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Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
Second Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan
Received 17 May 2003; received in revised form 18 May 2003; accepted 29 May 2003
Abstract
Alterations in immunological defense in the gut may lead to the bacterial infection that is frequently associated with cirrhosis of the liver.
The aim of this study was to investigate the changes in distribution and function of intestinal intraepithelial lymphocytes (IELs) in relation
to intestinal barrier dysfunction in experimental cirrhosis. Cirrhosis was induced in mice by treatment with carbon tetrachloride (CCl4 )
intraperitoneally with 5% alcohol in drinking water for 12 weeks. Bacterial translocation was assessed in mesenteric lymph nodes (MLNs)
by the transport of fluorescence-labeled latex beads and by bacteriological cultures. The lymphocyte subpopulation was compared in three
groups (cirrhosis, alcohol alone and controls). IFN- production from isolated IELs was determined by ELISA after stimulation with anti-CD3
or IL-12/IL-18. The total number of IELs significantly increased in the cirrhosis and alcohol groups. There was a preferential increase in
TCR+CD8+ population in the alcohol group, but no change in cirrhosis. Bacterial translocation was negative in the control group, and
a small number was noted in the alcohol group, whereas it was significantly noted in the cirrhosis group. Although the number of IEL was
significantly increased in the cirrhosis group, their proliferative response was decreased, and IFN- production from each IEL was markedly
diminished in either stimulation by anti-CD3 or IL-12/IL-18. These changes were more remarkable in the cirrhosis group than in the alcohol
group. In conclusion, bacterial translocation due to intestinal barrier dysfunction in cirrhosis may be closely correlated with the alteration of
the immune function in IELs.
2003 Elsevier B.V. All rights reserved.
Keywords: Intraepithelial lymphocytes; Interferon-; Liver cirrhosis; Alcohol; Bacterial translocation
1. Introduction
It is known that cirrhosis is associated with altered gastrointestinal functions such as decreased gut motility and
changes in absorptive capacity due to mucosal abnormalities and portal hypertension. These intestinal alterations may
induce failure in the gut barrier to exclude endogenous bacteria and toxins from portal and systemic circulation. These
changes may lead to complications such as spontaneous bacterial peritonitis (SBP), or the systemic inflammatory re Corresponding author. Tel.: +81-429-95-1211x2367;
fax: +81-429-96-5201.
E-mail addresses: miura@me.ndmc.ac.jp (S. Miura),
hishii@sc.itc.keio.ac.ip (H. Ishii).
1 Tel: +81-3-3353-1211x2260; fax: +81-3-3356-9654.
0165-2478/$ see front matter 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.imlet.2003.05.002
placed in 10% formalin, subsequently embedded in paraffin and stained with hematoxylineosin or Azan stain. The
small intestine specimen was taken from a location 5 cm toward the anal side from the pylorus (jejunum) and from a
location 5 cm toward the oral side from the ileocecal valve
(ileum). To evaluate the morphologic changes, each sample was fixed with 10% formalin, and stained with a hematoxylin and eosin solution. For light microscopic analysis, 10
well-oriented crypt-villus units were examined per slide under a microscope. Another part of the removed intestine was
fixed for 12 h at 4 C in periodatelysineparaformaldehyde
(PLP). Subsequently, these specimens were washed and dehydrated for 12 h with PBS containing 10, 15, and 20%
sucrose. After fixation, they were embedded in Tissue-Tek
O.C.T. compound (Sakura Fineteck Inc., USA) and frozen in
liquid nitrogen. Cryostat sections of frozen tissue were cut
at 7 m. Immunohistochemistry was done with the labeled
streptavidin biotin technique (LSAB). Primary antibodies
used in the immunostaining were monoclonal antibodies that
had reacted to CD4 (RM4-5, rat IgG2a PharMingen, San
Diego, CA), and CD8 (53-6.7, rat IgG2a PharMingen, San
Diego, CA). The tissue sections were incubated with adequately diluted primary antibodies overnight at 4 C, and
treated with subclass- and host-matched biotinylated antibodies for 1 h at room temperature. They were visualized by
streptavidinFITC. Rinsing with PBS containing 1% bovine
serum albumin was performed after each step. A cover slip
was applied using glycerol jelly. These sections were observed under an fluorescence microscope (BX60 Olympus,
Tokyo). The infiltrated cells were expressed as the number of CD4 and CD8 positive cells per mm in muscularis
mucosa.
2.3. Measurement of translocation of inert particles
and bacteria
To investigate the translocation of inert particles
from the gut lumen, groups of mice were given 1 m
fluorescein-labeled latex beads (Polysciences, Warrington,
PA) in drinking water (1 108 /ml) for 7 days before they
were to be sacrificed. After the mice were sacrificed, mesenteric lymph nodes (MLNs) were removed, fixed in O.C.T.
compound and frozen, then sectioned at a thickness of
10 m. The latex beads in sections of MLNs were observed
through a fluorescent microscope. Six sections per MLN
were examined, and the mean number of beads per section
was calculated. The control mice were also given beads
with the same method for 7 days, and the mean number of
beads per section of MLNs was calculated.
To measure bacterial translocation, organs were harvested aseptically and cultured for bacteria as previously
described by Spaecth et al. [16]. Briefly, the MLNs, spleen
and liver were removed, weighed, and homogenized in
sterile glass tissue grinders. An equal volume of tissue
homogenate (10 l) from various experimental groups was
cultured separately on blood agar plates to culture totally
3. Results
3.1. Histologic and immunohistochemical evaluation
There was a marked increase in collagen deposition in the
CCl4 exposed mouse livers, revealing a cirrhotic appearance
in the paraffin-embedded liver sections stained with Azan.
By 12-week exposure to CCl4 , about 72% of mice survived
and all of them developed cirrhosis (n = 86). Conversely,
in mouse livers with chronic alcohol exposure there was no
significant deposition of collagen. Fig. 1 shows morphometric analysis of intestinal morphology determined by light
microscopy in the jejunum and ileum of the small intestine.
The mucosal architecture in mice exposed to alcohol and/or
CCl4 was not significantly different from that of the controls,
and epithelial shedding was absent. However, there was a
significant decrease in the villus height of the jejunum and
ileum mucosa in both the alcohol and cirrhosis groups. Conversely, there was an increase in the crypt depth compared
with the control animals in both groups. In particular, there
was a significant increase in the crypt depth in the ileum of
the cirrhosis groups.
Fig. 2 compares the number of CD4 and CD8 positive
IELs (A) and LPLs (B) per mm in the muscularis mucosa of
the jejunum and ileum of the small intestine in the control,
alcohol, and cirrhosis groups. As we can see from Fig. 2A,
the number of CD8 positive IELs significantly increased in
Fig. 1. Villus height and crypt depth in two different regions (jejunum and ileum) of the small intestine determined by light microscopy. The mucosal
architecture was compared in three different groups (control, alcohol, and cirrhosis). () P < 0.05. Values are means S.E.M. of six animals.
Fig. 2. Lymphocyte subpopulations in the intestinal mucosa determined by immunohistochemistry. Data compares the number of CD4 and CD8 positive
IELs (A) and LPLs (B) per mm muscularis mucosa in the jejunum and ileum of the small intestine in alcohol and cirrhosis groups compared with the
control group. () P < 0.05. Values are means S.E.M. of six animals.
Table 1
Flow cytometric analysis on subpopulations of small intestinal (ileal) IELs
TCR+CD4+ (n = 3)
TCR+CD8+ (n = 3)
TCR+CD8+ (n = 3)
Control
group (%)
Alcohol
group (%)
Cirrhosis
group (%)
9.5 4.0
16.4 2.1
39.6 2.1
6.8 2.4
15.0 4.6
55.7 6.6
10.3 2.2
16.8 0.8
38.1 5.1
Table 2
Determination of bacterial translocation
Experimental groups
Liver (colony-forming
units (CFUs) in samples)
Spleen (colony-forming
units (CFUs) in samples)
Control group (n = 6)
Alcohol group (n = 6)
Cirrhosis group (n = 6)
None
None
5.4 0.8
None
None
5.2 1.0
None
3.0 0.6
31.0 5.0
compared with the control group. These suggest the possibility that there was an increase in the number of IELs in
the cirrhosis groups, while maintaining the same proportion
of subpopulations as the control groups.
3.3. Measurement of translocation of inert particles and
bacteria
Fig. 3A is a representative micrograph of the translocation of fluorescein-labeled latex beads taken up in the MLNs
of the cirrhosis groups (upper panel) and controls (lower
panel). As we can see from Fig. 3B there were few particles
taken up in the MLNs of the control groups, and a small
number of latex beads were translocated to the MLNs in
the alcohol groups. However, a large number of latex beads
were found to be transferred in the MLNs in the cirrhotic
group. Table 2 compares the detection of bacterial translocation in the splanchnic organs (MLNs, spleen and liver) in
the three groups. All cultures were negative in the control
groups, and there were no colony forming units (CFUs) in
any samples. However, a small number of CFUs were noted
in the MLNs of the alcohol groups, although these numbers
were small. There was a significant number of CFUs noted
in all cultures in the cirrhotic group, with a remarkable increase in the MLNs.
3.4. Cell proliferation assay of IELs
To determine whether the increased number of IELs in the
small intestine of the cirrhosis group was due to increased
proliferation, we evaluated the mitogen (PHA)-stimulated or
TCR-mediated (anti-CD3) IEL proliferative responses of the
three groups. As we can see from Fig. 4A, the IELs obtained
from the control groups responded to PHA stimulation at
levels different from the alcohol groups. Furthermore, the
IELs obtained from the cirrhosis groups did not proliferate as
much as the controls. Similarly, the proliferative responses
to anti-CD3 were significantly suppressed in the cirrhosis
and alcohol groups (Fig. 4B).
3.5. IFN- production from IELs stimulated with anti-CD3
or with combination of IL-12 and IL-18
Fig. 5A has the IFN- concentration in the culture media
after the cells were treated with anti-CD3. IFN- could not
be detected in the culture media after 3 days when the cells
had not been stimulated, but after stimulation with anti-CD3
the culture media of IELs from the control group contained
which have been discovered in patients with liver cirrhosis or experimental cirrhosis [3,25,26]. However, another
intriguing and possible etiologic factor is inappropriate immune activity, especially at the level of the intestinal mucosa, since the intestinal mucosal immune system functions
as the first line of defense against enteric bacteria. The lymphoid tissues associated with the intestine are continuously
exposed to antigens in the lumen of the gut. Under healthy
conditions, a few indigenous bacteria are known to continuously translocate to the mesenteric lymph node, but because
immune defenses are intact, these bacteria do not survive.
However, it is known that injuries, caused by such factors
as alcohol and burns [27,28], as well as specific conditions,
such as obstructive jaundice [29,30] or total parenteral nutrition [31,32], frequently disrupt effective mucosal defense
to intraluminal microorganisms. When these conditions occur, there is a suppression of intestinal immune response
against injury [27] or heightened immunologic activity and
activation within intramucosal lymphoid tissue [29]. In the
present study, we found that there was a marked impairment
in IFN- production in each IEL from cirrhotic intestines
compared with the controls or alcohol groups. The exact
mechanism for reduced IFN- production from IELs is not
known in cirrhostic mice, but this may not be due to the direct
toxic effect of CCl4 because our preliminary experiments
revealed that short term injection (2 weeks) of CCl4 did not
significantly affect IFN- production. Since it has been reported that IL-12/IL-18 induction is not overridden by the
TCR pathway [33], one can speculate that by cirrhotic condition the downstream common transcriptional pathway was
inhibited. Anyhow, such decreased IFN- production during
cirrhosis may affect the phagocytic ability of macrophages
and phagocytic cells and thus allow bacteria to multiply and
transfer to extraintestinal sites. IFN- produced by intestinal T cell helps in the resolution of Yersinia enterocolitia
infection [34]. In another study [35], IFN- was shown to
confer protection against the S. typhimurium invasion of epithelial cells and fibroblasts. We also found that the proliferating response of IELs to mitogenic stimuli was significantly
suppressed, suggesting an impaired response of IELs in the
intestinal mucosa of cirrhotic condition. This finding is in
accordance with the suppression in T cell mitogenesis that
has been recorded after exposure to alcohol [28]. In our previous study using cirrhotic rats, induced by CCl4 , we also
demonstrated a remarkable suppression in the appearance of
anti-cholera-toxin containing cells in the intestinal mucosa
and mesenteric lymph nodes [10]. Such combinatorial decreases in IFN- production, mitogenic ability and the production of antigen-specific antibodies will potentially result
in disrupting the hosts complex defense network, resulting
in immune dysfunctions against microorganisms.
Despite the significant inhibition of proliferating responses in lymphocytes, we found that there was a significant increase in the number of lymphocytes both in the
intraepithelial region as well as in the lamina proprial
regions in cirrhotic animals, without a change in the
10
Fig. 4. Proliferative response of IELs from small intestine induced by PHA (A) and anti-CD3 stimulation (B). Sixty-six hours after starting to culture
IELs in PHA (5 g/ml)-treated or anti-CD3 coated wells, the wells were pulsed with 0.5 Ci of 3 H-thymidine and 6 h later radioactivity of harvested
cells was evaluated. IELs from alcohol and cirrhosis groups are compared with control group. () P < 0.05 compared with controls. Mean S.E.M. of
four experiments.
Fig. 5. IFN- concentrations determined by ELISA in the culture media after IELs were stimulated with anti-CD3 (A) or combination of IL-12/IL-18 (B).
IELs from alcohol and cirrhosis groups are compared with control group after stimulation with plate-coated anti-CD3 (10 g/ml), or IL-12 (100 pg/ml)
and IL-18 (500 pg/ml) for 3 days. () P < 0.05. Mean S.E.M. of four experiments.
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