Dual Targeting of CCR2 and CCR5

Review
Dual targeting of CCR2 and CCR5:

therapeutic potential for immunologic and
cardiovascular diseases
Qihong Zhao1
Research and Development, Bristol-Myers Squibb, Princeton, New Jersey, USA
RECEIVED OCTOBER 9, 2009; REVISED FEBRUARY 19, 2010; ACCEPTED FEBRUARY 22, 2010. DOI: 10.1189/jlb.1009671
ABSTRACT
A cardinal feature of inflammation is the tissue recruitment of leukocytes, a process that is mediated predominantly by chemokines via their receptors on migrating cells. CCR2 and CCR5, two CC chemokine receptors, are important players in the trafficking of
monocytes/macrophages and in the functions of other
cell types relevant to disease pathogenesis. This review provides a brief overview of the biological actions
of CCR2 and CCR5 and a comprehensive summary of
published data that demonstrate the involvement of
both receptors in the pathogenesis of immunologic diseases (RA, CD, and transplant rejection) and cardiovascular diseases (atherosclerosis and AIH). In light of the
potential for functional redundancy of chemokine receptors in mediating leukocyte trafficking and the consequent concern over insufficient efficacy offered by
pharmacologically inhibiting one receptor, this review
presents evidence supporting dual targeting of CCR2
and CCR5 as a more efficacious strategy than targeting either receptor alone. It also examines potential
safety issues associated with such dual targeting. J.
Leukoc. Biol. 88: 4155; 2010.
Introduction
Tissue recruitment of leukocytes, a cardinal feature of inflammation, is mediated mainly by chemokines (chemotactic cytokines) via their receptors. The chemokine superfamily can be
categorized into four groups (CC, CXC, CX3C, and C), according to the number and spacing of conserved cysteines in
Abbreviations: AIAadjuvant-induced arthritis, AIHaccelerated intimal hyperplasia, ApoBtgapolipoprotein B transgenic, ApoEapolipoprotein E,

BOSbronchiolitis obliterans syndrome, CAVcardiac allograft vasculopathy, CDCrohns disease, CIAcollagen-induced arthritis, CsA
cyclosporine A, DNBSdinitrobenzenesulfonic acid, DSSdextran sodium
sulfate, GMME1GM-CSF fused to an N-terminal truncated form of
MCP-1, HFDhigh fat diet, KOknockout, LDLRlow-density lipoprotein
receptor, LIALPS-induced arthritis, MMPmatrix metalloprotease, PNA
CCR5antisense peptide nucleic acid, RArheumatoid arthritis,
SCWAstreptococcal cell wall-induced arthritis, shRNAshort hairpin RNA,
TBEVtick-borne encephalitis virus, VSMCvascular smooth muscle cell,
WNVWest Nile virus
0741-5400/10/0088-0041 Society for Leukocyte Biology
the amino acid sequence [1 4]. The effects of chemokines on

their target cells are mediated by receptors that belong to the
seven-transmembrane-spanning G protein-coupled receptor
family [1 4]. Besides their well-recognized role in leukocyte
recruitment, some chemokines and chemokine receptors impact other cellular functions such as activation, proliferation,
and differentiation [1 4]. In addition, a subset of chemokine
receptors plays a nonredundant role in infectious diseases, as
demonstrated by resistance to HIV/AIDS in people homozygous for CCR5 32 (a loss of function mutation) [59] and
resistance to Plasmodium vivax-induced malaria in people carrying a loss-of-expression mutation (T46C) in the gene promoter
of the Duffy antigen receptor for chemokines [10, 11]. As chemokine receptors are G protein-coupled receptors, many pharmaceutical/biotech companies have invested appreciable time
and efforts in developing small-molecule antagonists [12, 13].
As a result, two such antagonists have been approved by the
U.S. Food and Drug Administration: Maraviroc (a CCR5 antagonist) for the treatment of HIV/AIDS [14] and Plerixafor (a
CXCR4 antagonist) for use in combination with G-CSF to mobilize hematopoietic stem cells to the peripheral blood for collection and subsequent autologous transplantation in patients
with non-Hodgkins lymphoma and multiple myeloma [15].
However, for chronic inflammatory diseases, clinical trials with
antagonists of a single chemokine receptor (e.g., CCR1, CCR2,
or CCR5) have not proved successful [12, 13]. Considering the
complexity in the pathogenesis of these diseases and the potential for functional redundancy of chemokine receptors,
these failed clinical studies suggest that targeting a single receptor may not be adequate for efficacy in these chronic conditions.
CCR2 and CCR5 are two CC chemokine receptors that are
important players in the trafficking of monocytes/macrophages and in the functions of other cell types relevant to disease pathogenesis [16, 17]. Considering that several reviews
about targeting CCR2 or CCR5 alone have been published
previously, and clinical trials with agents against either receptor have, so far, afforded little efficacy in patients with chronic
1. Correspondence: Bristol-Myers Squibb, Rt. 206 and Province Line Rd.,
Princeton, NJ 08543, USA. E-mail: qihong.zhao@bms.com
Volume 88, July 2010
Journal of Leukocyte Biology 41
inflammatory diseases, this review focuses on dual targeting of

CCR2 and CCR5. It provides a brief overview of the biological
functions of CCR2 and CCR5 and a comprehensive summary
of published data demonstrating the involvement of both receptors in five immunologic and cardiovascular diseases. In
light of the potential for functional redundancy of chemokine
receptors in mediating leukocyte trafficking and the consequent concern over insufficient efficacy offered by pharmacologically inhibiting one receptor, the review presents evidence
supporting dual targeting of CCR2 and CCR5 as a more efficacious strategy than targeting either receptor alone. In addition, it examines potential safety issues associated with dual
targeting of CCR2 and CCR5.
BIOLOGICAL ACTIONS OF CCR2 AND

CCR5
The chemokine receptor CCR2 is expressed abundantly on the
so-called inflammatory subset of blood monocytes [18]. It is
also expressed on, and functions in, other immune/inflammatory cell types such as dendritic cells and memory Th1 cells
[19, 20]. CCR2 is a G protein-coupled receptor that binds
multiple ligands, known as macrophage chemoattractant proteins, including CCL2 (MCP-1), CCL8 (MCP-2), CCL7 (MCP3), and CCL13 (MCP-4). The relative contribution of each of
these ligands to CCR2-mediated in vivo function remains to be
elucidated [2, 3]. Of these ligands, MCP-1 is studied most extensively, and CCR2 is considered to be the exclusive receptor
for MCP-1 [2, 3]. The essential role of the CCR2/MCP-1 axis
in the tissue recruitment of monocytes/macrophages has been
demonstrated by studies in which CCR2 or MCP-1 has been
genetically ablated [2124], as well as studies in which CCR2
or MCP-1 is pharmacologically inhibited [25, 26]. KO mice of
CCR2 or MCP-1 are apparently healthy but exhibit an impaired ability to recruit monocytes/macrophages to sites of
inflammation [2730] and to produce cytokines such as TNF-
[31, 32]. The impaired recruitment of monocytes in CCR2 KO
mice or mice treated with a CCR2 antagonist can be explained
by reduced emigration of inflammatory monocytes from bone
marrow to blood [32] and reduced immigration of blood
monocytes from blood to inflamed tissues [3336]. Notably,
the CCR2/MCP-1 axis is not important for monocyte adhesion
but for migration into the inflamed tissue, such as atherosclerotic arteries [37]. Consistent with its key role in monocyte
trafficking, CCR2 has been shown to drive inflammation in a
number of animal models of diseases (see below). These include, but are not limited to, immunologic disorders such as
RA, CD, and transplant rejection, as well as cardiovascular diseases, including atherosclerosis and AIH.
Compared with CCR2, CCR5 is expressed predominantly on
macrophages differentiated from blood monocytes and Th1
cells activated in response to inflammatory stimuli [38]. It is
also expressed on nonimmune cells such as osteoclasts [39]
and VSMCs [40]. CCR5 is a G protein-coupled receptor that
binds multiple ligands, including CCL4 (MIP-1), CCL5
(RANTES), CCL3 (MIP-1), CCL8 (MCP-2), and CCL3L1
(MIP-1/LD78). Of these ligands, MIP-1 and MIP-1/
LD78 are considered to be selective to CCR5, yet RANTES is
42 Journal of Leukocyte Biology
the most extensively studied [4]. The relative contribution of

each of these ligands to CCR5-mediated in vivo function remains to be elucidated. Relative to CCR2, the in vivo function
of CCR5 is less well defined. CCR5 has been shown to contribute to the survival of macrophages during inflammation and
infection [41]. It may function to retain tissue macrophages in
inflamed tissue [42]. Also, CCR5 is important in Th1 cell recruitment and activation in inflammation [43]. For nonleukocyte cell types, CCR5 is important for the formation of osteoclasts [39], suggesting its contribution to RA pathogenesis by
affecting osteoclast function. In addition, engagement of
CCR5 on VSMCs by MIP-1 leads to their activation as measured by increased calcium flux and secretion of tissue factor
[40], suggesting its role in atherosclerosis and AIH. Consistent
with its role in these cell types, CCR5 has been shown to be an
important player in animal models of RA, CD, transplant rejection, atherosclerosis, and AIH.
PATHOGENIC ROLES OF CCR2 AND

CCR5 IN DISEASES
CCR2 and CCR5 modulate functions of monocytes/macrophages and Th1 cells as well as nonimmune cells and are both
involved in the pathogenesis of animal models of RA, CD,
transplant rejection, atherosclerosis, and AIH. For each of
these diseases, a brief overview of its pathogenesis and a summary of preclinical evidence, genetic and pharmacologic, for
CCR2 and CCR5 are provided in the following section. Where
available, clinical data related to intervention of CCR2 or
CCR5 are also discussed.
RA
RA is characterized clinically by joint pain, stiffness, and swelling as a result of synovial inflammation. Despite advances in
biologic therapies, such as TNF- blockers, significant unmet
medical need remains, including a significant percentage of
nonresponders and inadequate responders to each therapy
and the development of secondary and even tertiary resistance
to these therapies. Normal synovium contains a few cells and is
composed mostly of fibroblast-like cells, plus a smaller portion
of macrophage-like cells and other bone marrow-derived cells.
In contrast, the synovium in RA patients expands into a vascularized, invasive inflammatory mass (the pannus) that destroys
cartilage. Immune cell infiltrates in the pannus are predominantly CD4 T cells and macrophages, both of which exhibit
an activated phenotype. CD4 T cells are thought to contribute to the initiation of the disease, and macrophages contribute to the perpetuation of the disease. These cells produce
cytokines and MMPs, contributing to pain and inflammation
in the affected joints, as well as to the destruction of cartilage
matrix and bone. Consistent with the role of CCR2 and CCR5
in the tissue recruitment of monocyte/macrophages and Th1
cells and that of CCR5 in osteoclast formation, genetic and
pharmacological intervention of either receptor has been
shown to reduce disease in multiple preclinical models of RA.
www.jleukbio.org
Zhao Therapeutic potential of CCR2/CCR5 dual targeting
CCR2 inhibition in RA
Interventions, pharmacological and genetic, have been used to
validate the CCR2/MCP-1 axis in preclinical models of RA
(Table 1). Pharmacological inhibitors included antagonists of
CCR2 (peptidic and small-molecule), antibodies against CCR2
and MCP-1, and a mutant MCP-1, which inhibits binding of
endogenous MCP-1 to the endothelial surface. CCR2 KO mice
have also been used as a genetic approach.
Independent studies using three different antagonists of
CCR2 demonstrated the importance of the CCR2-MCP-1 axis
in mouse models of RA [26, 44, 45]. The first study used a
peptide antagonist of CCR2, 7-ND, a mutant MCP-1, in which
the N-terminal amino acids 2 8 have been deleted [44]. Administration of 7-ND substantially reduced the incidence and
progression of spontaneous arthritis enhanced by CFA in
MRL/lpr mice. The reduction in disease severity was seen in
preventative and therapeutic dosing of this antagonist, although less with therapeutic dosing. Disease reduction correlated with reduction in histopathological parameters: synovial
inflammation, synovial hyperplasia, pannus formation, and
bone destruction. The second study used an engineered GMCSF-MCP-1 fusokine GMME1, which potently inhibits CCR2mediated function [45]. GMME1 robustly reduced the incidence and severity of mouse CIA as well as synovial inflammation and cartilage erosion when administered therapeutically.
The third study used a small-molecule CCR2 antagonist
INCB3344 [26]. Therapeutic dosing of INCB3344 reduced disease severity of AIA in the rat by 75%, as assessed by the clinical score of swollen joints. This dosing scheme provided
plasma drug levels at trough that covered the chemotaxis IC70.
Histological evaluation showed significant inhibition of joint
inflammation (82%) and bone erosion (64%) in these animals.
Consistent with the results from studies with antagonists of
CCR2, blockade of MCP-1 following MCP-1 DNA vaccination
[46] or direct administration of anti-MCP-1 antibody [4750]
further demonstrated the importance of the MCP-1/CCR2 axis

in the pathology of experimental arthritis (Table 1). MCP-1
DNA vaccination robustly inhibited AIA in rat [46]. AntiMCP-1 antibodies were efficacious in multiple models: CIA in
rat, SCWA in rat, and LIA in rabbit [4750]. These anti-MCP-1
antibodies inhibited arthritis when dosed preventatively or
therapeutically. Reduction in clinical score correlated with that
in histopathological parameters such as synovial inflammation.
Furthermore, the importance of MCP-1 in the rat AIA model
was demonstrated with P8A-MCP-1, a mutated MCP-1, in which
proline at position 8 was changed to alanine, which displaces
endogenous MCP-1 from the endothelial surface and inhibits
monocyte infiltration into inflamed tissues [51]. Therapeutic
dosing of P8A-MCP-1 reduced clinical signs and histopathology
as measured by joint destruction, synovial lining, macrophage
infiltration, and bone erosion.
Although there is substantial pharmacological data supporting CCR2 in the pathogenesis of experimental arthritis, reports with CCR2 KO mice and anti-mouse CCR2 antibody
MC-21 provided contrasting results. Reports from the same
laboratory have described worsening of arthritis in three different models in CCR2-deficient mice relative to wild-type controls [86, 87]. The three models are CIA, collagen antibodyinduced arthritis, and Mycobacterium avium-induced arthritis.
The mechanism underlying the increased disease in CCR2deficient mice is not clear but was attributed by the authors to
the reduction of activation-induced cell death as a result of
CCR2 deficiency [86]. A separate study with the anti-mouse
CCR2 antibody MC-21 showed that i.p. injection of MC-21 at
500 g/mouse during the disease-initiation phase (Days 0 15)
improved clinical signs and histological scores of mouse CIA,
but the same treatment during the progression phase (Days
2136) exacerbated the disease [88]. The mechanism underlying the disease worsening by therapeutic dosing of MC-21 at
this dose is not clear and was attributed initially by the authors
to the inhibition of the so-called CCR2 regulatory T cell
Table 1. A Summary of Preclinical Studies Supporting a Role of CCR2/MCP-1 Axis in Diseases

Disease
Species
Model
RA
mouse, rat, rabbit
MRL/lpr arthritis, CIA, AIA,

SCWA, LIA
CD
Transplant rejection
(acute)
mouse
mouse
DSS colitis, DNBS colitis

Heart allograft, islet
allograft
(chronic)
Atherosclerosis
mouse, rat
CAV, BOS
mouse, rabbit, pig
AIH
mouse, rat, rabbit,

dog, monkey
ApoE/ mouse (HFD or

normal chow), LDLR/
mouse (HFD), ApoBtg
(HFD), LDLR/ rabbit
(normal chow), pig
(HFD)
Wire injury, vein grafting,
cuff placement balloon
injury, stenting
www.jleukbio.org
Intervention
7-ND MCP-1, GM-CSF-CCL2,
anti-CCR2 Ab, anti-MCP-1 Ab,
P8A-MCP1
CCR2 KO, MCP-1 KO
CCR2 KO, anti-MCP-1 Ab plus
rapamycin, CCR2 KO plus
rapamycin
7-ND MCP-1, CCR2 KO, MCP-1
KO, anti-MCP-1 Ab
CCR2 KO, MCP-1 KO, 7-ND
MCP-1, propagermium
CCR2 KO, MCP-1 KO, shRNA

CCR2, 7-ND MCP-1, anti-MCP-1
Ab, anti-CCR2 Ab
Reference
[26, 4452]
[53, 54]
[55, 56]
[5760]
[6171]
[7285]
function [88]. However, 3 years later, the same authors reported that the worsening of mouse CIA was a result of activation of CCR2-expressing basophils via CCR2 cross-linking by
MC-21, administered at high dose but not low dose [52]. In
contrast to high-dose MC-21, low-dose MC-21 reduced the disease when dosed therapeutically [52]. This is consistent with
the effect of other inhibitors of the CCR2/MCP-1 axis on experimental arthritis, when they are dosed therapeutically. In
summary, the majority of preclinical data from multiple studies supports the notion that pharmacological inhibition of
CCR2 reduces arthritis.
Clinically, three separate clinical studies in RA patients using
different interventions of CCR2-MCP-1 axis have been reported. The agents include Novartiss anti-MCP-1 antibody
(ABN912) [89], Millenniums humanized anti-CCR2 antibody
(MLN1202) [90], and Mercks small-molecule antagonist
(MK812). All three studies failed to demonstrate a clinical
benefit in RA patients. The basis for these failures is not completely clear. However, in the study with ABN912, a dose-dependent increase of plasma MCP-1 was observed [89]. This
increase was thought to be a result of the reduced clearance
of MCP-1 bound to ABN912, arguing that the agent was not
appropriate for testing the hypothesis. In the study with
MLN1202, the high dose (4 mg/kg) reached a trough plasma
concentration that provided 94% of CCR2 occupancy over the
treatment period yet offered no efficacy [90]. This suggests
that CCR2 is an irrelevant mechanism for RA, or even greater
receptor occupancy is needed. The details of the MK812 clinical study have not been published.
CCR5 inhibition in RA
Genetic (CCR5 KO) and pharmacological (CCR5 antagonist)
approaches were used to validate CCR5 in models of RA (Table 1). Two studies evaluated the effect of CCR5 deficiency on
mouse CIA. One study showed that loss of CCR5 did not affect
the incidence or severity of the disease [86], and the other
showed that loss of CCR5 reduced the incidence significantly
but not the severity of the disease [91]. The incidence reduction correlated with the lower plasma levels of IgG, especially
IgG2a and IgG2b, against collagen type II [91]. Notably, treatment with the CCR5 antagonist SCH-X immediately following
CIA induction reduced the clinical score, joint inflammation,
and bone/cartilage erosion significantly in a nonhuman primate (rhesus monkey) CIA model [92]. The treatment also
reduced systemic inflammation, as measured by C-reactive protein, and altered antibody response to type II collagen.
Clinically, the effect of CCR5 intervention has been studied
using gene polymorphism (CCR532) and CCR5 antagonists.
CCR532 is a 32-bp deletion within the coding sequence of
CCR5 that results in a complete loss of function of the receptor in homozygous individuals [93, 94]. Several studies have
shown that loss of function of CCR5 reduces incidence and/or
severity of human RA [9599]. Recent meta-analysis has confirmed the negative association between CCR532 and human
RA (odds ratio0.65; P0.0001) [100]. Also, a recent report
found a negative association between two functional polymorphisms in the CCR5 gene (32 and C-1835T) and juvenile RA
[101]. Importantly, although the frequency of homologous
32 deletion is low in RA patients versus normal controls, two

typical RA patients with homologous 32 deletion were identified in one study [99], raising a question of whether pharmacologic targeting of CCR5 will be effective in RA. Consistent
with this concern, two trials evaluating CCR5 antagonists in RA
have been reported to be ineffective in patients with RA. Details about the study design of these trials have not been published.
CCR2 and CCR5 dual inhibition in RA

Consistent with the notion that CCR2 and CCR5 are important
players in preclinical models of RA, mice treated with a smallmolecule antagonist of mouse CCR2, CCR5, and CXCR3
(TAK-779) were protected from CIA [102]. In addition, infiltration of leukocytes (including T cells) into the joints was robustly inhibited in TAK-779-treated mice. IC50 (nM) of TAK779 in inhibiting functions mediated by CCR2, CCR5, and
CXCR3 in mouse was reported to be 24, 369, and 236, respectively [103, 104]. One caveat with this study is that no pharmacokinetic/exposure data were provided, and therefore, the
relative inhibition of each of the three receptors by TAK-779
was unknown. This caveat applies to all other studies using
TAK-779 discussed in this review.
CD
CD is an uncontrolled, chronic mucosal inflammation in the
gastrointestinal tract. Despite advances in biologic therapies,
such as TNF- blockers, a significant, unmet medical need remains, as these therapies are suboptimal, not only for induction but also for maintenance and remission. In addition to
concerns over malignancy and serious infection associated with
administration of TNF- blockers, concomitant use of potent
immunosuppressants for helping maintain efficacy adds to the
safety burden. CD is a prototypic Th1-type granulomatous disease characterized by accumulation and activation of memoryeffector T cells and macrophages in the gut epithelium. Consistent with the role of CCR2 and CCR5 in the tissue recruitment of monocytes/macrophages and Th1 cells, genetic and
pharmacological interventions have demonstrated the importance of CCR2 and CCR5 in preclinical models of CD.
CCR2 inhibition in CD
CCR2 KO and MCP-1 KO mice have been used to demonstrate the importance of the CCR2/MCP-1 axis in models of
CD (Table 1). The models included DSS-induced acute colitis,
in which the immune cell infiltrates are predominantly neutrophils and monocyte/macrophages, as well as DNBS-induced
subacute colitis, where monocyte/macrophages and T cells
predominate. In one study [53], CCR2 KO mice were protected from DSS-induced intestinal adhesions, mucosal ulcerations, and colonic inflammation by Day 3, but the effect disappeared by Day 7. In these mice, increased levels of IL-10
and decreased levels of IFN- may have contributed to reduced inflammation. In a separate study about DNBS-induced
colitis [54], MCP-1 KO mice showed markedly reduced severity
of colitis, macroscopically and histologically, and decreased
www.jleukbio.org
mortality. This disease reduction was correlated with a downregulation of myeloperoxidase activity, decreased levels of IL1, IL-12p40, and IFN-, and reduced infiltration of CD3 T
cells, F4/80 macrophages, and 5-hydroxyltryptamine-expressing enterochromaffin cells in the colonic mucosa.
CCR5 inhibition in CD
Compared with wild-type mice, CCR5 KO mice are protected
from DSS-induced colitis macroscopically and histologically
[53] (Table 2). In these mice, mucosal mRNA expression of
IL-4, IL-5, and IL-10 was increased, whereas that of IFN- was
decreased, corresponding to a shift to a Th2 pattern of T cell
activation.
CCR2 and CCR5 dual inhibition in CD

Consistent with the notion that CCR2 and CCR5 are important
players in preclinical models of CD, mice treated with a smallmolecule antagonist of mouse CCR2, CCR5, and CXCR3
(TAK-779) were protected from DSS-induced colitis [104]. Furthermore, infiltration of monocyte/macrophages into the lamina propria was inhibited almost completely, and expression of
colonic IL-1 and IL-6 was decreased significantly in TAK-779treated mice.
TRANSPLANT REJECTION
Immunosuppressive drugs and ancillary care have led to outstanding short-term (1- to 3-year) patient and graft survival
rates. However, a significant, unmet medical need remains as a
result of such problems as poor, long-term (5 year) graft survival rates (e.g., 50% in the case of heart transplant) and
significant toxicities of the currently available immunosuppressive drugs. The mechanism for acute rejection is the ability of
T cells to recognize, via their antigen receptors, polymorphic
versions of a variety of proteins, such as the MHC. Chronic
allograft rejection is mediated by antigen-specific cellular and
humoral immunity and importantly, is often associated with
considerable transplant vasculopathy, one form of AIH that
limits the long-term survival of allografts. Consistent with the
role of CCR2 and CCR5 in the tissue recruitment of monocyte/macrophages and Th1 cells and that of CCR5 in activating VSMCs, genetic and pharmacological interventions have
demonstrated the importance of the two receptors in models
of allograft rejection, acute and chronic.
CCR2 inhibition in transplant rejection

Acute rejection. Genetic (CCR2 KO) and pharmacological (anti-MCP-1 antibody) approaches were used to support
the importance of the CCR2/MCP-1 axis in acute rejection
of allograft transplants (Table 1). In one study with the cardiac allograft model [55], CCR2-deficient mice had a modest prolongation of graft survival (13 days) relative to wildtype control (8 days), with an associated reduction of IFN-
and increase of IL-4, suggesting that allograft survival is related to the shift of Th1 to Th2 function. This study also
evaluated the effect of CCR2 deficiency on islet allograft
survival in a heterotopic islet cell allograft rejection model,
in which pancreatic islet allografts are placed under the kidney capsule of recipient mice rendered diabetic by streptozotocin [55]. When islet cells were transplanted into CCR2deficient recipients, a prolonged allograft survival (24 days)
was observed relative to that of wild-type mice (12 days). Of
note, this study did not examine the added effect of an immunosuppressant (e.g., CsA or rapamycin) on the survival
of heart or islet allograft. In a separate study using a similar
heterotopic islet cell allograft rejection model [56], islet
allograft survival in CCR2-deficient mice was not significantly different from that in wild-type littermates. However,
when CCR2-deficient recipients were treated with a subtherapeutic dose of rapamycin, the islet allograft was engrafted permanently (120 days). Wild-type littermates
treated with the same protocol did not exhibit any longterm graft survival; rejection occurred within 20 days [56].
Permanent engraftment (120 days) was also observed
when wild-type mice were treated with anti-MCP-1 antibody
plus rapamycin [56]. These data suggest a potential additive
benefit for intervention of the CCR2/MCP-1 axis with standard immunosuppressant treatment.
Table 2. A Summary of Preclinical Studies Supporting a Role of CCR5/RANTES Axis in Diseases

Disease
Species
Model
Intervention
RA
CD
(acute)
mouse, monkey
mouse
mouse, monkey
CIA
DSS colitis
Heart allograft, islet allograft
[91, 92]
[53]
[105111]
(chronic)
mouse, monkey
CAV
Atherosclerosis
mouse
AIH
mouse
ApoE/ mouse (HFD or

normal chow), LDLR/
mouse (HFD)
Wire injury
CCR5 KO, CCR5 antagonist

CCR5 KO
CCR5 KO, CCR5 KO plus CsA,
CCR5 KO plus rapamycin,
CCR5 antagonist
(small-molecule)
CCR5 KO plus CsA,
CCR5 KO plus rapamycin,
CCR5 KO plus anti-CXCR3 Ab,
Met-RANTES, CCR5 antagonist
CCR5 KO, Met-RANTES,
[44AANA47]-RANTES
CCR5 KO, Met-RANTES
[119121]
www.jleukbio.org
Reference
[105, 106, 112114]
[115118]
Chronic rejection. The importance of the CCR2-MCP-1

axis in models of chronic rejection of transplant has been
studied using genetic deletions of CCR2 and MCP-1, as well as
pharmacological interventions (CCR2 antagonism with 7-NDand anti-MCP-1-neutralizing antibody; Table 1). Two separate
studies evaluated the effect of intervention against the CCR2/
MCP-1 axis in two models of chronic rejection: cardiac allografts and aortic allografts. In the study with cardiac allografts
[57], intramuscular gene transfer of 7-ND MCP-1 reduced intimal hyperplasia by 39% and decreased infiltrates of F4/80
and CCR2 mononuclear cells. In contrast, in the study with
aortic allografts, CCR2 deficiency or MCP-1 deficiency did not
reduce intimal hyperplasia [58]. The basis for the difference
in the effects on neointima formation between the two studies
is not clear and is likely related to the difference in arterial
sites between the two models. This supposition is consistent
with the finding that CCR2 deletion reduced atherosclerotic
lesions in the aortic root but not in the thoracoabdominal
aorta (see Atherosclerosis below). Alternatively, potential Th2
switch, resulting from genetic deficiency of MCP-1 or CCR2
but not from pharmacological inhibition of the CCR2/MCP-1
axis, may account for the difference; a Th2-cytokine milieu is
considered to be detrimental to aortic allograft survival [59].
Two separate studies about chronic rejection of lung allograft evaluated the effects of intervention against the CCR2/
MCP-1 axis on BOS, the leading cause of mortality following
lung transplantation and contributing to the low allograft survival rate. The syndrome is characterized by a perivascular/
bronchiolar recruitment of mononuclear cells that ultimately
destroys the airway. In one study [60], following tracheal transplantation, recruitment of mononuclear cells was reduced significantly, and BOS was attenuated in CCR2-deficient mice
and in wild-type mice treated with a neutralizing anti-MCP-1
antibody. This finding was corroborated by another study using a rat heterologous s.c. model of BOS [122], in which neutralizing anti-MCP-1 antibody treatment reduced airway obstruction and airway epithelium loss by 80% and 50%, respectively.
CCR5 inhibition in transplant rejection

Acute rejection. Genetic and pharmacological interventions demonstrated the importance of CCR5 in acute rejection
of allografts (Table 2). With the cardiac allograft model, four
studies were reported. The first study showed that CCR5 deficiency or treatment with a neutralizing antibody against CCR5
prolonged allograft survival from 7 to 20 days and markedly
reduced infiltrating, activated mononuclear cells [105]. When
CsA was given to CCR5-deficient mice, permanent engraftment
(100 days) was observed. This is in contrast to the survival of
only 23 days observed when wild-type mice were given CsA
alone [105]. In the second study using a similar cardiac allograft model [106], mice treated with an antibody against
CCR5 combined with CsA had prolonged allograft survival (83
days) relative to CsA treatment alone (6 days). This effect was
associated with significant reduction of mononuclear cells in
the graft. In the other two studies from the same laboratory
using a model of heterotopic heart allograft transplantation
[107, 108], CCR5 deficiency only modestly prolonged the sur46 Journal of Leukocyte Biology
vival of cardiac allograft (by 23 days) but substantially decreased T cell and macrophage infiltrates at the time of rejection. The authors attributed this modest effect to the increased production of alloreactive antibodies in CCR5deficient mice [107, 108]. It is important to note that
treatment with CsA was not conducted in these two studies.
With the allograft pancreatic islet cell heterotopic model,
two studies were reported. In the first study [109], islet allografts, transplanted into CCR5-deficient mice, survived longer
(35 days) than those into wild-type littermates (10 days). The
prolonged allograft survival in CCR5-deficient mice was associated with a shift from a Th1 to a Th2 phenotype. In the second study [110], CCR5 function was blocked with PNA CCR5,
an PNA CCR5, in which the sugar phosphate backbone of the
natural nucleic acid has been replaced with a synthetic peptide
backbone. Islet allografts in mice treated with PNA CCR5 survived significantly longer (12 days) than those treated with
control (6.5 days). This prolongation was associated with decreased CCR5 expression and reduced T cell proliferation.
Notably, in a primate model of heterotopic allo-heart transplantation, monotherapy with a CCR5 antagonist from Merck
(Whitehouse Station, NJ, USA; CMPD 167) markedly attenuated recruitment of CCR5-positive leukocytes into the graft,
decreased peri-operative stress responses (e.g., fever and diminished activity), yet only marginally prolonged allograft survival [111].
Chronic rejection. The importance of CCR5 in chronic
rejection, especially CAV, is highlighted in four studies in
mouse and one study in nonhuman primate (Table 2). In the
first mouse study using a cardiac allograft transplant model
[105], treatment of CCR5-deficient mice with CsA at the
clinical dose caused permanent allograft survival (100
days), compared with only 2- to 3-day survival in the wildtype mice. Histological examination at Day 100 post-transplantation revealed no sign of vasculopathy in the CCR5deficient recipients, as measured by leukocyte infiltration,
interstitial fibrosis, and intimal hyperplasia [105]. In the
second mouse study using a similar model [106], allografts
treated with anti-CCR5-neutralizing antibody plus rapamycin
exhibited significantly prolonged survival (83 days), as compared with allografts treated with a control antibody plus
rapamycin (6 days). Treatment with an anti-CCR5 antibody
plus rapamycin also reduced the progression of chronic rejection significantly, as measured by lack of intimal lesions
at Day 90. In the other mouse studies, the effect of dual intervention of CCR5 and CXCR3 or of CCR5 and CCR1 on
CAV was evaluated. In a fully MHC-mismatched murine cardiac allograft model [112], treatment of CCR5/ mice
with anti-CXCR3 antibody led to permanent graft survival
(100 days) of the donor hearts with complete lack of intimal lesions. In a model in which B6.CH-2bm12 strain donor
hearts were transplanted heterotopically into C57BL/6 mice
(myosin heavy chain II mismatch) [113], blockade of CCR5
and CCR1 with Met-RANTES reduced intimal thickening
significantly with markedly decreased infiltration of CD4
and CD8 T cells and MOMA-2 monocytes/macrophages
in the allografts. In the nonhuman primate (cynomolgus
monkey) study using heterotopic allo-heart transplantation
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described above [111], treatment with the CCR5 antagonist

CMPD 167 plus CsA delayed alloantibody production and
suppressed vasculopathy up to Day 54, as measured by a
CAV scoring system for intimal lesions. In humans, graft
survival was significantly longer in patients homozygous for
CCR532 than those homozygous CCR5 wild-type and heterozygous CCR532 (hazard ratio0.367; P0.033) [114].
CCR2 and CCR5 dual inhibition in acute and chronic

rejection
Consistent with the role of CCR2 and CCR5 in models of
acute and chronic rejection, wild-type mice treated with the
small-molecule antagonist TAK-779 or CCR2-deficient mice
treated with TAK-779 exhibited prolonged survival of heart
and islet allografts. In the first study, TAK-779 treatment prolonged survival of both types of allografts and robustly inhibited CD4, CD8, and CD11c cellular infiltration [123]. Furthermore, TAK-779 treatment substantially attenuated the development of CAV, fibrosis, and cellular infiltration. The
second study evaluated the effects of CCR2 deficiency, CCR5
deficiency, and a combination of CCR2 deficiency with rapamycin or TAK-779 in a model of islet allografts [124]. CCR2 or
CCR5 deficiency modestly prolonged the survival from 11 to
16 days. The combination of CCR2 deficiency with rapamycin
further prolonged the survival (38 days) relative to either
treatment alone (16 days for CCR2 deficiency and 17 days for
rapamycin) [124]. However, the combination of CCR2 deficiency with TAK-779 did not prolong allograft survival, and the
authors concluded that dual inhibition of CCR2 and CCR5
had no synergistic effect [124]. Adding CsA or rapamycin to a
combination of CCR2 deficiency and TAK-779 was not conducted but would provide further proof of concept.
ATHEROSCLEROSIS
Cardiovascular disease continues to be the principal cause of
death in the Western world despite changes in lifestyle and
the increasing use of therapies to lower plasma lipids. Compelling evidence exists that vascular inflammation plays a key role
in atherosclerosis. Immune cells, particularly macrophages and
T cells, dominate in atherosclerotic lesions. In experimental
models of atherosclerosis, recruitment of macrophages and
Th1 cells plays a crucial role in the progression of atherosclerosis and destabilization of the vulnerable plaque. Consistent
with the role of CCR2 and CCR5 in the tissue recruitment of
monocyte/macrophages and Th1 cells and that of CCR5 in
the activation of VSMCs, genetic and pharmacological evidence exists, demonstrating the importance of the two receptors in multiple models of atherosclerosis.
CCR2 inhibition in atherosclerosis

With genetic and pharmacologic intervention, numerous studies in preclinical models of atherosclerosis have shown that
targeting the CCR2-MCP-1 axis reduces aortic root lesion size,
decreases macrophage infiltration, and also increases plaque
stability (Table 1). Notably, substantial reduction of lesional
macrophage content but not of plasma levels of lipids and li-
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poproteins was observed consistently when the CCR2-MCP-1

axis was inhibited.
Genetically, CCR2/ and MCP-1/ mice were used to
validate the CCR2-MCP-1 axis in mouse models of atherosclerosis: ApoE / mice, LDLR/ mice, and ApoBtg mice (Table 1). Two independent studies have shown that relative to
ApoE/ controls, CCR2//ApoE/ mice fed with a HFD
or normal chow have reduced aortic root lesions with decreased cellular infiltration [61, 62]. Smaller yet statistically
significant decreases in the size of early lesions were also observed in CCR2//ApoE/ mice, indicating a gene-dosage
effect [61, 62]. Interestingly, relative to CCR2//ApoE/
mice, CCR2//ApoE/ mice exhibited significantly reduced lesions at the aortic root but not at the thoracoabdominal aorta [63]. Deficiency of MCP-1 was shown to markedly
reduce lesion size and macrophage infiltrates in two models of
atherosclerosis: the LDLR/ and ApoBtg mice fed with a
HFD [64, 65]. Conversely, MCP-1 overexpression in ApoE/
mice increased the macrophage infiltrate and size of lesion
[125].
Pharmacologically, the importance of the CCR2-MCP-1 axis
in experimental atherosclerosis was demonstrated following
gene transfer of 7-ND in ApoE/ mice in three separate
studies [66 68]. HFD, normal chow, and normal chow plus
treatment with angiotensin II were used to modulate the formation of the atherosclerotic lesion, respectively. These studies
demonstrated consistently that 7-ND gene transfer reduced
lesions with decreased macrophage infiltration. Importantly, in
the 20- to 30-week-old ApoE/ mice with more advanced lesions, 7-ND gene transfer did not reverse the lesion, but it almost blocked its progression completely. Furthermore, it also
increased lesional collagen and elastin content, suggesting that
inhibition of the CCR2-MCP-1 axis by 7-ND stabilizes the
plaque.
The importance of the CCR2-MCP-1 axis in models of atherosclerosis was also demonstrated following treatment with
propagermanium, a polymer that selectively inhibits in vitro
chemotaxis in response to MCP-1 [126]. Although its exact
mechanism of action is not clear, it may target GPI-anchored
proteins that are functionally important for CCR2 [126]. This
compound reduced monocyte/macrophage infiltrates in thioglycollate-induced peritonitis in mice, a model for measuring
CCR2-dependent monocyte/macrophage recruitment [69].
Three studies evaluated the effect of propagermanium in
three different models of atherosclerosis: the ApoE/ mouse,
the LDLR/ rabbit, and the HFD-fed pig, and provided consistent evidence that propagermanium treatment reduces lesions with decreased macrophage infiltration [69 71].
Although there are robust genetic and pharmacological data
supporting the importance of CCR2 in experimental atherosclerosis, a study with CCR2//ApoE/ mouse bone marrow transplantation reached a different conclusion. The study
was designed to test the ability of CCR2/ leukocytes to reverse established atherosclerotic lesions in the ApoE/ background [127]. Bone marrow from CCR2//ApoE/ mice
was transferred into the ApoE/ mice (16 weeks old) that
had preformed lesions. Assessment of lesion size 9 weeks posttransplantation showed no effect of CCR2 deletion on the size
of advanced lesion, indicating that bone marrow progenitor

cell-derived CCR2 did not influence the progression of established lesions. A caveat with this study is that only cells newly
derived from bone marrow were examined. However, the results suggest that the CCR2-MCP-1 axis may have a diminished
role in advanced lesions in which the inflammatory process
may involve other pathways (e.g., CCR5-RANTES axis).
Clinical evidence has yet to be generated that inhibiting the
CCR2-MCP-1 axis provides a benefit, i.e., reduced morbidity
and mortality in patients with atherosclerosis. Encouragingly,
an initial analysis showed that MLN1202, a humanized antiCCR2 antibody, was well tolerated and met its primary endpoint of reducing C-reactive protein levels for months after a
single dose in 108 patients who were at high risk for atherosclerosis [128]. It will be interesting to see if continued treatment with MLN1202 provides clinical benefit for patients with
atherosclerosis.
CCR5 inhibition in atherosclerosis

Multiple lines of evidence have demonstrated that inhibition
of the CCR5-RANTES axis reduces lesion size, decreases macrophage infiltration, and increases plaque stability in animal
models of atherosclerosis (Table 2). Several studies have
shown that CCR5 deficiency reduced atherogenesis in
ApoE/ mice fed HFD or normal chow. One study evaluated
two separate lines of CCR5//ApoE/ mice [115]. Relative
to ApoE/ control mice, CCR5//ApoE/ mice exhibited
a reduction of lesion size at the aortic root by 50% and in the
thoracoabdominal aorta by 60% after 10 12 weeks of a highcholesterol diet, and the reductions were 50% and 80%, respectively, after 22 weeks of a high-cholesterol diet. The
CCR5//ApoE/ mice also exhibited reduced lesional macrophage content, decreased lesional expression of CD4, decreased Th1 T cell Ig and mucin domain 3 expression, increased expression of the anti-inflammatory cytokine IL-10,
and increased smooth muscle cell content. Consistent with
this, another study showed that relative to ApoE/ control
mice, CCR5//ApoE/ mice fed with normal chow for 26
weeks, 36 45 weeks, and 65 80 weeks, CCR5 deletion reduced
the aortic lesion by 13%, 46%, and 58%, respectively [116].
Yet, in a separate study, relative to ApoE/ control mice,
CCR5//ApoE/ mice fed with normal chow for 16 weeks
did not exhibit significant reduction in the size of the aortic
root lesions [129]. One possible explanation for this discrepancy is the difference in the length of the feeding with normal
chow and/or the stage of the lesions, especially considering
that the inhibitory effect of CCR5 deletion on atherogenesis
appears to be more pronounced on late-stage/advanced lesions. In summary, following HFD and normal chow, CCR5
deletion inhibits atherogenesis, especially at a late stage, and
this inhibition applies to lesion formation at aortic root and
thoracoabdominal aorta; in contrast, CCR2 deletion appears to
impact atherogenesis at the aortic root but not the thoracoabdominal aorta. Despite these preclinical data, the Offspring
Cohort of the Framingham Heart Study found no statistically
significant association between CCR532 homozygosity or heterozygosity and a lower risk of cardiovascular disease [130].
Pharmacologically, Met-RANTES, an antagonist of mouse

CCR5 and mouse CCR1 [131], reduced atherosclerosis in
LDLR/ mice fed with HFD. Given that CCR1-deficient mice
showed increased lesions in ApoE/ or in LDLR/ background [132, 133], the inhibitory effect of Met-RANTES on
atherosclerosis was most likely mediated by CCR5 antagonism.
Consistent with the results obtained with CCR5 KO studies,
Met-RANTES reduced lesions in the aortic root by 40% and
those in the thoracoabdominal aorta by 60% in LDLR/
mice fed a high-cholesterol diet for over 14 weeks [117]. MetRANTES also reduced lesional Mac-1 macrophage and CD4
T cells and increased smooth muscle cells, again in agreement
with the results of studies with CCR5 KO mice. In addition,
increased interstitial collagen content and reduced expression
of MMP-9 within the lesion suggest an added beneficial effect
of Met-RANTES (presumably via the CCR5 antagonism) on
the stability of atherosclerotic plaques. The effect of MetRANTES on the lesion in LDLR/ mice was corroborated
further by a more recent study using [44AANA47]-RANTES, an
inhibitor of RANTES oligomerization [118].
One study transplanted CCR5/ and CCR5/ bone marrow into lethally irradiated LDLR/ mice to assess the effect
of CCR5 deficiency on atherosclerosis [134]. CCR5 deficiency
in bone marrow-derived cells reduced macrophage content
and MMP-9 and increased IL-10 and collagen content in early
atherosclerosis. However, these effects were not sustained in
advanced atherosclerosis. Although this study questions the
importance of CCR5 in a late stage of atherosclerosis, two caveats should be considered. First, lethal irradiation kills only
high-proliferating cells, such as bone marrow-derived cells, but
not resident lesional macrophages, which are thought to express high levels of CCR5. Second, this procedure did not affect the function of CCR5-expressing cells that are not bone
marrow-derived, including CCR5-expressing VSMCs, which
may contribute to atherosclerosis.
CCR2 and CCR5 dual inhibition in atherosclerosis

No study directly evaluating the effect of dual inhibition of
CCR2 and CCR5 on atherosclerosis has been reported preclinically or clinically. Two related studies have been reported:
one with TAK-779, a small-molecule antagonist for mouse
CCR2, CCR5, and CXCR3 (a CXC chemokine receptor), and a
study with combined intervention of MCP-1, CX3CR1 (a receptor for fractalkine CX3CL1), and CCR5. Treatment of
LDLR/ mice on HFD with TAK-779 reduced the lesion in
the aortic root by 43% and in the carotid arteries by 68%
[135]. The number of T cells in the lesions was reduced by
95%, concurrent with 98% reduction in IFN-. In another
study with ApoE/ mice [136], a combined deficiency of
MCP-1 and CX3CR-1, together with Met-RANTES treatment,
led to abrogation of bone marrow monocytosis and to a
marked and additive 90% inhibition of atherogenesis at the
aortic sinus. Additionally, comparing the effect of dual deficiency of MCP-1 and CX3CR1 with either single deficiency,
the study showed an added effect with dual intervention [136].
The results from this study agree with those from an earlier
report showing that monocyte subsets differentially use CCR2,
CCR5, and CX3CR1 to accumulate with atherosclerotic
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plaques in ApoE/ mice [137]. Overall, these results support

the notion that each the three axes (MCP-1/CCR2, RANTES/
CCR5, and CX3CL1/CX3CR1) impacts the pathogenesis of
atherosclerosis independently. Notably, the importance of
CX3CR1 in atherosclerosis has been demonstrated in mouse
[138, 139] and in human [130]. Mechanistically, unlike CCR2
and CCR5, CX3CR1 functions as an adhesion molecule to
tether leukocytes to the inflamed vessels, and intervening
CX3CR1 reduces this tethering function, thus providing the
protective effect against atherosclerosis [130].
AIH
Intimal hyperplasia is the thickening of the intima of a
blood vessel as a result of smooth muscle cell migration
from the media and their subsequent proliferation in response to mechanical injury and altered hemodynamics. It
occurs pathologically in a variety of disease settings (e.g.,
atherosclerosis). Intimal hyperplasia also occurs after arteriovenous fistula for dialysis, coronary artery bypass grafting,
and allograft transplantation. Under these conditions, it is
now referred to as AIH, which is the major cause not only
for long-term vascular graft failure associated with these surgical procedures but also for chronic rejection of allografts.
The unmet medical need for AIH remains high, as no viable therapies are currently available. It has become increasingly evident that subendothelial inflammation, including
infiltration of monocytes/macrophages, is an integral part
of the pathogenesis of AIH. Consistent with the function of
CCR2 and CCR5 in the tissue recruitment of monocyte/
macrophages and that of CCR5 in the activation of VSMCs,
genetic and pharmacological evidence exists, demonstrating
the importance of CCR2 and CCR5 in multiple models of
AIH.
CCR2 inhibition in AIH

The role of CCR2 and MCP-1 has been studied extensively using genetic and pharmacological approaches in animal models
of AIH. These studies have provided consistent evidence that
inhibition of CCR2 reduces intimal hyperplasia (Table 1). Genetically, CCR2/ and MCP-1/ mice have been used to
demonstrate the importance of the CCR2/MCP-1 axis in
mouse models of AIH. Two studies using wire injury of the
femoral artery one with normolipidemic CCR2/ mice and
the other, hyperlipidemic CCR2//ApoE/ mice have
demonstrated that deletion of CCR2 reduces intimal hyperplasia (neointima) by 61% and 47%, respectively [72, 73]. Mechanistically, significant reduction of monocyte/macrophage content in the neointima was observed in one study. However, in
the other study, robust reduction of VSMC proliferation, but
not the monocyte/macrophage content, was detected. The
basis for this lack of reduction in the lesional content of
monocytes/macrophages in this study is not clear but is likely
related to the normolipidemic nature of the model, in which
there are too few macrophages to measure accurately. In the
same model of wire injury, MCP-1 deficiency reduced the neointima formation by 30% and appeared to reduce VSMC
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proliferation [74]. Most recently, in a vein-grafting model (venous interposition in the carotid artery) in ApoE*3-Leiden
mice fed with a HFD, local infection of the vein graft with lentivirus expressing shRNA-silencing CCR2 reduced vein-graft
thickening by 38% and was associated with reduced smooth
muscle cell migration and proliferation [75]. Although this
study provides interesting evidence that local inhibition of
CCR2 function can reduce AIH, it does not address the role of
CCR2 in the emigration of inflammatory monocytes from
bone marrow to blood.
Pharmacologically, a number of studies in multiple species
have evaluated the effect of intervening in the CCR2/MCP-1
axis in different models of AIH. In mice, two independent
studies have evaluated the effect of intervening in the CCR2/
MCP-1 axis on two different models of AIH: cuff placement on
femoral artery and vein graft [76, 77]. In both studies, gene
transfer of 7-ND MCP-1 (a peptide antagonist for CCR2) into
skeletal muscles reduced the neointima by 60% and 51%, respectively, and reduced neointimal monocyte/macrophage
content, cytokine production, and VSMC proliferation significantly. In rat normolipidemic models of balloon injury of the
carotid artery, two studies using gene transfer of 7-ND MCP-1
or anti-MCP-1 antibodies demonstrated reduction of neointima
of 60% and 56%, respectively, with significantly reduced neointimal monocyte/macrophage content, local cytokine production, and VSMC proliferation [78, 79]. In the rabbit, three
studies evaluated the effect of intervening in the CCR2/MCP-1
axis on AIH in two different hyperlipidemic models: balloon
injury of the carotid artery and stenting of the iliac artery. In
the balloon injury model [80], gene transfer of 7-ND MCP-1
into skeletal muscles reduced neointima by 40%. In the stenting model, coating stents with a biocompatible polymer containing a plasmid expressing the 7-ND MCP-1 gene reduced
the neointima by 23% and 44% in the two separate studies
[81, 82]. In all of these studies, significant reduction of neointimal monocyte/macrophage content was observed [80 82].
In the dog normolipidemic model of autologous vein graft,
jugular vein grafts were first transfected with adenovirus containing the 7-ND gene or a control vector and then interposed
into the carotid arteries. Gene transfer with 7-ND reduced the
neointima by 65% relative to vector controls and reduced
monocyte/macrophage content and VSMC proliferation significantly [83]. In the monkey, five studies evaluated the effect of
intervening in the CCR2/MCP-1 axis using an anti-CCR2 antibody or with 7-ND gene transfer on AIH. In the balloon injury
model, the anti-CCR2 antibody did not inhibit neointima formation, and in the model of stenting of the iliac artery, it reduced neointima by 46% and reduced macrophage content
significantly in the neointima [84]. Four additional monkey
studies evaluated the effect of 7-ND gene transfer on different
models of AIH, including arterial injury-induced cuff [76], balloon [79], and stenting of the iliac artery [82, 85]. 7-ND gene
transfer reduced neointima formation by 75%, 85%, 2530%,
and 50%, respectively, and reduced local inflammatory parameters significantly. The different magnitude of reduction in
neointima is likely related to the differences in how the arterial injury was induced, how long each study was carried out,
and how the 7-ND gene was delivered (Table 1). In summary,
genetic deletion and pharmacological inhibition of the CCR2/

MCP-1 axis reduced neointima in normolipidemic and hyperlipidemic models of AIH, induced by such insults as mechanical injuries, vein graft, and allograft vasculopathy in mouse,
rat, rabbit, dog, and monkey. Furthermore, the inhibitory effect of intervening on this axis on AIH (vasculopathy) was also
demonstrated in the context of chronic rejection (see Transplant Rejection above).
CCR5 inhibition in AIH

The importance of CCR5 in AIH pathogenesis was demonstrated with genetic (CCR5 KO) and pharmacological (MetRANTES) approaches (Table 2). In two studies using a hyperlipidemic (ApoE/) mouse model of wire-induced injury of
left carotid arteries, CCR5-deficient mice showed an 60%
reduction in neointima formation relative to the wild-type littermates [119, 120]. Also, CCR5 deficiency decreased neointimal macrophages and T cells substantially and led to a greater
than twofold increase in levels of the anti-inflammatory cytokine IL-10 in the neointima [119]. Pharmacologically, systemic
administration of Met-RANTES, a peptide antagonist for
mouse CCR5 and mouse CCR1 [131], reduced neointima formation by 35% and macrophage infiltrates by 50% in a
similar model of wire-injured carotid arteries [121]. Given that
CCR5 deficiency, but not CCR1 deficiency, reduced neointima
formation in the same study [119], the inhibitory effect of
Met-RANTES on neointima formation was most likely mediated by a CCR5 antagonism and not a CCR1 antagonism.
Taken together, genetic and pharmacologic evidence exists
demonstrating the importance of the CCR5/RANTES axis in
AIH. Furthermore, compelling evidence also exists in support
of a key role of this axis in AIH in the context of chronic rejection (see Transplant Rejection above).
RATIONALE FOR DUAL TARGETING OF

CCR2 AND CCR5
Complementary cellular distribution and differential cellular
functions of CCR2 and CCR5 provide a rationale that dual targeting of the two receptors will potentially provide greater efficacy than targeting either receptor alone. CCR2 and CCR5 are
expressed on different cell types in a complementary manner.
For monocyte/macrophages and T cells, the expression pattern for the two receptors appears to be complementary.
CCR2 is expressed predominantly on blood monocytes [18]
and memory T cells [20], whereas CCR5 is expressed on tissue
macrophages and activated T effector cells [38]. Recent studies have also shown that CCR2 and CCR5 are expressed on
separate subsets of blood monocytes [140]. In the case of nonleukocyte cell types, the expression pattern of the two receptors is distinct in that CCR5, but not CCR2, is expressed on
and functionally important for osteoclasts [39] and VSMCs
[40], which are considered to contribute to RA and atherosclerosis/AIH, respectively.
The complementary expression of the two receptors is supported further by recent in vitro and ex vivo evidence demonstrating a reciprocal pattern of expression and function for
CCR2 versus CCR5 during the differentiation of monocytes

into macrophages. In vitro studies have shown a down-regulation of CCR2 expression and an up-regulation of CCR5 expression during monocyte differentiation into macrophages
[141, 142]. This reciprocal pattern of expression of the two
receptors is paralleled by the reciprocity in their function; i.e.,
calcium flux and chemotaxis in response to MCP-1 are decreased, but that in response to RANTES or MIP-1 is increased [141, 142]. Furthermore, ex vivo studies with samples
from RA patients have shown substantially higher expression
of CCR2 than CCR5 on monocytes in peripheral blood [143].
In contrast, substantially higher expression of CCR5 relative to
CCR2 is observed on macrophages in synovial fluid [144].
Thus, this suggests down-regulation of CCR2 and up-regulation of CCR5, as monocytes migrate from blood into synovial
fluid and differentiate into synovial-fluid macrophages. The
reciprocal pattern of expression and function for CCR2 versus
CCR5 during monocyte differentiation implies that the two
receptors exert different effects on monocytes/macrophages,
and dual targeting may offer greater inhibition of their function than targeting either receptor alone. As discussed previously, CCR2 plays an essential role in mediating the migration
of monocytes from bone marrow to blood and from blood to
tissues. CCR5 functions to modulate the activation, survival,
and possible retention of macrophages in the tissue.
As discussed in Atherosclerosis above, a compelling example
of spatially dependent differences in the functions of the two
receptors has been provided by examining the effect of CCR2
deficiency and CCR5 deficiency on the atherosclerotic lesions
in aortic root versus the thoracoabdominal aorta in ApoE/
mice fed with HFD, one commonly used model of atherosclerosis. CCR2 deficiency reduced lesions in the aortic root by
50 75% by Weeks 513 of HFD [61, 62] but did not reduce
lesions in the thoracoabdominal aorta at all after 10 weeks of
HFD [63]. In contrast, following 10 or 12 weeks of HFD,
CCR5 deficiency reduced thoracoabdominal aortic lesions by
60% and aortic root lesions by 50%, respectively [115].
Practically, this rationale is supported further by the reports
of successful identification of potent dual antagonists against
CCR2 and CCR5 [13]. Fortuitously, the process of discovering
such dual inhibitors has been helped by the high molecular
homology (71% identical residues) shared between the two
receptors [144].
SAFETY CONCERNS OVER DUAL

TARGETING OF CCR2 AND CCR5
From the perspective of drug safety, data gleaned thus far
from the literature have suggested that a dual antagonist of
CCR2 and CCR5 may have several potential issues. First, CCR5
acts as one of the coreceptors for HIV entry into T cells and
macrophages [145], and CCR5 antagonists (e.g., Maraviroc)
have proven to be effective for the treatment of HIV [14].
This class of agents works specifically against CCR5-tropic viruses and is inactive against viruses that use CXCR4 as the coreceptor [14]. During infection, HIV starts out as CCR5-tropic
and only later does it switch to use CXCR4. Thus, concern
with this class of agents, albeit not yet realized, is that treat-
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ment with an antagonist against CCR5 or CCR2/5 can result

in coreceptor switching to CXCR4 [14]. To mitigate this potential risk, appropriate screening for HIV positivity and exclusion criteria should be implemented in the clinical studies
with a CCR2/5 dual antagonist. Second, an association between CCR5 deficiency and increased neuro-invasive diseases
induced by flaviviruses, WNV and TBEV, has been reported
from studies about CCR5-deficient mice (WNV) [146] and humans carrying a CCR5 32 homozygous allele (WNV and
TBEV) [147149]. This raises concern over the increased susceptibility to flaviviral infection following long-term use of an
antagonist of CCR5 or of CCR2/5. Although this increased
susceptibility has not yet been reported in HIV patients taking
a CCR5 antagonist as part of an anti-HIV regime, the consequences of long-term pharmaceutical intervention on CCR5 or
CCR2/5 should be assessed carefully through rigorous postmarketing surveillance. Third, it has been reported that deficiency of CCR2 and/or CCR5 can increase susceptibility to
several other microbial infections in mice summarized in two
recent reviews [150, 151]. It is important to note, however,
that most of these studies were conducted with microbial inocula, which are very high relative to naturally occurring infections. Clinical translation from these preclinical findings will
most likely be obtained empirically. Finally, a concern has also
been raised over a potential linkage between CCR2 and/or
CCR5 and hepatotoxicity as a result of reports of reduced liver
regeneration in mice deficient in CCR2 [152, 153] and CCR5
[154, 155], as well as increased serum liver enzymes in humans
treated with some CCR5 antagonists [156]. However, more
extensive clinical experience with CCR2 inhibitors (Phase II)
[90] and with a marketed CCR5 antagonist (Maraviroc) and
other late-stage CCR5 inhibitors has called into question any
direct linkage between CCR2/5 antagonism and hepatotoxicity.
CONCLUSION AND FUTURE PROSPECTS

Summarized in this review is a large body of preclinical literature/data providing genetic and pharmacologic evidence that
CCR2 and CCR5 are important players in the pathogenesis of
immunologic and cardiovascular diseases. Although clinical
proof-of-concept for a dual antagonist of the two receptors is
yet to be obtained, developing such a dual antagonist for its
broad clinical use holds great promise.
One intriguing question in studying chemokine receptormediated recruitment of leukocytes is why so many chemokines and chemokine receptors are needed for the recruitment of one leukocyte type. A simple answer to this apparent
redundancy question is that these receptors are required in a
spatially and temporally dependent manner to fine-tune the
recruitment of a leukocyte type into inflamed tissue. From the
perspective of drug discovery, targeting one chemokine receptor may not be sufficient to provide clinical benefit for patients with chronic inflammatory diseases, as has been suggested by failed clinical studies with selective inhibitors of the
CCR2-MCP-1 axis in RA. As presented in this review, strong
preclinical evidence exists for the spatially and temporally dependent modulation of expression and function of CCR2 and
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CCR5 and for the importance of both receptors in the pathogenesis of multiple diseases. Therefore, dual targeting of CCR2
and CCR5 should provide greater efficacy than targeting CCR2
or CCR5 alone, and a CCR2/CCR5 dual inhibitor has the potential for broad, clinical use. Not surprisingly, this opportunity is being seized by the pharmaceutical industry [13], so it
is reasonable to expect that such dual antagonists will soon be
evaluated in patients for efficacy and safety.
ACKNOWLEDGMENTS
The author thanks Drs. Phil Murphy, Paul Davies, Gary
Schieven, Luisa Salter-Cid, Timothy Reilly, Megan Wind-Rotolo, and Julie Carman for their comments about the manuscript.
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KEY WORDS:
chemokines chemokine receptors disease pathogenesis monocyte/macrophage recruitment

Dual Targeting of CCR2 and CCR5

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Dual Targeting of CCR2 and CCR5

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Review

Dual targeting of CCR2 and CCR5:

Abbreviations: AIAadjuvant-induced arthritis, AIHaccelerated intimal hyperplasia, ApoBtgapolipoprotein B transgenic, ApoEapolipoprotein E,

0741-5400/10/0088-0041 Society for Leukocyte Biology

the amino acid sequence [1 4]. The effects of chemokines on

Volume 88, July 2010

Journal of Leukocyte Biology 41

inflammatory diseases, this review focuses on dual targeting of

BIOLOGICAL ACTIONS OF CCR2 AND

Volume 88, July 2010

the most extensively studied [4]. The relative contribution of

PATHOGENIC ROLES OF CCR2 AND

Zhao Therapeutic potential of CCR2/CCR5 dual targeting

further demonstrated the importance of the MCP-1/CCR2 axis

Table 1. A Summary of Preclinical Studies Supporting a Role of CCR2/MCP-1 Axis in Diseases

mouse, rat, rabbit

MRL/lpr arthritis, CIA, AIA,

DSS colitis, DNBS colitis

mouse, rabbit, pig

mouse, rat, rabbit,

ApoE/ mouse (HFD or

CCR2 KO, MCP-1 KO, shRNA

Volume 88, July 2010

Journal of Leukocyte Biology 43

Volume 88, July 2010

32 deletion is low in RA patients versus normal controls, two

CCR2 and CCR5 dual inhibition in RA

Zhao Therapeutic potential of CCR2/CCR5 dual targeting

CCR2 and CCR5 dual inhibition in CD

CCR2 inhibition in transplant rejection

Table 2. A Summary of Preclinical Studies Supporting a Role of CCR5/RANTES Axis in Diseases

ApoE/ mouse (HFD or

CCR5 KO, CCR5 antagonist

Volume 88, July 2010

[105, 106, 112114]

Journal of Leukocyte Biology 45

Chronic rejection. The importance of the CCR2-MCP-1

CCR5 inhibition in transplant rejection

Volume 88, July 2010

Zhao Therapeutic potential of CCR2/CCR5 dual targeting

described above [111], treatment with the CCR5 antagonist

CCR2 and CCR5 dual inhibition in acute and chronic

CCR2 inhibition in atherosclerosis

poproteins was observed consistently when the CCR2-MCP-1

Journal of Leukocyte Biology 47

of advanced lesion, indicating that bone marrow progenitor

CCR5 inhibition in atherosclerosis

Volume 88, July 2010

Pharmacologically, Met-RANTES, an antagonist of mouse

CCR2 and CCR5 dual inhibition in atherosclerosis

Zhao Therapeutic potential of CCR2/CCR5 dual targeting

plaques in ApoE/ mice [137]. Overall, these results support

CCR2 inhibition in AIH

Volume 88, July 2010

Journal of Leukocyte Biology 49

genetic deletion and pharmacological inhibition of the CCR2/

CCR5 inhibition in AIH

RATIONALE FOR DUAL TARGETING OF

Volume 88, July 2010

CCR2 versus CCR5 during the differentiation of monocytes

SAFETY CONCERNS OVER DUAL

Zhao Therapeutic potential of CCR2/CCR5 dual targeting

ment with an antagonist against CCR5 or CCR2/5 can result

CONCLUSION AND FUTURE PROSPECTS