasms than in nonpathological skin [45]. Progesterone Progesterone receptor signaling is similar to that of estrogens. The progesterone receptor (PR) is a homo- or heterodimer composed of two receptor proteins, PR-A and PR-B, both binding

progesterone [102]. Both forms are derived from the same gene and their expression is regulated in many tissues by estrogens [78]. PR-B is a stronger activator of transcription and its activity is modulated by PR-A via formation of heterodimers or by interference with coactivators [78]. PRs are present in eccrine sweat and sebaceous glands and in the keratinocytes throughout the epidermis [102,115]. In 30% of cases of androgenetic alopecia, PR staining has been observed within the dermal papilla of hair follicles [115]. Despite those ndings, the role of progesterone on the skin and its appendages, and particularly on sebaceous gland activity, has not been elucidated as yet.

Summary Glucocorticoids and sex steroid hormones are involved in the regulation of several human skin functions. In general, steroid hormones exert their eects through intracellular receptors, the glucocorticoid (GR), mineralocorticoid (MR), androgen (AR), estrogen (ER), and progesterone (PR) receptors. These receptors belong to the superfamily of the nuclear receptors, together with the thyroid hormone and retinoid and vitamin D receptors. In recent years, studies have focused on the expression of steroid receptors in the skin and on the mechanisms through which steroid hormones exert their actions and inuence various skin diseases. Although great progress has been made, there are still a lot of issues to be explored and, most of all, new target-specic drugs to be invented. References
[1] Thiboutot D, Jabara S, McAllister JM, et al. Human skin is a steroidogenic tissue: steroidogenic enzymes and cofactors are expressed in epidermis, normal sebocytes, and an immortalized sebocyte cell line (SEB-1). J Invest Dermatol 2003;120: 90514. [2] Chen W, Thiboutot D, Zouboulis C. Cutaneous androgen metabolism: basic research and clinical perspectives. J Invest Dermatol 2002;119: 9921007. [3] Reichardt HM, Tronche F, Berger S, et al. New insights into glucocorticoid and mineralocorticoid signaling: lessons from gene targeting. Adv Pharmacol 2000;47:121. [4] De Bosscher K, Berghe WV, Haegeman G. The interplay between the glycocorticoid reseptor and nuclear factorkB or activator protein-1: molecular

510

STEFANAKI

et al [21] Belvisi MG, Wicks S, Battram CH, et al. Therapeutic benet of a dissociated glucocorticoid and the relevance of in vitro separation of transrepression from transactivation activity. J Immunol 2001; 166:197582. [22] Coghlan MJ, Kym PR, Elmore SW, et al. Synthesis and characterization of non-steroidal ligands for the glucocorticoid receptor: selective quinoline derivatives with prednisolone equivalent functional activity. J Med Chem 2001;44:287985. [23] Schacke H, Schottelius A, Docke WD, et al. Dissociation of trnasactivation from transrepression by a selective glucocorticoid receptor agonist leads to separation of therapeutic eects from side eects. Proc Natl Acad Sci USA 2004;101:22732. [24] Wel C, Nakane M, Stashko M, et al. Transactivation and repression properties of the novel nonsteroid glucocorticoid receptor ligand 2,5dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5(1-methylcycloxen-3-y1)-1H-[1]benzopyrano[3,4-F] quinoline (A276575) and its four stereoisomers. Mol Pharmacol 2002;62:297303. [25] Heck S, Kullman M, Gast A, et al. A distinct modulating domain in glucocorticoid receptor monomers in the repression of activity of the transcription factor AP-1. EMBO J 1994;13: 408795. [26] Liden J, Delauney F, Rafter I, et al. A new function for the C-terminal zinc nger of the glucocorticoid receptor. Repression of RelA transactivation. J Biol Chem 1997;272:2146772. [27] Hofmann TG, Hehner SP, Bacher S, et al. Various glucocorticoids dier in their ability to induce gene expression, apoptosis and to repress NF-kB-dependent transcription. FEBS Lett 1998;441:4416. [28] Breslin MB, Geng CD, Vedeckis WV. Multiple promoters exist in the human GR gene, one of which is activated by glucocorticoids. Mol Endocrinol 2001;15:138195. [29] Bamberger CM, Bamberger AM, De Castro M, et al. Glucocorticoid receptor b a potential endogenous inhibitor of glucocorticoid action in humans. J Clin Invest 1995;95:243541. [30] Lu NZ, Cidlowski JA. Translational regulatory mechanisms generate N-terminal glucocorticoid receptor isoforms with unique transcriptional target genes. Mol Cell 2005;18:33142. [31] Kino T, Chrousos GP. Glucocorticoid and mineralocorticoid receptors and associated diseases. Essays Biochem 2004;40:13755. [32] Charmandari E, Chrousos GP, Ichijo T, et al. The human glucocorticoid receptor (hGR) b isoform suppresses the transcriptional activity of hGRa by interfering with formation of active coactivator complexes. Mol Endocrinol 2005;19:5264. [33] Yudt MR, Jewell CM, Bienstock RJ, et al. Molecular origins for the dominant negative function of human glucocorticoid receptor b. Mol Cell Biol 2003;23:431930.

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13] [14]

[15]

[16]

[17]

[18]

[19]

[20]

mechanisms for gene repression. Endocr Rev 2003; 24:488522. Reichardt HM, Schutz G. GC-signaling: multiple variations of a common theme. Mol Cell Endocrinol 1998;146:16. Lifton RP, Ghavari AG, Geller DS. Molecular mechanisms of human hypertension. Cell 2001; 104:54556. Shafer-Korting M, Kleuser B, Ahmed M, et al. Glucocorticoids for human skin: new aspects of the mechanism of action. Skin Pharmacol Physiol 2005;18:10314. Necela BM, Cidlowski JA. Crystallization of the human glucocorticoid receptor ligand binding domain: a step towards selective glucocorticoids. Trends Pharmacol Sci 2003;24:5861. Chrousos GP, Kino T. Intracellular glycocorticoid signaling: a formerly simple system turns stochastic. Science 2005;304:4853. Kino T, De Martino MU, Charmandari E, et al. Tissue glucocorticoid resistance/hypersensitivity syndromes. J Steroid Biochem Mol Biol 2003;85: 45767. Paul-Clark MJ, Roviezzo F, Flower RJ, et al. Glucocorticoid receptor nitration leads to enhanced anti-inammatory eects of novel steroid ligands. J Immunol 2003;171:324552. Schacke H, Docke WD, Asadullah K. Mechanisms involved in the side eects of glucocorticoids. Pharmacol Ther 2002;96:2343. Barnes PJ. Molecular mechanisms of corticosteroids in allergic diseases. Allergy 2001;56:92836. Galon J, Franchimont D, Hiroi N, et al. Gene profling reveals unknown enhancing and suppressive actions of glucocorticoids on immune cells. FASEB J 2002;16:6171. Radoja N, Komine M, Jho SH, et al. Novel mechanism of steroid action in skin through glucocorticoid receptor monomers. Mol Cell Biol 2000;20: 432839. Wintereld L, Cather J, Cather J, et al. Changing paradigms in dermatology: nuclear hormone receptors. Clin Dermatol 2003;21:44754. Perez P, Page A, Brano A, et al. Altered skin development and impaired proliferative and inammatory responses in transgenic mice overexpressing the glucocorticoid receptor. FASEB J 2001;15: 20302. Oikarinen A, Haapasaari KM, Sutinen M, et al. The molecular basis of GC-induced skin atrophy: topical GC apparently decreases both collagen synthesis and corresponding collagen m-RNA level in human skin in vivo. Br J Dermatol 1998;139:110610. Kellendonk C, Tronche F, Reichardt H, et al. Mutagenesis of the glucocorticoid receptor in mice. J Steroid Biochem Mol Biol 1999;69:2539. Belvisi MG, Brown TG, Wicks S, et al. New glucocorticoids with an improved therapeutic ratio? Pulm Pharmacol Ther 2001;14:2217.

GLUCOCORTICOID AND SEX HORMONE RECEPTORS

511

[34] Spica I, Hammer S, Kleuser B, et al. Transcriptional activity of potent glucocorticoids: relevance of glucocorticoid receptor isoforms and drug metabolites. Skin Pharmacol Appl Skin Physiol 2003;16:14350. [35] Hamid QA, Wenzel SE, Hauk PJ, et al. Increased glucocorticoid receptor beta in airway cells of glucocorticoid-insensitive asthma. Am J Respir Crit Care Med 1999;159:16004. [36] Leung DY, Hamid Q, Vottero A, et al. Association of glucocorticoid insensitivity with increased expression of glucocorticoid receptor beta. J Exp Med 1997;186:156774. [37] Sousa AR, Lane SJ, Cidlowski JA, et al. Glucocorticoid resistance in asthma is associated with elevated in vivo expression of the glucocorticoid receptor beta-isoform. J Allergy Clin Immunol 2000;105:94350. [38] Kino T, Tulpakov A, Ichijo T, et al. G protein b interacts with the glucocorticoid receptor and suppresses its transcriptional activity in the nucleus. J Cell Biol 2005;169:88596. [39] Belgi G, Friedman PS. Traditional therapies: glucocorticoids, azathioprine, methotrexate, hydroxyurea. Clin Exp Dermatol 2002;27:54654. [40] Croxtal JD, Van Hal PT, Choudhury Q, et al. Different glucocorticoids vary in their genomic and non-genomic mechanism of action in A549 cells. Br J Pharmacol 2002;135:5119. [41] Barnes PJ, Adcock IM. How do corticosteroids work in asthma? Ann Intern Med 2003;139:35970. [42] Korting HC, Unholzer A, Schaer-Korting M, et al. Dierent skin thinning potential of equipotent medium-strength glucocorticoids. Skin Pharmacol Appl Skin Physiol 2002;15:8591. [43] Hanan J, Abrams B, Cherill R. Management of atopic dermatitis with prednicarbate emollient cream 0.1%, a nonhalogenated prednisolone derivative. Journal of Geriatric Dermatology 1994;2: 11927. [44] Schaer-Korting M, Korting HC, Kerscher MJ, et al. Prednicarbate activity and benet/risk ratio in relation to other topical glucocorticoids. Clin Pharmacol Ther 1993;54:44856. [45] Kariya Y, Moriya T, Suzuki T, et al. Sex steroid hormone receptors in human skin appendages and its neoplasms. Endocr J 2005;52:31725. [46] Fritsch M, Orfanos CE, Zouboulis CC. Sebocytes are the key regulators of androgen homeostasis in human skin. J Invest Dermatol 2001;116:793 800. [47] Homan R. Steroidogenic isoenzymes in human hair and their potential role in androgenetic alopecia. Dermatology 2003;206:859. [48] Rabe T, Grunwald K, Runnebaum B. Hyperandrogenism in women. In: Runnebaum B, Rabe T, editors. Gynecological endrocrinology and reproductive medicine. Vol 1. Berlin (Heidelberg): Springer-Verlag; 1994. p. 5912.

[49] Lubahn DB, Joseph DR, Sullivan PM, et al. Cloning of human androgen receptor complementary DNA and localization to the X chromosome. Science 1988;240:32730. [50] Lee HJ, Chang C. Recent advances in adrogen receptor action. Cell Mol Life Sci 2003;60:161322. [51] Hellwinkel OJC, Muller A, Struve D, et al. Inuence of androgens and age on androgen receptor and 5a-reductase II transcription. Eur J Endocrinol 2000;143:21725. [52] Quigley CA, De Bellis A, Marshke KB, et al. Androgen receptor defects: historical, clinical and molecular perspectives. Endocr Rev 1995;16:271321. [53] Beato M, Chavez S, Truss M. Transcriptional regulation by steroid hormones. Steroids 1996;61: 24051. [54] Roche PJ, Hoare SA, Parker MG. A consensus DNA-binding sit for the androgen receptor. Mol Endocrinol 1992;6:222935. [55] Claessens F, Verrijidt G, Schoenmakers E, et al. Selective DNA binding by the androgen receptor as a mechanism for hormone specic gene regulation. J Steroid Biochem Mol Biol 2001;76:2330. [56] Heinlein CA, Chang C. Androgen receptor coregualtors: an overview. Endocr Rev 2002;23: 175200. [57] Chang C, Saltzman A, Yeh S, et al. Androgen receptor: an overview. Crit Rev Eukaryot Gene Expr 1995;5:97125. [58] Azziz R, Carmina E, Sawaya ME. Idiopathic hirsutism. Endocr Rev 2000;21:34762. [59] Tilley WD, Marcelli M, McPhaul MJ. Recent studies on the androgen receptor: new insights into old questions. Mol Cell Endocrinol 1990;68:C7C10. [60] Edwards A, Hammond HA, Jin L, et al. Genetic variations at ve tetrameric tandem repeat loci in four human population groups. Genomics 1992; 12:24153. [61] Sawaya ME, Shalita AR. Androgen receptor polymorphisms (GAG repeat lengths) in androgenetic alopecia, hirsutism and acne. J Cutan Med Surg 1998;3:915. [62] Legro RS, Shahbahrami B, Lobo RA, et al. Size polymorphisms of the androgen receptor among female Hispanics and correlations with androgenic characteristics. Obstet Gynecol 1994;83: 7016. [63] Choudry R, Hodgins MB, Van der Kwast TH, et al. Localization of androgen receptors in human skin by immunohistochemistry: implications for the hormonal regulation of hair growth, sebaceous glands and sweat glands. J Endocrinol 1992;133: 46775. [64] Deplewski D, Roseneld RL. Role of hormones in pilosebaceous unit development. Endocr Rev 2000; 21:36392. [65] Inui S, Fukuzato Y, Nakajima T, et al. Androgeninducible TGF-b1 from balding dermal papilla cells exhibits epithelial cell growth: a clue to

512

STEFANAKI

et al [82] Kamat A, Hinshelwood MM, Murry BA, et al. Mechanisms in tissue-specic regulation of estrogen biosynthesis in humans. Trends Endocrinol Metab 2002;13:1228. [83] Klotz DM, Hewitt SC, Ciana P, et al. Requirements of estrogen receptor alpha in insulin like growth factor1 (IGF-1) induced uterine responses and in vivo evidence for IGF-1/estrogen receptor cross talk. J Biol Chem 2002;277:85317. [84] Filardo EJ. Epidermal growth factor receptor (EGFR) transactivation by estrogen via the G-protein-coupled receptor, GPR30; a novel signaling pathway with potential signicance for breast cancer. J Steroid Biochem Mol Biol 2002; 80:2318. [85] Peach K, Webb P, Kuiper GG, et al. Dierential ligand activation of estrogen receptors ER-a and ER-b at AP-1 sites. Science 1997;277:150810. [86] Ohnemus U, Uenalan M, Conrad F, et al. Hair cycle control by estrogens: catagen induction via estrogen receptor (ER)-a is checked by ER-b signaling. Endocrinology 2005;146:121425. [87] Petterson K, Delaunay F, Gustafson JA. Estrogen receptor b acts as a dominant regulator of estrogen signaling. Oncogene 2000;19:49708. [88] Saji S, Omoto Y, Shimizu C, et al. Expression of estrogen receptor (ER) b cx protein in ER-a positive breast cancer: specic correlation with progesterone receptor. Cancer Res 2002;62:484953. [89] Weihua Z, Ekman J, Almkvist A, et al. Involvement of androgen receptor in 17b estradiol induced proliferation in rat uterus. Biol Reprod 2002;67: 61623. [90] Ettinger B, Black DM, Mitlak BH, et al. Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifen: results from a 3-year randomized clinical trial. Multiple outcomes of Raloxifen Evaluation (MORE) Investigators. JAMA 1999;282:63745. [91] Ke HZ, Foley HA, Simmons V, et al. Long-term treatment of lasofoxifene preserves bone mass and bone strength and does not adversely aect the uterus in ovariectomized rats. Endocrinology 2004;145:19962005. [92] Labrie F, El-Alfy M, Berger L, et al. The combination of a novel selective estrogen receptor modulator with an estrogen protects the mammary gland and uterus in a rodent model: the future of postmenopausal womens health? Endocrinology 2003;144:47006. [93] Gustafson JA. Therapeutic potential of selective estrogen receptor modulators. Curr Opin Chem Biol 1998;2:50811. [94] Fitzpatrick LA. Selective estrogen receptor modulators and phytoestrogens: new therapies for the postmenopausal woman. Mayo Clin Proc 1999; 74:6017. [95] Beck V, Unterrieder E, Krenn L, et al. Comparison of standardized plant extracts for large scale use in

[66] [67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75] [76]

[77]

[78]

[79] [80]

[81]

understanding paradoxical eects of androgen on human hair growth. FASEB J 2002;16:19679. Raudrant D, Rabe T. Progesterones with antiandrogenic properties. Drugs 2003;63:46392. Diamanti-Kandarakis E. Current aspects of antiandrogen therapy in women. Curr Pharm Des 1999;5:70723. Battmann T, Bonls A, Branche C, et al. RU 58841 a new specic topical antiandrogen: a candidate of choice for the treatment of acne, androgenetic alopecia and hirsutism. J Steroid Biochem Mol Biol 1994;48:5560. De Brouwer B, Tetelin C, Leroy T, et al. A controlled study of the eects of RU 58841, a non-steroidal antiandrogen, on human hair production by balding scalp grafts maintained on testosteroneconditioned nude mice. Br J Dermatol 1997;137: 699702. Pan HJ, Wilding G, Uno H, et al. Evaluation of RU58841 as an anti-androgen in prostate PC3 cells and a topical anti-alopecia agent in the bald scalp of stumptailed macaques. Endocrine 1998;9:3943. Munster U, Nakamura C, Haberland A, et al. RU 58841-myristatedprodrug development for topical treatment of acne and androgenetic alopecia. Pharmazie 2005;60:812. Ashcroft GS, Mills SJ. Androgen receptor-mediated inhibition of cutaneous wound healing. J Clin Invest 2002;110:61524. Nelson LR, Bulun SE. Estrogen production and action. J Am Acad Dermatol 2001;45(Suppl): S11624. Hall G, Phillips TJ. Estrogen and skin: the eects of estrogen, menopause, and hormone replacement therapy on the skin. J Am Acad Dermatol 2005; 53:55568. Nilson S, Makela S, Treuter E, et al. Mechanisms of estrogen action. Physiol Rev 2001;81:153565. Shearman AM, Cupples LA, Demissie S, et al. Association between estrogen receptor alpha gene variation and cardiovascular disease. JAMA 2003;290:226370. Herrington DM, Vittingho E, Lin F, et al. Statin therapy, cardiovascular events, and total mortality in the Heart and Estrogen/Progestin Replacement Study (HERS). Circulation 2002;105:91722. Tungeon JL, McDonnel DP, Martin KA, et al. Hormone therapy: physiological complexity belies therapeutic simplicity. Science 2004;304:126973. Nardulli AM, Shapiro DJ. DNA binding by nuclear receptors. Receptor 1993;3:24755. Weisz A, Rosales R. Identication of an estrogen response element upstream of the human c-fos gene that binds estrogen receptor and the AP-1 transcription factor. Nucleic Acids Res 1990;18: 5097106. Losel RM, Falkenstein E, Feuring M, et al. Nongenomic steroid action: controversies, questions, and answers. Physiol Rev 2003;83:9651016.

GLUCOCORTICOID AND SEX HORMONE RECEPTORS

513

[96]

[97]

[98]

[99]

[100]

[101]

[102]

[103]

[104]

[105]

[106]

hormone therapy. J Steroid Biochem Mol Biol 2003;84:25968. Schmid D, Muggli R, Zulli E. Dermatological application of soy isoavones to skin ageing in post menopausal women. Cosmetics and Toiletries 2001;1:14651. Schmid D, Zulli E. Topically applied soy isoavones increase skin thickness. Cosmetics and Toiletries 2002;117:4550. Kanda N, Watanabe S. 17-b estradiol inhibits oxidative stress-induced apoptosis in keratinocytes by promoting bcl-2 expression. J Invest Dermatol 2003;121:15009. Kim SY, Kim SJ, Lee JY, et al. Protective eects of dietary soy isoavones against UV induced skin aging in the hairless mouse. J Am Coll Nutr 2004;23: 15762. Kang S, Chung JH, Lee JH, et al. Topical N-acetyl cysteine and genistein prevents ultraviolet-light-induced signaling that leads to photo-aging in human skin in vivo. J Invest Dermatol 2003;120:83541. Wei H, Saladi R, Lu Y, et al. Isoavone genistein: phytoprotection and clinical implications in dermatology. J Nutr 2003;133(Suppl):38119. Pelletier G, Ren L. Localization of sex steroid receptors in human skin. Histol Histopathol 2004; 19:62936. Thorton MJ, Taylor AH, Mulligan K. Oestrogen receptor beta is the predominant estrogen receptor in human scalp skin. Exp Dermatol 2003;12: 18190. Pelletier G, Labric C, Labrio F. Localization of oestrogen receptor a, oestrogen receptor b and androgen receptors in the rat reproductive organs. J Endocrinol 2000;165:35970. Callens A, Vaillant L, Lecompte P, et al. Does hormonal aging exist? A study of the inuence of dierent hormone therapy regimens on the skin of postmenopausal women using non-invasive measurement techniques. Dermatology 1996;193: 28994. Verdier-Sevrain S, Yaar M, Cantatore J, et al. Estradiol induces proliferation of keratinocytes via

[107]

[108]

[109]

[110]

[111]

[112]

[113]

[114]

[115]

receptor-mediated mechanisms. FASEB J 2004; 18:12524. Moverare S, Lindberg MK, Faegerman J, et al. Estrogen receptor a, but not b, is involved in the regulation of the hair follicle cycling as well as the thickness of epidermis in male mice. J Invest Dermatol 2002;119:10538. Surazynski A, Jarzabek K, Haczynski J, et al. Differential eects of estradiol and raloxifene on collagen biosythesis in cultured human skin broblasts. Int J Mol Med 2003;12:8039. Kahlert S, Nuedling S, Van Eickels M, et al. Estrogen receptor alpha rapidly activates the IGF-1 receptor pathway. J Biol Chem 2000;275:1844753. Ashcroft GS, Greenwell-Wild T, Horan MA, et al. Topical estrogen accelerates cutaneous wound healing in aged humans associated with an altered inammatory response. Am J Pathol 1999;155: 113746. Son ED, Lee JY, Lee S, et al. Topical application of 17b-estradiol increases extracellular matrix protein synthesis by stimulating TGF-b signaling in aged human skin in vivo. J Invest Dermatol 2005;124: 114961. Conrad F, Ohnemus U, Gerstmayer B, et al. Estrogens and human hair scalp hair growthdstill more questions than answers. J Invest Dermatol 2004; 122:8402. Conrad F, Ohnemus U, Bodo E, et al. Substantial sex-dependent dierences in the response of human scalp hair follicles to estrogen stimulation in vitro advocate gender-tailored management of female versus male pattern balding. Journal of Investigative Dermatology Symposium Proceedings 2005; 10(3):2436. Oh HS, Smart RC. An estrogen receptor pathway regulates the telogen-anagen hair follicle transition and inuences epidermal cell proliferation. Proc Natl Acad USA 1996;93:1252530. Wallace NB, Smaller BR. Estrogen and progesterone receptors in androgenetic alopecia versus alopecia areata. American Journal of Dermatology 1998;2:1603.

Dermatol Clin 25 (2007) 515523

The Vitamin D Receptor

Carsten Carlberg, PhDa,b,*, Sabine Seuter, PhDa
a

Life Sciences Research Unit, Universite` of Luxembourg, 162A, Avenue de la Faencerie, L-1511 Luxembourg b Department of Biochemistry, University of Kuopio, Post Box 1621, FIN-70211 Kuopio, Finland

The epidermis is self-renewing through a proliferation and dierentiation process of keratinocytes migrating from the stratum basale to the stratum corneum. An important regulator of keratinocyte growth is the nuclear hormone, 1a,25-dihydroxyvitamin D3 (1a,25[OH]2D3) [1]. The actions of 1a,25(OH)2D3 and its synthetic analogs are mediated by the nuclear receptor, vitamin D receptor (VDR), which is the only nuclear protein that binds the nuclear hormone with high anity (Kd 0.1 nM) [2]. This classies VDR into the classical endocrine receptor subgroup of the nuclear receptor superfamily, which also contains the receptors for the nuclear hormones retinoic acid, thyroid hormone, estradiol, progesterone, testosterone, cortisol, and aldosterol [3]. Most mammalian tissues, including keratinocytes, express the VDR, but the receptor also is found in many malignant tissues, including basaland squamous-cell carcinomas and melanomas [4]. The availability of mice lacking a functional VDR gene has expanded the understanding of the role of 1a,25(OH)2D3 in epidermal dierentiation. The most striking feature of the VDR-null mouse is the development of alopecia (likewise found in many patients who have mutations in the VDR) [5]; these mice also exhibit a defect in epidermal dierentiation. Unlike other phenotypic features of vitamin D resistance in mice lacking VDR, normalization of serum calcium fails to

correct the alopecia, dilated hair follicles, and dermal cysts. Moreover, VDR-null mice also show an increased susceptibility to tumor formation [6]. VDR ligands are important for the regulation of normal cellular growth and dierentiation and have a potential in the prevention and the treatment of hyperproliferative malignancies, such as cancer and psoriasis [7,8]. Rather often, however, transformed cells get resistant to these actions of 1a,25(OH)2D3, making the clinical application of VDR ligands dicult. Epidemiologic evidence supports the importance of adequate vitamin D3 nutrition for several types of cancer [9].

The nuclear receptor superfamily Nuclear receptors represent an important transcription factor family [10]. The 48 human members of this superfamily are the best characterized genes of approximately 3000 mammalian genes involved in transcriptional regulation [11]. Nuclear receptors modulate genes that aect processes as diverse as reproduction, development, inammation, and general metabolism. They have a modular structure onto which certain functions can be ascribed. The amino-terminus is of variable length and sequence in the dierent family members. It contains a transactivation domain, termed AF-1, which is recognized by coactivator (CoA) proteins or other transcription factors, often in a ligand-independent fashion. The central DNAbinding domain has two zinc-nger motifs common to the entire family. The carboxy-terminal ligand-binding domain (LBD), whose overall architecture is well conserved between the various family members, nonetheless diverges suciently to guarantee selective ligand recognition and
derm.theclinics.com

The Academy of Finland, the Finnish Cancer Organisation, and the Juselius Foundation supported the work. * Corresponding author. Life Sciences Research du Luxembourg, 162A, Avenue de la Unit, Universite Fa encerie, L-1511 Luxembourg, Luxembourg. E-mail address: carsten.carlberg@uni.lu (C. Carlberg).

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.004

516

CARLBERG & SEUTER

accommodate the broad spectrum of nuclear receptor ligand structures. The LBD consists of 250 to 300 amino acids in 11 to 13 a-helices [12]. Ligand binding causes a conformational change within the LBD, whereby, at least in the case of endocrine nuclear receptors, helix 12, the most carboxy-terminal a-helix, closes the ligand-binding pocket via a mouse-traplike intramolecular folding event [13]. The LBD also is involved in a variety of interactions with nuclear proteins, such as other members of the nuclear receptor superfamily and coregulator proteins.

Chromatin and cofactors The major protein constituents of chromatin are the four dierent histones that form a nucleosome, onto which DNA is wound. Covalent modications of the lysines at the amino-terminal tails of these histone proteins neutralize their positive charge, thus their attraction for the negatively charged DNA backbone is diminished [14]. As a consequence, the association between the histone and the DNA becomes less stable. This inuences the degree of chromatin packaging and regulates the access of transcription factors to their potential binding sites. When nuclear receptors are bound to REs in the regulatory regions of their target genes, they recruit positive and negative coregulatory proteins, referred to as CoAs [15] and corepressors (CoRs) [16], respectively. In a simplied view of nuclear receptor signaling, in the absence of ligand, the nuclear receptor interacts with CoR proteins, such as nuclear receptor corepressor (NCoR), silencing mediator of retinoic acid and thyroid hormone receptor (SMRT), hairless, and Alien, which in turn associate with histone deacetylases leading to a locally increased chromatin packaging [17,18]. The binding of ligand induces the dissociation of the CoR and the association of a CoA of the p160-family, such as SRC-1, TIF2, or receptor associated coactivator 3 (RAC3) [19]. Some CoAs have histone acetylase activity or are complexed with proteins harboring such activity and this results in the net eect of local chromatin relaxation [20]. In a subsequent step, ligand-activated nuclear receptors change rapidly from interacting with the CoAs of the p160 family to those of mediator complexes, such as Med1 [21]. The mediator complexes, which consist of approximately 15 to 20 proteins, build a bridge to the basal transcription machinery [22]. In this way, ligand-activated

nuclear receptors execute two tasks, modication of chromatin and regulation of transcription. Most coregulators are not exclusive to nuclear receptors and are used in a similar manner by other transcription factors. Based on their mode of action, coregulators can be classied into two main groups [23]. The rst group contains factors that covalently modify histones, such as acetylation/deacetylation and methylation/demethylation, a process that follows a precise, combinatorial histone code [14]. The second group of coregulators includes ATP-dependent chromatin remodeling factors that modulate promoter accessibility to transcription factors and to the basal transcriptional machinery [24]. The complex network of coregulators can be viewed as dening a cofactor code being characterized by distinct patterns of coregulator recruitment and by their regulated enzymatic activities. The histone code, therefore, can be considered in a continuum with the cofactor code, as histones are crucial targets for the enzymatic activities of coregulators and have a key role in specifying coregulator recruitment on the basis of the reading of the histone code. Cell- and time-specic expression patterns of some coregulators can produce distinct modalities of nuclear receptor transcriptional activity resulting from dierences in the relative CoR and CoA protein levels. This aspect may have some diagnostic and therapeutic value in dierent types of cancer [25]. Concerning skin cancer, the idea was developed that the stoichiometric ratio between CoAs of the p160 family and Med1 might regulate a 1a,25(OH)2D3-dependent balance between proliferation and dierentiation of keratinocytes [26]. The switch between gene repression and activation is more complex, however, than a simple alternative recruitment of two dierent regulatory complexes. Most coregulators are coexpressed in the same cell type at relatively similar levels, which raises the possibility of their concomitant recruitment to a specic promoter. This has been resolved by the mutually exclusive binding of CoAs and CoRs to ligand-bound and -unbound nuclear receptors, respectively. In this respect it is interesting that the CoR hairless is expressed in keratinocytes and interacts with several members of the nuclear receptor superfamily, including the VDR [27]. Therefore, repression and activation more likely is achieved by a series of sequential events that are mediated by multiple enzymatic activities that are promoter and cell-type specic. Transcriptional regulation is a highly dynamic event of rapid association and

THE VITAMIN D RECEPTOR

517

dissociation of proteins and their modication, including proteolytic degradation and de novo synthesis. A pattern of recruitment and release of cohorts of coregulatory complexes was demonstrated on a single region of the trefoil factor 1 promoter in breast cancer cells [28]. This study revealed detailed and coordinated patterns of coregulator recruitment and preferential selectivity for factors that have similar enzymatic activities. Some coregulators seemed to be redundant and dierent family members were equally capable of being recruited alternately to the promoter. 1a,25-dihydroxyvitamin D3 signaling Nuclear receptors mostly regulate transcription by binding directly to specic response elements (REs), either as a monomers, homodimers, or heterodimers. They also indirectly can inuence transcription through other DNA-binding transcription factors. VDR and several other nuclear receptors function as heterodimers with one of the three retinoid X receptors (RXRs) a, d, and g [29]. Primary 1a,25(OH)2D3-responding genes contain a specic VDR binding site in their promoter region, which is referred to as a 1a,25(OH)2D3 response element (VDRE). An essential prerequisite for the direct modulation of transcription by VDR ligands is the location of at least one activated VDR protein close to the transcription start site (TSS) of the respective primary target gene (Fig. 1). This is achieved, in most cases, through the specic binding of the DNA-binding domain

of the VDR to the major grove of a hexameric DNA sequence, referred to as core binding motif, with the consensus sequence RGKTSA (R A or G, K G or T, and S C or G) [2]. VDR has been shown to form homodimers [30,31] and heterodimers with the thyroid hormone receptor [32,33] and the retinoic acid receptor [34], but by far the strongest binding partner of VDR is RXR. The trimeric complex of VDR, RXR, and a VDRE can be considered a molecular switch for primary 1a,25(OH)2D3 responding genes. VDR-RXR heterodimers bind to VDREs formed by a direct repeat (DR) of two hexameric core binding motifs with three intervening nucleotides (DR3-type) [30] and to DR4-type REs along with other members of the nuclear receptor superfamily [35]. Also, eective VDR binding is observed on everted repeat (ER)-type REs with 6 to 9 spacing nucleotides (ER6, ER7, ER8, and ER9) [33,36]. In addition, secondary 1a,25(OH)2D3 responding genes contribute to the physiologic eects of 1a,25(OH)2D3, but their induction is delayed by a few hours or even days and probably are mediated by primary 1a,25(OH)2D3-responding gene products, such as a transcription factor or a coregulator protein [37]. Expression proling using microarray technology indicates that comparable numbers of genes are downregulated by 1a,25(OH)2D3 as they are upregulated by the hormone [38]. The mechanisms of the downregulation of genes by 1a,25(OH)2D3 are much less understood, but they also seem to require the binding of an agonist to the VDR. On the basis

Fig. 1. Overview of vitamin D3 metabolism and target gene regulation in keratinocytes.

518

CARLBERG & SEUTER

of many microarray screenings of dierent mammalian cells, the authors estimate the total number of primary 1a,25(OH)2D3 target genes to be 250.

the entire metabolic machinery to produce 1a,25(OH)2D3 from 7-dehydrocholesterol and are a target for that hormone. The expression and enzymatic activities of CYP27B1 and CYP24 also are regulated tightly in keratinocytes and coupled to the dierentiation of these cells.

Biologic synthesis and degradation of 1a,25-dihydroxyvitamin D3 Vitamin D can be obtained from the diet and by the action of sunlight on the skin. UVB radiation in excess is harmful, because it causes DNA damage that can result in cancer. Exposure of the skin to the UV rays of sunlight of a wavelength of approximately 295 nm, however, induces the photolytic conversion of 7-dehydrocholesterol to previtamin D3 followed by thermal isomerization to vitamin D3 (see Fig. 1) [39]. The rst step in the metabolic activation of vitamin D is hydroxylation of carbon 25 to 25-hydroxyvitamin D3 (25[OH]D3), which occurs primarily in the liver [40]. Several hepatic cytochrome P450s (CYPs) are shown to 25-hydroxylate vitamin D compounds, but the one encoded by the CYP2R1 gene seems most critical. The 25-hydroxylation of vitamin D is regulated poorly, so that the levels of 25(OH)D3 increase in proportion to vitamin D3 intake. Therefore, plasma 25(OH)D3 levels are used commonly as an indicator of vitamin D status [41]. The second step in vitamin D bioactivation, the formation of 1a,25(OH)2D3, occurs under physiologic conditions, mainly in the kidney [42]. 1a-hydroxylase (encoded by the gene CYP27B1), however, is expressed in many tissues (including keratinocytes), and extrarenally produced 1a,25(OH)2D3 primarily serves as an autocrine/ paracrine factor. Dietary calcium can regulate the 1a-hydroxylase directly through changes in serum calcium and indirectly by altering parathyroid hormone levels [43]. Feedback regulation by 1a,25(OH)2D3 limits its circulating levels to minimize the potential for vitamin D intoxication. The high potency of 1a,25(OH)2D3 in elevating serum calcium and phosphate levels requires a mechanism to attenuate its activity. This is accomplished within virtually all target cells by the 1a,25(OH)2D3-inducible 25(OH)D3-24-hydroxylase (encoded by the genes CYP24), which catalyzes a series of oxidation reactions at carbons 24 and 23, leading to side chain cleavage and inactivation [44]. In addition, 1a,25(OH)2D3 can be converted to the 1a,25(R)(OH)2D323(S),26-lactone, which has mild antagonist activity toward 1a,25(OH)2D3 action [45]. Keratinocytes have Vitamin D receptor target genes The essential role of 1a,25(OH)2D3 for mineral homeostasis and skeletal integrity is the result of its regulation of Ca2-transporting proteins and bone-forming enzymes, such as calbindin D9K, osteocalcin, and osteopontin [46]. Proliferation is inhibited by high doses of 1a,25(OH)2D3 by blocking cells at the G0/G1 to S transition. This block is presumed to occur because of an upregulation of the cyclin-dependent kinase inhibitors p21 [47] and p27. The p21(waf1/cip1) gene promoter has functional VDREs indicating that the regulation of at least this cell-cycle inhibitor is at the transcriptional level [48]. Another interesting 1a,25(OH)2D3-target gene is cyclin C. The cyclin C-CDK8 complex is found associated with the RNA polymerase II basal transcriptional machinery [49] and is considered as a functional part of those mediator protein complexes that are involved in gene repression [50]. The fact that the cyclin C gene, located in chromosome 6q21, is deleted in a subset of acute lymphoblastic leukemias, suggests its involvement in tumorigenesis [51]. The steady state mRNA expression levels of the CYP24 gene are very low in the absence of ligand, but it is up to 1000-fold induced by stimulation with 1a,25(OH)2D3 [52]. Most other known primary 1a,25(OH)2D3 target genes, such as cyclin C and p21(waf1/cip1), often show an inducibility of twofold or less after short-term treatment with 1a,25(OH)2D3 [37,53]. Both genes, however, have 10,000- to 100,000-fold higher basal expression levels compared with that of the CYP24 gene. Therefore, when the relative levels are taken into account, 2- to 20-fold more cyclin C and p21(waf1/cip1) than CYP24 mRNA molecules are produced after induction with 1a,25(OH)2D3. To understand the regulation of the CYP24, cyclin C, and p21(waf1/cip1) genes by 1a,25(OH)2D3 in more detail, the authors analyzed 7.1 to 8.4 kilobases (kb) of their promoters using chromatin immunoprecipitation assays, using in each case a set of 20 to 25 overlapping promoter regions [48,52,54]. The spatiotemporal, 1a,25(OH)2D3-dependent chromatin changes in

THE VITAMIN D RECEPTOR

519

the three gene promoters was studied by chromatin immunoprecipitation assays with antibodies against acetylated histone 4, VDR, RXR, and RNA polymerase II. Promising promoter regions then were screened in silico for putative VDREs, whose functionality was analyzed sequentially with gel shift, reporter gene, and re-chromatin immunoprecipitation assays. This approach identied four VDREs for the CYP24 and cyclin C genes and three VDRE-containing regions in the p21(waf1/cip1) gene promoter. Most of them, however, simultaneously are under the control of other transcription factors, such as p53 in case of the p21(waf1/cip1) gene [48], and, therefore, possess signicant basal levels of transcription. Consequently, the fold induction aorded by 1a,25(OH)2D3 stimulation is much less than for the CYP24 gene, despite the large numbers of new mRNA molecules that may be produced. Another interesting primary 1a,25(OH)2D3 target is the peroxisome proliferator activated receptor (PPAR) d gene, which carries a potent DR3-type VDRE in close proximity to its TSS [55]. PPARd and VDR proteins are widely expressed (ie, both also are found in keratinocytes) [56]. An apparent overlap in the physiologic action of the two nuclear receptors is their involvement in the regulation of cellular growth, particularly in neoplasms. At the example of prostate and breast cancer cells, it was already shown that they display a spectrum of sensitivities to the antiproliferative action of 1a,25(OH)2D3 [57] at the levels of expression of a number of genes, including that of VDR [58], CYP24 [59], and the CoR SMRT [25]. These genes have been used as markers for the ecacy of the treatment of these cancers with VDR agonists. Because epidemiologic studies have linked the incidence of prostate and breast cancer to low serum levels of 25(OH)D3 as a result of diet and environment [60,61], the initiation or progression of these cancers seems to relate to reduced dietary intake or cellular resistance to the antiproliferative eects of 1a,25(OH)2D3. High PPARd expression in tumor seems to be positive for the prognosis of the respective cancer [62]. In this way, the upregulation of PPARd expression can be considered a supportive action and part of the mechanism of the antiproliferative action of 1a,25(OH)2D3 and its synthetic analogs. An alternative approach to the identication of primary 1a,25(OH)2D3 target genes was performed with the six members of the insulin-like growth factor binding protein (IGFBP) gene

family. Here, rst an in silico screen was performed, which then was followed by the analysis of candidate 1a,25(OH)2D3-responsive sequences by gel shift, reporter gene, and re-chromatin immunoprecipitation assays [63]. Induction of gene expression was conrmed independently using quantitative real-time polymerase chain reaction techniques. Using this approach, the genes, IGFBP1, -3, and -5, were demonstrated to be primary 1a,25(OH)2D3 target genes. The in silico screening of the 174 kb of genomic sequence surrounding all six IGFBP genes identied 15 candidate VDREs, 10 of which were shown to be functional in chromatin immunoprecipitation assays. The in silico screening approach was not restricted to regulatory regions that comprise only maximal 2 kb of sequence up- and downstream of the TSS, as in a recent whole genome screen for regulatory elements [64], but involved up to 10 kb of anking sequences and intronic and intergenic sequences. Therefore, this approach revealed candidate VDREs that are located more than 30 kb distant from their target gene TSS. Based on the present understanding of enhancers, DNA looping and chromatin units being anked by insulators or matrix attachment sites, these distances are not limiting [65]. In normal keratinocytes, locally produced 1a,25(OH)2D3 induces several proteins directly involved in dierentiation. In addition, autocrine effects of 1a,25(OH)2D3 include increases in calcium receptor (CaR) and phospholipase C gene expression, which are required for modulation of keratinocyte dierentiation by calcium [66]. Moreover, 1a,25(OH)2D3 is mandatory for the maintenance of a steep calcium gradient within the epidermis that aects normal keratinocyte function, the integrity of the permeability barrier, and the recovery when the permeability barrier is disrupted.

1a,25-dihydroxyvitamin D3 analogs Most analogs of 1a,25(OH)2D3 developed to date are modied at their side chain. They were synthesized with the goal of improving the biologic prole of the natural hormone, which has been limited by its hypercalcemic eects for a therapeutic application in hyperproliferative diseases, such as psoriasis, dierent types of cancer [67], and bone disorders, such as osteoporosis [68]. The central step in 1a,25(OH)2D3 signaling is the conformational shift of VDRs LBD and the resulting exchange of protein-protein interaction

520

CARLBERG & SEUTER

partners [69,70]. Therefore, only those VDR ligands that cause both an ecient dissociation of CoRs from the receptor and a specic binding of CoAs nally lead to transcriptional activation (ie, act as agonists). Most of the 3000 known 1a,25(OH)2D3 analogs show an agonistic potential, but they dier greatly in their eciency [71]. Some of these agonists have been shown to be superagonists (ie, they act in living cells at clearly lower concentrations than 1a,25[OH]2D3). A few of these 1a,25(OH)2D3 analogs have been chosen for clinical trials. Although MC903 (calcipotriol) is not a superagonist, it is the clinically most successful analog being applied topically against keratinocyte dysfunction in psoriasis [72]. MC903 is a very low calcemic analog, mainly because systemically it clearly is metabolized more rapidly than the natural hormone [73]. Therefore, MC903 is active in keratinocytes and other dermal cells [74] but has only minor systemic eects; however, MC903 still is not a perfect drug, as it is not eective in all patients who have psoriasis and may cause skin irritations in some. Most 1a,25(OH)2D3 analogs currently in clinical trials are designed for oral administration against dierent types of cancer [75], osteoporosis [76], and immune disorders [77]. Experimental comparisons of the most prominent superagonists with 1a,25(OH)2D3 in dierent in vitro assays showed that all these VDR ligands have a half maximal eective concentration (EC50) value of approximately 0.1 nM for inducing the critical complex formation of between VDR, RXR, and DNA [7880]. In parallel, a comparison of many VDREs demonstrated that they dier in their anity for VDR-RXR heterodimers but show identical molecular action (ie, they all are activated with an EC50 value of approximately 0.1 nM) [80]. This suggests that on classical VDREs none of the superagonists is signicantly superior to the natural hormone in activating VDR-RXR heterodimers. This means that the denition of superagonist applies only to the in vivo prole, in which the natural hormone is at a disadvantage because of rapid metabolism via the induction of the CYP24 gene and other degradative pathways [81]. In most in vivo assays, the period of ligand treatment is much longer than in in vitro assays, so that only in living cells significant amount of 1a,25(OH)2D3 analog metabolism can be observed. In addition, cells contain intracellular vitamin D binding proteins that may be able to discriminate between dierent 1a,25(OH)2D3 analogs [82].

Compared to the natural agonist, some superagonists show RE selectivity and others seem to dierentiate more clearly between DNA-dependent (genomic) and DNA-independent (nongenomic) 1a,25(OH)2D3 signaling pathways than the natural hormone [83]. These relative dierences in EC50 values, however, are not larger than a factor of 10, so that promoter and pathway selectivities themselves are not sucient to explain the improved in vivo prole of superagonists in relation to 1a,25(OH)2D3. The available crystal structure data on the complexes of VDR with a collection of agonists [12,8486] have been unable to explain superagonistic actions in general and agonist-selective functional proles in particular. Reasons for the apparent unique way of agonist binding to the VDR-LBD could be that the crystal structures by their nature represent minimum energetic states of the dynamic complex between ligand and receptor. The authors aimed to overcome these problems by using the power of molecular dynamics simulations and a VDR-LBD complexed with a CoA peptide [87]. According to the structural analysis, the molecular origin of the superagonistic prole of MC1288 (20-epi 1a,25(OH)2D3) compared with 1a,25(OH)2D3 is a region between helices 6 and 7, which shows the most exible dierences between the simulated receptor-ligand complexes. This region is located opposite to helix 12 and both together serve as a roof to the ligand-binding pocket. The four hydrophobic residues V300, I310, L313, and L393 (helix 11) interact with A303 and L309, which the authors found most critical for the agonist-specic prole. Most of the distances between these six residues are shorter in the VDR-MC1288 complex compared with the VDR-1a,25(OH)2D3 complex, suggesting tighter packing in this region. From a mechanistic point of view, the two-side chain analog Gemini and its derivatives are interesting analogs, because their extra side chain increased the ligand volume in both of their conformations by approximately 20% and 25%, respectively. The complexes of VDR with the twoside chain analogs also showed at the region between helices 6 and 7 the most signicant dierences to the VDR-1a,25(OH)2D3 structure. In contrast, the ligand-binding pocket of MC1288-bound VDR was found 17.2% smaller than that of the complex with the natural hormone. Because the volume of the compound MC1288 is only 1.5% smaller than that of 1a,25(OH)2D3, the ligand-binding pocket of the

THE VITAMIN D RECEPTOR

521

MC1288 complex is with 66.5% more eciently lled than that of the natural hormone (55.9%). The volume of the MC1288 ligand-binding pocket was reduced, because most of the 40 residues forming the ligand-binding pocket moved toward the ligand. At present, the commercially most successful VDR ligand, MC903, is applied topically, whereas future analogs are designed for oral application and higher bioavailability. Present experience has demonstrated that the pharmacokinetic prole, such as cellular uptake, transport, and in particular metabolic stability, is a critical characteristic of a VDR ligand in determining its suitability for clinical application.

Summary The authors have begun to establish the intricate network of interactions, which are required to mobilize a single genes expression in response to 1a,25(OH)2D3. A larger challenge lies ahead, however, when confronted with the higher order of regulated networks of genes, where the sum eect of ligand treatment may reveal itself. In an eort to study this, the authors have started applying systems biology to the eld of nuclear receptor biology, through a European Union funded Marie Curie Research Training Network, Systems biology of nuclear receptors (NUCSYS) (www.uku./nucsys). References
[1] Bikle DD, Gee E, Pillai S. Regulation of keratinocyte growth, dierentiation, and vitamin D metabolism by analogs of 1,25-dihydroxyvitamin D. J Invest Dermatol 1993;101:7138. [2] Carlberg C, Polly P. Gene regulation by vitamin D3. Crit Rev Eukaryot Gene Expr 1998;8:1942. [3] Carlberg C. Mechanisms of nuclear signalling by vitamin D3. Interplay with retinoid and thyroid hormone signalling. Eur J Biochem 1995;231:51727. [4] Norman AW. Receptors for 1a,(2)5(OH)2D(3): past, present, and future. J Bone Miner Res 1998; 13:13609. [5] Kato S, Takeyama K-i, Kitanaka S, et al. In vivo function of VDR in gene expression-VDR knockout mice. J Steroid Biochem Mol Biol 1999;69: 24751. [6] Welsh J. Vitamin D and breast cancer: insights from animal models. Am J Clin Nutr 2004;80:1721S4S. [7] Carlberg C, Saurat JH. Vitamin D-retinoid association: molecular basis and clinical applications. J Investig Dermatol Symp Proc 1996;1:826.

[8] Carlberg C. The antiproliferative eect of vitamin D3 analogues. Dermatology 1996;192:1957. [9] Schwartz GG. Vitamin D and the epidemiology of prostate cancer. Semin Dial 2005;18:27689. [10] Chawla A, Repa JJ, Evans RM, et al. Nuclear receptors and lipid physiology: opening the X-les. Science 2001;294:186670. [11] Maglich JM, Sluder A, Guan X, et al. Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes. Genome Biol 2001;2:RESEARCH0029. [12] Rochel N, Wurtz JM, Mitschler A, et al. Crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell 2000;5:1739. [13] Moras D, Gronemeyer H. The nuclear receptor ligand-binding domain: structure and function. Curr Opin Cell Biol 1998;10:38491. [14] Jenuwein T, Allis CD. Translating the histone code. Science 2001;293:107480. [15] Aranda A, Pascual A. Nuclear hormone receptors and gene expression. Physiol Rev 2001;81:1269304. [16] Burke LJ, Baniahmad A. Co-repressors 2000. FASEB J 2000;14:187688. [17] Polly P, Herdick M, Moehren U, et al. VDR-Alien: a novel, DNA-selective vitamin D3 receptor-corepressor partnership. FASEB J 2000;14:145563. [18] Privalsky ML. The role of corepressors in transcriptional regulation by nuclear hormone receptors. Annu Rev Physiol 2004;66:31560. [19] Leo C, Chen JD. The SRC family of nuclear receptor coactivators. Gene 2000;245:111. [20] Castillo AI, Jimenez-Lara AM, Tolon RM, et al. Synergistic activation of the prolactin promoter by vitamin D receptor and GHF-1: role of coactivators, CREB-binding protein and steroid hormone receptor coactivator-1 (SRC-1). Mol Endocrinol 1999; 13:114154. [21] Rachez C, Suldan Z, Ward J, et al. A novel protein complex that interacts with the vitamin D3 receptor in a ligand-dependent manner and enhances transactivation in a cell-free system. Genes Dev 1998;12: 1787800. [22] Rachez C, Lemon BD, Suldan Z, et al. Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex. Nature 1999;398: 8248. [23] Perissi V, Rosenfeld MG. Controlling nuclear receptors: the circular logic of cofactor cycles. Nat Rev Mol Cell Biol 2005;6:54254. [24] Acevedo ML, Kraus WL. Transcriptional activation by nuclear receptors. Essays Biochem 2004;40: 7388. [25] Khanim FL, Gommersall LM, Wood VH, et al. Altered SMRT levels disrupt vitamin D3 receptor signalling in prostate cancer cells. Oncogene 2004;23: 671225. [26] Oda Y, Sihlbom C, Chalkley RJ, et al. Two distinct coactivators, DRIP/mediator and SRC/p160, are dierentially involved in vitamin D receptor

522

CARLBERG & SEUTER

[27]

[28]

[29] [30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40] [41]

[42]

transactivation during keratinocyte dierentiation. Mol Endocrinol 2003;17:232939. Hsieh JC, Sisk JM, Jurutka PW, et al. Physical and functional interaction between the vitamin D receptor and hairless corepressor, two proteins required for hair cycling. J Biol Chem 2003;278:3866574. Metivier R, Penot G, Hubner MR, et al. Estrogen receptor a directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 2003;115:75163. Mangelsdorf DJ, Evans RM. The RXR heterodimers and orphan receptors. Cell 1995;83:84150. Carlberg C, Bendik I, Wyss A, et al. Two nuclear signalling pathways for vitamin D. Nature 1993;361: 65760. Cheskis B, Freedman LP. Ligand modulates the conversion of DNA-bound vitamin D3 receptor (VDR) homodimers into VDR-retinoid X receptor heterodimers. Mol Cell Biol 1994;14:332938. Schra der M, Carlberg C. Thyroid hormone and retinoic acid receptors form heterodimers with retinoid X receptors on direct repeats, palindromes, and inverted palindromes. DNA Cell Biol 1994;13: 33341. Schra der M, Mu ller KM, Nayeri S, et al. VDR-T3R receptor heterodimer polarity directs ligand sensitivity of transactivation. Nature 1994;370:3826. Schra der M, Bendik I, Becker-Andre M, et al. Interaction between retinoic acid and vitamin D signaling pathways. J Biol Chem 1993;268:178306. Quack M, Carlberg C. Ligand-triggered stabilization of vitamin D receptor/retinoid X receptor heterodimer conformations on DR4-type response elements. J Mol Biol 2000;296:74356. Schra der M, Nayeri S, Kahlen JP, et al. Natural vitamin D3 response elements formed by inverted palindromes: polarity-directed ligand sensitivity of vitamin D3 receptor-retinoid X receptor heterodimer-mediated transactivation. Mol Cell Biol 1995;15:115461. Palmer HG, Sanchez-Carbayo M, Ordonez-Moran P, et al. Genetic signatures of dierentiation induced by 1a,25-dihydroxyvitamin D3 in human colon cancer cells. Cancer Res 2003;63:7799806. White JH. Proling 1,25-dihydroxyvitamin D3-regulated gene expression by microarray analysis. J Steroid Biochem Mol Biol 2004;8990, 23944. Holick MF, Frommer JE, McNeill SC, et al. Photometabolism of 7-dehydrocholesterol to previtamin D3 in skin. Biochem Biophys Res Commun 1977; 76:10714. Dusso AS, Brown AJ, Slatopolsky E, et al. Am J Physiol Renal Physiol 2005;289:F828. Holick MF. The cutaneous photosynthesis of previtamin D3: a unique photoendocrine system. J Invest Dermatol 1981;77:518. Fraser DR, Kodicek E. Unique biosynthesis by kidney of biologically active vitamin D metabolite. Nature 1970;288:7646.

[43] Omdahl JL, Gray RW, Boyle IT, et al. Regulation of metabolism of 25-hydroxycholecalciferol by kidney tissue in vitro by dietary calcium. Nat New Biol 1972;237:634. [44] Zierold C, Darwish HM, DeLuca HF. Identication of a vitamin D-response element in the rat calcidiol ([2]5-hydroxyvitamin D[3]) 24-hydroxylase gene. Proc Natl Acad Sci USA 1994;91:9002. [45] Ishizuka S, Norman AW. Metabolic pathways from 1a,25-dihydroxyvitamin D3 to 1a,25-dihydroxyvitamin D3-26,23-lactone. Stereo-retained and stereo-selective lactonization. J Biol Chem 1987;262:716570. [46] Sutton AL, MacDonald PN. Vitamin D: more than a bone-a-de hormone. Mol Endocrinol 2003;17: 77791. [47] Jiang H, Lin J, Su Z-z, et al. Induction of dierentiation in human promyelotic HL-60 leukemia cells activates p21, WAF1/CIP1, expression in the absence of p53. Oncogene 1994;9:3397406. [48] Sarama ki A, Banwell CM, Campbell MJ, et al. Regulation of the human p21(waf1/cip1) gene promoter via multiple binding sites for p53 and the vitamin D3 receptor. Nucleic Acids Res 2006;34:54354. [49] Rickert P, Seghezzi W, Shanahan F, et al. Cyclin C/ CDK8 is a novel CTD kinase associated with RNA polymerase II. Oncogene 1996;12:263140. [50] Bourbon HM, Aguilera A, Ansari AZ, et al. A unied nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymerase II. Mol Cell 2004;14:5537. [51] Li H, Lahti JM, Valentine M, et al. Molecular cloning and chromosomal localization of the human cyclin C (CCNC) and cyclin E (CCNE) genes: deletion of the CCNC gene in human tumors. Genomics 1996;32:2539. [52] Va isa nen S, Dunlop TW, Sinkkonen L, et al. Spatiotemporal activation of chromatin on the human CYP24 gene promoter in the presence of 1a,(2)5dihydroxyvitamin D(3). J Mol Biol 2005;350:6577. [53] Swami S, Raghavachari N, Muller UR, et al. Vitamin D growth inhibition of breast cancer cells: gene expression patterns assessed by cDNA microarray. Breast Cancer Res Treat 2003;80:4962. [54] Sinkkonen L, Malinen M, Saavalainen K, et al. Regulation of the human cyclin C gene via multiple vitamin D3-responsive regions in its promoter. Nucl Acids Res 2005;33:244051. [55] Dunlop TW, Va isa nen S, Frank C, et al. The human peroxisome proliferator-activated receptor d gene is a primary target of 1a,25-dihydroxyvitamin D3 and its nuclear receptor. J Mol Biol 2005; 349:24860. [56] Di-Poi N, Michalik L, Desvergne B, et al. Functions of peroxisome proliferator-activated receptors (PPAR) in skin homeostasis. Lipids 2004;39:10939. [57] Trump DL, Hershberger PA, Bernardi RJ, et al. Anti-tumor activity of calcitriol: pre-clinical and clinical studies. J Steroid Biochem Mol Biol 2004; 8990:51926.

THE VITAMIN D RECEPTOR

523

[58] Evans SR, Nolla J, Hanfelt J, et al. Vitamin D receptor expression as a predictive marker of biological behavior in human colorectal cancer. Clin Cancer Res 1998;4:15915. [59] Albertson DG, Ylstra B, Segraves R, et al. Quantitative mapping of amplicon structure by array CGH identies CYP24 as a candidate oncogene. Nat Genet 2000;25:1446. [60] Ingles SA, Coetzee GA, Ross RK, et al. Association of prostate cancer with vitamin D receptor haplotypes in African-Americans. Cancer Res 1998;58: 16203. [61] Hanchette CL, Schwartz GG. Geographic patterns of prostate cancer mortality. Evidence for a protective eect of ultraviolet radiation. Cancer 1992;70: 28619. [62] Reed KR, Sansom OJ, Hayes AJ, et al. PPARd status and Apc-mediated tumourigenesis in the mouse intestine. Oncogene 2004;23:89926. [63] Matilainen M, Malinen M, Saavalainen K, et al. Regulation of multiple insulin-like growth factor binding protein genes by 1a,(2)5-dihydroxyvitamin D(3). Nucleic Acids Res 2005;33:552132. [64] Xie X, Lu J, Kulbokas EJ, et al. Systematic discovery of regulatory motifs in human promoters and 30 UTRs by comparison of several mammals. Nature 2005;434:33845. [65] Ogata K, Sato K, Tahirov TH. Eukaryotic transcriptional regulatory complexes: cooperativity from near and afar. Curr Opin Struct Biol 2003;13: 408. [66] Bikle DD, Pillai S. Vitamin D, calcium, and epidermal dierentiation. Endocr Rev 1993;14:319. [67] Mrk Hansen C, Binderup L, Hamberg KJ, et al. Vitamin D and cancer: eects of 1,(2)5(OH)2D(3) and its analogs on growth control and tumorigenesis. Front Biosci 2001;6:D82048. [68] Brown AJ. Therapeutic uses of vitamin D analogues. Am J Kidney Dis 2001;38:S3S19. [69] Carlberg C. Molecular basis of the selective activity of vitamin D analogues. J Cell Biochem 2003;88: 27481. [70] Freedman LP. Multimeric coactivator complexes for steroid/nuclear receptors. Trends Endocrinol Metab 1999;10:4037. [71] Bouillon R, Okamura WH, Norman AW. Structurefunction relationships in the vitamin D endocrine system. Endocr Rev 1995;16:20057. [72] Bury Y, Ruf D, Mrk Hansen C, et al. Molecular evaluation of vitamin D3 receptor agonists designed for topical treatment of skin diseases. J Invest Dermatol 2001;116:78592. [73] Kissmeyer A-M, Binderup L. Calcipotriol (MC903): pharmacokinetics in rats and biological activities of metabolites. Biochem Pharmacol 1991;41:16016.

[74] Masuda S, Strugnell S, Calverley MJ, et al. In vitro metabolism of the anti-psoriatic vitamin D analog calcipotriol, in two cultured human keratinocyte models. J Biol Chem 1994;269:4794803. [75] Johnson CS, Hershberger PA, Bernardi RJ, et al. Vitamin D receptor: a potential target for intervention. Urology 2002;60:12330. [76] Eisman JA. Genetics of osteoporosis. Endocr Rev 1999;20:788804. [77] Mathieu C, Adorini L. The coming of age of 1,25dihydroxyvitamin D3 analogs as immunomodulatory agents. Trends Mol Med 2002;8:1749. [78] Herdick M, Bury Y, Quack M, et al. Response element- and coactivator-mediated conformational change of the vitamin D3 receptor permits sensitive interaction with agonists. Mol Pharmacol 2000;57: 120617. [79] Quack M, Carlberg C. Selective recognition of vitamin D receptor conformations mediates promoter selectivity of vitamin D analogs. Mol Pharmacol 1999;55:107787. [80] Toell A, Polly P, Carlberg C. All natural DR3-type vitamin D response elements show a similar functionality in vitro. Biochem J 2000;352:3019. [81] Jones G, Strugnell SA, DeLuca HF. Current understanding of the molecular actions of vitamin D. Physiol Rev 1998;78:1193231. [82] Wu S, Ren S, Chen H, et al. Intracellular vitamin D binding proteins: novel facilitators of vitamin D-directed transactivation. Mol Endocrinol 2000;14: 138797. [83] Carlberg C, Quack M, Herdick M, et al. Central role of VDR conformations for understanding selective actions of vitamin D3 analogues. Steroids 2001;66: 21321. [84] Tocchini-Valentini G, Rochel N, Wurtz JM, et al. Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands. Proc Natl Acad Sci USA 2001;98:54916. [85] Tocchini-Valentini G, Rochel N, Wurtz JM, et al. Crystal structures of the vitamin D nuclear receptor liganded with the vitamin D side chain analogues calcipotriol and seocalcitol, receptor agonists of clinical importance. Insights into a structural basis for the switching of calcipotriol to a receptor antagonist by further side chain modication. J Med Chem 2004;47:195661. [86] Eelen G, Verlinden L, Rochel N, et al. Superagonistic action of 14-epi-analogs of 1,25-dihydroxyvitamin D explained by vitamin D receptor-coactivator interaction. Mol Pharmacol 2005;67:156673. [87] Molnar F, Pera kyla M, Carlberg C. Vitamin D receptor agonists specically modulate the volume of the ligand-binding pocket. J Biol Chem 2006;281: 1051626.

Dermatol Clin 25 (2007) 525530

Retinoid Receptors
Batya B. Davidovici, MDa,*, Yalc in Tu zu n, MDb, Ronni Wolf, MDa
a b

The Dermatology Unit, Kaplan Medical Center, 76100 Rechovot, Israel Department of Dermatology, Istanbul University, 340 Istanbul, Turkey

In 1976, Michael Sporn and his colleagues [1] coined the term retinoid to refer to the naturally occurring compounds with vitamin A activity and to synthetic analogs of retinol; however, their structure had been determined in 1931 by Karrer and colleagues [2], who were awarded the Nobel prize for their discovery. Beginning in the 1960s, these compounds were assessed for multiple therapeutic uses, and their clinical benets were starting to amass. In 1969, Kligman and colleagues [3], and then other investigators, reported on the use of topical tretinoin for acne vulgaris, intensifying the interest in the diverse possibilities for the therapeutic use of retinoids in oral and topical formulations. An important cornerstone in the understanding of the mechanism of retinoic acid action was the discovery of intranuclear retinoic acid receptors (RARs) in 1987, by two independent groups [4,5]. This discovery demonstrated a retinoid-responsive transcription factor, giving rise to the concept that retinoids are like hormones. Thus, the diversity of the retinoids biologic eects is being translated increasingly to therapeutic applications in various elds of medicine. Further indications are being studied. This article focuses on the retinoid receptors to elucidate our understanding of their complex biologic activity that is reected in their therapeutic clinical eects as well as in their adverse reactions. Not all retinoids act through binding to nuclear receptors; thus, other supplementary mechanisms are still to be discovered.

What are retinoids? Retinoids covers the synthetic and natural forms of vitamin A (the term vitamin A includes preformed vitamin A alcohol, retinol and its aldehyde, retinal and its acid, transretinoic acid or tretinoin, and the provitamin b-carotene). Vitamin A cannot be synthesized in vivo by the human body; therefore, it is an essential nutrient that must be acquired through diet. Retinyl esters and b-carotene are the two major precursors of retinol that are present in our diet. About 50% of the intake of vitamin A is derived from animal products containing retinol and retinyl esters, whereas the remaining 50% is provided by plant carotenoids. In mammals, vitamin A exists in interconvertible forms as retinol (vitamin A alcohol), which is the main storage form; retinal (vitamin A aldehyde), which is necessary in its 11-cis-isomer form for visual function; and retinoic acid (vitamin A acid). There are three generations of synthetic retinoids. Manipulation of the polar end group and the polyene side chain of vitamin A forms the rst generation of retinoids (nonaromatic), which includes tretinoin (all-trans retinoic acid), isotretinoin (13-cis-retinoic acid), and alitretinoin (9-cis retinoic acid) [6]. The second-generation retinoids, etretinate and acitretin, also known as monoaromatic retinoids, are synthesized by replacing the cyclic end group of vitamin A with various substituted and nonsubstituted ring systems [6]. The third-generation retinoids, the polyaromatic (arotinoids) retinoids, including tazarotene (Tazorac), adapalene (Dierin), and bexarotene (Targretin), were developed by cyclizing the polyene chain. They bear little structural resemblance to retinol and qualify as a retinoid because
derm.theclinics.com

* Corresponding author. E-mail address: bdavidovici@yahoo.com (B.B. Davidovici).

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.016

526

DAVIDOVICI

et al

they share one or more functions of the parent compound. In summary, a retinoid can be dened as any molecule that, by itself or through metabolic conversion, binds to and activates the RARs, thereby eliciting transcriptional activation of retinoic acidresponsive genes that results in specic biologic responses.

Retinoids: activity therapeutics applications and associated side eects Retinoids have been shown to be ecacious for several skin conditions, including psoriasis, acne, ichthyosis, photoaging, and cancer, and to reduce skin atrophy caused by corticosteroid treatment for inammatory diseases [7]. Retinoids inhibit Kaposi sarcoma cell proliferation, and this eect is associated with favorable clinical results [811]. Other positive eects have been shown in skin T-cell lymphoma [12]. In addition to skin diseases, one of the most striking eects of retinoids is their ability to promote cellular dierentiation of acute promyelocytic leukemia cells in humans [13]. Retinoids also are used in chemoprevention of lung cancer and have been tested alone or in combination with interferon in cervical cancer [14]. Unfortunately, these benecial eects are associated with severe side eects, such as developmental abnormalities in embryos, hypertriglyceridemia, general mucocutaneous toxicity, and headache. Moreover, prolonged treatment with all-trans retinoic acid is associated with multiple side eects, such as bone pain, retinoic acid syndrome, renal failure, thrombosis, and metabolic resistance [15].

RAR, VDR, and TR bind to their elements with greater anity as heterodimers (two dierent monomers), unlike receptors for the steroid hormones, which form homodimers (two identical monomers). The key partner for heterodimerization and ultimate functioning of these receptors is the retinoid X receptor (RXR) [16]. RXR probably is an important regulatory protein, because it has the ability to heterodimerize with many other receptors in addition to RARs, VDRs, and TRs. The RARs and RXRs are complex classes of receptors, each composed of a, b, and g subtypes and each encoded by dierent genes. To increase the diversity further, each of the retinoid receptors has more than one isoform. RARs bind all-trans retinoic acid, whereas the only known physiologic ligand for RXR is 9-cis retinoic acid [17]. In the presence of retinoid, the RARs and RXRs can bind specic DNA regulatory sequences and, thereby, activate specic sets of genes. Some of the genes regulated are other DNA-binding proteins (ie, other regulatory proteins). According to this paradigm, retinoids may cause changes in the expression of other genes by increasing or suppressing the expression of other regulatory proteins. By altering the expression of growth factors, oncogenes, keratins, or transglutaminases, retinoids can exert widespread changes in growth or dierentiation, controlling such diverse processes as epithelial dierentiation (cornication), embryonic morphogenesis, and carcinogenesis. An understanding of the complex interactions among the various nuclear retinoid receptors, including those of other members of this superfamily, will enhance the understanding of retinoid action.

The retinoid receptors How do the retinoids exert such a vast inuence on dierent cell lines? The answer lies in the milestone discovery of RARs and the characterization of their molecular features. It revealed a resemblance of RARs to steroid, vitamin D (VDR), and thyroid hormone receptors (TR) [4,5]. These receptors characteristically bind to regulatory regions in DNA called hormone response elements, or target sequences, and activate gene transcription in a ligand-dependent manner. Thus, they are referred to as ligand (hormone-)dependent transcription factors. These nuclear receptors bind to their response elements as dimers.

Who are the major retinoid receptors in human skin? A systematic analysis of the relative levels of each RAR and RXR protein has been made in human skin. Human epidermis expresses RAR-a, RAR-g, RXR-a, and RXR-b mRNAs [18]. RXR-g mRNA is undetectable, and the transcript for RARb is barely detectable. A similar pattern is observed in cultured human keratinocytes and dermal broblasts. These relative levels of retinoid receptor mRNA mirror their relative protein levels. In nuclei from human epidermis, there are ve times more total RXRs than RARs [19]. Thus, RXRs do not seem to be limiting for heterodimerization with RARs. This assumes that the number of dierent

RETINOID RECEPTORS

527

RXR partners (TR, VDR, and other unknown orphan receptors that might heterodimerize with RXRs in human skin) is limited and that they are expressed at levels similar to, or less than, RARs; however, the extent to which competition among nuclear receptors for interaction with RXRs inuences RAR-mediated responses in skin is unknown. For RAR proteins, 87% are RAR-g, and 13% are RAR-a, with no detectable RAR-b. Of the RXR proteins, 90% are RXR-a. In gel shift studies performed on nuclei isolated from human skin and cultured keratinocytes, which in both cases are expressing their normal endogenous receptors, only RXR-a and RAR (mostly-g) heterodimers bind to retinoic acidresponse elements (RAREs) [19,20]. Neither RAR nor RXR homodimers are detectable. Therefore, normal human epidermis is regulated by RXR-a and RAR-g heterodimers. In terms of the ligands needed to activate RXR/RAR-bound RAREs, based on chloramphenicol acetyltransferase reporter gene activity in keratinocytes, the heterodimer requires only the RAR ligand. Furthermore, the presence of RXR ligand does not confer additional transactivation induced by RAR ligand. Therefore, at their physiologic levels, RARs and RXRs in human skin bind RAREs as heterodimers and stimulate these response elements in response to RAR, but not RXR, ligands; however, the RXR is key, because the heterodimer will not bind to the RARE without the presence of the RXR protein. The stimulation of target gene (RARE) transcription is by a process known as transactivation. Thus, the physiologic actions of retinoids are mediated primarily through RAR/RXR-mediated transactivation.

skin by all-trans retinoic acid application. Keratin 6 normally is not expressed in human skin; keratin 6 is expressed, however, in hyperproliferative skin diseases and in response to retinoids. Retinoids also can antagonize gene transcription (a process called transrepression). Accordingly, transrepression is a widespread mechanism for cross-talk between retinoid receptors and the transcription factor activator protein (AP)-1 (ie, inhibition of AP-1driven responses). The molecular mechanism of transrepression is believed to involve direct or indirect proteinprotein interactions between retinoid receptors and several AP-1 components or competition between retinoid receptors and AP-1, because a common factor (or factors) is required for their activities [27,28]. Like transactivation, transrepression is dependent on trans retinoic acid. Thus, there probably are three distinct mechanisms through which retinoid receptors modulate gene expression: transactivation through direct binding to RARE in target gene promoters, thereby stimulating basal transcriptional machinery; transrepression of AP-1 (and probably other transcription factors) through proteinprotein interactions; and competition with other nuclear receptors (eg, VDR, TR) for heterodimerization with RXR.

Receptor-selective retinoids The design of receptor-selective as well as function-specic retinoids is required to achieve an optimized therapeutic index of retinoids. But is it feasible? Retinoids that are selective for the RAR subtype and for RAR or RXR have been described [2931]. Thus, in principle, it is possible to design retinoids that exhibit a broad range of biologic activities. Given this possibility, how can these retinoids be put to their best practical advantage? Part of the answer to this complex question depends on whether RAR subtypes possess functional specicity. Studies with transfected receptors acting over isolated response elements support the view that they do; however, this remains to be demonstrated in vivo. RAR-g is the predominant RAR in skin, which is expressed in keratinocytes throughout the epidermis and in broblasts in the dermis [32]. Therefore, it is expected that RAR-gspecic retinoids would have potent eects on skin cells. One drawback to their use is the possibility of enhanced skin irritation and scaling, because it was shown that the intensity of these side eects of retinoids correlates

What are the target genes of retinoid receptors and how are they regulated by the retinoid receptors? To understand how retinoids exert their eects in the skin, it is crucial to know how retinoid receptors regulate gene expression and what genes are under their regulation. Several genes are regulated directly by functional RAREs in their promoters [21]. Of these, the best characterized are cellular retinoic acidbinding protein II (CRABP II) [22,23], cellular retinol-binding protein [24], and keratin 6 [25,26]. Each of these genes is induced in human

528

DAVIDOVICI

et al

with their ability to activate RAR-g [33]. Whether RAR-a, which is expressed at approximately one tenth of the level of RAR-g, also mediates side eects is not known. Treatment of mouse skin with an RXRselective retinoid was without detectable eect [34], suggesting that RXR homodimers do not transactivate genes in the skin. Perhaps the most promising new therapeutic uses of synthetic retinoids for skin diseases are as AP-1 antagonists. Immune cell activation and inammation are typical features of many common skin diseases. Transcription of several lymphokine and cytokine genes, which drive the hyperplastic inammatory response in skin, is regulated by transcription factors AP-1 and nuclear factor-kappa B (NF-kB). This repressive activity seems to be the basis for their benecial therapeutic eects as anti-inammatory or antiproliferative agents. Retinoids, like corticosteroids, are able to inhibit AP-1 [27,28], but unlike corticosteroids, they do not inhibit NF-kB in all cell lines [35,36]. Retinoid inhibition of AP-1 is mediated by the interaction of RAR and RXR with AP-1 components, which compose AP-1, to form an inactive complex. Thus, RARs possess two ligand-dependent functions: transactivation of genes containing RAREs and transrepression of genes containing AP-1 response elements. Retinoic acid activates both functions; however, some synthetic retinoids are able to distinguish between these two functions. Synthetic retinoids have been described that exhibit potent AP-1 transrepression, but negligible RARE transactivation, and vice versa [37]. Such retinoids should be useful in elucidating the roles of transactivation versus transrepression in mediating physiologic and pharmacologic responses of skin to retinoids. In addition, retinoids that preferentially transrepress AP-1 may prove to be useful anti-inammatory and antiproliferative therapeutic agents. Such retinoids also oer the interesting possibility of reduced side eects, compared with conventional retinoids, because of their inability to stimulate RAR (RAR-g is a mediator of retinoid irritation) transactivation activity. Finally, orphan receptors, such as chicken ovalbumin upstream promoter-transcription factor (COUP-TF), may function as negative regulators of retinoid signaling [38,39]. COUPTF, which is expressed in skin, inhibits the expression of RAR-dependent reporter genes in human keratinocytes. This most likely occurs as a result of competition of COUP-TF with RARs for

RARE binding. COUP-TF binds to RAREs and to other direct repeat-response elements recognized by VDR, TR, and other receptors. Each receptor functions as a heterodimer with RXR. Therefore, COUP-TF, which binds to DNA as homodimers, may inuence the activities of the subset of nuclear receptors that partner with RXR. Obviously, there is much to be learned about the function and regulation of COUP-TF and other orphan receptors in the skin. Examples of receptor-selective retinoids Synthetic molecules could be screened for their ability to bind and activate the specic receptors. Many of these compounds have no structural similarities to all-trans retinol or retinoic acid; however, by virtue of their ability to activate the receptor, they can mediate the retinoid eect and, thus, be considered retinoids. Adapalene is a synthetic retinoid that ts this description and has been used topically in humans. It has restricted receptor specicity, possessing poor anity for RAR-a, higher anity for RAR-b and -g, and no interaction with RXR-a. Although adapalene does not bind to CRABPs, it is capable of inducing CRABP II mRNA like all-trans retinoic acid [4042]. Unlike retinoic acid, however, adapalene does not induce clinical erythema, epidermal hyperplasia, or spongiosis following a short-term (4 days) occlusive treatment. Tazarotene is another synthetic molecule that has biologic activity topically. By itself, tazarotene does not bind to RARs or RXRs; however, its acid metabolite (AGN 190299) has a weak, but relative, receptor selectivity for RARs (RAR-bORAR-gORAR-a) [43] and is believed to be the principal active form. Bexarotene was the rst synthetic, highly selective RXR retinoid to be studied in humans; in 1999, it was the rst retinoid approved for the treatment of cutaneous T cell lymphoma (CTCL) as an oral capsule and a topical gel formulation [44,45]. Central hypothyroidism occurred in 40% of patients in the CTCL trials of bexarotene. This eect probably is mediated through suppression of thyrotropin b subunit secretion by the thyrotrope cell of the anterior pituitary, which expresses RXR-g [46]. Summary Retinoids are key regulators of dierentiation, proliferation, and inammation. Their successful

RETINOID RECEPTORS

529

use in the treatment of various skin diseases and neoplasias has revolutionized the practice of dermatology as well as oncology. Their enormous impact underscores the need for more mechanistic studies to identify new target genes and clarify the molecular details of retinoid action. This could pave the way for the synthesis of new pharmacologic tools with extended possible therapeutic repertoire in multiple pathologic states and the improvement of their therapeutic index.

References
[1] Sporn MB, Dunlop NM, Newton DL, et al. Prevention of chemical carcinogenesis by vitamin A and its synthetic analogs (retinoids). Fed Proc 1976;35(6): 13328. [2] Karrer P, Morf R, Schopp K. [The knowledge of vitamin A from sh oil]. Helv Chim Acta 1931;14: 103641 [in German]. [3] Kligman AM, Fulton JE Jr, Plewig G. Topical vitamin A acid in acne vulgaris. Arch Dermatol 1969;99: 46976. [4] Petkovich M, Brand NJ, Krust A, et al. A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature 1987;330:44450. re V, Ong ES, Segui P, et al. Identication of [5] Gigue a receptor for the morphogen retinoic acid. Nature 1987;330:6249. [6] Nguyen EH, Wolverton SE. Systemic retinoids. In: Wolverton SE, Wilkin JK, editors. Systemic drugs for skin diseases. Philadelphia: WB Saunders; 2000. p. 269310. [7] Schwartz E, Mezick JA, Gendimenico CJ, et al. In vivo prevention of corticosteroid-induced skin atrophy by tretinoin in the hairless mouse is accompanied by modulation of collagen, glycosaminoglycans, and bronectin. J Invest Dermatol 1994;102: 2416. [8] Saiag P, Pavlovic M, Clerici T, et al. Treatment of early AIDS-related Kaposis sarcoma with oral all-transretinoic acid: results of a sequential non-randomized phase II trial. Kaposis Sarcoma ANRS Study Group. Agence Nationale de Recherches sur le SIDA. AIDS 1998;12(16):216976. [9] Nagpal S, Cai J, Zheng T, et al. Retinoid antagonism of NF-IL6: insight into the mechanism of antiproliferative eects of retinoids in Kaposis sarcoma. Mol Cell Biol 1997;17(7):415968. [10] Corbeil J, Rapaport E, Richman DD, et al. Antiproliferative eect of retinoid compounds on Kaposis sarcoma cells. J Clin Invest 1994;93(5):19816. [11] Duvic M, Friedman-Kien AF, Looney DJ, et al. Topical treatment of cutaneous lesions of AIDSrelated Kaposis sarcoma using 9-cis-retinoic acid (alitretinoin) gel: results of phase 1 and 2 trials. Arch Dermatol 2000;136:14619.

[12] Zhang C, Duvic M. Retinoids: therapeutic applications and mechanisms of action in cutaneous T-cell lymphoma. Dermatol Ther 2003;16(4):32230. [13] Chomienne C, Fenaux P, Degos L. Retinoid dierentiation therapy in promyelocytic leukemia. FASEB J 1996;10(9):102530. [14] Nagpal S, Chandraratna RA. Recent developments in receptor-selective retinoids. Curr Pharm Des 2000;6(9):91931. [15] Wang ZY, Chen Z, Huang W, et al. Problems existing in dierentiation therapy of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA). Blood Cells 1993;19(3):63341. [16] Yu VC, Delsert C, Andersen B, et al. RXR b: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response element. Cell 1991;67:125166. [17] Levin AA, Sturzenbecker LJ, Kazmer S, et al. 9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR a. Nature 1992;355:35961. [18] Elder JT, Astrom A, Pettersson U, et al. Dierential regulation of retinoic acid receptors and binding proteins in human skin. J Invest Dermatol 1992;98: 6739. [19] Fisher GJ, Talwar HS, Xiao JH, et al. Immunological identication and functional quantitation of retinoic acid and retinoid X receptor proteins in human skin. J Biol Chem 1994;269:2062935. [20] Xiao JH, Durand B, Chambon P. Endogenous retinoic acid receptor retinoid X receptor heterodimers are the major functional forms regulating retinoidresponsive elements in adult human keratinocytes. J Biol Chem 1995;270:300111. [21] Armstrong RB, Ashenfelter KO, Eckho C, et al. General and reproductive toxicology of retinoids. In: Sporn MB, Roberts AB, Goodman DS, editors. The retinoids: biology, chemistry, and medicine. New York: Raven Press; 1994. p. 54572. [22] Sanquer S, Gilchrest BA. Characterization of human cellular retinoic acidbinding proteins -I and II: ligand binding anities and distribution in skin. Arch Biochem Biophys 1994;311:8694. [23] Astro m A, Tavakkol A, Pettersson U, et al. Molecular cloning of two human cellular retinoic acid binding proteins (CRABP). Retinoic acidinduced expression of CRABP-II but not CRABP-I in adult human skin in vivo and in skin broblasts in vitro. J Biol Chem 1991;266:176626. [24] Fisher GJ, Reddy AP, Datta SC, et al. All-trans retinoic acid induces cellular retinol-binding protein in vivo. J Invest Dermatol 1995;105:806. [25] Rosenthal DS, Griths CE, Yuspa SH, et al. Acute or chronic topical retinoic acid treatment of human skin in vivo alters the expression of epidermal transglutaminase, loricrin, involucrin, laggrin, and keratins 6 and 13 but not keratins 1, 10, and 14. J Invest Dermatol 1992;98:34350. [26] Rosenthal DS, Roop DR, Hu CA, et al. Changes in photo-aged human skin following topical

530

DAVIDOVICI

et al [37] Fanjul A, Dawson MI, Hobbs PD, et al. A new class of retinoids with selective inhibition of AP-1 inhibits proliferation. Nature 1994;372:10711. [38] Hirose T, Apfel R, Pfahl M, et al. The orphan receptor TAK1 acts as a repressor of RAR-, RXR- and T3R-mediated signaling pathways. Biochem Biophys Res Commun 1995;211:8391. [39] Cooney A, Tsai S, OMalley B, et al. Chicken ovalbumin upstream promoter transcription factor (COUP-IF) dimers bind to dierent GGTCA response elements, allowing COUP-IF to repress hormonal induction of the vitamin D3, thyroid hormone, and retinoic acid receptors. Mol Cell Biol 1992;12:415363. [40] Bernard BA. Adapalene, a new chemical entity with retinoid activity. Skin Pharmacol 1993;6(Suppl 1): 619. [41] Griths CE, Elder JT, Bernard BA, et al. Comparison of CD271 (adapalene) and all-trans retinoic acid in human skin: dissociation of epidermal eects and CRABP-II mRNA expression. J Invest Dermatol 1993;101:3258. [42] Elder JT, Kaplan A, Cromie MA, et al. Retinoid induction of CRABP II mRNA in human dermal broblasts: use as a retinoid bioassay. J Invest Dermatol 1996;106:51721. [43] Nagpal S, Athanikar J, Chandraratna RA. Separation of transactivation and AP1 antagonism functions of retinoic acid receptor alpha. J Biol Chem 1995;270:9237. [44] Trent JT, Romanelli P, Kerdel FA. Topical targretin and intralesional interferon alfa for cutaneous lymphoma of the scalp. Arch Dermatol 2002;138(11): 14213. [45] Zackheim HS, Kashani-Sabet M, Main S. Treatment of mycosis fungoides with topical corticosteroids: a second look. J Clin Dermatol 1998;1:1520. [46] Duvic M, Olsen EA, Omura GA, et al. A phase III, randomized, double-blind, placebo-controlled study of peldesine (BCX-34) cream as topical therapy for cutaneous T-cell lymphoma. J Am Acad Dermatol 2001;44(6):9407.

[27]

[28] [29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

application of all-trans retinoic acid. J Invest Dermatol 1990;95:5105. Saatcioglu F, Claret FX, Karin M. Negative transcriptional regulation by nuclear receptors. Semin Cancer Biol 1994;5:34759. Pfahl M. Nuclear receptor/AP-1 interaction. Endocr Rev 1993;14:6518. Chen JY, Penco S, Ostrowski J, et al. RAR-specic agonist/antagonists which dissociate transactivation and AP-1 transrepression inhibit anchorage-independent cell proliferation. EMBO J 1995;14:118797. Dawson MI, Jong L, Hobbs PD. Conformational eects on retinoid receptor selectivity. 2. Eects of retinoid bridging group on retinoid X receptor activity and selectivity. J Med Chem 1995;38:336883. Lehmann JM, Jong L, Fanjul A, et al. Retinoids selective for retinoid X receptor response pathways. Science 1992;258:19446. Tavakkol A, Griths CE, Keane KM, et al. Cellular localization of mRNA for cellular retinoic acidbinding protein II and nuclear retinoic acid receptor-gamma 1 in retinoic acid-treated human skin. J Invest Dermatol 1992;99:14650. Chen S, Ostrowski J, Whiting C, et al. Retinoic acid receptor gamma mediates topical retinoid ecacy and irritation in animal models. J Invest Dermatol 1995;104:77983. Gendimenico GJ, Stim TB, Corbo M, et al. A pleiotropic response is induced in F9 embryonal carcinoma cells and rhino mouse skin by all-transretinoic acid, a RAR agonist but not by SRi 11237, a RXR-selective agonist. J Invest Dermatol 1994; 102(5):67680. Scheinman RI, Gualberto A, Jewell CM, et al. Characterization of mechanisms involved in transrepression of NF-kappa B by activated glucocorticoid receptors. Mol Cell Biol 1995;15:94353. Caldenhoven E, Liden J, Wissink S, et al. Negative cross-talk between RelA and the glucocorticoid receptor: a possible mechanism for the antiinammatory action of glucocorticoids. Mol Endocrinol 1995;9:40112.

Dermatol Clin 25 (2007) 531540

Toll-Like Receptors in Dermatology

Martin Mempel, MDa,b,c,*, Behnam Naderi Kalali, MScia,b, Markus Ollert, MDa,b, Johannes Ring, MDa,c
Department of Dermatology and Allergy, Technical University Munich, Biedersteiner Strasse 29, 80802 Munich, Germany b Clinical Research Division of Molecular and Clinical Allergotoxicology, Technical University Munich, Biedersteiner Strasse 29, 80802 Munich, Germany c Division of Environmental Dermatology and Allergy, GSF National Research Center for Environment and Health/Technical University Munich, Biedersteiner Strasse 29, 80802 Munich, Germany
a

The human skin represents the rst line of defense against potentially hazardous environmental threats (ie, the infection by microbes, such as viruses, bacteria, and fungi). To fulll this crucial function and to maintain the integrity of the skin compartment, evolution has equipped the human immune system with a variety of sophisticated tools leading to an ecient defense system of responses to various infectious challenges. The role of the skin within the dierent defense lines is multifaceted. First, the skin has to discriminate rapidly between self and non-self and dangerous and nondangerous. In other words, the skin has to be capable of starting a sucient innate, nonspecic immune response as well as initiating and amplifying a robust specic immune response. Both tasks fundamentally depend on the capacity to identify pathogen-associated molecular patterns within a foreign antigenic structure and to induce a proinammatory state of the skin in response. This central role of the immune defense system is performed by the group of pathogenassociated pattern recognition receptors, among which the group of Toll-like receptors (TLRs) has evolved as the central family during the last years

[1]. Ten TLRs are identied in humans, all of which share similarities in their structure and function, but respond to dierent microbial components (Fig. 1) [2]. Identication of Toll receptors Historically, the family of Toll receptors was described originally in the Drosophila y, where its genetic disruption leads to an impairment in the dorsoventral rotation during embryogenesis [3,4]. The German word Toll (fantastic, marvelous) was used by the German research group that discovered this central function, a discovery that led to the Nobel prize [5]. Besides its function during embryogenesis, it soon became evident that the Toll receptors play a central role in the defense to fungi. In 1997, Janeway and colleagues [6] described the rst human homolog of the Toll receptor family, which is now known as TLR4. Because of the sequence homologies and the similar function in innate immune defense, this group of receptors has been classied as TLRs in mammals. The role of TLRs is pleiotropic and partially overlapping. Although originally considered as pure receptors for pathogen-associated molecules, evidence has been growing that there are a variety of endogenous ligands for the dierent TLRs helping to determine the degree of immune responses that are evoked after ligation of the respective TLR.
derm.theclinics.com

* Corresponding author. Klinik und Poliklinik fu r Dermatologie und Allergologie, der TU Mu nchen, Biedersteiner Str. 29, D-80802 Mu nchen, Germany. E-mail address: m.mempel@lrz.tum.de (M. Mempel).

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.014

532

MEMPEL

et al

Fig. 1. Cellular localization and ligands for the family of TLRs. While the surface-expressed TLRs recognize bacterial compounds, the intracellular receptors are active against virus-associated molecules. CD14, cluster of dierentiation 14; dsRNA, double-stranded RNA; LP, lipoprotein; LTA, lipoteichoic acid; LPS, lipopolysaccharide; MD2, myeloid dierentiation 2; PGN, peptidoglycan; ssRNA, single-stranded RNA.

Ligand specicity for Toll-like receptors Five TLRs (TLR1, -2, -4, -5, and -6) recognize bacterial cell wall components. Because of their expression on the cell surface and their extracellular domain, they are classied as extracellular TLRs [7]. TLR4 was the rst receptor to be identied in humans. This receptor recognizes lipopolysaccharides of gram-negative bacteria [6], as well as virally encoded proteins (eg, the F protein of respiratory syncytial virus [8]) and self-proteins (eg, various heat-shock proteins [9] and b-defensins [10]). Moreover, matrix proteins, such as bronectin and the plasma protein brinogen, also have been identied to signal through TLR4 [11]. TLR2 can form homodimers recognizing lipoteichoic acid and peptidoglycan from grampositive bacteria [12], phosphatidyl-di-mannosides from mycobacteria [13], porin from Neisseria, and measles virus hemagglutinin [14] or it can associate with TLR1 as the TLR1/TLR2 heterodimer

reacting to tri-acetylated lipoprotein from mycobacteria and the crucial mycobacterial 19-kd protein [15]. Furthermore, TLR2/TLR6 heterodimers are found in the recognition of diacetylated lipoproteins [16,17], the phenol-soluble modulin of Staphylococcus epidermidis [18], and zymosan from yeasts [19]. TLR5 is the receptor for bacterial agellin from various bacteria; it is found as a TLR5 homodimer and as the TLR4/TLR5 heterodimer reacting to agellin [20,21]. In contrast to the group of extracellular TLRs, TLR3, -7, -8, and -9 are located in the cytoplasm and depend on the capacity of the microbial compound to penetrate the cell membrane and to be sensed in an intracytoplasmic compartment [22]. Consequently, these receptors recognize double-stranded RNA (TLR3) [23], single-stranded RNA (TLR7 and 8) [2426], and unmethylated prokaryotic DNA sequences, so-called cytosinphosphatidyl-Guanosin (CpG) motifs predominantly found in bacterial genome (TLR9) [27].

TOLL-LIKE RECEPTORS IN DERMATOLOGY

533

Human TLR7 and -8 have a strong capacity to react in response to imidazoquinolone (eg, imiquimod, resiquimod), a feature that has been exploited extensively for the treatment of viral disease and cancerous transformation of keratinocytes [28]. TLR10 was discovered recently; its exact function and the corresponding ligand are not known (see Fig. 1) [29]. Signaling of Toll-like receptors The family of TLRs shows great similarities with the interleukin (IL)-1 receptor, especially in its intracytoplasmic domain with a highly conserved TollIL-1 receptor (TIR) motif [30]. Five adaptor molecules have been described for the TIR domain: myeloid dierentiation factor 88 (MyD88), TIR domain-containing adaptor protein (TIRAP), TIR-containing adaptor inducing interferon regulatory factor b (TRIF), TRIF-related adaptor molecule (TRAM), and sterile alpha and HEAT/ Armadillo motif (SARM); the rst four have activating functions and SARM inhibitory capacities [31]. The extracellular part is composed of repetitive leucine residues (leucine-rich repeat). The intracytoplasmic signaling structure is divided into MyD88-dependent and -independent pathways, both of which show recruitment of crucial adaptor molecules to the TIR motif [32]. Most TLRs (TLR1, -2, -4, -5, -6, -7, -8, and -9) use MyD88 to initiate a signaling cascade that leads to the downstream activation of kinases and results in the translocation of the central transcription factors nuclear factor-kappa B (NF-kB) and interferon regulatory factor (IRF)-3 [2]. To this end, MyD88 associates with TIRAP to form a complex that recruits Interleukin-1 receptorassociated kinase (IRAK) and subsequently tumor necrosis factor receptor associated factor (TRAF)-6, nally resulting in activation of the IKappa B kinase complex (Fig. 2). In MyD88-independent signaling, which is used by TLR3 and to a certain degree by TLR4, the adaptor molecule TRIF is recruited to the intracellular part of TLR3 directly [33] or to TLR4 by way of TRAM [34]. This leads to activation of tank binding kinase (TBK)-1 and TRAF-6, a crucial checkpoint for the induction of an NF-kBdominated immune response or an IRF-3dominated immune response with a type I interferon (IFN) activation pattern [35]. It has become clear recently that the recruitment of TRAF-6 and TBK-1 is mutually exclusive, most probably because of steric hindrances that lead to the exclusive activation of

the NF-kB or IRF-3 pathway, depending on the accessibility of the binding sites [17]. The divergent activation of these two transcription factors leads to a preferential state of proinammatory immune responses (as for NF-kB) or antiviral immune responses (as for IRF-3). This activation is under the negative regulatory control of SARM, an alternative adaptor molecule for the TIR domain (Fig. 2) [31].

Consequences of Toll-like receptor activation Activation of NF-kB is one of the central activation pathways after recognition of TLR ligands. This activation induces the production of various chemokines and cytokines and enhances the capacity of phagocytotic cells to ingest microbial compounds. Because some of the TLRs (ie, TLR2 and -4) are able to colocalize to phagosomes [36,37], an early contact of the immune system with potentially hazardous microbial antigens is provided. Within the phagosome, two types of innate receptors cooperate to determine the type and magnitude of the immune response: phagocytic receptors (eg, mannose receptors) and pathogen-associated molecular pattern receptors (eg, TLRs) [38]. This combination provides a maturation signal for the phagosomes and seems crucial to distinguish self-antigens (which should not be loaded on major histocompatibility complex [MHC] II molecules) and foreign antigens (which should be loaded eciently on MHC II molecules) [38]. In addition to triggering phagocytotic and maturation signals, the engagement of TLRs enhances costimulatory molecule expression, such as CD80 and CD86, providing a second signal for the full immune response [39,40]. Thus, the engagement of TLRs in the phagosome enhances the killing of captured microorganisms and the ecient degradation of the ingested proteins for presentation at the cell surface. The situation is dierent for the group of intracellular TLRs (TLR3, -7, -8, and -9) that are located on the endosomal membrane where they sense virus-derived pattern molecules and respond with the induction of antiviral genes, such as type I IFNs [22]. Besides the induction of soluble factors, the engagement of intracellular TLRs enhances the presentation of antigens by the MHC I pathway, leading to the activation of CD8 cells, which are the central weapon against virally infected cells [41,42]. In addition to its central role in triggering

534

MEMPEL

et al

Fig. 2. Signaling pathways for the dierent TLRs. Receptors recognizing bacterial compounds tend to activate NF-kB, whereas virus-response TLRs preferentially signal through IRFs.

and shaping the cellular immune response, TLR activation results in the production of antimicrobial defensins. Thus, several epithelial cell types, including airway epithelial cells and keratinocytes, react to the stimulation through TLR2 or -4 by the up-regulation of human b-defensin 2 (HBD-2) [43,44]. After the induction of HBD-2, this antimicrobial peptide can act as a ligand for TLR4 and thereby enhance the immune response. In addition to defensin production [10], TLR engagement induces reactive oxygen and nitrogen species that are crucial for the killing of intracellular pathogens, such as Mycobacterium tuberculosis. In contrast to murine cells, the induction of mycobacterial killing in human macrophages and dendritic cells requires the parallel engagement of the vitamin D receptor [45]. The most important feature of TLR activation, however, is the production of a proinammatory milieu, which is provided by certain cytokines and chemokines. These are predominantly tumor necrosis factor a and IL-12 for NF-kB signaling TLR ligands and IFNa/b for IRF-3 signaling TLR ligands. The combination of the induction of a robust cellular immune response, together with a rapid skewing of the immune response toward a T helper

(TH)-1dominated prole, have rendered TLR agonists interesting adjuvants for allergy treatment and the design of tumor vaccines [46]. Finally, the engagement of TLRs in some tissues leads to the induction of programmed cell death, using caspase 8 and the FAS-associated death domain protein. This proapoptotic eect has been shown for mycobacterial, bacterial, and mycoplasmal lipoproteins and signals through TLR2 and -4 [15,47]. In summary, the sensing of microbial patterns through TLRs leads to the induction of a robust immune response of the TH-1 type, including the induction of potent self-defense molecules and the ecient conversion of immature antigen-presenting cells in a fully equipped amplier of a cellular immune response. In tissues that are not capable of specic antigen presentation, TLR signaling also might induce apoptosis to minimize the spreading of infection. Toll-like receptors in the epidermis The response of human skin cells to TLR ligands depends strongly on the cell type participating in the immune response. In principle, three

TOLL-LIKE RECEPTORS IN DERMATOLOGY

535

major cell populations react to microbial stimuli within the epidermis: keratinocytes, antigen-presenting cells, and melanocytes. The repertoire of TLR expression, however, varies between the cells. For example, keratinocytes denitely express TLR1, -2, -3, and -5 [4850], whereas controversy exists about the expression of TLR4 and -9, which have been shown to be expressed in keratinocytes by some authors shown [51]. However, their denite role in skin defense in not yet clear. Langerhans cells express high levels of TLR2; intermediate levels of TLR3, -4, -8, and -10; and low levels (to none at all) of TLR1, -5, -6, -7, and -9 [52]. High functional responses are seen especially for the TLR3 ligand polyriboinosinic-polyribocytidylic acid (equivalent of viral double-stranded RNA), the TLR2 ligand peptidoglycan, and the TLR7/8 ligand R848 [53]. Melanocytes express TLR4 (at least) and seem to respond, by matrix metalloproteinase induction, to the corresponding ligands. In parallel to the diverse expression of the dierent TLRs within the epidermal cells, the pattern of response also diers remarkably. Keratinocytes respond with NF-kB translocation to the challenge by Candida albicans or Staphylococcus aureus, in the latter case in a TLR2-dependent way [49,51]. This translocation results in the transcription of proinammatory genes, such as IL-8, cyclooxygenase 2, and inducible nitric oxide synthase and the production of several chemokines [49]. This switch to a proinammatory status also enables keratinocytes to induce dendritic cell maturation, which, in turn, leads to better antigen presentation [54]. Dendritic cells also respond directly to TLR ligands by up-regulation of their costimulatory molecules and by the production of various cytokines. Ligands that signal through one of the extracellular TLRs favor a stronger up-regulation of CD80 and CD86, whereas ligands for the intracellular TLRs induce better up-regulation of MHC I and II molecules [53]. The capacity of melanocyte-derived cells to upregulate metalloprotease activity after stimulation by the TLR4 ligands small fragment hyaluronic acid fragments have been suggested to play a role in tumor progression by melanoma cells [55]. If is not known whether this ability also plays a role in nontransformed melanocytes. Toll-like receptors and dermatologic diseases Leprosy It soon became clear that the reaction of mycobacterial cell wall components in leprosy

and tuberculosis would involve TLRs. Because the two opposite immunologic response patterns in leprosy include a T-cell reactive, TH-1 dominated course of the disease and a T-cell anergic, TH-2 dominated humoral course, several groups have tried to identify ligands for the dierent TLRs and correlate the expression of the respective receptor with the pattern of disease [56,57]. These experiments revealed the 19- and 33-kd lipoproteins as ecient activators of human monocytes and dendritic cells through the TLR1/ TLR2 heterodimer [58]. Consequently, in the T-cell reactive form of leprosy, a higher expression of TLR1 and -2 is found at the site of inammation [57]. Thus, it is tempting to speculate that inecient up-regulation of TLRs or inecient signaling through the receptors accounts, at least in part, for the dierences in immune response between T-cell reactive and T-cell anergic patients. Engagement of TLRs in some tissues also leads to the induction of programmed cell death. This phenomenon might contribute to the typical nerve damage seen in leprosy, because Schwann cells have been described to express TLR2 and to respond to the 19-kd lipoprotein by apoptosis [59]. In this case, the better reactivity of patients would be accompanied by greater nerve damage and would result in worse outcome. Acne vulgaris Acne lesions show a multifaceted pathology. Besides comedo formation with increased sebum secretion and obstruction of the sebaceous glandular duct, a profound inammation of the pilosebaceous unit, especially in the papulopustular type, is a hallmark of the disease. In inammatory acne, the presence of Propionibacterium acnes contributes to the initiation and perpetuation of the inammation by inducing proinammatory cytokines and tissuedamaging enzymes, such as proteases and hyaluronidases [60]. The proinammatory response in acne lesions especially seems to depend on TLR2. In accordance, TLR2 expression has been identied at the site of inammation, suggesting a pathogenic role for the disease [61]. Clinically, this observation is reected by cases of acute exacerbation of acne in which the addition of anti-inammatory treatment to conventional strategies leads to faster improvement of symptoms. Lupus erythematosus In patients who have lupus, the loss of tolerance to autoantigens leads to the development of

536

MEMPEL

et al

antinuclear antibodies that are the diagnostic hallmarks of the disease. Recent studies in the lupus-prone lpr mouse and in puried human B cells and antigen-presenting cells showed that various substrates of these autoantibodies also are activators of TLR signaling and that a full stimulation of autoreactive B cells functions through a parallel engagement of Fcg receptors and TLRs [6267]. In this scenario, especially the endosomally located TLRs (TLR3, -7, -8, and -9), which are capable of recognizing nucleic acids, would initiate and perpetuate this autoimmune pathology by reacting with innate response patterns to the nucleic acid derivates and, at the same time, initiate a humoral response to the autoantigen [68]. Psoriasis vulgaris Psoriasis vulgaris is a chronic inammatory skin disease with two prominent pathologic ndings: lesional skin in psoriasis shows inltration of T cells and neutrophils without an evident stimulus, and areas of psoriatic inammation show a marked hyperproliferation with shortening of the epidermal turnover time. Because TLRs are implicated in both, the regulation of immune responses and the control of dierentiation versus apoptosis, this group of receptors has been investigated by several groups in patients who have psoriasis; however, knowledge is scarce concerning a functional role in the disease. In comparative immunostaining experiments, no substantial differences in TLR expression between lesional plaques and control skin were observed [69]. In psoriatic plaques, the expression of TLR2 seemed to be increased in the upper parts of the epidermis, whereas TLR5 was down-regulated in the basal keratinocytes. Whether these ndings have a functional relevance needs to be explored. Psoriasis is marked by a strong expression of antimicrobial defensins [70], which are inuenced, at least in part, by TLR signaling. Borreliosis and syphilis The causative agent for the development of Lyme disease is the spirochete Borrelia. Clinical manifestations are the presence of migrating erythema followed by arthritis, neural involvement, and acrodermatitis in the late stage. The membrane of Borrelia contains several components that are potential ligands for TLRs, such as the outer surface protein A, which has been shown to signal through TLR2/TLR6 heterodimers [16]

or through the TLR1/TLR2 heterodimer and the bacterial agellin, which is a ligand of TLR5 [20]. Consequently, polymorphisms in the TLR2 gene have been associated with late-stage Lyme disease [71]. The engagement of TLR2 seems to down-regulate the expression of TLR5 rendering the host less responsive to Borrelia [72]. The stimulation of TLR2 also seems to induce matrix metalloproteinases, a mechanism by which the migration of the agellate within the cutaneous tissue might be enhanced [73]. The situation is similar in syphilitic lesions caused by Treponema pallidum. For infection with this pathogen, the lipopeptides produced by the bacterium seem to be the strongest stimulus for immune cell proliferation. These lipopeptides have been described to signal through TLR2 and result in the production of IL-12 and the up-regulation of costimulatory molecules [74]. In parallel to Borrelia, Treponema pallidum produces a agellar lament consisting of polymerized agellin subunits. Experiments in knock-out mice revealed that agellin-dependent nitric oxide production required TLR5 and -4, most probably as a TLR4/TLR5 heterodimer [21]. Toll-like receptor polymorphisms and atopic eczema The increase in atopic diseases in the last decades has been associated with various changes in the daily lifestyle in Western civilizations. Although not all theories have persisted, a sound majority of researchers explain this increase by the hygiene hypothesis, according to which the lack of exposure to infectious stimuli (or the lack of appropriate TH-1prone signaling) leads to a TH-2directed immune state preferentially responding to various stimuli with a humoral response pattern [75]. Dierences in the functionality of TLRs have been postulated as markers of this inappropriate TH-1 response and have been analyzed in their association with atopic diseases. Several studies found associations for other diseases, especially for polymorphisms within the TLR2 and -4 gene [76], but not for atopic eczema [77]. As a consequence of these genetic studies, it must be concluded that such polymorphisms might, at some point, contribute to the TH-2 deviation of atopic diseases; however, most probably are not the central genetic factors. A dierent approach was taken in a recent publication in which the mRNA for TLR2 and -4 was compared for children in industrialized and rural areas [78];

TOLL-LIKE RECEPTORS IN DERMATOLOGY

537

however, no experiments on TLR functionality were included, so TLR signaling in atopy awaits a further evaluation. The hygiene hypothesis works better for explaining the increase in respiratory atopy, whereas there are no consistent data for atopic eczema [79]. Toll-like receptor ligands in dermatologic therapy Soon after the description of the proinammatory capacity of TLRs, various approaches were undertaken to incorporate these capacities into daily practice. They were primarily dermatologicallergologic approaches to use TLR ligands as therapeutics. For example, soon after the identication of monophosphoryl lipid A (MPLA) as an active component of lipopolysaccharide for TLR4 signaling, specic allergen preparations using MPLA in combination with allergen extracts were introduced successfully into specic immunotherapy [80]. Almost the same holds true for CpG motifs as ligands for TLR9, which are now used increasingly to enhance the activity of specic immunotherapy formulations [81]. Imidazoquinolones, such as imiquimod and R-848, have been introduced into dermatologic practice for viral warts and most recently for the treatment of epidermal skin cancers and precancerous lesions [82,83]. Because the imidazoquinolones signal through TLR7 and most probably also through TLR8, their activation prole strongly resembles that of activators of the endosomally located TLRs, with high induction of type I IFNs (eg, IFNa/b) and good induction of cellular immune responses against virally infected cells [26]. The clinical experience for TLR7 ligands in the treatment of genital viral warts has provided sucient evidence that the stimulation of immune cells is eective in combating the virally infected cells. Thereby, the induction of IFNa/b has a direct antiviral eect and, at the same time, enhances the induction of antiviral CD8 T cells [84]. Keratinocytes do not express TLR7 or -8; despite this fact, they are major targets for antihuman papillomavirus therapy because they are the principle infected cell population. This discrepancy can be explained by two recent investigations. Keratinocytes can up-regulate TLR7 when stimulated virally [85], and in addition to TLRs, adenosine receptors seem to play a crucial role in the recognition of imidazoquinolones in keratinocytes [86]. This also might be the reason for the good clinical eect in the treatment of supercial epidermal cancers in which the transformed

keratinocytes also are targeted directly or through the inltration of primary immune competent cells. Thus, the activation of immune cells and keratinocytes by two distinct mechanisms provides an ecient immune response suitable for the treatment of cutaneous warts and epidermal (pre) malignancies. Summary TLRs are central in the induction of a rapid and robust immune response to various microorganisms. An understanding of them will help to explain various dermatologic diseases and will assist in the design of new and ecient drugs in several areas. References
[1] Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006;124:783801. [2] Kawai T, Akira S. TLR signaling. Cell Death Dier 2006;13:81625. [3] Anderson KV, Jurgens G, Nusslein-Volhard C. Establishment of dorsal-ventral polarity in the Drosophila embryo: genetic studies on the role of the Toll gene product. Cell 1985;42:77989. [4] Anderson KV, Nusslein-Volhard C. Information for the dorsalventral pattern of the Drosophila embryo is stored as maternal mRNA. Nature 1984;311: 2237. [5] Cohen B. Nobel committee rewards pioneers of development studies in fruities. Nature 1995;377:465. [6] Medzhitov R, Preston-Hurlburt P, Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997;388:3947. [7] Kawai T, Akira S. Pathogen recognition with Tolllike receptors. Curr Opin Immunol 2005;17:33844. [8] Kurt-Jones EA, Popova L, Kwinn L, et al. Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nat Immunol 2000;1:398401. [9] Beg AA. Endogenous ligands of Toll-like receptors: implications for regulating inammatory and immune responses. Trends Immunol 2002;23:50912. [10] Biragyn A, Runi PA, Leifer CA, et al. Toll-like receptor 4-dependent activation of dendritic cells by beta-defensin 2. Science 2002;298:10259. [11] Okamura Y, Watari M, Jerud ES, et al. The extra domain A of bronectin activates Toll-like receptor 4. J Biol Chem 2001;276:1022933. [12] Schwandner R, Dziarski R, Wesche H, et al. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2. J Biol Chem 1999;274:174069.

538

MEMPEL

et al [27] Hemmi H, Takeuchi O, Kawai T, et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000; 408:7405. [28] Schiller M, Metze D, Luger TA, et al. Immune response modiersmode of action. Exp Dermatol 2006;15:33141. [29] Hasan U, Chaois C, Gaillard C, et al. Human TLR10 is a functional receptor, expressed by B cells and plasmacytoid dendritic cells, which activates gene transcription through MyD88. J Immunol 2005;174:294250. [30] Bowie A, ONeill LA. The interleukin-1 receptor/ Toll-like receptor superfamily: signal generators for pro-inammatory interleukins and microbial products. J Leukoc Biol 2000;67:50814. [31] Carty M, Goodbody R, Schroder M, et al. The human adaptor SARM negatively regulates adaptor protein TRIF-dependent Toll-like receptor signaling. Nat Immunol 2006;7:107481. [32] Hashimoto C, Hudson KL, Anderson KV. The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein. Cell 1988;52:26979. [33] Yamamoto M, Sato S, Hemmi H, et al. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 2003;301:6403. [34] Fitzgerald KA, Rowe DC, Barnes BJ, et al. LPSTLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF. J Exp Med 2003;198:104355. [35] Fitzgerald KA, McWhirter SM, Faia KL, et al. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol 2003;4: 4916. [36] Blander JM, Medzhitov R. Regulation of phagosome maturation by signals from toll-like receptors. Science 2004;304:10148. [37] Underhill DM, Ozinsky A, Hajjar AM, et al. The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature 1999;401:8115. [38] Blander JM, Medzhitov R. Toll-dependent selection of microbial antigens for presentation by dendritic cells. Nature 2006;440:80812. [39] Michelsen KS, Aicher A, Mohaupt M, et al. The role of toll-like receptors (TLRs) in bacteria-induced maturation of murine dendritic cells (DCS). Peptidoglycan and lipoteichoic acid are inducers of DC maturation and require TLR2. J Biol Chem 2001; 276:256806. [40] Tsuji S, Matsumoto M, Takeuchi O, et al. Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guerin: involvement of toll-like receptors. Infect Immun 2000;68:688390. [41] Heit A, Maurer T, Hochrein H, et al. Cutting edge: Toll-like receptor 9 expression is not required for CpG DNA-aided cross-presentation of

[13] Gilleron M, Quesniaux VF, Puzo G. Acylation state of the phosphatidylinositol hexamannosides from Mycobacterium bovis bacillus Calmette Guerin and mycobacterium tuberculosis H37Rv and its implication in Toll-like receptor response. J Biol Chem 2003;278:298809. [14] Bieback K, Lien E, Klagge IM, et al. Hemagglutinin protein of wild-type measles virus activates toll-like receptor 2 signaling. J Virol 2002;76:872936. [15] Lopez M, Sly LM, Luu Y, et al. The 19-kDa Mycobacterium tuberculosis protein induces macrophage apoptosis through Toll-like receptor-2. J Immunol 2003;170:240916. [16] Bulut Y, Faure E, Thomas L, et al. Cooperation of Toll-like receptor 2 and 6 for cellular activation by soluble tuberculosis factor and Borrelia burgdorferi outer surface protein A lipoprotein: role of Tollinteracting protein and IL-1 receptor signaling molecules in Toll-like receptor 2 signaling. J Immunol 2001;167:98794. [17] Kang SS, Kauls LS, Gaspari AA. Toll-like receptors: applications to dermatologic disease. J Am Acad Dermatol 2006;54:95183 [Quiz: 836]. [18] Hajjar AM, OMahony DS, Ozinsky A, et al. Cutting edge: functional interactions between toll-like receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin. J Immunol 2001;166:159. [19] Sato M, Sano H, Iwaki D, et al. Direct binding of Toll-like receptor 2 to zymosan, and zymosan-induced NF-kappa B activation and TNF-alpha secretion are down-regulated by lung collectin surfactant protein A. J Immunol 2003;171:41725. [20] Hayashi F, Smith KD, Ozinsky A, et al. The innate immune response to bacterial agellin is mediated by Toll-like receptor 5. Nature 2001;410: 1099103. [21] Mizel SB, Honko AN, Moors MA, et al. Induction of macrophage nitric oxide production by Gramnegative agellin involves signaling via heteromeric Toll-like receptor 5/Toll-like receptor 4 complexes. J Immunol 2003;170:621723. [22] Wagner H. The immunobiology of the TLR9 subfamily. Trends Immunol 2004;25:3816. [23] Alexopoulou L, Holt AC, Medzhitov R, et al. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 2001;413:7328. [24] Diebold SS, Kaisho T, Hemmi H, et al. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 2004;303: 152931. [25] Heil F, Hemmi H, Hochrein H, et al. Species-specic recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 2004;303:15269. [26] Hemmi H, Kaisho T, Takeuchi O, et al. Small antiviral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol 2002;3:196200.

TOLL-LIKE RECEPTORS IN DERMATOLOGY

539

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

DNA-conjugated antigens but essential for crosspriming of CD8 T cells. J Immunol 2003;170:28025. Zaks K, Jordan M, Guth A, et al. Ecient immunization and cross-priming by vaccine adjuvants containing TLR3 or TLR9 agonists complexed to cationic liposomes. J Immunol 2006;176:733545. Becker MN, Diamond G, Verghese MW, et al. CD14-dependent lipopolysaccharide-induced betadefensin-2 expression in human tracheobronchial epithelium. J Biol Chem 2000;275:297316. Hertz CJ, Wu Q, Porter EM, et al. Activation of Toll-like receptor 2 on human tracheobronchial epithelial cells induces the antimicrobial peptide human beta defensin-2. J Immunol 2003;171:68206. Liu PT, Stenger S, Li H, et al. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science 2006;311:17703. Dabbagh K, Lewis DB. Toll-like receptors and T-helper-1/T-helper-2 responses. Curr Opin Infect Dis 2003;16:199204. Into T, Kiura K, Yasuda M, et al. Stimulation of human Toll-like receptor (TLR) 2 and TLR6 with membrane lipoproteins of Mycoplasma fermentans induces apoptotic cell death after NF-kappa B activation. Cell Microbiol 2004;6:18799. Ko llisch G, Kalali BN, Voelcker V, et al. Various members of the Toll-like receptor family contribute to the innate immune response of human epidermal keratinocytes. Immunology 2005;114:53141. Mempel M, Voelcker V, Ko llisch G, et al. Toll-like receptor expression in human keratinocytes: nuclear factor kappaB controlled gene activation by Staphylococcus aureus is toll-like receptor 2 but not toll-like receptor 4 or platelet activating factor receptor dependent. J Invest Dermatol 2003;121:138996. Miller LS, Sorensen OE, Liu PT, et al. TGF-alpha regulates TLR expression and function on epidermal keratinocytes. J Immunol 2005;174:613743. Pivarcsi A, Bodai L, Rethi B, et al. Expression and function of Toll-like receptors 2 and 4 in human keratinocytes. Int Immunol 2003;15:72130. Renn CN, Sanchez DJ, Ochoa MT, et al. TLR activation of Langerhans cell-like dendritic cells triggers an antiviral immune response. J Immunol 2006;177: 298305. Terhorst D, Kalali BN, Weidinger S, et al. Monocyte-derived dendritic cells from highly atopic individuals are not impaired in their pro-inammatory response to toll-like receptor ligands. Clin Exp Allergy 2007;37:38190. Lebre MC, Antons JC, Kalinski P, et al. Doublestranded RNA-exposed human keratinocytes promote Th1 responses by inducing a Type-1 polarized phenotype in dendritic cells: role of keratinocyte-derived tumor necrosis factor alpha, type I interferons, and interleukin-18. J Invest Dermatol 2003;120:9907. Voelcker V, Gebhardt C, Averbeck M, et al. Small fragments of hyaluronic acid (HA) induce metalloprotease- and cytokine upregulation in human

[56]

[57]

[58]

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

melanoma cells [abstract]. J Invest Dermatol 2006; 126:S30. Krutzik SR, Modlin RL. The role of Toll-like receptors in combating mycobacteria. Semin Immunol 2004;16:3541. Krutzik SR, Ochoa MT, Sieling PA, et al. Activation and regulation of Toll-like receptors 2 and 1 in human leprosy. Nat Med 2003;9:52532. McIntur JE, Modlin RL, Kim J. The role of tolllike receptors in the pathogenesis and treatment of dermatological disease. J Invest Dermatol 2005; 125:18. Oliveira RB, Ochoa MT, Sieling PA, et al. Expression of Toll-like receptor 2 on human Schwann cells: a mechanism of nerve damage in leprosy. Infect Immun 2003;71:142733. Vowels BR, Yang S, Leyden JJ. Induction of proinammatory cytokines by a soluble factor of Propionibacterium acnes: implications for chronic inammatory acne. Infect Immun 1995;63: 315865. Kim J, Ochoa MT, Krutzik SR, et al. Activation of toll-like receptor 2 in acne triggers inammatory cytokine responses. J Immunol 2002;169:153541. Barrat FJ, Meeker T, Gregorio J, et al. Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus. J Exp Med 2005; 202:11319. Boule MW, Broughton C, Mackay F, et al. Toll-like receptor 9-dependent and -independent dendritic cell activation by chromatin-immunoglobulin G complexes. J Exp Med 2004;199:163140. Ding C, Wang L, Al-Ghawi H, et al. Toll-like receptor engagement stimulates anti-snRNP autoreactive B cells for activation. Eur J Immunol 2006;36: 201324. Ehlers M, Fukuyama H, McGaha TL, et al. TLR9/ MyD88 signaling is required for class switching to pathogenic IgG2a and 2b autoantibodies in SLE. J Exp Med 2006;203:55361. Lau CM, Broughton C, Tabor AS, et al. RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement. J Exp Med 2005;202:11717. Savarese E, Chae OW, Trowitzsch S, et al. U1 small nuclear ribonucleoprotein immune complexes induce type I interferon in plasmacytoid dendritic cells through TLR7. Blood 2006;107:322934. Rahman AH, Eisenberg RA. The role of toll-like receptors in systemic lupus erythematosus. Springer Semin Immunopathol 2006;28:13143. Baker BS, Ovigne JM, Powles AV, et al. Normal keratinocytes express Toll-like receptors (TLRs) 1, 2 and 5: modulation of TLR expression in chronic plaque psoriasis. Br J Dermatol 2003;148:6709. Ong PY, Ohtake T, Brandt C, et al. Endogenous antimicrobial peptides and skin infections in atopic dermatitis. N Engl J Med 2002;347:115160.

540

MEMPEL

et al [79] Ring J, Przybilla B, Ruzicka T, editors. Handbook of atopic eczema. New York: Springer, Berlin; 2006. [80] Mothes N, Heinzkill M, Drachenberg KJ, et al. Allergen-specic immunotherapy with a monophosphoryl lipid A-adjuvanted vaccine: reduced seasonally boosted immunoglobulin E production and inhibition of basophil histamine release by therapy-induced blocking antibodies. Clin Exp Allergy 2003;33:1198208. [81] Creticos PS, Schroeder JT, Hamilton RG, et al. Immunotherapy with a ragweed-toll-like receptor 9 agonist vaccine for allergic rhinitis. N Engl J Med 2006;355:144555. [82] Kodner CM, Nasraty S. Management of genital warts. Am Fam Physician 2004;70:233542. [83] Schon MP, Schon M. Immune modulation and apoptosis induction: two sides of the antitumoral activity of imiquimod. Apoptosis 2004;9:2918. [84] Wille-Reece U, Wu CY, Flynn BJ, et al. Immunization with HIV-1 Gag protein conjugated to a TLR7/8 agonist results in the generation of HIV-1 Gag-specic Th1 and CD8 T cell responses. J Immunol 2005;174:767683. [85] Kalali NB, Ko llisch G, Bauer S, et al. Innate immune response of human keratinocytes to doubled stranded RNA (dsRNA). J Invest Dermatol 2006; 126:S57 [abstract]. [86] Schon MP, Schon M, Klotz KN. The small antitumoral immune response modier imiquimod interacts with adenosine receptor signaling in a TLR7- and TLR8-independent fashion. J Invest Dermatol 2006;126:133847.

[71] Schroder NW, Heine H, Alexander C, et al. Lipopolysaccharide binding protein binds to triacylated and diacylated lipopeptides and mediates innate immune responses. J Immunol 2004;173:268391. [72] Cabral ES, Gelderblom H, Hornung RL, et al. Borrelia burgdorferi lipoprotein-mediated TLR2 stimulation causes the down-regulation of TLR5 in human monocytes. J Infect Dis 2006;193:84959. [73] Behera AK, Hildebrand E, Uematsu S, et al. Identication of a TLR-independent pathway for Borrelia burgdorferi-induced expression of matrix metalloproteinases and inammatory mediators through binding to integrin alpha 3 beta 1. J Immunol 2006;177:65764. [74] Bouis DA, Popova TG, Takashima A, et al. Dendritic cells phagocytose and are activated by Treponema pallidum. Infect Immun 2001;69:51828. [75] Ring J, Darsow U, Jacob T, et al. Eczema and dermatitis. Allergy & Clinical Immunology International - Journal of the World Allergy Organization 2005;17:1925. [76] Eder W, Klimecki W, Yu L, et al. Toll-like receptor 2 as a major gene for asthma in children of European farmers. J Allergy Clin Immunol 2004;113:4828. [77] Weidinger S, Novak N, Klopp N, et al. Lack of association between Toll-like receptor 2 and Tolllike receptor 4 polymorphisms and atopic eczema. J Allergy Clin Immunol 2006;118:2779. [78] Lauener RP, Birchler T, Adamski J, et al. Expression of CD14 and Toll-like receptor 2 in farmers and non-farmers children. Lancet 2002;360: 4656.

Dermatol Clin 25 (2007) 541557

Melanocyte Receptors: Clinical Implications and Therapeutic Relevance

J. Andrew Carlson, MDa,*, Gerald P. Linette, MD, PhDb, Andrew Aplin, PhDc, Bernard Ng, MDa, Andrzej Slominski, MD, PhDd
a

Division of Dermatopathology and Dermatology, Department of Pathology and Laboratory Medicine, Albany Medical College MC-81, 47 New Scotland Ave., Albany, NY 12208, USA b Division of Oncology, Washington University School of Medicine, Campus Box 8056, 660 S. Euclid Avenue, St Louis, MO 63110, USA c Center for Cell Biology and Cancer Biology, Albany Medical College, MC-165, 47 New Scotland Ave., Albany, NY 12208, USA d Department of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, 930 Madison Ave., Memphis, TN 38163, USA

Melanocyte biology Melanocytes are an intraepidermal population of dendritic cells responsible for the production of melanin, a pigment that varies from yellow to brown to black pigment that after transfer to neighboring keratinocytes acts both as an endogenous screen and a buering system against harmful ultraviolet (UV) wavelengths in sunlight [1]. Skin pigmentation has both individual and societal implications. The cosmetic desire for increased pigmentation (tanning) has resulted in many deleterious alterations including hastened skin ageing with wrinkles and poikiloderma and an increase in lentigines, melanocytic nevi, and melanoma. Focal or widespread loss of normal pigmentation not only renders individuals extraordinarily vulnerable to the harmful eects of sunlight (eg, increased risk of skin cancer in albinism), but it can also result in severe emotional

Reviewed work was supported in part by National Institutes of Health (NIH) grants AR047079 and AR052190 from National Institute of Arthritis and Muskuloskeletal and Skin Disease and University of Tennessee Cancer pilot grant to Andrzej Slominski, and NIH grant R01-GM067893 to Andrew Aplin. * Corresponding author. E-mail address: carlsoa@mail.amc.edu (J.A. Carlson).

stress and, in some societies, ostracism and discrimination (eg, vitiligo). Melanocytes are derived from the neural crest and are located along the basal layer of the epidermis and within the hair follicle, predominately the basal layer of the hair bulb matrix [1,2]. By the 50th day of intrauterine life, melanocytes can be detected in the epidermis; their migration to the epidermis and survival is dependent on receptor tyrosine kinase (RTK) c-Kit and its ligand stem cell factor (SCF) within the epidermis [3,4]. Mutations of the c-Kit gene lead to patches of hypopigmentation caused by lack of melanocyte migration, termed piebaldism [5]. Another important signaling molecule in melanocyte migration and development is Wnt5a, which signals via the Frizzled-5 receptor [6]. Overexpression of Wnt5a/Frizzled is found in melanomas and associated with increased cell motility and invasiveness [7,8]. Skin keratinocytes obtain melanin pigment from melanocytes, and keratinocytes provide the necessary microenvironment for melanocyte survival, proliferation, dierentiation, and migration via production of ligands that interact with melanocyte receptors [1,911]. The epidermal melanin unit denotes the symbiotic relationship between one melanocyte transporting melanin via its dendritic processes to approximately 36 keratinocytes [10]. Melanocytes are located on the basement membrane among basal keratinocytes at
derm.theclinics.com

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.005

542

CARLSON

et al

ratio of 1 melanocyte per 5 basal keratinocytes in hematoxylin and eosinstained histologic sections. This balance is maintained through regulated induction of melanocyte division. During childhood as the skin surface expands, throughout adulthood to maintain melanocyte numbers, and in response to exposure to sunlight or skin wounding, melanocytes are stimulated to proliferate at a low rate. Melanocyte proliferation entails uncoupling from keratinocytes, loss of their dendrites, cell division, migration along the basement membrane, then recoupling with keratinocytes to form the epidermal melanin unit. Keratinocytes regulate melanocyte growth and expression of melanocyte cell surface receptors via cell adhesion and growth factors, which include E-cadherin, P-cadherin, and desmoglein that are regulated through growth factors such as hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and endothelin-1 (produced by broblasts or keratinocytes). Morphogens such as Notch receptors and their ligands also play a role in maintaining melanocyte function and morphology [12]. Loss of keratinocyte regulation characterizes the development of melanoma and is seen in the down-regulation of E-and P-cadherins, up-regulation of melanocyte-melanocyte and melanocyte cellbroblast adhesion molecules such as Mel-CAM and N-cadherin, expression of cell-matrix adhesion molecules such as avb3 integrins and increased elaboration of metallo-proteinases [10]. The importance in growth factor signaling in producing the malignant phenotype has been shown in experimental models where increased expression of basic broblastic growth factor (bFGF), HGF, SCF, and endothelin-3 coupled with UV radiation produced invasive and in situlike tumors [13,14]. Melanins are polymorphous and multifunctional biopolymers, whose biosynthesis involves a metabolic pathway beginning with the oxidation of tyrosine to L-DOPA, followed by a series of divergent steps that give rise to a brown-black pigment (eumelanin) composed predominantly of indolic units and a yellow to reddish-brown pigment (pheomelanin) having a backbone of benzothiazine units [1,2]. Most of human skin and hair pigmentation involves a combination of these pathways giving rise to mixtures of varying composition [1,2]. The phenotypic expression of this is generally classied according to the clinical Fitzpatrick skin types 1 through 6 and emphasizes the inverse relationship between the degree of pigmentation and solar damage to the skin, including

photocarcinogenesis. The functions of melanin pigments include protection from UV light, control of vitamin D3 synthesis, and local thermoregulation [1,15,16]. Melanogenesis is under complex regulatory control by multiple agents interacting via pathways activated by receptor-dependent and -independent mechanisms, in hormonal, autocrine, paracrine, or intracrine fashion [1]. Because of the multidirectional nature and heterogeneous character of the melanogenesis-modifying agents, its controlling factors are not organized into simple linear sequences, but they interact instead in a multidimensional network, with extensive functional overlapping with connections arranged both in series and in parallel [1,2]. The most important positive regulator of melanogenesis is the MC1 receptor with its ligands melanocortins and ACTH, whereas among the negative regulators, agouti protein stands out, determining intensity of melanogenesis and also the type of melanin synthesized [1,17]. Solar UV light is one of the main culprits in the etiology of skin cancers, and skin pigmentation and melanin content are principal determinants of the susceptibility to melanoma and other sun-induced skin cancers [1, 1822]. In general, individuals with fair skin who burn rather than tan when exposed to sun are at high risk for melanoma [19,21]. Mutations and polymorphisms of melanocortin-1 receptor type 1 gene (MCR1) play a role in skin cancer [2325]. Other important risk factors for skin cancer include DNA repair capacity, because melanoma patients have a lower DNA repair capacity than the general population, which may be in part related to loss-of-function mutations in the MCR1 [26,27]. Indeed, there exists a strong association between BRAF mutations and germline defects in melanocortin receptor type 1 (MC1R) and incidence of melanoma with no evidence of chronic sun damage [27]. Melanoma, like other cancers, arises because of accumulation of mutations in genes crucial for cell proliferation, cell dierentiation, and cell death [2830]. The factors inuencing development and progression of melanoma include tumor initiation (mutations, loss of heterozygosity, gene amplication, gain and loss of chromosomes, gene silencing by methylation), growth (loss of cell cycle control, growth factors, neovascularization), resistance to apoptosis (inactivation of cell death pathways, gain of anti-apoptotic and survival factors), invasion and metastasis (cell motility, cell adhesion, proteolytic enzymes), and escape from immune surveillance (loss or gain of

MELANOCYTE RECEPTORS

543

immune regulators) [29]. Melanocyte receptors that engage with tumor suppressor, oncogene, cell adhesion and cell-motility, and apoptotic pathways are all potential targets for oncogenic events or therapeutic agents. Tumor suppressor pathways most commonly inactivated in melanoma are the p16INK4/cyclindependent kinases 4 and 6/retinoblastoma protein and p14ARF/human double minute/p53 pathways, which control the G1 stage of the cell cycle [31,32]. Ras and its eector pathways Raf-MAPK kinaseERK and PI3K-Akt are most commonly activated [33,34]. Melanoma also shares many characteristics in common with developmental precursors, stem cells, or melanoblasts, to melanocytes that include activation of developmental signaling pathways [12,35,36]. Specically, signaling by receptor tyrosine kinases (RTK) (eg, c-Kit), the Wnt signaling pathway, melanocortin signaling pathway (a-MSH/MC1-R/cAMP), as well as loss of the p16INK4a cyclin-dependent kinase inhibitor are pathways that impact on the expression or function of Mitf, which plays an essential role in melanocyte development and survival [1,37,38]. Lastly, down-regulation of death receptors such as Fas also play a role in melanoma progression [39,40].

Cell surface melanocyte receptors Fig. 1 outlines key membrane bound melanocyte receptors and cell cycle pathways involved in melanocyte physiology and/or are activated or disrupted in melanoma. Table 1 lists melanocyte receptors that have both clinical relevance and potential as therapeutic targets to augment normal melanocyte function or manage melanocytic pathology. Among those are receptors coupled to G-proteins (G-protein coupled receptors [GPCR]) or having tyrosine kinases activities (receptor tyrosine kinases [RTK]). Other receptors of importance are death receptors, nuclear receptors such as the vitamin D receptor, and cell adhesion molecules, which can activate critical cell cycle, survival, and migration pathways. G-protein coupled receptors G-protein-coupled receptors, which initiate cellular signaling, constitute the largest class of cell-surface localized receptors whose genes account for about 5% of the human genome [41]. The ligands of GPCRs are chemically diverse and include neurotransmitters, hormones,

phospholipids, odorants, and, in phototransduction, photons. Receptor agonists, in some cases, can activate multiple members of GPCR families, such as the neurotransmitter catecholamines that can activate nine distinct members of the adrenergic family of GPCRs. Cytoplasmic domains of activated GPCRs transduce signals to activate guanine nucleotide exchange of one or more heterotrimeric G proteins found on the inner leaet of the cell membrane. Activation stimulates the exchange of bound GDP for GTP by the G-protein a (Ga) subunits and the functional dissociation of the Gbg complex. Ga and the Gbg complexes then act to regulate the activity of members of the larger family of G-protein eector molecules, including adenylyl cyclase, phospholipase Cb, cyclic nucleotide phosphodiesterases, and various ion channels. Among the numerous factors regulating melanocyte/melanoma behavior, the proopiomelanocortin (POMC)-derived peptides MSH, ACTH, and b-endorphin appear to be the most important [1,2,11,17]. In this context, and of particular interest, is the demonstration of MC1R activity up-regulation by UV light, indicating a novel site for physicochemical interactions with the environment [1,22]. Other important regulators of normal and malignant melanocyte activities include endothelines, histamine, eocosanoids, catecholamines, corticotrophin-releasing hormone (CRH), serotonin, and melatonin acting through corresponding GPCRs [1,11,17,42,43]. There is also substantial overlapping in the regulation of melanocyte behavior by the above factors [1]. GPCRs are considered among the most desirable targets for drug development, and many GPCRs, such as endothelin receptors, chemokine receptors, metabotropic glutamate receptors, and protease-activated receptors have been regulated in human cancers [44]. Using microarray data, some GPCRs, such as endothelin receptor A (ENRA), may be involved in early tumor progression, and others, such as CXCR4, may play a critical role in tumor invasion and metastasis [44]. Receptors for proopiomelanocortin-derived peptides The precursor protein POMC produces many biologically active peptides via a series of enzymatic steps in a tissue-specic manner, yielding a, b, g-MSH, ACTH) and b-endorphin [17]. The a, b, g-MSH and ACTH peptides bind with dierent anities to the extracellular G-proteincoupled MCR1-5 [1,17,45]. Of those, the most important role in the regulation of the melanocyte behavior

Fig. 1. Outline of the major cell surface receptor-mediated signaling pathways important in the dierentiation, survival, and function of melanocytes. In melanoma, regulatory disruption via activating mutations or gene amplications of oncogenes or deletion, epigenetic silencing, or inactivating mutations of tumor suppressor genes of either upstream or down stream constituents of these pathways can be commonly found. Mitogen-activated protein kinase (MAPK) pathway and phosphatidylinositol 3 kinase (PI3K)-Akt pathway are 2 crucial pathways activated by numerous growth factors and cell surface receptors. Binding of growth factors to their respective receptors leads to, via adapter proteins (not shown), activation of RAS proteins, which phosphorylate the mitogen-activated protein kinase (MEK) kinases, which then act on extracellular-related kinase (ERK) kinases. ERK kinases also interact with the PI3K-AKT pathway. Phosporylated ERK kinases (ERK-P) translocate to the nucleus and activate transcription factors, which promote cell cycle progression and proliferation. The PI3K-AKT pathway mediates cell survival signaling via growth factors such as PDGF, NGF, and IGF-1. Phosphatase and tensin homolog (PTEN) inhibits growth factor signaling by inactivating phosphatidylinositol triphosphate (PIP3) generated by PI3K. Activated PI3K converts the plasma membrane lipid phosphatidylinositol 4,5-bisphosphonate to PIP3, which acts as a second messenger leading to the phosphorylation AKT and subsequent up-regulation of cell cycle, growth, and survival proteins. AKT can also up-regulate mTOR (mammalian target of rapamycin), S6K, and NFkb leading to cell growth and inhibition of apoptosis. Wnt-Frizzled-b-catenin pathway. In the Wnt-Frizzled-b-catenin pathway, b-catenin plays a central role in cell adhesion and cell signaling. Wnt ligands activate the G-proteincoupled receptor, Frizzled, which blocks the breakdown of b-catenin by inactivation of the kinase GSK3. b-catenin accumulates in the cytoplasm and translocates to the nucleus, where it binds to LEFTCF transcription factors and increases the expression of several genes, including microphthalmia transcription factor (MITF) important in melanocyte survival and matrix metalloproteinases (MMP) crucial for cell invasion. JAK/Stat pathway can be activated by cytokines as well as growth factors such as EGF. JAK-STAT pathway is mediated by Janus kinase (JAK), a tyrosine kinase that phosphorylates STAT proteins localized to the plasma membrane. Phosphorylated STAT proteins are translocated to the nucleus where they activate gene transcription leading to inhibition of apoptosis and angiogenesis. a-MSH-MC1R or Microphthalmia-associated transcription factor (MITF) pathway. MITF is regulated at both the transcriptional level via activation of melanocortin receptor 1 (MCR1) pathway and at the posttranslation level via ERK phosphorylation (not shown). MC1R activates cyclic AMP (cAMP), which activates the camp response-element binding protein (CREB) leading to increased expression of MITF. MITF stimulates melanin production and cell cycle arrest in normal melanocytes, and in melanoma cells, it protects against apoptosis and adds chemotherapy resistance. (Courtesy of B. NG, MD, Albany, NY; Copyright 2006).

MELANOCYTE RECEPTORS

545

is played by MCR1. Although there are reports showing MCR2 expression on melanocytes [46,47], its role in normal and malignant melanocytes is unclear. Concerning b-endorphin, recent reports have shown that the cutaneous b-endorphin/m-opiate receptor system is functionally active via its ability to up-regulate melanocyte dendricity, proliferation, and pigmentation [48]. Melanocortin receptor type The MC1R (activated with similar eciency by a-MSH and ACTH) shows widespread cutaneous expression and inuence behavior of both melanocytes and keratinocytes as well as the skin immune system [1,17,26,45,49]. Mutations in the MC1R gene lead to fair skin and red hair in humans, which is also seen with inactivating human POMC gene mutations. MC1R mutant receptor expression changing the receptor activity is also listed as one of etiologic factors responsible for an increased incidence of the melanoma and nonmelanoma skin cancers [23,24]. The MC1R has pleiotropic eects including the modulation of a wide range of immune actions such as anti-inammatory actions, expression of adhesion molecules, and expression inammatory transcription factors (down-regulation of NFkB) [1,50]. It has been proposed that these actions would be consistent with a cytoprotective role for this hormone in protecting skin cells from exogenous stress, such as ultraviolet radiation (UVR), exposure to biological agents, or oxidative stress. In addition to actions on normal skin cells, MC1R ligands also modulate both cutaneous and uveal melanoma cell behavior. With respect to melanoma, it is intriguing to see reports showing that while a-MSH has the potential to retard metastatic spread it can also reduce the immune response against melanoma (for example through down-regulation of adhesion molecules) [45]. Thus, role of the MC1R extends far beyond cutaneous and hair pigmentation and includes modulation of immune system, cell viability, cell dierentiation, and ligand-induced activation of detoxication system in melanocytes [1,26,45]. However, progression of melanoma may owe at least some of its success to the protective role of a-MSH [45]. Cutaneous pigmentation is determined by the amounts of eumelanin and pheomelanin synthesized by epidermal melanocytes, which is also regulated by MC1R [1]. The human MC1R gene is highly polymorphic, and certain allelic variants of the gene are associated with red hair

phenotype, melanoma, and nonmelanoma skin cancer [24,51]. The impact of specic polymorphisms in the MC1R on the responses of melanocytes to melanotropins and UV radiation have been investigated [26,52]. Human melanocytes homozygous for Arg160Trp, heterozygous for Arg160Trp and Asp294His or for Arg151Cys and Asp294His substitutions, but not melanocytes homozygous for Val92Met substitution, in the MC1R, showed a signicantly reduced response to its specic ligands [53,54]. Presence of a nonfunctional MC1R increased in their sensitivity to the cytotoxic eect of UV radiation, suggesting that loss-of-function mutations in the MC1R gene sensitize melanocytes to the DNA damaging eects of UV radiation, thereby increasing risk of melanoma development [53]. Targeting melanocortin receptors Both natural-occurring and synthetic peptides have been used as inhibitors or agonists to identify the pathophysiologic role of MCR in dierent tissues [55]. For systemic processes, MCR inhibitors, mainly via prevention of NFkB activation, have been proposed as modulators of the immune system to dampen damage from inammation, ischemia and infection, or as suppressors of appetite to prevent obesity. Radioconjugates of a cyclic peptide analog of a-MSH (eg, coupled with 111In-DOTA) are being studied as selective agents for early detection of melanoma using positron-emission tomography imaging and as potential therapeutic agents [56,57]. The Y86 conjugate appears promising based on high selective uptake in B16 melanoma with rapid clearance from normal tissues [57]. Corticotrophin-releasing hormone receptor Melanocytes and melanoma cells express G proteincoupled CRH-R1 responding to CRH and urocortin peptides (exogenous or produced locally) through activation of cAMP, IP3, and Camediated pathways to modify the melanocyte phenotype [30,58,59]. In both normal and immortalized melanocytes, CRH inhibited cell proliferation in serum-containing medium, while inhibiting early and late apoptosis in serum free media [58]. Concerning melanoma cells, the eect was heterogenous depending on cell line [58,60]. The variability in CHR action on melanoma cells could be explained by co-expression of alternatively spliced CRH-R1 isoforms on the same cells that would modify the action of the CRH-R1a isoform [30,59]. Of signicance, an antimelanoma eect

546

Table 1 Melanocyte receptors: clinical relevance and potential therapeutic strategies Receptors avb3 integrin Function Dimer that forms cell-matrix adhesion molecule. MPAK and AKT activation; regulates cell motility, survival, and dierentiation. Regulates proliferation, dierentiation, and immune functions. Death receptors induce apoptosis. Pathologic mechanism Increased expression in melanoma, facilitating invasion and angiogenesis Activating mutation in minority of melanomas, mostly mucosal or lentigo maligna. Not described Antagonist Radiolabeled conjugates, Cilengitide (EMD 121,974) Imatinib, genitib, erlotinib SCF Agonist Clinical target Metastatic melanoma

c-Kit

Metastatic melanoma

CRH-R1

Antalarmin, a-helical CRH

Death receptors, DR3, DR4, Fas

Loss of expression or mutation contributes to immune evasion. Down-regulated in melanoma. Activated in melanoma because of increased expression of Wnt5a leads to melanoma invasiveness and motility. Gene polymorphisms aect hair and skin color and response to UVR. Loss of melatonin or its receptors may facilitate melanoma development and progression. Bosentan

Endothelin receptor

Frizzled

GPCR induces melanocyte proliferation and melanin synthesis. G protein coupled receptor activates AKT pathway, regulates motility.

CRH, urocortin and modied CRH and urocortin peptides Fas-Ligand, TRAIL; HGS-ETR1, -2, -2J PRO1762, CD95-Fc, TRA-8 EDN-1, EDN-2, EDN-3

Metastatic melanoma
CARLSON

Metastatic melanoma

et al Metastatic melanoma

Wnt5a

Metastatic melanoma

Melanocortin receptor (MC1R)

Pigmentation, anti-inammatory, anti-pyretic. GPCR and nuclear receptors aid in homeostasis and protect against radiation damage.

Agouti protein

Melatonin receptors

a-MSH ACTH O b-MSHOO g-MSH, radiolabeled a-MSH conjugate Melatonin

Tanning response, melanoma

Melanoma

MelCam

Cell-cell adhesion molecule.

Metabotropic glutamate receptor (Grm1) N-cadherin

Notch receptors

Receptor protein kinases

VEGF receptor

Vitamin D receptor

GPCR, which activates MPAK and AKT pathways. Cell-cell adhesionmelancoyte-broblast adhesion. MPAK and AKT activation regulates cell growth, survival, and dierentiation. MPAK and AKT activation regulates cell growth, survival and dierentiation. RTK, activates MPAK pathway and leads to angio-/lymphogenesis Regulates cell growth, survival.

Up-regulated in melanoma, aids in metastatic spread and growth at distant sites. Possibly up-regulated in melanoma. Increased expression in melanoma. Up-regulates melanoma cell adhesion via N-cadherin. Activated or mutated in some melanomas by paracrine and autocrine mechanisms. Increase expression in melanoma along with VEGF Polymorphosisms related to risk for melanoma and melanoma survival.

Blocking antibodies

Metastatic melanoma

Bay 36-7620

Glutamate

Metastatic melanoma

Blocking antibodies

Metastatic melanoma

Jagged 1-2, Delta1-3

Metastatic melanoma

Imatinib, genitib, erlotinib

bFGF, HGF, IGF-1, VEGF

Metastatic melanoma
MELANOCYTE RECEPTORS

BAY 43-9006 (sorafenib)

VEGF-1, -2,-3

Metastatic melanoma

Vitamin D3

Melanoma

Novel pharmacologic agent in development or clinical trials indicated in bold italics. Abbreviations: SCF, stem cell factor; UVR, ultraviolet radiation; VEGFR, vascular endothelial growth factor receptor.

547

548

CARLSON

et al

for selective CRH-R1 agonists has already been observed in experimental models of melanoma in vivo [60]. Accordingly, selective targeting of CRH-R1 has been proposed for the treatment of malignant tumors that include melanoma (patent WO0153777). Endothelin receptors Endothelins (EDN-1, EDN-2, EDN-3) are paracrine signal peptides that bind to at least two subtypes of GPCR (EDNRA and EDNRB) [36,61,62]. Endothelins induce normal human melanocyte proliferate, produce melanin, and aect chemokinesis, chemotaxis, and dendricity. Keratinocytes produce EDN-1 after exposure to UVB. In melanoma cell lines, EDN induces down-regulation of E-cadherin expression and up-regulates N-cadherin, increases avb3 integrin expression and tumor proteolytic activity; these events enhance melanoma cell adhesion, migration, and invasiveness. The selective blockade of EDNRB results in inhibition of focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) phosphorylation and cell proliferation induced by EDN [62]. EDN-1 also acts as a autocrine/paracrine growth factor or an antiapoptotic factor in human cancers, and blockade of ET-1 receptors can sensitize human tumor cells to apoptosis [26,63]. In an experimental study, bosentan, a dual EDNR(A/B)-receptor antagonist, decreased melanoma cell viability and DNA synthesis and induced melanoma cell apoptosis in dened human melanoma cells [63]. In addition, the eects of bosentan and alkylating agents were additive in melanoma cells [63]. Melatonin receptors Melatonin is an indole with pleiotropic bioactivities, which are mediated through interactions with high-anity membrane bound or nuclear receptors or through nonreceptor actions. Because melatonin receptors are expressed in melanocytes and melanoma cells, these have the potential to mediate phenotypic actions on cellular proliferation and dierentiation [16,42,43,64]. In addition, its receptor-independent activity suggests that melatonin could also have a protective role against UV-induced pathology [42,43,65]. Both biosynthetic and biodegradative pathways for melatonin have been initially characterized in whole human skin and in melanoma cells [42,43,66,67]. Melatonin has also been reported to exhibit tumorostatic properties in dierent tumor models

that include melanomas [42,43]. In addition, several clinical studies have reported positive results with melatonin in patients with metastatic malignant melanoma. Most recently, melatonin has been shown to have tumorostatic eect in human melanoma cell lines of dierent behavior [65]. The intensity of the oncostatic response to melatonin was related to the cell-linespecic pattern of melatonin cell surface and nuclear receptor expressions [65]. Thus, targeting melatonin receptors may represent a realistic approach in melanoma therapy [42]. Frizzled-5 receptor Frizzled receptors are GPCR that transduce signals upon Wnt binding leading to stabilization of b-catenin. In the absence of Wnt signaling, cytoplasmic b-catenin is degraded via an ubiquitin-mediated pathway, after phosphorylation by glycogen synthase kinase-b (GSK3b) [68]. Upon stabilization, b-catenin accumulates in the cytoplasm, some of which translocates into the nucleus where it participates in transcription via ternary complex factors (TCFs), lymphoid-specic DNA-binding proteins, regulating genes involved in development and tumorigenesis such as c-Myc, matrix metalloproteinase, and cyclin D1 [68]. In melanoma, gene expression proling has shown increased expression of Wnt5a/Frizzled in subsets of tumors [69]; this increased expression of Wnt and its receptor Frizzled correlates with histologic features of invasiveness [7,70]. Metabotropic glutamate receptor 1 Metabotropic glutamate receptor 1 (GRM1) is GPCR, the ligand of which is glutamate that was identied as aberrantly expressed in transgenic mouse model of spontaneous melanoma [71]. Its activity, whether agonist or constitutively induced, can be blocked by the inhibitor BAY 36-7620; thus, this receptor could be a target for therapy if it plays a role in human melanomas. Chemokine receptors Chemokines belong to a superfamily of small, cytokine-like molecules characterized by four conserved cysteine residues and by their capacity for binding to particular GPCR [72]. Chemokines regulate the directional migration of leukocytes; thus, they play an important role in infection and inammation. These molecules also play a broader role in the biological process as exemplied by the CXC chemokine receptor CXCR4 and its ligand CXCL12 (also known as

MELANOCYTE RECEPTORS

549

stromal-derived factor-1): CXCR4 is linked to human immunodeciency virus-1 (HIV-1) infection (serving as a coreceptor required for entry of HIV-1 into T cells), hematopoietic stem cell mobilization [73], and developmental processes such as embryogenesis, organogenesis, and angiogenesis. In addition, CXCR4 has been implicated in tumor progression where it is highly expressed in breast cancer cells. In vivo neutralization of CXCR4 by monoclonal antibodies (mAbs) signicantly impaired metastasis of breast cancers to regional lymph nodes and lung, indicating the involvement of CXCR4 in selective metastases of breast cancer to certain organs [74]. In melanoma [75], expression of CXCR4 in vivo selectively enhances the metastatic potential of melanoma cells to lung, but not to other organs. For primary cutaneous melanoma, CXCR4 is a prognostic marker indicative of a high risk of relapse; therefore, patients expressing this receptor warrant close follow-up [76]. Based on the above, chemokine receptors would be an attractive target to regulate the migration of tumor cells in vivo. Small molecular inhibitors of CXCR4 such as AMD3100 (byciclam) that are available orally, have been developed and may be of value in the treatment of metastatic melanoma [77]. Receptor tyrosine kinases Protein receptor kinases, which catalyze the phosphorylation of specic tyrosine residues on their substrate proteins is another major cell signaling paradigm. Protein receptor kinases are composed of both receptor tyrosine kinases (RTKs) and nonreceptor tyrosine kinases (NRTKs). These enzymes are involved in cellular signaling pathways that regulate key cell functions such as cellular proliferation, dierentiation, and apoptosis [41]. Examples of RTKs include the cell surface receptors for insulin (IR), insulinlike growth factor-I (IGF-IR), and epidermal growth factor (EGFR). NRTKs are made up of nine distinct families: Src, Jak, Abl, Fak, Fps, Csk, Syk, Pyk2, and Btk. These families share proteinprotein interaction domains (eg, SH2 [Src homology 2] and SH3 domains) that mediate many of their actions. Protein tyrosine receptor kinases have an extracellular ligand binding domain, a transmembrane domain, and an intracellular catalytic domain. Activation of the receptor is achieved by ligand binding to the extracellular domain, which induces dimerization of the receptors. Receptors then are able to autophosphorylate

tyrosine residues outside the catalytic domain, which form SH2 or phosphotyrosine binding (PTB) sites. The SH2 and PTB domains serve as docking sites for the recognition and recruitment of SH2-domaincontaining proteins (eg, NRTK Src and Ras). c-Kit (CD117) C-Kit is an RTK for SCF, which is a growth factor for melanocytes that aects melanogenesis, proliferation, migration, and survival. Activation of c-Kit signicantly promotes migration of the melanocytes both in vitro and in vivo, suggesting that, in mammalian melanocytes, activation of the cKit is primarily responsible for transmission of promigration signals, which may antagonize proliferation and melanogenesis [78]. In melanoma, the unregulated activity of c-Kit may be caused by overexpression, autocrine loops, or mutational activation; thus, targeting c-Kit with antagonists such as imatinib (Gleevec) is a potentially exploitable target in metastatic melanoma. Nevertheless, a recent clinical trial report concluded that imatinib as a single agent had no activity in patients with relapsed, refractory metastatic melanoma [79]. It appears that c-Kit was expressed at low levels on only a small minority of metastatic melanomas accounting for, in part, the lack of imatinib activity. Vascular endothelial growth factor, hepatocyte growth factor, insulinlike growth factor-1, basic broblastic growth factor, and eye-derived growth factor receptors All of these growth factor receptors have a tyrosine kinase activity and activate signaling pathways that alter gene expression patterns and induce proliferation. In many cancers, both the overexpression of the growth factor and the receptor, besides mutations at the cytoplasmic tyrosine kinase domain, contribute to constitutive signaling; thus, these receptors make attractive targets for targeted therapies [80]. For example, in the transition from radial to vertical growth phase, melanoma as well as angiogenesis is heralded by both the expression and release of vascular endothelial growth factor (VEGF), which facilitates both growth of new blood and the tumor [29,81,82]; inhibition of VEGF receptor (VEGFR) by sorafenib (formally known as BAY 43-9006 [83,84] in combination with antibodies that block VEGF such as bevacizumab might represent important combination therapy in metastatic melanoma patients. Early clinical

550

CARLSON

et al

development of sorafenib given with carboplatin and paclitaxel to patients with relapsed, refractory metastatic melanoma shows striking activity with prolonged progression-free survival (PFS; median PFS, 10 months) [85,86]. Randomized phase III clinical trials are underway in North America, Europe, and Australia evaluating this combination in metastatic melanoma. Death receptors Death receptors are transmembrane proteins that belong to the tumor necrosis factor (TNF) family of receptors [87]. They are activated by specic extracellular or type II transmembrane family member ligands, that rapidly trigger cellular death via an intracellular proteolytic cascade, which leads to irreversible cleavage of proteins involved maintenance of vital cell functions [39,87]. In melanocytes, 3 death receptor family members appear to be prominent: Fas (CD95) and TNFrelated apoptosis-inducing ligand cell receptors 1 and 2 (TRAIL-R1 [DR-4] and TRAIL-R2 [DR5], also known as DR4/TNFRSF10A and DR5/ TNFRSF10B, respectively) [40,88,89]. Agents that can trigger Fas or TRAIL-mediated cell death may prove eective in the treatment of metastatic melanoma [89,90]. Blockade of expression of death receptor ligands, such as PD-L1 (programmed death-ligand #1, member of the B7 family of costimulatory molecules), which appear to inhibit human tumor-specic T cell responses, is also another target for melanoma therapy [91]. Hersey and associates have characterized death receptor expression by human melanoma in detail and most melanoma lines examined express DR4 and DR5 [92,93]. However, many melanoma lines that express DR4/DR5 are not susceptible to TRAIL-mediated apoptosis suggesting additional determinants that down-regulate sensitivity such as protein kinase C (PKC) activation [92]. Recent data conrm DR5 expression at relatively high levels in approximately 75% of primary cutaneous melanoma; interestingly, DR4 expression was signicantly lower in both primary lesions and metastatic melanoma [88]. Novel therapeutics (recombinant ligands and monoclonal antibodies) specic for DR4/DR5 are currently in early-phase clinical trials. Fas (also known as APO-1 or CD95) is a type II membrane receptor of the TNF family and is known to be the receptor for Fas ligand (FasL). FasL engagement (or monoclonal antibody crosslinking) of cell surface

Fas triggers tumor cell death by apoptosis in a variety of experimental systems. Expression of Fas by human cutaneous melanomas has been documented in approximately 60% of cases [94]; only a small minority (3 of 44 primary cutaneous melanomas) had detectable mutations in Fas [95]. A survey of 13 established human melanoma lines showed low-level Fas expression in 5 of 13 lines [40]; however, this has been challenged because another group has reported high cell surface Fas expression on 15 of 17 melanoma lines examined [96]. The signicance of these ndings remains unclear. Cell adhesion receptors Melanocyte homeostasis is also governed by intercellular communication via cell-cell adhesions and cell-extracellular matrix (ECM) adhesion [10,97]. In this manner, transmembrane cell adhesion receptors inuence whether a cell remains quiescent or proliferates, dierentiates, or undergoes apoptosis; thus, the nature of cell-cell and cell-ECM interactions are integral in the regulation of homeostasis and tissue phenotype [98]. Alterations in the expression and/or adhesive interactions of cell adhesion receptors can trigger unchecked proliferation and altered invasive properties of melanocytes. In the development of melanoma, accumulating evidence has shown the importance of the tumor microenvironment on the behavior of malignant cells, where interactions between melanoma cells and stromal cells create a context that promotes the transitions from normal to benign to in situ to locally invasive to metastatic lesions [10,29,99]. Alterations in cell adhesion receptors are important in the processes underlying these transitions. Specically, melanoma progression is associated with the loss of E-cadherin function and with up-regulation or induction of N-cadherin, MelCAM, and integrin aVb3. Cadherins These calcium-dependent transmembrane proteins modulate cell-cell adhesion [100]. Alterations in the form and expression of cadherins appear critical in melanoma progression. Frequently, a switch from E- to N-cadherin expression occurs during the transition from radial to vertical growth phase. Loss of E-cadherin expression appears to be an important way that melanocytic cells detach from basal keratinocytes. Re-expression of E-cadherin in melanoma cells reduces their tumorigenicity in vivo [101]. N-cadherin

MELANOCYTE RECEPTORS

551

expression is frequently up-regulated in melanoma cells and allows for interactions with broblasts and endothelial cells in the dermis [102]. In this manner, enhanced N-cadherin expression promotes melanoma cell migration and survival. avb3-integrin Integrins are heterodimeric transmembrane glycoproteins that participate in cell adhesion, migration, and proliferation. In particular the vitronectin/bronectin receptor avb3 is up-regulated in melanoma progression [103]. Expression of the b3 subunit is controlled by the RAFMEK-ERK1/2 pathway that is highly active in melanoma cells because of either mutational activation of BRAF or RAS [104]. Antagonists to avb3 integrin can block tumor-associated angiogeneisis and cause tumor regression [105]. This integrin has an Arg-Gly-Asp (RGD) binding site that can be targeted by radiolabeled peptides [56,105] or inhibited by the cyclic peptide cilengitide (EMD 121,974) [106]. Expression of aVb3 integrin allows melanoma cells to bind to and localize matrix metalloproteinase (MMP)-2 (aka, type 4 collagenase/gelatinase A), which is suspected to augment the metastatic capability of melanoma cells by degrading the basement membrane zone and surrounding tissues. The development of agents that can block these interactions, such as antibodies, could stop metastatic spread of melanoma [107]. Recent studies have generated avb3-targeting nanoparticle to deliver a kinasedefective form of C-RAF to angiogenic blood vessels in mice [108]. It would be of considerable interest if a similar nanoparticle could be generated to block B-RAF signaling in avb3-expressing melanoma cells. MelCAM MelCAM, a member of the IgG superfamily of cell adhesion molecules [100], is another good marker of tumor progression in human melanoma [109111]. It has been primarily linked to a role in invasion and metastasis. MelCAM expression in nontumorigenic melanoma cells mediates invasion of melanoma cells in a human skin reconstruct system [112] and tumorigenicity in nude mice [113]. Additionally, reduced MelCAM expression or antibody-mediated function blocking of MelCAM inhibits melanoma cell tumorigenicity and metastasis [112,114]. MelCAM participates in both homotypic and heterophilic interactions, although in the latter case the nature of the ligand remains to be dened [107,115,116]. MelCAM

has a reciprocal regulation loop with the serine/ threonine kinase, AKT. Inhibition of elevated AKT activity in human melanoma cell lines substantially reduced the expression of MelCAM; conversely, overexpression of constitutively active AKT up-regulated the levels of MelCAM [117]. In addition, overexpression of MelCAM in melanoma cells activated endogenous AKT and inhibited the proapoptotic protein BAD, leading to increased survival under stress conditions. Studies on melanoma cells implanted in nude mice have shown blocking antibodies to MelCAM inhibit cell growth and metastasis [114]. Therefore, the MelCAM-AKT signaling axis in melanoma is a potential target for therapy. Notch receptors In humans, the Notch family consists of 4 transmembrane receptors (Notch1-4) and 5 ligands (Jagged 1-2, Delta1-3). Notch receptors 1 and 4 may be up-regulated in melanoma [118,119]. Ligand binding leads to metalloproteinase- and gsecretasemediated proteolysis and cleavage of Notch1 intracellular from the plasma membrane [120,121]. Notch1 is translocated to the nucleus, where it associates with transcription factors RBP-Jk/CSL and mastermind-like (MAML) to form a heteromeric complex that mediates the transcription of genes in the hairy enhancer of split (HES). Notch receptors and their ligands regulate cell fate specication, dierentiation, proliferation, and survival during cell-cell contact. In melanoma, Notch1 signaling drives the vertical growth phase of melanoma toward more aggressive phenotypes via activation of MAPK and PI3K-Akt pathways, increased tumor cell adhesion, and expression of N-cadherin [121]. Blocking of Notch signaling involved in melanoma progression may be a potential target of therapy in metastatic melanoma. Initial studies addressing this issue have shown that a g-secretase inhibitor compound induced apoptosis in melanoma cell lines [122]. Nuclear receptors Estrogen androgen receptors Normal melanocytes and melanoma cells have been shown to express functional receptors for steroidal sex hormones (reviewed in [1]). Androgen action appears to stimulate melanin pigmentation; however, cell culture studies showed conicting results. Nevertheless, melanocytes

552

CARLSON

et al

from genital skin do express androgen receptors and can transform testosterone to dihydrotestosterone (DHT). Similarly, genital melanocytes do express functional estrogen receptors; however, estrogen has shown inconsistent eects on proliferation and tyrosinase activity of cultured human foreskin melanocyte [1]. On the other hand, patients with elevated serum estrogen concentrations tend to have increased skin pigmentation, suggesting that estradiol may be involved in the pathogenesis of melasma (chloasma). These diverse reports suggest that the eects of androgens and estrogens on melanocyte activity may be inuenced by multiple factors, such as culture conditions, sex, age, and anatomic localization [1]. Concerning melanoma, it is know that its incidence rates rise in women until about age 50, and recent studies have found a correlation between estrogen receptor a (ERa) and melanoma progression [123]. Additional experimental studies are required to determine whether estrogen and androgen receptors could serve as a target for melanoma therapy. Glucocorticoids receptors Both inhibition and stimulation of melanin synthesis have been reported after glucocorticoid treatment, and this class of nuclear receptors may also be involved in melanocyte development (reviewed in [1]). The glucocorticoid receptors (GR) are expressed in the majority of human melanomas, with higher expression levels in metastatic sites, and, notably, glucocorticoids can inhibit growth of experimental melanomas in mice and GR-expressing human melanoma cells in culture [1]. However, potent immunosuppressive properties of glucorticoids could serve as a limiting factor in such a therapeutic approach, but one study found that glucocorticoids did not aect adoptive cell transfer (ACT)-based immunotherapy regimen in transplanted B16 melanoma [124]. Finally, epidemiologic studies showed that glucocorticoid-based therapy appeared to be protective against melanoma incidence in a Mediterranean population [125]. Thus, glucocorticoids and their receptors could be of help in melanoma therapy; however, further studies are needed to better dene this issue. Vitamin D3 receptors Vitamin D3 is formed in the skin by UVB-mediated photolysis of 7-dehydrocholesterol with following thermal isomerization [126]. To exert

its bioregulatory action, vitamin D3 must be converted to its active form 1,25-dihydoxycholecalciferol locally (skin) or at the systemic level (liver and kidney) [126]. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3; calcitriol] is inactivated through hydroxylation at position 24 [126]. 1,25(OH)2D3 has pleiotropic activities that, in addition to regulation of calcium metabolism, also acts as a potent regulator of cell growth, dierentiation, apoptosis, and immune and endocrine functions [126 129]. The signaling pathway of 1,25(OH)2D3 uses the vitamin D nuclear receptor (VDR), which is a transcription factor for 1,25(OH)2D3 target genes that have a major inhibitory eect on the G1/S checkpoint of the cell cycle by up-regulating the cyclin-dependent kinase inhibitors p27 and p21, and by inhibiting cyclin D1. Indirect mechanisms include up-regulation of transforming growth factor-beta and down-regulation of the epidermal growth factor receptor. 1,25(OH)2D3 may induce apoptosis either indirectly through effects on the IGFR and TNF-a or more directly via the Bcl-2 family system, the ceramide pathway, the death receptors (eg, Fas), and the stress-activated protein kinase pathways (Jun N terminal kinase and p38). Taking into consideration well-documented antitumorigenic properties of vitamin D3, it is becoming evident that targeting VDR may serve as a potential adjuvant therapy of melanoma [130]. Specically, melanocytes and melanoma cells do express functionally active VDR, which apparently mediate vitamin D3 antimelanoma eects (reviewed in [1,16]). Thus, the antiproliferative and prodierentiation eects of 1,25(OH)2D3 have been shown in cultured melanocytes, MM cells, and MM xenografts. Furthermore, melanoma patients may present with lower levels of 1,25(OH)2D3 in their sera [131], and polymorphisms at the VDR has been suggest to inuence susceptibility to malignant melanoma [132,133]. Finally, epidemiologic studies by some investigators found that melanoma patients do better if exposed to solar radiation after removal of the tumor, an indirect indication of a benecial role for vitamin D metabolites during disease progression [134]. However, other investigators have questioned this correlation [135]. Nevertheless, there is sucient experimental evidence for the use of vitamin D3 or its analogs in melanoma therapy. Unfortunately, potential use of vitamin D in therapy of melanoma may be limited, because of well-documented toxicity (hypercalcemia) of vitamin D3 derivatives when used at pharmacologic concentrations. Nevertheless, this cytoxicity

MELANOCYTE RECEPTORS

553

(hypercalcemic eect) can be decreased or eliminated by cleavage or modication of the side chain [136]. Most recently, it has been uncovered that the classical enzyme of steroidogenesis cytochrome P450scc can also cleave the side chain of 7-DHC to produce 7-dehydropregnenolone (7-DHP) identifying a novel metabolic pathway producing 5,7 diene hydroxysteroids that after UVB exposure could generate secosteroidal vitamin Dlike (VitDL) products [137,138]. Moreover, vitamin D3, ergosterol (provitamin D2) and vitamin D2 are additionally metabolized by the same P450scc producing hydroxyderivatives [42,139,140], some of which induce dierentiation and inhibit proliferation of skin cells [139,141]. Thus, new family of vitamin D derivatives was identied that could be tested for its potential antimelanoma activity.

Summary Cell surface and nuclear receptors and adhesion molecules are important regulators of critical pathways in normal and pathologic melanocytes. Antagonists and agonists of these proteins oer a novel, disease-specic means of targeted therapy. Much of the current development of anticancer therapies tries to target causative proteins in a specic manner to minimize side eects, some melanocyte receptors discussed in this review may become the targets for therapy of metastatic melanoma in the near future. References
[1] Slominski A, Tobin DJ, Shibahara S, et al. Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 2004;84(4):1155228. [2] Slominski A, Wortsman J, Plonka PM, et al. Hair follicle pigmentation. J Invest Dermatol 2005; 124(1):1321. [3] Holbrook KA. Melanocytes in human embryonic skin and fetal skin: review and new ndings. Pigment Cell Res 1998;1(Suppl):617. [4] Yoshida H, Kunisada T, Grimm T, et al. Review: melanocyte migration and survival controlled by SCF/c-kit expression. J Invest Dermatol Symp Proc 2001;6(1):15. [5] Fleischman RA, Saltman DL, Stastny V, et al. Deletion of the c-kit protooncogene in the human developmental defect piebald trait. Proc Natl Acad Sci U S A 1991;88(23):108859. [6] Christiansen JH, Coles EG, Wilkinson DG. Molecular control of neural crest formation, migration and dierentiation. Curr Opin Cell Biol 2000; 12(6):71924.

[7] Weeraratna AT, Jiang Y, Hostetter G, et al. Wnt5a signaling directly aects cell motility and invasion of metastatic melanoma. Cancer Cell 2002;1(3): 27988. [8] Bachmann IM, Straume O, Puntervoll HE, et al. Importance of P-cadherin, beta-catenin, and Wnt5a/frizzled for progression of melanocytic tumors and prognosis in cutaneous melanoma. Clin Cancer Res 2005;11(24 Pt 1):860614. [9] Kunisada T, Yamazaki H, Hayashi SI. Review: ligands for receptor tyrosine kinases expressed in the skin as environmental factors for melanocyte development. J Investig Dermatol Symp Proc 2001;6(1):69. [10] Haass NK, Herlyn M. Normal human melanocyte homeostasis as a paradigm for understanding melanoma. J Investig Dermatol Symp Proc 2005;10(2): 15363. [11] Slominski A, Paus R, Schadendorf D. Melanocytes as sensory and regulatory cells in the epidermis. J Theor Biol 1993;164(1):10320. [12] Moriyama M, Osawa M, Mak SS, et al. Notch signaling via Hes1 transcription factor maintains survival of melanoblasts and melanocyte stem cells. J Cell Biol 2006;173(3):3339. [13] Berking C, Takemoto R, Satyamoorthy K, et al. Induction of melanoma phenotypes in human skin by growth factors and ultraviolet B. Cancer Res 2004;64(3):80711. [14] Noonan FP, Dudek J, Merlino G, et al. Animal models of melanoma: an HGF/SF transgenic mouse model may facilitate experimental access to UV initiating events. Pigment Cell Res 2003;16(1):1625. [15] Wood JM, Jimbow K, Boissy RE, et al. Whats the use of generating melanin? Exp Dermatol 1999; 8(2):15364. [16] Slominski A, Wortsman J. Neuroendocrinology of the skin. Endocr Rev 2000;21(5):45787. [17] Slominski A, Wortsman J, Luger T, et al. Corticotropin releasing hormone and proopiomelanocortin involvement in the cutaneous response to stress. Physiol Rev 2000;80(3):9791020. [18] Diepgen TL, Mahler V. The epidemiology of skin cancer. Br J Dermatol 2002;146(Suppl 61):16. [19] Gilchrest BA, Eller MS, Geller AC, et al. The pathogenesis of melanoma induced by ultraviolet radiation. N Engl J Med 1999;340(17):13418. [20] Kennedy C, ter Huurne J, Berkhout M, et al. Melanocortin 1 receptor (MC1R) gene variants are associated with an increased risk for cutaneous melanoma which is largely independent of skin type and hair color. J Invest Dermatol 2001;117(2):294300. [21] Carlson JA, Slominski A, Linette GP, et al. Biomarkers in melanoma: predisposition, screening and diagnosis. Expert Rev Mol Diagn 2003;3(2): 16384. [22] Slominski A, Pawelek J. Animals under the sun: eects of ultraviolet radiation on mammalian skin. Clin Dermatol 1998;16(4):50315.

554

CARLSON

et al cell signaling and regulation. Trends Endocrinol Metab 2006;17(2):4854. Slominski A, Fischer TW, Zmijewski MA, et al. On the role of melatonin in skin physiology and pathology. Endocrine 2005;27(2):13748. Slominski A, Wortsman J, Tobin DJ. The cutaneous serotoninergic/melatoninergic system: securing a place under the sun. FASEB J 2005; 19(2):17694. Li S, Huang S, Peng SB. Overexpression of G protein-coupled receptors in cancer cells: involvement in tumor progression. Int J Oncol 2005;27(5): 132939. Eves PC, MacNeil S, Haycock JW. alpha-Melanocyte stimulating hormone, inammation and human melanoma. Peptides 2006;27(2):44452. Slominski A, Ermak G, Mihm M. ACTH receptor, CYP11A1, CYP17 and CYP21A2 genes are expressed in skin. J Clin Endocrinol Metab 1996; 81(7):27469. Hunt G, Donatien PD, Lunec J, et al. Cultured human melanocytes respond to MSH peptides and ACTH. Pigment Cell Res 1994;7(4):21721. Kauser S, Schallreuter KU, Thody AJ, et al. Regulation of human epidermal melanocyte biology by beta-endorphin. J Invest Dermatol 2003;120(6): 107380. Manna SK, Sarkar A, Sreenivasan Y. Alpha-melanocyte-stimulating hormone down-regulates CXC receptors through activation of neutrophil elastase. Eur J Immunol 2006;36(3):75469. Moustafa M, Szabo M, Ghanem GE, et al. Inhibition of tumor necrosis factor-alpha stimulated NFkappaB/p65 in human keratinocytes by alphamelanocyte stimulating hormone and adrenocorticotropic hormone peptides. J Invest Dermatol 2002;119(6):124453. Healy E. Melanocortin 1 receptor variants, pigmentation, and skin cancer susceptibility. Photodermatol Photoimmunol Photomed 2004;20(6):2838. Abdel-Malek ZA, Kadekaro AL, Kavanagh RJ, et al. Melanoma prevention strategy based on using tetrapeptide {alpha}-MSH analogs that protect human melanocytes from UV-induced DNA damage and cytotoxicity. FASEB J 2006;20(9):15613. Scott MC, Wakamatsu K, Ito S, et al. Human melanocortin 1 receptor variants, receptor function and melanocyte response to UV radiation. J Cell Sci 2002;115(Pt 11):234955. Abdel-Malek Z, Scott MC, Suzuki I, et al. The melanocortin-1 receptor is a key regulator of human cutaneous pigmentation. Pigment Cell Res 2000; 13(Suppl 8):15662. Getting SJ. Targeting melanocortin receptors as potential novel therapeutics. Pharmacol Ther 2006;111(1):115. Weiner RE, Thakur ML. Radiolabeled peptides in oncology: role in diagnosis and treatment. BioDrugs 2005;19(3):14563.

[23] Han J, Kraft P, Colditz GA, et al. Melanocortin 1 receptor variants and skin cancer risk. Int J Cancer 2006;119(8):197684. [24] Rees JL. The genetics of sun sensitivity in humans. Am J Hum Genet 2004;75(5):73951. [25] Hauser JE, Kadekaro AL, Kavanagh RJ, et al. Melanin content and MC1R function independently affect UVR-induced DNA damage in cultured human melanocytes. Pigment Cell Res 2006;19(4):30314. [26] Kadekaro AL, Wakamatsu K, Ito S, et al. Cutaneous photoprotection and melanoma susceptibility: reaching beyond melanin content to the frontiers of DNA repair. Front Biosci 2006;11:215773. [27] Landi MT, Bauer J, Pfeier RM, et al. MC1R germline variants confer risk for BRAF-mutant melanoma. Science 2006;313(5786):5212. [28] Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100(1):5770. [29] Carlson JA, Ross JS, Slominski A, et al. Molecular diagnostics in melanoma. J Am Acad Dermatol 2005;52(5):74375 [quiz: 7758]. [30] Slominski A, Wortsman J, Pisarchik A, et al. Cutaneous expression of corticotropin-releasing hormone (CRH), urocortin, and CRH receptors. FASEB J 2001;15(10):167893. [31] Chin L. The genetics of malignant melanoma: lessons from mouse and man. Nat Rev Cancer 2003; 3(8):55970. [32] Sharpless E, Chin L. The INK4a/ARF locus and melanoma. Oncogene 2003;22(20):30928. [33] Daniotti M, Oggionni M, Ranzani T, et al. BRAF alterations are associated with complex mutational proles in malignant melanoma. Oncogene 2004; 23(35):596877. [34] Gorden A, Osman I, Gai W, et al. Analysis of BRAF and N-RAS mutations in metastatic melanoma tissues. Cancer Res 2003;63(14):39557. [35] Grichnik JM, Burch JA, Schulteis RD, et al. Melanoma, a tumor based on a mutant stem cell? J Invest Dermatol 2006;126(1):14253. [36] Lahav R. Endothelin receptor B is required for the expansion of melanocyte precursors and malignant melanoma. Int J Dev Biol 2005;49(23):17380. [37] Goding CR. Melanocyte development and malignant melanoma. Forum (Genova) 2000;10(3): 17687. [38] Widlund HR, Fisher DE. Microphthalamia-associated transcription factor: a critical regulator of pigment cell development and survival. Oncogene 2003;22(20):303541. [39] Wehrli P, Viard I, Bullani R, et al. Death receptors in cutaneous biology and disease. J Invest Dermatol 2000;115(2):1418. [40] Bullani RR, Wehrli P, Viard-Leveugle I, et al. Frequent downregulation of Fas (CD95) expression and function in melanoma. Melanoma Res 2002; 12(3):26370. [41] Gavi S, Shumay E, Wang HY, et al. G-protein-coupled receptors and tyrosine kinases: crossroads in

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

MELANOCYTE RECEPTORS

555

[57] McQuade P, Miao Y, Yoo J, et al. Imaging of melanoma using 64Cu- and 86Y-DOTAReCCMSH(Arg11), a cyclized peptide analogue of alpha-MSH. J Med Chem 2005;48(8):298592. [58] Slominski A, Zbytek B, Pisarchik A, et al. CRH functions as a growth factor/cytokine in the skin. J Cell Physiol 2006;206(3):78091. [59] Slominski A, Zbytek B, Zmijewski M, et al. Corticotropin releasing hormone and the skin. Front Biosci 2006;11:223048. [60] Carlson KW, Nawy SS, Wei ET, et al. Inhibition of mouse melanoma cell proliferation by corticotropin-releasing hormone and its analogs. Anticancer Res 2001;21(2A):11739. [61] Eberle J, Weitmann S, Thieck O, et al. Downregulation of endothelin B receptor in human melanoma cell lines parallel to dierentiation genes. J Invest Dermatol 1999;112(6):92532. [62] Rosano L, Spinella F, Genovesi G, et al. Endothelin-B receptor blockade inhibits molecular eectors of melanoma cell progression. J Cardiovasc Pharmacol 2004;44:S1369. [63] Berger Y, Bernasconi CC, Juillerat-Jeanneret L. Targeting the endothelin axis in human melanoma: combination of endothelin receptor antagonism and alkylating agents. Exp Biol Med (Maywood) 2006;231(6):11119. [64] Slominski A, Pisarchik A, Zbytek B, et al. Functional activity of serotoninergic and melatoninergic systems expressed in the skin. J Cell Physiol 2003; 196(1):14453. [65] Fisher T, Zmijewski M, Zbytek B, et al. Oncostatic eects of indole melatonin and expression of its cytosolic and nuclear receptor in cultured human melanoma cell lines. Int J Oncol 2006;29: 66572. [66] Slominski A, Pisarchik A, Semak I, et al. Serotoninergic and melatoninergic systems are fully expressed in human skin. FASEB J 2002;16(8): 8968. [67] Slominski A, Semak I, Pisarchik A, et al. Conversion of L-tryptophan to serotonin and melatonin in melanoma cells. FEBS Lett 2002;511:1026. [68] Weeraratna AT. A Wnt-er wonderlandthe complexity of Wnt signaling in melanoma. Cancer Metastasis Rev 2005;24(2):23750. [69] Bittner M, Meltzer P, Chen Y, et al. Molecular classication of cutaneous malignant melanoma by gene expression proling. Nature 2000;406(6795): 53640. [70] Pham K, Milovanovic T, Barr RJ, et al. Wnt ligand expression in malignant melanoma: pilot study indicating correlation with histopathological features. Mol Pathol 2003;56(5):2805. [71] Namkoong J, Martino JJ, Chen S. From existing therapies to novel targets: a current view on melanoma. Front Biosci 2006;11:208192. [72] Baggiolini M. Chemokines in pathology and medicine. J Intern Med 2001;250(2):91104.

[73] Broxmeyer HE, Orschell CM, Clapp DW, et al. Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist. J Exp Med 2005;201(8): 130718. [74] Muller A, Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature 2001;410(6824):506. [75] Murakami T, Maki W, Cardones AR, et al. Expression of CXC chemokine receptor-4 enhances the pulmonary metastatic potential of murine B16 melanoma cells. Cancer Res 2002;62(24):732834. [76] Scala S, Ottaiano A, Ascierto PA, et al. Expression of CXCR4 predicts poor prognosis in patients with malignant melanoma. Clin Cancer Res 2005;11(5): 183541. [77] Scala S, Giuliano P, Ascierto PA, et al. Human melanoma metastases express functional CXCR4. Clin Cancer Res 2006;12(8):242733. [78] Alexeev V, Yoon K. Distinctive role of the cKit receptor tyrosine kinase signaling in mammalian melanocytes. J Invest Dermatol 2006;126(5):110210. [79] Wyman K, Atkins MB, Prieto V, et al. Multicenter Phase II trial of high-dose imatinib mesylate in metastatic melanoma: signicant toxicity with no clinical ecacy. Cancer 2006;106(9):200511. [80] Perona R. Cell signalling: growth factors and tyrosine kinase receptors. Clin Transl Oncol 2006;8(2): 7782. [81] Simonetti O, Lucarini G, Brancorsini D, et al. Immunohistochemical expression of vascular endothelial growth factor, matrix metalloproteinase 2, and matrix metalloproteinase 9 in cutaneous melanocytic lesions. Cancer 2002;95(9):196370. [82] Graells J, Vinyals A, Figueras A, et al. Overproduction of VEGF concomitantly expressed with its receptors promotes growth and survival of melanoma cells through MAPK and PI3K signaling. J Invest Dermatol 2004;123(6):115161. [83] Adnane L, Trail PA, Taylor I, et al. Sorafenib (BAY 43-9006, Nexavar((R))), a Dual-Action Inhibitor That Targets RAF/MEK/ERK Pathway in Tumor Cells and Tyrosine Kinases VEGFR/ PDGFR in Tumor Vasculature. Methods Enzymol 2005;407:597612. [84] Sharma V, Kaur I, Kumar B. Calcipotriol versus coal tar: a prospective randomized study in stable plaque psoriasis. Int J Dermatol 2003;42(10):8348. [85] Flaherty KT, Brose M, Schuchter L, et al. Phase I/II trial of BAY 43-9006, carboplatin (C) and paclitaxel (P) demonstrates preliminary antitumor activity in the expansion cohort of patients with metastatic melanoma. J Clin Oncol 2004;22(14S):7507. [86] Flaherty KT. Chemotherapy and targeted therapy combinations in advanced melanoma. Clin Cancer Res 2006;12(7 Pt 2):2366s70s. [87] Bouralexis S, Findlay DM, Evdokiou A. Death to the bad guys: targeting cancer via Apo2L/TRAIL. Apoptosis 2005;10(1):3551.

556

CARLSON

et al [103] Albelda SM, Mette SA, Elder DE, et al. Integrin distribution in malignant melanoma: association of the beta 3 subunit with tumor progression. Cancer Res 1990;50(20):675764. [104] Woods D, Cherwinski H, Venetsanakos E, et al. Induction of beta3-integrin gene expression by sustained activation of the Ras-regulated Raf-MEKextracellular signal-regulated kinase signaling pathway. Mol Cell Biol 2001;21(9):3192205. [105] Haubner R, Wester HJ, Burkhart F, et al. Glycosylated RGD-containing peptides: tracer for tumor targeting and angiogenesis imaging with improved biokinetics. J Nucl Med 2001;42(2):32636. [106] Eskens FA, Dumez H, Hoekstra R, et al. Phase I and pharmacokinetic study of continuous twice weekly intravenous administration of Cilengitide (EMD 121974), a novel inhibitor of the integrins alphavbeta3 and alphavbeta5 in patients with advanced solid tumours. Eur J Cancer 2003;39(7): 91726. [107] McGary EC, Lev DC, Bar-Eli M. Cellular adhesion pathways and metastatic potential of human melanoma. Cancer Biol Ther 2002;1(5):45965. [108] Hood JD, Bednarski M, Frausto R, et al. Tumor regression by targeted gene delivery to the neovasculature. Science 2002;296(5577):24047. [109] Lehmann JM, Riethmuller G, Johnson JP. MUC18, a marker of tumor progression in human melanoma, shows sequence similarity to the neural cell adhesion molecules of the immunoglobulin superfamily. Proc Natl Acad Sci U S A 1989;86(24): 98915. [110] Shih IM, Elder DE, Hsu MY, et al. Regulation of Mel-CAM/MUC18 expression on melanocytes of dierent stages of tumor progression by normal keratinocytes. Am J Pathol 1994;145(4):83745. [111] Shih IM, Elder DE, Speicher D, et al. Isolation and functional characterization of the A32 melanomaassociated antigen. Cancer Res 1994;54(9): 251420. [112] Satyamoorthy K, Muyrers J, Meier F, et al. MelCAM-specic genetic suppressor elements inhibit melanoma growth and invasion through loss of gap junctional communication. Oncogene 2001; 20(34):467684. [113] Xie S, Luca M, Huang S, et al. Expression of MCAM/MUC18 by human melanoma cells leads to increased tumor growth and metastasis. Cancer Res 1997;57(11):2295303. [114] Mills L, Tellez C, Huang S, et al. Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma. Cancer Res 2002;62(17):510614. [115] Shih IM, Speicher D, Hsu MY, et al. Melanoma cell-cell interactions are mediated through heterophilic Mel-CAM/ligand adhesion. Cancer Res 1997;57(17):383540. [116] Johnson JP, Bar-Eli M, Jansen B, et al. Melanoma progression-associated glycoprotein MUC18/

[88] McCarthy MM, Divito KA, Sznol M, et al. Expression of tumor necrosis factor-related apoptosisinducing ligand receptors 1 and 2 in melanoma. Clin Cancer Res 2006;12(12):385663. [89] Kurbanov BM, Geilen CC, Fecker LF, et al. Ecient TRAIL-R1/DR4-mediated apoptosis in melanoma cells by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). J Invest Dermatol 2005;125(5):10109. [90] Fischer U, Schulze-Ostho K. Apoptosis-based therapies and drug targets. Cell Death Dier 2005;12(Suppl 1):94261. [91] Blank C, Kuball J, Voelkl S, et al. Blockade of PD-L1 (B7-H1) augments human tumor-specic T cell responses in vitro. Int J Cancer 2006;119(2): 31727. [92] Wu JJ, Zhang XD, Gillespie S, et al. Selection for TRAIL resistance results in melanoma cells with high proliferative potential. FEBS Lett 2005; 579(9):19404. [93] Zhang XD, Franco A, Myers K, et al. Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma. Cancer Res 1999;59(11):274753. [94] Redondo P, Solano T, Vazquez B, et al. Fas and Fas ligand: expression and soluble circulating levels in cutaneous malignant melanoma. Br J Dermatol 2002;147(1):806. [95] Shin MS, Park WS, Kim SY, et al. Alterations of Fas (Apo-1/CD95) gene in cutaneous malignant melanoma. Am J Pathol 1999;154(6):178591. [96] Eberle AN, Froidevaux S. Radiolabeled alphamelanocyte-stimulating hormone analogs for receptor-mediated targeting of melanoma: from tritium to indium. J Mol Recognit 2003;16(5):24854. [97] Haass NK, Smalley KS, Li L, et al. Adhesion, migration and communication in melanocytes and melanoma. Pigment Cell Res 2005;18(3):1509. [98] Bissell MJ, Radisky DC, Rizki A, et al. The organizing principle: microenvironmental inuences in the normal and malignant breast. Dierentiation 2002;70(910):53746. [99] Ruiter D, Bogenrieder T, Elder D, et al. Melanoma-stroma interactions: structural and functional aspects. Lancet Oncol 2002;3(1):3543. [100] Aplin AE, Howe A, Alahari SK, et al. Signal transduction and signal modulation by cell adhesion receptors: the role of integrins, cadherins, immunoglobulin-cell adhesion molecules, and selectins. Pharmacol Rev 1998;50(2):197263. [101] Hsu M, Andl T, Li G, et al. Cadherin repertoire determines partner-specic gap junctional communication during melanoma progression. J Cell Sci 2000;113(Pt 9):153542. [102] Hsu MY, Wheelock MJ, Johnson KR, et al. Shifts in cadherin proles between human normal melanocytes and melanomas. J Investig Dermatol Symp Proc 1996;1(2):18894.

MELANOCYTE RECEPTORS

557

[117]

[118]

[119]

[120] [121]

[122]

[123]

[124]

[125]

[126] [127] [128] [129]

MCAM mediates homotypic cell adhesion through interaction with a heterophilic ligand. Int J Cancer 1997;73(5):76974. Li G, Kalabis J, Xu X, et al. Reciprocal regulation of MelCAM and AKT in human melanoma. Oncogene 2003;22(44):68919. Nickolo BJ, Hendrix MJ, Pollock PM, et al. Notch and NOXA-related pathways in melanoma cells. J Investig Dermatol Symp Proc 2005;10(2):95104. Nickolo BJ, Osborne BA, Miele L. Notch signaling as a therapeutic target in cancer: a new approach to the development of cell fate modifying agents. Oncogene 2003;22(42):6598608. Miele L. Notch signaling. Clin Cancer Res 2006; 12(4):10749. Liu ZJ, Xiao M, Balint K, et al. Notch1 signaling promotes primary melanoma progression by activating mitogen-activated protein kinase/ phosphatidylinositol 3-kinase-Akt pathways and up-regulating N-cadherin expression. Cancer Res 2006;66(8):418290. Qin JZ, Stennett L, Bacon P, et al. p53-independent NOXA induction overcomes apoptotic resistance of malignant melanomas. Mol Cancer Ther 2004; 3(8):895902. Mori T, Martinez SR, ODay SJ, et al. Estrogen receptor-alpha methylation predicts melanoma progression. Cancer Res 2006;66(13):66928. Hinrichs CS, Palmer DC, Rosenberg SA, et al. Glucocorticoids do not inhibit antitumor activity of activated CD8 T cells. J Immunother 2005;28(6): 51724. Landi MT, Baccarelli A, Calista D, et al. Glucocorticoid use and melanoma risk. Int J Cancer 2001; 94(2):3023. Holick MF. Vitamin D: a millenium perspective. J Cell Biochem 2003;88(2):296307. Holick MF. Evolution and function of vitamin D. Recent Results Cancer Res 2003;164:328. Bikle DD. Vitamin D and skin cancer. J Nutr 2004; 134(Suppl 12):3472S8S. Bikle DD, Oda Y, Xie Z. Vitamin D and skin cancer: a problem in gene regulation. J Steroid Biochem Mol Biol 2005.

[130] Osborne JE, Hutchinson PE. Vitamin D and systemic cancer: is this relevant to malignant melanoma? Br J Dermatol 2002;147(2):197213. [131] Cornwell ML, Comstock GW, Holick MF, et al. Prediagnostic serum levels of 1,25-dihydroxyvitamin D and malignant melanoma. Photodermatol Photoimmunol Photomed 1992;9(3):10912. [132] Hutchinson PE, Osborne JE, Lear JT, et al. Vitamin D receptor polymorphisms are associated with altered prognosis in patients with malignant melanoma. Clin Cancer Res 2000;6(2):498504. [133] Halsall JA, Osborne JE, Potter L, et al. A novel polymorphism in the 1A promoter region of the vitamin D receptor is associated with altered susceptibility and prognosis in malignant melanoma. Br J Cancer 2004;91(4):76570. [134] Berwick M, Armstrong BK, Ben-Porat L, et al. Sun exposure and mortality from melanoma. J Natl Cancer Inst 2005;97(3):1959. [135] Lim HW, Gilchrest BA, Cooper KD, et al. Sunlight, tanning booths, and vitamin D. J Am Acad Dermatol 2005;52(5):86876. [136] Holick MF, Garabedian M, Schnoes HK, et al. Relationship of 25-hydroxyvitamin D3 side chain structure to biological activity. J Biol Chem 1975; 250(1):22630. [137] Slominski A, Zjawiony J, Wortsman J, et al. A novel pathway for sequential transformation of 7-dehydrocholesterol and expression of the P450scc system in mammalian skin. Eur J Biochem 2004;271(21):417888. [138] Slominski A. Neuroendocrine system of the skin. Dermatology 2005;211(3):199208. [139] Slominski A, Semak I, Zjawiony J, et al. Enzymatic metabolism of ergosterol by cytochrome p450scc to biologically active 17alpha, 24-dihydroxyergosterol. Chem Biol 2005;12(8):9319. [140] Slominski A, Semak I, Zjawiony J, et al. The cytochrome P450scc system opens an alternate pathway of vitamin D3 metabolism. FEBS J 2005;272(16): 408090. [141] Slominski A, Semak I, Wortsman J, et al. An alternative pathway of vitamin D metabolism. FEBS J 2006;273(13):2891901.

Dermatol Clin 25 (2007) 559562

Langerhans Cell Receptors

Arieh Ingber, MDa,b,*
a

Department of Dermatology, Hadassah University Hospital, Ein Kerem, Jerusalem 91120, Israel b Hebrew University, Jerusalem, Israel

Langerhans cells (LC) are a subtype of dendritic cells (DCs), which reside in the epidermis. LCs are antigen-presenting cells that originate in bone marrow and enter the epidermis through blood vessels [1,2]. In vitro and in vivo studies have demonstrated that the major function of LCs is to provide signals for immune responses in the epidermis against a variety of antigens, including contact allergens, microorganisms, and tumor antigens [3,4]. The skin is the largest organ of the human body and is the rst barrier against hazards to the living organism. LCs play a major role in the functioning of the skins immune system in terms of innate and adaptive immune responses [5,6]. Innate immunity is dened as a fast and immediate defense response against pathogens such as viruses and bacteria, whereas adaptive immunity is mediated by T and B cells and is a slower immune response [7]. LCs exhibit a variety of antigen receptors that are able to respond to a wide range of antigens. Within the last two decades, these receptors have been the subject of considerable research. This article focuses on the rapidly growing body of knowledge with respect to the functions of LC receptors. Langerin (CD207) Langerin is a type II transmembrane cell surface receptor on LCs and is restricted to

* Department of Dermatology, Hadassah University Hospital, Ein Kerem, Jerusalem 91120, Israel. E-mail address: arieh@hadassah.org.il

them. Langerin is a 40-kD protein and a C-type lectin with mannose-binding specicity [8]. It was postulated that the major function of immature LCs found in the skin is to capture antigens. The captured antigens are recognized by LCs via internalization and processing. Langerin plays an important role in the internalization of cell surface antigens. LCs migrate to regional lymph nodes and mature into antigen-presenting cells [9,10]. Langerin is rapidly able to internalize from the surface into Birbeck granules in LCs [11]. It has been suggested that the process of antigen capture, internalization, processing, migration to lymphoid tissue, and maturation of LCs is a consequence of langerin function. Although langerin expression is down-regulated upon LC maturation, detectable levels of langerin remain in LCs once they reach cutaneous lymph nodes [12]. Recent studies on knockin mice expressing enhanced green uorescent protein have revealed two langerin-positive dendritic cell subsets in cutaneous lymph nodes. The main subset (90%) matches that expected for LCs, and the second subset (10%) shows a phenotype expected in blood-derived DCs found in cutaneous lymph nodes (CD8a). The levels of langerin are lower in the second subset than in the main subset [13]. Dermal-derived DCs migration to cutaneous lymph nodes is faster than LC-derived DCs. Thus, after epicutaneous application of uorescent dye (TRITC) into dermal-derived DCs, the dye is found in draining cutaneous lymph nodes as early as 24 hours after skin painting and peaks on day 2, whereas in LC-derived DCs the dye is found 4 days after skin painting. The presumption is that it takes longer for LCs to reach the lymph nodes because they rst need to detach from neighboring keratinocytes [13,14].
derm.theclinics.com

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.019

560

INGBER

DC-LAMP (CD208) DC-LAMP is a 70- to 90-kD antigen and a member of the LAMP (lysosome associated membrane protein) family. It is specically expressed by mature DCs located in lymphoid tissues. DC-LAMP has an important role in the processing of exogenous antigen [15].

Toll-like receptors Toll is a receptor that is expressed by insects. Toll plays an essential role in insect immunity response against fungal pathogens. Toll-like receptors (TLRs) are transmembrane glycoprotein receptors that recognize microorganisms [23]. Thirteen TLR5 have been identied. Only TLRs 1 through 10 are expressed in humans [24,25]. It has been shown that imiquimod enhances immune responses by eecting LC5 and TLR. Topically applied imiquimod increases LC migration into regional cutaneous lymph nodes and enhances antigen presentation, contact hypersensitivity reaction, and treatment ecacy. TLR7 ligands were recently recognized as powerful players in vaccination [5,26]. Fc3Ri, inammatory dendritic epidermal cells, and Langerhans cell Fc3Ri is a high-anity receptor for immunoglobulin E. The presence of Fc3Ri receptor on LCs at antigen presentation in the lymph node is associated with Th2-type immune responses characterized by interleukin (IL)-4, IL-5, and IL-13producing T cells [27]. Aggregation of Fc3Ri receptor on LCs induces the release of various chemotactic factors, such as IL-16 and monocyte chemotactic protein [28]. Inammatory dendritic epidermal cells (IDECs) present at inammatory sites in the skin. IDECs serve as an amplier for allergic inammatory reactions. Stimulation of Fc3Ri on IDECs induces the release of IL-12 and IL-18 and enhances the T-helper type I cells. Fc3Ri and IDEC play an important role in the pathogenesis of atopic dermatitis [27]. Atopy patch tests with aeroallergens have been used as an experimental model for atopic dermatitis [29]. It has been demonstrated that 72 hours after the allergen challenge a high number of IDECs invade the epidermis and Fc3Ri is up-regulated. Th2-type immune response is introduced, and atopic dermatitis skin lesions develop. Fc3Ri bearing DCs maintain the inammatory reaction in the skin of patients who have atopic dermatitis [30]. Recent studies have shown that tacrolimus down-regulates the expression of Fc3Ri on DCs and the number of IDEC in skin lesions in atopic dermatitis [31]. Summary LCs play an important role in epidermal resistance against a variety of antigens via signals

DC-SIGN (CD209) The contact between DCs and resting T cells in the lymphoid tissue is essential in initiating an immune response. The expression of ICAM-3 on resting T cells is important in the rst contact with DCs [16]. Recently, a novel type II transmembrane protein, DCs-specic C-type lectin DC-SIGN, has been identied. DC-SIGN binds to ICAM-3 and mediates adhesion with T cells [16]. It is speculated that DC-SIGN is a stabilizer of the DCT cell contact zone [17]. DS-SIGN also binds to the HIV-1 envelope glycoprotein gp-120 but does not function as a receptor for viral entry into DCs. Instead, DCSIGN allows DCs to carry HIV-1 through the lymphatic as a Trojan horse [18].

DEC-205 (CD205) DEC-205 is an endocytic receptor that can be used by DCs to direct captured antigens from the extracellular space to an antigen-processing compartment. High-level expression of DEC-205 is associated with a more eective antigen presentation. In rabbits, the antibody binding to T-cell hybridomas is 100-fold more ecient in the presence of DEC-205 [19]. DEC-205 is a marker for LCs and helps to discriminate LCs from dermal DCs. LCs express high levels of DEC-205, whereas DCs express low levels of DEC-205 [20].

Macrophage mannose receptor (DC206) Macrophage mannose receptor is a 175-kD receptor, type I membrane molecule. It has an Nterminal, cysteine-rich domain adjacent to a type II bronectin and light C-type carbohydrate recognition domains [21]. These domains enable the binding of various ligand (viruses, bacteria) to the receptor. The mannose receptor is predominantly expressed by macrophages but also by hepatic and lymphatic endothelia and DCs. [22].

LANGERHANS CELL RECEPTORS

561

for immune responses. This is achieved by a cascade of receptors that enhance antigen recognition, intralization, processing, migration to regional lymph nodes, antigen presentation, and activation of rest t lymphocytes. The main LC receptors are Langerin, DC-LAMP, DEC-205, DC-SIGN, macrophage mannos receptors, TLRs, Fc3Ri, and IDECs. References
[1] Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392(6675): 24252. [2] Cella M, Sallusto F, Lanzavecchia A. Origin, maturation and antigen presenting function of dendritic cells. Curr Opin Immunol 1997;9(1):106. [3] Knight SC, Hunt R, Dore C, et al. Inuence of dendritic cells on tumor growth. Proc Natl Acad Sci USA 1985;82(13):44957. [4] Nair S, Babu JS, Dunham RG, et al. Induction of primary, antiviral cytotoxic and proliferative responses with antigens administrated via dendritic cells. J Virol 1993;67(7):40629. [5] Schiller M, Metze D, Luger TA, et al. Immune response modiers: mode of action. Exp Dermatol 2006;15(5):33141. [6] Krogsgaard M, Davis MM. How T cells see antigen. Nat Immunol 2005;6(3):23945. [7] Janeway CA Jr, Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002;20:197216. [8] Valladeau J, Ravel O, Dezutter-Dambuyant C, et al. A novel C-type lectin specic to Langerhans cells, is and endocytic receptor that induces the formation of Birbeck granules. Immunity 2000;12(1):7181. [9] Figdor CG, van Kooyk Y, Adema GJ. C-type lectin receptor on dendritic cells and Langerhans cells. Nat Rev Immunol 2002;2(2):7784. [10] Hunger RE, Sieling PA, Ochoa MT, et al. Langerhans cells utilize CD1a and langerin to eciently present nonpeptide antigens to T cells. J Clin Invest 2004;113(5):7018. [11] Mc Dermott R, Ziylan U, Spehner D, et al. Birbeck granules are subdomain of endosomal recycling compartment in human epidermal Langerhans cells, which form where langerin accumulates. Mol Biol Cell 2002;13(1):31735. [12] Stoitzmer P, Holzmann S, McLellan AD, et al. Visualization and characterization of migratory Langerhans cells in murine skin and lymph nodes by antibodies against langerin/CD207. J Invest Dermatol 2003;120(2):26674. [13] Kissenpfennig A, Malissen B. Langerhans cells: revisiting the paradigm using genetically engineered mice. Trends Immunol 2006;27(3):1329. [14] Kissenpfennig A, Henri S, Dubois B, et al. Dynamics and function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24] [25]

[26]

[27]

[28]

from slower migrating Langerhans cells. Immunity 2005;22(5):64354. de saintVis B, Vincent J, Vandenabeele S, et al. A novel lysosome-associated glycoprotein, DCLAMP, induced upon DC maturation is transiently expressed in MHC class II compartment. Immunity 1998;9(3):32536. Geijtenbeek TB, Torensma R, van Vliet SJ, et al. Identication of DC-SIGN, a novel dendritic cellspecic ICAM-3 receptor that supports primary immune responses. Cell 2000;100(5):57585. de la Rosa G, Yanez-Mo M, Samaneigo R, et al. Regulated recruitment of DC-SIGN to cell-cell contact regions during zymosan-induced human dendritic cell aggregation. J Leukoc Biol 2005;77(5): 699709. Martin MP, Lederman MM, Hutcheson HB, et al. Association of DC-SIGN promoter polymorphism with increased risk for parenteral, but not mucosal, acquisition of human immunodeciency virus type1 infection. J Virol 2004;78(24):140536. Jiang M, Swiggard WJ, Heuer C, et al. The receptor DEC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen presentation processing. Nature 1995;375(6527):1515. Kronin Y, Wu L, Gong S, et al. DEC-205 as a marker of dendritic cells with regulatory eects on CD8 T cell responses. Int Immunol 2000;12(5):7315. Wileman TE, Lennartz MR, Stahl PD. Identication of the macrophage mannose receptor as a 175kDa membrane protein. Proc Natl Acad Sci USA 1986;83(8):2505. Taylor PR, Martinez-Pomares L, Stacey M, et al. Macrophage receptors and immune recognition. Annu Rev Immunol 2005;23:90144. Akira S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett 2003;85(2):8595. Akira S. Mammalian Toll-like receptors. Curr Opin Immunol 2003;15(1):511. Beutler B. Innate immune responses to microbial poisons: discovery and function of the Toll-like receptors. Annu Rev Pharmacol Toxicol 2003;43: 60928. Levy O, Zarember KA, Roy RM, et al. Selective impairment of TLR-mediated innate immunity in human newborns: neonatal blood plasma reduces monocyte TNF-alpha induction by bacterial lipopeptides, lipopolysaccharide and imiquimod, but preserves the response to R-848. J Immunol 2004; 173(7):462734. Novak N, Bieber T. The role of dendritic cell subtypes in the pathophysiology of atopic dermatitis. J Am Acad Dermatol 2005;53(2 Suppl 2):S1716. Gieger E, Magerstaedt R, Wessendorf JH, et al. IL-4 induces the intracellular expression of the alpha chain of the high-anity receptor for IgE in in vitro-generated dendritic cells. J Allergy Clin Immunol 2000;105(1 Pt 1):1506.

562 [29] Ring J, Darsow U, Behrendt H. Role of aeroallergens in atopic eczema: proof of concept with the atopy patch test. J Am Acad Dermatol 2001; 45(1 Suppl):S4952. [30] Kerschenlohr K, Decard S, Przybilla B, et al. Atopy patch test reactions show a rapid inux of inammatory dendritic epidermal cells in patients with

INGBER

extrinsic atopic dermatitis and patients with intrinsic atopic dermatitis. J Allergy Clin Immunol 2003; 111(4):86974. [31] Panhans-Gross A, Novak N, Kraft S, et al. Human epidermal Langerhans cells are targets for the immunosuppressive macrolide tacrolimus (FK506). J Allergy Clin Immunol 2001;107(2):34552.

Dermatol Clin 25 (2007) 563575

Cutaneous Mast Cell Receptors

Michihiro Hide, MD, PhDa,*, Yuhki Yanase, PhDa,b, Malcolm W. Greaves, MD, PhD, FRCPc
a

Department of Dermatology, Programs for Biomedical Research, Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan b Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32, Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan c National University of Singapore, National Skin Centre, 1, Mandalay Road, Singapore, 308205, Singapore

Dierentiation Since the discovery of mast cells by Paul Ehrlich in 1877, mast cells had been believed to be derived from undierentiated mesenchymal cells [1] until Kitamura and colleagues [2] found that mast cells are derived from committed progenitor cells in bone marrow. The mast cell progenitors circulate as mononuclear leukocytes, which express CD34 and kit, receptor for stem cell factor (SCF), but lack characteristic secretory granules and high-afnity IgE receptor (Fc3RI) [3]. The progenitor cells migrate into tissues and complete their dierentiation to mature mast cells, which express tryptase, KIT, and Fc3RI under the inuence of SCF. SCF is expressed constitutively by endothelial cells, broblasts, and other stromal cells and acts as the ligand of KIT, which is highly expressed in mast cells. Tissue mast cells reside closely with these stromal cells, producing SCF. Two types of mast cells, MCT and MCTC [4], exist in humans. MCT and MCTC are dierent in their granular neutral proteases, tissue localizations, and functions. MCT, which possess only tryptase as a major component of secretary granules, are considered as the human counterpart of mucosal-type mast cells of rodents. MCTC, which contain tryptase, chymase, carboxypeptidase [5], and cathepsin G [6] in their granules, are considered as the human counterpart of connective tissue-type mast cells of rodents. MCT reside in intestinal mucosa and lung, whereas MCTC are

located in skin and intestinal submucosa [4]. The functional dierence between two types of mast cells is that MCTC degranulate in response to the basic secretagogues such as substance P (SP) and compound 48/80 [7] and C5a and C3a anaphylatoxin [8], whereas MCT do not respond to them. Functions Mast cells, together with basophils, are known for their central role in type I hypersensitivity because of their surface expression of Fc3RIs. The interaction of an antigen with IgE-occupied Fc3RI results in the activation of downstream signaling molecules that lead to the release of preformed or de novo synthesized mediators [9]. Histamine and serotonin, which are preformed and stored in the secretory granules and released in minutes by Fc3RI aggregation, contribute to the development of the immediate-phase reactions, such as the contraction of smooth muscle, vascular dilatation, and extravasation of plasma into dermis. Eicosanoids, prostaglandins, and leukotrienes (LTs), which are synthesized from arachidonic acid after the activation of mast cells, act as potent chemotactic factors of leukocytes (eg, LTB4) and contribute to the development of the intermediate-phase reactions. Subsequently, within hours, cytokines such as interleukin (IL)-3, -4, -5, -6; granulocytemacrophage colony stimulating factor; and tumor necrosis factor (TNF) are synthesized and released from antigen-activated mast cells [10,11]. These proinammatory cytokines possess chemotactic activity to inammatory cells such as eosinophils and contribute to the late-phase reactions. The
derm.theclinics.com

* Corresponding author. E-mail address: mhide@hiroshima-u.ac.jp (M. Hide).

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.013

564

HIDE

et al

cytotoxic granular proteins from eosinophils and other kinds of granulocytes have an impact on the development of inammation of allergic diseases, such as atopic asthma and atopic dermatitis. Historically, mast cells have been intensively investigated from the viewpoint of IgE-mediated immediate hypersensitivity. However, mast cells express numerous receptors other than Fc3RI and could be activated or inactivated by these receptors (Table 1). Moreover, recent studies have shown that mast cells are involved in innate and acquired immunity [12], wound healing [13], cancer development, autoimmune diseases [14], and neurogenic disorders due to psychological stress [15]. We describe the intracellular signal transductions mediated by Fc3RI as a basic mechanism of mast cell activation and inactivation and the details of individual receptors. Intracellular signal transductions for the activation of mast cells Mast cells are activated by a variety of stimuli via receptors expressed on their plasma membrane. The intracellular signal transductions for mast cell activation or inactivation have been revealed in most detail for those triggered by the activation of Fc3RI (Fig. 1). Fc3RI is tetrameric receptor composed of the IgE-binding a chain, the membranetetraspanning b chain, and the disulde-linked homodimer of the g chains [16]. The b and g chains possess the immunoreceptor tyrosine-based activation motif (ITAM). Upon aggregation of receptors, ITAMs in the b and g chain are tyrosine phosphorylated by Src kinases, including Lyn [17]. When phosphorylated, the b- and g-chain ITAMs provide binding sites for the SH2 domains of Lyn and Syk, respectively. The recruited and activated Syk directly or indirectly phosphorylates the linker for activation of T cells (LAT), SH2-containing leukocyte-specic protein of 76 kDa (SLP-76), src homology 2 domain-containing (SHC), Vav, the guanosine nucleotide exchange facots for Rho/ Rac proteins, phospholipase C (PLC)-g1, and PLC-g2 [18]. The critical tyrosine-phosphorylated PLC-g1 and PLC-g2 translocate to the plasma membrane and are allowed to access phosphatidylinositol-4,5-bisphosphate (PIP2). PIP2 is catalyzed by PLCs, and the resultant products, inositol-1,4,5trisphosphate (IP3) and diacylglycerol, promote the increase of cytosolic calcium levels and the activation of protein kinase C (PKC), respectively. IP3 initiates the calcium signal by binding with IP3

receptor on the endoplasmic reticulum to release calcium, and the resulting depletion of intracellular calcium store causes the sustained calcium entry from outside of the cell [19]. The PKCs involved in the antigen-activated signaling pathway are composed of the classical (bI, bII) and the novel (d, 3, and q) isoforms [17]. These PKCs are activated by diacylglycerol binding with their C1 domain or by calcium binding with their C2 domain [20], which lead to degranulation. Syk activates the mitogen-activated protein kinase (MAPK) signaling cascades through the activation of the GTP exchange factors Sos and Vav. The activation of Sos results in the activation of Erk via Ras small GTPase [21], whereas the direct activation of Vav by Syk results in the activation of JNK via Rho family GTPase, Rac1/cdc42 [22]. The activation of Erk is indispensable for the activation of cytosolic phospholipase A2 on the release of eicosanoids [23]. Erk and JNK control the synthesis of proinammatory cytokines such as TNF-a through activating transcription factors such as nuclear factor (NF)-kB and activator protein1 [24]. Inhibitory signals in mast cell signal transductions The aggregation of Fc3RI activates not only the activating signaling pathways described previously but also activates inhibitory signals. Upon the Fc3RI crosslink, the immunoreceptor tyrosine-based inhibitory motifs (ITIMs) are phosphorylated by src kinases and provide the binding site to the inhibitory phosphatases, such as SH2-domain containing tyrosine phosphatase-1/2 and SH2-containing inositol phosphatase-1 [17]. The recruited and activated SH2-domain containing tyrosine phosphatase (SHP)-1 is reported to dephosphorylate ITAMs of the Fc receptors, Syk, leukocyte-specic protein of 76 kDa, and Vav [25], whereas SH2-containing inositol phosphatase-1 dephosphorylates PIP3 to PIP2 and thus negatively regulates the eect of the phosphatidylinositol 3-kinase [26]. Receptors for mast cell activation Fc3RI Crosslinkage of Fc3RI causes the most potent and variable responses of all kinds of mast cells. The binding of multivalent antigens to antigenspecic IgE bound to Fc3RI result in the cross-linking of Fc3RI. The addition of abundant

CUTANEOUS MAST CELL RECEPTORS

565

Table 1 Receptors expressed on human mast cells, functions, or intracellular signal transductions in skin or other organs Expression Receptor Activation receptors Fc3RI c-kit (CD117) Skin [97,82] Other organs Universal Lung [97,82], uterus [97], tonsillar [97], heart [97], renal [97], BMMC [97], synovial [97], HMC-1 [82] Intestinal [41], CBMC [102] Pulmonary MC [48], intestinal () [49] HMC-11,2,4 [99] Initiating signal Src family tyrosin kinase, syk Src family kinase [33,105], PI3K [33], PLCg [105], Btk [35] JAK Ga q [106], PLC [ H1; Gq/11 [107], H2; Gas [107], H4; Gi/os [107] PI3K [57], PLCg [57] Gq/G11 [108] [Ca2] [ Gai [109] [Ca2] [

IL-4R NK-1 Histamine receptor

[98] [46] H2 [99] H4 [99] [59] [100,61] [97,82]

NGFR PAR-2 C5aR (CD88)

CBMC [103], HMC-1 [59] Lung [104], tonsil [100], HMC-1 [61] Heart [97], synovial [97], lung (MCTC) [65], CBMC [64], basophil [6], HMC-1 [82] CBMC [64], HMC-1 [64] TLR2, CBMC [70], lung [69]

C3aR TLR

[63] TLR2 [69]

Gai [110] [Ca2] [ TLR2; MyD88 [111], TRAF [111], IRAK [111], TIRAP [111] TLR3; TRIF [111], TLR4; MyD88 [111], TRAF [111], IRAK [111], TIRAP [111], TRIF [111] SHIP1 (ITIM) [74] SHIP1 (ITIM) [81] Dephosphorylation of Src family kinase [112,83], JAK [112,84] Integrin binding to ECM substrates [85,87] Unknown basophil activation marker G-protein couple [90] [Ca2] [ PLA [

TLR3 [69] TLR4 [69] TLR9 [69] Inhibitory receptors FcgRII (CD32) Siglec3 (CD33) CD45

TLR3, lung [69] TLR4, CBMC [70], lung [69]

[97,82] [82] [97,82]

CBMC [79], HMC-1 [79], basophil [82] Lung [82], HMC-1 [82] Lung [97], uterus [97], tonsillar [97], heart [97], renal [97], BMMC [97], HMC-1 [97] Lung [97], uterus [97], tonsillar [97], renal [97], BMMC [97], HMC-1 [82] CBMC, lung [96], basophil [101] Lung [82], HMC-1 [92]

Activated markers LAMP3 (CD63)

[97,82]

CD203c CD69

[101] [97,82]

Abbreviations: BMMC, bone marrow-derived mast cell; CBMC, cord blood-derived mast cell; ECM, extracellular matrix; HMC-1, human leukemic mast cells; JAK, janus family tyrosine kinase; MC, mast cell; PLA, phospholipase A; PLC, phospholipase C; TLR, Toll-like receptor.

monovalent haptens, which bind to IgE and prohibit their cross-linkage, immediately terminate intracellular calcium signaling, exocytosis, and ruing of the plasma membrane [27].

Therefore, the role of IgE has been understood as a connector between antigens and Fc3RI. In contrast to this traditional view, recent studies revealed that mast cells could be stimulated by the

566

HIDE

et al

Antigen

Antigen

Y
ITIM

FcRIIb
PI(345)IP3 PI(45)P2

Y Y
ITAM ITAM Lyn

FcRI CRAC
PI(45)P2 PLC DAG IP3 SLPLAT ITAM ITAM

Ca2+
SCF SCF

c-kit

PI(34)P2 ITIM

PLC PI3K JAK Ca2+ Ca2+

ITAM ITAM

SHC Grb2 Soc

SHIP1 PLC
PKB/Akt

Syk

76

PI3K SHC Sos Vav

BTK

Grb2

IP

3R

Ca2+

Ras

NF-B

Ras

ER
Rho family PKC

[Ca2+]i

STAT MAPK

MAPK

Calmodurin

Membrane ruffling spreading

PLD
Calcineurin

Growth, Proliferation, Survival

Cytokine production

Degranulation

Fig. 1. Major pathways of the intracellular signal transduction in mast cells. Mast cells are activated by a variety of stimuli via receptors expressed on their plasma membrane. The intracellular signal transductions for mast cell activation or inactivation have been revealed in most detail for those triggered by the activation of Fc3RI.

binding of monomeric IgE to Fc3RI, resulting in mast cell survival, degranulation, and release of cytokines [28,29]. We have demonstrated that the mast cells derived from human skin degranulate in response to IgE (Fig. 2). In a subpopulation of patients who have chronic urticaria, immunoglobulin (Ig)G autoantibodies directed against the a-chain of Fc3RI have been demonstrated to be pathogenic. These autoantibodies cause activation of dermal mast cells and basophils by cross-linking Fc3RI in a complement-dependent manner in most cases [3032]. Kit Together with its ligand SCF, the receptor tyrosine kinase kit is a key controlling receptor for a number of cells, including hematopoietic stem cells, mast cells, melanocytes, and germ cells. The kit signaling plays a pivotal role in the growth, homing, dierentiation, and survival of these cells [33]. Gain-of-function mutations in c-kit, the gene for kit protein, have been described in a number of human cancers, including testicular germinomas, acute myeloid leukemia, and gastrointestinal

stromal tumors. Stimulation of kit by its ligand leads to the dimerization of the receptors, the induction of their intrinsic tyrosine kinase activity, and the phosphorylation of key tyrosine residues within the receptor. These phosphorylated tyrosine residues serve as docking sites for a number of signal transduction molecules containing the Src homology 2 domain or phosphotyrosine binding domain, which is thereby recruited to the receptor and activated many times through phosphorylation by the receptor [34]. Consequently, the Ras-ERK cascade, the janus family tyrosine kinase (JAK)-STAT system, the PI3K/ Akt system, and PLCg-mediated calcium mobilization are activated [34]. SCF-dependent activation of kit is crucial for the growth, dierentiation, survival, and homing of mast cells [33]. Simultaneous addition of SCF and antigen to their membrane-bound IgE enhances the degranulation from human mast cells and markedly increases the mRNA or protein concentrations of multiple cytokines in the cells [24], although SCF alone does not induce degranulation [33]. Iwaki and colleagues [35] reported that Btk plays a crucial role in the amplication

CUTANEOUS MAST CELL RECEPTORS

567

or the proliferation of mast cells, but it strongly enhances the proliferation of intestinal mast cells in synergy with SCF [40]. Moreover, the addition of IL-4 to the culture of mast cells with SCF supports the chymase-negative MCT subtype, whereas SCF by itself supports the predominance of the triptase/chymase positive MCTC. The proliferation and cytokine expression by human mast cells in response to IL-4 are regulated more by a signaling pathway involving ERK1/2 and c-Fos than STAT6 [41]. Polymorphisms of a subunit of IL-4R gene, including a gain-of-function mutation, are reported to be associated with atopic eczema and asthma [42,43]. There are other reports that deny such an association or functions of IL-4 receptors on B cells and T cells [44,45].
Fig. 2. SP and monomeric IgE induce degranulation of human skin mast cells. SP and monomeric IgE released b-hexosaminidase from human skin mast cells. Mast cells were collected from human skin tissues obtained from residual tissue after routine surgery with informed consent and cultured with recombinant human SCF as described by Kambe and colleagues [7]. Cymase and tryptase were stained as described by Irani and colleagues [113]. Two lots of IgE isolated from human IgE myeloma were obtained from Chemicon (Temecula, CA) and Calbiochem (San Diego, CA). Degranulation was assessed by measuring the release of b-hexosaminidase. This study was approved by Ethical Committee of Hiroshima University (No. 365).

Neurokinin-1 receptor SP is a neuropeptide that is classied as a tachykinin. Together with tachykinin A and tachykinin B, SP acts primarily via neurokinin-1 and -2 receptors (NK1R and NK2R). It is abundant in the periphery and in the central nervous system, and the release of SP from nerves is induced by danger signals or stress [46]. NK1 receptor is selective for SP; the relative anities for neurokinin A and neurokinin B are 100- and 500-fold lower, respectively [47]. Human pulmonary mast cells have also been reported to release histamine on SP stimulation [48]. Highly puried mast cells from human intestinal tissues have been shown to be resistant to SP stimulation and to lack SP receptors [49]. The studies revealed that mast cell responsiveness to SP stimulation may dier substantially among species and mast cell subsets [46]. The expression of NK1 receptor on skin mast cells is controversial. Guhl and colleagues [46] showed the expression of NK1 receptor on human skin mast cells using a polymerase chain reaction method and uorescence-activated cell sorter analysis. They also showed that histamine release from the cells stimulated with SP was blocked by NK1R antagonist. SP does not trigger cytokine transcription, including TNF-a, IL-6, and IL-8, in highly puried skin mast cell supernatant. SP may stimulate human mast cells to participate in acute responses but seems unlikely to contribute signicantly to a late-phase response, such as those found in most allergic settings, because the latter rely on mast cellderived cytokines [46]. On the other hand, Okabe and colleagues [50] showed that SP induces not only histamine release, but also the release of TNF-a and

of Fc3RI-mediated activation by kit in bone-marrow-derived mast cells. At a molecular level, the causative factors are likely to be multiple. An increased expression of SCF and point mutations in c-kit, which lead to disinhibition of mast cell proliferation, have been observed in some cases of usually benign and indolent mast cell proliferative disease in the skin, termed urticaria pigmentosa [36,37]. IL-4R IL-4 is synthesized and released by CD4T cells, Th2 cells, basophils, and mast cells [38]. IL-4 activates JAK/signal transducers and activators of transcription (STAT6) pathway via the IL4 receptor. In many types of cells, STAT6 plays an essential role in the production of cytokines in response to IL-4. Nabeshima and colleagues [39] recently demonstrated that IL-4 modulates the histamine content of mouse mast cells in a mast cell/broblast coculture system through a STAT6 signaling pathway in broblasts. In humans, IL-4 by itself has no eect on the survival

568

HIDE

et al

LTB4 [51] from human skin slices via MAPK. The discrepancy between these studies may be explained by a heterogeneity of mast cells due to genetic background or their microenvironments. Okabe and colleagues [52] recently showed that mouse bone marrowderived mast cells became responsive to SP when cocultured with broblasts. They release histamine and LTB4 in response to SP. The sensitivity of such mast cells to inhibit MAPK is somewhat dierent among three strains ofmice tested. Furthermore, the degrees of TNF-a and LTB4 release from human skin tissues are largely heterogeneous among donor subjects. Skin obtained from 2 out of 10 [50] and 15 out of 23 [51] subjects did not release TNF-a and LTB4 in response to SP, whereas all skin samples released histamine. The NK1 receptor expressed on dermal mast cells may play a central role in itching in inammatory skin disease. The activated dermal mast cell releases tryptase, which activates proteinase activated receptor2 (PAR2) on adjacent C-neurone terminals. This leads to release from the terminal of the neuropeptide SP, which causes mast cell activation via the NK1 receptor. This receptor and PAR2 are potential targets for novel anti-itch therapies [53]. Histamine receptor Histamine is synthesized and released by basophils, mast cells, lymphocytes, nerve cells, and entrochroman-like cells of stomach. Its actions are mediated by four subtypes of G-coupled protein receptors termed the H1, H2, H3, and H4 receptors. The distribution of the receptor expression depends on tissues. The receptor functions are dierent between the subtypes. In addition to releasing histamine, mast cells have receptors for histamine on their cell surface. Although all H1H4 receptors are detected on human mast cells, only H2 and H4 receptors are expressed on human skin mast cells. Cytokine release from human leukemic mast cells (HMC-1) and basophils is inhibited by the H1 or H2 receptor antagonist [54]. They showed no eect on calcium ux in resting or stimulated cells. On the other hand, histamine H4 receptor mediates chemotaxis and calcium mobilization of HMC-1 [55]. Although precise functions of these receptors in human skin mast cells are unknown, ligation of the rodent H4 receptor results in intense scratching [56]. Thus, the H4 receptor may be relevant to histamine-induced pruritus in skin diseases (eg, in atopic dermatitis).

Nerve growth factor receptor Nerve growth factor (NGF) is a neurotrophic factor that plays essential roles in neuronal dierentiation, proliferation, and survival [57]. NGF stimulates a wide variety of inammatory cells involved in allergic reactions, including mast cells [57]. Biologic actions of NGF are mediated through two types of specic receptors with distinct anities: the low-anity P75 neurotrophin receptors and the high-anity tyrosine kinase TrkA receptors [57]. In neuronal cells, the interaction of NGF with TrkA receptors induces receptor homodimerization and autophosphorylation, which further initiate the activation of PI3kinase, PLCg, PKC, and MAPK [58]. In mast cells, TrkA is preferentially expressed and causes the exocytosis in response to NGF via the activation of Tyrosine kinase, PLC, PI3K, and PKC but not MAPK [57]. Serum levels of NGF are increased in patients who have atopic dermatitis, causing increased secretion of tryptase by mast cells. In particular, levels of neurotrophin-4, probably derived from keratinocytes, are elevated, suggesting the involvement of neurotrophins in the pathophysiology of atopic dermatitis [59,60]. PAR2 PAR2 is a member of PAR family, which consists of PAR1 to -4, and belongs to the large family of 7-transmembrane-region receptors that couple to guanine nucleotide-binding proteins and are activated by serine protease [61]. PAR1, -3, and -4 are receptors for thrombin and expressed on platelets, whereas is PAR2 is distributed in a wide variety of tissues, including gastrointestinal, circulatory, and respiratory organs and skin. In the skin, PAR2 is expressed on keratinocytes, activated endothelial cells, sensory nerves, and mast cells, thus being involved in cutaneous inammatory responses [61]. Trypsin, tryptase, and specic agonists for PAR-2 induce Ca2 mobilization in primary skin mast cells, and agonists of PAR-2 induce the release of histamine from primary skin mast cells and HMC-1 [61]. PAR-2 could be a target for novel anti-itch therapy [53]. C3aR, C5aR (CD88) The complement-activated products C3a and C5a display powerful biological activities that lead to inammatory sequelae. They are strong chemoattractants, being involved in the recruitment of inammatory cells, such as neutrophils, eosinophils, monocytes, and T lymphocytes; in the

CUTANEOUS MAST CELL RECEPTORS

569

activation of phagocytic cells; and in the release of granule-based enzymes and generation of oxidants, all of which may contribute to innate immune functions or tissue damage [62]. C3a and C5a induce a rapid release of histamine from human skin mast cells [63] and are highly active mediators in stimulating chemotaxis and mobilization of cytosolic-free [Ca2]i of the human cell line HMC-1, cord bloodderived mast cell (CBMC) and of cutaneous mast cells [64]. C5a induces the degranulation of mast cells in skin or heart but not in lung. Oskeritzian [65] showed that the receptor for C5a (C5aR) is expressed on MCTC in skin and activated by C5a. On the other hand, lung-derived human mast cells are predominantly MCT. Less than 10% of them express C5aR, and they show little response to C5a. MCTC cells separated by cell sorting from lung responded to C5a, indicating that C5aR functionally distinguishes the MCTC type from the MCT type of human mast cells. According to Harmann and colleagues, the capability of C3a to induce degranulation of HMC-1 or CBMC is more potent than that of C5a. C3a, but not C5a, induces a robust production of chemokine monocyte chemoattractant protein1 and T-cell expressed and secreted RANTES/CCL5 via the activation of ERK pathway from human mast cell line, LAD2. C5a also induces the release of plasminogen activator inhibitor 1 from HMC-1 and basophils [66]. IgG-dependent histamine release from basophils in chronic urticaria is augmented by C5a. This augmentation is abolished by using an antibody to the C5a receptor [67]. Toll-like receptors Toll-like receptors (TLRs) are a family of pattern recognition receptors known to play an important role in host defense [68]. Since Medzhitov and colleagues found a human counterpart encoding TLR4 to Toll transmembrane receptor express in Drosophila in 1997, 10 TLR have been identied in humans. Human skin mast cells express TLR2, TLR3, and TLR4 on their membrane [69]. TLR2 TLR2, in complex with TLR1 or TLR6, recognizes gram-positive, bacterial-derived lipopeptides, peptidoglycan (PGN), and zymosan, the cell wall component of yeast [70]. When stimulated with Staphylococcus aureus PGN (a TLR2 activator) for 24 h, CBMC produced signicant levels of granulocyte-macrophage colony stimulating factor

and IL-1b [70]. High doses of PGN may marginally enhance degranulation of cord-blood derived mast cells [70]. TLR3 Human mast cells release interferon-g in response to polyI:C, a double-stranded RNA and a ligand for TLR3. The ligation of TLR3 by polyI:C neither induces the degranulation for itself nor enhances that mediated by IgE receptors on CBMC and HMC-1 [71]. The adhesion to extracellular matrix via integrin is known to potentiate mast cell responses mediated by IgE. The ligation of TLR3 decreases the adhesion of human mast cells to bronectin and vironectin. These effects on mast cells may help their chemotaxis for the site of inammation. TLR4 TLR4 is the major signaling molecule for most types of lipopolysaccharide (LPS) [70]. LPS (TLR4) and PGN (TLR2) induce the signicant release of not only TNF-a but also of IL-5, IL-10, and IL-13 by human CBMC [72]. Thus, TLR2 and TLR4 pathways may lead to pro-Th2 immune responses [72]. TLR6 TLR6 has been revealed to be expressed on CBMC, but it is not as well understood as other members of the TLR family. Studies with TLR6and TLR2-decient mice indicate that TLR2 is essential for the response to bacterial lipoproteins. The binding of microorganisms to TLR triggers intracellular signaling through MyD88dependent or MyD88-independent pathways. The activation of TLR2 homodimer or TLR2TLR6 heterodimer induces NF-kB activation through the association of adaptor molecules such as MyD88, TIRAP, and IRAP. TLR3 also induces NF-kB activation but via a MyD88-independent pathway. This pathway mainly leads to the activation of IRF3 though TRIF and IKK3 signal. The stimulation of TLR4 facilitates the activation of the MyD88-dependent and MyD88independent pathways. Recent work suggests that the activation of TLR2 and TLR4 on mast cells by peptidoglycan from S aureus leads to secretion by mast cells of TNF-a, IL-4, IL-5, and IL-13. This may help explain the familiar clinical observation that secondary infection of the aected skin in atopic dermatitis by S aureus leads to intense pruritus and deterioration of the dermatitis [73].

570

HIDE

et al

Receptors to inhibit mast cell activation Mast cells express a number receptors to inhibit the activation of mast cells. Final cellular reactions are determined by the balance of signaling induced by these receptors [74]. The inhibitory receptors traverse the plasma membrane once, and their N-terminal extracellular domains consist of C-type lectin domains (eg, mast cell functionassociated antigen and CD72) or Ig-like domains (eg, FcgRIIB, Siglec family, signal regulatory proteina , leukocyte Ig-like receptor, and killer inhibitory receptor). Each of these receptors contains at least one cytoplasmic ITIM that becomes phosphorylated after coaggregation with Fc3RI and attenuates immunoreceptor-induced signaling events through the recruitment of specic SH2containing phosphatases (eg, SHP-1, SHP-2, SHIP, or SHIP2) [9]. Among these inhibitory receptors, it has been demonstrated that human skin MCs express FcgRIIB and Siglec 3. In addition, human skin MCs express a transmembrane protein tyrosine phosphatase, CD45, which can negatively regulate cytokine-dependent activation of hematopoietic cells. FcgRIIb FcgRII (CD32) is a singlechain, low-anity receptor for the Fc portion of IgG. Three genes in humans (FCGRIIA, FCGRIIB, and FCGRIIC) and one in mice (FcgrIIb) encode the dierent subtypes [75]. FcgRIIB (CD32b) contains an ITIM domain, whereas FcgRIIA and FcgRIIC associate with ITAM domain. FcgRIIB has been shown to inhibit signaling events when coaggregated with Fc3RI and the SCF receptor, c-kit, on mast cells. On coaggregation with Fc3RI, the FcgRIIB ITIM is tyrosyl-phospholylated by the src family proteintyrosin kinase Lyn and recruits SH2 domain-containing inositol 5 phosphatase, SHIP, that hydrolyzes the PI3K-generated second messenger, PI(3,4,5)P3 to PI(3,4)P2 [76,77]. It has been proposed that coaggregation by a bifunctional fusion protein of Fc3RIa and FcgRIIB would cause an inhibition of dermal mast cell functions and could represent a novel approach to the treatment of mast celldriven cutaneous diseases, such as urticaria [78]. Zhao and colleagues [79] recently reported that FcgRIIA, but not FcgRIIB, is constitutively and functionally expressed on human skin-derived mast cells. The cross-linking of FcgRIIA leads to degranulation and secretion of newly generated lipid mediators and cytokines.

Siglec 3 (CD33) Siglecs are members of the Ig superfamily that bind to sialic acid and are mainly expressed by cells of the hematopoietic system [80]. Eleven Siglecs have been reported. With the exception of Siglecs 1 and 4, the cytoplasmic domains of the other nine Siglecs contain one or more ITIM or ITIM-like motifs [81]. These properties suggest that Siglecs may be involved in the regulation of cellular activation initiated by receptors containing the ITAM domain. It has been reported that human mast cells express Siglec 3(CD33) [82], but their function has not been elucidated. CD45 CD45 is a transmembrane protein tyrosine phosphatase that regulates Src family kinase in hematopoietic cells [83]. It can directly dephosphorylate and inactivate JAK-family kinase and thus negatively regulate JAK-STAT signaling in response to multiple cytokines including IL-3, IL-4, and interferon-g [84]. Regarding mast cells, a loss of CD45 expression leads to an increased proliferation of murine bone marrow-derived mast cell (BMMC)s in response to IL-3 [84]. It has been also reported that CD45 is essential for ZAP-70, but not for Syk, to reconstitute Fc3RIinitiated degranulation signals in mast cells [84]. Activation markers Several surface receptors have been identied on the surface of basophils. It has not been well proved whether these are applicable to mast cells. CD63 (LAMP-3) CD63 belongs to the newly identied transmembrane 4 superfamily of membrane proteins that are characterized by four transmembrane domains [85]. CD63 is translocated from the cytosol to the plasma membrane of human basophils in response to the activation of Fc3RI [86]. This receptor interacts with the a3, a4, and a6 chains of b1 integrins and modulates cellular adhesion [87]. Anti-CD63 antibody inhibits Fc3RI-mediated degranulation of RBL-2H3 rat mast cells via the inhibition of cell adhesion to bronectin and vitronectin [87]. CD63 has also been shown to regulate cell development, proliferation, motility, and adhesion in a human melanoma cell line [85]. The expression of CD63 was increased in mast cells of patients who had indolent mastocytosis [88]. Thus, CD63 may be used as markers

CUTANEOUS MAST CELL RECEPTORS

571

of activation for human skin mast cells and basophils. Basophil CD63 expression on leukocytes from atopic donors that had been incubated with sera from patients who had chronic urticaria has been proposed as a sceening test for the presence of anti-Fc3R1 autoantibodies in these sera [89]. CD69 CD69 is a type II transmembrane C-type lectin broadly expressed in human leukocytes [90]. It is induced early after activation of T-lymphocytes, NK cells, and other cells of hematopoietic origin [91]. It has been also reported to be overexpressed in mast cells in the bone marrow of patients who have indolent systemic mast cell diseases [92]. Anti-CD69 monoclonal antibodies induce a rise of intracellular calcium concentration, the activation of phospholipase A2, the induction of cytokine secretion, and cell proliferation in RBL-2H3 cells. They induce a moderate degree of serotonin release and a high amount of TNFa production, as compared with that induced by the activation of Fc3RI. Furthermore, the eects via Fc3RI and those via CD69 on TNF-a production are partially additive. Thus, CD69 could be involved in the induction of the late-phase reactions mediated by mast cells through TNF-a release [90]. CD203c CD203c is an ectoenzyme expressed only on basophils, mast cells, and their CD34 progenitor cells in the peripheral blood [93]. CD203c may be a better basophil activation marker than other reported molecules because of its specicity [94]. The cross-linking of Fc3RI up-regulates the expression of CD203c on cell surface. The up-regulation of CD203c on peripheral blood basophils is used to evaluate IgE-mediated hypersensitivity to latex and aeroallergen. It has recently been shown that anti-FceRI autoantibodies in sera from patients who have chronic autoimmune urticaria cause increased expression by basophils of CD 203c when incubated with donor whole blood. This procedure has been proposed as a method to identify antiFc3RI autoantibodies in patients who have chronic urticaria [95]. Regarding mast cells, neoplastic mast cells in systemic mastocytosis have been shown to express increased levels of CD203c [93]. Schernthaner reported that the expression of CD203c on human mast cells grown from cord bloodderived CD34 hematopoietic progenitor cells decreased

along with the maturation [96]. Taken together, CD203c on mast cells may be related to their immaturity rather than their activation [96].

References
[1] Kitamura Y, Ito A. Mast cell-committed progenitors. Proc Natl Acad Sci U S A 2005;102(32): 1112930. [2] Kitamura Y, Shimada M, Hatanaka K, et al. Development of mast cells from grafted bone marrow cells in irradiated mice. Nature 1977;268(5619): 4423. [3] Li L, Krilis SA. Mast-cell growth and dierentiation. Allergy 1999;54(4):30612. [4] Irani AA, Schechter NM, Craig SS, et al. Two types of human mast cells that have distinct neutral protease compositions. Proc Natl Acad Sci U S A 1986; 83(12):44648. [5] Irani AA, Goldstein SM, Wintroub BU, et al. Human mast cell carboxypeptidase: selective localization to MCTC cells. J Immunol 1991; 147(1):24753. [6] Schechter NM, Irani AA, Sprows JL, et al. Identication of a cathepsin G-like proteinase in the MCTC type of human mast cells. J Immunol 1990; 145(8):265261. [7] Kambe N, Kambe M, Kochan JP, et al. Human skin-derived mast cells can proliferate while retaining their characteristic functional and protease phenotypes. Blood 2001;97(7):204552. [8] Fu reder W, Agis H, Willheim M, et al. Dierential expression of complement receptors on human basophils and mast cells. J Immunol 1995;155(6): 315260. [9] Galli SJ, Kalesniko J, Grimbaldeston MA, et al. Mast cell as tunable eector and immunoregulatory cells: recent advances. Annu Rev Immunol 2005;23:74986. [10] Bradding P, Holgate ST. Immunopathology and human mast cell cytokines. Crit Rev Oncol Hematol 1999;31(2):11933. [11] Kobayashi H, Ishizuka T, Okayama Y. Human mast cells and basophils as sources of cytokines. Clin Exp Allergy 2000;30(9):120512. [12] Marshall JS. Mast-cell response to pathogens. Nat Rev Immunol 2004;4(10):78799. [13] Puxeddu I, Piliponsky AM, Bachelet I, et al. Mast cells in allergy and beyond. Int J Biochem Cell Biol 2003;35(12):16017. [14] Benoist C, Mathis D. Mast cells in autoimmune disease. Nature 2002;420(6917):8758. [15] Arck PC, Slominski A, Theoharides TC, et al. Neuroimmunology of stress: skin takes center stage. J Invest Dermatol 2006;126(8):1697704. [16] Blank U, Rivera J. The ins and outs of IgE-dependent mast-cell exocytosis. Trends Immunol 2004; 25(5):26673.

572

HIDE

et al for a distinct subset of chronic urticaria patients? J Clin Invest 1995;96(6):260612. Ferrer M, Nakazawa K, Kaplan AP. Complement dependence of histamine release in chronic urticaria. J Allergy Clin Immunol 1999;104(1): 16972. Gilllan AM, Tkaczyk C. Integrated signalling pathways for mast-cell activation. Nat Rev Immunol 2006;6(3):21830. Ronnstrand L. Signal transduction via the stem cell factor receptor/c-Kit. Cell Mol Life Sci 2004; 61(1920):253548. Iwaki S, Tkaczyk C, Satterthwaite AB, et al. Btk plays a crucial role in the amplication of Fc3RImediated mast cell activation by kit. J Biol Chem 2005;280(48):4026170. Longley BJ Jr, Morganroth GS, Tyrrell L, et al. Altered metabolism of mast-cell growth factor (c-kit ligand) in cutaneous mastocytosis. N Engl J Med 1993;328(18):13027. Nagata H, Okada T, Worobec AS, et al. c-kit mutation in a population of patients with mastocytosis. Int Arch Allergy Immunol 1997;113(13): 1846. Nelms K, Keegan AD, Zamorano J, et al. The IL-4 receptor: signaling mechanisms and biologic functions. Annu Rev Immunol 1999;17:70138. Nabeshima Y, Hiragun T, Morita E, et al. IL-4 modulates the histamine content of mast cells in a mast cell/broblast co-culture through a Stat6 signaling pathway in broblasts. FEBS Lett 2005; 579(29):66538. Lorentz A, Bischo SC. Regulation of human intestinal mast cells by stem cell factor and IL-4. Immunol Rev 2001;179:5760. Lorentz A, Wilke M, Sellge G, et al. IL-4-induced priming of human intestinal mast cells for enhanced survival and Th2 cytokine generation is reversible and associated with increased activity of ERK1/2 and c-Fos. J Immunol 2005;174(11):67516. Hershey GK, Friedrich MF, Esswein LA, et al. The association of atopy with a gain-of-function mutation in the alpha subunit of the interleukin-4 receptor. N Engl J Med 1997;337(24):17205. Shirakawa I, Deichmann KA, Izuhara I, et al. Atopy and asthma: genetic variants of IL-4 and IL-13 signalling. Immunol Today 2000;21(2): 604. Dupre D, Audrezet MP, Ferec C. Atopy and a mutation in the interleukin-4 receptor gene. N Engl J Med 2000;343(1):6970. Franjkovic I, Gessner A, Konig I, et al. Eects of common atopy-associated amino acid substitutions in the IL-4 receptor alpha chain on IL-4 induced phenotypes. Immunogenetics 2005;56(11):80817. Guhl S, Lee HH, Babina M, et al. Evidence for a restricted rather than generalized stimulatory response of skin-derived human mast cells to substance P. J Neuroimmunol 2005;163(12):92101.

[17] Rivera J, Gilllan AM. Molecular regulation of mast cell activation. J Allergy Clin Immunol 2006;117(6):121425. [18] Siraganian RP. Mast cell signal transduction from the high-anity IgE receptor. Curr Opin Immunol 2003;15(6):63946. [19] Patterson RL, van Rossum DB, Nikolaidis N, et al. Phospholipase C-g: diverse roles in receptor-mediated calcium signaling. Trends Biochem Sci 2005; 30(12):68897. [20] Spitaler M, Cantrell DA. Protein kinase C and beyond. Nat Immunol 2004;5(8):78590. [21] Jabril-Cuenod B, Zhang C, Scharenberg AM, et al. Syk-dependent phosphorylation of Shc. J Biol Chem 1996;271(27):1626872. [22] Teramoto H, Salem P, Robbins KC, et al. Tyrosine phosphorylation of the vav proto-oncogene product links Fc3RI to the Rac-JNK pathway. J Biol Chem 1997;272(16):107515. [23] Zhang C, Baumgartner RA, Yamada K, et al. Mitogen-activated protein (MAP) kinase regulates production of tumor necrosis factor-a and release of arachidonic acid in mast cells. J Biol Chem 1997;272(20):13397402. [24] Hundley TR, Gilllan AM, Tkaczyk C, et al. Kit and Fc3RI mediate unique and convergent signals for release of inammatory mediators from human mast cells. Blood 2004;104(8):24107. [25] Veillette A, Latour S, Davidson D. Negative regulation of immunoreceptor signaling. Annu Rev Immunol 2002;20:669707. [26] Huber M, Helgason CD, Damen JE, et al. The src homology 2-containing inositol phosphatase (SHIP) is the gatekeeper of mast cell degranulation. Proc Natl Acad Sci U S A 1998;95(19): 113305. [27] Maeyama K, Hohman RJ, Ali H, et al. Assessment of IgE-receptor function through measurement of hydrolysis of membrane inositol phospholipids: new insights on the phenomena of biphasic antigen concentration-response curves and desensitization. J Immunol 1988;140(11):391927. [28] Pandy V, Mihara S, Fensome-Green A, et al. Monomeric IgE stimulates NFAT translocation into the nucleus, a rise in cytosol Ca2, degranulation, and membrane ruing in the cultured rat basophilic leukemia-2H3 mast cell line. J Immunol 2004;172(7):404858. [29] Kawakami T, Kitaura J. Mast cell survival and activation by IgE in the absence of antigen: a consideration of the biologic mechanisms and relevance. J Immunol 2005;175(7):416773. [30] Hide M, Francis DM, Grattan CE, et al. Autoantibodies against the high-anity IgE receptor as a cause of histamine release in chronic urticaria. N Engl J Med 1993;328(22):1599604. [31] Fiebiger E, Maurer D, Holub H, et al. Serum IgG autoantibodies directed against the alpha chain of Fc3RI: a selective marker and pathogenetic factor

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

CUTANEOUS MAST CELL RECEPTORS

573

[47] Gerard NP, Garraway LA, Eddy RL Jr, et al. Human substance P receptor (NK-1): organization of the gene, chromosome localization, and functional expression of cDNA clones. Biochemistry 1991; 30(44):106406. [48] Heaney LG, Cross LJ, Stanford CF, et al. Substance P induces histamine release from human pulmonary mast cells. Clin Exp Allergy 1995;25(2): 17986. [49] Bischo SC, Schwengberg S, Lorentz A, et al. Substance P and other neuropeptides do not induce mediator release in isolated human intestinal mast cells. Neurogastroenterol Motil 2004;16(2):18593. [50] Okabe T, Hide M, Koro O, et al. Substance P induces tumor necrosis factor-a release from human skin via mitogen-activated protein kinase. Eur J Pharmacol 2000;398(2):30915. [51] Okabe T, Hide M, Koro O, et al. The release of leukotriene B4 from human skin in response to substance P: evidence for the functional heterogeneity of human skin mast cells among individuals. Clin Exp Immunol 2001;124(1):1506. [52] Okabe T, Hide M, Hiragun T, et al. Bone marrow derived mast cell acquire responsiveness to substance P with Ca(2) signals and release of leukotriene B(4) via mitogen-activated protein kinase. J Neuroimmunol 2006;181(12):112. [53] Steinho M, Vergnolle N, Young SH, et al. Agonists of proteinase-activated receptor 2 induce inammation by a neurogenic mechanism. Nat Med 2000;6(2):1518. [54] Lippert U, Moller A, Welker P, et al. Inhibition of cytokine secretion from human leukemic mast cells and basophils by H1- and H2-receptor antagonists. Exp Dermatol 2000;9(2):11824 [Erratum in: Exp Dermatol 2002;11(4):386]. [55] Hofstra CL, Desai PJ, Thurmond RL, et al. Histamine H4 receptor mediates chemotaxis and calcium mobilization of mast cells. J Pharmacol Exp Ther 2003;305(3):121221. [56] Bell JK, McQueen DS, Rees JL. Involvement of histamine H4 and H1 receptors in scratching induced by histamine receptor agonists in Balb C mice. Br J Pharmacol 2004;142(2):37480. [57] Stempelj M, Ferjan I. Signaling pathway in nerve growth factor induced histamine release from rat mast cells. Inamm Res 2005;54(8):3449. [58] Huang EJ, Reichardt LF. Trk receptors: roles in neuronal signal transduction. Annu Rev Biochem 2003;72:60942. [59] Groneberg DA, Serowka F, Peckenschneider N, et al. Gene expression and regulation of nerve growth factor in atopic dermatitis mast cells and the human mast cell line-1. J Neuroimmunol 2005;161(12):8792. [60] Grewe M, Vogelsang K, Ruzicka T, et al. Neurotrophin-4 production by human epidermal keratinocytes: increased expression in atopic dermatitis. J Invest Dermatol 2000;114(6):110812.

[61] Moormann C, Artuc M, Pohl E, et al. Functional characterization and expression analysis of the proteinase-activated receptor-2 in human cutaneous mast cells. J Invest Dermatol 2006;126(4): 74655. [62] Guo RF, Ward PA. Role of C5a in inammatory responses. Annu Rev Immunol 2005;23:82152. [63] el-Lati SG, Dahinden CA, Church MK. Complement peptides C3a- and C5a-induced mediator release from dissociated human skin mast cells. J Invest Dermatol 1994;102(5):8036. [64] Hartmann K, Henz BM, Kruger-Krasagakes S, et al. C3a and C5a stimulate chemotaxis of human mast cells. Blood 1997;89(8):286370. [65] Oskeritzian CA, Zhao W, Min HK, et al. Surface CD88 functionally distinguishes the MCTC from the MCT type of human lung mast cell. J Allergy Clin Immunol 2005;115(6):11628. [66] Wojta J, Kaun C, Zorn G, et al. C5a stimulates production of plasminogen activator inhibitor-1 in human mast cells and basophils. Blood 2002;100(2): 51723. [67] Kikuchi Y, Kaplan AP. A role for C5a in augmenting IgG-dependent histamine release from basophils in chronic urticaria. J Allergy Clin Immunol 2002;109(1):1148. [68] Akira S. Toll-like receptor signaling. J Biol Chem 2003;278(40):381058. [69] Kulka M, Metcalfe DD. TLR3 activation inhibits human mast cell attachment to bronectin and vitronectin. Mol Immunol 2006;43(10): 157986. [70] McCurdy JD, Olynych TJ, Maher LH, et al. Cutting edge: distinct Toll-like receptor 2 activators selectively induce dierent classes of mediator production from human mast cells. J Immunol 2003; 170(4):16259. [71] Kulka M, Alexopoulou L, Flavell RA, et al. Activation of mast cells by double-stranded RNA: evidence for activation through Toll-like receptor 3. J Allergy Clin Immunol 2004;114(1):17482. [72] Varadaradjalou S, Feger F, Thieblemont N, et al. Toll-like receptor 2 (TLR2) and TLR4 dierentially activate human mast cells. Eur J Immunol 2003;33(4):899906. [73] Supajatura V, Ushio H, Nakao A, et al. Dierential responses of mast cell Toll-like receptors 2 and 4 in allergy and innate immunity. J Clin Invest 2002; 109(10):13519. [74] Bruhns P, Fremont S, Daeron M. Regulation of allergy by Fc receptors. Curr Opin Immunol 2005; 17(6):6629. [75] Stefanescu RN, Olferiev M, Liu Y, et al. Inhibitory Fc gamma receptors: from gene to disease. J Clin Immunol 2004;24(4):31526. [76] Hermes B, Zuberbier T, Haas N, et al. Decreased cutaneous of stem cell factor and of the p75NGF receptor in urcaria. Br J Dermatol 2003;148(3): 4117.

574

HIDE

et al for hematopoietic cells. Immunol Today 1994; 15(10):47983. Diaz-Agustin B, Escribano L, Bravo P, et al. The CD69 early activation molecule is overexpressed in human bone marrow mast cells from adults with indolent systemic mast cell disease. Br J Haematol 1999;106(2):4005. Hauswirth AW, Florian S, Schernthaner GH, et al. Expression of cell surface antigens on mast cells: mast cell phenotyping. Methods Mol Biol 2006; 315:7790. Boumiza R, Monneret G, Forissier MF, et al. Marked improvement of the basophil activation test by detecting CD203c instead of CD63. Clin Exp Allergy 2003;33(2):25965. Yasnowsky KM, Dreskin SC, Efaw B, et al. Chronic urticaria sera increase basophil CD203c expression. J Allergy Clin Immunol 2006;117(6): 14304. Schernthaner GH, Hauswirth AW, Baghestanian M, et al. Detection of dierentiation- and activation-linked cell surface antigens on cultured mast cell progenitors. Allergy 2005;60(10): 124855. Valent P, Schernthaner GH, Sperr WR, et al. Variable expression of activation-linked surface antigens on human mast cells in health and disease. Immunol Rev 2001;179:7481. Babina M, Guhl S, Starke A, et al. Comparative cytokine prole of human skin mast cells from two compartments: strong resemblance with monocytes at baseline but induction of IL-5 by IL-4 priming. J Leukoc Biol 2004;75(2):24452. Lippert U, Artuc M, Grutzkau A, et al. Human skin mast cells express H2 and H4, but not H3 receptors. J Invest Dermatol 2004;123(1):11623. He SH, Xie H, Fu YL. Activation of human tonsil and skin mast cells by agonists of proteinase activated receptor-2. Acta Pharmacol Sin 2005;26(5): 56874. Ghannadan M, Hauswirth AW, Schernthaner GH, et al. Detection of novel CD antigens on the surface of human mast cells and basophils. Int Arch Allergy Immunol 2002;127(4):299307. Ochi H, De Jesus NH, Hsieh FH, et al. IL-4 and -5 prime human mast cells for dierent proles of IgEdependent cytokine production. Proc Natl Acad Sci U S A 2000;97(19):1050913. Tam SY, Tsai M, Yamaguchi M, et al. Expression of functional TrkA receptor tyrosine kinase in the HMC-1 human mast cell line and in human mast cells. Blood 1997;90(5):180720. He S, Aslam A, Gaca MD, et al. Inhibitors of tryptase as mast cell-stabilizing agents in the human airways: eects of tryptase and other agonists of proteinase-activated receptor 2 on histamine release. J Pharmacol Exp Ther 2004; 309(1):11926.

[77] Rauh MJ, Kalesniko J, Hughes M, et al. Role of Src homology 2-containing-inositol 5-phosphatase (SHIP) in mast cells and macrophages. Biochem Soc Trans 2003;31(Pt 1):28691. [78] Tam SW, Demissie S, Thomas D, et al. A bispecic antibody against human IgE and human FcgRII that inhibits antigen-induced histamine release by human mast cells and basophils. Allergy 2004; 59(7):77280. [79] Zhao W, Kepley CL, Morel PA, et al. FcgRIIa, not FcgRIIb, is constitutively and functionally expressed on skin-derived human mast cells. J Immunol 2006; 177(1):694701. [80] Li L, Yao Z. Mast cell and immune inhibitory receptors. Cell Mol Immunol 2004;1(6):40815. [81] Crocker PR, Varki A. Siglecs, sialic acids and innate immunity. Trends Immunol 2001;22(6):33742. [82] Ghannadan M, Baghestanian M, Wimazal F, et al. Phenotypic characterization of human skin mast cells by combined staining with toluidine blue and CD antibodies. J Invest Dermatol 1998;111(4): 68995. [83] Siraganian RP, Zhang J, Suzuki K, et al. Protein tyrosine kinase Syk in mast cell signaling. Mol Immunol 2002;38(1618):122933. [84] Irie-Sasaki J, Sasaki T, Matsumoto W, et al. CD45 is a JAK phosphatase and negatively regulates cytokine receptor signalling. Nature 2001;409(6818): 34954. [85] Radford KJ, Thorne RF, Hersey P. Regulation of tumor cell motility and migration by CD63 in a human melanoma cell line. J Immunol 1997;158(7): 33538. [86] Grutzkau A, Smorodchenko A, Lippert U, et al. LAMP-1 and LAMP-2, but not LAMP-3, are reliable markers for activation-induced secretion of human mast cells. Cytometry A 2004;61(1): 628. [87] Kraft S, Fleming T, Billingsley JM, et al. AntiCD63 antibodies suppress IgE-dependent allergic reactions in vitro and in vivo. J Exp Med 2005; 201(3):38596. [88] Escribano L, Orfao A, Diaz Agustin B, et al. Human bone marrow mast cells from indolent systemic mast cell disease constitutively express increased amounts of the CD63 protein on their surface. Cytometry 1998;34(5):2238. [89] Gyimesi E, Sipka S, Danko K, et al. Basophil CD63 expression assay on highly sensitized atopic donor leucocytes-a useful method in chronic autoimmune urticaria. Br J Dermatol 2004;151(2):38896. [90] Sancho D, Santis AG, Alonso-Lebrero JL, et al. Functional analysis of ligand-binding and signal transduction domains of CD69 and CD23 C-type lectin leukocyte receptors. J Immunol 2000; 165(7):386875. [91] Testi R, DAmbrosio D, De Maria R, et al. The CD69 receptor: a multipurpose cell-surface trigger

[92]

[93]

[94]

[95]

[96]

[97]

[98]

[99]

[100]

[101]

[102]

[103]

[104]

CUTANEOUS MAST CELL RECEPTORS

575

[105] Kitamura Y, Hirotab S. Kit as a human oncogenic tyrosine kinase. Cell Mol Life Sci 2004;61(23): 292431. [106] Monastyrskaya K, Hostettler A, Buergi S, et al. The NK1 receptor localizes to the plasma membrane microdomains, and its activation is dependent on lipid raft integrity. J Biol Chem 2005; 280(8):713546. [107] Akdis CA, Simons FE. Histamine receptors are hot in immunopharmacology. Eur J Pharmacol 2006; 533(13):6976. [108] Steinho M, Buddenkotte J, Shpacovitch V, et al. Proteinase-activated receptors: transducers of proteinase-mediated signaling in inammation and immune response. Endocr Rev 2005;26(1):143. [109] Auger GA, Smith BM, Pease JE, et al. The use of membrane translocating peptides to identify sites

[110]

[111] [112]

[113]

of interaction between the C5a receptor and downstream eector proteins. Immunology 2004;112(4): 5906. Elsner J, Oppermann M, Czech W, et al. C3a activates the respiratory burst in human polymorphonuclear neutrophilic leukocytes via pertussis toxin-sensitive G-proteins. Blood 1994;83(11):332431. Takeda K, Akira S. TLR signaling pathways. Semin Immunol 2004;16(1):39. Huntington ND, Tarlinton DM. CD45: direct and indirect government of immune regulation. Immunol Lett 2004;94(3):16774. Irani AM, Bradford TR, Kepley CL, et al. Detection of MCT and MCTC types of human mast cells by immunohistochemistry using new monoclonal anti-tryptase and anti-chymase antibodies. J Histochem Cytochem 1989;37(10):150915.

Dermatol Clin 25 (2007) 577588

Receptors of Eccrine, Apocrine, and Holocrine Skin Glands

Uwe Wollina, MDa,*, Mohamed Badawy Abdel-Naser, MDb, Ruta Ganceviciene, MDc,d, Christos C. Zouboulis, MDc,e
Department of Dermatology and Allergology, Academic Teaching Hospital Dresden-Friedrichstadt, Friedrichstrasse 41, 01067 Dresden, Germany b Faculty of Medicine, Ain Shams University Hospital, 4 Al Rahman Tower 11281, El Sawah Square, Cairo, Egypt c Laboratory for Biogerontology, Dermato-Pharmacology and Dermato-Endocrinology, Institute of Clinical Pharmacology and Toxicology, Charite Universitaetsmedizin Berlin, Campus Benjamin Franklin, 12200 Berlin, Germany d Department of Dermatology, Vilnius University Hospital, Santariskiu Klinikos, Vilnius, Lithuania e Department of Dermatology, Venereology, and Allergology, Dessau Medical Center, 06847 Dessau, Germany
a

Eccrine, apocrine, and holocrine glands belong to the group of specialized miniorgans of skin known as skin appendages. Eccrine glands develop from the sweat gland anlagen in the palmoplantar skin between 14 to 16 weeks estimated gestational age (EGA). Canalization of the dermal component (ie, coils and dermal duct) is complete by 16 weeks EGA. Canalization of the epidermal duct or acrosyringium is not complete until 22 weeks EGA. The specic end product of eccrine glands is sweat. They do not function in utero but start functioning immediately after birth. Apocrine glands usually arise from the upper portion of hair follicles not before the fth month of EGA. They function transiently during the third trimester and become quiescent in the neonate. Their function reappears during puberty. Their secretions play a role in sexuality. Apocrine glands, such as Molls glands of the eyelid, contribute to ocular host defense. The largest apocrine gland, however, is the breast gland, which is not discussed in this article. The development of holocrine sebaceous glands is associated closely with the formation of hair follicles during embryonic life. They rest until puberty. Their specic end product is sebum [1,2].
* Corresponding author. E-mail address: wollina-uw@khdf.de (U. Wollina).

Sex steroid receptors Sex-steroid receptors are distributed widely among human tissues. As an example of their action, the estrogen receptors (ERs) are discussed in more detail. Estrogens eects are genomic or nongenomic. Estrogens mediate their genomic activity by interaction with and activation of specic nuclear ERs. The ER-ligand complex binds to specic DNA sequences within the regulatory region of the target genes. By interaction with other cellular components, these target genes are activated or suppressed. This is known as the classical pathway. The nonclassical pathway of genomic action also can be mediated by interactions with ERs, in this case with membrane-bound ERs localized in the caveolae. Another option is the binding to G-proteincoupled receptor. This interaction activates kinases that act on nuclear transcription factors (ie, nongenomic) [3,4]. Two isoforms of ERs are known (ie, ER-a and ER-b). Whereas a large number of natural and synthetic estrogens show a similar binding anity to both receptors, other ligands are more selective, such as genistein or daidzein, which activate ER-b much more strongly. ER immunostaining usually is seen in a nuclear pattern [5]. ER-a is expressed exclusively in basal and partially dierentiated cells of normal sebaceous glands but not in sebaceous tumors. ER-a is seen in
derm.theclinics.com

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.001

578

WOLLINA

et al

only a small portion of the eccrine gland epithelia with a weak cytoplasmic staining. ER-b is expressed by sebaceous glands and cultured human sebocytes in the same distribution as ER-a [6]. Human eccrine sweat glands express ER-b. In mouse sebaceous glands there is a gender dierence in the expression of ERs and androgen receptors (ARs) [7] but in human skin there is no gender difference in the pattern or distribution of ERs [8]. Treatment of gonadectomized male mice with prostaglandin E2 leads to expression of ER-a [7]. ER-b expression is diminished in many human eccrine neoplasms [9,10]. A case of a 45-year-old woman who had a signet cell carcinoma of the eccrine sweat gland of the eyelid, however, showed positivity for ER and progesterone receptor (PR) [11]. Cases of ER-positive malignant eccrine spiradenoma also are described [12]. In the axillary apocrine glands, a strong nuclear expression of ER-a is seen in the secretory epithelium [13]. Sebaceous glands belong to the peripheral intracrine tissues in which estrogens are formed by transformation of prohoromone, dehydroepiandrosterone (DHEA), locally. There is no DHEA receptor in human tissue but DHEA is a major source of estrogen in women. Oral DHEA supplementation in postmenopausal women who have mostly sebostatic skin is capable of increasing sebum production by almost 80% after 12 months. Apart from this, other benecial cutaneous eects are documented, such as improved skin hydration, reduced facial pigmentation, and increased skin thickness [14,15]. PRs a and b have been identied in human skin. In normal sebaceous glands, PRa and PRb are expressed by basal and dierentiated sebocytes [9]. PR expression has been observed in syringoma, suggesting the possibility of hormonal control of this benign eccrine tumor [16,17]. ARs are expressed strongly by all sebocytes, in eccrine and apocrine sweat glands [18]. In sebaceous glands, undierentiated basal sebocytes express AR in higher quantity than dierentiated sebocytes [19]. In eccrine and apocrine sweat glands, luminal cells of the secretory portion are variably AR reactive [20]. In male mice, but not in female animals, gonadectomy leads to a diminution of the AR expression by sebaceous glands [7]. In benign eccrine tumors, the expression of AR is diminished [9]. In apocrine carcinoma, AR and AR messenger RNA (mRNA) may be absent [21]. ARs are expressed by sebaceous and sweat glands within nevus sebaceous of Jadassohn [22]. Papillary hidradenomas may overexpress AR [23].

In addition, AR is expressed by the secretory epithelium of axillary apocrine glands in correlation to their secretory activity [13]. An exception to this rule is the apocrine gland of the eyelid (ie, Molls gland). Not all primates express ARs in this gland [24]. In patients who have nonautoimmune dry eyes with meibomian gland dysfunction, the level of bioavailable testosterone is reduced signicantly [25]. The available data suggest that all adnexal glands of human skin interact with sex steroids. This can be of developmental importance, such as for sebaceous glands during puberty, or of regulatory importance, such as in apocrine glands, where phermones are produced under the inuence of androgens [2,13]. Extramammary Pagets disease is an uncommon neoplasm of the skin with apocrine dierentiation. Eighty percent of these tumors and many of the lymph node metastases are AR positive [26]. These tumor cells are characterized, however, by a consistent lack of ERs and PRs [27]. In the case of acne, the adenarche (ie, the starting point of endogenous androgen production) sebaceous glands increase their volume and their sebum production [2,28]. A major stimulus for this is the DHEA, later on testosterone plays a major role. There is no acne without androgens. In women who have acne, the androgen eects can be balanced with lower doses of estrogen combined with progestins, such as levonogestrel, desogestrel, norgestinate, gestodene, cyproterone acetate, chlormadinone acetate, and drospirenone [29]. Studies of oral contraceptives show them to be ecient and safe in women, reducing inammatory and noninammatory lesions [29,30]. In therapeutically resistant cystic acne of men and women, an androgen excess occurs. DHEA sulfate and 17-0H-progesterone levels are higher and sex-hormonebinding globulin is lower in aected subjects compared with controls. Therefore, low-dose dexamethasone can be used for 6 months to decrease DHEA sulfate [31]. In hyperandrogenism, with the triade of acne, alopecia, and hypertrichosis, contraceptives and antiandrogens are the cornerstone of treatment [32]. In ER-positive sweat gland adenocarcinoma, the use of hormone-blocking agents, such as tamoxifen, has been discussed for the treatment of metastasizing disease [12,33,34]. Luteinizing hormone/human chorionic gonadotrophin receptor (LH/hCG-R) was detected in skin by immunohistochemistry and reverse transcription polymerase chain reaction. In eccrine

RECEPTORS OF SKIN GLANDS

579

sweat glands, LH/hCG-R is localized in clear cells that are involved in uid and ion transport. Changes in sweating during puberty and menopause might be related to changes in LH levels. In the secretory coils, cells adjacent to the basement membrane were stained with monoclonal antibody against LH/hCG-R. In sebaceous glands, thin cells with round nuclei close to the duct were stained intensively, whereas polyhedral sebocytes with fat droplets showed a faint staining [35]. Mineralocorticoid and glucocorticoid receptors Mineralocorticoid receptor (MCR) mediates aldosterone actions on salt and water balance in restricted target cells. MCR is a member of the nuclear receptor superfamily and acts as a ligandinduced transcription factor modulating the expression of specic proteins involved in physiologic responses to aldosterone [36]. Two human MCR isoforms are identied (ie, a and b). Both isoforms are coexpressed in sweat gland ducts. In patients who have abnormal mineralocorticoid status, the expression may be compromised. In primary hyperaldosteronism and Liddle syndrome, MCRb but not MCRa expression in sweat glands is reduced markedly [37]. The enzyme, 11-b-hydroxysteroid dehydrogenase type II, is shown to confer specicity on MCR by inactivating glucocorticoids. Colocalization of the enzyme and the MCR is seen in a majority of sodium-transporting epithelia. In skin, MCR is expressed in basal cell of the sweat ducts but not in apocrine or sebaceous glands. The dehydrogenase is localized in luminal epithelia of the eccrine duct [38]. Like ARs or PRs, unliganded glucocorticoid receptors (GCRs) are located in the cytoplasm attached to heat shock proteins (hsp 90, 70, and 56). When hormones bind to GCRs, hsps are released and, through an energy-dependent process, the hormone receptor complexes form homo- or heterodimers. The zinc ngers of their DNA domains slot into the glucocorticoid response elements in the DNA helix where they exert activation of suppression of protein synthesis. In addition, nongenomic eects of glucocorticoids are mediated by interaction with surface proteins and lipids [39]. GCRs immunoreactivity is found in sweat gland and sebaceous gland epithelia [40]. This probably explains the induction of steroidinduced acne.

Orphan nuclear hormone receptors Nuclear hormone receptors are important in the regulation of epithelial cell dierentiation and lipid metabolism. Dierent receptors are identied in human skin. Some of them play a crucial role in lipid metabolism of sebaceous glands. In sebaceous glands maintained ex vivo and in human sebocyte cultures, peroxisome proliferationactivated receptors (PPARs) (types a, d, and g), retinoid X receptor (RXR)-a, liver X receptor-a, and pregnane X receptor but not farnesoid X receptor could be detected (Fig. 1) [4143]. All PPARs subtypes also are expressed in sebaceous glands in vivo. Furthermore, DAX1 and SF1 are expressed by sebaceous glands and cultured human sebocytes and eccrine sweat glands in human skin [44,45]. PPARa and PPARg ligands inhibited the lipid synthesis in sebaceous glands maintained ex vivo [42] but stimulated lipogenesis in diabetic patients in vivo [46]. In agreement with the in vivo data, natural and some synthetic PPARa, g, and a/d agonists increased lipid synthesis in human sebocytes cultures (SZ95 and SEB-1 cell lines) [43,46,47]. Investigations on human SZ95 sebocytes in vitro suggest that PPARg activation by ultraviolet light B irradiation and PPARg-dependent cyclooxygenase-2 activation are important regulators of cellular responses to oxidative stress [48]. Inhibition of the synthesis of the natural PPARa agonist leukotriene B4 in vivo led to signicant improvement of inammatory acne lesions and seborrhoea [4951]. In sebocyte development, there is a limited cooperation between PPAR and RXR agonists. In vitro, a submaximal dose of rexinoidda RXR agonistdaugmented the stimulation of sebocyte dierentiation by PPAR agonists [52]. The development of new RXR agonists might lead to cancer prevention for diseases, such as Muir-Torre syndrome, where sebaceous tumors arise on skin [53]. Neurotrophic factor receptor Ret is the specic receptor for glial cell line derived neurotrophic factor (GDNF), a factor initially identied as a neurotrophic factor for doaminergic neurons. Ret was found in rats in the epithelial cells of the secretory duct and the coiled secretory terminal of eccrine sweat glands. Electron microscopy showed that Ret immunoreactivity was localized in the basal area of secretory cells adjacent

580

WOLLINA

et al

Fig. 1. Expression of PPARa, PPARd, and PPARg1 in normal human sebaceous glands. Strong expression of PPARg1 and weaker of PPARa and PPARd are seen. Skin biopsies were xed in spirit-formol (9:1) xative for 12 hours at 4 C, rinsed in 0.01-M phosphate-buered saline (PBS) (pH 7.6), embedded in paran, and sectioned (2 to 3 Um thick). Sections were then heated to 60 C to melt paran and deparanized in xylene, followed by a rehydration for 1 minute through a graded series of ethanol. The sections were then incubated with PPARg monoclonal antibody for 1 hour and PPARd or PPARg1 overnight (from Santa Cruz, Heidelberg, Germany) at room temperature. Before incubation with the PPARd primary antibody, high-pressure boiling of the specimens for 3 minutes in a pressure cooker with citrate buer was performed. Primary monoclonal antibodies were added at dilutions 1:100 (PPARa) or 1:50 (PPARd, PPARg1). The negative control consisted of sections incubated with antibody diluent instead of the primary antiserum. Subsequently, the sections were incubated with a biotinylated secondary antibody (diluted 1:500) for 30 minutes and then with a streptavidine enzyme conjugate. Finally, the sections reacted with a fuchsin substrate chromogen system for 510 minutes and were couterstained with Mayers hematoxylin. (From Alestas T, Ganceviciene R, Fimmel S, et al. Enzymes involved in the biosynthesis of leukotriene B4 and prostaglandin E2 are active in sebaceous glands. J Mol Med 2006;84:80; with kind permission from Springer Science and Business Media.)

to myoepithelial cells. The ligand GDNF was observed in most myoepithelial cells of the secretory coils and in a few nerve bers innervating sweat glands. It is suggested that GDNF plays a trophic role for sweat glands [54]. Another receptor of the GDNF family is glial cell line-derived neurotrophic factor-family receptor (GFR) a2, a receptor that is important for the development of parasympathetic and enteric neurons. In a GFR a2-knockout mouse model, the density of sympathetic sweat gland innervation was reduced by 50% to 70% [55]. Vanilloid and cannabinoid receptors The family of vanilloid receptors (VRs) includes VR1, vanilloid-like proteins 1 and 2, and VR 50 -splice variant. In skin, the VR1 is distributed widely in human tissues. It is a nonselective cation channel that binds capsaicin. VR1 is activated by endogenous factors, such as increase of the body temperature or decrease of pH or anandamine compounds. VR1 immunoreactivity is seen in dierentiated sebocytes and the secretory portion of eccrine sweat glands. They seem to be

involved in neurogenic inammation, temperature, and pH control [56]. Cannabinoid receptors mediate the psychopharmacologic action of marijuana and are localized in the central and the peripheral nervous systems, as well as on cells of the immune system. CB1 and CB2 also are expressed by sebocytes and eccrine sweat glands [57]. Endovanilloids, cannabinoids, and opioids function as intracutaneous itch mediators and likely are involved in skin-derived, pruritogenic pruritus [58,59]. The use of specically designed ligands to such receptors will open a new avenue for the treatment of itch.

Nicotinic and muscarinic acetylcholine receptors Acetylcholine (Ach) has two types of receptors, nicotinic and muscarinic. The nicotinic ACh receptor (nAChR) is a 290-kd protein that consists of a ring of ve similar subunits. Seventeen nAChR subunits are identied: a110, b14, g, d and 3. They form ion channels with dierent functional and pharmacologic characteristics.

RECEPTORS OF SKIN GLANDS

581

Nicotin is an ACh antagonist and interacts with all AChR types except a9 subunits [51]. Five subtypes of muscarinic AChR (mAChR) are known. The mAChRs are expressed by keratinocytes in human epidermis and skin glands. The mAChRs are involved in the early steps of embryologic dierentiation of eccrine sweat glands. During development, the innervation of eccrine sweat glands undergoes a striking change from noradrenergic to cholinergic function. In rat skin it was observed that mAChRs are detectable as early as postnatal day 4 when glands begin to form. Within the next 2 weeks, m3 AChR protein and mRNA increase. m3 AChR expression does not depend on intact gland innervation. The m3 AChR is coupled to phospholipase C [60]. Muscarinic blockage during development or in adult animals results in the loss of secretory responsiveness, which is regulated by innervation [61]. In postnatal life, ACh is the major transmitter for sudomotoric activity and seems to be of importance for sebaceous gland activity. In rat skin, m2 AChR is expressed by nerve bers that innervate eccrine sweat glands [62] but m3 AChR seems to be the major receptor subtype in the regulation of sweat gland secretion [63]. Scopolamin is a substance with similar binding anities to all of the ve known muscarinic receptor subtypes. By 0.6-mg scopolamine given orally, a signicant reduction of sweat gland activity occurs as measured by skin conductance in normal adult volunteers [64]. In undierentiated sebocytes, the nAChR subunits a3, a9, b4, and mAChR subtypes m2m5 are found. Mature sebocytes express a7, b2, b4, m2, and m4 AChR. In eccrine sweat glands, the a3, a7, and m2m5 AChR are most prominent in myoepithelial cells whereas a9, b2, m1, m3, and m4 AChR are present in acinar epithelia [65]. The expression of a7 AChR is abolished in the epidermis adjacent to the acrosyringium in palmoplantar pustulosis, a disease associated with nicotine abuse [66]. Dysfunction of mAChR is a possible cause of idiopathic sudomotor failure (a disease with sudden onset, concomitant sharp pain, or cholinergic urticaria over the entire body), elevated serum IgE, lack of autonomic dysfunction other than generalized anhidrosis, and marked response to steroids. Although thermoregulatory sweating is absent, emotional sweating is preserved [67]. Botulinum toxin is the most potent pharmacologic inhibitor of focal hyperhidrosis. It blocks specic intracellular transport proteins

of ACh but itself does not interact with any AChR [68]. Goldstein and colleagues [69] reported a patient who had frequent episodes of pain, sweating, and ushing bilaterally in the hairless skin of the ophthalmic and maxillary distributions of the trigeminal nerve. The patient reported benet from anticholinergic quarternary amine trospium chloride whereas intravenous edrophonium evoked pain and sweating. The authors concluded that facial pain and sweating can result from by occupation of muscarinic cholinergic receptors after ACh release from local nerves. Angiotensin II receptors The tissue response to angiotensin IIdan octapeptide that regulates blood pressuredis mediated by specic cell surface receptors, AT1 and AT2. AT1 is predominant in the vascular system. AT1 is expressed by eccrine and apocrine sweat glands. In apocrine glands, luminal and periluminal cells of the acrosyringium are AT1 positive. The luminal cells of the dermal duct express AT1 whereas the basal cells, secretory cells, and myoepithelial cells are negative. The whole gland is AT2 negative. In eccrine sweat glands, the complete acrosyringium and the luminal cells of the dermal duct are AT1 positive and the remaining cellular elements negative. AT2 is not expressed by eccrine sweat glands [70]. AT1 also is found in sebaceous glands [71]. In eccrine poroma, some of the tumor cells in cell strands and cells surrounding luminal structures are AT1 positive; AT2 is not expressed [70]. Melanocortin receptors Melanocortins regulate immune functions, pigmentation, lipid metabolism, and adrenal hormone secretion. Melanocortins include adrenocorticotrophic hormone, a-melanocyte-stimulating hormone (aMSH), b-lipotrophin, and endorphins. Melanocortins bind to specic G-protein coupled receptors. Five dierent melanocortin receptor (MR) subtypes are known [72]. MR-1 transcripts and immunoreactivity are seen in sebocytes in vitro and in vivo. Sebum production can be increased by aMSH. By interaction of aMSH with MR-1, interleukin (IL)-8 expression of sebocytes is modulated. Immunoelectronmicroscopy demonstrates MR-1 along the cell surface and intracytoplasmatically within tubular endosomes, suggesting the internalization of MR-1 [73].

582

WOLLINA

et al

MR-1 is expressed by a variety of sebaceous malformations, hamartomas, and neoplasms, such as sebaceoma, cylindroma, and extramammary Pagets disease. MR-1 is found in eccrine sweat glands in dermal ducts and secretory epithelia. Eccrine tumors, such as syringoma or hidroacanthoma simplex, also express this type of MR [74]. MR-5 is expressed by dierentiating, lipidladen sebaceous cells but not in basal sebocytes. Therefore, MR-5 seems to represent a sebocyte dierentiation marker [75]. In transgenic mice, a targeted disruption of MR-5 resulted in a marked decrease in sebum production by sebaceous glands. MR-5 also is expressed by eccrine sweat glands [76]. Others could not identify MR-5 immunoreactivity in sebaceous glands [73]. Because melanocortins can suppress inammation by prevention of nuclear factor kB activation, MR ligands are of potential creating new anti-inammatory acne drugs [77].

Further neuropeptide receptors Vasoactive intestinal peptide receptor (VIP-R) has been localized by immunohistochemistry in acinar cells of the secretory coil and in luminal cells of the duct in eccrine sweat glands. It has been suggested that VIP is involved in regulation of chloride reabsorption from primary sweat at the ductal segment [78]. Further analysis revealed that two functional receptor types are expressed, namely a high-anity VIP-R (kd 0.582.4 kd) and a low-anity type (kd 175288 kd) (Fig. 2) [79]. VIP-R immunoreactivity is seen in

cystic structures of steatocystoma multiplex [80] but is absent in normal sebaceous glands [81]. Corticotropin-releasing hormone (CRH) is the most proximal element of the hypothalamic-pituitary-adrenal axis, and it acts as central coordinator for neuroendocrine and behavioral responses to stress. In human skin, the CRH receptor (CRH-R) 1 is expressed in all major cellular populations of epidermis, dermis, and subcutis with CRH-R1a the most prevalent isoform. The CRH-R2 gene is expressed solely in hair follicle keratinocytes and papilla broblasts, whereas CRH-R2 antigen is localized predominantly in hair follicles, sebaceous and eccrine glands, and muscle and blood vessels [82,83]. Investigations on the expression of CRH, CRH-binding protein (CRH-BP), and CRHRs were performed in SZ95 sebocytes in vitro and normal and acne-involved sebaceous glands in vivo [83,84]. CRH, CRH-BP, CRH-R1, and CRH-R2 were detectable in SZ95 sebocytes at the mRNA and protein levels with CRH-R1 the predominant type (CRH-R1/CRH-R2 2) (Fig. 3). CRH was biologically active on human sebocytes. It induced a biphasic increase of synthesis of sebaceous lipids with a maximum stimulation at 107 Mol (M) and upregulated mRNA levels of 3b-hydroxysteroid dehydrogenase/D(54)isomerase, although it did not aect cell viability, cell proliferation, or IL-1 beta-induced IL-8 release. Testosterone at 107 M downregulated CRH-R1 and CRH-R2 mRNA expression at 6 to 24 hours, and growth hormone switched CRH-R1 mRNA expression to CRH-R2 at 24 hours. Based on these ndings, CRH may be an autocrine hormone for human sebocytes that

Fig. 2. Expression of VIP-R. Immunoperoxidase staining with VIP-R specic monoclonal antibody 109.10 (Immunotech, Marseille, France) diluted 1:50. (A) Periglandular cells and cutaneous nerve bers around the sebaceous gland were stained whereas the sebocytes remained negative. (B) VIP-R expression in the most mature layer of epithelial cells of steatocystoma multiplex.

RECEPTORS OF SKIN GLANDS

583

Fig. 3. Expression of CRH, CRH-BP, CRH-R1, and CRH-R2 in normal human sebaceous glands. CRH barely is expressed, whereas strong expression of CRH-BP and CRH-R2 and weaker of CRH-R1 are detected. Skin biopsies were xed in spirit-formol (9:1) xative for 12 hours at 4 C, rinsed in 0.01-M PBS (pH 7.6), embedded in paran, and sectioned (2 to 3 Um thick). Sections then were heated to 60 C to melt paran and deparanized in xylene and rehydrated for 1 minute through a graded series of ethanol. The tissue sections then were incubated with diluted 1:50 primary antibodies (Santa Cruz, Heidelberg, Germany) in a humidied chamber for 30 minutes at room temperature. The optimal concentration of both the primary and secondary antibody was predetermined by titration assay. The negative control consisted of sections incubated with antibody diluent instead of the primary antiserum. Subsequently, the sections were incubated with a biotinylated secondary antibody (diluted 1:500) for 15 minutes and then with a streptavidine enzyme conjugate for 10 minutes. Finally, the sections reacted with a fuchsin substrate-chromogen system for 510 minutes and counterstained with Mayers hematoxylin.

exerts homeostatic lipogenic activity, whereas testosterone and growth hormone induce CRH negative feedback. The ndings implicate CRH in the clinical development of acne, seborrhea, and other skin disorders associated with alterations in lipid formation of sebaceous origin [84]. Although CRH does not modify the IL-6 beta-induced IL-8 release, it is able to directly enhance Il-6 and IL-8 release from human sebocytes, an activity mediated by CRH receptors [85]. In vivo, CRH, CRH-BP, CRH-R1, and CRHR2 are expressed in sebaceous glands at the protein level, whereas increased expression of CRH was detected only in acne-involved sebaceous glands [83]. The latter data indicate a proinammatory activity of CRH on the sebaceous gland. Adrenomedullin (AM) is a regulatory peptide that is synthesized and secreted by a large number of cells and tissues. AM is a potent vasodilator and exerts other functions, such as regulating cell growth and antimicrobial defense. Two receptors, L1 and calcitonin receptor-like receptor (CRLR), which are able to bind AM, have been cloned and characterized. Strong immunoreactivity for AM and its receptors is present in sweat and sebaceous glands. Immunoreactivity for the CRLR reveals a similar expression pattern to that seen for AM.

AM expression is not markedly upregulated in acne lesions, suggesting a minor role for this antimicrobial peptide in acne. AM and its receptors are colocalized in the same compartments and cell types of the skin. This nding is consistent with a proposed autocrine/paracrine mechanism in the physiology of AM [86]. Sweat glands express higher levels of substance P binding sites. Functional analysis suggests high anity receptors [87] Gastrin-releasing peptide receptors have been identied in eccrine sweat glands and sebaceous glands [88]. Somatostatin is a proinammatory neuropeptide. The somatostatin receptor is expressed by sweat glands but not sebaceous glands [89]. Cytokine receptors IL-13 is involved in various chronic infectious and in autoimmune diseases, in asthma and brosis [90,91]. It exerts its function after binding to the specic IL-13 receptor (IL-13R). IL-13 has two types of receptor: a heterodimer, composed of IL-13Ra1 and IL-4Ra, and the IL-13Ra2, a decoy receptor [92]. The IL-13R is found in sebaceous glands and eccrine sweat gland of skin. It is suggested that the

584

WOLLINA

et al

receptor might play a functional role in the inammatory cascade of atopic dermatitis [93]. IL-13 and IL-13R are increased in lesional skin of bleomycin-induced scleroderma in mice [94]. Spontaneous scleroderma in humans shows a remarkable impact of skin gland architecture. It is yet not clear, however, whether or not IL-13 and IL-13R of these glands contribute to dermal brosis and sclerosis. Cytokine receptors have been identied in clear cells and duct epithelia of eccrine sweat glands, such as IL-1aR, IL-1bR, IL-6R, and tumor necrosis factor a-R. Inammatory skin, as in cutaneous leishmaniasis, shows minor modications of the staining intensity in particular for IL-6R [95].

Transforming growth factor-beta and morphogenetic protein receptors The transforming growth factor-beta (TGF-b) superfamily is involved in growth and dierentiation. In mammals, TGF-b is found in three highly homologous isoforms that exert their eects via heteromeric complexes of type-I and type-II receptors (TGF-b receptor proteins type I and type II [TbRI and TbRII]). TGF-b regulates the growth and metabolism of various cell types,

including keratinocytes. These growth factors signal through transmembrane serine/threonine kinase receptors. Activation of type I receptor kinase phosphorylates intracellular signaling proteins named Smads. In normal human skin, immunoreactive TGFb2, but not TGF-b1, is detected predominantly in the epidermis, hair follicles, and sebaceous glands. The epidermal expression of TbRI and TbRII is very weak in the majority of normal skin samples. In addition, no expression of TGF-b2 is detected in the eccrine secretory portion or in eccrine spiradenoma, but it is detected in the upper eccrine ducts and in eccrine poroma [96]. In an immunohistochemical study of human eccrine sweat glands, expression of TGFb-RI was restricted to myoepithelial cells, whereas bone morphogenetic protein-receptor (BMPR) type IA was expressed selectively by dermal duct and acrosyringium. BMPR type IB monoclonal antibody gave a faint staining of secretory epithelium and myoepithelial cells. It is suggested that myoepithelial cells and duct cells are major targets of the TGF-b signaling pathway [97]. The authors investigated the expression of TGF-b1, -b2, and -b3; latent TGF-bbinding protein (LTBP); and TbRI and TbRII during induced hair growth in C57 BL-6 mice. They have found a positive reactivity for LTBP and TbRI

Table 1 Some implications of ligand-receptor interactions of skin glands in clinical dermatology Receptor type Estrogen Pathogenic and diagnostic relevance Y in eccrine neoplasms Therapeutic relevance Hormone replacement therapy for menopausal women; hormone therapy for hyperandrogenism and female acne ? Dexamethasone treatment of cystic acne; antiandrogens for female acne ? ? Chemoprevention of cancer ? Potential for new itch and pain therapy Pain and antihydrotic therapy Potential for anti-inammatory drugs ? Potential new acne treatments Antimicrobial substances ? ?

Progesteron Androgen Mineralocorticoid Glucocorticoid Orphan nuclear Neurotrophic factor Vanilloid, cannabinoid, opioid Nicotinic/muscarinic Melanocortin VIP Corticotropin-releasing hormone Adrenomedullin IL-13 TGF-b

[ in syringoma [ in cystic acne ? Steroid acne Acne, seborrhoea ? Itch, pain Hyperhidrosis, pain [ in hamartomas and tumors Variable expression in eccrine tumors ? ? Fibrosis, sclerosis Promotes growth of sebaceous glands

RECEPTORS OF SKIN GLANDS

585

in sebocytes. TbRII was found in sebaceous glands without signicant variations during the hair cycle. A bidirectional interaction of sebocytes and hair follicle epithelium in the TGF-b/LTBP seems possible. Sebocytes can be considered to be a target for TGFs, because they express both TbRI and TbRII. The general properties of TGF-b as a growth inhibitor of epithelial cells may suggest involvement in either the abrogation of extensive growth at the end of anagen or the initiation of catagen for the follicle epithelium and growth control for sebaceous glands [98]. Clinical implications and therapeutic relevance The knowledge of specic ligand-receptor interactions during development and function of skin glands not only is of academic interest but also this knowledge has implications for diagnostics and treatments. The expression of ligands and receptors is a tool in dermatopathology of adnexal tumors. Available treatments of female acne, hyperandrogensism, focal hyperhidrosis, itch, and pain are in part based on ligand-receptor eects. Further substances and indications are on the horizon. A summary is provided in Table 1. Summary The skin is the largest independent peripheral endocrine organ and exerts a great complexity of interaction with hormones, neuropeptides, PPAR ligands, growth factors, and cytokines [99,100]. Knowledge of receptor expression and receptorligand function oers new insight into pathologic conditions and opens new opportunities in drug development. References
[1] Loomis CA, Birge MB. Fetal skin development. In: Eicheneld LF, Frieden IJ, Esterly NB, editors. Textbook of neonatal dermatology. Philadelphia: WB Saunders; 2001. p. 117. [2] Zouboulis CC, Fimmel S, Ortmann J, et al. Sebaceous glands. In: Hoath SB, Maibach HI, editors. Neonatal skindstructure and function. 2nd edition. New York: Marcel Dekker; 2003. p. 5988. [3] Thornton MJ. The biological actions of estrogens on skin. Exp Dermatol 2002;11:487502. vrain S, Bonte F, Gilchrest B. Biology of [4] Vevrier-Se estrogens in skin: implications for aging skin. Exp Dermatol 2006;15:8394. [5] De Angelis M, Stossi F, Waibel M, et al. Isocoumarins as estrogen receptor beta selective ligands: isomers from isoavone phytoestrogens

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

and their metabolites. Bioorg Med Chem 2005; 23:652942. Makrantonaki E, Adjaye J, Herwig R, et al. Agespecic hormonal decline is accompanied by transcriptional changes in human sebocytes in vitro. Aging Cell 2006;5:33144. Azzi L, El-Alfy M, Labrie F. Gender dierences and eects of sex steroids and dehydroepiandrosterone on androgen and oestrogen a receptors in mouse sebaceous glands. Br J Dermatol 2006;154: 217. Thornton MJ, Taylor AH, Mulligan K, et al. Oestrogen receptor beta is the predominant oestrogen receptor in human scalp skin. Exp Dermatol 2003;12:18190. Kariya Y, Moriya T, Suzuki T, et al. Sex steroid hormone receptors in human skin appendage and its neoplasms. Endocr J 2005;52:31725. Hu S, Bakshandeh H, Kerdel FA, et al. Eccrine syringobroadenoma of clear cell variant: an immunohistochemical study. Am J Dermatopathol 2005;27:22831. nocarciBetis F, Hofman V, Lagier J, et al. Ade ` re de type nome sudoral primitive de la paupie ` cellules en bague a ` chaton. J Fr Ophtalmol eccrine a 2002;25:54751. Mirza I, Kloss R, Sieber SC. Malignant eccrine spiradenoma. Arch Pathol Lab Med 2002;126: 5914. Beier K, Ginez I, Schaller H. Localization of steroid hormone receptors in the apocrine sweat glands of the human axilla. Histochem Cell Biol 2005;123:615. Baulien E-E, Thomas G, Legrain S, et al. Dehydroepiandrosterone (DHEA), DEHEA sulphate, and aging: contribution of the DHEAge study to a sociabiological issue. Proc Natl Acad Sci USA 2000;97:427984. langer A, et al. Is dehyLabrie F, Lun-The V, Be droepiandrosterone a hormone? J Endocrinol 2005; 187:16996. Wallace ML, Smoller BR. Progesterone receptor positivity supports hormonal control of syringomas. J Cutan Pathol 1995;22:4425. Timpanidis PC, Lakhani SR, Groves RW. Progesterone receptor-positive syringoma associated with diabetes. J Am Acad Dermatol 2003;48(Suppl 5): S1034. rouanne B, et al. InhiFimmel S, Saborowski A, Te bition of the androgen receptor by antisense oligonucleotides regulates the biological activity of androgens in SZ95 sebocytes. Horm Metab Res 2007;39:14956. Choudhry R, Hodgins MB, Van der Kwast TH, et al. Localization of androgen receptors in human skin by immunohistochemistry: implications for the hormonal regulation of hair growth, sebaceous glands and sweat glands. J Endocrinol 1992;133: 46775.

586

WOLLINA

et al [36] Verrey F. Transcriptional control of sodium transport in tight epithelia by adrenal steroids. J Membr Biol 1995;144:93110. [37] Zennaro M-C, Farman N, Bonvalet J-P, et al. Tissue-specic expression of a and b messenger ribonucleic acid isoforms of the human mineralocorticoid receptor in normal and pathological states. J Clin Endocrinol Metab 1997;82:134552. [38] Hirasawa G, Sasano H, Takahasi K, et al. Colocalization of 11 beta-hydroxysteroid dehydrogenase type II and mineralocorticoid receptor in human epithelia. J Clin Endocrinol Metab 1997;82:385963. [39] Nussey S, Whitehead S. Endocrinology. An integrated approach. Oxford (UK): BIOS Scientic Publisher; 2001. [40] Karstila T, Rechardt L, Honkaniemi J, et al. Immunocytochemical localization of glucocorticoid receptor in rat skin. Histochemistry 1994;102: 3059. [41] Chen W, Yang C-C, Sheu H-M, et al. Expression of peroxisome proliferator-activated receptor and CCAAT/enhancer binding protein transcription factors in cultured human sebocytes. J Invest Dermatol 2003;121:4417. [42] Downie MM, Sanders DA, Maier LM, et al. Peroxisome proliferation-activated receptor and farnesoid X receptor ligands dierentially regulate sebaceous dierentiation in human sebaceous glands cultures in vitro. Br J Dermatol 2004;151: 76675. [43] Alestas T, Ganceviciene R, Fimmel S, et al. Enzymes involved in the biosynthesis of leukotriene B4 and prostaglandin E2 are active in sebaceous glands. J Mol Med 2006;84:7587. [44] Patel MV, McKay IA, Burrin JM. Transcriptional regulators of steroidogenesis, DAX-1 and SF-1, are expressed in human skin. J Invest Dermatol 2001; 117:155965. [45] Chen W, Yang C, Liao CY, et al. Expression of sexdetermining genes in human sebaceous glands and their possible roles in pathogenesis of acne. J Eur Acad Dermatol Venereol 2006;20:84652. [46] Trivedi NR, Cong Z, Nelson AM, et al. Peroxisome proliferator-activated receptors increase human sebum production. J Invest Dermatol 2006; 126:20029. bel A, Seltmann H, Fimmel S, et al. Dierenti[47] Wro ation and apoptosis in human immortalized sebocytes. J Invest Dermatol 2003;120:17581. [48] Zhang Q, Seltmann H, Zouboulis CC, et al. Involvement of PPARgamma in oxidative stressmediated prostaglandin E(2) production in SZ95 human sebaceous gland cells. J Invest Dermatol 2006;126:428. [49] Zouboulis CC, Nestoris S, Adler YD, et al. A new concept for acne therapy: a pilot study with zileuton, an oral 5-lipoxygenase inhibitor. Arch Dermatol 2003;139:66870.

[20] Shikata N, Kurokawa I, Andachi H, et al. Expression of androgen receptors in skin appendage tumors: an immunohistochemical study. J Cutan Pathol 1995;22:14953. [21] Bratthauer GL, Lininger RA, Man YG, et al. Androgen and estrogen receptor mRNA status in apocrine carcinomas. Diagn Mol Pathol 2002;11: 1138. [22] Hamilton KS, Johnson S, Smoller BR. The role of androgen receptors in the clinical course of nevus sebaceous of Jadassohn. Mod Pathol 2001;14: 53942. [23] Odani A, Campanati A. Papillary hidradenoma: immunohistochemical analysis of steroid receptor prole with a focus on apocrine dierentiation. J Clin Pathol 1999;52:82932. [24] Stoeckelhuber M, Stoeckelhuber BM, Welsch U. Apocrine glands in the eyelid of primates contribute to the ocular host defense. Cells Tissues Organs 2004;176:18794. [25] Tamer C, Oksuz H, Sohut S. Androgen status of the nonautoimmune dry eye. Ophthalmic Res 2006;38:2806. [26] Fujimoto A, Takata M, Hatta N, et al. Expression of structurally unaltered androgen receptor in extramammary Pagets disease. Lab Invest 2000; 80:146571. [27] Diaz de Leon E, Carcangiu ML, Prieto VG, et al. Extramammary Paget disease is characterized by the consistent lack of estrogen and progesterone receptors but frequently expresses androgen receptor. Am J Clin Pathol 2000;113:5725. [28] Zouboulis CC. Acne: sebaceous gland action. Clin Dermatol 2004;22:3606. [29] Huber J, Walch K. Treating acne with oral contraceptives: use of lower doses. Contraception 2006; 73:239. [30] Katsabas AD, Stefanaki C, Cunlie WJ. Guidelines for treating acne. Clin Dermatol 2004;22:43944. [31] Marynick SP, Chakmakjian ZH, McAree DL, et al. Androgen excess in cystic acne. N Engl J Med 1983;308:9816. [32] Shaw JC. Acne: eect of hormones on pathogenesis and treatment. Am J Clin Dermatol 2002;3: 5718. [33] Schro der U, Dries V, Klussmann JP, et al. Successful adjuvant tamoxifen therapy for estrogen receptorpositive metastasizing sweat gland adenocarcinoma: need for a clinical trial? Ann Otol Rhinol Laryngol 2004;113:2424. [34] Daniel SJ, Nader R, Kost K, et al. Facial sweat gland carcinoma metastasizing to neck nodes: a diagnostic and therapeutic challenge. Arch Otolaryngol Head Neck Surg 2001;127:14958. duri G, Pissard S, et al. Luteiniz[35] Venencie PY, Me ing hormone/human chorionic gonadotrophin receptors in various epidermal structures. Br J Dermatol 1999;141:43846.

RECEPTORS OF SKIN GLANDS

587

[50] Zouboulis CC, Saborowski A, Boschnakow A. Zileuton, an oral 5-lipoxygenase inhibitor, directly reduces sebum production. Dermatology 2005;210: 368. [51] Misery L. Nicotine eects on skin: are they positive or negative? Exp Dermatol 2004;13:66570. [52] Kim MJ, Deplewski D, Ciletti N, et al. Limited cooperation between peroxisome proliferatorsactivated receptors and retinoid X receptor antagonists in sebocyte growth and development. Mol Genet Metab 2001;74:3629. [53] Lippman SM, Lotan R. Advances in the development of retinoids as chemopreventive agents. J Nutr 2000;130:479S82S. [54] Yoshida T. Immunohistochemical localization of glial cell line-derived neurotrophic factor and its receptor Ret in the rat sweat gland. Kurume Med J 2004;51:193202. [55] Hiltunen PH, Airaksinen MS. Sympathetic cholinergic target innervation requires GDNF family receptor GFR alpha 2. Mol Cell Neurosci 2004; 26:4507. [56] Sta nder S, Moormann C, Schumacher M, et al. Expression of vanilloid receptor subtype 1 in cutaneous sensory nerve bers, mast cells, and epithelial cells of appendage structures. Exp Dermatol 2004; 13:12939. [57] Sta nder S, Schmelz M, Metze D, et al. Distrubution of cannabinoid receptor 1 (CB1) and 2 (CB2) on sensory nerve bers and adnexal structures in human skin. J Dermatol Sci 2005;38:17788. [58] Paus R, Schmelz M, Biro T, et al. Frontiers in pruritus research: scratching the brain. J Clin Invest 2006;116:117486. [59] Ikoma A, Steinho M, Sta nder S, et al. The neurobiology of itch. Nat Rev Neurosci 2006;7:53547. [60] Grant MP, Landis SC. Developmental expression of muscarinic cholinergic receptors and coupling to phospholipase C in rat sweat glands are independent of innervation. J Neurosci 1991;11:377282. [61] Landis SC. Development of muscarinic receptors and regulation of secretory responsiveness in rodent sweat glands. Life Sci 1999;64:3815. [62] Haberberger RV, Bodenbenner M. Immunohistochemical localization of muscarinic receptors (M2) in the rat skin. Cell Tissue Res 2000;300:38996. [63] Schiavone A, Brambilla A. Muscarinic M3 receptors mediate secretion from sweat glands in the rat. Pharmacol Res 1991;23:2339. [64] Golding JF, Stott JR. Comparison of the eects of a selective muscarinic receptor antagonist and hyoscine (scopolamine) on motion thickness, skin conductance and heart rate. Br J Clin Pharmacol 1997;43:6337. [65] Kurzen H, Berger H, Ja ger C, et al. Phenotypical and molecular proling of the extraneuronal cholinergic system of the skin. J Invest Dermatol 2004;123:93749.

[66] Hagforsen E, Edvinsson M, Nordlind K, et al. Expression of nicotinic receptors in the skin of patients with palmoplantar pustulosis. Br J Dermatol 2002;146:38391. [67] Nakazato Y, Tamura N, Ohkuma A, et al. Idiopathic pure sudomotor failure: anhidrosis due to decits in cholinergic transmission. Neurology 2004;63:147680. [68] Haider A, Solish N. Focal hyperhidrosis: diagnosis and treatment. Can Med Assoc J 2005;172: 6975. [69] Goldstein DS, Pechnik S, Moak J, et al. Painful sweating. Neurology 2004;63:14715. [70] Takeda H, Kondo S. Immunohistochemical study of angiotensin receptors in normal human sweat glands and eccrine poroma. Br J Dermatol 2001; 144:118992. [71] Steckelings UM, Wollschla ger T, Peters J, et al. Human skin: source and target organ for angiotensin II. Exp Dermatol 2004;13:14854. [72] Millington GWM. Propiomelanocortin (POMC): the cutaneous roles of its melanocortin products and receptors. Clin Exp Dermatol 2006;31: 40712. [73] Bo hm M, Schiller M, Sta nder S, et al. Evidence for expression of melanocortin-1 receptor in human sebocytes in vitro and in vivo. J Invest Dermatol 2002;118:5339. [74] Sta nder S, Bo hm M, Brzoska T, et al. Expression of melanocortin-1 receptor in normal, malformed and neoplastic skin glands and hair follicles. Exp Dermatol 2002;11:4251. [75] Zhang L, Li WH, Anthonavage M, et al. Melanocortin-5 receptor: a marker of human sebocyte differentiation. Peptides 2006;27:41320. [76] Thiboutot D, Sivarajah A, Gilliand K, et al. The melanocortin 5 receptor is expressed in human sebaceous glands and rat preputial glands. J Invest Dermatol 2000;115:6149. [77] Gatting SJ. Targeting melanocortin receptors as potential novel therapeutics. Pharmacol Ther 2006;111:115. [78] Kummer W, Herbst WM, Heym C. Vasoactive intestinal polypeptide receptor-like immunoreactivity in human sweat glands. Neurosci Lett 1990; 110:23943. [79] Heinz-Erian P, Paul S, Said SI. Receptor for vasoactive intestinal peptide on isolated human sweat glands. Peptides 1986;7(Suppl 1):1514. [80] Wollina U, Bocker T, Castelli E. Talgdru sentumoren. In: Garbe C, Dummer R, Kaufmann R, et al, editors. Dermatologische Onkologie. Berlin: Springer-Verlag; 1997. p. 53247. [81] Wollina U, Paus R, Feldrappe S. Sequential expression of glutathione-S-transferase isoenzymes during hair anagen growth in mice and their relationship to phosphotyrosinase and VIP receptor. Histol Histopathol 1995;10:3945.

588

WOLLINA

et al [92] Arima K, Sato K, Tanaka G, et al. Characterization of the interaction between interleukin-13 and interleukin-13 receptors. J Biol Chem 2005;280: 2491522. [93] Akaiwa M, Yu B, Umeshita-Suyama R, et al. Localization of human interleukin 13 receptor in non-haematopoietic cells. Cytokine 2001;13:7584. [94] Matsushita M, Yamamoto T, Nishioka K. Upregulation of interleukin-13 and its receptor in a murine model of bleomycine-induce scleroderma. Int Arch Allergy Immunol 2004;135:34856. [95] Ahmed AA, Nordlind K, Schultzberg M, et al. Proinammatory cytokines and their corresponding receptor proteins in eccrine sweat glands in normal and cutaneous leishmaniasis human skin. An immunohistochemical study. Exp Dermatol 1996;5:2305. [96] Furue M, Kato M, Nakamura K, et al. Dysregulated expression of transforming growth factor beta and its type-I and type-II receptors in basalcell carcinoma. Int J Cancer 1997;71:5059. [97] Wollina U, Lange D, Ten Dijke P, et al. Eccrine sweat glands: expression of transforming growth factor-beta and bone morphogenetic protein type I receptors and their intracellular Smad proteins. Acta Derm Venereol 1999;79:1836. [98] Wollina U, Lange D, Funa K, et al. Expression of transforming growth factor beta isoforms and their receptors during hair growth phases in mice. Histol Histopathol 1996;11:4316. [99] Zouboulis CC. Human skin: an independent peripheral endocrine organ. Horm Res 2000;54: 23042. [100] Zouboulis CC. The human skin as a hormone target and an endocrine gland. Hormones 2004;3: 926.

[82] Slominski A, Pisarchik A, Tobin DJ, et al. Dierential expression of a cutaneous corticotropin-releasing hormone system. Endocrinology 2004;145:94150. [83] Ganceviciene R, Marciukaitiene I, Graziene V, et al. New accents in the pathogenesis of acne vularis. Acta Medica, in press. [84] Zouboulis CC, Seltmann H, Hiroi N, et al. Corticotropin-releasing hormone: an autocrine hormone that promotes lipogenesis in human sebocytes. Proc Natl Acad Sci USA 2002;99:714853. [85] Krause K, Schnitger A, Fimmel S, et al. Corticotropin-releasing hormone skin signalling is receptormediated and is predominant in the sebaceous glands. Horm Metab Res 2007;39:16670. [86] Mu ller FB, Mu ller-Rover S, Korge BP, et al. Adrenomedullin: expression and possible role in human skin and hair growth. Br J Dermatol 2003;148:308. [87] Deguchi M, Niwa M, Shigematsu K, et al. Specic [125I] Bolton-Hunter substance P binding sites in human and rat skin. Neurosci Lett 1989;99:28792. [88] Staniek V, Misery L, Peguet-Navarro J, et al. Expression of gastrin-releasing peptide receptor in human skin. Acta Derm Venereol 1996;76:2826. [89] Misery L, Bourchanny D, Kanitakis J, et al. Modulation of substance P and somatostatin receptors in cutaneous lymphocytic inammatory and tumoral inltrates. J Eur Acad Dermatol Venereol 2001;15:23841. [90] Krause S, Behrends J, Borowski A, et al. Blockade of interleukin-13-mediated cell activation by a novel inhibitory antibody to human IL-13 receptor alpha1. Mol Immunol 2006;43:1799807. [91] Fichtner-Feigl S, Strober W, Kawakami K, et al. IL-13 signaling through the IL13alpha2 receptor is involved in induction of TGF-beta1 production and brosis. Nat Med 2006;12:99106.

Dermatol Clin 25 (2007) 589595

Disorders of Cell Kinetics and Dierentiation

Yalc in Tu zu n, MDa,*, Neslihan Dolar, MDa, Sadiye Keskin, MDa, Ronni Wolf, MDb
a

Department of Dermatology, Cerrahpasa Medical Faculty, Istanbul University, 34098 Istanbul, Turkey b Dermatology Unit, Kaplan Medical Center, Rechovot, 76100, Israel

The epidermis is a multilayered keratinizing squamous epithelium. The stratied squamous epidermis is composed of a proliferative basal layer containing the self-sustaining stem cell component and spinous and granular cells that express a tightly regulated set of proteins and lipids that eventually are woven into the cornied cell envelope of the corneocyte, providing the skin with its mechanical and chemical barrier [1]. Besides keratinocytes, there are three other important cell populations in the epidermis: melanocytes, Langerhans cells or epidermal antigen processing cells, and Merkel cells, which appear to function as mechanoreceptors interacting with peripheral nerves. Epidermal cell kinetics All tissues in the human body are divided into three groups according to their regeneration properties. 1. Static tissues that are formed by nonregenerative cells, ie, the central nervous system. 2. Tissues that are normally nonregenerative but become regenerative as a response to injury, ie, hepatic cells. 3. Tissues that are always regenerative, ie, the epidermis and gastrointestinal mucosae. The cell cycle that has a signicant role in the regeneration of cells is divided into four phases.

* Corresponding author. E-mail address: yalcintuzun@yahoo.com (Y. Tu zu n).

The rst phase is the resting phase called gap1 (G1) phase and follows mitosis. The duration of this phase is variable but is the longest. After the G1 phase, cells enter the S phase, which consists of DNA replication as a response to some unknown signals. The duration of the S phase is relatively constant in mammalian tissues and is approximately 7 to 16 hours in dividing keratinocytes. During this phase, DNA products that are ready to replicate are formed. After this phase is the G2 phase, which lasts 6 to 8 hours and consists of the nal preparations to cell division. The last phase is mitosis, which is the shortest phase of the cell cycle. After this phase, two daughter cells are produced. There is an additional phase termed G0. Some cells pass into this phase from G1 or G2 phases. Cells that do not enter the cell cycle in this phase can enter the active cell cycle as a response to appropriate signals. Entry of cells to phase G1 is controlled by cyclin-dependent kinases and their inhibitors to stop growth, which is important for keratin dierentiation [1,2]. The kinetics of cell transformation in skin is the basis of physiologic and pathologic conditions including skin cancer, wound healing, and psoriasis. The skin is a continuously self-renewing organ, with its complex, multilayered kinetic organization. The epidermis contains mainly keratinocytes, but there are also dendritic and stem cells and possibly a pool of quiescent cells. Keratinocytes originate in the basal layer of the epidermis, through division of a population of stem cells. The daughter cells then divide several times as transit amplifying cells, which lose their proliferative capacity and dierentiate into postmitotic and keratin-rich cells, which migrate upward to the spinous layer of the epidermis. As
derm.theclinics.com

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.007

590

ZU N TU

et al

these cells migrate to the granular layer, they loose their nuclei and become anuclear and form the uppermost corneum layer, which periodically sheds. Each stem cell gives rise to a number of transient amplifying cells that, after a few cycles of mitosis, become terminally dierentiated, nonmitotic keratinocytes. Each stem cell and all its progeny have been referred to as the epidermal proliferative unit [1,3]. During the transition to corneocytes commonly referred to as terminal dierentiation, keratinocytes die in a tightly controlled fashion [4]. This type of cell death is known as apoptosis or programmed cell death, and it has a major role in the dierentiation of the epidermis. Apoptosis mediates several important physiologic and pathologic processes. Granular cell nuclei in the epidermis contain a larger amount of fragmented DNA than basal and spinous cell nuclei, and granular cells undergo a type of apoptosis [5]. Caspases are the key enzymes of apoptosis. They cleave their substrates specically at 4 amino-acid motifs leading to the inactivation and degradation of a large number of regulatory and structural proteins. In addition, caspases have a role in regulating and amplifying apoptotic signals [4]. Turnover of the epidermis allows the organ to maintain its barrier function, repair injured skin in wound healing, and receive the signals that stimulate or inhibit cell proliferation. The isolation of epidermal stem cells has been hampered by diculties in nding specic and sensitive markers such as those present in hematopoetic stem cells. Initially, keratin-19 was thought to be such a marker, but then it was found in many nonlabeled cells too. It was thought that only stem cells would have high telomerase activity. However, telomerase activity is higher in rapidly dividing cells than in stem cells. The other postulated stem cell markers have included keratin-15, c-myc, a noncadherin-associated b catenin, p63, the antigen recognized by monoclonal antibodies 10G7 and C8 144B, low expression of E-cadherin, and the high expression of delta 1 [1]. There are also uncertainties regarding the extent of keratinocyte turnover in various skin diseases. Hyperproliferation may represent a risk factor for skin cancer and occurs in some physiologic conditions such as wound healing and altered permeability barrier function as well as pathologic conditions such as psoriasis. In addition to the eects on keratinocyte proliferation of chemopreventive and antipsoriatic agents, they also can change cell turnover [3]. Thus, cell cycle and keratinocyte turnover have signicant roles

in pathogenesis of skin diseases and response to therapy.

Disorders of cell kinetics and dierentiation Psoriasis Psoriasis vulgaris is a hyperproliferative disorder of keratinocytes, characterized by both hyperplasia of the spinal layer and incomplete dierentiation of the granular and cornied layer (agranulosis and parakeratosis). The mechanism of keratinocyte proliferation in psoriasis is not known completely. In the histologic evaluation of early psoriatic lesions, it was shown that the activation of T lymphocytes and macrophages stimulate epidermal hyperproliferation [5]. In one study, Hatta and colleagues [6] assessed the proliferative activity of stimulated and unstimulated epidermis by DNA ow cytometry, mitotic index, the brodeoxyuridine labeling index, and the immunohistochemical analysis of the cell proliferation markers, Ki 67 and DNA polymerase a. The study found that although the baseline proliferative activity of uninvolved skin does not dier between psoriasis patients and normal individuals, the proliferative response to tape stripping is signicantly greater in psoriasis patients than in controls. This dierence may be because of overactivation of keratinocyte growth stimulatory pathways involving transforming growth factoralpha (TGFa) overexpression in psoriasis patients. In the psoriatic epidermis, there are a lot of regulators of proliferation compared with the normal epidermis. In recent studies it was found that the apoptotic inhibitors of basal cell carcinoma Bcl-2 and BclX1 were synthesized in high levels from all layers of psoriatic epidermis. Recently in the study by Pena and colleagues [7], which was made on rat epidermis, it was concluded that high levels of BclX1 and BCLX2 protect the cell against destruction after ultraviolet (UV) radiation, but it has no role on the dierentiation of epidermis. Finally it was shown that BclX1 has a role on hyperproliferation but has no role on the dierentiation of keratinocytes [5,7,8]. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP biotin nick-end labeling (TUNEL) method has been used widely for the detection of free 30 -OH DNA ends occurring during apoptosis. There are clear parallels between keratinocyte terminal dierentiation and

DISORDERS OF CELL KINETICS AND DIFFERENTIATION

591

apoptosis, and the former has been proposed to be a form of apoptosis [5]. Some studies that compare normal epithelia and psoriatic epithelia by using this method clearly showed a marked increase in the number of free 3-OH DNA ends in the condensed chromatin of granular cell nuclei in the normal epidermis, whereas apoptotic characteristics such as reduced cell volume, membrane blebbing, and apoptotic bodies were not detected in any layer of the psoriatic epidermis [5,6]. Psoriatic epidermis is characterized by marked epidermal hyperplasia, accompanied by regular elongation of the rete ridges and incomplete differentiation. In the study of Kawashima and colleagues [5], the expression level of Ki-67, a marker for detecting cells in S, G2, and M phases of the cell cycle was signicantly higher in psoriatic epidermis than in normal epidermis. With this, they tried to explain epidermal hyperplasia in psoriasis. Egr1, which protects cells against apoptosis and stimulates cell proliferation, was found higher in lesions of chronic plaque psoriasis. Also p12, p14, and p16, which regulate replication of keratinocytes, were found higher in these lesions [5]. Molecules such as cytokeratin 6, 16, 17, skin-associated antileukoprotease, psoriasin, TGFa, amphiregulin, epidermal growth factor receptor, interleukin 1ra (IL-1ra), IL-1b, IL 6, IL 8, Gro a/b/g, and bronectin are expressed in the psoriatic plaque, but they are absent in normal skin [9,10]. The psoriatic plaque in itself has considerable heterogeneity. There are some histologic and biochemical dierences between the margin and center of the psoriatic lesions. Mommers and colleagues [11] found that the quantitative parameters showed large interindividual variability; however, the percentage of dierentiated cells was signicantly higher in the margin than in the center. They measured keratin 10 levels with ow cytometry. A statistically signicant dierence in the percentage of keratin 10positive cells was shown between the center and the margin (which is important for cell dierentiation). Henrich Koebner rstly described in 1878 the Koebner phenomenon that psoriatic lesions formed in nonsymptomatic skin after skin trauma. During this transformation, some histologic and molecular changes can be seen. In the epidermis, although thymidine units, S, G2, M phase, ornithine decarboxylase activity, phospholipase A2 activity, and arachidonic acid release increases, protein kinase C activation decreases [9].

Microvascular changes are also important in epidermal proliferation in pathogenesis of psoriasis [12]. These changes occur early in the formation of a psoriatic plaque. Microvascular changes within lesional skin include pronounced dilatation, tortuosities, increased permeability, and endothelial cell proliferation within the venous limb of capillaries in the dermal papilla. Especially endothelial cells on venules are more sensitive to angiogenic factors [12,13]. Vascular proliferation in psoriasis usually forms under the inuence of angiogenic factors, which originate from the epidermis. These factors include TGFa, tumor necrosis factor (TNF)a, and platelet-derived endothelial cell growth factor/thymidine phosphorylase [12]. Nickolo and colleagues [14] have also suggested that their ndings of increased levels of IL-8 and reduced levels of thrombospondin-1, a naturally occurring inhibitor of angiogenesis, may contribute to the expansion of the vasculature seen in psoriatic skin [15]. A new mediator that is not an enzyme has been identied in the 1970s, shows no protein character, has low molecular mass, and originates from tumor tissue. Endothelial cell-stimulating angiogenesis factor (ESAF) [12] specially stimulates the proliferation of pericytes and microvascular endothelial cells but possesses no mitogenic activity on aortic, large vein endothelial cells, broblasts, or any other cell type. Its stimulatory eect on endothelial cells is synergistic with basic broblast growth factor. Serum levels of ESAF have been found to be elevated in conditions in which active neovascularization is occurring. The angiogenic potential of vascular endothelial growth factor (VEGF) acts as a potent and selective mitogen for endothelial cells in vitro. In the study of Brushan and colleagues [12], the production and distribution of levels of ESAF and VEGF was studied in the skin and serum of patients with psoriasis and in normal controls. Tissue levels of these angiogenic factors were found to be signicantly elevated in plaques of psoriasis compared with normal control skin. It was concluded that angiogenic factors are more than VEGF in psoriatic skin and stimulate neovascularization. Investigation of cell kinetics helps us to understand the pathogenesis of diseases and mechanisms of eect of therapeutic agents. Understanding cell proliferation and dierentiation in psoriasis enables us to nd new therapeutic agents. Topical corticosteroids are the mainstay in the treatment of psoriasis. They eect by inhibiting

592

ZU N TU

et al

epidermal proliferation, enhancing normal dierentiation, and inhibiting inammation. Besides these favorable eects, it is known that corticosteroids have side eects such as skin atrophy, striae, telangiectasia, perioral dermatitis, steroid acne, and suppression of the hypothalamicpituitary-adrenal axis [16,17]. Since the rst report on the benecial eects of vitamin D3 in psoriasis by Morimoto and colleagues, new analogs have been developed to decrease hormonal eects on calcium phosphate homeostasis and maintain eects on keratinocyte proliferation and dierentiation. Vitamin D3 and its analogs inhibit keratinocyte proliferation and induce terminal dierentiation, so calcipotriol enhances dierentiation and reduces epidermal hyperproliferation [13,14,18]. The only adverse eect is local skin irritation. Studies conrm that the combination of calcipotriol and a topical corticosteroid is clinically more eective compared with monotherapy with calcipotriol or a corticosteroid, because corticosteroids have potent antiinammatory eects, and vitamin D3 interferes predominantly with epidermal proliferation and keratinization [13,19]. With combination therapy the percentage of vimentin-positive cells (inammatory cells) is reduced more than the number of keratinocytes. After treatment there is a marked decrease in vimentin-positive cells and a marked increase in keratin 10positive cells (which is a sign of cell dierentiation) and slight increase in the cells S, G2, and M phase [16]. Many systemic agents are widely used in the treatment of psoriasis. They provide good control of psoriasis in the majority of patients. Methotrexate is a competitive inhibitor of the enzyme dihydrofolate reductase, which is responsible for the conversion of folic acid to reduced folate with cofactors such as tetrahydrofolate. Reduced folates are required for metabolic transfer of 1-carbon units in various biochemical reactions. The inhibition of thymidylate synthesis appears to be the most important eect exerted by methotrexate, which results in inhibition of DNA synthesis and arrest of cell division in the S phase. Methotrexate inhibits replication and function of T and B cells and suppresses the secretion of various cytokines such as IL-1, interferon-g and TNF. Methotrexate also suppresses epidermal cell division in psoriasis [20]. The mechanism of action of biologic therapies diers from the currently used systemic therapies by attacking specic steps in the pathogenesis pathway of psoriasis. Accordingly, researchers are now targeting these pathogenetic steps, resulting

in more specic therapies and minimizing or limiting the disruption to overall immune function observed with more traditional agents. Approximately 50 biologics are currently being evaluated for their ecacy in treating psoriasis. Etanercept is a recombinant TNFa receptor fusion protein, consisting of two extracellular ligand binding domains of the human p75 TNFa receptor fused to the Fc portion of human IgG1 [21]. The primary action of etanercept is to bind and inactivate soluble and cell bound TNF and lymphotoxin alpha [22]. Iniximab is a chimeric TNFa monoclonal antibody combining a human IgG Fc portion and a murine variable region, which in vivo neutralizes soluble TNFa and blocks TNFa bound to cell membranes [23]. Alefacept is a dimeric human fusion protein composed of the protein LFA3 fused to the Fc portion of IgG1. Alefacept reduces the number of circulating CD45RO T cells as well as CD4 and CD8 T cells. It does not however aect CD%RA T cells or B cells and is likewise ineective with respect to primary or secondary immune responses [23]. Efaluzimab is a humanized monoclonal IgG1 antibody against CD11a. By binding to CD11a and inhibiting LFA-1/ICAM-1 interactions, efaluzimab inhibits binding of T cells to vascular endothelial cells, inhibits T cell tracking into the dermis, and prevents activation of T cells. The net eect is to reduce the release of inammatory cytokines in the skin, resulting in the improvement of psoriasis [22]. Onercept is a recombinant unmodied fully human soluble type 1 TNF receptor, which acts as an anti-TNF agent. However, the ndings of T cells and macrophages in the dermis of plaques before pathologic changes in the epidermis have pointed to the role of the immune system, particularly aberrant T cell proliferation in the development of this disorder. It is now understood that an unidentied antigen activates native Langerhans cells in the epidermis and these cells subsequently migrate to the lymph nodes, where Langerhans MHC-bound antigen binds to and activates T cell receptors. In addition to the bound antigen, other costimulatory interactions, such as those between lymphocyte function antigen and CD2, intercellular adhesion molecule-1, and CD28 are required. The inhibition of these specic costimulatory signals is currently the main focus of immunotherapy research for psoriasis [23].

DISORDERS OF CELL KINETICS AND DIFFERENTIATION

593

In psoriasis, there is hyperproliferation and altered keratinization, including an increase of cycling keratinocytes and enhanced expression of involucrin, epidermal fatty acid binding protein (E-FABP), skin-derived antiproteinase (SKALP), and cytokeratin 16. In psoriasis and other skin diseases with changes in epidermal growth, disturbances in epidermal proliferation and dierentiation may be associated with an impaired skin barrier function. In acute and cumulative experimentally induced skin irritation, an increase of the number of proliferative cells, as measured by Ki-67 staining; an increase in the expression of involucrin and E-FABP; and variable SKALP expression in association with impaired barrier function were found [24]. Pityriasis rubra pilaris Pityriasis rubra pilaris (PRP) is a disease marked by hyperproliferation and dermal inammation. The etiology of PRP is unknown and has long been a matter of debate. Microscopically, the features of PRP include irregular acanthosis, acantholytic dyskeratosis, hypergranulosis, and mild chronic inammatory inltrate in the upper dermis. Alternating vertical and horizontal parakeratosis in the interfollicular stratum corneum is said to be characteristic [2527]. Electron microscopy reveals changes in the epidermis: decreased numbers of tonolaments and desmosomes, enlarged intercellular spaces, and signs of ultrastructural parakeratosis of the stratum corneum, including lipidlike vacuoles, incomplete keratinization and nuclear remnants. The corneocytes are fusiform with numerous pits. There is an abnormally high uptake of radiolabeled amino acids and injected in PRP lesions, suggesting increased epidermal replacement, similar to ndings in psoriatic skin. Subsequently, autoradiography studies using tritiated thymidine showed an increased labeling index of epidermal cells compared with normal reecting increased cell proliferation [25]. Results of Western blot analysis of the skin of familial PRP patients suggested keratin 17 expression, which has been found in psoriatic skin and basal cell carcinoma but is not detected in normal epidermis. Keratins 6 and 16 were also expressed, a nonspecic nding indicating keratinocyte activation [25,28]. Lichen planus Keratinocytes in normal human epidermis undergo an orderly pattern of maturation during

their migration from the basal layer through the spinous and granular layers to the cornied layer. This progression is accompanied by changes in the synthesis of keratins and involucrin. Dierent distributions have also been found in dierent layers of the epidermis. In the basal epidermal layer, basic keratin 5 and acidic K14 are produced, and under normal circumstances suprabasal cells synthesize the dierentiation-related keratin pair, K1 and K10. In hyperproliferative diseases such as psoriasis and skin tumors, K6 and K16 are produced, and this keratin pair is also synthesized during wound healing [29]. Lichen planus (LP) is a chronic skin disorder characterized by cutaneous inammation and abnormal epidermal growth and keratinization. The inltration of T lymphocytes into the epidermis in the condition implies an immune response targeted to the epidermis. HLA-DRpositive keratinocytes overlying the dermal inltrate also suggest gamma interferon release from activated T lymphocytes, and therefore an immunologic mechanism may be responsible for the expression of LP [30]. There is a close similarity between the histologic appearances of discoid lupus erythematosus (DLE) and LP. The epidermis in these dermatoses shows orthohyperkeratosis and hydropic degeneration of the basal cell layer, and because of the morphologic resemblance, it is suggested that these dermatoses may share similar pathophysiologic mechanisms. It was presumed that the epidermal turnover was decreased in these dermatoses; however, measurements of cell proliferation in DLE by tritiated thymidine uptake and Ki-67stained nuclei and in LP by tritiated thymidine uptake have shown an increase in cell proliferation. It is presumed, therefore, that the epidermal reparative response to erosive damage to the basal compartment is very active in these diseases. Some investigations have found that the expression of dierentiation-specic keratins (1 and 10) is disturbed in psoriatic epidermis. However in DLE and LP the dierentiation-specic keratins were unaected and were expressed normally. These keratins are the markers for early dierentiated keratinocytes, suggesting that the early dierentiation phase seems to proceed normally in DLE and LP. In contrast, involucrin, a marker for terminal keratinization was expressed in the lower epidermal cell layers in contrast to the expression pattern in normal epidermis, and this staining pattern was also seen in psoriatic epidermis. The increased involucrin expression in psoriatic and wounded epidermis is considered to be

594

ZU N TU

et al

caused by a recovery mechanism. Keratinocytes in hyperproliferative conditions complete their keratinization process in a much shorter time than usual so that involucrin is expressed much earlier or more precociously than in normal epidermis. This is considered the means by which the epidermis recovers in a limited time. This led to the proposal that the increased involucrin expression in DLE and LP is caused by a recovery mechanism similar to that occurring in wound healing, because the destruction of the basal layer in DLE and LP may be regarded as equivalent to a micro-wound. Keratin 6 and 16 have been referred to as markers of hyperproliferative or activated keratinocytes, and it has been reported that the expression of these keratins is induced by epidermal growth factor and TGFa at the transcriptional level. In the lesions of DLE and LP, it is suggested that keratinocytes may be activated in response to the damage in the basal layer and that keratin 6, 16, and 17 are expressed as a consequence of the increased rate of cell cycling [29]. A study comparing epidermal cell characteristics as assessed by the ow cytometry of LP lesional skin and of normal epidermis and psoriatic lesional skin, reveals that the percentage of cells in S and G2M phase and the percentage of vimentin positive cells in lesional LP is intermediate between the value observed in normal and in psoriatic lesional skin. The percentage of keratin 10positive cells in LP is equal to the percentage of keratin 1positive cells in normal skin [30]. Skin tumors Seborrheic keratosis, actinic keratosis, Bowens, disease and squamous cell carcinoma are neoplasms of epidermal keratinocytic derivation. Information on cell kinetics may be a useful adjunct to routine histopathology in the understanding of the nature of tumors, including the relationship between benign and malignant tumors and also to prognosis [31]. Proliferation cell nuclear antigen (PCNA) is a 36-kD nonhistone nuclear polypeptide present in nuclei throughout the cell cycle. It is synthesized in late G1 and S phase levels, therefore correlating with the cells proliferative state. Apoptosis-related antigen (LeY) is a glycoprotein that has been found to be highly expressed in a variety of cancer cells and on the cell surface of virus-infected T lymphocytes. Apoptotic cells are often found in tumor tissues, helping to dene the balance of the tumor cell population. Apoptosis is also found in tissues

characterized by rapid cell turnover rates. There is a relationship between cell growth and apoptosis, or PCNA and LeY expression. In the normal epidermis, both PCNA and LeY expression is very low. In the neoplasms, both PCNA and LeY expression is higher than in the normal skin, and the distribution of staining was dierent depending on the type of the neoplasm and the degree of potential malignancy. In general, the percentage of PCNA and LeY expression increases progressively from seborrheic keratosis to actinic keratosis to Bowens disease to squamous cell carcinoma. It is probable, however, that in such benign tumors, proliferation and dierentiation (or apoptosis) occur equivalently, helping to keep the balance of the tumor cell population, but in malignant tumors, the balance of proliferation and dierentiation may be lost [31]. The kinetics of cells, keratin, and lipids in the skin could provide useful information about skin biology in health and disease. The kinetics of skin cell turnover are of interest for a variety of physiologic and pathologic conditions. There are also uncertainties regarding the extent of keratinocyte turnover in various skin diseases. Hyperproliferatiation may represent a risk factor for skin cancer and occurs in physiologic conditions such as wound healing.

References
[1] Kimyai-Asadi A, Jih MH, Freedberg IM, et al. Epidermal cell kinetics, epidermal dierentiation and keratinization. In: Freedberg IM, Eisen AZ, Wol K, editors. Fitzpatricks dermatology in general medicine. 6th edition. New York: Mc Graw Hill; 2004. p. 8999. [2] Jetten AM, Harvat BL. Epidermal dierentiation and squamous metaplasia: from stem cell to cell death. J Dermatol 1997;24:71125. [3] Hsieh EA, Chai CM, Lumen BO, et al. Dynamics of keratinocytes in vivo using H2O labelling: a sensitive marker of epidermal proliferation state. J Invest Dermatol 2004;123:5306. [4] Rendl M, Ban J, Mrass P, et al. Caspase-14 expression by epidermal keratinocytes is regulated by retinoids in dierentiation-associated manner. J Invest Dermatol 2002;119:11505. [5] Kawashima K, Doi H, Ito Y, et al. Evaluation of cell death and proliferation in psoriatic epidermis. J Dermatol Sci 2004;35:20714. [6] Hatta N, Takata M, Kawara S, et al. Tape stripping induces marked epidermal proliferation and altered TGF-a expression in non-lesional psoriatic skin. J Dermatol Sci 1997;14:15461.

DISORDERS OF CELL KINETICS AND DIFFERENTIATION

595

[7] Pena JC, Fuhs E, Thompson CB. Bcl-X expression inuences keratinocyte cell survival but not terminal dierentiation. Cell Growth Dier 1997;8: 61929. [8] Gandarillas A, Goldsmith LA, Gschmeisser S, et al. Evidence that apoptosis and terminal dierentiation of epidermal keratinocytes are distinct process. Exp Dermatol 1999;8:719. [9] Kerkhof PCM, Gerritsen MJP, Jong EMGJ. Transition from symptomless to lesional psoriatic skin. Clin Exp Dermatol 1996;21:3259. [10] Franssen MEJ, Zeeuwen PLJM, Vierwinden G, et al. Phenotypical and functional dierences in germinative subpopulations derived from normal and psoriatic epidermis. J Invest Dermatol 2005;124: 37383. [11] Mommers JM, Rossum MM, Hooijdonk CAEM, et al. Quantication pf epidermal cell populations in the centre and margin of stable psoriatic plaques. Arch Dermatol Res 1999;291:8892. [12] Brushan M, Mclaughlin B, Weiss JB, et al. Levels of endothelial cell stimulating angiogenesis factor and vascular endothelial growth factor are elevated in psoriasis. Br J Dermatol 1999;141:105460. [13] Creamer D, Allen MH, Sousa A, et al. Localization of endothelial proliferation and microvascular expansion in active plaque psoriasis. Br J Dermatol 1997;136:85965. [14] Nickolo BJ, Mitra RS, Vrani J, et al. Aberrant production of interleukin-8 and thrombospondin-1 by psoriatic keratinocytes mediates angiogenesis. Am J Pathol 1994;144:8208. [15] Weiss JB, Brown RA, Kumar S, et al. An angiogenic factor isolated from tumours. A potent low molecular weight compound. Br J Cancer 1979;40:4936. [16] Rossum MM, Erp PEJ, Kerkhof PCM. Treatment of psoriasis with a new combination of calcipotriol and betamethasone dipropionate: a ow cytometric study. Dermatology 2001;203:14852. [17] de Jong EM, Mork NJ, Seijger MMB, et al. The combination of calcipotriol and methotrexate compared with methotrexate and vehicle in psoriasis: results of a multicentre placebo-controlled randomized trial. Br J Dermatol 2003;148:31825.

[18] Christophers E, Mrowietz U. Psoriasis. In: Freedberg IM, Esen AZ, Wol K, et al, editors. Fitzpatricks dermatology in general medicine. 6th edition. New York: Mc Graw Hill; 2004. p. 40725. [19] Lebwohl M. Topical application of calcipotriene and corticosteroids: combination regimens. J Am Acad Dermatol 1997;37:558. [20] Yamauchi Ps, Rizk D, Kormeili T, et al. Current systemic therapies for psoriasis: where are we now? J Am Acad Dermatol 2003;49:6677. [21] Lebwohl M. Psoriasis. Lancet 2003;361:1197204. [22] Gladman DD. Psoriatic arthritis. Dermatol Ther 2004;17:5036. [23] Stebbins WG, Lebwohl MG. Biologics in combination with nonbiologics: ecacy and safety. Dermatol Ther 2004;17:43240. [24] Mail Le TK, Schalkwijk J, Kerkhof PCM, et al. A histological and immunohistochemical study on chronic irritant contact dermatitis. Am J Contact Dermat 1998;9:238. [25] Albert MR, Mackool BT. Pityriasis rubra pilaris. Int J Dermatol 1999;38:111. [26] Lever WF, Schaumburg-Lever G. Histopathology of the skin. 7th edition. Philadelphia: JB Lipincott; 1990 p. 176. [27] Goldsmith LA, Baden HP. Lichen rubra pilaris. In: Freedberg IM, Esen AZ, Wol K, et al, editors. Fitzpatricks dermatology in general medicine. 6th edition. New York: Mc Graw Hill; 2004. p. 4424. [28] Vanderhooft SL, Francis JS, Holbrook KA, et al. Familial pityriasis rubra pilaris. Arch Dermatol 1995;131:44853. [29] Ichikawa E, Watanabe S, Takahashi H. Keratin and involucrin expression in discoid lupus erythematosus and lichen planus. Arch Dermatol Res 1997; 289:51926. [30] Glade CP, Vleuten VD, Erp V, et al. The epidermis of chronic idiopathic lichen planus during topical treatment with the vitamin D3 analogue KH 1060. Clin Exp Dermatol 1998;23:114. [31] Tsuji T, Kitajima S, Koashi Y. Expression of proliferating cell nuclear antigen (PCNA) and apoptosis related antigen (LeY) in epithelial skin tumors. Am J Dermatopathol 1998;20:1649.

Dermatol Clin 25 (2007) 597603

Vesicular and Bullous Disorders: Pemphigus

Adone Baroni, MD, PhDa, Alessandro Lanza, DDSb, Nicola Cirillo, DDSb, Giampiero Brunetti, MDa,*, Eleonora Ruocco, MD, PhDa, Vincenzo Ruocco, MDa
b a Department of Dermatology, Second University of Naples, Via Sergio Pansini, 5, 80131-Napoli, Italy Department of Odontostomatology, Second University of Naples, Via Sergio Pansini, 5, 80131-Napoli, Italy

Among the bullous autoimmune disorders, pemphigus is the only disorder to have a good relevance with known cutaneous receptors. Pemphigus is a chronic, autoimmune disease involving the skin and Malpighian mucous membranes. Pemphigus leads to progressive blistering and subsequent erosions. The main clinical subtypes are pemphigus vulgaris (PV) with the pemphigus vegetans variant, pemphigus foliaceus, and pemphigus erythematosus. Genetic factors have an essential role. Exogenous factors, such as drugs, physical agents, viruses, hormones, food, and emotional stress, often play a triggering eect. Pemphigus immunoglobulin (Ig)G can cause direct inhibition of desmogleins adhesive function through steric hindrance. Dsg3 can act as a receptor by triggering complex intracellular events, including changes in intracellular calcium concentration, phosphokinase C, and p38 mitogen-activated protein kinase activation, transcriptional regulation, and proteinase activity, all causing desmosome disassembly. Detachment of keratinocytes from one another could also occur as a consequence of the activation of the apoptotic machinery by Fas-FasL pathway. Furthermore, a subset of pemphigus autoantibodies is thought to recognize and block keratinocyte acetylcholine receptors, which are known to play a crucial role in mediating keratinocytekeratinocyte cohesion. Thus, acantholysis in pemphigus seems to result

* Corresponding author. E-mail address: bgiampy@inwind.it (G. Brunetti).

from cooperative and nely coordinated biological signals, which regulate cell adhesion. Routine laboratory examinations for diagnostic purposes are the Tzanck test, standard histology, direct immunouorescence (DIF), indirect immunouorescence (IIF), and ELISA assays. A novel therapeutic approach based on the antiacantholytic eect of cholinomimetics drugs has been proposed. The demonstration that pyridostigmine bromide and carbachol can improve or cases prevent the acantholysis in vivo provides new prospects for the nonhormonal treatment of patients who have pemphigus. A role for receptors in the autoimmune blistering diseases has not been clearly elucidated. Among these disorders, pemphigus represents an exception in that, although the binding of pemphigus IgG to desmogleins is likely to be the main pathogenic event, the specicity of antigen(s) targeted by pemphigus IgG and the role of receptor-mediated apoptosis are subjected to dispute. Hence, we underline the role that receptors are thought to play in pemphigus. Pemphigus is a chronic, autoimmune, blistering disease involving the skin and Malpighian mucous membranes. Of the several clinical subtypes, the major ones are PV with the pemphigus vegetans variant, pemphigus foliaceus with the two variants endemic pemphigus foliaceus (Brazilian pemphigus foliaceus, or fogo selvagem meaning wild re) and pemphigus erythematosus, and paraneoplastic pemphigus. When the outbreak of pemphigus is facilitated by one or more environmental triggers, the disease is usually labeled as induced pemphigus [1].
derm.theclinics.com

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.07.001

598

BARONI

et al

Etiopathogenesis Genetic factors play an essential role. There is a well established human leukocyte antigen (HLA) association with DR4, DR14, and DQ1, DQ3 genes in most forms of pemphigus, whereas an HLA-DR1 gene is linked to the endemic foliaceus form. Genetic factors may determine susceptibility to the disease, but its outbreak possibly occurs when predisposed individuals are exposed to the appropriate inducing agents, which may be known (induced pemphigus) or unknown (idiopathic pemphigus). In several cases, the outbreak of pemphigus is facilitated by exogenous triggering factors, such as drugs (penicillin, pyrazolone or their derivatives, and interferons), physical agents (burns, ionizing radiations), viruses (Herpesviridae family), hormones (pregnancy, Graves disease), food (garlic, leek),or emotional stress [1]. In the majority of patients, no inducing agent can be detected, so the cause of the disease, leading to the production of pathogenic pemphigus antibodies (IgG, occasionally IgA), usually remains unknown. An intriguing hypothesis has suggested that the horror autotoxicus of pemphigus could stem from changes in serum concentration of the recently discovered Dsg3, the circulating 30 kDa immunodominant fraction of Dsg3 [2]. The binding of the autoantibodies to their targets generates a plethora of biological eects due to their direct interference with desmogleins adhesive function and to more complex events involving intracellular pathways that modify proteases activity or calcium metabolism, leading to loss of cellcell adhesion. Recently, a pivotal role in generating keratinocyte dyshesion has been suggested for the apoptosis [3,4], but further studies to conrm this hypothesis are required (see section on Antigens and specic receptors). It has also been speculated that pemphigus autoantibodies directly interfere with the molecules and peculiar enzyme activities (transglutaminase) responsible for cellcell adhesion, thereby leading to acantholysis [5].

Clinical presentation PV, the most common type of pemphigus, presents in middle-aged and genetically predisposed individuals who have oral lesions in two thirds of patients, in contrast to pemphigus foliaceus in which mucous membrane involvement is usually absent. Oral blisters are fragile and

rupture readily, leaving erosions that heal with diculty. Flaccid bullae develop over several sites of the skin (trunk, scalp, and exures). The blistering is not always obvious, and often lesions consist of crusted erosions. Left untreated, the disease progresses with an almost always fatal outcome owing to uncontrolled uid and protein loss or opportunistic infection. In pemphigus vegetans, pustular lesions and hypertrophic granulations or vegetations develop at the mucosal margins and on the intertriginous areas (axillae and, especially in women, inguinal regions and submammary folds). Pemphigus foliaceus and pemphigus erythematosus are diseases of middle age, whereas the endemic form, clustered in jungle areas of Brazil, Colombia, Paraguay, and Peru and possibly precipitated by a biting black y (Simulium nigrimanum), frequently aects children and adults. An unusual incidence of pemphigus foliaceus has been reported in young Tunisian women. The onset is frequently localized to the scalp and face, slowly spreading over the chest and upper back. Scattered, scaly, and crusted lesions, intermingled with a few small accid bullae, involve the seborrheic areas and rarely extend, except in the endemic form in which the patient may become erythrodermic. Pemphigus erythematosus may show additional clinical lesions of lupus erythematosus. These diseases are usually more benign than PV, and spontaneous remissions can occur. Paraneoplastic pemphigus is a distinctive form of pemphigus associated with lymphoproliferative disease, thymomas, and sarcomas. Clinical presentation is polymorphous and may mimic erythema multiforme, with target oral and palmoplantar lesions or lichen planus pemphigoides with severe mucosal erosions and large blisters on the trunk. The prognosis is poor, being conditioned by the coexisting neoplastic disease. The clinical pattern of induced pemphigus is variable. Pemphigus foliaceus and pemphigus erythematosus are the most common presentations induced by drugs containing a sulfhydryl group (thiol drugs) (eg, penicillamine and captopril) and by some physical agents (eg, sunburn). The vulgaris form, often heralded by a herpetiform presentation, can be induced by nonthiol drugs (eg, pyrazolon derivatives and progesterone) and by dierent triggering factors (eg, radiotherapy, thermal burns, and viruses [especially Herpetoviridae]). The course is unpredictable. Many patients who have the foliaceus and erythematosus forms recover spontaneously when the inducing factor

VESICULAR AND BULLOUS DISORDERS: PEMPHIGUS

599

is removed (induced pemphigus proper). In most patients who have the vulgaris form, the disease continues its course despite removal of any inducing factor (triggered pemphigus).

Pathology The hallmark of histologic changes in pemphigus is the intraepithelial cleft that results from cellcell dyshesion (acantholysis) with the formation of bullae containing isolated, round, acantholytic (or Tzanck) cells. The cleft is suprabasal in PV and pemphigus vegetans and subcorneal in the pemphigus foliaceus subtypes. A mild to moderate inammatory inltrate may be seen in the upper dermis. In paraneoplastic pemphigus, dyskeratotic keratinocytes and basal cell vacuolization with interface dermatitis are histologic ndings. Eosinophils in the dermis characterize the early stages of drug-induced pemphigus.

Antigens and specic receptors Almost all patients who have pemphigus have pathogenic circulating IgG autoantibodies, which are detectable in the serum at dierent concentrations (or titers) by IIF, that bind to normal components of keratinocyte cell membrane belonging to the cadherin supergene family. The pemphigus antigens have lately been identied as desmoglein 3 (Dsg3) (130 kDa) for PV and desmoglein 1 (Dsg1) (160 kDa) for pemphigus foliaceus. They are a type I transmembrane glycoproteins that mediate intercellular adhesion by engaging calcium-dependent interactions with apposed cells [6]. Some putative non-Dsg pemphigus antigens represent other adhesion molecules, such as plakoglobin [7], desmocollins [8], desmoplakin [9], and collagen XVII/BP180 [10]. Although desmogleins cannot be considered as receptors stricto sensu, binding of PV IgG to Dsg3 has been demonstrated to trigger transduction of intracellular signals, including PKC and p38 mitogen-activated protein kinase activation, transcriptional regulation, and proteinase activity, all causing desmosome disassembly [1115]. It has been supposed that PV IgG binding to Dsg3 may interfere with the stability of the cadherin/catenin complex and contribute to the acantholytic process by inducing dissociation of Dsg3 from plakoglobin (PG) (also called gamma catenin) [16]. Cytoplasmatic accumulation of PG should

determine its delocalization inside the nucleus [17,18], where catenins form a complex with transcriptional factors such as lymphoid enhancer binding factor/T-cell factor, thus playing a transcriptional role (for review, see Ref. [19]). This hypothesis is conrmed by some experimental evidence: Intracellular localization of plakoglobin is altered in PV [18], keratinocytes PG/ do not exhibit tonolaments retraction and loss of cell adhesion after PV-IgG incubation [17], and plakoglobin could promote expression of urokinase plasminogen activator receptor [20,21]. The reduction of Dsg3 half-life observed after exposure to PV serum [22] could be considered as a mechanism of receptorial down-regulation in response to blocking autoantibodies (Fig. 1). Other pemphigus antigens include keratinocyte acetylcholine receptors (AChR) [23,24]. Approximately 85% of patients who have pemphigus develop antibodies against the a9 acetylcholine receptor, a 50-kDa homopentamer that regulates adhesion in epithelial cells [25], and pemphaxin, a 75-kDa novel annexin (also known annexin 31 or ANXA9) that can act as an acetylcholine receptor [24]. Human epidermal keratinocytes are members of a nonneuronal signaling network mediating intercellular communication in the skin in which the cytotransmitter acetylcholine (ACh) acts as a local hormone or a cytokine. Nonneuronal ACh is synthesized, secreted, and degraded by keratinocytes and exerts a plethora of biological eects on these cells because it can simultaneously activate dierent intracellular signal transduction cascades [2629]. Binding of ACh to the cell membrane elicits several biochemical events, including the biological sum, which, when taken together with cumulative eects of other hormonal and environmental stimuli, determines several complex modications [30,31]. It has been reported that ACh is responsible for dramatic eects on keratinocyte proliferation, migration, and dierentiation, more than for intercellular adhesion and for other sublayers [32,33]. In several cell types, activation of AChRs aects signaling pathways regulating the expression or function of adhesion molecules [34,35] and the protein phosphorylation status [36,37]. Nguyen and colleagues [38], by investigating the mechanisms that mediate cholinergic control of cellcell adhesion, have shown that keratinocyte cholinergic receptors can regulate desmosomal adhesion by altering the level of expression of Dsg1 and Dsg3 and the phosphorylation status of Dsg3.

600

BARONI

et al

Fig. 1. Intracellular pathways triggered by pemphigus serum in keratinocytes involve receptorial and nonreceptorial signaling (see text for details). ACh, acetylcholine; AChR, acetylcholine receptor; cAMP, cyclic adenosin monophosphate; CM, cell membrane; DAG, diacylglycerol; Dpk, desmoplakin; Dsc, desmocollins; Dsg, desmoglein; FasL, Fas ligand; IP3, inositol 1,4,5-triphosphate; KIF, keratin intermediate laments; mAChR, muscarinic acetylcholine receptors; MAPK, mitogen-activated protein kinase; nAChR, nicotinic acetylcholine receptors; PG, plakoglobin; PKC, protein kinase C; Pkp, plakophilins; PLC, phospholipase C; pPG, phosphorilated plakoglobin; PV-IgG, pemphigus vulgaris-IgG; uPA, urokinase plasminogen activator; uPAR, urokinase plasminogen activator receptor. (Adapted from Cirillo N, Femiano F, Gombos G, et al. Matrix metalloproteinase 9 is the outer executioner of desmoglein 3 in apoptotic keratinocytes. Oral Dis 2007;13:3415; with permission.)

The ability of cholinomimetics drugs such as carbachol and pyridostigmine bromide to improve or prevent acanthohlysis in vivo [39] suggests that the ACh and its receptors could be involved in the pathomechanism of acantholysis, giving us new prospects for the nonhormonal treatment of patients who have pemphigus (Box 1) [40]. Whether desmogleins and AChRs directly alter the dynamic of desmosome assembly and disassembly, other receptors involved in the acantholysis could drive cellcell detachment by triggering the extrinsic apoptotic pathway [41]. In pemphigus, this receptor-mediated cell death seems to be induced by the activation of membrane FasR

through the binding to its ligand (FasL). FasL levels are markedly increased in sera from patients who have pemphigus; this has been demonstrated to trigger apoptosis through the activation of caspase 8. Recent works have reported that the extrinsic or death receptor-mediated pathway and the intrinsic, mitochondria-dependent pathway could be involved in pemphigus-associated cell death [42]. The nding that caspase3 [43] and matrix metalloproteinase 9 [44] directly cleaves Dsg3 and other desmosomal proteins, thereby causing apoptotic disruption of desmosomes, further suggests a strict correlation between loss of keratinocyte adhesion and apoptosis. Further

VESICULAR AND BULLOUS DISORDERS: PEMPHIGUS

601

Box 1. Experimental evidence showing the involvement of acetylcholine receptors in acantholysis Acetylcholine a9 receptor is targeted by pemphigus vulgaris antibodies. Pemphigus vulgaris antibody identies pemphaxin. Cholinomimetic drugs exhibit antiacantholytic activity. Cholinergic antagonists induce in vitro acantholysis. Eighty-ve percent of patients who have pemphigus develop antibodies against keratinocyte AChR. Epithelial acetylcholine receptors regulate cell adhesion. studies will clarify whether apoptosis represents the cause of acantholysis or whether cellcell detachment induces anoikis. Laboratory diagnosis Routine laboratory examinations for diagnostic purposes are the Tzanck test, standard histology, DIF, and IIF. Smears taken by scraping the bottom of a bulla or an erosion show typical acantholytic cells (Tzanck test). Standard histology of a fresh bulla reveals suprabasilar (PV) or subcorneal (pemphigus foliaceus) splitting with formation of intraepithelial cavities. DIF shows the intercellular deposition of IgG and C3 in the perilesional epithelium. IIF (using monkey or guinea-pig esophagus) is the classic method to detect and titer pemphigus antibodies in serum. This test is also useful to monitor the course of pemphigus and to adjust the treatment because antibody titers usually parallel disease activity. An ELISA kit for detection and titration of anti-Dsg3 and anti-Dsg1 antibodies has recently become available. In particular cases and when the diagnosis remains uncertain, more sophisticated laboratory investigations (eg, immunoblotting, immunoprecipitation) are needed. HLA typing is useful to search for pemphigus-prone genes in the patients genotype.

Treatment Systemic steroids and other immunosuppressive agents are the rst-line treatment of pemphigus.

Prednisone or deazacort are given initially in a high dose (100200 mg/d), often with azathioprine (2.5 mg/kg/d) or cyclophosphamide (13 mg/ kg/d) or mycophenolate mofetil (1 g twice per day). Once blistering has been controlled, the steroid dosage can gradually be lowered. Treatment usually needs to be continued for several years, which leads to the fact that mortality and morbidity are more likely to be due to side eects of the steroid and immunosuppressive therapy than to the disease itself. In mild cases, and especially in pemphigus erythematosus, topical or intralesional steroid therapy alone may be attempted. Alternative treatment regimens are pulse methylprednisolone therapy; corticotropin; antimalarials; high-dose intravenous IgG [45]; rituximab [46]; and oral tetracycline combined with nicotinamide, plasmapheresis, extracorporeal photopheresis, and immunoadsorption with specic adsorbers [47]. Patients in remission should be discouraged from taking unnecessary drugs that carry a risk of pemphigus induction. Exposure to the sun and other UV sources requires special caution because these agents may facilitate relapses of pemphigus. The same can be said for intensive and prolonged emotional stress. Patients should be advised to have a balanced diet and to avoid eating foods that contain garlic, onion, and leek because these plants (belonging to the genus Allium) contain allyl compounds, which have a proven acantholytic potential [48]. Numerous studies on keratinocyte AChRs have allowed the development of novel therapies with promising results based on the antiacantholytic eects of cholinomimetics drugs, such as pyridostigmine bromide and carbachol [39], which are acetylcholinesterase inhibitors and act as indirect acetylcholine agonists. This novel therapeutic approach is supported by the reduced risk of developing pemphigus in current smokers and in exsmokers [49]. Cigarette smoke contains the cholinergic agonist nicotine that may interfere with the activity of the newly described antibody to the keratinocyte acetylcholine receptor. Treatment with pyridostigmine bromide (360 mg/d) in patients who have pemphigus keeps the disease under control at a lower dose of systemic glucocorticosteroids, which, although lifesaving, may cause severe adverse eects [39]. One of the hypothetical mechanisms that could explain the antiacantholytic eect of pyridostigmine bromide and carbachol is the direct competition of these drugs with pemphigus IgG for binding to keratinocytes. Pemphigus IgG can cause the blockage of AChR

602

BARONI

et al inositol 1,4,5-triphosphate in DJM-1 cells, a squamous cell carcinoma line. J Invest Dermatol 1995; 104:337. Osada K, Seishima M, Kitajima Y. Pemphigus IgG activates and translocates protein kinase C from the cytosol to the particulate/cytoskeleton fractions in human keratinocytes. J Invest Dermatol 1997;108: 4827. Berkowitz P, Hu P, Liu Z, et al. Desmosome signaling. Inhibition of p38 MAPK prevents pemphigus vulgaris IgG-induced cytoskeleton reorganization. J Biol Chem 2005;280:2377884. Wang X, Bregegere F, Frusic-Zlotkin M, et al. Possible apoptotic mechanism in epidermal cell acantholysis induced by pemphigus vulgaris autoimmunoglobulins. Apoptosis 2004;9:13143. Seishima M, Satoh S, Nojiri M, et al. Pemphigus IgG induces expression of urokinase plasminogen activator receptor on the cell surface of cultured keratinocytes. J Invest Dermatol 1997;109:6505. Aoyama Y, Owada MK, Kitajima Y. A pathogenic autoantibody, pemphigus vulgaris-IgG, induces phosphorylation of desmoglein 3, and its dissociation from plakoglobin in cultured keratinocytes. Eur J Immunol 1999;29:223340. Caldelari R, de Bruin A, Baumann D, et al. A central role for the armadillo protein plakoglobin in the autoimmune disease pemphigus vulgaris. J Cell Biol 2001;153:82334. Mignogna MD, Pannone G, Lo Muzio L, et al. Catenin dislocation in oral pemphigus vulgaris. J Oral Pathol Med 2001;30:26874. Lanza A, Cirillo N, Femiano F, et al. How does acantholysis occur in pemphigus vulgaris: a critical review. J Cutan Pathol 2006;33:40112. Lo Muzio L, Pannone G, Staibano S, et al. Strict correlation between uPAR and plakoglobin expression in pemphigus vulgaris. J Cutan Pathol 2002; 29:5408. Mann B, Gelos M, Siedow A, et al. Target genes of beta-catenin T cell factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas. Proc Natl Acad Sci U S A 1999;96:16038. Cirillo N, Femiano F, Gombos F, et al. Serum from pemphigus vulgaris reduces desmoglein 3 half-life and perturbs its de novo assembly to desmosomal sites in cultured keratinocytes. FEBS Lett 2006; 580:327681. Bastian BC, Nuss B, Romisch J, et al. Autoantibodies to annexins: a diagnostic marker for cutaneous disorders? J Dermatol Sci 1994;8:194202. Nguyen VA, Ndoye A, Grando SA. Pemphigus vulgaris antibody identies pemphaxin, a novel keratinocyte annexin-like molecule binding acetylcholine. J Biol Chem 2000;275:2946676. Nguyen VT, Ndoye A, Grando SA. Novel human alpha9 acetylcholine receptor regulating keratinocyte adhesion is targeted by pemphigus vulgaris autoimmunity. Am J Pathol 2000;157:137791.

with subsequent reduction of calcium inux. Hence, cholinomimetic agents can ameliorate intracellular calcium metabolism, thus preventing the eects of pemphigus IgG. Cholinergic agonists can have a protective eect against pemphigus acantholysis and up-regulate the expression of adhesion molecules, protecting them from pemphigus IgG-induced phosphorylation. Elucidation of the cholinergic control of keratinocytes adhesion has a potential for the development of treatment regimens using safer drugs to control blistering in a variety of other skin diseases.

[12]

[13]

[14]

References
[1] Ruocco V, Ruocco E. Pemphigus and environmental factors. G Ital Dermatol Venereol 2003;138:299309. [2] Lanza A, Femiano F, De Rosa A, et al. The N-terminal fraction of desmoglein 3 encompassing its immunodominant domain is present in human serum: implications for pemphigus vulgaris autoimmunity. Int J Immunopathol Pharmacol 2006;19:319. [3] Baroni A, Buommino E, Paoletti I, et al. Pemphigus serum and captopril induce heat shock protein 70 and inducible nitric oxide synthase overexpression, triggering apoptosis in human keratinocytes. Br J Dermatol 2004;150:107080. [4] Pelacho B, Natal C, Espana A, et al. Pemphigus vulgaris autoantibodies induce apoptosis in HaCaT keratinocytes. FEBS Lett 2004;566:610. [5] Esposito C, Lombardi ML, Ruocco V, et al. Implication of tissue transglutaminase and desmoplakin in cell adhesion mechanism in human epidermis. Mol Cell Biochem 2000;206:5765. [6] Garrod DR, Merritt AJ, Nie Z. Desmosomal cadherins. Curr Opin Cell Biol 2002;14:53745. [7] Korman NJ, Eyre RW, Klaus-Kovtun V, et al. Demonstration of an adhering-junction molecule (plakoglobin) in the autoantigens of pemphigus foliaceus and pemphigus vulgaris. N Engl J Med 1989;321:6315. [8] Dmochowski M, Hashimoto T, Garrod DR, et al. Desmocollins I and II are recognized by certain sera from patients with various types of pemphigus particularly Brazilian pemphigus foliaceus. J Invest Dermatol 1993;100:3804. [9] Mimouni D, Foedinger D, Kouba DJ, et al. Mucosal dominant pemphigus vulgaris with anti-desmoplakin autoantibodies. J Am Acad Dermatol 2004;51: 627. [10] Schumann H, Baetge J, Tasanen K, et al. The shed ectodomain of collagen XVII/BP180 is targeted by autoantibodies in dierent blistering skin diseases. Am J Pathol 2000;156:68595. [11] Seishima M, Esaki C, Osada K, et al. Pemphigus IgG, but not bullous pemphigoid IgG, causes a transient increase in intracellular calcium and

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

VESICULAR AND BULLOUS DISORDERS: PEMPHIGUS

603

[26] Ndoye A, Buchli R, Greenberg B, et al. Identication and mapping of keratinocyte muscarinic acetylcholine receptor subtypes in human epidermis. J Invest Dermatol 1998;111:4106. [27] Sharma G, Vijayaraghavan S. Nicotinic receptor signaling in nonexcitable cells. J Neurobiol 2002;53: 52434. [28] Grando SA, Kawashima K, Wessler I. Introduction: the non-neuronal cholinergic system in humans. Life Sci 2003;72:200912. [29] Kurzen H, Berger H, Jager C, et al. Phenotypical and molecular proling of the extraneuronal cholinergic system of the skin. J Invest Dermatol 2004;123: 93749. [30] Grando SA, Pittelkow MR, Shultz LD, et al. Pemphigus: an unfolding story. J Invest Dermatol 2001;117:9905. [31] Kurzen H, Schallreuter KU. Novel aspects in cutaneous biology of acetylcholine synthesis and acetylcholine receptors. Exp Dermatol 2004;13:2730. [32] Grando SA, Horton RM, Mauro TM, et al. Activation of keratinocyte nicotinic cholinergic receptors stimulates calcium inux and enhances cell dierentiation. J Invest Dermatol 1996;107:4128. [33] Zia S, Ndoye A, Lee TX, et al. Receptor-mediated inhibition of keratinocyte migration by nicotine involves modulations of calcium inux and intracellular concentration. J Pharmacol Exp Ther 2000;293: 97381. [34] Williams CL, Hayes VY, Hummel AM, et al. Regulation of E cadherin-mediated adhesion by muscarinic acetylcholine receptors in small cell lung carcinoma. J Cell Biol 1993;121:64354. [35] Conroy WG, Ogden LF, Berg DK. Cluster formation of alpha7-containing nicotinic receptors at interneuronal interfaces in cell culture. Neuropharmacology 2000;39:2699705. [36] Nakayama H, Numakawa T, Ikeuchi H, et al. Nicotine-induced phosphorylation of extracellular signal-regulated protein kinase and CREB in PC12h cells. J Neurochem 2001;79:48998. [37] Dajas-Bailador FA, Soliakov L, Wonnacott S. Nicotine activates the extracellular signal-regulated kinase 1/2 via the alpha7 nicotinic acetylcholine receptor and protein kinase A, in SH-SY5Y cells and

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

hippocampal neurones. J Neurochem 2002;80: 52030. Nguyen VT, Arredondo J, Chernyavsky AI, et al. Keratinocyte acetylcholine receptors regulate cell adhesion. Life Sci 2003;72:20815. Nguyen VT, Arredondo J, Chernyavsky AI, et al. Pemphigus vulgaris acantholysis ameliorated by cholinergic agonists. Arch Dermatol 2004;140: 32734. Grando SA. New approaches to the treatment of pemphigus. J Investig Dermatol Symp Proc 2004;9: 8491. Puviani M, Marconi A, Cozzani E, et al. Fas ligand in pemphigus sera induces keratinocytes apoptosis through the activation of caspase-8. J Invest Dermatol 2003;120:1647. Arredondo J, Chernyavsky AI, Karaouni A, et al. Novel mechanisms of target cell death and survival and of therapeutic action of IVIg in pemphigus. Am J Pathol 2005;167:153144. Weiske J, Schoneberg T, Shroder W, et al. The fate of desmosomal proteins in apoptotic cells. J Biol Chem 2001;276:4117581. Cirillo N, Femiano F, Gombos G, et al. Matrix metalloproteinase 9 is the outer executioner of desmoglein 3 in apoptotic keratinocytes. Oral Dis 2007; 13:3415. Sami N, Qureshi A, Ruocco E, et al. Corticosteroid-sparing eect of intravenous immunoglobulin therapy in patients with pemphigus vulgaris. Arch Dermatol 2002;138:115862. Dupuy A, Viguier M, Bedane C, et al. Treatment of refractory pemphigus vulgaris with rituximab (antiCD20 monoclonal antibody). Arch Dermatol 2004; 140:916. Lu ftl M, Stauber A, Mainka A, et al. Successful removal of pathogenic autoantibodies in pemphigus by immunoadsorption with a tryptophan-linked polyvinylalcohol adsorber. Br J Dermatol 2003; 149:598605. Ruocco E, Aurilia A, Ruocco V. Precautions and suggestions for pemphigus patients. Dermatology 2001;203:2017. Sullivan TP, Elgart GW, Kirsner RS. Pemphigus and smoking. Int J Dermatol 2002;41:52830.

Dermatol Clin 25 (2007) 605612

Atopic Dermatitis: an Update and Review of the Literature

, MD, PhDa,*, Ronni Wolf, MDb,c Jasna Lipozen cic
a

University Department of Dermatology and Venereology, Zagreb University Hospital Center and School of Medicine, University of Zagreb, Salata 4, 10000 Zagreb, Croatia b Hebrew UniversityHadassah Medical School, Jerusalem, Israel c Dermatology Unit, Kaplan Medical Center, Rechovot 76100, Israel

Atopic dermatitis (AD) (eczema) is a chronically relapsing inammatory skin disease associated with erythema; scaly and oozing plaques on the forehead and face, neck, hands, and exural areas; and severe pruritus [1,2]. AD aects 2% to 5% of the general population, with 10% to 20% or more occurrences in infants and children and 1% to 3% in adults [3]. There is a wide variation in the prevalence of AD in dierent populations of the world, and it appears to be increasing [4,5]. Eczematous skin changes (infantile eczema) of early childhood may dier from common atopic dermatitis in terms of lack of family background of atopy, absence of signs of type I allergies, clinical course, and failure to respond promptly to topical treatment. Self-healing, the susceptibility to allergy, and the characteristic immune prole of lesional skin and peripheral blood are hallmarks of AD. They, together with the two dierent types of AD (intrinsic and extrinsic) require new denitions and reassessment. According to the recent ndings of a search for susceptibility genes, it appears that nonatopic dermatitis should be considered as a distinct entity of childhood eczema. The aected youngsters show dry ichthyosiform and eczematous skin changes at an early age with absence of sensitization to common allergens [6], and 33% of them should complete clearing within 5 years of age without skin lesions.

* Corresponding author. E-mail address: jasna.lipozencic@zg.htnet.hr ). (J. Lipozen cic

The optimal protocol for management in children has still not been established [7]. The diagnostic criteria that were recommended by the American Academy of Dermatology at the 2003 consensus conference are currently used by many clinicians for diagnosis and treatment of children and adults [810]. Moreover, the mechanisms underlying the pathogenesis of AD remain unclear, although numerous studies have demonstrated the integral involvement of immunopathology, genetic predisposition, and emotional and environmental stimuli in AD development and progression [8]. With the intent of bringing uniformity to the diagnosis of AD, Hanin and Rajka [11] proposed a number of diagnostic criteria based on their clinical experience. Williams and colleagues [12,13] coordinated a UK working party and rened Hanin and Rajkas criteria for AD, which were suitable for hospital and epidemiological settings. De and colleagues [14] found statistical advantage in favor of Hanin and Rajkas criteria (sensitivity 96.4%, specicity 93.75%) compared with the UK working partys diagnostic criteria (sensitivity 86.14%, specicity 95.83%) that reached a level of signicant less than .005. They noted that dermatitis in classical distribution, its chronic relapsing nature, dry skin, and infra-orbital folds had relative values and these features were helpful in discriminating cases of AD from controls. The terminology of AD is controversial, with many dierent names used in dierent countries, among them neurodermitis, constitutionalis atopica, and atopic eczema. Johansson and colleagues [15] and the European Academy of Allergology
derm.theclinics.com

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.009

606

 & WOLF LIPOZENCIC

and Clinical Immunology (EAACI) proposed a new term, atopic eczema/dermatitis syndrome (AEDS), which was divided into nonallergic and allergic types, the latter applying to immunoglobulin (Ig)E-associated allergic AEDS. They revised the nomenclature of AD and atopy and called only the IgE-associated forms of the diseases true AD [16]. It is hoped that the term eczema will replace the current term AD. Several genetic analyses have identied dierent chromosome regions with a linkage to AD features: Th2 cell cytokine genes on 5q31-33, on 1q21, 3q21, 17q25, and 20p, which are closely related to some major psoriasis loci [17,18]. Further genetic regions associated with AD features include gene polymorphisms, activator of transcription (STAT)-6; the proximal promoter of regulated on activation, normal T-cell expressed and secreted (RANTES); interleukin (IL)-4; IL-4 Ra; and transforming growth factor (TGF)-b [5,19 21]. An association of one region intron 2 polymorphisms (rs 324,011) with total serum IgE and a STAT-6 risk haplotype for elevated IgE in white adults was also proven [21]. Candidate genes found in regions (3q21, 5q31-33, and 11q13) code for various immunomodulators, including costimulatory proteins (CD80 and CD86) involved in T-cell activation (3q21); IL-3, -4, -5, and -11; granulocyte-macrophage colony-stimulating factor (GM-CSF) (5q31-33); and beta subunit of the high-anity IgE receptors (11q13) [8]. Finally, a genetic linkage was shown to contribute to immunologic abnormalities of AD pathogenesis [22].

Immunologic background Skin lesions in AD result from disturbed cellular immunity, including epidermal Langerhans cells (LCs) with IgE receptors of high and low anity and humoral factors (increased production of IgE and collaboration with T-helper [Th] subsets and LCs, with resultant high production of related cytokines). There are activated Th1 cells with increased production of interferon (IFN)-g in acute AD skin lesions binding to keratinocytes and, consequently, inammatory skin changes in the disease [22]. A relative imbalance between Th1 and Th2 subsets of CD4 T cells producing cytokines are indicative of prominent immune disorders in AD [22]. Autoreactivity to human proteins in patients with AD has been postulated as a decisive

pathogenetic factor for AD. Several investigations have looked into the question of whether the stress-inducible enzyme, manganese superoxide dismutase (MnSOD) of human and fungal origin, might act as an autoallergen in AD. SchmidGlendelmeier and colleagues [23] investigated 69 AD patients with scoring atopic dermatitis (SCORAD) 27 and demonstrated that human MnSOD may play a role as an autoallergen in a subset of these patients, including those with nonatopic eczema. The authors showed that such sensitization may be induced primarily by exposure to environmental fungal MnSOD of Malassezia sympodialis by molecular mimicry leading to cross-reactivity [23]. Genetic and environmental factors play a pivotal role in triggering AD. Research has focused on patterns of cytokines and chemokines reecting a deviated immune response in AD and on the involvement of various cells and the epidermal barrier. The ndings on T cells with regulatory features as well as on IgE-mediated autoreactivity will give insight into the defective tolerance of AD patients [24]. Imbalance of Th1 and Th2 in AD depends on polymorphism in the IL-18 gene on peripheral mononuclear cells, which reacts after stimulation with superantigens through up-regulation of IL-18 and down-regulation of IL-12 [24]. Substance P, nerve growth factor (NGF), and vasoactive intestinal polypeptides (VIP) are increased in the blood of AD patients. Cytokines and chemokines are also key factors in the pathogenesis of AD. There is a Th2 cytokine prole of IL-4, IL-5, and IL-13 in the skin in the acute phase of AD, while Th1/0 with IFN-g, IL-12, and GM-CSF prevail in the chronic phase. Moreover, eosinophilic cationic protein (ECP) and IL-16 are elevated in the acute AD phase, and IL-10 plays an important immunoregulatory role in atopic as well as in nonatopic eczema [24]. In lesional AD skin, there are two types of the high-anity receptors for IgE-bearing myeloid dendritic cells (DC), ie, LCs and inammatory dendritic epidermal cells (IDECs), each of which displays a dierent function in the pathophysiology of AD [10]. Specically, LCs play a predominant role in the initiation of the allergic immune response and conversion of prime na ve T cells into the T cell of the Th2 type with high amounts of IL-4. Furthermore, stimulation of high-anity receptors for IgE on the surface of LCs by allergens induces the release of chemotactic signals and recruitment of IDECs and T cells in vitro. Stimulation of highanity IgE receptor (FceRI) on IDECs leads to

ATOPIC DERMATITIS

607

the release of high amounts of proinammatory signals, which contribute to allergic immune response. Keratinocytes play a role in innate immunity by expressing toll-like receptors and by producing antimicrobial peptides in response to invading microbes [25]. AD keratinocytes secrete a unique prole of chemokines and cytokines. Apoptosis of keratinocytes is a crucial event in the formation of eczema (spongiosis in AD). The expression of dierent immunologic parameters has been studied in AD patients since immune dysregulation is a possible key defect in AD. Regulatory T cells (Tregs) or Th3 cells (CD25/CD4) can suppress Th1 as well as Th2 cells. Superantigens of Staphylococcus aureus cause defects in Tregs function and promote skin inammation [24]. Autoallergens (eg, Homs 1-5 and DSF 70) are atopy-related autoantigens (ARA) in the setting of AD and other atopic diseases. IgE autoreactivity appears very early (during the rst year of life) and is associated with ares in AD. Adhesion molecules may play an important role in the homing of T-cell subsets into allergen-exposed skin of atopic individuals. High expressions of adhesion molecules, especially intercellular adhesion molecules (ICAM)-1 and ICAM-3, E-selectin, and L-selectin, in skin lesions of AD patients revealed that they may play an important role in the pathogenesis of AD and may be of clinical relevance for the management of AD. It is important not to ignore various molecules expressed on LCs, such as Ecadherin, a homophilic adhesion molecule, and others [26]. AD is product of an interaction between various susceptibility genes, host and environmental factors, infectious agents, defects in skin barrier function, and immunological responses [27]. Activation of T lymphocytes, DCs, macrophages, keratinocytes, mast cells, and eosinophils are characteristic of AD skin inammatory responses [10]. Histopathology Clinically unaected skin in AD is still not normal. It has a greater irritant skin response than healthy skin with microscopically sparse perivascular T-cell inltrate, eosinophils, and macrophages [26]. There is marked inltration of CD4 activated memory T cells, of LCs (to a lesser extent), of IDECs, and of macrophages in lesional acute AD skin lesions. In chronic AD skin lesions there is chronic inammation with

increased collagen deposition in the dermis, and macrophages dominate in the dermal mononuclear cell inltrate with eosinophils and smaller numbers of T cells [10,26]. IgE and IgE receptors Serum IgE levels are elevated in about 80% of adult AD patients, who also show sensitization against airborne and food allergens and/or concomitant allergic respiratory allergy [10,28]. The subtype of AD with normal serum IgE levels has a late onset (older than 20 years) and a lack of these sensitizations. Some of these patients, however, have IgE sensitization against microbial antigens (S aureus enterotoxins and Candida albicans or Malassezia sympodialis) with low IgE serum levels and without any detectable sensitizations. These cases develop into the extrinsic variant of AD with increasing IgE serum levels and sensitizations against airborne and food allergens later in life. Expression of high-anity IgE receptor (Fc3RI) can be found in the epidermal skin lesions of patients with AD because of higher IgE-binding capacity of the DCs in their skin and blood [10]. Skin barrier disfunction AD is characterized by dry skin and increased transepidermal water loss even in nonlesional skin, and fewer ceramides in the cornied envelope of lesional and nonlesional skin are found AD patients [10,29]. Changes in the stratum corneum pH in AD skin may impair lipid metabolism in the skin. Such alterations allow penetration and susceptibility of irritants and allergens, triggering the inammatory response, cutaneous hyperreactivity, inammation, and skin damage characteristic of AD [10]. Filaggrin deciency leads to mild or severe ichthyosis vulgaris. Impaired keratinocyte dierentiation and barrier formation allow increased transepidermal water loss, and entry of allergens, antigens, and chemicals from the environment in AD [30]. Diagnosis Well-dened diagnostic criteria are important in the diagnosis of AD and diagnostic criteria developed by Hanin and Rajka [11] are widely accepted. Skin biopsies are not essential for the diagnosis but they can exclude other diagnosis in adults. In the dierential diagnosis, combination forms with components of atopic, contact, and

608

 & WOLF LIPOZENCIC

irritative eczema are important. Atopic eczema of the hands and the feet must be dierentiated from psoriasis, keratodermas in the palms and soles, and tinea. The dierential diagnosis of acute AD with intense erythema of the skin, together with exudation or blistering, for example, diers from dierential diagnoses of the chronic lichenied form. Diagnostic tests The investigation of exacerbating factors in AD involves a patient history, specic skin and blood tests, and challenge tests, depending on the degree of the disease severity and on the suspected factors involved. The atopy patch test (APT) was introduced to assess sensitization to inhalant allergens in AEDS patients. Fuiano and Incorvaia [31] recently conrmed the high value of APT in patients with mite-induced AEDS and suggested that its routine use might also improve the diagnosis of respiratory allergy to house-dust mites. Cytokine responses to allergens can be detected in cord blood mononuclear cells (CBMC), suggesting allergen priming already in utero. Nilsson and colleagues [32] found that 53 of 82 patients with AEDS had signicantly lower numbers of IFN-g-producing CBMC after stimulation with various antigens than their non-AEDS counterparts. They found a low number of IL-12-producing CBMC to be associated with IgE sensitization during early childhood and that a reduced number of IFN-g-producing CBMC promotes the development of AEDS during the rst 2 years of life [32]. The APT is primarily a tool for investigating the mechanisms of eczema in the skin. It can, however, also reveal sensitization in patients with AD and might identify a subgroup of AD patients [10]. Sensitization to inhalant allergens (eg, dust, mites, animal dander, and pollen) can be detected by skin patch test (SPT)s (if the skin is free from eczema) or by measuring specic IgE antibodies. Contact sensitization to topical medications frequently occurs in AD patients, especially in adults. The possibility of contact allergy needs to be ruled out by patch testing [10,33]. Treatment Topical Management of AD is a clinical challenge. Basic therapy of AD should comprise optimal skin care, addressing the skin barrier defect with regular use

of emollients and skin hydration, along with identication and avoidance of specic and nonspecic trigger factors [10]. Nonspecic irritants include contactants, such as clothing. Irritating factors include soap and hot water during showering or bathing. Mild syndets with an adjusted pH value (acidied to pH 5.5 to 6.0) should be used for cleansing. There are a number of therapeutic agents for use according to disease severity: basic therapy for solely dry skin, low-to-mid potency topical corticosteroids (TCs) and/or topical calcineurin inhibitors (TCIs) for mild to moderate AD, mid-high potency TCs/TCIs for moderateto-severe AD, and systemic therapy (eg, cyclosporine A or phototherapy) for recalcitrant, severe AD. A combination of dierent topical agents may be indicated. Systemic treatment options are recommended in cases of severe AD. There is severe dryness of the skin in AD because of increased transepidermal water loss caused by a dysfunction of the skin barrier [10]. The regular use of emollients is important for addressing this problem and, together with skin hydration, it represents the mainstay of the general management of AD. Emollients should be applied continuously, even if no actual inammatory skin lesions are apparent. Emollients containing polidocanol are effective in reducing pruritic symptoms. Urea is used for intensive hydration of the skin, while salicyl acid can be added to an emollient for the treatment of chronic hyperkeratotic lesions. Topical corticosteroids Topical corticosteroids have been for several decades the mainstay of AD therapy and other inammatory disorders. TCs are still important in the treatment of acute are-ups. Dierent therapeutic schemes have been established for the topical administration of TCs: intermittent use may be as eective as an initial therapy with a high-potent steroid followed by a time-dependent dose reduction or switch to a lower potent preparation. The choice of an adequate vehicle is important to achieve the optimal therapeutic eect. Treatment with TCs contributes to a reduction of skin colonization with S. aureus and may aect a further trigger factor of AD. Only mild-to-moderately potent preparations should be used on genital, facial, or intertriginous skin areas in short-term application, and only mild-to-moderately potent steroid preparations should be used in children. During acute ares, steroids should be used in combination with emollient skin care to avoid steroid overuse and steroid-related side eects [10].

ATOPIC DERMATITIS

609

Topical calcineurin inhibitors Topical immunomodulators are a relatively new class of medications for the treatment of AD. They are eective for the treatment of AD, but are also eective in a number of other steroid-responsive dermatoses. Tacrolimus (Protopic) ointment is a topical formulation of FK506, and pimecrolimus cream (Elidel) of a new oral ascomycin derivate [34]. Tacrolimus and pimecrolimus are selectively inhibitors of the activation of T cells by inhibiting phosphatase calcineurin. After T-lymphocyte activation (after interaction of costimulatory ligands on antigen-presenting cells with T-cell receptor) this increases the levels of free calcium within the cell, which binds to calmodulin and in turn activates calcineurin. Tacrolimus and pimecrolimus inhibit calcineurin, thus preventing the dephosphorylation activity of phosphatase and the production and release of inammatory cytokines and T-cell proliferation [34]. It is important in ADS treatment to use pimecrolimus and tacrolimus according guidelines and recommendations to include optimal skin care, identication of triggers, treatment of infections, and avoidance of allergens [35]. The Food and Drug Administration (FDA) raised concerns about potential malignancy risk that can be observed with increased use of these drugs [36]. Pimecrolimus and tacrolimus are steroid-free, anti-inammatory topical medications for the treatment of AD. In the United States and Europe, pimecrolimus cream (1%) and tacrolimus ointment (0.03%) are government-approved for the treatment of AD from the age of 2 years, while tacrolimus ointment 0.1% is approved only in adults. The anti-inammatory potency of 0.1% tacrolimus ointment is similar to a corticosteroid with moderate potency. Both agents proved to be eective, with a good safety prole for a treatment period of up to 2 years with pimecrolimus and up to 4 years with tacrolimus [10,37]. Current FDA guidelines recommend that topical pimecrolimus and tacrolimus are indicated for the short-term or intermittent long-term treatment of AD in patients 2 years of age or older who are unresponsive to or intolerant of other conventional therapies or in whom other therapies are inadvisable because of potential cancer risk [35]. Wet-wrap therapy A wet layer of cotton dressing in combination with antiseptics or topical corticosteroid (TCs) has been shown to be benecial in cases of exacerbated AD skin lesions [10,38].

Topical antimicrobial therapy Topical antiseptics, such as triclosan or chlorhexidine, oer the advantage of a low sensitizing potential and low resistance rate. They can be used in emollients or as a supplement in wetwrap dressing therapy. The combination of a topical antimicrobial agent to a topical steroid preparation has been shown to result in greater clinical improvement over a topical steroid alone [10]. A topical antibiotic treatment might be benecial for the treatment of mild and localized forms of this secondary infection. Topical fusidic acid has proved to be very eective against S aureus (application for periods of no longer than about 2 weeks is advisable). Other secondary infections caused by yeasts, dermatophytes, or streptococci have also been implicated as trigger factors in AD and should be treated [10]. Systemic management Systemic antibiotic treatment is indicated for widespread bacterial secondary infection (primarily S. aureus). First- or second-generation cephalosporins or semisynthetic penicillins administered for 7 to 10 days are usually eective. Clindamycin or oral fusidic acid are possible alternatives in cases of penicillin or cephalosporin allergy. Maintenance antibiotic therapy, however, should be avoided since it might result in colonization by methicillin-resistant organisms. Infection with herpes simplex virus (Kaposis varicelliform eruption) is a severe and life-threatening complication of AD and requires a systemic antiviral treatment with acyclovir or other antiviral agents (eg, valacyclovir) [10]. Systemic corticosteroids In cases of acute are-up, patients may benet from a short course of systemic therapy with corticosteroids, but long-term use in adults and any use in children should be avoided. Cyclosporin A Cyclosporin is immunosuppressive and antiinammatory drug. T-cell receptor activation causes release of intracellular calcium that in turn binds to calmodulin and activates calcineurin. The calcineurin complex then dephosphorylates the nuclear factor of activated T cells (NFATc), which migrate into the nucleus and make a complex that is a transcription factor for inammatory cytokines (eg, IL-2). Cyclosporin binds to cyclophilin (intracytoplasmic proteinsimmunophylin), which

610

 & WOLF LIPOZENCIC

blocks the dephosphorylation of NFAT, resulting in a decrease of T-helper cells (CD4) and cytotoxic (CD8) in the epidermis [39]. Cyclosporin A (CyA) inhibits calcineurin-dependent pathways, resulting in reduced levels of proinammatory cytokines, such as IL-2 and IFN-g. CyA is eective in treatment for both adult and childhood AD. Because of the possible side eects, particularly renal toxicity, the use of CyA should be limited to patients with severe refractory disease. The treatment may be administered in the form of a short- or longterm therapy with high-dose (3 to 5 mg/kg/d) or low-dose (2.5 mg/kg/d) regimens, depending on the individual patients medical condition. If the therapy has failed after 3 months of the maximum dose of CyA (5 mg/kg/day), it should be discontinued [39]. Azathioprine Azathioprine (Imuran) is a well-known systemic immunosuppressive agent from 1959 that aects purine nucleotide synthesis and is eective in severe recalcitrant AD [10]. It has a number of side eects including myelosuppression, hepatotoxicity, and susceptibility for infection. The recommended dosage of azathioprine for dermatologic indications is 1 to 3 mg/kg daily but it should be determined based on thiopurine methyltransferase levels (TPMT). Knowing the baseline TPMT activity is clinically useful in most patients who receive azathioprine and to monitoring during the therapy [39]. Benets may not be apparent until 2 to 3 months after treatment onset. Antihistamines The therapeutic value of antihistamines seems to reside principally in their sedative properties, and they are useful as a short-term adjuvant to topical treatment during relapses associated with severe pruritus. The second-generation antihistamines, cetirizine hydrochloride, loratadine, and fexofenadine hydrochloride have distinct eects because of their longer half-lives. Desloratadine has proven eectiveness in controlling pruritus. Nonsedating antihistamines seem to have only very modest value in atopic eczema. Leukotriene antagonists Leukotriene antagonists (montelukast and zarlukast) are useful for the treatment of asthma and allergic rhinitis. In AD therapy they are not fully elucidated. Zarlukast is approved in AD and asthma for adolescents and adults [5]. In

chronic AD montelukast achieved little success. Montelukast administered 5 mg daily for 4 weeks in a clinical double-blind study of moderate to severe AD in young patients (6 to 16 years) showed a signicant decrease in disclosed severity, but in another study with severe AD and dierent doses (5 mg, 10 mg, 20 mg) there was partial improvement (relief of pruritus; and erythema) in few patients. AD patients failed to show any benet from leukotriene receptor antagonist therapy [5]. Phototherapy The following therapy options may be used for AD: broad-band UVB (280 to 320 nm), narrowband UVB (311 to 313 nm), UVA (320 to 400 nm), UVA1 (340 to 400 nm), PUVA, and PUVA bath. Combinations of UVB and TCs and UVB with UVA as well as UVA1 medium- and highdose therapy are useful in AD therapy [40]. In the pediatric population, UV therapy should be restricted to children older than 12. Interferon therapy Interferons (IFN) are a family of secretory glycoproteins produced by most eukaryotic cells triggered by a variety of viral and nonviral inducers. In dermatologic disorders there are three forms of IFN (IFN-a2a, -a2b, and g) with antiviral, antiproliferative, and immunoregulatory clinical eects [34]. Evidence of reduced production of IFN-g in vitro by mononuclear cells of patients with AD, and the suppression of IL-4-mediated IgE stimulation by IFN-g has prompted evaluation of the use of IFN in AD. A randomized placebo-controlled double-blind multicenter trial that studied the eects of daily subcutaneous injection (50 mg/m2) of recombined IFN-g achieved in 45% - 57% (10-100 mg/m2) a signicant clinical response [34]. Therapy with IFN-a has been investigated in patients with AD with varied and contradictory results [34]. Bioengineered immunomodulators Most of the new approaches aim at inhibiting components of the allergic inammatory response, including cytokine modulation (eg, tumor necrosis factor [TNF] inhibitors), blockade of inammatory cell recruitment (chemokine receptor antagonists, cutaneous lymphocyte antigen inhibitors), and inhibition of T-cell activation (alefacept and efalizumab) [10]. Bioengineered immunomodulators are in a clinical trial phase for AD treatment. Agents that interfere with T-cell

ATOPIC DERMATITIS

611

activation or T-cell tracking in the skin (alefacept, efalizumab) are eective in the treatment of psoriasis. They change the immune prole from Th1 to Th2, or block cytokines. These agents are currently in clinical trials for psoriasis and psoriatic arthritis. Iniximab (Remicade) is a chimeric (human-mouse) monoclonal antibody and its target is human TNF-a. Iniximab binds to soluble forms of TNF-a and blocks the interaction of TNF-a with TNF-a receptors. Inliximab is approved for the treatment of Crohns disease and rheumatoid arthritis, pyoderma gangrenosum, and psoriasis [34]. All have no place in the therapy of AD. IgE-blocking antibody IgE-blocking antibody omalizumab (Xolair) is a recombinant human monoclonal antibody that targets specic antihuman IgE drugs (binds free serum IgE and avoids binding to Fc3RI receptors as well as Fc3RII [CD23] receptors on mast cells, basophiles, and antigen-presenting cell surfaces, which stops release of proinammatory mediators). Omalizumab is for use in adults and children older than 12 years with asthma and AD for 3 months; subcutaneous injections of 0.015 mg/kg IgE and 0.03 mg/kg each 2 weeks or 4 weeks. The role of omalizumab in dermatology and for AD is probably best directed toward patients who have high levels of IgE and in whom the IgE is an etiologic factor for their disease [41]. Conclusion Although there has been an enormous progress in our understanding of the molecular biology of AD in the past decade, it seems that we are only scraping the tip of the iceberg, particularly concerning therapy of the disease. Management currently focuses on avoidance of triggers, use of skin hydration, and reduction of skin inammation. There are no disease-modifying drugs. Summary We described atopic eczema/dermatitis from the aspects of immunologic background, genetics, skin barrier dysfunction, IgE receptors, and triggers of AD (including allergens, microorganisms, and autoantigens). We have also reviewed diagnostic procedures, treatment modalities with topical treatment (emollients, topical corticosteroids,

topical calcineurin inhibitors, wet wrap therapy, and topical antimicrobial therapy) systemic management (antimicrobials, systemic corticosteroids, cyclosporine A, azathioprine, antihistamines), and phototherapy. Finally, we have discussed primary and secondary prevention and have emphasized the role of the dierent cell receptors and their up-and down-regulation in this setting. References
J, Bobek D, Jakic Razumovic J. The [1] Lipozen cic presence of surface CD30 on T cells in atopic dermatitis. Acta Dermatovenerol Croat 2003;11:14552. L, Lipozen J, Jakic Razumovic J. Atopic [2] Lugovic cic dermatitis: immunophenotyping of inammatory cells in skin lesions. Int J Dermatol 2001;40:48994. [3] Ring J, Huss-Marp J. Atopic eczema. Karger Gazette 2004;79. [4] Schultz Larsen F, Hanin JM. Epidemiology of atopic dermatitis. Immunol Allergy Clin North Am 2002;22:124. [5] Mo hrenschlager M, Darsow U, Schnopp C, et al. Atopic eczema: whats new? J Eur Acad Dermatol Venereol 2006;20:50313. [6] Fo lster-Holst R, Steinsland K, Lange I, et al. Verlauf des eczema infantum. Hautarzt 1999;50(Suppl 1): 108. [7] Taieb A. The natural history of atopic dermatitis. J Am Acad Dermatol 2001;45:S46. [8] Turner JD, Schwartz RA. Atopic dermatitis. A clinical challenge. Acta Dermatovenerol Alp Panonica Adriat 2006;15:5968. [9] Eicheneld L, Hanin J, Luger T, et al. ICCAD II faculty. Consensus conference on pediatric atopic dermatitis. J Am Acad Dermatol 2003;49:108895. [10] Akdis CA, Akdis M, Bieber T, et al. European Academy of Allergology; Clinical Immunology/American Academy of Allergy, Asthma and Immunology/ PRACTALL Consensus Group. Diagnosis and treatment of atopic dermatitis in children and adults: European Academy of Allergology and Clinical Immunology/American Academy of Allergy, Asthma and Immunology/PRACTALL consensus report. Allergy 2006;61:96987. [11] Hanin J, Rajka G. Diagnostic features of atopic dermatitis. Acta Derm Venereol Suppl (Stockh) 1980;92:447. [12] Williams HC, Burney PG, Pembroke AC, et al. The UK working partys diagnostic criteria for atopic dermatitis. III. Independent hospital validation. Br J Dermatol 1994;131:40616. [13] Williams HC, Burney PG, Pembroke AC, et al. Validation of UK diagnostic criteria for atopic dermatitis in a population setting. Br J Dermatol 1996;135: 127. [14] De D, Kanwar AJ, Handa S. Comparative ecacy of Hanin and Rajkas criteria and the UK working

612

 & WOLF LIPOZENCIC

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

partys diagnostic criteria in diagnosis of atopic dermatitis in a hospital setting in North India. J Eur Acad Dermatol Venereol 2006;20:8539. Johansson SG, Hourihane JO, Bousquet J, et al. EAACI (the European Academy of Allergology and Clinical Immunology) nomenclature task force. A revised nomenclature for allergy. An EAACI position statement from EAACI nomenclature task force. Allergy 2001;56:81324. Johansson SG, Bieber T, Dahl R, et al. Revised nomenclature for allergy for global use: report of the nomenclature review committee of the world allergy organization, October 2003. J Allergy Clin Immunol 2004;113:8326. Cookson WO, Ubhi B, Lawrence R, et al. Genetic linkage of atopic dermatitis to psoriasis susceptibility loci. Nat Genet 2001;27:3723. Cookson WO, Moatt MF. The genetics of atopic dermatitis. Curr Opin Allergy Clin Immunol 2002; 2:3837. Tamura K, Suzuki M, Arakawa H, et al. Linkage and association studies of STAT6 gene polymorphisms and allergic diseases. Int Arch Allergy Immunol 2003;131:338. Novak N, Kruse S, Kraft S, et al. Dichotomic nature of atopic dermatitis reected by combined analysis of monocyte immunophenotyping and single nucleotide polymorphisms of the interleukin-4/interleukin-13 receptor gene: the dichotomy of extrinsic and intrinsic atopic dermatitis. J Invest Dermatol 2002;119:8705. Weidinger S, Klopp N, Wagenpfeil S, et al. Association of a STAT 6 haplotype with elevated serum IgE levels in a population based cohort of white adults. J Med Genet 2004;41:65863. Lugovic L, Lipozencic J, Jakic-Razumovic J. Prominent involvement of activated Th1-subset of T-cells and increased expression of receptor for IFNgamma on keratinocytes in atopic dermatitis acute skin lesions. Int Arch Allergy Immunol 2005;137: 12533. Schmid-Glendelmeier P, Fluckiger S, Disch R, et al. IgE-mediated and T cell-mediated autoimmunity against manganese superoxide dismutase in atopic dermatitis. J Allergy Clin Immunol 2005;115:106875. Hinz T, Staudacher A, Bieber T. Neues in der Pathophysiologie der atopischen Dermatitis. Hautarzt 2006;57:56774. Sayama K, Komatsuzawa H, Yamasaki K, et al. New mechanisms of skin innate immunity: ASK1mediated keratinocyte dierentiation regulates the expression of beta-defensins, LL37, and TLR2. Eur J Immunol 2005;35:188695. L, Lipozen J, Jakic -Razumovic J. The Lugovic cic role of adhesion molecules in atopic dermatitis. Acta Dermatovenerol Croat 2006;14:27. Novak N, Bieber T, Leung DY. Immune mechanisms leading to atopic dermatitis. J Allergy Clin Immunol 2003;112:S12839.

[28] Novak N, Bieber T. Allergic and non-allergic forms of atopic disease. J Allergy Clin Immunol 2003;112: 25262. [29] Sator PG, Schmidt JB, Honigsmann H. Comparison of epidermal hydration and skin surface lipids in healthy individuals and in patients with atopic dermatitis. J Am Acad Dermatol 2003;48: 3528. [30] Irvine AD, Mc Lean WHI. Breaking the (un)sound barrier: laggrin is a major gene for atopic dermatitis. J Invest Dermatol 2006;126:12002. [31] Fuiano N, Incorvaia C. Value of skin prick test and atopy patch test in mite-induced respiratory allergy and/or atopic eczema/dermatitis syndrome. Minerva Pediatr 2004;56:53740. [32] Nilsson C, Larsson AK, Hoglind A, et al. Low numbers of interleukin-12-producing cord blood mononuclear cells and immunoglobulin E sensitization in early childhood. Clin Exp Allergy 2004;34: 37380. [33] Echechipia S, Gomez B, Lasa E, et al. Epicutaneous test with inhalers in the study of atopic dermatitis. An Sist Sanit Navar 2003;26(Suppl 2):317. [34] Berman B, De Araujo T, Lebwohl M. Immunomodulators. In: Bolognia JL, Jorizzo JL, Rapini RP, editors. Dermatology. Edinburgh (Scotland): Mosby; 2003. p. 203353. [35] Fonacier L, Spergel J, Charlesworth EN, et al. Report of the topical calcineurin inhibitor task force of the American College of Allergy, Asthma and Immunology and the American Academy of Allergy, Asthma and Immunology. J Allergy Clin Immunol 2005;115:124953. [36] Spergel JM, Leung DYM. Safety of topical calcineurin inhibitors in atopic dermatitis: evaluation of the evidence. Curr Allergy Asthma Rep 2006;6: 2704. [37] Wahn U, Bos JD, Goodeld M. Flare reduction in eczema with lidel (children) Multicenter Investigator Study Group. Ecacy and safety of pimecrolimus cream in the long-term management of atopic dermatitis in children. Pediatrics 2002; 110(1 Pt 1):e2. [38] Foelster-Holst R, Nagel F, Zoellner P, et al. Ecacy of crisis intervention treatment with topical corticosteroid prednicarbate with and without partial wetwrap dressing in atopic dermatitis. Dermatology 2006;212:669. [39] Wolverton S, Darst M. Systemic drugs. In: Bolognia JL, Jorizzo JL, Rapini RP, editors. Dermatology. Edinburgh (Scotland): Mosby; 2003. p. 20717. [40] Scheinfeld NS, Tutrone WD, Weinberg JM, et al. Phototherapy of atopic dermatitis. Clin Dermatol 2003;21:2418. [41] Scheinfeld N. Omalizumab: a recommbinat humanized monoclonal IgE-blocking antibody. Dermatol Online J 2005;11(1):2. Available at: http://dermatology. cdlib.org/111/reviews/omalizumab/scheinfeld.html.

Dermatol Clin 25 (2007) 613623

Contact Dermatitis
Nanna Fyhrquist-Vanni, PhDa, Harri Alenius, PhDa, Antti Lauerma, MD, PhDb,*
a

Unit of Excellence for Immunotoxicology, Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, FIN-00250 Helsinki, Finland b Control of Hypersensitivity Diseases, Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, FIN-00250 Helsinki, Finland

Contact dermatitis (CD) is one of the most common skin diseases, with a lifetime prevalence of over 30% [1]. It has a chronic course with relapses occurring at every contact with the hapten, and disease management is complicated by lack of eective and reliable diagnosis. Currently, there is no cure for CD. CD represents the most common cause of occupational skin diseases in industrial countries and, thus, is of major importance in occupational medicine. By denition, CD comprises acute and chronic manifestations of irritant and allergic CD (ACD). Irritant CD mainly is a result of toxicity to epidermal cells, resulting in inammation largely mediated by innate immunity. ACD, alternatively, is an adaptive immune response toward chemicals penetrating the skin. In common with other forms of allergy, ACD progresses in two phases. During the initial sensitization phase, the host is immunized to the allergen. After re-exposure to the allergen, a more rapid and potent secondary immune response is mounted. The second, elicitation or eector phase, manifests in ACD, and the T cells are key mediators of the reaction. On activation, the T cells release cytokines, chemokines, and cytotoxins, which leads to stimulation of local blood vessels, recruitment of immune cells, such as macrophages and eosinophils, and subsequent amplication of the response. T-cell activation is tightly regulated, and these processes,

which include many receptors, are potential targets for manipulation by drugs [2]. Currently, the mainstay of CD treatment is topical corticosteroids, which act via glucocorticoid receptors. Topical steroids have a large array of side eects, including skin atrophy. Newer antiinammatory treatments (ie, topical immunomodulators [TIMs], tacrolimus and pimecrolimus) seem to have limited potential in CD, especially in irritant CD, where they are not eective. Therefore, in this review, the emphasis is on potential new receptor targets in CD. As receptors represent potential for soluble ligand-like interventions, they are attractive candidates for therapy in this often chronic disease. This overview of CD pathophysiology, especially that of ACD, considers step by step the future possibilities of interventions in these often chronic and disabling diseases. Irritation of the skin Irritant CD can start from many causes. These include, for example, repeated washing of the hands and direct contact with irritants. Irritant dermatitis is a major cause of occupational hand eczema. The barrier function of the skin is of obvious importance: irritant CD usually starts with removal of lipids from stratum corneum, which leads to barrier disturbance and increased transepidermal water loss, followed by dryness of the skin. These disturbances cause direct activation of the immune system, especially innate mechanisms. All contact allergens also are irritants. The typical contact allergen usually is a small and
derm.theclinics.com

This work was supported by Grant No. 213072 and 214479 from the Academy of Finland. * Corresponding author. E-mail address: antti.lauerma@ttl. (A. Lauerma).

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.002

614

FYHRQUIST-VANNI

et al

highly reactive molecule, which readily penetrates the upper layers of the skin. Contact allergens also impair skin barrier function in terms of irritation, resulting in activation of innate immunity.

Initiation of contact dermatitisdcytokines and their receptors The inammatory response of irritant and ACD shares a similar clinical picture. Although the processes mediating these two diseases are dierent, they have several mechanisms in common. During the initiation of irritant CD, and the induction and elicitation of ACD, there is a similar pattern of cytokine release, possibly caused by danger signals induced by irritants and allergens. Hapten or irritant exposure causes epidermal cells to secrete interleukin (IL)-1b, IL-1a [3], tumor necrosis factor alpha (TNF-a), C-X-C chemokine ligand CXCL10 (also known as IFN-ginducible protein-10 [IP-10]), CXCL2 (macrophage inammatory protein-2 [MIP-2]), and interferon-g (IFN-g) [4]. The primary cellular sources of these cytokines are the keratinocytes (TNF-a and IL-1a), dendritic cells (DCs) (IL-1b), natural killer (NK) cells, and inltrating lymphocytes (IFN-g) [4]. Exactly which receptors and pathways of signal transduction mediate this early wave of cytokine release is unknown. One clue comes from recent ndings that the NOD-like receptor (NLR), NALP3, which is part of the caspase-1 activating inammasome complex and one of the key drivers of IL-1b, is essential to the development of contact hypersensitivity (CHS) in mice. In this study, the lack of NALP3 and its adaptor molecule, apoptosisassociated speck-like protein containing a caspase recruitment domain, reduced CHS in gene-manipulated mice [5]. NLRs, such as NALP3, Toll-like receptors (TLRs), and retinoic acid-inducible geneIlike receptors [6], collectively designated pattern recognition receptors, likely are the rst-line receivers of danger signals during the early immune response. Recent ndings indicate that the NLRs, NALP3 and NALP1, are crucial for detecting infection and tissue injury [6]. Langerhans cells (LCs) play a central role during the initiation of CHS [7]. When skin has been exposed to haptens, LCs take up and process them into haptenated peptides and migrate into secondary lymph nodes. In the lymph nodes, LCs present the haptens to na ve, antigen-specic T cells and, in addition to the antigen-specic signal, provide the T-cells with costimulatory and polarizing signals.

The activated T-cells expand clonally and dierentiate into eector cells, and the T-cells that express cutaneous lymphocyte-associated antigen (CLA) have the potential to migrate to skin. Activated T-cells release several cytokines and chemokines, which lead to leukocyte inltration and inammation. The triggering of LC migration requires several signals that induce maturation and promote their migratory capacity. Several cytokines most probably are involved, and studies using neutralization of various cytokines and cytokine-decient animals have provided some insight. IL-1b production plays a central role in the sensitization phase of human ACD [3,8,9], and mice treated with IL-1bneutralizing antibodies or IL-1bdecient mice have impaired CHS [8,9]. Also, TNF-a knockout mice have impaired CHS responses. The impairment might be related to impaired LC maturation and migration and direct impairment of proinammatory cytokines. This gives a role for their receptors as possible targets for therapy. Innate immune cells, such as NK cells, DCs, and macrophages, have major inuence on the skewing of the adaptive immune response toward a T helper type 1 (Th1) or T helper type 2 (Th2) response. Activated pattern recognition receptors induce DCs and macrophages to produce IL-12 and IL-18. Acting in concert with IFN-g, these cytokines are critical for the development of polarized Th1 responses [10]. Mice decient in IL-18, which is a proinammatory cytokine, have impaired CHS responses. In general, ACD is considered a Th1-mediated response. Therefore, it does not come as a surprise that mice lacking IFN-g do not have CHS responses [11]. In addition to Th1 cytokines, however, Th2 cytokine RNA is increased in experimental CHS in mice (Nanna Fyhrquist-Vanni, PhD, and colleagues, personal communication, 2006), and Th2 cells have been isolated from human ACD lesions [12]. Whether or not Th2-type cytokines mediate CHS or only play a down-regulatory role remains to be unraveled. A proinammatory role for Th2 cytokines is advocated by the fact that mice lacking IL-4 (ie, have impaired Th2 immunity) do have impaired CHS responses [13]. Likewise, STAT6-decient mice display decreased CHS [14]. In contrast, mice lacking IL-10 have an enhanced CHS response [15]. IL-10 at rst glance seems a good candidate for the treatment of CHS, as it is immunosuppressive in mice. When

CONTACT DERMATITIS

615

human volunteers were given intravenous infusions of IL-10, however, their IFN-ginduced proinammatory cytokines and chemokines increased. This shows that IL-10 has signicant overall immunostimulatory properties in humans in vivo [16]. IL-17 is a cytokine that is expressed by a new subtype of T cells, the Th17 cells. It also is important in CHS responses, as shown by impairment of CHS in mice lacking the IL-17 gene [17]. IL-23, IL-6, and transforming growth factor-b (TGF-b) are important drivers of Th17 polarization in vitro [18]. Unexpected results also can be seen in knockout animal studies. IL-12 is a powerful Th1polarizing cytokine, but mice lacking it still have a normal CHS [19]. Alternatively, mice lacking IL-3 display impaired CHS [20]. Leukoyte recruitment in contact dermatitisdchemokines and their receptors One to two days after the onset of the inammatory response in ACD, the dermis and partly the epidermis contain a leukocyte inltrate. These inltrates cause the clinical signs of ACD, including heat, edema, itching, vesicles, and bulla in intense inammation. For the inltrates to form, they need leukocytes from the blood circulation. Cell tracking into the skin is mediated by adhesion molecules, chemokines, and matrix metalloproteinases. All of these may be interesting targets when designing new strategies of treating CD. Leukocyte trac represents a key element in the regulation of all immune responses. The complex migratory behavior of DCs, T cells, and B cells is regulated by a exible code provided by the large family of chemokines and their receptors [21]. Chemokines signal through G-protein coupled receptors, which can be blocked selectively by small-molecule antagonists. This provides an opportunity for the development of therapeutic compounds that suppress the recruitment of a particular kind of leukocyte [22]. Leukocyte extravasation is regulated by selectins, chemokines, and integrins. The interaction between selectins and their carbohydrate ligands allows rolling of leukocytes on the vascular endothelium, leading to exposure of chemokine receptors to their ligands displayed on the surface of endothelial cells. The triggering of chemokine receptors results in the rapid activation of integrins, leading to rm adhesion and transmigration [21]. Monocytes and immature DC express receptors for inammatory chemokines, such as C-X-C

chemokine receptor 1 (CXCR1), C-C chemokine receptor 1 (CCR1), CCR2, and CCR5. CXCR1 and CCR2 may be important particularly because they mediate arrest of rolling monocytes under ow condition. As DCs mature, there is a complete loss in responsiveness to inammatory chemokines resulting from loss of corresponding receptors on the cell surface. At the same time, receptors for lymphoid chemokines, such as CXCR4, CCR4, and CCR7, are upregulated. CCR7 upregulation results in responsiveness to C-C chemokine ligand 21 (CCL21/secondary lymphoid tissue chemokine [SLC]) and CCL19 (EBV1 ligand chemokine [ELC]), which are produced by lymphatic endothelial cells and interdigitating DC, respectively. CCR7 has a prominent role in driving DC migration to the lymph nodes [21,23,24]. DC migration is central to CHS initiation; mice that are decient in CCR7 [25] and mice whose interaction between the CCR7 receptor and its ligands is impaired pharmacologically [26,27] have their CHS responses suppressed. DCs also are an abundant source of chemokines. After stimulation with lipopolysaccharide, DCs produce an initial burst of inammatory chemokines, such as CCL3 (macrophage inammatory protein-1a [MIP-1a]), CCL4 (macrophage inammatory protein-1b [MIP-1b]), and CXCL8 (IL-8), followed by CCL5 (regulated on activation normal T-cell expressed and secreted [RANTES]) and CCL2 (monocyte chemotactic protein-1 [MCP-1]). At later time points, DCs produce mainly lymphoid chemokines, such as CCL19, CCL17 (thymus- and activation-regulated chemokine [TARC]), CCL22 (macrophage-derived chemokine [MDC]), and CCL18 (pulmonary and activation-regulated chemokine [PARC]). CCL22, which is upregulated on mouse LCs once they reach the lymph node, is attractive for recently activated T cells [28]. CCL22 and CCL17 bind to the chemokine receptor, CCR4, which is expressed on a subset of memory T cells but not on naive T cells. CCR4 is rapidly upregulated after T-cell activation [26]. CCR5 is expressed on the surface of Th1 cells, monocytes, macrophages, and, to a lower extent, on Th2 cells. The relative importance of CCR5 in Th1 reactions is not clear. Mice decient in CCR5 have enhanced CHS responses [29], suggesting a downmodulating role for CCR5 in CHS responses. CCR6, which is involved in the recruitment of CD4 IL-10 producing T regulatory cells (Tregs), also seems to have a suppressive eect: CHS is enhanced and persistent in CCR6-decient

616

FYHRQUIST-VANNI

et al

mice [30]. Thus, these two receptors are candidates for future anti-inammatory therapy. Th2 cytokines, IL-4 and IL-13, stimulate the production of CCL11 (eotaxin) and CCL22, which is antagonized by Th1 cytokine, IFN-g. IFN-g in turn induces chemokines, CXCL10, CXCL9 (monokine induced by IFN-g [MIG]), and CCL5, which are antagonized by IL-4 [21]. CXCL10, which is a ligand for CXCR3 and is expressed by activated T cells, has a role in CHS; knockout mice without CXCL10 have an impaired CHS response. Keratinocytes produce CCL27 (cutaneous T cell-attracting chemokine [CTACK]), which is the ligand of CCR10. CCR10 is expressed on CLA eector or memory T cells. Neutralization of the interaction of skin-specic chemokine CCL27 and its receptor, CCR10, on CLA-positive skin-specic T cells results in impairment of CHS [31].

Capture of allergens for antigen presentation ACD is a specic immune response to haptens bound to cutaneous carrier proteins. A chemical allergen that gains access to the viable epidermis interacts either directly with cutaneous DC, or more likely, conjugated to proteins that the DCs internalize [32]. At present, the nature of carrier proteins is not known. Therefore, interventions to the formation of hapten-carrier complexes are not feasible. Alternatively, steps involving the capture of antigens are well characterized and may provide possibilities for interventions. DCs continuously sample their environment by pinocytosis (eg, uptake of uid-phase components). In addition, they express several receptors for receptor-mediated endocytosis, macropinocytosis, or phagocytosis of exogenous antigens. Such receptors are C-type lectin receptors, immunoglobulin receptors (FcgRI, FcgRIII, and FcgRII), and complement receptors (C3 and C4) [33]. These receptors facilitate eective presentation of antigen and activation of the immune system. Some receptors cause induction of tolerance, however, either by blocking maturation of DCs or via induction of signals that render DC tolerogenic. Tolerogenic DCs in turn induce Tregs that actively suppress the immune response by various means [34]. The C-type lectins bind sugar residues present on pathogens and proteins. They possess dierent numbers of carbohydrate recognition domains, ranging from one single domain to up to 10

dierent carbohydrate recognition domains, allowing the binding of specic sugars [33]. DEC205 (CD205) is a potent antigen receptor that is expressed only by DCs. DEC-205 is internalized into coated pits and transports ligands to late endosomal, major histocompatibility complex (MHC) class II compartments. Here, peptides are generated and loaded onto MHC class II molecules, after which DEC-205 recycles to the surface of DCs for new rounds of antigen uptake [34]. DEC-205 is the most eective antigen receptor on DC that has been shown to mediate antigen presentation without further activation of DC. Targeting of DC in the steady state by antigen linked to DEC-205 antibodies in mice resulted in the disappearance of antigen-specic T-cells [35] and induction of Tregs [36]. Even though DC immunotherapy has been introduced in clinics, this approach is far away from clinical applications in disease, such as allergy, because of inadequate patient safety. The most successful clinical trials so far have been done with DCs that are matured and activated appropriately [37]. Immunoglobulin receptors (Fc receptors [FcRs]) mediate endocytosis of antigen-antibody complexes. Monocyte-derived human DCs express mainly FcgRII (CD32) and FcaR (CD89), LCs express FcgRI and Fc3RI (CD23), and blood DCs express FcgRII and FcgRI [33]. FcRs are not recycled as are lectins but degraded together with their ligands in the endosomal compartments [34]. The engagement of FcRs result either in immune activation or inhibition. The inhibitory FcgIIB receptor harbors a cytoplasmic immunoreceptor tyrosine-based inhibition motif, which, via several processes, inhibits DC maturation and the activation of T cells. Coligation of Fc3RI and FcgRIIb can diminish eector cell activation in vitro [38] and the extent of the allergic response in vivo [39]. Recently, Zhu and colleagues [40] were able to suppress IgE- and allergen-induced human mast cell and basophil activation with a chimeric protein containing cat allergen and enough of the Fcg portion of IgG to engage FcgRIIb. This approach may provide a more eective and safer alternative to direct immunization of allergy patients with allergens as a means of immunotherapy. DCs also express receptors for heat shock proteins (hsp), gp96 and hsp70, which mediate the internalization of hsp-peptide complexes, and DCs may phagocytose apoptotic cells through receptors, CD36 and integrin avb5 [41,42]. The uptake of apoptotic material does not lead to DC activation, and antigens from this material

CONTACT DERMATITIS

617

are presented in a tolerogenic fashion [43]. Liu and colleagues [44] showed that when chicken ovalbumin was encapsulated into apoptotic cells, it would, instead of inducing an immune response, lead to the deletion of antigen-specic T cells and induction of tolerance.

Maturation of dendritic cells for antigen presentation Immature DCs are ecient at internalizing antigen but inecient at presenting antigen. Soon after encountering a danger or other signal, which induces maturation, however, antigen takeup, antigen transport, and processing are modied. This process may provide another step where induction and elicitation of CHS could be prevented or suppressed in terms of therapy. During maturation, the cell surface expression of most antigen surface receptors on the DC is decreased, and macropinocytosis and phagocytosis are downmodulated. At the same time, MHC molecules are delivered to the surface more rapidly, and surface expression of T-cell costimulatory molecules increases [33]. Maturation of DCs also induces modications in the expression of chemokine receptors and adhesion molecules and changes in the cytoskeleton organization. These all contribute to the migration of DCs to secondary lymph organs, which is where DCs present antigens and initiate adaptive immunity. In addition to presenting antigens, DCs modify the outcome of the immune response by secreting cytokines. At least ve types of receptors induce DC maturation: TLRs, cytokine receptors, TNFreceptor family molecules, FcRs, and sensors of cell death. Pathogenic compounds, such as bacterial cell walls, unmethylated CpG, and double-stranded RNA, induce DC maturation via TLR activation. Inammatory mediators, such as TNF-a, IL-1b, and prostaglandin E2, induce DC maturation [10]. Furthermore, CD40 ligand (CD40-L, also known as CD154) [45], Fas ligand (FasL) [46], OX40 [47] on T cells, and engagement of most FcRs [48,49] trigger DC maturation. Finally, necrotic cells that release hsps gp96, hsp90 and hsc70 induce DC maturation [50].

Presentation of antigensdrole of receptors Presentation of antigens to T-cell receptors represents the next step for possible interventions.

DCs process engulfed antigens into peptide fragments and load them onto MHC class I or II complex molecules. MHC class I is recognized by the CD8 coreceptor on cytotoxic T cells, and MHC class II by the CD4 coreceptor on helper T cells [33]. A strict compartmentalization of the biogenesis of MHC class I and II molecules accounts for dierential loading of antigen onto MHC molecules. MHC class I molecules collect peptides derived from proteins synthesized in the cytosol, whereas MHC class II molecules collect peptides in the vesicular system. According to this model, CD8 cytotoxic T cells, which bind MHC class I molecules, would respond to endogenous antigen only and CD4 helper T cells would respond to endogenous and exogenous antigen. Cross presentation of internalized antigen by MHC class I molecules, however, does occur. The major route of internalization, which results in ecient cross presentation, probably is phagocytosis [33]. In addition to MHC class I and II, there is a third class of MHC molecules involved in antigen presentation to T cells: the CD1 proteins. The CD1d molecule complexed with a synthetic lipid antigen, a-galactosylceramide, activates NK killer T (NKT) cells in humans and mice [51]. The natural ligands of CD1d probably are lipid antigens. Once activated, NKT cells produce a strong and rapid cytokine response [51]. These cells have an essential role in the initiation processes of CHS: NKT celldecient mice (Ja18/, CD1d/) fail to elicit CHS responses [52]. The initiation of the adaptive immune response takes place in the draining lymph nodes. Fully mature DCs produce chemokines, such as CCL18 (DC chemokine 1 [DC-CK1]) and CCL19 (MIP3b), which attract naive T cells [53]. During the initial encounter between a DC and T cell, adhesion molecules DC-SIGN and CD54 (ICAM-1) interact with ICAM-3 and CD11a/CD18 to retain the T cell and approximate the two cell types [54]. The antigen-recognition molecule of the T cell is the T-cell receptor (TCR). This immunoglobulin-related protein recognizes short peptide fragments, which are bound to MHC molecules on the surface of antigen-presenting cells (APCs). The TCR consists of two dierent polypeptide chains, the a and b chains. A minority of T cells bears an alternative receptor made up of a dierent pair of polypeptide chains, called g and d. In general, gd T cells have a regulatory inuence on immune responses and ab on T cell development. It has been demonstrated, however, that they are

618

FYHRQUIST-VANNI

et al

involved in CHS reactions, playing a crucial role by assisting the eector ad T cells [55].

Costimulation receptors and ligands Presentation of hapten-carrier complexes to T-cell receptors is not sucient for mounting an immune response in ACD. T-cell activation requires two independent signals [56]. In addition to TCR ligation, T cells need a so-called costimulatory signal, which stimulates T cells in conjunction with the antigen-specic stimuli, through engagement of particular costimulatory receptors on the surface of T cells. In the absence of costimulatory signals, T cells fail to respond. Thus, the interaction of costimulatory receptors and ligands represents the next potential target for manipulation by drugs, as tolerance might be achieved by blocking costimulation. The best-characterized pathway of T-cell costimulation involves the CD28 receptor. CD28 binds to costimulatory molecules B7-1 (CD80) and B7-2 (CD86). The structurally related glycoproteins, B7-1 and B7-2, are expressed on the surface of APCs, and their expression is enhanced in the presence of immune stimulating agents, such as microbes and cytokines. The CD28 receptor is expressed constitutively on all T cells in mice and on 95% of CD4 T cells and 50% of CD8 T -cells in humans [2]. Costimulatory signals via the CD28 receptor promote expansion of antigen-stimulated T cells and their dierentiation into eector and memory cells. CD28 signals enhance the production of IL-2 and other cytokines and promote cell-survival, energy metabolism, and cell-cycle progression [2]. CD28 signals promote expansion of antigenstimulated T cells and their dierentiation into eector and memory cells. CD28 signals and subsequent production of IL-2 also are important for the development and survival of Tregs in the thymus and in the periphery. Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is an additional receptor for B7 molecules. The CD28-related protein is expressed constitutively on the surface of Tregs and induced on activated T cells. Contrary to CD28, CTLA-4 delivers an inhibitory signal to the activated T cell and thus is involved in the induction and maintenance of T-cell tolerance. This was conrmed by the phenotype of mice decient in CTLA-4, which is characterized by massive T-cell activation and proliferation and T-cellmediated tissue damage.

CTLAIg is a potent inhibitor of CD28-mediated T-cell costimulation and is the rst of its kind brought to the market. It was recently approved as treatment of rheumatoid arthritis [57]. A third CD28-related protein, inducible costimulator (ICOS), is induced on activated T cells, and binds its ligand, LICOS, which is distinct from B7-1 and B7-2. LICOS is produced on activated DCs, monocytes, and B cells. Its contribution to immune responses has yet to be dened. Activated T cells express several proteins that contribute to the sustaining and modifying of the costimulatory signal. One such protein is CD40-L, which binds to CD40 on APCs. Signals to CD40 on DCs increase the expression of B7 (CD80 and CD86) molecules and the production of cytokines (eg, IL-12), which further intensies the interaction between T cells and APCs [58]. Other members of the TNF family, such as OX40 (CD134), 4-1BBL, CD70, and the cytokine LIGHT (lymphotoxin homolog, inducible and competes with HSV glycoprotein D for HveA and is expressed on T-lymphocytes), also provide costimulation on TCR stimulation, leading to full and eective T-cell responses [59]. Recent ndings indicate that the CD28-B7-2 pathway of costimulation is required for priming of T cells for CHS responses [60]. In contrast, CD40-CD154 interaction is dispensable [61]. CD40 engagement, however, enhances LC priming of IFN-gproducing T cells, using a mechanism distinct from increased IL-12 production [62].

T-cell contribution to the contact hypersensitivity response: ab T cells, gd T cells, and natural killer T cells The next step for therapeutic interventions in CHS is the action of antigen-specic T cells. As there are several subtypes of T cells, an interesting approach in treatment of CHS could be rebalancing the amounts and activities of these subtypes. CHS long was considered mediated by ab T cells alone, until several experiments demonstrated that NKT cells, B1 cells, and gd T cells also are essential to the CHS response [55,63,64]. The NKT cells are activated by glycolipid ligands that are released during immunization and rapidly begin to secrete IL-4. NKT cellssecreted IL-4 coactivates CS-initiating B1 cells, which produce hapten-specic IgM antibodies that enter the circulation. Immunization also activates gd T cells, which are mobilized from the spleen. At secondary

CONTACT DERMATITIS

619

hapten challenge, local B1 cellderived IgM complexes with antigen to activate complement C5 and the subsequent generation of C5a. C5a activates C5a receptors on mast cells and platelets to induce release of vasoactive serotonin, TNF-a, and possibly leukotriene and prostaglandin, which act to recruit eector ab T cells. The gd T-cell subset provides help to the eector ab T cells to optimize the response [65]. Finally, the eector ab T cells attract bone marrowderived leukocytes for elicitation of the 24- to 48-hour CHS response [66]. The relative importance of these subsets of leukocytes is underscored by the nding that mice decient in NKT cells [52], B1 cells [67], complement C5 [68], C5 receptors [69], and mast cells [70] have impaired CHS. CD8 cytotoxic and CD4 helper T cells in contact hypersensitivity The respective roles of CD4 helper T cells and CD8 cytotoxic T cells in the development of the CHS reactions is a matter of debate in the literature. Several experimental studies by independent investigators from the past 10 years have implied that CHS responses to strong haptens are mediated by CD8 type-1 T cells, whereas CD4 T cells would have a regulatory role. In some instances, however, especially those where there is a decient CD8 T-cell pool, CD4 T cells can be eector cells of CHS [71]. Moreover, CHS to DNFB and oxazolone was impaired in CD4-decient mice (CD4/) [7274], suggesting that CD4 T cells either are important for the full development of CHS or behave as eector cells of CHS. In recent experiments, the authors have demonstrated that CD4 T cells alone can elicit full CHS responses in RAG decient mice by adoptive transfer, using oxazolone as antigen. By contrast, oxazolone-specic CD8 T cells were not able to adoptively transfer CHS responses in the RAG/ mice (Nanna Fyhrquist-Vanni and colleagues, unpublished results, 2006). These results support earlier studies, which suggest CD4 help is critical for secondary expansion of the CD8 T cell pool [75,76]. Yet, the respective roles of the two subtypes of T cells CHS responses need to be claried further. Downregulation of contact dermatitis by T regulatory cells and apoptosis The course of acute CD is self-limiting. If an allergen or irritant encounter skin repetitively,

however, chronic dermatitis may ensue. In cases of irritant dermatitis, however, the cessation of irritation usually results in healing of the dermatitis within approximately 2 months. In cases of ACD, some allergens may cause longer-lasting dermatitis. Examples of these are chromium and rubber allergens. As physiologic downregulation of CD is a process leading to the same result, healthy skin, which is what treatments aim for, these processes may provide possibilities for interventions in CD. Tregs and their functional role in health and disease have been given considerable attention during the past 10 years. Because of the absence of a specic cell surface marker, Tregs remained obscure for several decades. Soon after, they were proved essential to self-tolerance [77] and their role in autoimmune disease [78] became apparent. Several studies imply that Tregs also may play a role in asthma and allergy [12,7981], mediating a protective eect through inhibiting the activity of T cells [82], DCs [12], B-cells, basophils, and eosinophils [83]. The mechanisms by which Tregs exert their inhibitory eects are unknown; however, some Treg subtypes seem to act through cell contact and others through the release of anti-inammatory cytokines, such as IL-10 and TGF-b [82]. Cell surface receptors, such as CTLA-4, PD-1, membrane-bound TGF-b, and IL-10 and their respective receptors may be involved in Treg-mediated suppression [83]. When the inammation has terminated, the activated T cells undergo programmed cell death or apoptosis. Apoptosis can be induced by several mechanisms. The interaction of the receptor molecule, Fas, on T cells with its ligand, FasL, is well documented. FasL is a member of the TNF family and Fas is a member of the TNF receptor family. Both of these are induced normally during the adaptive immune response. A certain subtype of DCs express FasL, which enables them to kill Fas-bearing, activated T cells, opening up another possibility for therapy. Paradoxically, these molecules that are involved in the clearance of the mediators of inammation are involved equally in misoriented killing of cells during disease. Cytotoxic molecules, such as perforin, granzyme B, and FasL, also are crucial for the expression of CHS. Mice decient in perforin and FasL fail to mount CHS reactions to haptens [84]. These molecules are upregulated during T-cell activation and reect the activities of cytotoxic killer T cells that mediate apoptosis of keratinocytes in acute ACD lesions [85,86].

620

FYHRQUIST-VANNI

et al

Clinical implications and therapeutic relevance of receptor-ligand interventions The most abundant data on the eect of a receptor-ligand pair on inammatory responses comes from studies using receptor- or ligandgenedecient mice. It has to be taken into account, however, that the mice are born without the particular gene and the developmental dierences in these mice aect the result. A more realistic model for possible drug development is silencing of genes by RNA interference, the use of specic antibodies, or small-molecule peptides aimed at interfering with receptor function. Recent experience from an antibody-based drug therapy directed against a single receptor, this time CD28, shows that such therapies may, despite thorough preclinical and animal testing, give surprising and serious unwanted results. Six volunteers were given TGN141, modied antibody against CD28 at 1/500 of the dose found nontoxic in animals. All volunteers had a cytokine release syndrome within minutes from starting the trial, including symptoms of angioedema and systemic inammation [87]. It is not yet known whether or not the experiment has hurt the volunteers health permanently. Therefore, future drug trials need to be done with more care when single receptors are targeted.

use, however, is limited, as cyclosporine has a wide array of serious side eects [89,90]. A new treatment of chronic CD is 9-cisretinoid acid (alitretinoin). It binds to retinoid X receptors and to all retinoid acid receptors. Its mechanism of action is not yet well understood. It was shown eective in chronic hand dermatitis in a double-blind, placebo-control trial [91]. Other receptor mediated treatments used in CD include antihistamines, which bind to the histamine 1-receptor. These drugs do not, however, have clinical ecacy in CD and are not shown to directly reduce the itch that is involved in the symptoms [92].

References
[1] Kadyk DL, McCarter K, Achen F, et al. Quality of life in patients with allergic contact dermatitis. J Am Acad Dermatol 2003;49(6):103748. [2] Sharpe AH, Abbas AK. T-cell costimulationdbiology, therapeutic potential, and challenges. N Engl J Med 2006;355(10):9735. [3] Nakae S, Naruse-Nakajima C, Sudo K, et al. IL-1 alpha, but not IL-1 beta, is required for contactallergen-specic T cell activation during the sensitization phase in contact hypersensitivity. Int Immunol 2001;13(12):14718. [4] Enk AH, Katz SI. Early molecular events in the induction phase of contact sensitivity. Proc Natl Acad Sci USA 1992;89(4):1398402. [5] Sutterwala FS, Ogura Y, Szczepanik M, et al. Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity 2006;24(3):31727. [6] Creagh EM, ONeill LA. TLRs, NLRs and RLRs: a trinity of pathogen sensors that co-operate in innate immunity. Trends Immunol 2006;27(8):3527. [7] Reis e Sousa C. Dendritic cells as sensors of infection. Immunity 2001;14(5):4958. [8] Enk AH, Angeloni VL, Udey MC, et al. An essential role for Langerhans cell-derived IL-1 beta in the initiation of primary immune responses in skin. J Immunol 1993;150(9):3698704. [9] Shornick LP, De Togni P, Mariathasan S, et al. Mice decient in IL-1beta manifest impaired contact hypersensitivity to trinitrochlorobenzone. J Exp Med 1996;183(4):142736. [10] Banchereau J, Briere F, Caux C, et al. Immunobiology of dendritic cells. Annu Rev Immunol 2000;18: 767811. [11] Saulnier M, Huang S, Aguet M, et al. Role of interferon-gamma in contact hypersensitivity assessed in interferon-gamma receptor-decient mice. Toxicology 1995;102(3):30112. [12] Cavani A, Nasorri F, Prezzi C, et al. Human CD4 T lymphocytes with remarkable regulatory functions

Current receptor-specic treatments for contact dermatitis The current repertoire of treatments for CD includes glucocorticoids and TIMs. Glucocorticoids exert their eect via intracellular glucocorticoid receptors. Glucocorticoids have wide eects on inammatory processes. They are ecient treatment, but their use is limited because of the possibility of skin atrophy and tachyphylaxis, which results in lack of eect. Also, their use often is limited, as patients often are overestimating these risks on their own. Oral glucocorticoids provide the most eective treatment choice in CD, but their use is limited because of side eects [88]. TIMs, tacrolimus and pimecrolimus, bind to FK-binding receptor, which activates calcineurin phosphatase, resulting in suppression of T-cell activation. TIMs do not cause skin atrophy and the likelihood of tachyphylaxis is smaller in them, making long-term therapy a possibility. TIMs do not, however, have treatment eect in irritant dermatitis. Oral cyclosporine binds to cyclophilin, which also activates calcineurin phosphatase. Its

CONTACT DERMATITIS

621

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

on dendritic cells and nickel-specic Th1 immune responses. J Invest Dermatol 2000;114(2):295302. Traidl C, Jugert F, Krieg T, et al. Inhibition of allergic contact dermatitis to DNCB but not to oxazolone in interleukin-4-decient mice. J Invest Dermatol 1999;112(4):47682. Yokozeki H, Ghoreishi M, Takagawa S, et al. Signal transducer and activator of transcription 6 is essential in the induction of contact hypersensitivity. J Exp Med 2000;191(6):9951004. Berg DJ, Leach MW, Kuhn R, et al. Interleukin 10 but not interleukin 4 is a natural suppressant of cutaneous inammatory responses. J Exp Med 1995;182(1):99108. Mocellin S, Marincola F, Rossi CR, et al. The multifaceted relationship between IL-10 and adaptive immunity: putting together the pieces of a puzzle. Cytokine Growth Factor Rev 2004;15(1):6176. Nakae S, Komiyama Y, Nambu A, et al. Antigen-specic T cell sensitization is impaired in IL-17-decient mice, causing suppression of allergic cellular and humoral responses. Immunity 2002;17(3):37587. Veldhoen M, Hocking RJ, Atkins CJ, et al. TGFbeta in the context of an inammatory cytokine milieu supports de novo dierentiation of IL-17-producing T cells. Immunity 2006;24(2):17989. Gorbachev AV, DiIulio NA, Fairchild RL. IL-12 augments CD8 T cell development for contact hypersensitivity responses and circumvents antiCD154 antibody-mediated inhibition. J Immunol 2001;167(1):15662. Mach N, Lantz CS, Galli SJ, et al. Involvement of interleukin-3 in delayed-type hypersensitivity. Blood 1998;91(3):77883. Sallusto F, Mackay CR, Lanzavecchia A. The role of chemokine receptors in primary, eector, and memory immune responses. Annu Rev Immunol 2000;18:593620. Pease JE, Williams TJ. Chemokines and their receptors in allergic disease. J Allergy Clin Immunol 2006; 118(2):30518 [quiz: 31920]. Sallusto F, Schaerli P, Loetscher P, et al. Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation. Eur J Immunol 1998;28(9):27609. Sozzani S, Allavena P, DAmico G, et al. Dierential regulation of chemokine receptors during dendritic cell maturation: a model for their tracking properties. J Immunol 1998;161(3):10836. Forster R, Schubel A, Breitfeld D, et al. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 1999;99(1):2333. DAmbrosio D, Iellem A, Bonecchi R, et al. Selective up-regulation of chemokine receptors CCR4 and CCR8 upon activation of polarized human type 2 Th cells. J Immunol 1998;161(10):51115. Saeki H, Moore AM, Brown MJ, et al. Cutting edge: secondary lymphoid-tissue chemokine (SLC) and

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes. J Immunol 1999;162(5):24725. Tang HL, Cyster JG. Chemokine up-regulation and activated T cell attraction by maturing dendritic cells. Science 1999;284(5415):81922. Zhou Y, Kurihara T, Ryseck RP, et al. Impaired macrophage function and enhanced T cell-dependent immune response in mice lacking CCR5, the mouse homologue of the major HIV-1 coreceptor. J Immunol 1998;160(8):401825. Varona R, Villares R, Carramolino L, et al. CCR6decient mice have impaired leukocyte homeostasis and altered contact hypersensitivity and delayedtype hypersensitivity responses. J Clin Invest 2001; 107(6):R3745. Homey B, Alenius H, Muller A, et al. CCL27CCR10 interactions regulate T cell-mediated skin inammation. Nat Med 2002;8(2):15765. Kimbe I, Basketter DA, Gerberick GF, et al. Allergic contact dermatitis. Int Immunopharmacol 2002; 2(23):20111. Guermonprez P, Valladeau J, Zitvogel L, et al. Antigen presentation and T cell stimulation by dendritic cells. Annu Rev Immunol 2002;20:62167. Mahnke K, Knop J, Enk AH. Induction of tolerogenic DCs: you are what you eat. Trends Immunol 2003;24(12):64651. Bonifaz L, Bonnyay D, Mahnke K, et al. Ecient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8 T cell tolerance. J Exp Med 2002;196(12):162738. Mahnke K, Qian Y, Knop J, et al. Induction of CD4/CD25 regulatory T cells by targeting of antigens to immature dendritic cells. Blood 2003; 101(12):48629. Figdor CG, de Vries IJ, Lesterhuis WJ, et al. Dendritic cell immunotherapy: mapping the way. Nat Med 2004;10(5):47580. Galli SJ, Kalesniko J, Grimbaldeston MA, et al. Mast cells as tunable eector and immunoregulatory cells: recent advances. Annu Rev Immunol 2005;23:74986. Takai T, Ono M, Hikida M, et al. Augmented humoral and anaphylactic responses in Fc gamma RII-decient mice. Nature 1996;379(6563):3469. Zhu D, Kepley CL, Zhang M, et al. A novel human immunoglobulin Fc gamma Fc epsilon bifunctional fusion protein inhibits Fc epsilon RI-mediated degranulation. Nat Med 2002;8(5):51821. Thery C, Amigorena S. The cell biology of antigen presentation in dendritic cells. Curr Opin Immunol 2001;13(1):4551. Albert ML, Pearce SF, Francisco LM, et al. Immature dendritic cells phagocytose apoptotic cells via alphavbeta5 and CD36, and cross-present antigens

622

FYHRQUIST-VANNI

et al

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53] [54]

[55]

to cytotoxic T lymphocytes. J Exp Med 1998;188(7): 135968. Verbovetski I, Bychkov H, Trahtemberg U, et al. Opsonization of apoptotic cells by autologous iC3b facilitates clearance by immature dendritic cells, down-regulates DR and CD86, and up-regulates CC chemokine receptor 7. J Exp Med 2002; 196(12):155361. Liu K, Iyoda T, Saternus M, et al. Immune tolerance after delivery of dying cells to dendritic cells in situ. J Exp Med 2002;196(8):10917. Caux C, Massacrier C, Vanbervliet B, et al. Activation of human dendritic cells through CD40 crosslinking. J Exp Med 1994;180(4):126372. Rescigno M, Piguet V, Valzasina B, et al. Fas engagement induces the maturation of dendritic cells (DCs), the release of interleukin (IL)-1beta, and the production of interferon gamma in the absence of IL-12 during DC-T cell cognate interaction: a new role for Fas ligand in inammatory responses. J Exp Med 2000;192(11):16618. Ohshima Y, Tanaka Y, Tozawa H, et al. Expression and function of OX40 ligand on human dendritic cells. J Immunol 1997;159(8):383848. Regnault A, Lankar D, Lacabanne V, et al. Fcgamma receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization. J Exp Med 1999;189(2): 37180. Jurgens M, Wollenberg A, Hanau D, et al. Activation of human epidermal Langerhans cells by engagement of the high anity receptor for IgE, Fc epsilon RI. J Immunol 1995;155(11):51849. Basu S, Binder RJ, Suto R, et al. Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-kappa B pathway. Int Immunol 2000;12(11):153946. Kronenberg M. Toward an understanding of NKT cell biology: progress and paradoxes. Annu Rev Immunol 2005;23:877900. Campos RA, Szczepanik M, Itakura A, et al. Cutaneous immunization rapidly activates liver invariant Valpha14 NKT cells stimulating B-1 B cells to initiate T cell recruitment for elicitation of contact sensitivity. J Exp Med 2003;198(12): 178596. Cyster JG. Chemokines and cell migration in secondary lymphoid organs. Science 1999;286(5447):2098102. Geijtenbeek TB, Torensma R, van Vliet SJ, et al. Identication of DC-SIGN, a novel dendritic cellspecic ICAM-3 receptor that supports primary immune responses. Cell 2000;100(5):57585. Yokozeki H, Watanabe K, Igawa K, et al. Gammadelta T cells assist alphabeta T cells in the adoptive transfer of contact hypersensitivity to para-phenylenediamine. Clin Exp Immunol 2001; 125(3):3519.

[56] Bretscher P, Cohn M. A theory of self-nonself discrimination. Science 1970;169(950):10429. [57] Bluestone JA, St Clair EW, Turka LA. CTLA4Ig: bridging the basic immunology with clinical application. Immunity 2006;24(3):2338. [58] Lambrecht BN. The dendritic cell in allergic airway diseases: a new player to the game. Clin Exp Allergy 2001;31(2):20618. [59] Sato T, Ishii N, Murata K, et al. Consequences of OX40-OX40 ligand interactions in langerhans cell function: enhanced contact hypersensitivity responses in OX40L-transgenic mice. Eur J Immunol 2002;32(11):332635. [60] Xu H, Heeger PS, Fairchild RL. Distinct roles for B7-1 and B7-2 determinants during priming of eector CD8 Tc1 and regulatory CD4 Th2 cells for contact hypersensitivity. J Immunol 1997;159(9): 421726. [61] Gorbachev AV, Heeger PS, Fairchild RL. CD4 and CD8 T cell priming for contact hypersensitivity occurs independently of CD40-CD154 interactions. J Immunol 2001;166(4):232332. [62] Gorbachev AV, Fairchild RL. CD40 engagement enhances antigen-presenting langerhans cell priming of IFN-gamma-producing CD4 and CD8 T cells independently of IL-12. J Immunol 2004;173(4): 244352. [63] Ptak W, Askenase PW. Gamma delta T cells assist alpha beta T cells in adoptive transfer of contact sensitivity. J Immunol 1992;149(11):35038. [64] Tsuji RF, Szczepanik M, Kawikova I, et al. B celldependent T cell responses: IgM antibodies are required to elicit contact sensitivity. J Exp Med 2002;196(10):127790. [65] Askenase PW. Yes T cells, but three dierent T cells (alphabeta, gammadelta and NK T cells), and also B-1 cells mediate contact sensitivity. Clin Exp Immunol 2001;125(3):34550. [66] Askenase PW, Szczepanik M, Itakura A, et al. Extravascular T-cell recruitment requires initiation begun by Valpha14 NKT cells and B-1 B cells. Trends Immunol 2004;25(8):4419. [67] Szczepanik M, Akahira-Azuma M, Bryniarski K, et al. B-1 B cells mediate required early T cell recruitment to elicit protein-induced delayedtype hypersensitivity. J Immunol 2003;171(11): 622535. [68] Tsuji RF, Geba GP, Wang Y, et al. Required early complement activation in contact sensitivity with generation of local C5-dependent chemotactic activity, and late T cell interferon gamma: a possible initiating role of B cells. J Exp Med 1997;186(7): 101526. [69] Tsuji RF, Kawikova I, Ramabhadran R, et al. Early local generation of C5a initiates the elicitation of contact sensitivity by leading to early T cell recruitment. J Immunol 2000;165(3):158898. [70] Askenase PW, Van Loveren H, Kraeuter-Kops S, et al. Defective elicitation of delayed-type

CONTACT DERMATITIS

623

[71]

[72]

[73]

[74]

[75] [76]

[77]

[78]

[79]

[80]

[81]

hypersensitivity in W/Wv and SI/SId mast cell-decient mice. J Immunol 1983;131(6):268794. Saint-Mezard P, Berard F, Dubois B, et al. The role of CD4 and CD8 T cells in contact hypersensitivity and allergic contact dermatitis. Eur J Dermatol 2004;14(3):1318. Kondo S, Beissert S, Wang B, et al. Hyporesponsiveness in contact hypersensitivity and irritant contact dermatitis in CD4 gene targeted mouse. J Invest Dermatol 1996;106(5):9931000. Wang B, Fujisawa H, Zhuang L, et al. CD4 Th1 and CD8 type 1 cytotoxic T cells both play a crucial role in the full development of contact hypersensitivity. J Immunol 2000;165(12):678390. Saint-Mezard P, Chavagnac C, Vocanson M, et al. Decient contact hypersensitivity reaction in CD4-/- mice is because of impaired hapten-specic CD8 T cell functions. J Invest Dermatol 2005; 124(3):5629. Bevan MJ. Helping the CD8() T-cell response. Nat Rev Immunol 2004;4(8):595602. Sun JC, Williams MA, Bevan MJ. CD4 T cells are required for the maintenance, not programming, of memory CD8 T cells after acute infection. Nat Immun 2004;5(9):92733. Sakaguchi S, Sakaguchi N, Asano M, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of selftolerance causes various autoimmune diseases. J Immunol 1995;155(3):115164. Gambineri E, Torgerson TR, Ochs HD. Immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance (IPEX), a syndrome of systemic autoimmunity caused by mutations of FOXP3, a critical regulator of T-cell homeostasis. Curr Opin Rheumatol 2003;15(4):4305. Akbari O, Freeman GJ, Meyer EH, et al. Antigenspecic regulatory T cells develop via the ICOSICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity. Nat Med 2002;8(9):102432. Ling EM, Smith T, Nguyen XD, et al. Relation of CD4 CD25 regulatory T-cell suppression of allergen-driven T-cell activation to atopic status and expression of allergic disease. Lancet 2004; 363(9409):60815. Cavani A, Mei D, Guerra E, et al. Patients with allergic contact dermatitis to nickel and nonallergic

[82]

[83]

[84]

[85]

[86]

[87]

[88]

[89]

[90]

[91]

[92]

individuals display dierent nickel-specic T cell responses. Evidence for the presence of eector CD8 and regulatory CD4 T cells. J Invest Dermatol 1998;111(4):6218. OGarra A, Vieira P. Regulatory T cells and mechanisms of immune system control. Nat Med 2004; 10(8):8015. Taylor A, Verhagen J, Akdis CA, et al. T regulatory cells in allergy and health: a question of allergen specicity and balance. Int Arch Allergy Immunol 2004;135(1):7382. Kehren J, Desvignes C, Krasteva M, et al. Cytotoxicity is mandatory for CD8() T cell-mediated contact hypersensitivity. J Exp Med 1999;189(5): 77986. Trautmann A, Akdis M, Kleemann D, et al. T cellmediated Fas-induced keratinocyte apoptosis plays a key pathogenetic role in eczematous dermatitis. J Clin Invest 2000;106(1):2535. Yawalkar N, Hunger RE, Buri C, et al. A comparative study of the expression of cytotoxic proteins in allergic contact dermatitis and psoriasis: spongiotic skin lesions in allergic contact dermatitis are highly inltrated by T cells expressing perforin and granzyme B. Am J Pathol 2001;158(3): 8038. Suntharalingam G, Perry MR, Ward S, et al. Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. N Engl J Med 2006;355(10):101828. Del Rosso J, Friedlander SF. Corticosteroids: options in the era of steroid-sparing therapy. J Am Acad Dermatol 2005;53(1 Suppl 1):S508. Beck LA. The ecacy and safety of tacrolimus ointment: a clinical review. J Am Acad Dermatol 2005; 53(2 Suppl 2):S16570. Grassberger M, Steinho M, Schneider D, et al. Pimecrolimudan anti-inammatory drug targeting the skin. Exp Dermatol 2004;13(12):72130. Ruzicka T, Larsen FG, Galewicz D, et al. Oral alitretinoin (9-cis-retinoic acid) therapy for chronic hand dermatitis in patients refractory to standard therapy: results of a randomized, double-blind, placebo-controlled, multicenter trial. Arch Dermatol 2004;140(12):14539. ODonoghue M, Tharp MD. Antihistamines and their role as antipruritics. Dermatol Ther 2005; 18(4):33340.

Dermatol Clin 25 (2007) 625632

Reticulohistiocytosis
vio Barbosa Luz, MD, PhDa, Patricia Shu Kurizky, MDb, Fla Marcia Ramos-e-Silva, MD, PhDc,*
General Policlinics of Rio de Janeiro/Carlos Chagas Medical Post-Graduation Institute, Rua Desembargador Izidro 28/1001, 20521-160 Rio de Janeiro, Brazil b Post-Graduation Course in Dermatology, Sector of Dermatology, Universidade Federal Fluminense, i, Brazil Rua Henrique Cordeiro 120/603 Bl. 01, 22631-450 Rio de Janeiro, Nitero c Sector of Dermatology and Post-Graduation Course, University Hospital and School of Medicine, Federal University of Rio de Janeiro, Rua Dona Mariana 143/C-32, 22280-020 Rio de Janeiro, Brazil
a

Reticulohistiocytoses (RHCs) consist of a group of proliferative 2macrophage diseases, and can be divided into three groups: multicentric reticulohistiocytosis (MR), diuse cutaneous RHC, and solitary reticulohistiocytoma.

Multicentric reticulohistiocytosis MR constitutes a rare systemic histiocytic disease that was described rst in 1937 by Weber and Freudenthal [3]. In 1949, Portugal and colleagues [4], in Brazil, proved its histiocytic nature, and in 1954, Goltz and Laymon [5] introduced the present nomenclature, dierentiating it from the purely cutaneous forms. The disease is found mainly in white patients (80% of the cases), with a slight predominance in women (1.85:1), and an average age of 50 years, but cases of MR have been reported in children [2]. Clinically, it is characterized by an insidious and progressive onset; 40% of the cases begin with articular symptoms, 30% begin with cutaneous symptoms, and 29% begin with both [2]. Evolution is marked by periods of remission and recurrence, and between 11% [2] and 45% [6] of patients develop erosive deforming arthritis. The cutaneous form is composed of papules and nodules, ranging from a few millimeters to 2 cm, which are rounded, translucent, and yellowpinkish or yellow-brownish. The most aected sites include the hands (80%), face (65%), upper limbs (51%), trunk (49%), lower limbs, ears and mucosa (30%), and neck (23%); some patients have more than one aected region [2]. Coalescence of multiple nodules in the face results in facies leonina. In a review of 96 cases, the pathognomonic signal of the bead collar, which consists of a linear grouping of small papules in perionychia, was found in 27%, papillary xanthelasma was found in 17%, and vermicular lesions
derm.theclinics.com

Etiopathogenesis Caro and Senear [1] suggested that RHC represents a single pattern of inammatory response to several stimuli other than a true neoplasia. The association of MR with autoimmune and neoplastic diseases has been reported [2]. Because several cytokines and growth factors usually are secreted in those diseases, it is possible that an abnormal histiocytic proliferation is triggered by such substances. The rarity of the disease, the presence of a specic type of histiocyte, and the absence of infectious agents and family cases suggest individual predispositiondprobably not inheriteddthrough a mutation or defect in the formation of a specic type of histiocyte. The authors believe that RHC originates by proliferation and dierentiation of an anomalous histiocytic clone in response to stimuli, probably lymphocytic, which are still unknown. Some peculiarities of each group are discussed below.

* Corresponding author. E-mail address: ramos.e.silva@dermato.med.br (M. Ramos-e-Silva).

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.015

626

LUZ

et al

in the nostrils were found in 15% [2,6]. Ulceration is rare, and other atypical ndings include hypochromic plaques, telangiectasia, erythroderma, nodules larger than 1 cm in size, and the Koebner phenomenon [7]. The oral (Fig. 1) and the nasal are the most affected mucosa; the tongue also is aected, and there are reports of vulvar and perianal lesions and voice changes caused by alteration of the vocal cords [8]. The chronic symmetric diuse polyarteritis of destructive course is the classic osteoarticular manifestation. The most aected articulations include the hands (75%), knees (65%), wrists (46%), shoulders (36%), and ankles (31%), often with more than one articulation aected simultaneously [2,6]. The typical radiologic aspect consists of well-delimited periarticular erosions associated with juxta-articular reabsorption [2,9]. That erosion probably results from proteases secreted by activated histiocytes, mainly urokinases, as suggested by Lotti and colleagues [10]. Polyarteritis remains active for periods that vary from 6 to 8 years, developing with stabilization (40%), improvement (10%), or progression to mutilating osteoarthropathy (50%) [11]. Other manifestations described in the literature and that emphasize the multisystemic character of the disease include weight loss, itching, weakness, cardiovascular symptoms, myalgia, fever, anorexia, dysphagia, lymphadenopathy, rhinorrhea, supraglottic mass, conjunctivitis and episcleritis, increased lacrimation, hepatosplenomegaly, and increase in the size of the submandibular salivary glands [2]. Despite not being considered a paraneoplastic disease, 25% of the cases present with malignancy [2]. The neoplasias described include breast cancer

[12], ovarian cancer [13], endometrial cancer [14], pleural mesothelioma [15], lymphoma [16], melanoma [17], stomach cancer [10], and penis cancer [18]. Associations also are found with systemic diseases, including Sjo grens syndrome and other autoimmune and thyroid diseases [2,19]. The dierential diagnosis includes sarcoidosis, Zayds family dermatoarthritis, lepromatous leprosy, and other histiocytoses, mainly those of Langerhans cells. Immunohistochemistry can help to dierentiate between MR and xanthogranuloma, which is positive for factor XIIIa, and nodular tenosynovitis, which is positive for actin. Rheumatoid arthritis and psoriatic arthritis are the main dierential diagnoses from the rheumatologic point of view. Diuse cutaneous reticulohistiocytosis Described rst in 1969 by Filgueira and colleagues [20] under the name rheumatoid histiocytoma, it was recognized as an exclusively cutaneous MR variety in 1979 by Piette and colleagues [21]. It especially aects patients older than 60 years, with a denite prevalence among women, and is restricted to the skin [22]. It is characterized by diuse, symmetric, usually asymptomatic papules or small nodules that are located mainly on the trunk (Fig. 2) [23], with some reports of itching. The conuence of lesions on the face may result in facies leonina, similar to MR [22]. Xanthogranuloma is the main dierential diagnosis both clinically and histopathologically. It is considered by some investigators to be part of the same nosologic spectrum [24]. Other diseases that should be dierentiated are sarcoidosis,

Fig. 1. Lysosomal intracytoplasmic marking of CD68 in MR cells (400).

Fig. 2. MAC387 small histiocytes in between the inltrate with many multinucleated and oncocytic macrophages in MR (400).

RETICULOHISTIOCYTOSIS

627

leprosy, mastocytosis, granuloma annulare, xanthoma, and lymphoma. Solitary reticulohistiocytoma Zak [25], in 1950, described four cases of solitary lesions that presented histopathologic aspects that were similar to the case described in 1934 by Montgomery and OLeary [26] as multiple ganglioneuromas of the skin. Because of its similarity to histiocytoma, Zak [25] preferred the name reticulohistiocytoma of the skin. It is more frequent in adults, with an average age of 50 years, despite reports of lesions in infants, including those still nursing [27], and pregnant women [28]. The clinical presentation is not characteristic; nodules or papules, located mainly on the head, range in color from yellowish to brownish, sizes range from a few millimeters to a little more than 2 centimeters, and the consistency can be variable. The dierential diagnosis includes xanthogranuloma, nodular tenosynovitis, dermatobroma, brolipoma, Hashimoto and Pritzkers disease, Spitzs nevus, mastocytoma, and pyogenic granuloma.

thrombomodulin. Small, activated histiocytes are MAC387 positive. Lymphocytes, mainly CD4, are found in MR inltrates [29].

Cellular receptors in reticulohistiocytosis MR is the most investigated form of RHC, probably because of the rarity of the other forms. Analyses of the markers and cytokines involved in its pathogenesis are important to the understanding of RHC histogenesis and provide the basis for the development of eective therapeutics. CD68 AND MAC387: evidence of macrophage maturation in multicentric reticulohistiocytosis CD68 is a lysosomal protein that is highly specic to macrophages, and it is the most important immunohistochemistry marker in MR [2]. Positive CD68 cells are found in great quantity and correspond, in most cases, to the oncotic histiocytes and giant multinuclear cells (Fig. 3) [29]. MAC387 is a monoclonal antibody that recognizes the leukocitary protein L1 (calprotectin) that is expressed in neutrophils and monocytes. The monocytes tend to lose calprotectin gradually after migration from the blood vessels to the tissues, which makes it a useful marker for the detection of macrophages that have been derived from the blood recently [32]. In addition to being used widely in the phenotypical characterization of myelomonocytic cells in situ, Goebeler and colleagues [33], through Western blot, observed

Histopathology and immunohistochemistry Numerous giant multinuclear cells and oncotic histiocytes, so called for being similar to thyroid oncocytes, typically are found. These histiocytes have eosinophilic and nely granular cytoplasm, resembling ground glass. The lymphocytes tend to be numerous, mainly in recent lesions, and, with evolution, broblasts can be observed, with the development of brosis [29]. This was called reticulohistiocytic granuloma by Purvis and Helwig [30] in 1954, despite some investigators contesting the use of that terminology. Microscopy conrms the diagnosis of RHC, relying on the clinical-pathologic correlation to dene its exact form [31]. The unique and common histopathologic characteristics allow the classication of MR, diuse cutaneous RHC, and solitary reticulohistiocytoma as parts of a specic nosologic spectrum of non-Langerhans histiocytosis; however, the absence of reports on the evolution of one form into another and the expanded knowledge about the clinical presentations suggest that the dierent nomenclatures should be retained. MR histiocytic cells are positive for vimentin, CD68, and CD45; negative for S-100 protein, CD34, and XIIIa factor; and weak reactors for

Fig. 3. Diuse cutaneous reticulohistiocytosis. Papules and tubercles with lichenoid aspect on trunk and arms.

628

LUZ

et al

that this antibody is directed against the calciumbinding MRP14 protein. The presence of antigen MAC387 in small histiocytes, found in a review of 23 MR cases [29], may indicate that those cells represent collateral elements in the formation of the disease or a maturation stage of the cells causing the disease. Eagle and colleagues [34] observed a similar pattern of marking for MAC387, describing it as found only in the nonactivated histiocytes of the inltrate (Fig. 4). The presence of cells that present doublemarking for CD68 and MAC387 strengthens the hypothesis that they represent variations of the phenotypic expression of the MR cells, probably due to a maturation process of those cells [29]. The natural history of the MR lesions presupposes the transformation of small histiocytes into oncocytes and of those into giant cells. Observation of small MAC387 histiocytes, large oncocytic histiocytes and giant CD68 cells, and histiocytes with intermediate sizes of cytoplasm expressing both antigens, suggests that the maturation process of those cells should be accompanied by a modication in their antigenic expression. Taking into account the fact that this maturation results in a loss of the phagocyte activity of the cells [35] and that MAC387 is found in granulocytes and activated macrophages [36,37], it is possible to presume that small activated histiocytes, as the result of some unknown incentive, proliferate and undergo alterations until changing into giant cells lled with hypofunctional lysosomes.

The role of thrombomodulin and histiocytes in multicentric reticulohistiocytosis Thrombomodulin is a transmembrane anticoagulant glycoprotein that regulates thrombin. Thrombomodulin dermal positive cells do not express factor XIIIa, CD34, or CD68 and are found in normal skin, mainly in the papillary and upper reticular dermis [38]. The study of those cells in granuloma annulare showed that they probably act in the evolutionary process of the lesion, whereas factor XIIIa cells would participate in tissue recovery [39]. In MR, thrombomodulin can be observed in the dendritic cells and in the histiocyte periphery. It is possible that such cells have a role in MR similar to the one that they have in granuloma annulare. Their expression in other histiocytoses has not been described [29]. An unpublished study by the authors demonstrated thrombomodulin expression in all RHCs, strengthening the hypothesis of a common origin for those diseases. Other markers for histiocytosis Factor XIIIa, considered useful in the diagnosis of xanthogranuloma, is a transglutaminase activated by thrombin and present in dermal dendrocytes [22]. CD34 usually is found in endothelial cells and in a specic group of dendritic cells, called dendrocytes type II by Doval and Toribio [40], and helps in the identication of a tumor of probable histiocytic origin, dermatobrosarcoma protuberans [41]. CD1a is a marker found in Langerhans cells, veil cells, and indeterminate dendritic cells [42]; it is important in the diagnosis of Langerhans cell histiocytosis and indeterminate cell histiocytosis. Factor XIIIa-, CD34-, and CD1a-positive cells tend to be negative in MR, and the occasional presence of those cells does not seem to play a relevant role in RHC [29]. The role of receptors and molecules in reticulohistiocytosis therapeutics Some therapies for RHC have been described; however, the results are not always satisfactory. Antiproliferative drugs, cyclophosphamide and methotrexate, psoralens and UV-A radiation, and topical nitrogen mustard and hydroxychloroquine may result in improvement of MR lesions [2]. In diuse cutaneous RHC, the lesions tend to appear and disappear spontaneously, with reports of complete spontaneous remission [43]. The treatments described in the literature generally

Fig. 4. MR. Perioral micropapules, nodules in a linear disposition on gingiva, vermicular lesions on nostrils, and xanthelasma-like lesions.

RETICULOHISTIOCYTOSIS

629

are not totally eective, but satisfactory results have been obtained with vinblastine [44]. The solitary reticulohistiocytoma, which presents as a single lesion in most cases, is treated easily with removal without the need for safety margins; there are not many studies regarding its treatment. Therapeutics that are addressed to cellular receptors constitute a growing eld in medicine, showing positive results in diseases that are dicult to control, like psoriasis [45] and lymphomas [46]. With regard to histiocytic diseases, Braiteh and colleagues [47] described an eective treatment of Erdheim-Chester disease, a nonLangerhans histiocytosis, with interferon-a. Despite the mechanism not having been claried, the investigators suggested a probable action in the maturation and activation of dendritic cells, immune-mediated histiocyte destruction, or a direct antiproliferative eect. Gorman and colleagues [48] demonstrated the presence of several cytokines (eg, tumor necrosis factor a [TNF-a] and interleukin [IL]-1b and -12) in the macrophages of the synovial tissue in a patient who had MR. TNF-a increases the response in the acute phasedthe number of molecules of adhesion and of proinammatory cytokinesd besides stimulating the production of proteases by the macrophages, inducing the destruction of bones and cartilages. De Rycke and colleagues [49] studied the synovial macrophages involved in the pathogenesis of autoimmune chronic arthritis, observing that treatment in vivo with antiTNF-a caused a rapid and pronounced effect, reducing macrophage inltration. Those two observations justify the use of drugs against that cytokine in the treatment of RHC. Etanercept is a fusion protein that blocks TNF-a activity by attaching to the receptor. It has ve indications approved by the US Food and Drug Administration: rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and moderate to severe psoriasis. Other applications in dermatology include granulomatous diseases, collagenases, and vasculitis. A patient who had Langerhans cell histiocytosis showed improvement with this immunomodulator, and, recently, some patients who had MR also had a good response to treatment [5053]. Iniximab is an antiTNF-a chimerical monoclonal IgG antibody (human/rat), rst developed for the treatment of rheumatoid arthritis and Crohns disease [45]. Its use in MR has been

reported, with rapid improvement of the patients, mainly in relation to the cutaneous lesions [19,54].

Other therapeutic possibilities not tested in reticulohistiocytosis related to receptors and molecules Adalimumab is the rst entirely human anti TNF-a antibody that has been used successfully in the treatment of pyoderma gangrenosum [55]. This immunomodulator has not been used in RHC, probably because of its high cost. Celacade, a new and nonpharmacologic immunomodulating therapy, was described experimentally as being capable of increasing the anti-inammatory cytokines and suppressing the secretion of proinammatory cytokines, among them IL-1b, which is increased in MR [56]. The peroxisome-proliferating activated gamma receptors (PPAR-g) are receptors that are expressed in adipocytes, but also were found recently in macrophages. Studies suggested that agonists of PPAR-g would have some eect on the suppression of inammation mediated by macrophages. Pioglitazone constitutes an agonist of that receptor; it is used in the treatment of type 2 diabetes and is considered safe and well tolerated in most patients. Side eects include edema, a slight decrease in hemoglobin and red cell count related to blood dilution, reduction of blood pressure, and weight gain [57]. Gregorio and colleagues [58] showed that it reduces the expression of CD68 cells and monocyte chemoattractant protein-1 of macrophages and serous levels of TNF-a. Although not tested, this seems to be an interesting form of intervention in RHC. Another receptor, the receptor for sphingosine1-phosphate, can be found on the surface and cytoplasm of CD68-positive macrophage and endothelial cells; its agonist, in addition to its known lymphopenic action, seems to increase macrophage homing in the lymph nodes of rats [59]. The treatment of venous grafts with transforming growth factor (TGF)-b1 antisense reduced hyperplasia of the innermost layer in reconstructions of heart arteries. Wol and colleagues [60] observed that it reduced the expression of TGF-b1 and positive CD68 cells in the media and adventitia layers of the arteries, possibly showing a benecial action in RHC. Sasahira and colleagues [61] studied the eects of IL-15 and TGF-a in colon cancer tissue; these

630

LUZ

et al and atypical histological features. Proc R Soc Med 1937;30:5226. Portugal H, Fialho F, Milano A. Generalized giantcell histiocytomatosis. Revista Argentina de Dermatologia 1944;28:12144. Goltz RW, Laymon CW. Multicentric reticulohistiocytosis of the skin and synovia. Arch Dermatol 1954;69(6):71731. Barrow MV, Holubar K. Multicentric reticulohistiocytosisda review of 33 patients. Medicine 1969; 48(4):287305. Aldridge RD, Main RA, Daly BM. The Koebners response in multicentric reticulohistiocytosis. Cutis 1984;34(1):7880. Malhotra R, Pribitkin EA, Bough D Jr, et al. Upper airway involvement in multicentric reticulohistiocytosis. Otolaryngol Head Neck Surg 1996;114(4): 6614. Ho SG, Yu RC. A case of multicentric reticulohistiocytosis with multiple lytic skull lesions. Clin Exp Dermatol 2005;30(5):5158. Lotti T, Santucci M, Casigliani R, et al. Report of three cases with the evaluation of tissue proteinase activity. Am J Dermatopathol 1988;10(6): 497504. Lesher JL, Allen BS. Multicentric reticulohistiocytosis. J Am Acad Dermatol 1984;11(4):71323. Catterall MD, White JE. Multicentric reticulohistiocytosis and malignant disease. Br J Dermatol 1978; 98(2):2214. Kamel H, Gibson G, Cassidy M. Case report: the CT demonstration of soft tissue involvement in multicentric reticulohistiocytosis. Clin Radiol 1996; 51(6):4401. Malik MK, Regan L, Robinson-Bostom L, et al. Proliferating multicentric reticulohistiocytosis associated with papillary serous carcinoma of the endometrium. J Am Acad Dermatol 2005;53(6):10759. Honeybourne D, Kellett JK. A mesothelioma presenting with multicentric reticulohistiocytosis. Postgrad Med J 1985;61(711):579. Magnin PH, Morgenfeld MC. [The reticulohistiocytosis and the internal cancer]. Revista Argentina de Dermatologia 1987;68:758 [in Spanish]. Gibson C, Cassidy M, OConnell P, et al. Multicentric reticulohistiocytosis associated with recurrence of malignant melanoma. J Am Acad Dermatol 1995;32(1):1346. Lambert CM, Nuki G. Multicentric reticulohistiocytosis with arthritis and cardiac inltration: regression following treatment for underlying malignancy. Ann Rheum Dis 1992;51(6):8157. Lee MW, Lee EY, Jeong YII, et al. Successful treatment of multicentric reticulohistiocytosis with a combination of iniximab, prednisolone and methotrexate. Acta Derm Venereol 2004;84(6):4789. Filgueira AL, Fraga S, Marques AS. [Rheumatoid histiocytomatosis]. Revista Dermatologica Ibero Latino-Americana 1969;3:25560 [in Portuguese].

factors promoted the depletion of macrophages associated with the tumors. The genes for MRP8 and MRP14, the latter recognized by antibodies anti-MAC387, are expressed in several subpopulations of dendritic cells, in dierent intensities, depending on the degree of maturation. Kumar and colleagues [62] observed that the expression of those proteins is increased after exposure to IL-10. The investigators suggested that, despite their unknown functions, they probably are related to maturation of dendritic cells, and it is possible to act on the synthesis of T cells under the inuence of that interleukin. Lugering and colleagues [63], however, studying combined treatment with IL-4 and -10, observed suppression of MRP14 secretion and biologically activated MRP8/14 (antigen 27E10) in peripheral monocytes. Based on the absence of response in macrophages that were cultivated for more than 7 days, the investigators suggested that more differentiated and activated cells tend to lose their ability to respond to the anti-inammatory cytokines. Thus, treatment with combined cytokines would be more ecient in the control of the progression of chronic inammatory processes [63]. This observation suggests that a treatment addressing immature MAC387 and CD3 cells could have a signicant role in the control of RHC. Summary RHCs are rare diseases of unknown etiology. Identication of the cell receptors and cytokines that are related to the pathogenesis of RHC has supplied important data with regard to the histogenesis of these diseases. In the future, the better identication of these cell receptors and cytokines will help the classication and nosological positioning of the clinical variants of the RHC, which are still considered controversial by several investigators. The development of therapies to address those molecules constitutes an important step in the optimization of treatment for RHCs. References
[1] Caro MR, Senear FE. Reticulohistiocytoma of the skin. AMA Arch Derm Syphilol 1952;65(6):70113. [2] Luz FB, Gaspar AP, Kalil-Gaspar N, et al. Multicentric reticulohistiocytosis: review of the literature. J Eur Acad Dermatol Venereol 2001;15(6):52431. [3] Weber FP, Freudenthal W. Nodular non-diabetic cutaneous xanthomatosis with hypercholesterolemia

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11] [12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

RETICULOHISTIOCYTOSIS

631

[21] Piette F, Thomas P, Hildebrand HF, et al. [Multiple reticulo-histiocytoma]. Ann Dermatol Venereol 1979;106(67):597601 [in French]. [22] Luz FB, Gaspar AP, Kalil-Gaspar N, et al. [The histiocytes and the non-Langerhans histiocytoses in dermatology]. Anais Brasileiros de Dermatologia 2003;78(1):99118 [in Portuguese]. [23] McKenna DB, Mooney EE, Young MM, et al. Multiple cutaneous reticulohistiocytosis. Br J Dermatol 1998;139(3):5446. [24] Zelger BW, Sidoro A, Orchard G, et al. A new unifying concept. Am J Dermatopathol 1996;18(5): 490504. [25] Zak FG. Reticulohistiocytoma (ganglioneuroma) of the skin. Br J Dermatol 1950;62:3514. [26] Montgomery H, OLeary PA. Multiple ganglioneuromas of the skin. Arch Dermatol Syphilol 1934;29: 2652. [27] Garcia JV, Campos SJ, de Anda G, et al. [Reticulohistiocytic grunloma]. Archivos Argetinos de Dermatologia 1990;40:2559 [in Spanish]. [28] Hunt SJ, Shin SS. Solitary reticulohistiocytoma in pregnancy: immunohistochemical and ultrastructural study of a case with unusual immunophenotype. J Cutan Pathol 1995;22(2):17781. [29] Luz FB, Gaspar AP, Kalil-Gaspar N, et al. Immunohistochemical prole of multicentric reticulohistiocytosis. SkinMed 2005;4(2):717. [30] Purvis WE, Helwig EB. Reticulohistiocytic granuloma (reticulohistiocytoma) of the skin. Am J Clin Pathol 1954;24(9):100515. [31] Snow JL, Muller AS. Malignancy-associated multicentric reticulohistiocytosis: a clinical, histological and immunophenotypic study. Br J Dermatol 1995;133(1):716. [32] Otani I, Mori K, Sata T, et al. Accumulation of MAC387 macrophages in paracortical areas of lymph nodes in rhesus monkeys acutely infected with simian immunodeciency virus. Microbes Infect 1999;1(12):97785. [33] Goebeler M, Roth J, Teigelkamp S, et al. The monoclonal antibody MAC387 detects an epitope on the calcium-binding protein MRP14. J Leukoc Biol 1994;55(2):25961. [34] Eagle RC Jr, Penne RA, Hneleski IS Jr. Eyelid involvement in multicentric reticulohistiocytosis. Ophthalmology 1995;102(3):42630. [35] Heathcote JG, Guenther LC, Wallace AC. Multicentric reticulohistiocytosis: a report of a case and a review of the pathology. Pathology 1985;17(4):6018. [36] Cerio R, Spaull J, Oliver GF, et al. A study of factor XIIIa and MAC 387 immunolabeling in normal and pathological skin. Am J Dermatopathol 1990;12(3): 22133. [37] OLaughlin S, Braverman M, Smith-Jeeries M, et al. Macrophages (histiocytes) in various reactive and inammatory conditions express dierent antigenic phenotypes. Hum Pathol 1992;23(12): 14108.

[38] Cuzzi-Maya T, Sidbury R, Epstein WL, et al. Thrombomodulin expression on dermal cells in normal psoriatic skin. Arch Dermatol Res 1998;290(5): 2339. [39] Soub CRW, Rochael MC, Cuzzi T. [Granuloma annulare: tissular distribution of XIIIa dermal dendrocytes, thrombomodulin dermal cells, and CD68 macrophages]. Anais Brasileiros de Dermatologia 2003;78(3):28998 [in Portuguese]. [40] Doval IG, Toribio J. [The histocyte: new concepts]. Actas Dermo-silogracas 1997;88:64962 [in Spanish]. [41] Rudolph P, Schubert B, Wacker H-H, et al. Immunophenotyping of dermal spindle cell tumors: diagnostic value of monocyte marker Ki-M1p and histogenetic considerations. Am J Surg Pathol 1997;21(7):791800. [42] Lappin MB, Kimber I, Norval M. The role of dendritic cells in cutaneous immunity. Arch Dermatol Res 1996;288(3):10921. [43] Goette DK, Odom RB, Fitzwater JE. Diuse cutaneous reticulohistiocytosis. Arch Dermatol 1982; 118(3):1736. [44] Winkelmann RK, Hu CH, Kossard S. Response of nodular non-X histiocytosis to vinblastine. Arch Dermatol 1982;118(11):91391. [45] Williams JD, Griths CE. Cytokine blocking agents in dermatology. Clin Exp Dermatol 2002;27(7): 58590. [46] DeNardo GL, Tobin E, Chan K, et al. Direct antilymphoma eects on human lymphoma cells of monotherapy and combination therapy with CD20 and HLA-DR antibodies and 90Y-labeled HLADR antibodies. Clin Cancer Res 2005;11(19):70759. [47] Braiteh F, Boxrud C, Esmaeli B, et al. Successful treatment of Erdheim-Chester disease, a non-Langerhans-cell histiocytosis, with interferon-a. Blood 2005;106(9):29924. [48] Gorman JD, Danning C, Schumacher HR, et al. Multicentric reticulohistiocytosis: case report with immunohistochemical analysis and literature review. Arthritis Rheum 2000;43(4):9308. [49] De Rycke L, Baeten D, Foell D, et al. Dierential expression and response to anti-TNFalpha treatment of inltrating versus resident tissue macrophage subsets in autoimmune arthritis. J Pathol 2005;206(1): 1727. [50] Scheinfeld N. The medical uses and side eects of etanercept with a focus on cutaneous disease. J Drugs Dermatol 2004;3(6):6539. [51] Lovelace K, Loyd A, Adelson D, et al. Etanercept and the treatment of multicentric reticulohistiocytosis. Arch Dermatol 2005;141(9):11678. [52] Kovach BT, Calamia KT, Walsh JS, et al. Treatment of multicentric reticulohistiocytosis with etanercept. Arch Dermatol 2004;140(8):91921. [53] Matejicka C, Morgan GJ, Schlegelmilch JG. Multicentric reticulohistiocytosis treated successfully with an anti-tumor necrosis factor agent: comment on the

632

LUZ

et al [59] Singer II, Tian M, Wickham LA, et al. Sphingosine1-phosphate agonists increase macrophage homing, lymphocyte contacts, and endothelial junctional complex formation in murine lymph nodes. J Immunol 2005;175(11):715161. [60] Wol RA, Malinowski RL, Heaton NS, et al. Transforming growth factor-beta1 antisense treatment of rat vein grafts reduces the accumulation of collagen and increases the accumulation of h-caldesmon. J Vasc Surg 2006;43(5):102836. [61] Sasahira T, Sasaki T, Kuniyasu H. Interleukin-15 and transforming growth factor alpha are associated with depletion of tumor-associated macrophages in colon cancer. J Exp Clin Cancer Res 2005;24(1):6974. [62] Kumar A, Steinkasserer A, Berchtold S. Interleukin10 inuences the expression of MRP8 and MRP14 in human dendritic cells. Int Arch Allergy Immunol 2003;132(1):407. [63] Lugering N, Kucharzik T, Lugering A, et al. Importance of combined treatment with IL-10 and IL-4, but not IL-13, for inhibition of monocyte release of the Ca(2)-binding protein MRP8/14. Immunology 1997;91(1):1304.

[54]

[55]

[56]

[57]

[58]

article by Gorman, et al. Arthritis Rheum 2003; 48(3):8646. Sellam J, Deslandre CJ, Dubreuil F, et al. Refractory multicentric reticulohistiocytosis treated with iniximab: two cases. Clin Exp Rheumatol 2005;23(1): 979. Hubbard VG, Friedmann AC, Goldsmith P. Systemic pyoderma gangrenosum responding to iniximab and adalimumab. Br J Dermatol 2005;152(5): 105961. Bolton AE. Biologic eects and basic science of a novel immune-modulation therapy. Am J Cardiol 2005;95:24C9C. Belcher G, Lambert C, Edwards G, et al. Safety and tolerability of pioglitazone, metformin, and gliclazide in the treatment of type 2 diabetes. Diabetes Res Clin Pract 2005;70(1):5362. Gregorio GB, Yao-Borengasser A, Rasouli N, et al. Expression of CD68 and macrophage chemoattractant protein-1 genes in human adipose and muscle tissuesdassociation with cytokine expression, insulin resistance, and reduction by pioglitazone. Diabetes 2005;54(8):230513.

Dermatol Clin 25 (2007) 633641

UV Light and Its Interaction with Cutaneous Receptors

Hagit Matz, MD
Department of Dermatology, Tel Aviv Sourasky Medical Center, 6 Weizmann Street, Tel Aviv 64239, Israel

Phototherapy (UV-A and UV-B) has become one of the most commonly used modalities for the treatment of a variety of skin diseases, although the action mechanisms have not been fully understood. Inhibition of DNA synthesis by UV radiation may be one of the therapeutic eects in proliferating skin diseases; however, phototherapy is also used for the treatment of allergic or autoimmune diseases. The capacity of UV radiation to aect the skin immune system was rst recognized in the early 1970s in numerous studies [1,2]. Therapeutic photoimmunology The skin is an important immunologic organ whose constitutive cells are involved in immune reactions [3]. UV-B, UV-A, and psoralen-UV-A (PUVA) radiation may have similar or even identical immunosuppressive consequences. The therapeutic relevance is determined by the physical properties of the type of UV employed [4]. UV-B mainly aects the epidermis and papillary dermis and is associated with keratinocyte, Langerhans cell, and T-cell depletion. UV-A radiation can penetrate more deeply into the dermis and thereby aect dermal broblasts, dermal dendritic cells, endothelial cells, and skin inltrating inammatory cells such as T lymphocytes, mast cells, and granulocytes [2]. Photoimmunologic eects of therapeutic relevance fall into three major categories: (1) eects on the production of soluble mediators, (2) modulation of the expression of cell surface associated molecules, and (3) induction of apoptosis in pathogenetically relevant cells.
E-mail address: e_h_matz@netvision.net.il

Eects on soluble mediators The capacity to modulate the production of soluble immunomodulatory mediators has been demonstrated for UV-B, UV-A, and UV-A1 radiation [5,6]. The benecial eects can be attributed, at least in part, to the induction of mediators with anti-inammatory or immunosuppressive properties or both. Anti-inammatory/immunosuppressive eects UV-B, UV-A, and UV-A1 suppress the production of proinammatory cytokines. In vitro studies employing cultured human keratinocytes have demonstrated that UV-B and, to some extent, UV-A radiation are capable of inducing the production of cytokines, neuropeptides, and prostanoids. UV-B and UV-A1 radiation have the capacity to increase signicantly IL-10 mRNA and protein expression in cultured normal human keratinocytes, and IL-10 protein expression is increased in human skin following in vivo UV irradiation [7,8]. IL-10 is a keratinocyte-derived cytokine of particular therapeutic relevance. IL-10 is functionally dened by its capacity to suppress the production of interferon gamma (IFN-g) by T lymphocytes of the T helper 1 subtype. Successful phototherapy (UV-A1 or UV-A/UV-B) of atopic dermatitis is associated with downregulation of IFN-g expression [9], and this eect may be explained, at least in part, by phototherapy-induced expression of IL-10 and subsequent paracrine suppression of IFN-g production. Another example is in vitro synthesis of the neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH). In vitro exposure of human keratinocytes to UV-B or UV-A1 radiation
derm.theclinics.com

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.006

634

MATZ

increases the synthesis of proopiomelanocorticotropin-derived peptides including alpha-MSH [5]. Alpha-MSH has anti-inammatory eects such as the inhibition of IL-1 or tumor necrosis factor (TNF)-alpha and immunosuppressive eects such as the inhibition of cell-mediated immune response. A third example is UV-B and UV-A induced production of prostaglandins in epidermal keratinocytes [10]. Prostaglandin (PGE2) is a potent immunosuppressant that aects the expression of costimulatory molecules on the surface of antigen-presenting cells, thereby preventing the activation of selected T-cell subsets (especially Th1 cells) [11]. UV radiation also aects Langerhans cells. Langerhans cells are an important cellular source of immunosuppressive prostanoids. UV irradiation of human dendritic cells markedly induced cyclooxygenase activity and caused the production and release of signicant amounts of PGE and thromboxane [11]. Benecial eects observed after UV-A1 phototherapy in scleroderma patients have been attributed to the production of cytokines, which upregulate matrix metalloproteinase expression in human skin [12]. Successful UV-A1 phototherapy of patients with localized scleroderma was associated with an up to 20-fold induction of matrix metalloproteinase 1 (MMP-1) expression in sclerotic skin lesions [12]. In these patients, skin sclerosis is due to increased collagen production and deposition. Phototherapy-induced softening and disappearance of skin lesions may result from induction of the MMP-1 protease. UV-A1 radiation induces MMP-1 protease, in part, by an autocrine mechanism and not only directly. UV-A1 also induces cytokines IL-1 and IL-6 [13] from human dermal broblasts. Existing studies have demonstrated that UVirradiated epidermal keratinocytes release cytokines that indirectly promote MMP-1 production in dermal broblasts. UV-A irradiation dose dependently increased MMP-1 but not MMP-2 production in human skin broblasts, and IL-1a and IL-1b also promoted MMP-1 but not MMP-2 production. Both IL-1a and IL-1b activated MAP kinase, signicantly elevating mRNA expression. Cell culture medium from UV-Birradiated keratinocytes increased MMP-1 production in UV-Airradiated broblasts, and IL-1ra dose dependently inhibited MMP-1 production. IL1ra dose dependently inhibited c-Jun mRNA expression of broblasts with no signicant eect

on c-Fos mRNA expression. UV-Birradiated keratinocytes promoted MMP-1 production in UV-Airradiated broblasts in a paracrine manner, whereas IL-1ra reduced MMP-1 production through inhibiting c-Jun mRNA expression. IL-1 has an important role in the dermal collagen degradation associated with UV-induced premature aging of the skin, and IL-1ra may be applied for the prevention and treatment of photoaging [14]. The depletion of the resident T-cell population in the skin may be due to a combination of UV-B induced apoptosis and decreased recruitment from the blood due to lower expression of the required adhesion and homing molecules. UV-B treatment can alter the expression of adhesion molecules by blood lymphocytes. UV-B irradiation can also inuence the cytokine production of circulating T cells. Four patients with active chronic plaque psoriasis were treated daily with narrow-band UV-B irradiation, and blood samples were obtained before treatment and weekly thereafter for 2 weeks. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured with a streptococcal superantigen or a conventional streptococcal antigen preparation, and cell culture supernatants were assayed for various cytokines. When stimulated with the superantigen, PBMCs from UV-Btreated psoriasis patients secreted greater amounts of the antiinammatory cytokine IL-10 and showed markedly decreased production of IL-1a, IL-1b, IL-2, IL-5, and IL-6 when compared with the pretreatment values. The production of IFN-g, IL-8, and IL-12p70 was also decreased but did not reach statistical signicance. The combination of UV-B induced apoptosis, increased secretion of anti-inammatory cytokines, and decreased trafcking of cells to the skin may help to explain the benecial eects of UV-B treatment on psoriasis and why disease remission can sometimes be sustained for a prolonged period [15]. Exposure to UV radiation inhibits contact sensitization to haptens applied not only to the irradiated skin area but also to the nonirradiated distant skin when the exposure dose is relatively high or the application skin area is large. In addition, hapten-specic tolerance develops by the generation of suppressor T cells. Phototherapy is also useful for immediate type hypersensitivity such as urticaria. The action mode may be the inhibitory eects of UV radiation on histamine release from mast cells. The results obtained from these experiments suggest

UV LIGHT AND ITS INTERACTION

635

that phototherapy exerts its anti-allergic and antiinammatory eects through immunosuppression [3]. Regulation of proteolytic enzymes by UV-inducible cytokines The migration of melanocyte precursors (melanoblasts) from the outer root sheath of hair follicles into clinically depigmented epidermis is crucial to the repigmentation of vitiliginous skin treated with photochemotherapy (PUVA), but such migratory cells must penetrate extracellular matrix tissue barriers in vivo. MMP-2 (metalloproteinase), MMP-9, and MT1-MMP transcripts are expressed by melanoblasts, but only MMP-2 is secreted and activated in the extracellular environment. Although the therapeutic ecacy of PUVA in stimulating repigmentation of vitiliginous skin might derive from direct eects of UV-A or 8-methoxypsoralen (8-MOP), studies have shown that keratinocyte-derived factors induced by UV radiation, especially alpha-MSH, have a major role in regulating melanocyte function. MMP-2 synthesis and secretion were induced by 8-MOP, alpha-MSH, or both. This induction of MMP-2 resulted in signicant increases of migration of melanoblasts on laminin or laminin-5 substrates. Taken together; these results suggest the importance of MMP-2 in melanoblast migration and in the response of vitiligo to PUVA therapy [16]. Reactive oxygen species generated in the skin by UV irradiation promote photoaging and photocarcinogenesis. The manganese (Mn) superoxide dismutase (SOD) is a primary antioxidant enzyme that crucially contributes to the homeostasis of oxygen radicals within the mitochondria and critically participates in the control of senescence and tumor generation. Repetitive UV-B exposure, as practiced for light hardening during phototherapy for various photodermatoses, can enhance the adaptive antioxidant response by upregulating MnSOD activity in the epidermal or dermal skin compartment. Irradiation of epidermal cells with UV-B induced a release of soluble factors that amplied MnSOD activity in broblasts via a paracrine mechanism [17]. Eects on cell surface receptors UV-B, UV-A, and PUVA radiation can directly aect the expression and function of cell surface receptors including adhesion molecules, cytokines, and growth factor receptors.

Modulation of adhesion molecule expression In many skin diseases there is an increased expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of epidermal keratinocytes [18]. The ICAM-1 molecule is functionally dened by its capacity to serve as a counter-receptor for the lymphocyte function associated antigen-1 (LFA-1), which is present on the surface of leukocytes. There is strong in vitro and in vivo evidence that ICAM-1/LFA-1 mediated cell-cell adhesion is an important prerequisite for the generation and maintenance of a variety of inammatory and immune reactions in the skin [1923]. In healthy human skin, keratinocytes express little or no ICAM-1 on their surface. In contrast, in inamed skin, keratinocyte ICAM-1 expression is markedly upregulated. Stimulation of keratinocytes by proinammatory cytokines including IFN-g, TNF-beta, and TNF-alpha is responsible for this upregulation. Cytokine-induced ICAM-1 expression may be eciently inhibited by irradiation of cultured keratinocytes with sublethal doses of UV-B or UV-A. This anti-inammatory property of UV radiation is also observed in vivo [24,25]. Exposure of human skin to suberythemal doses of UV-B was sucient to eectively suppress upregulation of keratinocyte ICAM-1 expression induced by rh-IFN-g injection. UV-B induced suppression of ICAM-1 is transient in nature, because 24 hours after irradiation, signicant induction of keratinocyte ICAM-1 expression was observed. If UV-B irradiation was repeated 24 hours after the rst exposure, reinduction of ICAM-1 suppression was achieved, indicating that a maximal antiinammatory eect required repetitive exposure to UV-B phototherapy. Many observations indicate that UV-B radiation suppresses the upregulation of cytokineinducible genes including HLA class II molecules and IL-7 [26]. This observation suggests that UVB induces a mechanism that in a general way prevents transcription of inducible genes. Treatment of psoriasis with PUVA is associated with a marked reduction in keratinocyte ICAM-1 expression in lesional skin [18]. There is no convincing evidence that PUVA, similar to UV-B and UV-A, is capable of directly modulating cytokine induced or keratinocyte ICAM-1 expression. PUVA therapy induced downregulation of keratinocyte ICAM-1 expression may best be explained by an indirect mechanism, that is, the

636

MATZ

reduction of cytokine-producing, skin-inltrating inammatory cells, which can also result in reduced ICAM-1 expression. In addition to its eects on keratinocytes, UVB has been found to signicantly suppress adhesion molecule expression in antigen-presenting cells such as monocytes or epidermal Langerhans cells [27,28]. UV-B induced inhibition of adhesion molecule expression is of functional relevance because the resulting alteration of the costimulatory repertoire of antigen-presenting cells seems to cause anergy in eector Th1 cells and preferential activation of regulatory Th2 cells. Eighty percent of T lymphocytes that inltrate psoriatic lesions express the surface glycoprotein cutaneous lymphocyte-associated antigen compared with less than 20% of cells in the blood. Exposure to UV-B is an eective treatment for psoriasis. PBMCs from psoriatic patients were stained for adhesion molecules and stimulated with streptococcal antigens before and once weekly during 3 weeks of UV-B treatment. During treatment, a marked and progressive decrease was observed in expression of the cutaneous lymphocyte-associated antigen and in very late antigen-4alpha by T cells. This decrease correlated closely with clinical improvement (Psoriasis Area and Severity Index). T-cell expression of intercellular adhesion molecule-1 was not signicantly aected during the treatment, and no change was observed in the activation markers CD25 and CD69 or in lymphocyte proliferation after stimulation with streptococcal antigens or superantigens. These ndings suggest that UV-B treatment of psoriasis is associated with a marked reduction in the expression of skin-homing molecules by circulating T cells. This reduction may be relevant to its therapeutic eect [29]. Targeting of cytokine and growth factor receptors The regulation of keratinocyte IL-1 receptor expression has a major impact on the course of inammatory reactions in the skin. Keratinocyte secreted IL-1a is one of the key cytokines in the initiation of cutaneous inammation. Human keratinocytes express two dierent receptor molecules for IL-1: IL-receptor type I (IL-1 RI) and IL-1 receptor type II (IL-1 RII). IL-1 RI serves as a signaling receptor, whereas IL-1 RII functions as a decoy receptor limiting or suppressing IL-1 mediated tissue responses. UV-B radiation regulates IL-1 RI and IL-1 RII expression dierentially in human keratinocytes [30]. Expression of IL-1RII is rapidly and

dramatically induced after UV-B radiation, whereas IL-1RI expression decreases at the same time (although at a later point it gradually increases). It has been proposed that UV-B radiation may limit excessive responses to IL-1 stimulation of keratinocytes under inammatory conditions by two complementary mechanisms: (1) increased expression of the decoy receptor IL-1RII and (2) decreased expression of the signaling molecule IL-1RI. Downregulation of the signaling receptor by UV-B radiation is not specic for IL-1a but may also be observed for other cytokines including TNF-alpha. In vitro exposure of human keratinocytes to sublethal doses of UV-B initially decreased mRNA and protein expression of the 55-kDa TNF receptor, which was subsequently followed by TNF receptor re-expression, eventually exceeding baseline levels [31]. At time points of decreased TNF receptor expression, TNF responsiveness of UV-Birradiated keratinocytes was signicantly reduced. UV-B did not aect the release of soluble TNF receptors from human keratinocytes. UV-B or UV-A1 radiation also failed to modulate the production of soluble ICAM-1 molecules produced by human keratinocytes [32]. In addition to cytokine receptors, growth factor receptors such as the epidermal growth factor receptor (EGFR) appear to be important target molecules for UV radiation as well as PUVA treatment. EGF is a growth factor for keratinocytes. EGFR expression and function are thought to be of central importance within the signal transduction cascade relevant for UV radiation induced gene expression. UV irradiation rapidly activates EGFR. EGFR activation is strongly mitogenic in many cell types including keratinocytes of the skin. UVinduced cutaneous proliferation results from EGFR activation [33]. EGFR may be an appropriate target for the chemoprevention of UVinduced skin cancer [34]. It has been proposed that PUVA induced inhibition of EGF binding might contribute to the benecial eects induced by PUVA therapy in psoriasis, which is characterized by keratinocyte hyperproliferation. Induction of apoptosis in skin inltrating cells T cells have an increased susceptibility toward UV radiation induced apoptosis when compared with other cell populations such as monocytes or

UV LIGHT AND ITS INTERACTION

637

keratinocytes. Morita and colleagues [35] were the rst to demonstrate that the induction of apoptosis in skin inltrating T cells is the basic mechanism in UV-A phototherapy of atopic dermatitis, leading to T-cell depletion from eczematous skin. Atopic dermatitis may be viewed as a T cellmediated skin disease in which activation of T-helper cells by inhalant allergens and other atopens leads to T-cell cytokine production and the subsequent development of eczema. This process involves an early initiation phase that is dominated by the expression of Th2-like cytokines, which is then switched into a second later phase with predominance of the Th1-like cytokine IFN-g [36]. According to this theory, IFN-g is responsible for the development and maintenance of clinically apparent eczema. Successful phototherapy of atopic dermatitis with UV-A1 radiation is associated with a marked reduction in the number of skin inltrating T cells and subsequent downregulation of IFN-g expression in lesional atopic skin [9]. Morita and colleagues demonstrated that UV-A1 phototherapy induced apoptosis in T-helper cells present in the dermal compartment of atopic eczema. After a few exposures of patients to single doses of UV-A radiation, apoptotic T cells were present in lesional atopic skin. Continuation of UV-A1 phototherapy led to a gradual increase in the number of apoptotic T-helper cells and a subsequent reduction of the inammatory inltrate and improvement of clinical symptoms [4,37,38]. UV-B irradiation also induced T-cell apoptosis. Apoptotic T cells were demonstrated in lesional psoriatic skin of patients undergoing UV-B phototherapy. Induction of T-cell apoptosis was observed regardless of whether broad-band or narrow-band UV-B was employed. Nevertheless, 311 nm UV-B radiation penetrates into deeper dermal levels; therefore, apoptosis occurs in both epidermal and dermal T cells. These dierences at least partially explain the clinical observation that narrow-band UV-B phototherapy is superior to broad-band UV-B phototherapy for the treatment of psoriasis [39]. Induction of T-cell apoptosis is thought to be a key mechanism in PUVA therapy. Evidence for the appearance of apoptotic T cells under PUVA therapy has thus far been provided for peripheral zary syndrome blood T cells in patients with Se undergoing extracorporeal photopheresis [40]. The induction of apoptotic cells is not an immunologically null event but most likely has immunosuppressive consequences. Phagocytosis of

apoptotic cells has profound eects on mediator production by macrophages [41]. After phagocytosis of apoptotic T cells, macrophage production of the anti-inammatory immunosuppressive cytokine IL-10 increases, whereas the production of proinammatory cytokines such as TNF-alpha, IL-1, and IL-2 is downregulated [41,42]. Further studies have demonstrated that inhibition of the production of proinammatory cytokines is mediated through the autocrine production of TGF-beta [43]. In addition, there is increased production of selected chemokines, in particular Mip-1-alfa and Mip-2. These studies provide a rationale to explain how extracorporeal photopheresis through the induction of apoptosis in only a small percentage of circulating T cells exerts immunosuppressive eects, as evidenced by the successful use of this modality in transplantation immunology. The mechanisms by which UV-A1 and UV-B radiation induce T-cell apoptosis dier markedly. In general, UV-A1 can cause preprogrammed cell death (early apoptosis), which is protein synthesis independent, as well as programmed cell death (late apoptosis), which requires de novo protein synthesis [44]. In contrast, UV-B irradiation as well as PUVA exclusively induce late apoptosis [45]. Morita and colleagues [35] have demonstrated that UV-A1 radiation can cause both early and late apoptosis and that UV-A1 radiation induced singlet oxygen generation is the initiating event leading to T-cell apoptosis. Singlet oxygen production induced the expression of Fas-ligand molecules on the surface of UV-A1 irradiated T cells. Subsequent binding of Fas ligand to Fas on the same or neighboring T cells was then shown to be responsible for T-cell apoptosis. The key role of singlet oxygen in eliciting early apoptosis in human T cells has been corroborated in an independent study employing Jurkat cells. UV-A1 radiation/singlet oxygen has been postulated to act on mitochondria and induce Jurkat cell apoptosis by opening the megachannel and by decreasing the mitochondrial membrane potential [45]. The capacity to induce early apoptosis in mammalian cells seems to be highly specic for UV-A1 radiation and singlet oxygen. From a phototherapeutic point of view, this qualitative dierence suggests that UV-A1 phototherapy is superior to UV-B or PUVA therapy for skin disease in which the induction of apoptosis in pathogenetically relevant cells is of critical importance. This assumption is supported by the observation

638

MATZ

that UV-A1 radiation, but not PUVA, is capable of inducing apoptosis in skin inltrating mast cells of patients with urticaria pigmentosa. As a consequence, UV-A1 phototherapy but not PUVA was associated with mast cell depletion from skin and longer lasting remission periods in these patients [46,47]. As is true for UV-A1 radiation and cutaneous T-cell lymphoma [48], in vitro studies indicate that malignant T cells are exquisitely sensitive to UV-A1 radiation induced apoptosis; therefore, UV-A1 phototherapy might prove to be at least equivalent, if not superior, to PUVA for this indication [49]. The lesional skin of patients with atopic dermatitis showed a signicant increase in p53positive dermal cells, apparently lymphocytes, after UV-A1 irradiation. In addition, some basal keratinocytes showed slight positive staining for p53 after treatment. By contrast, the expression of the bcl-2 gene in predominantly dermal lymphocytes was signicantly downregulated after UVA1 irradiation. The increase in p53-positive cells and decrease in bcl-2positive cells were closely linked to a signicant reduction in dermal T cells (CD3) and a substantial clinical improvement in skin condition. Medium-dose UV-A1 irradiation of atopic dermatitis skin lesions led to a marked modulation of the expression of p53 and bcl-2, which have a key role in regulating UV-A1 induced apoptosis [50]. Both T-cellderived skin diseases, atopic dermatitis and cutaneous T-cell lymphoma, exhibit an increased pre-therapeutic number of bcl-2 positive cells. After medium-dose UV-A1 phototherapy, the substantial improvement of the skin condition was linked to a signicant decrease of the dermal bcl-2positive cell count. Because the bcl-2 protein is known to act as an apoptosis inhibitor, its pre-therapeutic increase may provide the persistent cutaneous inammatory reaction in T-cellderived skin diseases. Moreover, the posttherapeutic reduction in bcl-2positive cells might represent a key mechanism of medium-dose UVA1 phototherapy [51]. UV-A1 aects the vascular dysregulation that is a primary pathogenetic factor of systemic sclerosis. Pre- and post-therapy skin biopsies of four patients with systemic sclerosis were evaluated immunohistochemically for angiostatic, angiogenic, and angioapoptotic features. These evaluations revealed a partial pre-therapy loss of endothelial CD31 and CD34 expression and a post-therapy increase in CD34 cells. Simultaneously, VEGF and M30

CytoDEATH immunolabeling demonstrated UV-A1 induced neovascularization and decreased endothelial apoptosis. These results suggest that UV-A1 irradiation exerts its positive eects in systemic sclerosis by a modulation of endothelial regulation/transformation besides the proposed induction of T-cell apoptosis and release of collagenases [52]. Photochemotherapy inhibits angiogenesis and induces apoptosis of human microvascular endothelial cells in vitro, which may be a possible mechanism of photochemotherapy in the treatment of psoriasis [53]. Narrow-band UV-B causes apoptosis in T lymphocytes but its eects on keratinocytes are unknown. Two types of cultured human keratinocytes, primary and immortalized, were exposed to narrow-band and broad-band UV-B and tested for apoptosis. Both UV-B light sources induced apoptosis in keratinocytes as determined by the presence of DNA ladders, although narrow-band UV-B required approximately tenfold higher doses (narrow-band UV-B, 1000 mJ/cm2 versus broad-band UV-B, 125 mJ/cm2). By comparison, lower doses of narrow-band UV-B (750 mJ/cm2) induced apoptosis in T lymphocytes, suggesting cell type specicity for narrow-band UV-B induced apoptosis. Approximately 50% or more of the cells underwent apoptosis when exposed to narrow-band or broad-band UV-B. Electron micrographs showed that narrowband UV-Birradiated keratinocytes showed marked chromatin condensation, extensive cytoplasmic vacuolization, and fragmentation of the nuclear envelope [54].

Photobiologic aspects of photochemotherapy The immunomodulatory eects of phototherapeutic relevance, the induction of IL-10 or suppression of ICAM-1 induction, may be achieved by both UV-B and UV-A1 irradiation. The photobiologic mechanisms responsible for these immunomodulatory eects dier greatly depending on the type of UV radiation used [55]. Immunomodulation induced by UV-B results from radiation induced generation of DNA photoproducts, in particular thymine dimers. UV-B induced inhibition of IFN-gstimulated keratinocyte ICAM-1 expression was associated with the formation of signicant numbers of thymine dimers in the irradiated human skin [25]. Topical application of a DNA repair enzyme encapsulated

UV LIGHT AND ITS INTERACTION

639

into liposomes decreased the number of thymine dimerpositive keratinocytes in irradiated skin by about 40% to 50% and completely prevented UV-B induced inhibition of ICAM-1. Identical results were obtained from in vitro studies in which the role of thymine dimer formation for UV-B induced IL-10 expression in cultured murine keratinocytes was assessed [56]. The precise reason for the discrepancy between the partial reversal in thymine dimers and the total prevention of immunomodulatory eects such as IL-10 synthesis or inhibition of ICAM-1 induction is currently unknown. It has been postulated that gene-specic repair mechanisms may explain this phenomenon. The formation of thymine dimers appears to be of central importance for UV-B radiation induced therapeutic eects, but UV-B radiation might also have cell membrane eects independent of DNA damage. In contrast to UV-B, UV-A1 radiation induced immunomodulatory eects are thought to be based on oxidative mechanisms [32]. The generation of singlet oxygen has a prominent role. This conclusion is based on the observation that UVA induced gene events such as the regulation of ICAM-1 or collagenase I expression can be (1) inhibited by singlet oxygen quenchers, (2) enhanced through strategies that result in an increase in the half-life of singlet oxygen, and (3) mimicked by stimulation of unirradiated cells with singlet oxygen generating systems [57]. Singlet oxygen is not only an important mediator for UV-A induced gene regulatory events but also has a key role in UV-A induced immunomodulation. The photobiologic basis for PUVA-induced immunosuppressive eects has not yet been characterized [58]. The ecacy of photochemotherapy may not simply be attributed to antiproliferative eects but most likely involves immunomodulatory consequences. The interplay of the various photobiologic pathways is not completely understood. Many factors including immune suppression, the alteration of cytokine expression, and cell cycle arrest may contribute to the suppression of disease activity. References
[1] Kripke ML. Immunologic mechanisms in UV radiation carcinogenesis. Adv Cancer Res 1981;34: 69106. [2] Krutmann J. Therapeutic photoimmunology: photoimmunological mechanisms in photo (chemo)

[3] [4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

therapy. J Photochem Photobiol B 1998;44(2): 15964. Horio T. Indications and action mechanisms of phototherapy. J Dermatol Sci 2000;1(Suppl 23):S1721. Everett M, Yeargers E, Sayre R, et al. Penetration of epidermis by ultraviolet rays. Photochem Photobiol 1966;5:53342. Luger TA, Schwartz T. Eects of UV light on cytokines and neuroendocrine hormones. In: Krutmann J, Elmets CA, editors. Photoimmunology. Oxford (UK): Blackwell Science; 1995. p. 5576. Morita A, Grewe M, Ahrens C, et al. Ultraviolet A1 radiation eects on cytokine expression in human epidermoid carcinoma cells. Photochem Photobiol 1997;65:6305. Grewe M, Gyufko K, Krutmann J. Interleukin-10 production by cultured human keratinocytes: regulation by ultraviolet B and ultraviolet A1 radiation. J Invest Dermatol 1995;104:36. Grewe M, Duvic M, Aragane Y, et al. Lack of induction of IL-10 expression in human keratinocytes. J Invest Dermatol 1996;106:13301. Grewe M, Gyufko K, Schopf E, et al. Lesional expression of interferon-gamma in atopic eczema. Lancet 1994;343:256. Grewe M, Trefzer U, Ballhorn A, et al. Analysis of the mechanism of ultraviolet B radiation induced prostaglandin E2 synthesis by human epidermoid carcinoma cells. J Invest Dermatol 1993;101:52831. Grewe M, Stege H, Vink A, et al. Inhibition of intercellular adhesion molecule-1 (ICAM-1) expression in ultraviolet B-irradiated human antigen-presenting cells is restored after repair of cyclobutane pyrimidine dimers. Exp Dermatol 2000;9(6):42330. Stege H, Berneburg M, Humke S, et al. High dose ultraviolet A1 (UVA1) radiation therapy for localized scleroderma. J Am Acad Dermatol 1997;36:93843. Wlascheck M, Heinen G, Poswig A, et al. UVA-induced autocrine stimulation of broblast derived collagenase/MMP-1 by interrelated loops of interleukin-1 and interleukin-6. Photochem Photobiol 1994;59:5506. Wang X, Bi Z, Chu W, et al. IL-1 receptor antagonist attenuate MAP kinase/AP-1 activation and MMP1 expression in UVA-irradiated human broblasts induced by culture medium from UVB-irradiated human skin keratinocytes. Int J Mol Med 2005; 16(6):111724. Sigmundsdottir H, Johnston A, Gudjonsson JE, et al. Narrow-band UVB irradiation decreases the production of pro-inammatory cytokines by stimulated T cells. Arch Dermatol Res 2005;297(1):3942. Lei TC, Vieira WD, Hearing V. In vitro migration of melanoblasts requires matrix metalloproteinase-2: implications to vitiligo therapy by photochemotherapy. Pigment Cell Res 2002;15(6):42632. Naderi-Hachtroudi L, Peters T, Brenneisen P, et al. Induction of manganese superoxide dismutase in

640 human dermal broblasts: a UV-B mediated paracrine mechanism with the release of epidermal interleukin 1 alpha, interleukin 1 beta, and tumor necrosis factor alpha. Arch Dermatol 2002;138(11): 14739. Lisby S, Ralfkier E, Rothlein R, et al. Intercellular adhesion molecule-1 (ICAM-1) expression correlated to inammation. 1989;120:47984. Krutmann J, Czech W, Diepgen T, et al. High dose UVA1 therapy in the treatment of patients with atopic dermatitis. J Am Acad Dermatol 1992;26: 22530. Krutmann J. Phototherapy for atopic dermatitis. Dermatol Ther 1996;1:2431. Krutmann J. High dose UVA-1 therapy for inammatory skin disease. Dermatol Ther 1997;4:1238. Krutmann J, Diepgen TL, Luger TA, et al. High dose UVA-1 therapy for atopic dermatitis: results of a multicenter trial. J Am Acad Dermatol 1998; 38:58993. Krutmann J. Therapeutic photomedicine: phototherapy. In: Freedberg IM, Eisen AB, Wol K, et al, editors. Fitzpatricks dermatology in general medicine. 5th edition. New York: Mcgraw-Hill; 1999. p. 28709. Roza L, Stege H, Krutmann J. Role of UV-induced DNA damage in phototherapy. In: Honigsmann H, Jori G, Young AR, editors. The fundamental bases of phototherapy. Milano (TX): OEMF spa; 1996. p. 14552. Stege H, Rosa L, Vink AA, et al. Enzyme plus light therapy to repair DNA damage in ultraviolet-Birradiated human skin. Proc Natl Acad Sci U S A 2000;97:17905. Khan IU, Boehm KD, Elmets CA. Modulation of IFN-gamma-induced HLA-DR expression on the human keratinocyte cell line SCC-13 by ultraviolet radiation. Photochem Photobiol 1993;57:1036. Krutmann J, Khan IU, Wallis RS, et al. The cell membrane is a major locus for ultraviolet-B-induced alterations in accessory cells. J Clin Invest 1990;85: 152936. Simon JC, Krutmann J, Elmets CA, et al. Ultraviolet B irradiated antigen presenting cells display altered accessory signaling for T-cell activation: relevance to immune responses initiated in the skin. J Invest Dermatol 1992;98:66S9S. Sigmundsdottir H, Gudjonsson JE, Valdimarsson H. The eects of ultraviolet B treatment on the expression of adhesion molecules by circulating T lymphocytes in psoriasis. Br J Dermatol 2003;148(5): 9961000. Grewe M, Gyufko K, Budnik A, et al. Interleukin-1 receptors type I and type II are dierentially regulated in human keratinocytes by ultraviolet B radiation. J Invest Dermatol 1996;107:86571. Trefzer U, Brockhaus M, Lotscher H, et al. The human 55-kd tumor necrosis factor receptor is regulated in human keratinocytes by TNF-alfa and bu

MATZ

[32]

[18]

[33]

[19]

[20] [21] [22]

[34]

[35]

[23]

[36]

[37]

[24]

[38]

[25]

[39]

[26]

[40]

[27]

[41]

[28]

[42]

[29]

[43]

[30]

[44]

[31]

[45]

ultraviolet B radiation. J Clin Invest 1993;92: 46270. Krutmann J. Regulatory interactions between epidermal cell adhesion molecules and cytokines. In: Luger TA, Schwartz T, editors. Epidermal growth factors and cytokines. New York: Dekker; 1994. p. 41532. EL-Abseri TB, Putta S, Hansen LA. Ultraviolet irradiation induces keratinocyte proliferation and epidermal hyperplasia through the activation of the epidermal growth factor receptor. Carcinogenesis 2006;27(2):22531. EL-Abaseri TB, Fuhrman J, Trempus C, et al. Chemoprevention of UV light induced skin tumorigenesis by inhibition of the epidermal growth factor receptor. Cancer 2005;65(9):395865. Morita A, Werfel T, Stege H, et al. Evidence that singlet oxygen induced human T-helper cell apoptosis in the basic mechanism of ultraviolet-A radiation phototherapy. J Exp Med 1997;186:17638. Grewe M, Bruijnzeel-Koomen CAFM, Schopf E, et al. A role for Th1 and Th2 cells in the immunopathogenesis of atopic dermatitis. Immunol Today 1998;19:35961. Bevilaqua PM, Edelson RL, Gasparro FP. High performance liquid chromatography analysis of 8-methoxypsoralen monoadducts and crosslinks in lymphocytes and keratinocytes. J Invest Dermatol 1991;97:1515. Epstein JH. UVL-induced stimulation of DNA synthesis in hairless mouse epidermis. J Invest Dermatol 1968;52:4458. Picot E, Meunier L, Picot-Deheze ML, et al. Treatment of psoriasis with a 311 nm UVB lamp. Br J Dermatol 1992;127:50912. Yoo EK, Rook AH, Elenitas R, et al. Apoptosis induction by ultraviolet light A and photochemotherapy in cutaneous T-cell lymphoma: relevance to mechanism of therapeutic action. J Invest Dermatol 1996;107:23542. Voll RE, Hermann M, Roth EA, et al. Immunosuppressive eects of apoptotic cells. Nature 1997;390: 3501. Fadok VA, Bratton DL, Konowal A, et al. Macrophages that have ingested apoptotic cells in vitro inhibit proinammatory cytokine production through autocrine/paracrine mechanisms involving TGFbeta, PGE2 and PAF. J Clin Invest 1998;101:8906. McDonald PP, Fadok VA, Bratton D, et al. Transcriptional and translational regulation of inammatory mediator production by endogenous TGF-beta in macrophages that have ingested apoptotic cells. J Immunol 1999;163:616472. Godar DE. Preprogrammed and programmed cell death mechanisms of apoptosis: UV induced immediate and delayed apoptosis. Photochem Photobiol 1996;63:82530. Godar DE. UVA1 radiation mediates singlet-oxygen and superoxide-anion production which trigger

UV LIGHT AND ITS INTERACTION

641

[46]

[47]

[48]

[49]

[50]

[51]

[52]

two dierent nal apoptotic pathways: the S and P site of mitochondria. J Invest Dermatol 1999;112: 312. Stege H, Schopf E, Ruzicka T, et al. High dose UVA1 for urticaria pigmentosa. Lancet 1996;347: 64. Guhl S, Stefaniak R, Strathmann M, et al. Bivalent eect of UV light on human skin mast cells low-level mediator release at baseline but potent suppression upon mast cell triggering. J Invest Dermatol 2005; 124(2):4536. Plettenberg H, Stege H, Megahed M, et al. Ultraviolet A1 (340400 nm) phototherapy for cutaneous Tcell lymphoma. J Am Acad Dermatol 1999;41:4750. Morita A, Yamauchi Y, Yasuda Y, et al. Malignant T cells are exquisitely sensitive to ultraviolet A-1 (UVA-1) radiation induced apoptosis. J Invest Dermatol 2000;122(2):47783. Breuckmann F, Pieck C, Kreuter A, et al. Opposing eects of UVA1 phototherapy on the expression of bcl-2 and p53 in atopic dermatitis. Arch Dermatol Res 2001;293(4):17883. Breuckmann F, Von-Kobyletzki G, Avermaete A, et al. Ecacy of ultraviolet A1 phototherapy on the expression of bcl-2 in atopic dermatitis and cutaneous T-cell lymphoma in vivo: a comparison study. Photodermatol Photoimmunol Photomed 2002; 18(5):21722. Breuckmann F, Stuecker M, Altmeyer P, et al. Modulation of endothelial dysfunction and apoptosis:

[53]

[54]

[55]

[56]

[57]

[58]

UVA1-mediated skin improvement in systemic sclerosis. Arch Dermatol Res 2004;296(5):2359. Deng H, Yan CL, Hu Y, et al. Photochemotherapy inhibits angiogenesis and induces apoptosis of endothelial cells in vitro. Photodermatol Photoimmunol Photomed 2004;20(4):1919. Auero BM, Talwar H, Young C, et al. Narrow band UVB induces apoptosis in human keratinocytes. J Photochem Photobiol B 2006;82(2): 1329. Krutmann J, Grewe M. Involvement of cytokines, DNA damage, and reactive oxygen species in ultraviolet radiation-induced modulation of intercellular adhesion molecule-1 expression. J Invest Dermatol 1995;105:67S70S. Nishigori C, Yarosh DB, Ultrich SE, et al. Evidence that DNA damage triggers interleukin to cytokine production in UV-irradiated murine keratinocytes. Proc Natl Acad Sci U S A 1996;93: 103549. Wlaschek M, Briviba K, Stricklin GP, et al. Singlet oxygen may mediate the ultraviolet A-induced synthesis of interstitial collagenase. J Invest Dermatol 1995;104:1948. Grether-Beck S, Olaizola-Horn S, Schmitt H, et al. Activation of transcription factor AP-2 mediates UVA radiation and singlet oxygen induced expression of the human intercellular adhesion molecule-1 gene. Proc Natl Acad Sci U S A 1996;93: 1458691.

Dermatol Clin 25 (2007) 643653

Beta Adrenergic Receptors in Keratinocytes

Raja K. Sivamani, MSa, Susanne T. Lam, BAa, R. Rivkah Issero, MDa,b,*
a

Department of Dermatology, University of California, Davis, School of Medicine, Dermatology Research, TB 192, One Shields Avenue, Davis, CA 95616, USA b Dermatology Service, Department of Veterans Aairs, Northern California Health Care System, Mather, CA 95655, USA

Of the several identied classes of adrenergic receptors (alpha and beta, and their subtypes), it is of particular interest to note that human keratinocytes, the cells the make up the majority of the epidermis, express only beta2 adrenergic receptors (beta2AR) [14]. The beta adrenergic receptor is a classic seven-transmembrane G-proteincoupled receptor. These receptors serve as the endogenous receptor to the catecholamine hormones norepinephrine and epinephrine, and can be further subdivided into beta1, beta2, and beta3 subtypes, based on their dierential pharmacological response to catecholamines and specic antagonists, as well as dierences in their protein sequences [57]. The rst studies to suggest the presence of betaAR in the epidermis were functional ones that demonstrated an increase in levels of cAMP in keratinocytes after stimulation with nonspecic betaAR agonists [8,9]. Subsequently, work using agonists and antagonists with specicity for the dierent adrenergic receptor subtypes demonstrated that the increase in keratinocyte intracelluar cAMP levels was observed only with agents specic for the beta2AR, suggesting that keratinocytes mainly expressed this receptor subtype [2]. This nding was conrmed by later studies that investigated binding anities of selective agonists and antagonists [1012], radioligand-binding assays
These authors contributed equally to this work. * Corresponding author. Department of Dermatology, University of California, Davis, School of Medicine, Dermatology Research, TB 192, One Shields Avenue, Davis, CA 95616. E-mail address: rrissero@ucdavis.edu (R.R. Issero).

of epidermal membrane fragments [3], and autoradiographic mapping of the beta adrenergic receptor subtypes on epidermal keratinocytes [4]. Since only the beta2AR is expressed in keratinocytes, this review will focus on this receptor subtype and its physiological role in the epidermis. The beta2AR transduces signals via a G-proteincoupled signaling cascade that classically includes adenylate cyclase-mediated increases in cAMP, although cAMP independent signaling cascades have recently been described and their regulatory roles are becoming clearer [13]. Stimulation of the beta2AR leads to a transient increase in the keratinocyte intracellular calcium concentration [14], and this likely occurs through several signaling cascades (Fig. 1). Activation of the cAMP-dependent pathway by direct activation of adenylate cyclase has been shown to reproduce the increase in intracellular calcium that is seen with ligand stimulation of the beta2AR [15,16]. Although beta2AR activation in other cell types, notably cardiac myocytes, results in modulation of activity of voltage-gated calcium channels [17,18], only recently has the presence of a voltage-gated calcium channel been observed in keratinocytes [19]. Thus, the question of whether or not the beta2AR-induced calcium transient in keratinocytes is mediated by this channel has not yet been addressed. The rise in intracellular calcium induced by beta2AR activation in keratinocytes may also be the result of release of intracellular stores, as suggested by Koizumi and colleagues [15,20], and this intracellular release may in turn trigger calcium-activated calcium inux through as yet unidentied store-operated calcium channel within the keratinocyte membrane (reviewed in
derm.theclinics.com

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.012

644

SIVAMANI

et al

Fig. 1. Keratinocyte beta2 adrenergic receptor signaling pathways. a, cAMP dependent signaling. b, PP2A dependent pathway. c, IP3 and PKC dependent pathway. d, pathway for beta2 adrenergic receptor blockade. The signaling depicted in the diagram is unique for keratinocytes. In other cell types, agonist activation of the receptor results in dierent downstream eector cascades. Dotted lines indicate signaling pathways where causal associations have been demonstrated but denitive mechanisms have not yet been dened (described in text).

reference [21]). Regardless of the specic channels that mediate the calcium inux secondary to beta2AR stimulation, it is clear that an increase in intracellular calcium results from the activation of the beta2AR, and that this increase can alter keratinocyte dierentiation. Increases in the intracellular calcium concentration inhibit keratinocyte proliferation while increasing phenotypic dierentiation [2224]. And indeed, Mammone and colleagues [25] demonstrated that activation of the beta2AR on a human keratinocyte cell line results not only in increases in cAMP levels but also in increased expression of keratins 1 and 10, involucrin, and transglutaminase, all markers of terminal dierentiation in keratinocytes. Taken together, the evidence suggests that beta2AR activation can up-regulate keratinocyte dierentiation by the cAMP-mediated signaling pathway, but does not rule out contributions of the less classical non-cAMPmediated signaling pathway. Future work will be needed to clarify the role of each signaling arm in the dierentiation process. In the epidermis, calcium concentrations are lower in the basal layer and increase toward the stratum corneum [26,27], correlating with the level of keratinocyte dierentiation. Interestingly, it has been shown that keratinocytes are capable of producing epinephrine [28,29], an endogenous

adrenergic agonist with selectivity for the beta2AR, and basal layer keratinocytes produce higher levels of epinephrine than suprabasal keratinocytes [29]. Thus, it has been proposed that epinephrine activates keratinocyte beta2AR to modulate calcium inux and begin the dierentiation cascade crucial to the native architecture of the epidermis [29]. The beta2AR desensitizes upon repeated activation through several mechanisms, including down-regulation of the number of beta2AR receptors (reviewed in references [30,31]). Indeed, beta2AR expression is more highly expressed at the basal layers of the epidermis and decreases in expression toward the stratum corneum [12], suggesting that epinephrine may be activating the receptor to increase intracellular calcium levels and induce dierentiation. However, further studies will be needed to determine how essential the autocrine epinephrine/ beta2AR signaling cascade is for keratinocyte differentiation and formation of the epidermal architecture. Several cAMP-independent signaling pathways have also been described for the beta2AR in many cell types, and this is true in keratinocytes as well. Epinephrine stimulation of the beta2AR has been shown to increase the activity of protein kinase C and formation of inositol-1,4,5-trisphosphate [20], which can stimulate calcium release from internal

BETA ADRENERGIC RECEPTORS IN KERATINOCYTES

645

reserves. In keratinocytes, beta2AR activation results in a decrease in the activity of extracellular signal-related kinase (ERK), mediated not by the cAMP-dependent pathway, but rather by its dephosphorylation, which is accomplished through the action of beta2AR-induced activation of the serine/threonine phosphatase PP2A [32,33]. This is in contradistinction to beta2AR activation in multiple other cell types, which typically results in ERK phosphorylation and activation [3437]. Classical, cAMP-dependent signaling with beta2AR activation has also been described in keratinocytes [38,39]. Thus, both cAMP-dependent and-independent signal transduction govern keratinocyte physiology, and continuing investigations will clarify which pathway controls which cellular functions.

Atopic dermatitis Numerous disparate theories have been proposed for the pathogenesis of atopic dermatitis in the past decades. Although the denitive mechanism of atopic dermatitis remains uncertain, the possible role of beta adrenoceptors has been implicated for many years. As early as 1968, Szentivanyi [40] hypothesized that the exaggerated vascular activity and increased sensitivity of pilomotor smooth muscle seen in the skin of patients with atopic dermatitis could be attributed to a decreased response to beta adrenergic receptor blockade, and he suggested that this was like the result of a deciency of adenylate cyclase. An alternative explanation for the reduced cAMP responsiveness to beta adrenergic agonists (such as isoproterenol) seen in leukocytes from atopic individuals has been proposed by Grewe and colleagues [41] and Hanin [42] who showed an increase in cAMP-phosphodiesterase activity in these patients leukocytes. In vitro studies by Reed and colleagues [43] showed that beta adrenergic agonists inhibited DNA synthesis in normal epidermis but had no inhibitory eect on DNA synthesis of either lichenied or normal-appearing skin in atopic dermatitis patients. Failure to evoke normal inhibition of cell division of basal cells thus seems plausible in the pathogenesis of epidermal hyperproliferation seen in atopic dermatitis. While some investigators proposed that there might be lower densities of beta2 adrenergic receptors in undierentiated keratinocytes [44], there is limited evidence of this, and in fact, other investigators have found normal adrenoceptor

densities but lower receptor binding anities, albeit on lymphocytes [45]. Nonetheless, these early studies have provided provocative evidence suggesting the involvement of beta adrenoceptors as contributing to the pathogenesis of atopic dermatitis. Focusing on the keratinocyte defects, Schallreuter and colleagues [44] have demonstrated that cell extracts from epidermal suction blister roofs of the uninvolved skin of atopic dermatitis patients have a signicantly increased concentrations of norepinephrine and the cofactor (6R)-Lerythro-5,6,7,8,-tetrahydrobiopterin (6BH4), required for the conversion of L-phenylalanine to L-tyrosine, and for the subsequent conversion of L-tyrosine to L-dopadtwo of the early steps in the catecholamine synthetic pathway (Fig. 2), compared with aged-matched controls. In addition, in this study there was a parallel threefold induction of the catecholamine degrading enzyme monoamine oxidase-A (MAO-A) activity with half the normal activity of the enzyme phenylethanolamine-N-methyl transferase (PNMT) required for conversion of norepinephrine into epinephrine (see Fig. 2) in patients with atopic dermatitis. A prior study demonstrated an increase in the catecholamine degrading enzyme catechol-o-methyl transferase (COMT) levels in atopic epidermis [46]. Although circulating levels of norepinephrine have been reported to be between 1.5- and 2.5-fold increased in patients with atopic dermatitis as compared with controls [44,47], given the increase in the catecholamine degrading enzymes COMT and MAO-A in atopic epidermis, the source of the circulating catecholamines is likely not epidermal. Taken together these results support earlier observations of a perturbed catecholamine/beta adrenoceptor signaling in atopic dermatitis. Moreover, since gene polymorphisms of the beta2 adrenergic receptor gene have been found to inuence the structure and function of the receptor in other tissues [48], it is possible that these may also contribute to the altered responses in the beta2AR pathway seen in atopic epidermis. In a small preliminary study of four patients, this was found to be the case, with a single nucleotide polymorphism of the beta2AR noted in keratinocytes isolated from these patients, and the investigators propose that this can alter binding and perhaps function in aected keratinocytes [44]. Recently, Maestroni [49] investigated the interplay between the beta adrenergic system and the immune system. He found that beta2AR activation in skin and dendritic cells results in

646

SIVAMANI

et al

Fig. 2. Catecholamine synthesis pathway in atopic dermatitis and vitiligo. The solid arrows represent the normal pathway in the synthesis of norepinephrine and epinephrine from L-phenylalanine. The dashed arrows represent how this pathway is modulated in atopic dermatitis and vitiligo except that epidermal increases in the level COMT and MAO-A have only been shown for atopic dermatitis.

down-regulation of inammatory cytokine production and receptor expression, leading to reduced TH1 priming. He thus postulated that the beta adrenergic system plays a key role in modulating cytokine production and antigen presentation and can contribute to pathologic disorders of the skin such as atopic dermatitis. Overall, the evidence is suggestive of a defect in the beta2AR system as contributing to the pathogenesis of atopic dermatitis, but the exact biochemical pathways remain to be claried. Further investigation of beta adrenoceptors of the skin and their interaction with the immune system may oer new therapeutic approaches to atopic dermatitis.

Vitiligo Many theories have been proposed for the pathogenesis of vitiligo, including genetic, autoimmune, and oxidative damage etiologies (reviewed in references [13,50,51]). One pathogenic mechanism advanced by the work of Schallreuter and colleagues implicates the beta2AR signaling pathway and catecholamine synthetic network within the epidermis (recently reviewed in reference [13]). Both keratinocytes and melanocytes express beta2AR [4,10,11,52], both cell types

have the enzymatic machinery for catecholamine synthesis [52,53], and indeed, both have been demonstrated to generate norepinephrine (keratinocytes, but not melanocytes, can generate epinephrine) [5254]. Stimulation of the beta2AR on melanocytes results in an increase in intracellular levels of cAMP, and subsequently, increased melanogenesis [52]. Since the normal epidermal keratinocytes can endogenously generate epinephrine, they may be providing local stimulation of this beta2AR-mediated pathway for melanogenesis in the normal melanocyte. In vitiligo, there is accumulating evidence generated by the Schallreuter group that implicates both the catecholamine synthetic pathway and subsequent beta2AR signaling in the pathogenesis of the disease [5558]. First, it is interesting to note that systemic beta blockers can lead to exacerbation of vitiligo in some patients [55]. Since beta2AR-stimulation activates melanogenesis in vitro, it is tempting to propose that blockade of beta2AR on melanocytes in vivo may contribute to the decrease in melanin synthesis seen in vitiliginous skin. Second, patients with vitiligo exhibit increased plasma and urine concentrations of norepinephrine and its degradation products [5659]. One possible source may be from the keratinocytes within the vitiliginous areas that have

BETA ADRENERGIC RECEPTORS IN KERATINOCYTES

647

increased levels of 6BH4 (see Fig. 2) [57,58]. This defect, along with a concomitantly noted reduction in the activity of PNMT, in keratinocytes of aected vitiliginous epidermis (see Fig. 2) [57] may explain, in part, the accumulation of norepinephrine. Third, epidermal keratinocytes derived from the aected skin of patients with vitiligo (and not the clinically healthy appearing skin in these patients), demonstrate increased expression of the beta2AR in culture [12]. The expression of these receptors on keratinocytes is regulated by extracellular levels of calcium [12] as well as by 6-biopterin, the redox product of 6BHR, which is increased in the epidermis in patients with vitiligo (reviewed in references [13,60]). Since beta2AR activation by catecholamines also induces calcium inux into keratinocytes [14], one might have anticipated that intracellular calcium levels are increased in keratinocytes in vitiliginous epidermis. However, a defect in calcium inux has been demonstrated in keratinocytes derived from involved vitiliginous epidermis [61], although these data may be represented by a limited number of patient samples. How, or if, this possible defect in keratinocyte calcium ux plays a role in the pathogenesis of vitiligo, is not clear at this time. Nevertheless, the cumulative evidence points to an associated defect in the catecholamine synthetic pathway and to abnormal beta2AR expression in the vitiliginous epidermis, and it is likely that continued investigation will uncover the mechanisms by which these converge to dysregulate melanogenesis in vitiligo. Pharmacological manipulation of the beta2AR may then oer another tool for the treatment of vitiligo.

Psoriasis Psoriasis is yet another disease in which the beta2AR has been implicated in its pathogenesis. Studies by Voorhees and colleagues and Flaxman and Harper [8,9,6264] over 30 years ago demonstrated the regulation of keratinocyte proliferation by intracellular levels of cAMP, and that elevation of intracellular cAMP by catecholamine stimulation resulted in a decrease in proliferation. Work by these laboratories also suggested that a decrease in the ability of psoriatic keratinocytes to respond to beta adrenergic agonists with an increase in cAMP could be, in part, responsible for the increase in cell proliferation characteristic of the disease [6568]. These studies relied on keratome sections of psoriatic epidermis, but a later

study by Iizuka and colleagues [69] demonstrated similar ndings in epidermis microdissected from involved psoriatic skin free of stratum corneum, dermis, and appendages. These preparations also generated decreased levels of cAMP after stimulation with epinephrine, when compared with epidermal preparation microdissected from uninvolved skin. Generation of cAMP in response to other activators of adenylate cyclase, such as histamine, was intact, suggesting that the defect in the psoriatic epidermis resided in the beta adrenergic receptor pathway. Similar ndings were obtained by other investigators measuring the generation of cAMP in response to beta adrenergic agonist stimulation of cell membrane fractions derived from psoriatic and normal epidermis [70]. This reduced response is likely a function of decreased expression of receptors on keratinocytes from involved psoriatic epidermis, as has been demonstrated by direct binding studies [71]. Consistent with these ndings is the more recent report of decreased expression of beta 2 adrenergic receptor mRNA in involved psoriatic epidermis shown by reverse transcriptase polymerase chain reaction (RT-PCR) [72]. Together, these ndings suggest that the down-regulation of the number of beta adrenergic receptors, rather than an inherent defect in the receptor itself, is the mechanism that is responsible for the reduced beta-adrenergic responsiveness seen in psoriatic epidermis. This decreased response to endogenous agonists then results in a decrease in intracellular cAMP and thus an increase in keratinocyte proliferation. Adding support to the hypothesis that beta adrenergic receptor dysregulation contributes to the pathogenesis of psoriasis, are the multiple reports that beta adrenergic antagonists can exacerbate the disease. Systemic use of beta blockers, a common approach for hypertension, or even their topical use in eyedrops for glaucoma, has been reported to either exacerbate existing psoriasis, or to initiate the disease (reviewed in references [73,74]). The drug-induced psoriasis often remits when the drug is withdrawn. A guinea pig model for psoriasis has been proposed, using animals treated with systemic beta blockers [75,76], although not every histologic nding of the disease is replicated in this model [77]. It has been speculated that in addition to the direct eect of the beta blockers on keratinocyte proliferation, these drugs may be inducing an immunologic component that exacerbates the disease [78] and, in fact, in patients who have had psoriatic exacerbations in response to treatment with the beta

648

SIVAMANI

et al

blocker propanol, a lymphocytic hypersensitivity to the drug has been demonstrated [79,80]. Recently, data suggesting that beta2AR polymorphisms may contribute to the pathogenesis of some psoriatics by altering beta2AR has been reported [81]. Polymorphisms that involve amino acid substitutions at position 16 and 27 have previously been shown to alter functional properties of beta2AR in humans causing beta2AR desensitization. The polymorphic Gly16 beta2AR demonstrates prolonged down-regulation following long-term agonist exposure as compared with the wild-type Arg16 receptor [82]. Interestingly, in a recent study of 50 subjects, it was found that the Arg16 allele frequency was signicantly higher in the sporadic psoriasis group as compared with controls [81]. Since increased circulating levels of catecholamines have been observed in psoriatic patients [83], and a 10-fold increase in the expression of the epinephrine synthetic enzyme PNMT is also found in basal keratinocytes in involved psoriatic epidermis [84], it is tempting to propose that long-term exposure to increased levels of catecholamines, in the circulation or locally derived by the keratinocytes themselves, in combination with increased desensitization of beta2AR in individuals with the Arg16 allele, may predispose to psoriasis. In view of the noted abnormalities in the beta2AR response in psoriatic epidermis, it is somewhat surprising that pharmacologic approaches addressing this target have not been vigorously pursued. One US patent for the use of isoproterenol for the treatment of psoriasis was approved in 1977 [85], but no commercial product appears to have resulted as a consequence. However, it may be that currently used therapies also work by modifying this signaling pathway. For example, vitamin D, currently used as a topical treatment of psoriasis, has been shown to increase the generation of cAMP in response to betaAR agonists [86]. Glucocorticoids, the mainstay of topical therapy for psoriasis, increase both the expression of beta2AR in keratinocytes, and the generation of cAMP in response to agonists [87]. UVB irradiation, another mainstay in the treatment of psoriasis, has been shown to increase beta2AR-mediated cAMP accumulation [88]. Thus it is possible that the various eective treatments for psoriasis in some way improve the disease by modulating the beta2AR response in keratinocytes. Since the safety proles of beta agonists are well documented, it may be worthwhile revisiting this pharmaceutical class of drugs for

ecacy in the treatment of psoriasis, perhaps as adjunctive therapies.

Wound healing The identication of beta AR on keratinocytes in the 1970s was followed by studies that focused on correlating their activation and subsequent generation of cAMP with cellular consequences. Thus, early studies correlated beta AR agonist stimulation and intracellular increases of cAMP in keratinocytes with a decrease in cell proliferation [9,62]. Studies by Dunlap [89], and Donaldson and Mahan [90], demonstrated that incubation of newt keratinocytes with beta agonists inhibited their migration, but other studies failed to conrm the presumed relationship between the increase in intracellular cAMP and inhibition of cell migration in either human [91] or newt keratinocytes [89,92,93]. Recent work in our laboratory has focused on the role of the beta2AR in keratinocyte migration, and particularly with respect to cutaneous wound repair. We found that activation of the beta2AR by isoproterenol both increased intracellular cAMP concentrations and inhibited keratinocyte migration in a concentration-dependent manner [32]. However, we demonstrated that the beta2AR-mediated decrease in migration is not regulated by the induced increase in cAMP, but rather by a cAMP-independent signaling pathway (see Fig. 1). Evidence for this conclusion included the ndings that adenylate cyclase inhibitors that decreased intracellular cAMP failed to prevent the inhibition of keratinocyte migration induced by isoproterenol, and agents that directly activate adenylate cyclase, such as forskolin, induced an increase in intracellular concentrations of cAMP yet had no inhibitory eect on migration. In keratinocytes, activation of the beta2AR results in dephosphorylation and inactivation of phosphoERK MAP kinase, through the activation of the protein phosphatase PP2A [33]. ERK is activated by wounding [94] and is required for wound repair both in conuent keratinocyte cultures [95] and in human epidermis [96,97]. Thus, inactivation of ERK by its dephosphorylation as a direct consequence of activation of the beta2AR results in a reduced capacity of the keratinocytes to migrate. This inability to migrate in the presence of beta agonists then translates to in an inability to reepithelialize a cutaneous wound, either in ex vivo cultured human skin, or in vivo in a murine tailwound model [54]. Taken together, these studies

BETA ADRENERGIC RECEPTORS IN KERATINOCYTES

649

demonstrate that beta2AR agonists impair cutaneous wound epithelialization. The nding that beta2AR agonists impair wound healing prompted the logical question of whether beta2AR antagonists (beta blockers) can, on the other hand, improve it. This is indeed the case. Cultured human keratinocytes treated with beta2AR antagonists demonstrate an increase in migratory speed, and this translates to better ability to reepithelialize cutaneous wounds in either ex vivo organ cultured human skin [28], or in vivo in murine wounded skin [98,99]. As might be anticipated, in keratinocytes, beta blockers increase the activation of the pro-motogenic ERK and its phosphorylation [28]. If beta blockers can improve wound epithelialization, the question of what endogenous ligand they are blocking naturally ensues. Two sources of ligand can be proposed. Circulating levels of epinephrine are increased posttrauma [100,101] and thus exogenously added beta blockers could be preventing their action on beta2AR in keratinocytes, thereby enhancing migratory speed and wound epithelialization. Alternatively, since keratinocytes have the enzymatic machinery to generate catecholamines [1,28], they can, and do synthesize endogenous epinephrine [28,53], which could be locally secreted into the wound and function in an autocrine manner. In either scenario, the pharmacologic use of beta blockers would result in the observed increase in wound epithelialization. Other studies have also provided additional evidence that beta blockers may be benecial to cutaneous wound repair. Recently, Denda and colleagues [102] reported that beta2AR antagonists could repair the epidermal permeability barrier, a function required for the restoration of epidermal integrity after wounding. Additionally, beta blockers are also widely used in the post-burn wound recovery period, and a retrospective outcome analysis by Arbabi and colleagues [103] has

demonstrated a shorter time for burn wound healing in a cohort of patients that received beta blockers during their hospital stay. Thus, there are suggestions in the literature that both the endogenous beta2AR signaling network and exogenously supplied beta2AR antagonists may modulate wound repair in the clinical setting. Interestingly, preliminary epidemiologic studies have suggested a decrease in the incidence of one type of chronic wound, venous stasis ulcers, in patients in whom beta blockers are prescribed [104]. Clearly, this provocative nding will need further investigation to determine if systemic or topically administered beta blockers can be useful therapeutic approaches to healing chronic wounds. A more systematic epidemiological analysis of wound healing in cohorts of patients treated with beta2AR antagonists is warranted, and a clinical trial of these agents in patients with chronic wounds could help answer the question of clinical usefulness of this class of drugs. These studies may be on the horizon.

Perspective Beta2 adrenergic receptors were identied in keratinocytes more than 30 years ago, but their function in the epidermis continues to be elucidated. Abnormalities in their expression, signaling pathway, or in the generation of endogenous catecholamine agonists by keratinocytes have been implicated in the pathogenesis of cutaneous diseases such as atopic dermatitis, vitiligo, and psoriasis (Table 1). Although the keratinocyte defects appear to be similar in some of these diseases, it may be that in patients with an associated defect in melanocyte beta2AR signaling, vitiligo ensues, while in patients with associated abnormalities in lymphocytic immunologic function, atopic dermatitis is the outcome. New studies indicate that the beta2AR also modulates

Table 1 Proposed modulation of the keratinocyte beta2 adrenergic receptor and the catecholamine synthesis pathway in selected dermatological diseases Disorder Atopic dermatitis Vitiligo Psoriasis Beta2AR expression/ activation NA [ Y 6BH4 [ [ NA PNMT activity Y Y [ Norepinephrine [ [ NAa Epinephrine Y Y NAa

Abbreviation: NA, not yet denitively addressed. a Elevated circulating levels of norepinephrine and epinephrine have been reported in psoriasis but epidermal or keratinocyte levels have not yet been studied.

650

SIVAMANI

et al [11] Steinkraus V, Korner C, Steinfath M, et al. High density of beta 2-adrenoceptors in a human keratinocyte cell line with complete epidermal dierentiation capacity (HaCaT). Arch Dermatol Res 1991; 283(5):32832. [12] Schallreuter KU, Wood JM, Pittelkow MR, et al. Increased in vitro expression of beta 2-adrenoceptors in dierentiating lesional keratinocytes of vitiligo patients. Arch Dermatol Res 1993;285(4): 21620. [13] Grando SA, Pittelkow MR, Schallreuter KU. Adrenergic and cholinergic control in the biology of epidermis: physiological and clinical signicance. J Invest Dermatol 2006;126(9):194865. [14] Koizumi H, Yasui C, Fukaya T, et al. Beta-adrenergic stimulation induces intracellular Ca increase in human epidermal keratinocytes. J Invest Dermatol 1991;96(2):2347. [15] Yasui C, Koizumi H, Fukaya T, et al. Adenylate cyclase induces intracellular calcium increase in single human epidermal keratinocytes measured by uorescence microscopy using Fura 2-AM. Br J Dermatol 1992;127(6):58994. [16] Osawa Y, Koizumi H, Fukaya T, et al. Adenylate cyclase induces intracellular Ca2 increase in single human epidermal keratinocytes of the epidermal sheet as measured by digital imaging microscopy using Fura 2-AM. Arch Dermatol Res 1991;283(2):915. [17] Findlay I. Physiological modulation of inactivation in L-type Ca2 channels: one switch. J Physiol 2004;554(Pt 2):27583. [18] Zhou YY, Cheng H, Bogdanov KY, et al. Localized cAMP-dependent signaling mediates beta 2-adrenergic modulation of cardiac excitation-contraction coupling. Am J Physiol 1997;273(3 Pt 2):H16118. [19] Denda M, Fujiwara S, Hibino T. Expression of voltage-gated calcium channel subunit alpha1C in epidermal keratinocytes and eects of agonist and antagonists of the channel on skin barrier homeostasis. Exp Dermatol 2006;15(6):45560. [20] Koizumi H, Tanaka H, Ohkawara A. Beta-adrenergic stimulation induces activation of protein kinase C and inositol 1,4,5-trisphosphate increase in epidermis. Exp Dermatol 1997;6(3):12832. [21] Mauro T. The discovery channel: CRACking the code of calcium inux. J Invest Dermatol 2003;121(1):IXX. [22] Hennings H, Michael D, Cheng C, et al. Calcium regulation of growth and dierentiation of mouse epidermal cells in culture. Cell 1980;19(1):24554. [23] Boyce ST, Ham RG. Calcium-regulated dierentiation of normal human epidermal keratinocytes in chemically dened clonal culture and serum-free serial culture. J Invest Dermatol 1983;81(1 Suppl): 33s40s. [24] Tsao MC, Walthall BJ, Ham RG. Clonal growth of normal human epidermal keratinocytes in a dened medium. J Cell Physiol 1982;110(2):21929.

keratinocyte migration, and thus can function to regulate wound re-epithelialization. Cumulatively, these studies suggest a role for beta2AR active drugs in the management of cutaneous disease and wound healing. It is interesting to note that there appear to be only three US patents registered for the use of beta adrenergic agents for skin disease: one for beta agonists for the treatment of psoriasis [85], one for beta agonists to decrease skin edema in acute surgical wounds [105], and a third for the topical use of beta agonists in cosmetics or topical medications to decrease substance P-mediated pain or irritation [106]. Thus, ample opportunity exists for development of beta2AR active drugs as therapeutic agents for a number of dermatological indications. References
[1] Schallreuter KU. Epidermal adrenergic signal transduction as part of the neuronal network in the human epidermis. J Investig Dermatol Symp Proc 1997;2(1):3740. [2] Orenberg EK, Pfendt EA, Wilkinson DI. Characterization of alpha- and beta-adrenergic agonist stimulation of adenylate cyclase activity in human epidermal keratinocytes in vitro. J Invest Dermatol 1983;80(6):5037. [3] Steinkraus V, Steinfath M, Korner C, et al. Binding of beta-adrenergic receptors in human skin. J Invest Dermatol 1992;98(4):47580. [4] Steinkraus V, Mak JC, Pichlmeier U, et al. Autoradiographic mapping of beta-adrenoceptors in human skin. Arch Dermatol Res 1996;288(9):54953. [5] Frielle T, Daniel KW, Caron MG, et al. Structural basis of beta-adrenergic receptor subtype specicity studied with chimeric beta 1/beta 2-adrenergic receptors. Proc Natl Acad Sci U S A 1988;85(24): 94948. [6] Lands AM, Arnold A, McAuli JP, et al. Dierentiation of receptor systems activated by sympathomimetic amines. Nature 1967;214(88):5978. [7] Zheng M, Zhu W, Han Q, et al. Emerging concepts and therapeutic implications of [beta]-adrenergic receptor subtype signaling. Pharmacol Ther 2005; 108(3):25768. [8] Powell JA, Duell EA, Voorhees JJ. Beta adrenergic stimulation of endogenous epidermal cyclic AMP formation. Arch Dermatol 1971;104(4):35965. [9] Flaxman BA, Harper RA. In vitro analysis of the control of keratinocyte proliferation in human epidermis by physiologic and pharmacologic agents. J Invest Dermatol 1975;65(1):529. [10] Gazith J, Cavey MT, Cavey D, et al. Characterization of the beta-adrenergic receptors of cultured human epidermal keratinocytes. Biochem Pharmacol 1983;32(22):3397403.

BETA ADRENERGIC RECEPTORS IN KERATINOCYTES

651

[25] Mammone T, Marenus K, Maes D, et al. The induction of terminal dierentiation markers by the cAMP pathway in human HaCaT keratinocytes. Skin Pharmacol Appl Skin Physiol 1998;11(3): 15260. [26] Menon GK, Elias PM. Ultrastructural localization of calcium in psoriatic and normal human epidermis. Arch Dermatol 1991;127(1):5763. [27] Menon GK, Grayson S, Elias PM. Ionic calcium reservoirs in mammalian epidermis: ultrastructural localization by ion-capture cytochemistry. J Invest Dermatol 1985;84(6):50812. [28] Pullar CE, Rizzo A, Issero RR. Beta-adrenergic receptor antagonists accelerate skin wound healing: evidence for a catecholamine synthesis network in the epidermis. J Biol Chem 2006;281(30):2122535. [29] Schallreuter KU, Lemke KR, Pittelkow MR, et al. Catecholamines in human keratinocyte dierentiation. J Invest Dermatol 1995;104(6):9537. [30] Broadley KJ. Review of mechanisms involved in the apparent dierential desensitization of beta1and beta2-adrenoceptor-mediated functional responses. J Auton Pharmacol 1999;19(6):33545. [31] Johnson M. Beta2-adrenoceptors: mechanisms of action of beta2-agonists. Paediatr Respir Rev 2001;2(1):5762. [32] Chen J, Homan BB, Issero RR. Beta-adrenergic receptor activation inhibits keratinocyte migration via a cyclic adenosine monophosphate-independent mechanism. J Invest Dermatol 2002;119(6):12618. [33] Pullar CE, Chen J, Issero RR. PP2A activation by beta2-adrenergic receptor agonists: novel regulatory mechanism of keratinocyte migration. J Biol Chem 2003;278(25):2255562. [34] Kim N, Kim H, Youm JB, et al. Site specic dierential activation of ras/raf/ERK signaling in rabbit isoproterenol-induced left ventricular hypertrophy. Biochim Biophys Acta 2006;1763(10):106775. [35] Zheng M, Hou R, Han Q, et al. Dierent regulation of ERK1/2 activation by beta-adrenergic receptor subtypes in adult mouse cardiomyocytes. Heart Lung Circ 2004;13(2):17983. [36] Shenoy SK, Drake MT, Nelson CD, et al. G protein-independent ERK1/2 activation by the beta2 adrenergic receptor. J Biol Chem 2006;281(2): 126173. [37] Yeh CK, Ghosh PM, Dang H, et al. Beta-adrenergic-responsive activation of extracellular signalregulated protein kinases in salivary cells: role of epidermal growth factor receptor and cAMP. Am J Physiol Cell Physiol 2005;288(6):C135766. [38] Pullar CE, Issero RR. Cyclic AMP mediates keratinocyte directional migration in an electric eld. J Cell Sci 2005;118(Pt 9):202334. [39] Pullar CE, Issero RR, Nuccitelli R. Cyclic AMPdependent protein kinase A plays a role in the directed migration of human keratinocytes in a DC electric eld. Cell Motil Cytoskeleton 2001;50(4): 20717.

[40] Szentivanyi A. The beta adrenergic theory of the atopic abnormality in bronchial asthma. J Allergy 1968;42:20332. [41] Grewe SR, Chan SC, Hanin JM. Elevated leukocyte cyclic AMP-phosphodiesterase in atopic disease: a possible mechanism for cyclic AMP-agonist hyporesponsiveness. J Allergy Clin Immunol 1982; 70(6):4527. [42] Hanin JM. Pharmacophysiology of atopic dermatitis. Clin Rev Allergy 1986;4(1):4365. [43] Reed CE, Busse WW, Lee TP. Adrenergic mechanisms and the adenyl cyclase system in atopic dermatitis. J Invest Dermatol 1976;67(3):3338. [44] Schallreuter KU, Pittelkow MR, Swanson NN, et al. Altered catecholamine synthesis and degradation in the epidermis of patients with atopic eczema. Arch Dermatol Res 1997;289(12):6636. [45] Pochet R, Delaspesse G, DeManbuege J. Characterization of beta adrenoceptors on intact circulating lymphocytes from patients with atopic dermatitis. Acta Derm Venereol Suppl (Stockh) 1980;92:269. [46] Bamshad J. Catechol-o-methyl transferase in skin of patients with atopic dermatitis. J Invest Dermatol 1969;52(1):1002. [47] Buske-Kirschbaum A, Geiben A, Hollig H, et al. Altered responsiveness of the hypothalamus-pituitary-adrenal axis and the sympathetic adrenomedullary system to stress in patients with atopic dermatitis. J Clin Endocrinol Metab 2002;87(9): 424551. [48] Small KM, McGraw DW, Liggett SB. Pharmacology and physiology of human adrenergic receptor polymorphisms. Annu Rev Pharmacol Toxicol 2003;43:381411. [49] Maestroni GJ. Sympathetic nervous system inuence on the innate immune response. Ann N Y Acad Sci 2006;1069:195207. [50] Le Poole IC, Wankowicz-Kalinska A, van den Wijngaard RM, et al. Autoimmune aspects of depigmentation in vitiligo. J Investig Dermatol Symp Proc 2004;9(1):6872. [51] Njoo MD, Westerhof W. Vitiligo. Pathogenesis and treatment. Am J Clin Dermatol 2001;2(3):16781. [52] Gillbro JM, Marles LK, Hibberts NA, et al. Autocrine catecholamine biosynthesis and the beta-adrenoceptor signal promote pigmentation in human epidermal melanocytes. J Invest Dermatol 2004;123(2):34653. [53] Schallreuter KU, Wood JM, Lemke R, et al. Production of catecholamines in the human epidermis. Biochem Biophys Res Commun 1992;189(1): 728. [54] Pullar CE, Grahn JC, Liu W, et al. Beta2-adrenergic receptor activation delays wound healing. FASEB J 2006;20(1):7686. [55] Schallreuter KU. Beta-adrenergic blocking drugs may exacerbate vitiligo. Br J Dermatol 1995; 132(1):1689.

652

SIVAMANI

et al [71] Steinkraus V, Steinfath M, Stove L, et al. Betaadrenergic receptors in psoriasis: evidence for downregulation in lesional skin. Arch Dermatol Res 1993; 285(5):3004. [72] Takahashi H, Kinouchi M, Tamura T, et al. Decreased beta 2-adrenergic receptor-mRNA and loricrin-mRNA, and increased involucrin-mRNA transcripts in psoriatic epidermis: analysis by reverse transcription-polymerase chain reaction. Br J Dermatol 1996;134(6):10659. [73] OBrien M, Koo J. The mechanism of lithium and beta-blocking agents in inducing and exacerbating psoriasis. J Drugs Dermatol 2006;5(5):42632. [74] Tsankov N, Kazandjieva J, Drenovska K. Drugs in exacerbation and provocation of psoriasis. Clin Dermatol 1998;16(3):33351. [75] Gaylarde PM, Brock AP, Sarkany I. Psoriasiform changes in guinea-pig skin from propranolol. Clin Exp Dermatol 1978;3(2):15760. [76] Van de Kerkhof P. Psoriasiform changes in guineapig skin from propanalol. Clin Exp Dermatol 1979; 4(2):2678. [77] Wolf R, Shechter H, Brenner S. Induction of psoriasiform changes in guinea pig skin by propranolol. Int J Dermatol 1994;33(11):8114. [78] Wolf R. A new concept in the pathogenesis of druginduced psoriasis. Med Hypotheses 1986;21(3): 2779. [79] Halevy S, Livni E. Psoriasis and psoriasiform eruptions associated with propranololdthe role of an immunological mechanism. Arch Dermatol Res 1991;283(7):4723. [80] Halevy S, Livni E. Beta-adrenergic blocking drugs and psoriasis: the role of an immunologic mechanism. J Am Acad Dermatol 1993;29(3):5045. [81] Ozkur M, Erbagci Z, Nacak M, et al. Association of the Arg16Gly polymorphism of the beta-2-adrenergic receptor with psoriasis. J Dermatol Sci 2004;35(2):1624. [82] Liggett SB. Beta(2)-adrenergic receptor pharmacogenetics. Am J Respir Crit Care Med 2000;161(3 Pt 2):S197201. [83] Schmid-Ott G, Jacobs R, Jager B, et al. Stress-induced endocrine and immunological changes in psoriasis patients and healthy controls. A preliminary study. Psychother Psychosom 1998;67(1):3742. [84] Johansson O, Olsson A, Enhamre A, et al. An immunohistochemical study on catecholamine synthesizing enzymes and neuropeptides of the skin. Acta Derm Venereol 1987;67(1):17. [85] Nelson EL. Nelson Research & Development Company, assignee. Method for treatment of psoriasis. US patent 4038417, 1977. [86] Takahashi H, Tamura T, Iizuka H. 1,25-Dihydroxyvitamin D3 increased beta-adrenergic adenylate cyclase response of fetal rat keratinizing epidermal cells (FRSK cells). J Dermatol Sci 1996;11(2):1218. [87] Takahashi H, Iizuka H. Regulation of beta 2-adrenergic receptors in keratinocytes: glucocorticoids

[56] Salzer BA, Schallreuter KU. Investigation of the personality structure in patients with vitiligo and a possible association with impaired catecholamine metabolism. Dermatology 1995;190(2):10915. [57] Schallreuter KU, Wood JM, Ziegler I, et al. Defective tetrahydrobiopterin and catecholamine biosynthesis in the depigmentation disorder vitiligo. Biochim Biophys Acta 1994;1226(2):18192. [58] Schallreuter KU, Wood JM. The importance of -phenylalanine transport and its autocrine turnover to -tyrosine for melanogenesis in human epidermal melanocytes. Biochem Biophys Res Commun 1999;262(2):4238. [59] Cucchi ML, Frattini P, Santagostino G, et al. Catecholamines increase in the urine of non-segmental vitiligo especially during its active phase. Pigment Cell Res 2003;16(2):1116. [60] Hasse S, Gibbons NC, Rokos H, et al. Perturbed 6-tetrahydrobiopterin recycling via decreased dihydropteridine reductase in vitiligo: more evidence for H2O2 stress. J Invest Dermatol 2004;122(2): 30713. [61] Schallreuter KU, Pittelkow MP. Defective calcium uptake in keratinocyte cell cultures from vitiliginous skin. Arch Dermatol Res 1988;280(3): 1379. [62] Harper RA, Flaxman BA. Eect of pharmacological agents on human keratinocyte mitosis in vitro. II. Inhibition by catecholamines. J Cell Physiol 1975;86(2 Pt 1):2939. [63] Voorhees JJ, Duell EA, Bass LJ, et al. Role of cyclic AMP in the control of epidermal cell growth and dierentiation. Natl Cancer Inst Monogr 1973;38: 4759. [64] Voorhees JJ, Duell EA, Kelsey WH. Dibutyryl cyclic AMP inhibition of epidermal cell division. Arch Dermatol 1972;105(3):3846. [65] Voorhees JJ, Duell EA. Psoriasis as a possible defect of the adenyl cyclase-cyclic AMP cascade. A defective chalone mechanism? Arch Dermatol 1971;104(4):3528. [66] Voorhees JJ, Duell EA. Imbalanced cyclic AMPcyclic GMP levels in psoriasis. Adv Cyclic Nucleotide Res 1975;5:73558. [67] Voorhees JJ, Duell EA, Bass LJ, et al. Decreased cyclic AMP in the epidermis of lesions of psoriasis. Arch Dermatol 1972;105(5):695701. [68] Voorhees JJ, Duell EA, Bass LJ, et al. The cyclic AMP system in normal and psoriatic epidermis. J Invest Dermatol 1972;59(1):11420. [69] Iizuka H, Umeda K, Koizumi H, et al. Epinephrine-induced cyclic AMP accumulation in the psoriatic epidermis. Acta Derm Venereol 1981;61(5): 3915. [70] Mahrle G, Orfanos CE. [Lack of beta-adrenergic stimulation of membrane bound adenyl cyclase in psoriasis as compared to normal epidermis (authors transl)]. Arch Dermatol Res 1975;253(2): 195202 [in German].

BETA ADRENERGIC RECEPTORS IN KERATINOCYTES

653

[88]

[89]

[90]

[91]

[92]

[93]

[94]

[95]

[96]

increase steady-state levels of receptor mRNA in foetal rat keratinizing epidermal cells (FRSK cells). Br J Dermatol 1991;124(4):3417. Iizuka H, Kajita S, Ohkawara A. Ultraviolet radiation augments epidermal beta-adrenergic adenylate cyclase response. J Invest Dermatol 1985; 84(5):4013. Dunlap MK. Cyclic AMP levels in migrating and non-migrating newt epidermal cells. J Cell Physiol 1980;104(3):36773. Donaldson DJ, Mahan JT. Inuence of catecholamines on epidermal cell migration during wound closure in adult newts. Comp Biochem Physiol C 1984;78(2):26770. Iwasaki T, Chen JD, Kim JP, et al. Dibutyryl cyclic AMP modulates keratinocyte migration without alteration of integrin expression. J Invest Dermatol 1994;102(6):8917. Dunlap MK, Donaldson DJ. Eect of cAMP and related compounds on newt epidermal cell migration both in vivo and in vitro. J Exp Zool 1980; 212(1):139. Donaldson DJ, Dunlap MK, Mahan JT. Eects of concanavalin A and cholera toxin on epidermal cAMP and migration rate during wound closure in adult newts. Comp Biochem Physiol C 1984; 79(2):2438. Matsubayashi Y, Ebisuya M, Honjoh S, et al. ERK activation propagates in epithelial cell sheets and regulates their migration during wound healing. Curr Biol 2004;14(8):7315. Davido MS, Ungefroren H, Middendor R, et al. Catecholamine-synthesizing enzymes in the adult and prenatal human testis. Histochem Cell Biol 2005;124(34):31323. McCaig CD, Rajnicek AM, Song B, et al. Controlling cell behavior electrically: current views and future potential. Physiol Rev 2005;85(3):94378.

[97] Providence KM, Higgins PJ. PAI-1 expression is required for epithelial cell migration in two distinct phases of in vitro wound repair. J Cell Physiol 2004; 200(2):297308. [98] Pullar CE, Rizzo AE, Issero RR. Beta-adrenergic receptor antagonists accelerate skin wound healing: evidence for a catecholamine synthesis network in the epidermis [abstract]J Invest Dermatol 2006;126:64. [99] Sivamani RK, Pullar CE, Griths B, et al. Beta2-adrenergic receptor blockade accelerates burn wound healing [abstract]J Invest Dermatol 2006;126:59. [100] Sedowoa K, Barclay C, Quaba A, et al. The systemic stress response to thermal injury in children. Clin Endocrinol (Oxf) 1998;49(3):33541. [101] Wilmore DW, Long JM, Mason AD Jr, et al. Catecholamines: mediator of the hypermetabolic response to thermal injury. Ann Surg 1974;180(4): 65369. [102] Denda M, Fuziwara S, Inoue K. Beta2-adrenergic receptor antagonist accelerates skin barrier recovery and reduces epidermal hyperplasia induced by barrier disruption. J Invest Dermatol 2003;121(1):1428. [103] Arbabi S, Ahrns KS, Wahl WL, et al. Beta-blocker use is associated with improved outcomes in adult burn patients. J Trauma 2004;56(2):2659 [discussion: 26971]. [104] Margolis D, Issero RR, Hofstad O. Beta adrenergic drug use is associated with decreased incidence of venous leg ulcers [abstract]J Invest Dermatol 2006;126:49. [105] McDonald DM. The Regents of the University of California, assignee. Use of formoterol for treatment of tissue injury. US patent 5135954, 1992. [106] Leclaire J, de Lacharriere O, Breton L, et al, assignee. Cosmetic/pharmaceutical compositions comprising b-adrenergic agonists/substance P antagonists. US patent 5958432, 1999.

Dermatol Clin 25 (2007) 655662

Receptors in Skin Ageing and Antiageing Agents

Ilaria Ghersetich, MD*, Michela Troiano, MD, Vincenzo De Giorgi, MD, Torello Lotti, MD
Department of Dermatology, University of Florence, Via Lorenzo il Magnico 104, 50129 Florence, Italy

Chronologic ageing and photoageing Ageing is a complex and multifactorial process that occurs in all individuals at a variable rate; it is inuenced by genetic, environmental, and hormonal factors that result in several functional and esthetic changes in the skin. This process can be divided into intrinsic or chronologic ageing due to the passage of the time that occurs in the protected as well as the sun-exposed area and extrinsic or photoageing caused by the superimposition of chronic sun damage on intrinsically aged skin. Chronologic ageing [1] results in only subtle changes, such as a slight atrophy, a loss of elasticity, ne wrinkling, and deepening of expression wrinkles. Those changes also are evident histopathologically. The epidermis appears atrophic and attened. In the dermis, broblasts, inammatory cells, and the microvasculature are reduced signicantly, and the collagen bundles become thick and disoriented [2]. Photoageing is the consequence of chronic sun exposure and manifests clinically with ne and coarse wrinkling, roughness, dryness, laxity, shallowness, pigmentary mottling, telangiectasia, and, in some cases, preneoplastic and neoplastic changes [3]. Photoaged skin is typied histopathologically by acanthosis, loss of epidermal polarity, a basket weave appearance of the epidermis, keratinocyte atypia, irregularly dispersed melanocytes, reduction and alteration in collagen, twisted and dilated microvasculature, and, most of all, deposition of abnormal elastoid material

in the upper dermis (elastosis). Many of the functions of skin that decline with age show an accelerated decline in photoaged skin (Figs. 1 and 2) [4]. Factors involved in ageing: genetic, environmental, and hormonal mechanisms, focusing on cutaneous receptors In the skin, genetic, environmental, and hormonal mechanisms likely contribute to the ageing process; however, there are dierent theories about ageing [4]. Genetic factors: telomere theory The rst theory states that ageing is a preordained process that is determined genetically. Support for this theory comes from telomere lengths, the terminal portions of chromosomes that shorten at every cell cycle. By maintaining a constant telomere length, telomerase ensures retention of chromosomal stability attributed to functional telomeres and alters the cells biologic clock [5]; however, when telomeres reach a critical length, cell cycle arrest or apoptosis occurs [6]. Furthermore, primary cell cultures cannot continue to replicate indenitely, which is believed by some investigators to be a cancer-prevention strategy [7]. It has been proposed that telomerase activity is critical in the control of ageing. For example, telomerase-null mice exhibit a decreased life span after multiple generations, correlating with telomere length, and a decreased ability to respond to stress (eg, wound healing) [8]. Transduction of aged human broblasts with telomerase immortalizes the cells and reverses the aged phenotype when the broblasts are used to construct skin
derm.theclinics.com

* Corresponding author. E-mail address: i.ghersetich@dermatology.it (I. Ghersetich).

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.018

656

GHERSETICH

et al

Fig. 1. Chronologic aging.

equivalents [9]. The proliferative capability of human broblasts from donors of varying ages correlates well with telomere length. [10]. Nevertheless, in human epidermis, only basal keratinocytes express telomerase, whereas dierentiating suprabasal cells and the underlying dermal broblasts are telomerase negative [11]. Gilhar and colleagues [12] observed that there is no correlation between epidermal telomerase levels and epidermal ageing, suggesting that in a self-renewing tissue with slowly cycling stem cells, telomerase is not a limiting factor in ageing. Environmental factors: UV Another theory suggests that ageing is largely due to cumulative environmental damage [413].

Fig. 2. Photoaging.

In the skin, the major environmental inuence is sun exposure. It is estimated that approximately 50% of UV-induced damage is from the formation of free radicals. Free radicals can be generated from oxygen during normal metabolism and likely contribute to this process [14]. Organisms have evolved cellular-defense systems against the toxicity of free radicals, particularly oxygenbased free radicals, or reactive oxygen species (ROS). Longer-lived species have higher degrees of enzymatic protection against ROS [15]. The activity of antioxidant enzymes [13] and the levels of nonenzymatic antioxidants decline with age, allowing oxidative damage to occur. UV radiation can induce ROS, which damage cellular DNA, lipids, and proteins [16], accelerating the skin-ageing process. These DNA mutations may be related clinically to specic signs of photoageing, such as wrinkling, an increase in elastin, and collagen damage [17]. Sun exposure also has eects on pigmentation, causes vascular alteration and immunosuppression (which may have implications in reduced cutaneous tumor surveillance), alters the expression of selected genes, and has eects on the extracellular matrix. Recent studies demonstrated that UV irradiation alters the relative proportion of extracellular components (collagen, elastin, and proteoglycans) or degradative pathways that render these molecules nonfunctional [18]. Molecular alterations in the dermis include a decrease in collagen synthesis, induction of matrix metalloproteinases (MMPs), abnormal accumulation of elastic bers, and proteoglycans [19]. Transforming growth factor (TGF)-b, a multifunctional cytokine that regulates cell proliferation and dierentiation, tissue remodelling, and repair, is an important factor that is involved in the synthesis of extracellular matrix proteins [20]. TGF-b is a potent growth inhibitor of the epidermis, whereas in the dermis, TGF-b acts as a positive growth factor, inducing the synthesis of extracellular matrix proteins. TGF-b is known to stimulate broblast proliferation in the dermis, induce the synthesis and secretion of the major extracellular matrix proteins [21], and down-regulate the expressions of proteolytic enzymes (eg, collagenase and stromelysin) that degrade extracellular matrix proteins [22]. TGF-b rst binds to TGF-b type II receptor (TbRII), resulting in the formation of a heteromeric complex between TGF-b type I (TbRI) receptor and TbRII. Overexpression of TbRI or TbRII increases collagen promoter activity in broblasts [23]; however Quan and

RECEPTORS IN SKIN AGEING AND ANTIAGEING AGENTS

657

colleagues [24] showed that UV irradiation could cause down-regulation of TbRII receptors mRNA and protein and induction of Smad7 mRNA and protein in human skin, causing a decrease in collagen production. Activator protein (AP)-1 also is involved in this UV-mediated down-regulation of collagen synthesis. AP-1 is composed of two subunits: the constitutively expressed c-Fos and the UV-inducible c-Jun [25,26]. Overexpression of the c-jun component of AP-1 in cultured broblasts can decrease expression of type I collagen. Moreover, AP-1 controls the transcription of MMPs, enzymes that are responsible for degradation of the extracellular matrix. The MMPs include metalloproteinase-1 (a collagenase), MMP-3 (stromelysin), and MMP-9 (92-kd gelatinase). MMP expression is localized in epidermal keratinocytes and dermal broblasts [18]. Iron is required for the activation of MMP-1, the major metalloproteinase responsible for collagen degradation; however, MMP up-regulation can occur after a minimal dose of UV, resulting in a further reduction of collagen. Another important eect caused by UV is its eect on gene expression. Ageing and photoageing alter the expression of selected genes that are implicated in growth, dierentiation, immunomodulation, and UV response in human epidermis

[27]. This may explain, in part, the predisposition to photocarcinogenesis in the chronically sunexposed skin of (old) individuals (Table 1). Receptors involved in the hormonal mechanisms Estrogens have a key role in skin-ageing homeostasis as evidenced by the accelerated decline in skin appearance seen in the perimenopausal years. Estrogens have a profound inuence on skin. They aect several skin functions, such as hair growth, pigmentation, vascularity, elasticity, and water-holding capacity, as well as cellular functions, including proliferation, morphogenesis, differentiation, and apoptosis. In particular, it was shown by reverse transcriptase-polymerase chain reaction that nuclear estrogen receptor (ER)a and -b mRNA are expressed in human dermal broblasts, strongly suggesting that estradiol mediates its eects on the dermis through direct regulation of the broblast function mediated by ERs [28]. Since 1941, it has been observed that postmenopausal women who have osteoporosis have skin that is noticeably atrophied [29]. During the menopausal years, the loss of estrogen production accelerates skin changes [30]. The epidermis becomes thinner, and re-epithelialization is retarded. The dermis also becomes thinner and loses its

Table 1 Eect of ageing and sun exposure on the genetic response of cultured keratinocytes Gene product c-fos Major function Initial cell response after stimulation to divide or dierentiate Expression required for cell division Response to stress and injury Immune modulation, keratinocyte and broblast mitogen Immune modulation, keratinocyte mitogen, tissue remodelling (collagenase induction) Competitive inhibitor of IL-1a and IL-1b Involved in dierentiation Response to UV exposure Induced Eect of ageing Less inducible Eect of photoageing More inducible

c-myc GADD IL-1a

Down-regulated Induced No eect or induced

None None None

None None None

IL-1b

Induced

None

None

IL-1 receptor antagonist SRP2

No eect Induced

Increased expression Increased expression

Decreased expression Decreased expression

Abbreviations: GADD, growth arrest and DNA damage; SRP, serine/arginine rich protein.

658

GHERSETICH

et al

elasticity. The skin wrinkling and increased looseness of the skin after menopause correspond to changes in collagenous and elastic bers, which were reported to be due to estrogen deciency [31]. A signicant reduction in skin collagen has been demonstrated to start at menopause. More recently, Anito and colleagues [32] showed that skin collagen decline was correlated closely to the years following menopause. They showed that postmenopausal women had decreased amounts of types I and III collagen as well as a decreased ratio of types III/I collagen compared with premenopausal women. Several controlled studies also have shown the eects of estrogen on the thickness of skin collagen. By skin biopsy, Castelo-Branco and colleagues [33] showed an increase in skin collagen content of 1.8% to 5.1% with oral and transdermal hormone replacement therapies over 12 months. Estrogens cause an increase in collagen [33] and glycosaminoglycans [34] in the dermis, which may explain the decrease in skin wrinkling with estrogen treatment. Estrogens also aect the dermal water-holding capacity [35].

Those cytokines/receptors that increase with barrier perturbation in young skin also increase with barrier perturbation in aged skin, and those that decrease in young skin also do so in aged skin. Aged skin begins with fewer ligands and more receptors, however, and after permeability barrier perturbation, aged skin compensates with a greater modulation in the agonist (IL-1a) and lesser changes in the antagonists IL-1Ra and IL-1R T2, alterations that would enhance the IL-1mediated response. These compensatory responses to permeability barrier perturbation do not suce, however, to normalize permeability barrier homeostasis in aged epidermis. Moreover, IL-1a dysregulation in aged epidermis, under basal and stimulated conditions, could explain the decreased mitogenesis and lipid synthesis that occur in aged epidermis [37]. At the same time, TNF-a shows an increase in young and aged epidermis following permeability barrier perturbation, with no dierences in the absolute levels of young versus aged. These results conrm that selective alterations occur in the expression of the IL-1 family of cytokines in response to permeability barrier perturbation in aged epidermis [37].

Cytokines and cytokine receptors The ageing process has been dened as a progressive loss of function, an increased environmental vulnerability, and a decreased homeostatic capacity [27]. Such changes are well documented in ageing skin and, in general, are exacerbated in photoaged skin [36]. On the contrary, young cells are sensitive to environmental signals (eg, growth factor stimulation, contact inhibition, and UV irradiation) and respond appropriately through the activation of membrane receptors or various signal transduction pathways. For example, aged epidermal permeability barrier has a diminished capacity for recovery when it is stressed, compared with young epidermis. Ye and colleagues [37] hypothesized that cytokine dysregulation plays a key role in permeability barrier abnormality in aged epidermis. Normally, the acute permeability barrier disruption stimulates the release of prestored interleukin (IL)-1a and increased production of potentially regulatory cytokines, including IL-1a and TNF-a in the epidermis. A qualitatively, but not quantitatively, similar response can be observed in aged skin.

Treatment of photoageing targeting selected skin receptors Retinoids The term retinoid was coined to dene a mixture of naturally occurring molecular compounds with vitamin A activity and synthetic analogues of vitamin A (retinol) [38]. This denition, based on molecular structure and biologic activity, however, had a clear limitation. Based on current knowledge, retinoid can be dened as any molecule that binds to and activates nuclear retinoic acid receptors (RARs), by itself or through metabolic conversion, thereby eliciting transcriptional activation of retinoic acidresponsive genes that result in specic biologic responses [39]. Therefore, drugs with the ability to bind a receptor and to activate genes, even those with no chemical similarity to all-trans retinoic acid (TRA), can be considered retinoids. For years, retinoids have been the mainstay of topical therapy for the prevention and treatment of photoageing. Tretinoin (TRA), a nonselective agent that actives all RARs directly and retinoic X receptors indirectly, improved the clinical signs of

RECEPTORS IN SKIN AGEING AND ANTIAGEING AGENTS

659

photoageing in controlled clinical trials [40]. The benets of retinoids are believed to be mediated, at least in part, by their eects on collagenase induction. Pretreatment with TRA inhibits UVmediated induction of c-jun protein, AP-1, and MMP [26]. Pretreatment also reduces loss and accelerates recovery of RAR-g and RXR-a following UV exposure [41]; however, when topical tretinoin is applied to human skin, any collagen deciency existing in photoaged skin is remedied at least partially, and the skin is primed to prevent further matrix degradation induced by solar UV. Tazarotene is a second-generation retinoid that binds RAR-g and -b [42]. Like tretinoin, tazarotene is eective in the treatment of photodamage. Reduced atypia and restoration of keratinocyte polarity also have been noted after tazarotene therapy. In a 24-week randomized, controlled, double-blind study, treatment with 0.1% tazarotene resulted in signicant improvement in numerous clinical assessments of photodamage. Additional clinical improvement occurred during an open-label extension and had not reached a plateau by week 52 [43]. Compared with a standard dose of tretinoin, a high-dose regimen of tazarotene produced more rapid improvements in ne wrinkling and mottled pigmentation [44]. Tazarotene also is a strong irritant and, like tretinoin, is believed to inhibit AP-1dependent gene expression [45]. An active area of research is the development of receptor-selective retinoids to optimize therapy and minimize side eects [42]. Up-regulation of retinoic acid-response elements and the antagonizing actions of AP-1 are not linked [45], which suggests that receptor-selective retinoids hold promise. Therefore, retinoids have become essential in the treatment and prevention of photoageing. Antioxidants Numerous antioxidants have been analyzed for their ability to prevent or reverse clinical signs associated with photoageing secondary to ROS. Strategies using endogenous skin antioxidants and plant-derived or chemical compounds have been examined. Topical vitamin C Vitamin C, a potent antioxidant, has been shown to prevent erythema and sunburn cell formation after UV exposure. Vitamin C also can up-regulate collagen and tissue inhibitor of

metalloproteinases synthesis in human skin. Because of the short half-life of vitamin C, skin care formulations commonly include its derivatives, which do not penetrate the skin as readily; however, in a 6-month, double-blind, placebocontrolled, randomized trial examining the use of a topical vitamin C compound, a signicant decrease in wrinkles was found when measured by optical prolometry [46]. Prolometry is a technique that uses skin replicas measured with special image-processing software (optical prolometry) or laser (laser prolometry) to obtain an objective quantication of the skin surface topography. Oral supplements Oral supplements oer a systemic method to treat photoageing. An oral supplement containing a combination of 1-proline, 1-lysine, manganese, copper, zinc, quercetin, grape seed extract, N-acetyl D-glucosamine, and glucosamine sulfate improved wrinkles by 34% in a pilot study when measured by optical prolometry [47]. Another oral supplement composed of vitamin E, vitamin C, carotenoid, selenium, and proanthocyanidin led to a signicant decrease in the induction of MMPs after UV exposure. A reduction in UV-induced erythema also was noted, but it did not reach statistical signicance [48]. CoQ10 CoQ10 is a component of the mitochondrial electron transport chain; it also acts as an antioxidant in the skin, with 10-fold higher levels in the epidermis than the dermis [49]. Topical CoQ10 use led to signicant reductions in wrinkles measurements assessed by optical prolometry in a vehicle-controlled 6-month pilot study [50]. a-Lipoic acid a-Lipoic acid is an antioxidant and anti-inammatory agent that was shown to reduce the production of transcription factors, such as nuclear factor-kappa B (NF-kB), and indirectly aect the gene expression of inammatory cytokines. Treatment with a-lipoic acid led to significant improvements in clinical and objective measurements of photoageing, including laser prolometry [51]. Estrogens Skin tissue is an active target of estrogens, as demonstrated by estrogen receptors determination [52]. Estrogen replacement therapy improves the

660

GHERSETICH

et al

various symptoms of skin ageing. For example, oral estrogen therapy caused thickening of the skin in postmenopausal women [53] and prevented the age-related reduction in cutaneous elasticity, thereby inhibiting skin slackness and reducing the number and depth of wrinkles [54]. Also, the topical application of estrogen cream improved the external appearance of postmenopausal facial skin; elasticity and rmness improved markedly, and wrinkle depth and number decreased [55]. These benecial eects of estrogen may result from increased collagen or other matrix proteins. Son and colleagues [56] investigated the eects of 17b-estradiol on the expression of extracellular matrix. Topical application increased the expression of type I procollagen mRNA and protein, reduced MMP-1 protein levels, increased the expression of TGF-b1 mRNA, tropoelastin, and brillin-1 mRNA, elastic bers, keratinocyte proliferation, and epidermal thickness in aged human skin. Growth factors and cytokines Topical application of a cream containing a combination of growth factors and cytokines was evaluated in a pilot study for its eect on photoaged skin. Most patients showed clinical improvement in at least one facial area, and there was a signicant change in objective measurements by optical prolometry. In addition, new collagen formation was observed in biopsy specimens [57]. If aberrant cytokine signaling contributes to the pathogenesis of the aged epidermal permeability barrier abnormality, correction of the cytokine abnormality should ameliorate the structural, functional, and biochemical abnormalities in the aged epidermal permeability barrier. Future studies will test whether stimulation of cytokine production in the aged epidermis results in improved barrier homeostasis [37]. New compounds and putative skin receptors The focuse-rich polysaccharide FROP-3 increases glycosaminoglycan biosynthesis in broblast cultures. In a pilot study examining skin surface microrelief, the pattern of ne wrinkling found in skin of any age, a cream containing FROP-3 showed a 10- to 15-year decrease in apparent age after 4 weeks of treatment in most patients [58].

Skin surface microrelief changes predictably with age, with younger persons having a regular pattern of ne, thin lines. Wrinkles become deeper and thicker with increasing age. In a 5-week pilot study, an extract of date palm kernel reduced wrinkles by optical prolometry and visual assessment compared with placebo [59]. Oligodeoxynucleotide technology Oligodeoxynucleotide technology uses synthetic decoy cis elements to block the binding of transcription factors to promoter regions of target genes. An NF-kB oligodeoxynucleotide has been developed and was shown to reduce UV-induced inammatory changes (eg, swelling, leukocyte inltration, epidermal hyperplasia, and accumulation of proinammatory cytokines) when applied topically to mice [60]. This experiment focused on the role of NF-kB in sunburn. Because NF-kB also has a role in MMP induction and photoageing, modication of this pathway may have a future preventive role in photoageing.

Summary Skin ageing is an irreversible process during which ultrastructural and physiologic alterations occur. Dermatology has focused a lot of attention on the reversal of signs of ageing and photodamage with the purpose of achieving cosmetic benets and preventing photocancerogenesis. Recent advances in skin biology have claried the mechanisms by which photoageing occurs and have given rise to new treatments to prevent and reverse this process. The understanding of the role of key receptors involved in the complex pathomechanism of skin ageing probably will lead to the development of new therapeutic agents in the nearest future.

References
[1] Fenske NA, Lober CW. Structural and junctional changes in aging human skin. J Am Acad Dermatol 1986;15:57185. [2] Lavker RM, Zheng PS, Dong G. Aged skin: a study by light, transmission electron and scanning electron microscopy. J Invest Dermatol 1987;88(Suppl 3): S4451. [3] Green LJ, McCormick A, Weinstein GD. Photoaging and the skin: the eects of tretinoin. Dermatol Clin 1993;11:97105.

RECEPTORS IN SKIN AGEING AND ANTIAGEING AGENTS

661

[4] Yaar M, Gilchrest BA. Aging of skin. In: Freedberg IM, Eisen AZ, Wol K, et al, editors. Fitzpatricks dermatology in general medicine. New York: McGraw-Hill; 2003. p. 138698. [5] Taylor RS, Ramirez RD, Ogoshi M, et al. Detection of telomerase activity in malignant and non-malignant skin conditions. J Invest Dermatol 1996;106:75965. [6] Vaziri H, Benchimol S. From telomere loss to p53 induction and activation of DNA-damage pathway at senescence: the telomere loss/DNA damage model of cell aging. Exp Gerontol 1996;31:295301. [7] Campisi J. Replicative senescence: an old lives tale? Cell 1996;84:497500. [8] Rudolph KL, Chang S, Lee HW, et al. Longevity stress response and cancer in aging telomerase-decient mice. Cell 1999;96:70112. [9] Funk WD, Wang CK, Shelton DN, et al. Telomerase expression restores dermal integrity to in vitro-aged broblasts in a reconstituted skin model. Exp Cell Res 2000;258:2708. [10] Allsopp RC, Vaziri H, Patterson C, et al. Telomere length predicts replicative capacity of human broblasts. Proc Natl Acad Sci USA 1992;89:101148. [11] Harle-Bachor C, Boukamp P. Telomerase activity in the regenerative basal layer of the epidermis in human skin and in immortal and carcinoma-derived skin keratinocytes. Proc Natl Acad Sci USA 1996; 93:647681. [12] Gilhar A, Ullman Y, Karry R, et al. Cutaneous biology. Ageing of human epidermis: the role of apoptosis, Fas and telomerase. Br J Dermatol 2004;150: 5663. [13] Yasui H, Sakurai H. Age-dependent generation of reactive oxygen species in the skin of live hairless rats exposed to UVA light. Exp Dermatol 2003;12: 65561. [14] Harman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol 1956;11:298300. [15] Tolmaso JM, Ono T, Cutler RG. Superoxide dismutase: correlation with life-span and specic metabolic rate in primate species. Proc Natl Acad Sci USA 1980;77:277781. [16] Berlett BS, Stadtman ER. Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 1997;272:203136. [17] Kochevar IE. Molecular and cellular eects of UV radiation relevant to chronic photodamage. In: Gilchrest BA, editor. Photodamage. Cambridge (UK): Blackwell Science; 1995. p. 5167. [18] Fisher GJ, Wang ZQ, Datta SC, et al. Pathophysiology of premature skin aging induced by ultraviolet light. N Engl J Med 1997;337:141929. [19] Yin L, Morita A, Tsuiji T. Skin aging induced by ultraviolet exposure and tobacco smoking: evidence from epidemiological and molecular studies. Photodermatol Photoimmunol Photomed 2001;17: 17883. [20] Massague J. TGF-beta signal transduction. Annu Rev Biochem 1998;67:75391.

[21] Massague J. The transforming growth factor beta family. Annu Rev Cell Biol 1990;6:597641. [22] Eickelberg O, Kohler E, Reickhenberger F, et al. Extracellular matrix deposition by primary human lung broblasts in response to TGF-b1 and TGF-b3. Am J Physiol 1999;276:81424. [23] Kawakami T, Ihn H, Xu W, et al. Increased expression of TGF-b receptors by scleroderma broblasts: evidence for contribution of autocrine TGF-b signaling to scleroderma phenotype. J Invest Dermatol 1998;110:4751. [24] Quan T, He T, Voorhees JJ, et al. Ultraviolet irradiation blocks cellular responses to transforming growth factor beta by down-regulating its type-II receptor and inducing Smad7. J Biol Chem 2001;276: 2634956. [25] Fisher GJ, Talwar HS, Lin J, et al. Retinoic acid inhibits induction of c-jun protein by ultraviolet radiation that occurs subsequent to activation of mitogen-activated protein kinase pathways in human skin in vivo. J Clin Invest 1998;101:143240. [26] Fisher GJ, Datta S, Talwar HS, et al. Molecular basis of sun-induced premature skin ageing and retinoid antagonism. Nature 1996;379:3359. [27] Gilchrest BA, Garmyn M, Yaar M. Aging and photoaging aect gene expression in cultured human keratinocytes. Arch Dermatol 1994;130:826. [28] Haczynski J, Tarkowski R, Jarzabek K, et al. Human cultured skin broblasts express estrogen receptor alpha and beta. Int J Mol Med 2002;10(2): 14953. [29] Albright F, Smith PH, Richardson A. Postmenopausal osteoporosis-its clinical features. JAMA 1941;116:246574. [30] Punnonen R, Vaajalahti P, Teisala K. Local oestriol treatment improves the structure of elastic bers in the skin of postmenopausal women. Ann Chir Gynaecol Suppl 1987;202:3941. [31] Schmidt JB, Binder M, Macheiner W, et al. Treatment of skin aging with topical estrogens. Int J Dermatol 1996;35:66974. [32] Anito P, Palomba S, Sorrentino C, et al. Eects of postmenopausal hypoestrogenism on skin collagen. Maturitas 1999;33:23947. [33] Castelo-Branco C, Duran M, Gonzales-Merlo J. Skin collagen and bone changes related to age and hormone replacement therapy. Maturitas 1992; 15(2):10311. [34] Grosman N, Hvidberg E, Schou J. The eect of osteogenic treatment on the acid mucopolysaccharide pattern in the skin of mice. Acta Pharmacol Toxicol 1971;30:45864. [35] Holbrook KA, Wol K. Structure and development of skin. In: Fitzpatrick TB, Eisen AZ, Wol K, et al, editors. Fitzpatricks dermatology in general medicine. 3rd edition. New York: McGraw-Hill; 1987. p. 93131. [36] Gilchrest BA. Ageing. J Am Acad Dermatol 1984; 11(5 Pt 2):9957.

662

GHERSETICH

et al antioxidative combination of vitamins E and C, carotenoids, selenium and proanthocyanidins. Skin Pharmacol Appl skin Physiol 2002;15:30715. Shindo Y, Witt E, Han D, et al. Enzymic and nonenzymic antioxidants in epidermis and dermis of human skin. J Invest Dermatol 1994;102:1224. Hoppe U, Bergemann J, Diembeck W, et al. Coenzyme Q10, a cutaneous antioxidant and energizer. BioFactors 1999;9:3718. Beitner H. Randomized, placebo controlled, double blind study on the ecacy of a cream containing 5% alpha lipoic acid related to photoaging of facial skin. Br J Dermatol 2003;149:8419. Schmidt JB, Lindmaier A, Spona J. Hormone receptors in pubic skin of premenopausal and postmenopausal females. Gynecol Obstet Invest 1990;30: 97100. Brincat M, Moniz CJ, Studd JW, et al. Long-term effects of the menopause and sex hormones on skin thickness. Br J Obstet Gyneacol 1985;92:2569. Pierard GE, Letawe C, Dowlati A, et al. Eect of hormone replacement therapy for menopause on the mechanical properties of skin. J Am Geriatr Soc 1995;43:6625. Credi P, Faivre B, Agache P, et al. Eect of a conjugated estrogen cream (Premarin) cream on ageing facial skin. A comparative study with placebo cream. Maturitas 1994;19:21123. Son ED, Lee JY, Lee S, et al. Topical application of 17beta-estradiol increases extracellular matrix protein synthesis by stimulating tgf-Beta signaling in aged human skin in vivo. J Invest Dermatol 2005; 124:114961. Fitzpatrick RE, Rostan EF. Reversal of photodamage with topical growth factors: a pilot study. J Cosmet Laser Ther 2003;5:2534. Robert C, Robert AM, Robert L. Eect of a focuserich polysaccharide preparation on the age-dependent evolution of the skin surface micro-relief. Pathol Biol 2003;51:58690. Bauza E, Dal Farra C, Berghi A, et al. Date palm kernel extract exhibits antiaging properties and signicantly reduces skin wrinkles. Int J Tissue React 2002;24:1316. Abeyama K, Eng W, Jester JV, et al. A role for NF-kB dependent gene transactivation in sunburn. J Clin Invest 2000;105:17519.

[37] Ye J, Garg A, Calhoun C, et al. Alterations in cytokine regulation in aged epidermis: implications for permeability barrier homeostasis and inammation. IL-1 gene family. Exp Dermatol 2002;11:20916. [38] Sporn MB, Dunlop NM, Newton DL, et al. Relationships between structure and activity of retinoids. Nature 1976;263:1103. [39] Kang S, Voorhees JJ, editors. Fitzpatricks dermatology in general medicine. 6th edition. New York: McGraw-Hill; 2003. [40] Weiss JS, Ellis CN, Headington JT, et al. Topical tretinoin improves photodamaged skin: a doubleblind, vehicle-controlled study. JAMA 1988;259: 52732. [41] Wang Z, Boudjelal M, Kang S, et al. Ultraviolet irradiation of human skin causes functional vitamin A deciency, preventable by all-trans retinoic acid pre-treatment. Nat Med 1999;5:41822. [42] Nagpal S, Chandraratna RAS. Recent developments in receptor-selective retinoids. Curr Pharm Des 2000;6:91931. [43] Phillips TJ, Gottlieb AB, Leyden JJ, et al. Ecacy of 0.1% tazarotene cream for the treatment of photodamage: a 12-month multicenter, randomized trial. Arch dermatol 2002;138:148693. [44] Kang S, Leyden JJ, Lowe NJ, et al. Tazarotene cream for the treatment of facial photodamage: a multicenter, investigator-masked, randomised, vehicle-controlled, parallel comparison of 0.01%, 0. 025%, 0.05%, and 0.1% tazarotene creams with 0. 05% tretinoin emollient cream applied once daily for 24 weeks. Arch Dermatol 2001;137:1597604. [45] Napgal S, Athanikar J, Chandraratna RAS. Separation of transactivation and AP1 antagonism functions of retinoic acid receptor alpha. J Biol Chem 1995;270:92379. [46] Humbert PG, Haftek M, Creidi P, et al. Topical ascorbic acid on photoaged skin. Clinical, topographical and ultrastructural evaluation: doubleblind study vs. placebo. Exp Dermatol 2003;12: 23744. [47] Murad H, Tabibian MP. The eect of an oral supplement containing glucosamine, amino acids, minerals, and antioxidants on cutaneous aging: a preliminary study. J Dermatolog Treat 2001;12:4751. [48] Greul AK, Grundmann JU, Heinrich F, et al. Photoprotection of UV-irradiated human skin: an

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

Dermatol Clin 25 (2007) 663676

Bacterial and Viral Skin Diseases

Eleonora Ruocco, MD, PhDa, Giovanna Donnarumma, MDb, Adone Baroni, MD, PhDa, Maria Antonietta Tufano, MDb,*
a

Department of Dermatology, Second University of Naples, Via Pansini 5, 80131 Naples, Italy b Department of Experimental Medicine, Microbiology and Clinical Microbiology Section, Second University of Naples, Via Costantinopoli 16, 80138 Naples, Italy

Unimpaired skin protects the underlying tissue and is an excellent frontline defense against the invasion of pathogenous microorganisms thanks also to the presence of the skin microbiota. Throughout the life of an individual the skin is colonized by a number of microorganisms that can vary from a few hundred per cm2 on the dry surfaces of the forearm and back, where the prevalent bacterium is Staphylococcus epidermidis, to 10,000 per cm2 on the damp areas, such as the armpits and groin, where propionibacteria predominate, but where corynebacteria and negative coagulase staphylococci can also be observed. What is surprising is that the skin microbiota is made up of microorganisms belonging to a very limited number of species and that the microbial load of the healthy skin is kept consistent, considering the large variety and number of potential colonizers to which the skin is prey. It is not surprising, however, that the skin, the barrier between the body and the environment, is the site of frequent infections [1]. The integrity of this barrier is inuenced by the degree of scaling but also by several factors whose alterations upset the environmental balance of the resident ora and predispose the subject to infection [2]. Moreover, the skin displays microbicidal activity even when its physical integrity is impaired [3]. It contains the bioactive molecules, among which antimicrobial peptides such as defensins and cathelicidins are of critical importance to the host defense against microbial invasion [4].
* Corresponding author. E-mail address: mariaan.tufano@unina2.it (M.A. Tufano).

At least two dierent populations of microorganisms can be found in the skin microbiota: a permanent or resident ora, which is always present, and a temporary or transient ora, which settles on the human skin only for a certain period of time. The permanent ora is a stable population of microorganisms that is more or less regularly present on the skin in considerable numbers and does not usually comprise pathogenous microorganisms. The permanent ora is consistently present and if removed reforms within 24 to 72 hours. It is made up of aerobic or microaerophil microorganisms. The main bacterium resident on free skin (ie, nonfollicular) is S epidermidis; in the area of the follicular ostium, with a reduced oxygen supply, are mainly present Propionibacterium acnes, Propionibacterium granulosum, and Propionibacterium avidum. Species of Peptococcus, which are anaerobic staphylococci, are present in 20% of humans, especially on the forehead and in the elbow crease. Gram-positive liophil bacteria belonging to the genus Corynebacterium settle in sebum-rich areas, whereas bacteria belonging to the genus Brevibacterium are present in particularly humid areas. Gram-negative bacteria are not normal components of the skin ora. The microorganisms that make up the transient ora settle only temporarily on the skin from the external environment or from the adjacent mucosal areas. Unlike in the resident ora, the bacterial species present are numerous, since most microorganisms can at least temporarily survive on the skin. They cannot, however, multiply and, therefore, colonize the skin. In some subjects, Staphylococcus aureus is present in the nose and peri-anal area and can spread to other areas of the skin. Gram-negative
derm.theclinics.com

0733-8635/07/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2007.06.008

664

RUOCCO

et al

bacteria can also be isolated, particularly those belonging to the Enterobacteriaceae and Pseudomonaceae families. The former are part of the normal intestinal ora, the latter are found in the environment, most readily in damp places [5]. It is therefore evident that possible colonization and invasion by pathogenous microorganisms is counteracted both by the host defenses and by the resident ora, which contrast colonization by other microorganisms competing for nutrients or producing peptides with antimicrobial activity [2]. Most skin infections are therefore self-limiting in healthy subjects. Infections arising in a healthy skin are dened as primary infections. They are usually caused by a single microbial species; the entry point of the germ is often unknown, although a slight trauma is probably implicated. Instead, infections dened as secondary develop on preexisting skin lesions, which, therefore, facilitate the entry of the microorganisms. Atopic dermatitis, psoriatic lesions and other eczematous disorders are the most common examples, but also prone to this complication are surgical or injury wounds, burns, insect bites or stings, ulcers, and areas of maceration [6,7]. Secondary infections, usually polymicrobial, are generally caused by microorganisms that in themselves have little pathogenic power. When the humoral and cell immune defenses are low, secondary infections arise more readily and develop more rapidly [8]. Once the skin barrier has been penetrated, the microorganisms belonging to the resident ora, mainly coagulase negative anaerobic staphylococci, can cause infections, especially skin abscesses. However, also responsible for skin infections are microorganisms that, acquired from the environment, are temporarily part of the skin ora, for example S aureus and Streptococcus pyogenes. In general, a skin infection can follow three dierent events (Fig. 1):  a lesion of the skin that favors infection from the outside;  skin manifestations of systemic infections, which can spread through the blood from the site of infection to the skin or by direct invasion/penetration;  skin damage caused by toxins.

Bacterial infections Bacterial infections can be classied on the basis of the site involved, although some skin

infections with a bacterial etiology can involve more than one part of the body. Thus, infections with a bacterial etiology associated with an inammatory process limited to the hair follicle are classied as folliculitis. They are characterized clinically by the presence of abscesses and the formation of typical papules or pustules. Impetigo, erysipelas, and cellulitis are widespread infections. Impetigo is an infection limited to the epidermis and characterized by a bullous rash that evolves in crusts and pustules. Erysipelas is an acute erythematous infection that spreads rapidly and is usually associated with systemic symptoms. If the lesion is located in the subcutaneous fat and mainly involves the derma, it is called cellulitis. Both infections are associated with an intense inammatory process. Infections characterized by rapidly progressive cellulitis that causes extensive damage to the tissue below the derma, in particular to the muscular tissue, and impairs the blood ow are known as necrotizing infections, subsequent to which necrotizing fasciitis and gas gangrene (infections not considered of dermatological competence) arise. The microorganisms most commonly involved in skin infections of a bacterial etiology are listed in Table 1. Gram-positive cocci are responsible for most skin infections and often the same microorganism can cause dierent infections according to the dierent layers of skin that it is able to colonize. Staphylococci are generally aerobic, grampositive cocci catalase-positive, which appear in irregular, so-called grapelike clusters under the microscope, although single and paired cells are the most common in a uid culture. The dominant species of staphylococcus on the skin is S epidermidis on the face and chest, with a lesser but still substantial role for Staphylococcus hominis. In recent years there has been a much greater appreciation of the role of the normal skin ora in infection. Because of the normal colonization of skin by coagulase-negative staphylococci it is dicult to be sure that small, localized lesions such as folliculitis are really caused by these organisms [9]. There are a number of miscellaneous infections attributed to coagulase-negative staphylococci. S epidermidis and S hominis, coagulase-negative staphylococci, are now well-established pathogens in certain areas of the skin, while S aureus, a species of coagulase-positive staphylococci, remains a potent pathogen able to exhibit new antibiotic resistance patterns and to

INFECTIOUS SKIN DISEASES

665
microorganism

microorganism

toxin or immuno-complex

blood vessel Systemic infection Toxin-mediated infection Epithelial cells invasion

Dermis cells invasion PAPILLOMA

leukocytes

MACULA

PAPULE

BLISTER

ULCER

Fig. 1. Mucocutaneous lesion pathogenesis.

continue to infect both the immunocompetentand incompetent-host [10]. S aureus permanently colonizes the moist squamous epithelium of the anterior nostrils of 20% of the population, and is transiently associated with another 60% [11]. Occasionally, the organism can cause supercial skin infections. Primary staphylococcal infections of the skin are chiey boils, foruncles and other localized pustular lesions, and impetigo plus its more severe manifestation, scalded skin syndrome [12]. S aureus expresses a wide range of secreted and cell-surfaceassociated virulence factors, including surface proteins that promote adhesion to damaged tissue and to the surface of the host cells [13], which bind proteins in the blood to help evade immune responses and promote iron uptake [14]. Most strains express a polysaccharide capsule [15]. Moreover, S aureus has been regarded as a noninvasive pathogen but it is now evident that the bacterium can invade many types of

host cells by a mechanism involving the formation of a bronectin bridge between the bacterial bronectin-binding proteins and host a5b1 integrin molecules, which triggers internalization [16,17]. The ability of S aureus to cause infection seems to depend on the ability of the organism to produce a cocktail of enzymes or toxins that contribute to the appearance of disease. The microorganism can secrete a range of extracellular enzymes such as proteases, a hyaluronidase, a lipase, and a nuclease that facilitate tissue destruction and the spread of membrane-damaging toxins, which cause cytolytic eects on the host cells and tissue damage, and superantigens, which contribute to the symptoms of septic shock [18]. Toxic shock syndrome (TSS) is characterized by fever, headache, and confusion, with an erythematous rash resembling scarlet fever and desquamation in the later stages. The symptoms are also usually accompanied by diarrhea, vomiting, and hypotensive shock. TSS is caused by

666 Table 1 Main bacteria that involve the skin Microorganisms Staphylococcus aureus Diseases

RUOCCO

et al

Streptococcus pyogenes

Corynebacterium minutissimum Corynebacterium diphteriae Pseudomonas aeruginosa Propionibacterium acnes Proteus Mycobacterium ulcerans

Toxic shock syndrome Pyoderma Impetigo Furuncle Scarlet fever Streptococcal toxic shock syndrome Pyoderma Erysipelas Necrotizing fasciitis Erythrasma Cutaneous diphtheria Septicemia Ulcer Acne Folliculitis Folliculitis Buruli ulcer

exotoxins, the most common of which is toxic shock syndrome toxin 1 (TSST-1), produced mainly by strains of phage-group I S aureus. This toxin acts as a superantigen, stimulating the production of T cells and the release of cytokines [19]. Scalded skin syndrome or Ritter disease is also caused by a strain of toxigenous S aureus [20]. The toxin implicated in this syndrome is known as exfoliating toxin or scalded skin toxin. Initially the skin lesions may be mild, but the toxin causes the destruction of the desmosomes and the detachment of the supercial layer of the epidermis. It is generally regarded as a sporadic disease with most cases in children aged 0.5 to 2 years. Few adult patients are reported, chiey in the immunosuppressed, although cases in immunocompetent adults are known [21]. Impetigo is characterized by golden, stuck-on crusts or blisters (bullae); the blisters are most probably caused by small amounts of epidermolytic toxin or by the toxin in an otherwise resistant host [22]. About three quarters of the cases are in patients younger than 20, with about 35% in those younger than 10. In Europe it is chiey a staphylococcal disease, though one third of lesions have both S aureus and S pyogenes. American experience would suggest a predominantly streptococcal background, although there are signs that this may be changing. In AIDS patients an extensive atypical intertriginous form of

bullous impetigo has been reported as part of AIDS-related pruritis [23]. Besides infections such as boils or impetigo, S aureus also colonizes and aggravates lesions such as those of atopic dermatitis. Some studies indicate that when the density of S aureus exceeds a certain level, such as 106/cm2, an exudative or impetiginized form of lesion occurs. The reason for the overgrowth of S aureus in atopic dermatitis and not in diseases such as psoriasis is not known [24,25]. Protein A elicits a much less vigorous response in atopics than in normal skin or psoriatics, but this may be the result rather than a cause of colonization. Attention has recently focused on the skin lipids and there is some evidence that fatty acids, which may control staphylococcal colonization, are decient in atopics [4,26]. The therapy of choice for staphylococcal infections is usually a penicillase-resistant penicillin. The administration of erythromycin is frequent, although an increase in the frequency of erythromycin-resistant strains has been reported. Meticillin-resistant S aureus (MRSA) is a problem particularly for hospitals; vancomycin is the only drug available but strains resistant to it are also emerging (vancomycin intensive S aureus [VISA]) [27,28]. Skin infection with streptococci covers a range from simple colonization to primary and secondary infections; the skin provides an important portal of entry for systemic infection by these organisms. Streptococci are gram-positive, spherical, aerobic, and facultatively anaerobic bacteria, arranged in chains or pairs. They are nonsporing, catalase negative, oxidase negative, and mainly nonmotile. Hemolysis on blood-agar culture provides a useful division of streptococci into those that are hemolytic (beta hemolytic, showing a zone of complete hemolysis, and alpha hemolytic or viridans streptococci, showing a zone of incomplete hemolysis) and nonhemolytic (those with no eect on red blood cells). Hemolytic streptococci are further divided according to the carriage of polysaccharide or teichoic acid or Lanceeld group antigens. Direct streptococcal infection of the skin may take on a number of forms [29,30]. S pyogenes (group A hemolytic streptococcus) remains the major streptococcal pathogen in these infections but nongroup A streptococci also play a part and have become more commonly recognized with the widespread use of modern, rapid laboratory test kits. S pyogenes is the etiological agent of streptococcal impetigo and erysipelas, skin pathologies also

INFECTIOUS SKIN DISEASES

667

caused by S aureus. Infection generally arises from contact with infected skin lesions in other individuals. Once S pyogenes has colonized the skin, it invades the epithelium through small wounds, with consequent development of the lesion. A complex interaction of bacterial and host defense factors underlies the initiation, development, and clinical manifestations of streptococcal infection [31]. The M-protein molecule is known to be a major virulence factor of streptococci. It is present as a double-stranded coiled-coil structure projecting from the cell surface. The functional properties of M-protein include binding of brinogen, bronectin, and b2-microglobulin; adherence to host cells; interference with complement deposition; and the conferring of resistance to phagocytosis [32]. The quantity of M protein expressed on the cell surface appears to be an important factor in the pathogenesis: freshly isolated strains of group A, C, and G streptococci, particularly those from invasive infections, are often rich in this substance [33], and serotypes of S pyogenes such as M1, which express large quantities, are commonly associated with an invasive disease [34]. Enhancement of M-protein expression may be a factor underlying the increased virulence observed when streptococci are rapidly passed from host to host. The binding of brinogen and bronectin by streptococcal surface structures may play an important part in the attachment of microorganisms to wounds and clots in the rst stages of colonization and infection [35]. Other projecting cell-surface molecules with similarities to M protein, such as F-protein, in their cell-wall attachment structure show further functions including the inactivation of complement C5a, a major signal substance for the chemotactic attraction of leukocytes, and binding to the Fc portion of immunoglobulin (Ig) G and IgA antibodies. Teichoic acids also contribute to the virulence of S pyogenes by helping the microorganism bind to the epithelial cells [36]. A streptococcal skin infection develops 24 to 48 hours from the penetration of the skin and stimulates a marked inammatory response. S pyogenes elaborates a series of toxic products and enzymes, like hyaluronidase, that helps the microorganism to spread in the tissues. Lymphatic system involvement is common, which causes lymphadenitis and lymphangitis. Skin infection by S pyogenes may be complicated by nonsuppurative sequelae such as nephritis and scarlet fever; conversely, streptococcal infection at other sites in the body may lead to skin

manifestations, such as in rheumatic fever and acute guttate psoriasis. These conditions will be discussed later in this article. Streptococcal infections of the mouth; alimentary, respiratory, and genitourinary tracts; or deeper structures will not be discussed here except insofar as they are relevant to the skin [37]. Many streptococcal infections are toxin-mediated. Lysogenic strains of S pyogenes produce one or more types of pyrogenous exotoxins (previously called erythrogenic), like SPE-A, SPE-B, and SPE-C, which act on the blood vessels of the skin and cause a diffuse rash arising in association with streptococcal pharyngitis accompanied by scarlet fever. The exotoxin that causes scarlet fever is generally SPE-A [38]. Clinical manifestations similar to staphylococcal toxic shock syndrome are often supported by streptococcal toxins, particularly the pyrogenous exotoxin A, generally produced by M1, M3, or M5 phagotypes of the S pyogenes strains. Streptococcal toxic shock syndrome (STSS) varies somewhat from staphylococcal or classic forms. In most cases the primary site of infection is the skin, often in surgical wounds. In other cases STSS follows chickenpox or can infect immunodepressed patients. These clinical pictures often contrast with those seen in patients with staphylococcal toxic shock syndrome, in whom the primary infection is often subclinical [39]. Necrotizing fasciitis is an acute or subacute infection that spreads above the fascial planes causing thrombosis of the vessels and necrosis of the dermis and subcutaneous fat. It is usually caused by hemolytic or anaerobic streptococci or S aureus [40]. The disease may follow a trivial or unapparent injury to the skin and presents initially with cellulitis, which quickly develops a dusky discoloration, hemorrhagic bullae, and underlying areas of necrosis. There is risk of septicemia and rapid death. Necrotizing fasciitis is most commonly seen in elderly patients, often in association with serious preexisting medical disorders [41]. The rationale of antimicrobial therapy for streptococcal infections of the skin is to hasten the resolution of the lesion, to reduce the risks of suppurative and nonsuppurative complications, and reduce the chances of transmission of infection to others. Streptococci are still remarkably sensitive to penicillin. Many alternative drugs are available including erythromycin, tetracycline, and cephalosporin [42]. Other gram-positive bacteria belonging to the skin microbiota are coryneform bacteria. They are

668

RUOCCO

et al

considered responsible for skin odor and can be associated with pathologies of the skin. This heterogeneous group of microorganisms includes both aerobic and anaerobic pleomorphic bacteria that do not form spores. Because of their similarity to the diphtheria bacillus, these microorganisms were formerly referred to as diphtheroids. In cutaneous infections, coryneform bacteria are clearly involved both as a primary pathogen and a secondary superinfection of other cutaneous infections such as syphilis and streptococcal pyoderma. There are several cutaneous lesions from which coryneforms can be recovered and in which they are seen as playing important pathophysiological roles. These include trichomycosis axillaris, erythrasma, interdigital toe-web-space infections, acne, and pitted keratolysis [43]. About 20% of the population is colonized by Corynebacterium minutissimum, which causes erythrasma only in some cases [44]. Erythrasma is a supercial cutaneous infection ranging from low-grade scaling to thickly macerated areas of the skin. The preferred sites are the skin folds; predisposing factors are obesity, diabetes, and hyperhydrosis. Most infections show a typical reddish uorescence with Woods light. The uorescence a result of a production of porphyrins, which uoresce under long-wave ultraviolet light [45]. Corynebacterium diphtheriae is not an inhabitant of normal skin, although it may be recovered from intact skin under epidemic conditions. This microorganism is more commonly found on mucous membranes [46]. The skin may be the primary portal of entry; the microorganisms can be auto- or hetero-inoculated in an otherwise insignicant wound; there is a high frequency of asymptomatic carriers. Strains of C diphtheriae have been divided into gravis, intermedius, and mitis types on the basis of physiological, morphological, and molecular characteristics. Its pathogenic properties depend on the ability to produce toxins; only the strains infected by the pro-phage b have the tox gene and produce the exotoxin. C diphtheriae infections are more common in tropical and subtropical areas but epidemics have been described in temperate climates like North America. Erosive, ulcerative lesions with thick crusts are the most common clinical ndings [47]. The key therapy is the administration of the antitoxin, which should be administered early on. Antibiotic therapy can be performed with penicillin G or erythromycin [48].

Anaerobic coryneforms recovered from human skin belong above all to the genus Propionibacterium, and P acnes is the most numerous species. This bacterium plays an important role in acne but is not considered the cause. P acnes proliferates and generates inammation-provoking substances, resulting in the disruption of the follicular epithelium and progressive inammation as the contents of the follicle are injected into the dermis. In addition to its role in acne, P acnes is also a frequent opportunistic pathogen. Some authors [49,50] report the isolation of P acnes from an infected wound and in osteomyelitis and endocarditis. There are several reports of meningitis and botryomycosis due to P acnes. The antibiotics used to treat acne include tetracycline and erythromycin [51]. Gram-negative bacterial skin infections are much less frequent than gram-positive infections, but have considerable clinical importance. Pseudomonas aeruginosa is the cause of some supercial infections with particular characteristics. The microorganism readily colonizes damp environmental pockets and, as such, some areas of the body. In recent years there has been an increase in the cases of folliculitis from P aeruginosa in people attending saunas, Jacuzzis, and swimming pools. The skin rash is itchy papules or pustules characteristically distributed in the areas rich in apocrine and eccrine sweat glands. There may be associated symptoms of the infection in other areas, eg, mastodynia and earache. These disorders tend to have a spontaneous recovery but the use of quinolones may be useful. The genus Pseudomonas is frequently isolated from surgical wounds, varicose ulcers, bedsores, and burns, particularly during and after antibiotic therapy. The presence alone of Pseudomonas in these sites is a sign of infection, but the real danger is that the germ may multiply in depth and cause bacteremia [52]. Other gram-negative microorganisms can cause folliculitis during antibiotic treatment for acne, usually with tetracycline. When in young people receiving therapy for acne there is an increase in the pustular lesions, a gram-negative superinfection of the follicules should be suspected, in particular by Proteus or Pseudomonas. It is often sucient to suspend tetracycline, but it is often necessary to switch to another antimicrobial according to the sensitivity studies. After surgery or traumas associated with contamination of the wound, serious skin infections by mixed aerobic or anaerobic ora can occur [53]. These

INFECTIOUS SKIN DISEASES

669

are generally cellulitis with diuse necrosis of the skin and subcutaneous layers, at times extending to the muscles. These infections are extremely dicult to classify because of overlapping of the sites involved, of the germs responsible, and of the clinical manifestations. Many of these infections are severe, rapidly progressive, and associated with high mortality. The ora is composed of clostridia, other anaerobes, enterobacteria, streptococci, and staphylococci. The therapy for these syndromes is surgery with intense antibiotic support. Because the infections are caused by mixed aerobic anaerobic ora, broad-spectrum therapy is indicated, such as aminoglycosides plus clindamycin. Metronidazole, clindamycin, piperacillin, and cefoxitin are useful against anaerobes, and imipenem, ceftazidime, ciprooxacin, and combinations containing beta lactamase inhibitors and the association of piperacillin and tazobactam are eective against aerobes and facultative bacteria [54]. Warty skin lesions may fallow the inoculation of opportunist mycobacteria into supercial abrasions. These atypical mycobacterial infections of skin are caused by a group of nontuberculous mycobacterial microorganisms [55]. Mycobacterium marinum is the most common and causes cutaneous infection in immunocompetent individuals. This microorganism is found in fresh or salt water, or sh, thus shermen and those who keep sh are at higher risk [56]. Other pathogens include Mycobacterium avium-intracellular, Mycobacterium ulcerans, Mycobacterium chelonae, and Mycobacterium fortuitum. The infection is mainly via inoculation or trauma. M chelonae and M fortuitum are associated with injection and occur more often in immunocompromised individuals. Immunosuppression is associated with disseminated disease [57]. M ulcerans disease (Buruli ulcer) is an important health problem in several West African countries. It is prevalent in scattered foci around the world, predominantly in riverine areas with a humid, hot climate. M ulcerans infection leads to necrosis of subdermal tissue and secondary skin ulceration [58]. The choice of therapy will depend on the site and nature of the infection, the species of causative organism and the presence of any underlying predisposing condition. Moreover, the in vitro susceptibility to antimicrobial drugs varies considerably both between and within the species of mycobacteria. In general, however, drug sensitivity tests have not proved helpful; the combinations of drugs to which the bacilli are resistant

in vitro often prove eective in vivo. Therapy is therefore usually empirical and often based on anecdotal evidence or retrospective surveys [59]. Viral infections Viruses cannot be considered a component of the normal ora of the skin but the skin is a frequent site of manifestations of viral infection. The lesions may be limited or widespread as part of a systemic infection. Many common systemic virus infections are clinically apparent, mainly as a generalized maculopapular skin rash. For these virus infections, after an initial replication phase in or close to the site of infection, generally the oropharynx, there is systemic viremia with seeding of the skin. It is probable that the rash is immunemediated. In other cases, the skin lesion caused by the virus is a vesicular lesion. In these cases the skin lesions are the sites of viral replication and are infectious [60]. The microorganisms most commonly involved in skin infections of a viral etiology are listed in Table 2. During the past decade, increasing attention has been paid to papillomaviruses and the conditions they cause. This interest is largely because of advances that have been made in the detection and characterization of nucleic acids, as papillomavirus cannot be isolated in any routine cultures, and because of its association with cervical carcinoma. Human papillomaviruses (HPV) contain double-stranded DNA and are classied into types on the basis of their nucleotide sequences. There are now at least 90 human HPV types, sequentially numbered from 1, with some further divided into subtypes alphabetically if distinctive endonuclease restriction patterns are seen [61]. The types of HPV are associated with the particular consequences of infection. Infection is primarily of the squamous epithelium, and although there are many manifestations, the common wart (verruca vulgaris) and plantar warts are the usual presentations. The principal sites are on the hands and feet, and they are a major cause of dermatological consultation. Plantar warts are most commonly caused by HPV-1, -2, -4, -31, and -32. The virus enters through skin abrasions and infects the cells of the basal layers of the skin. There is no diusion to the deep tissues. Viral replication is slow and is closely dependent on the dierentiation of the host cells. Viral DNA is present in the basal cells, but the viral antigens and the infecting virus are produced only when the cells start to become squamous and

670 Table 2 Main viruses that involve the skin Virus family Herpetoviridae Virus genus Herpesvirus

RUOCCO

et al

Virus and disease Herpes simplex Varicella-zoster Herpesvirus type 6 Molluscum contagiosum Papillomavirus Coxsackievirus A, B Echovirus Measles Rubella Parvovirus B19 Stomatitis, genital herpes, etc. Chickenpox, Zoster Roseola infantum Warts Hand, foot, and mouth disease

Poxviridae Papovaviridae Picornaviridae Paramyxoviridae Togaviridae Parvoviridae

Molluscipoxvirus Papillomavirus Enterovirus Morbillivirus Rubivirus Parvovirus

Erythema infectiosum, aplastic crisis

keratinized once they reach the surface. The incubation period is usually about 4 to 6 months, with transmission by direct contact or by fomite. The natural history is of regression, with about two thirds resolving within 2 years. Other benign skin lesions include at warts (HPV-3 and -12) and butchers warts (HPV-7) [62]. Epidermodyslasia verruciformis is a rare condition. It is an HPV-specic disorder of cell-mediated immunity resulting in disseminated warty lesions that persist for life. An autosomal recessive mode of inheritance is probable. Up to 23 types of HPV infect these patients, such as HPV-5, -8, -9, and -12, and most do not cause lesions in healthy subjects [63]. Genital warts have attracted great attention because of their association with genital cancer. The common type of genital wart is the condyloma acuminatum, which is due to infection with HPV-6 or -11. The incubation period is about 1 to 6 months and may occur not only on the external genitalia, but also on the mucosal surfaces of the vagina and urethra. They rarely become malignant and usually spontaneously resolve. Infection may often be asymptomatic, so the possibilities of transmission are enhanced since the infected person does not seek treatment. The malignant potential of papillomaviruses has been recognized for many years, but it is only in the past 15 years that involvement in carcinoma of the cervix has been demonstrated The detection of HPV DNA by hybridization techniques has implicated HPV, particularly HPV-16 and -18, in a range of cervical lesions from carcinoma in situ to invasive cervical carcinoma. In malignant cells, the HPV genoma has been integrated into the cell genome, rather than remaining extrachromosomal. These HPV types also occur commonly

in sexually active women with no cervical lesions, so the role of the detection of HPV-16 and -18 in the management of women with a clinically and cytologically normal cervix remains to be dened [64]. Papillomaviruses have also been implicated in human malignancies of the skin, such as squamous cell carcinoma in patients with epidermodysplasia verruciformis, and a similar carcinoma may occur in immunocompromised renal transplant recipients, who have a high risk of developing warts [65]. Herpes simplex virus (HSV) is probably the most common virus infecting the human skin. HSV is divided into types 1 and 2 according to a number of features including DNA fragment size after endonuclease restriction analysis. As with all herpes viruses, they are large, enveloped virions with an icosahedral nucleocapsid consisting of 162 capsomeres, arranged around a linear, double-stranded DNA core. The DNAs of HSV1 and HSV-2 are largely colinear, and considerable homology exists between the HSV-1 and HSV-2 genomes. These homologous sequences are distributed over the entire genomic map, and most of the polypeptides specied by one viral type are antigenically related to polypeptides of the other viral type. This results in considerable cross-reactivity between the HSV-1 and HSV-2 glycoproteins, although unique antigenic determinants exist for each virus. Viral surface glycoproteins mediate HSV attachment to and penetration into the cells and provoke host immune responses. Eleven glycoproteins of HSV have been identied (gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, and gM), with a 12th predicted (gN). gD is the most potent inducer of neutralizing

INFECTIOUS SKIN DISEASES

671

antibodies and appears to be related to viral entry into a cell; gB is also required for infectivity. Antigenic specicity is provided by gG, with the resulting antibody response, thus allowing the distinction between HSV-1 (gG-1) and HSV-2 (gG-2) [66,67]. HSV-1 is generally associated with oro-labial disease, with most infections occurring during childhood, and HSV-2 with genital disease with infection following sexual debut [68]. However, it is possible for HSV-2 to cause oro-labial herpes and HSV-1 to cause genital herpes [68]. Primary infection with HSV-1 usually occurs in childhood and is often asymptomatic, although it can present as stomatitis, occasionally severe. After primary oral infection, the virus becomes latent in the neurons of the trigeminal ganglia, with reactivation likely later in life. The natural history is of a few hours of nonspecic tingling, followed by the development of vesicles, scabbing, and resolution over a few days. Two biologic properties of HSV that directly inuence human disease are latency and neurovirulence. During HSV infection, virions are transported by retrograde ow along axons that connect the point of entry into the body to the nuclei of sensory neurons. Viral multiplication occurs in a small number of sensory neurons, and the viral genome then remains in a latent state lifelong. With periodic reactivation brought on by events such as physical or emotional stress, fever, UV light, and tissue damage, the virus is transported back down the axon to replicate again at or near the original point of entry into the body. Such reactivation can result in clinically apparent disease (lesions) or a clinically inapparent (asymptomatic or subclinical) infection. The mechanisms by which HSV establishes latency are currently under investigation. Neurovirulence refers to the anity with which HSV is drawn to and propagated in neuronal tissue. This can result in profound disease with severe neurological sequelae, as is the case with neonatal HSV central nervous system (CNS) disease and with herpes simplex encephalitis in older children and adults [69]. Although there is debate about the usefulness of antivirals, such as acyclovir, continuous oral therapy is often recommended for herpetic infections to suppress a recurrence [60]. Molluscum contagiosum is a disease caused by a poxvirus of the Molluscipox virus genus that produces a benign self-limited papular rash of multiple umbilicated cutaneous tumors. This

common viral disease is conned to the skin and mucous membranes. It has an incubation period from 1 week to 6 months. The lesions vary in number from one to many hundreds, and develop from papules to esh-colored or pearly nodules, which often have an umbilicated center. Systemic symptoms are rare. In adults they usually occur on the trunk, genital area, and thighs, and infection may be sexually transmitted. In children, in whom infection is more common, the lesions are predominantly on the trunk and proximal extremities. The disease usually lasts 6 to 9 months, although individual lesions persist for only about 2 months. The virus has not been cultured and, if necessary, a specic diagnosis can be attained by electron microscopy [70]. Measles, rubella, parvovirus B19, and some enteroviruses produce generalized maculo-papular rashes and it may not be easy to distinguish between them clinically, particularly in infections of the latter three. Transmission is by the respiratory route for measles, rubella, and parvovirus B19, but is fecal-oral for the enteroviruses. Measles virus, a member of the Morbillivirus genus, is an enveloped virus with a nonsegmented negative-strand RNA genome. It has two envelope glycoproteins, the hemoagglutinin (H) and the fusion (F) protein, which are responsible for receptor binding and membrane fusion, respectively. The measles virus causes a common childhood disease with high fever and typical skin rash. Measles has an incubation period of about 10 to 11 days, the rash being preceded by a 2- to 3-day prodrome of fever, coryza, and conjunctivitis, with the appearance of the characteristic elevated white Kopliks spots on the mucosa of the mouth. The rash usually starts around the head and spreads to the trunk and extremities, with resolution after a few days [71]. Complications are not uncommon, arising in about 3% to 4% of cases, and include bacterial pneumonia and otitis media. Postinfectious encephalitis occurs at a rate of about 1 in 2000 to 5000 cases and carries signicant mortality. Subacute sclerosing panencephalitis (SSPE) occurs after an interval of some years in about one in a million cases. It reects a persistent infection with measles virus. Patients present with intellectual impairment and progress to motor dysfunction, coma, and death. In the immunocompromised child, measles may occur without a rash but lead to encephalitis or giant-cell pneumonia, which have high fatality rates [72].

672

RUOCCO

et al

The use of live attenuated vaccines given in the second year of life has led to a great decline in incidence of measles in countries with a high uptake. Despite the introduction of measles vaccine in the late 1960s, biannual epidemics were still occurring up to the late 1980s. The introduction of mumps, measles, and rubella (MMR) vaccine in 1988, together with more active vaccination campaigns, have led to an increased uptake of vaccine. This follows the example set by the United States, where measles is now an uncommon disease, although it still presents occasional problems of institutional outbreaks in adolescents who are not immunized or have vaccine failure [73]. Rubella virus is a positive-sense, singlestranded RNA virus belonging to the Togaviridae family of the genus Rubivirus [74]. Infection with rubella virus usually results in a mild disease that only rarely produces signicant sequelae. Rubella has a somewhat longer incubation period of 16 to 17 days and, particularly in children, is a mild disease, often asymptomatic. It is transmitted through aerosol and is less contagious than measles, but more than parotitis. The virus initially multiplies in the local lymphoid tissues, then spreads to the spleen and distant lymph nodes. Continuous multiplication in these tissues brings on, a week after infection, a viremic phase and an onset of the virus in the respiratory tract and in the skin and, in some cases in the placenta, joints, and kidneys. The pink macular rash usually starts on the face, spreads centrifugally, and lasts only 2 to 3 days. The major complication of rubella is intrauterine infection and fetal damage if infection occurs during the rst 4 months of pregnancy. Persistent infection with rubella virus can lead to immunopathological problems presenting after birth, such as pneumonia. No eective antiviral treatment exists. Live attenuated rubella vaccines have been available since the 1970s to prevent congenital rubella. It is given by parenteral route, generally associated with the vaccine for measles and parotitis [75]. The human parvovirus B19 belongs to the genus Erythrovirus and is the only member of the family Parvoviridae to cause a wide range of human diseases in both children and adults [76]. The virus is nonenveloped, and its genome consists of a linear, single-stranded DNA molecule of approximately 5600 nucleotides with terminal palindromic inverted sequences of 383 nucleotides at both ends. Infection with parvovirus B19 cannot be dierentiated clinically from rubella in

the adult. In the child, but not in the adult, the rash, erythema infectiosum (or Fifth disease), is usually accompanied by a malar erythema leading to the alternative name of slapped cheek syndrome. In patients with hemolytic anemia, such as sickle cell disease and hereditary spherocytosis, the transient inhibitory eect of parvovirus B19 on the bone marrow may lead to aplastic crisis. Infection during pregnancy does not lead to fetal damage, although hydrops fetalis, intrauterine death, and stillbirth may occur after infection in the second trimester [77]. No vaccine is available. Human herpes type 6 (HHV-6) is the sixth member of the herpes virus family that includes herpes simplex virus 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), and Epstein-Barr virus. It is a member of the beta-herpes virus subfamily, of the Roseolavirus genus. HHV-6 is an enveloped DNA virus of 160 to 200 nm in diameter and approximately 167 kbp in length. The sequence dierence between variant A and B is about 4%, and the two can be dierentiated by restriction fragment length polymorphism, monoclonal antibodies, polymerase chain reaction (PCR) and in vitro growth characteristics [78]. The isolation and characterization of HHV-6 was rst reported in 1986 using cultures of peripheral blood leukocytes (PBL) from patients with acquired immune deciency syndrome (AIDS) and lymphoproliferative disease. HHV-6 preferentially infects CD4 T lymphocytes, but can also infect other cell lines of epithelial, broblastic, and neuronal origin with dierent eciency. The clinical syndrome of exanthem sabitum (or roseola infantum, sixth disease) was rst described in 1913; however, its etiological link to HHV-6 was rst identied in 1988. Approximately 50% to 60% of children are infected by HHV-6 by 12 months of age and almost all children are infected by the age of 2 to 3 years. This illness is characterized by a high fever that resolves with the appearance of a pinkish macular rash. Studies have implicated HHV-6 as an etiological agent of meningitis and encephalitis in the immunocompromised [79]. Antiviral agents that have been found to be eective in vitro against HHV-6 include ganciclovir, foscarnet, and cidofovir. In otherwise healthy children HHV-6 infection is usually self-limited and no antiviral treatment is needed [80]. Varicella-zoster virus (VZV) is a highly cellassociated member of the Herpesviridae family and one of the eight herpes viruses to infect

INFECTIOUS SKIN DISEASES

673

humans. It is a double-stranded DNA virus and is most closely related to herpes simplex virus types 1 and 2. These viruses rapidly proliferate and invade and destroy the infected cells. The virus is ubiquitous in most populations worldwide and the primary infection causes varicella, more commonly known as chickenpox. VZV is neurotropic and establishes latency in sensory neurons. Reactivation from latency, usually during periods of impaired cellular immunity, causes herpes zoster (shingles). Varicella is a common childhood infection with an incubation period of 14 to 16 days. A shortened incubation period can be encountered especially in immunocompromised patients. The skin lesions progress rapidly through the stages of macules to papules to vesicles that rapidly burst and form a crust. The lesions appear in series; therefore, all stages in their genesis can be seen at any one time. Patients with varicella are generally considered to be infectious 2 days before the appearance of the rash and 7 days after the onset, when the vesicles have crusted. In children with normal cellular immunity, varicella is usually a benign and selflimiting illness. In adults, however, varicella presents a higher risk of complications such as viral pneumonia, encephalitis, and skin sepsis. Varicella in the rst 5 months of pregnancy may occasionally cause fetal damage [81]. Herpes zoster mainly aects a single dermatome of the skin. It may occur at any age but the majority of patients are older than 50. The latent virus reactivates in a sensory ganglion and tracks down the sensory nerve to the appropriate segment. The lower cervical, thoracic, and lumbar posterior root ganglia are most commonly involved. The rash is commonly preceded by paresthesia, burning pains, and tenderness of the skin. The trigeminal ganglion is another common site of reactivation and the ophthalmic branch of this nerve is 20 times more likely to be involved than the other 2 branches. Facial palsy associated with vesicles in the external auditory meatus is known as the Ramsay-Hunt syndrome and is thought to be a form of zoster involving the VIIth nerve. The skin lesions are usually accompanied by local pain, which often precedes the lesions, but pain may occur without visible lesions (zoster sine herpete). The most severe complication in healthy individuals is persistent pain at the site of the lesions that may last for several months [82]. Some benet may be had from early use of acyclovir, but whether this inuences postherpetic neuralgia has been debated.

Zoster in pregnancy presents no hazard to the fetus or neonate [83]. Last, it is known that immunocompromised patients present many additional problems regarding viral skin infections, but it is not known whether this is a consequence of therapy or of underlying problems, such as lymphoma or HIV infection. These infections, such as HSV and VZV with a latent state are more likely to reactivate and disseminate rather than remain as a localized lesion. Varicella and measles may be life threatening. Rubella does not seem to present any particular risk, and parvovirus B19 and enteroviruses only rarely present problems. The dissemination of warts can occur in molluscum contagiosum. In conclusion, the past decade has seen exciting developments in identifying the etiological agents for syndromes manifesting in the skin, but there are still others for which a viral origin seems likely. References
[1] Kazmierczak AK, Szewczyk EM. Bacteria forming a resident ora of the skin as a potential source of opportunistic infections. Pol J Microbiol 2004; 53(4):24955. [2] Hartmann AA. The inuence of various factors on the human resident skin ora. Semin Dermatol 1990;9(4):3058. [3] Zaslo M. Antimicrobial peptides of multicellular organisms. Nature 2002;415:38995. [4] Baroni A, Orlando M, Donnarumma G, et al. Tolllike receptor 2 (TLR2) mediates intracellular signalling in human keratinocytes in response to Malassezia furfur. Arch Dermatol Res 2005;10:19. [5] Elsner P. Antimicrobials and the skin physiological and pathological ora. Curr Probl Dermatol 2006; 33:3541. [6] Hoer U, Gloor M, Peters G, et al. Qualitative and quantitative investigations on the resident bacterial skin ora in healthy persons and in the non-aected skin of patients with seborrheic eczema. Arch Dermatol Res 1980;268(3):297312. [7] Marina SS, Bocheva GS, Kazanjieva JS. Severe bacterial infections of the skin: uncommon presentations. Clin Dermatol 2005;23(6):6219. [8] Edlich RF, Winters KL, Britt LD, et al. Bacterial diseases of the skin. J Long Term E Med Implants 2005;15(5):499510. [9] Akiyama H, Kanzaki H, Tada J, et al. Coagulasenegative staphylococci isolated from various skin lesions. J Dermatol 1998;25(9):5638. [10] Noble WC. Skin bacteriology and the role of Staphylococcus aureus in infection. Br J Dermatol 1998; 139(Suppl 53):912.

674

RUOCCO

et al [29] Bridge PD, Sneath PH. Numerical taxonomy of Streptococcus. J Gen Microbiol 1983;129(3): 56597. [30] Lawrence J, Yajko DM, Hadley WK. Incidence and characterization of beta-hemolytic Streptococcus milleri and dierentiation from S pyogenes (group A), S equisimilis (group C), and large-colony group G streptococci. J Clin Microbiol 1985; 22(5):7727. [31] Schlievert PM, Bohach GA, Ohlendorf DH, et al. Molecular structure of Staphylococcus and Streptococcus superantigens. J Clin Immunol 1995;15: 4S10S. [32] Carlsson F, Sandin C, Lindahl G. Human brinogen bound to Streptococcus pyogenes M protein inhibits complement deposition via the classical pathway. Mol Microbiol 2005;56(1):2839. [33] Cole JN, McArthur JD, McKay FC, et al. Trigger for group A streptococcal M1T1 invasive disease. FASEB J 2006;20(10):17457. [34] Chatellier S, Ihendyane N, Kansal RG, et al. Genetic relatedness and superantigen expression in group A streptococcus serotype M1 isolates from patients with severe and nonsevere invasive diseases. Infect Immun 2000;68(6):352334. [35] Courtney HS, Hasty DL, Dale JB. Anti-phagocytic mechanisms of Streptococcus pyogenes: binding of brinogen to M-related protein. Mol Microbiol 2006;59(3):93647. [36] Sandin C, Carlsson F, Lindahl G. Binding of human plasma proteins to Streptococcus pyogenes M protein determines the location of opsonic and nonopsonic epitopes. Mol Microbiol 2006;59(1):2030. [37] Stevens DL. Invasive group A streptococcal disease. Infect Agents Dis 1996;5(3):15766. [38] Kazmi SU, Kansal R, Aziz RK, et al. Reciprocal, temporal expression of SpeA and SpeB by invasive M1T1 group a streptococcal isolates in vivo. Infect Immun 2001;69(8):498895. [39] Baxter F, McChesney J. Severe group A streptococcal infection and streptococcal toxic shock syndrome. Can J Anaesth 2000;47(11):112940. [40] Everest E. Group A streptococcal fasciitis. Crit Care Resusc 1999;1(1):638. [41] Crum NF, Wallace MR. Group B streptococcal necrotizing fasciitis and toxic shock-like syndrome: a case report and review of the literature. Scand J Infect Dis 2003;35(1112):87881. [42] Currie BJ. Group A streptococcal infections of the skin: molecular advances but limited therapeutic progress. Curr Opin Infect Dis 2006;19(2):1328. [43] Kazmierczak AK, Szarapinska-Kwaszewska JK, Szewczyk EM. Opportunistic coryneform organisms residents of human skin. Pol J Microbiol 2005;54(1): 2735. [44] Golledge CL, Phillips G. Corynebacterium minutissimum infection. J Infect 1991;23(1):736. [45] Holdiness MR. Management of cutaneous erythrasma. Drugs 2002;62(8):113141.

[11] Peacock SJ, de Silva I, Lowy FD. What determines nasal carriage of Staphylococcus aureus? Trends Microbiol 2001;9:60510. [12] Lowy FD. Staphylococcus aureus infections. N Engl J Med 1998;339:52032. [13] Foster TJ, Hook M. Surface protein adhesins of Staphylococcus aureus. Trends Microbiol 1998;6: 4848. [14] Skaar EP, Schneewind O. Iron-regulated surface determinants (Isd) of Staphylococcus aureus: stealing iron from heme. Microbes Infect 2004;6:3907. [15] ORiordan K, Lee JC. Staphylococcus aureus capsular polysaccharides. Clin Microbiol Rev 2004;17: 21834. [16] Schwarz-Linek U, Hook M, Potts JR. The molecular basis of bronectin-mediated bacterial adherence to host cells. Mol Microbiol 2004;52:63141. [17] Schwarz-Linek U, Werner JM, Pickford AR, et al. Pathogenic bacteria attach to human bronectin through a tandem b-zipper. Nature 2003;423: 17781. [18] Dinges MM, Orwin PM, Schlievert PM. Exotoxins of Staphylococcus aureus. Clin Microbiol Rev 2000;13:1634. [19] Murray RJ. Recognition and management of Staphylococcus aureus toxin-mediated disease. Intern Med J 2005;35(2):10619. [20] Lina G, Gillet Y, Vandenesch F, et al. Toxin involvement in staphylococcal scalded skin syndrome. Clin Infect Dis 1997;25(6):136973. [21] Dondorp AM, Veenstra J, van der Poll T, et al. Activation of the cytokine network in a patient with AIDS and the recalcitrant erythematous desquamating disorder. Clin Infect Dis 1994;18(6): 9425. [22] Akiyama H, Morizane S, Yamasaki O, et al. Assessment of Streptococcus pyogenes microcolony formation in infected skin by confocal laser scanning microscopy. J Dermatol Sci 2003;32(3):1939. [23] Hirschmann JV. Impetigo: etiology and therapy. Curr Clin Top Infect Dis 2002;22:4251. [24] Chiller K, Selkin BA, Murakawa GJ. Skin microora and bacterial infections of the skin. J Investig Dermatol Symp Proc 2001;6(3):1704. [25] Akiyama H, Hamada T, Huh WK, et al. Confocal laser scanning microscopic observation of glycocalyx production by Staphylococcus aureus in skin lesions of bullous impetigo, atopic dermatitis and pemphigus foliaceus. Br J Dermatol 2003;148(3): 52632. [26] Travers JB, Norris DA, Leung DY. The keratinocyte as a target for staphylococcal bacterial toxins. J Investig Dermatol Symp Proc 2001;6(3):22530. [27] Veien NK. The clinicians choice of antibiotics in the treatment of bacterial skin infection. Br J Dermatol 1998;139(53):306. [28] Thestrup-Pedersen K. Bacteria and the skin: clinical practice and therapy update. Br J Dermatol 1998; 139(53):13.

INFECTIOUS SKIN DISEASES

675

[46] Funke G, von Graevenitz A, Clarridge JE 3rd, et al. Clinical microbiology of coryneform bacteria. Clin Microbiol Rev 1997;10(1):12559. [47] Connell TG, Rele M, Daley AJ, et al. Skin ulcers in a returned traveller. Lancet 2005;365:1925. [48] von Hunolstein C, Alfarone G, Scopetti F, et al. Molecular epidemiology and characteristics of Corynebacterium diphtheriae and Corynebacterium ulcerans strains isolated in Italy during the 1990s. J Med Microbiol 2003;52(2):1818. [49] Do TT, Strub WM, Witte D. Subacute Propionibacterium acnes osteomyelitis of the spine in an adolescent. J Pediatr Orthop B 2003;12(4):2847. [50] Pan SC, Wang JT, Hsueh PR, et al. Endocarditis caused by Propionibacterium acnes: an easily ignored pathogen. J Infect 2005;51(4):22931. [51] Oprica C, Nord CE. ESCMID Study Group on Antimicrobial Resistance in Anaerobic Bacteria. European surveillance study on the antibiotic susceptibility of Propionibacterium acnes. Clin Microbiol Infect 2005;11(3):20413. [52] Tariq S, Hameed S, Tyrer M, et al. Pseudomonal sepsis with subcutaneous nodules in an HIV-infected individual with high CD4 counts. J Infect 2005; 51(5):41920. [53] Jones ME, Karlowsky JA, Draghi DC, et al. Epidemiology and antibiotic susceptibility of bacteria causing skin and soft tissue infections in the USA and Europe: a guide to appropriate antimicrobial therapy. Int J Antimicrob Agents 2003;22(4): 40619. [54] Grieco T, Cantisani C, Innocenzi D, et al. Acute generalized exanthematous pustulosis caused by piperacillin/tazobactam. J Am Acad Dermatol 2005;52(4):7323. [55] Street ML, Umbert-Miller IJ, Roberts GD, et al. Nontuberculous mycobacterial infections of the skin. Report of fourteen cases and review of the literature. J Am Acad Dermatol 1991;24:20815. [56] Ena P, Sechi LA, Saccabusi S, et al. Rapid identication of cutaneous infections by nontubercular mycobacteria by polymerase chain reaction-restriction analysis length polymorphism of the hsp65 gene. Int J Dermatol 2001;40:4959. [57] van der Werf TS, Stienstra Y, Johnson RC, et al. Mycobacterium ulcerans disease. Bull World Health Organ 2005;83(10):78591. [58] Ang P, Rattana-Apiromyakij N, Goh CL. Retrospective study of Mycobacterium marinum skin infections. Int J Dermatol 2000;39:3437. [59] Ho MH, Ho CK, Chong LY. Atypical mycobacterial cutaneous infections in Hong Kong: 10-year retrospective study. Hong Kong Med J 2006;12(1): 216. [60] Park KC, Han WS. Viral skin infections: diagnosis and treatment considerations. Drugs 2002;62(3): 47990. [61] Haas S, Park TW, Voigt E, et al. Detection of HPV 52, 58 and 87 in cervicovaginal intraepithelial lesions

[62] [63]

[64]

[65]

[66]

[67]

[68]

[69] [70] [71]

[72]

[73]

[74]

[75]

[76] [77]

of HIV infected women. Int J Mol Med 2005;16(5): 8159. Akgul B, Cooke JC, Storey A. HPV-associated skin disease. J Pathol 2006;208(2):16575. Ortak T, Uysal AC, Alagoz MS, et al. Epidermodysplasia verruciformis: an unusual presentation. Dermatol Surg 2006;32(2):3026. Giovannelli L, Lama A, Capra G, et al. Detection of human papillomavirus DNA in cervical samples: analysis of the new PGMY-PCR compared to the hybrid capture II and MY-PCR assays and a twostep nested PCR assay. J Clin Microbiol 2004; 42(8):38614. Purdie KJ, Surentheran T, Sterling JC, et al. Human papillomavirus gene expression in cutaneous squamous cell carcinomas from immunosuppressed and immunocompetent individuals. J Invest Dermatol 2005;125(1):98107. Looker KJ, Garnett GP. A systematic review of the epidemiology and interaction of herpes simplex virus types 1 and 2. Sex Transm Infect 2005;81(2): 1037. Laerty WE, Downey L, Celum C, et al. Herpes simplex virus type 1 as a cause of genital herpes: impact on surveillance and prevention. J Infect Dis 2000; 181:14547. Lowhagen GB, Tunback P, Bergstrom T. Proportion of herpes simplex virus (HSV) type 1 and type 2 among genital and extragenital HSV isolates. Acta Derm Venereol 2002;82:11820. David WK. Neonatal herpes simplex infection. Clin Microbiol Rev 2004;17(1):113. Hanson D, Diven DG. Molluscum contagiosum. Dermatol Online J 2003;9(2):2. Yanagi Y, Takeda M, Ohno S, et al. Measles virus receptors and tropism. Jpn J Infect Dis 2006;59(1): 15. Kuhne Simmonds M, Jin L, Brown DW. Measles viral load may reect SSPE disease progression. Virol J 2006;3(1):49. Friederichs V, Cameron JC, Robertson C. Impact of adverse publicity on MMR vaccine uptake: a population based analysis of vaccine uptake records for one million children, born 1987-2004. Arch Dis Child 2006;91(6):4658. Murphy FA, Fauquet CM, Bishop DHL, et al. In: Virus taxonomy, 6th report of the International Committee on Taxonomy of Viruses (ICTV). Arch Virol 1995;10:1586. Wilson KM, Di Camillo C, Doughty L, et al. Humoral immune response to primary rubella virus infection. Clin Vaccine Immunol 2006;13(3):3806. Young NS, Brown KE. Parvovirus B19. N Engl J Med 2004;350:58697. Sanabani S, Neto WK, Pereira J, Sabino EC. Sequence variability of human erythroviruses present in bone marrow of Brazilian patients with various Parvovirus B19-related hematological symptoms. J Clin Microbiol 2006;44(2):6047.

676

RUOCCO

et al [81] Ku CC, Besser J, Abendroth A, et al. VaricellaZoster virus pathogenesis and immunobiology: new concepts emerging from investigations with the SCIDhu Mouse Model. J Virol 2005;79(5): 26518. [82] Bosnjak L, Jones CA, Abendroth A, et al. Dendritic cell biology in herpesvirus infections. Viral Immunol 2005;18(3):41933. [83] Holten KB. Treatment of herpes zoster. Am Fam Physician 2006;73(5):8824.

[78] Yoto Y, Qiu J, Pintel DJ. Identication and characterization of two internal cleavage and polyadenylation sites of Parvovirus B19 RNA. J Virol 2006; 80(3):16049. [79] Abdel-Haq NM, Asmar BI. Human Herpes Virus 6 (HHV6) infection. Indian J Pediatr 2004;71(1):8996. [80] Yoshida M, Yamada M, Tsukazaki T, et al. Comparison of antiviral compounds against human herpes virus 6 and 7. Antiviral Res 1998;40(12): 7384.