Alteration of Intestinal Intraepithelial Lymphocytes and Increased

Immunology Letters 90 (2003) 311

Alteration of intestinal intraepithelial lymphocytes and increasedbacterial translocation in a murine model of cirrhosis

Toshiaki Inamura a, Soichiro Miura b,, Yoshikazu Tsuzuki b, Yuriko Hara a,Ryota Hokari b, Toshiko Ogawa a, Ken Teramoto a, Chikako Watanabe a,

Hisashi Kobayashi a, Hiroshi Nagata a, Hiromasa Ishii a,1a Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan

b Second Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, JapanReceived 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 relationto 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 threegroups (cirrhosis, alcohol alone and controls). IFN- production from isolated IELs was determined by ELISA after stimulation with anti-CD3or IL-12/IL-18. The total number of IELs significantly increased in the cirrhosis and alcohol groups. There was a preferential increase inTCR+CD8+ population in the alcohol group, but no change in cirrhosis. Bacterial translocation was negative in the control group, anda small number was noted in the alcohol group, whereas it was significantly noted in the cirrhosis group. Although the number of IEL wassignificantly increased in the cirrhosis group, their proliferative response was decreased, and IFN- production from each IEL was markedlydiminished in either stimulation by anti-CD3 or IL-12/IL-18. These changes were more remarkable in the cirrhosis group than in the alcoholgroup. In conclusion, bacterial translocation due to intestinal barrier dysfunction in cirrhosis may be closely correlated with the alteration ofthe 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 gas-trointestinal functions such as decreased gut motility andchanges in absorptive capacity due to mucosal abnormali-ties and portal hypertension. These intestinal alterations mayinduce failure in the gut barrier to exclude endogenous bac-teria and toxins from portal and systemic circulation. Thesechanges may lead to complications such as spontaneous bac-terial peritonitis (SBP), or the systemic inflammatory re-

Corresponding author. Tel.: +81-429-95-1211x2367;fax: +81-429-96-5201.

E-mail addresses: [email protected] (S. Miura),[email protected] (H. Ishii).

1 Tel: +81-3-3353-1211x2260; fax: +81-3-3356-9654.

sponse syndrome, sepsis, and multiple organ failure [14].Although key steps in the pathogenesis of SBP have yet tobe elucidated, it is evident that the gut is a major sourceof bacteria in SBP [5]. Histopathologic studies have showntranslocations of bacteria, such as Candida albicans andEscherichia coli, by direct penetration of enterocytes asso-ciated with disruptions of the basal membrane [6]. Majorfactors that could contribute to bacterial translocation mightbe the suppression of immune defense, which might play apivotal role with other factors, such as physical disruptionof the mucosal barrier and intestinal overgrowth of bacteria.

In liver cirrhosis, there are a variety of systemic immuno-logical abnormalities, such as polyclonal hypergamma-globulinemia, auto-antibody production, decreased cellularimmunity, and decreased natural killer activity [7,8]. Inaddition to these systemic immunological changes, the

0165-2478/$ see front matter 2003 Elsevier B.V. All rights reserved.doi:10.1016/j.imlet.2003.05.002

4 T. Inamura et al. / Immunology Letters 90 (2003) 311

alteration of local immunity in the intestinal mucosa couldbe postulated [9,10], because gut-associated lymphoid tis-sue, as an independent immune organ, provides indispens-able immunologic protection against resident microbial floraand infectious pathogens [11,12]. In villous mucosa, laminapropria T and B lymphocytes compose the non-aggregatedlymphoid tissue acting as effector cells, which control andproduce IgA. Intestinal intraepithelial lymphocytes (IELs)have intimate cellular and molecular cross-talk with intesti-nal epithelial cells [13,14] as do a very large cell populationin the epithelial layer of the small intestine, with pheno-typic and functional features distinct from those of cells inperipheral lymphoid tissues. Because the IEL is the layerof the gastrointestinal immune system closest to the intesti-nal lumen and a rich source of cytokines, we hypothesizedthat the IEL changes during the development of cirrhosis.However, there has been a paucity of data relating to themorphological and functional changes of IELs in the in-testinal mucosa of liver cirrhosis. Recently, Alpini et al.established a murine model for cirrhosis by administeringcarbon tetrachloride (CCl4) and ethyl alcohol [15]. As thisis a reproducible model of cirrhosis in mice, we used thismodel to look at the effect of experimental cirrhosis on theintestinal barrier function against bacteria and the intestinalimmunity focusing especially on the phenotypic composi-tion of the IEL and the altered cytokine production within it.We demonstrated here that the decreased immune functionof IELs could be mutually related to bacterial translocationin cirrhosis.

2. Materials and methods

2.1. Induction of cirrhosis in mice (animal model)Male C3H mice (2025 g) were purchased from Charles

River (Tokyo, Japan), maintained in a temperature-controlledenvironment with a 12 h:12 h lightdark cycle and fed stan-dard chow ad libitum. To induce cirrhosis, the mice wereinjected intraperitoneally (IP) twice weekly with 0.1 ml of25% CCl4 in olive oil and they consumed 5% alcohol (byvolume) in their drinking water for 12 weeks (cirrhosisgroup) according to the procedure detailed by Alpini et al.[15]. Another group of mice received only olive oil IP and5% alcohol in drinking water (alcohol group). The controlgroups were treated with material in the same manner butwithout CCl4 or alcohol. Body weight and food intake weremonitored daily. These animals were used for the experi-ments that followed 12 weeks after being exposed to CCl4and alcohol. All procedures carried out on the animalswere approved by the Animal Research Committee of KeioUniversitys School of Medicine.

2.2. Histologic evaluation and immunohistochemistry

Specimens for light microscopy were taken from the liverand the small intestine. A few blocks from each liver were

placed in 10% formalin, subsequently embedded in paraf-fin and stained with hematoxylineosin or Azan stain. Thesmall intestine specimen was taken from a location 5 cm to-ward the anal side from the pylorus (jejunum) and from alocation 5 cm toward the oral side from the ileocecal valve(ileum). To evaluate the morphologic changes, each sam-ple was fixed with 10% formalin, and stained with a hema-toxylin and eosin solution. For light microscopic analysis, 10well-oriented crypt-villus units were examined per slide un-der a microscope. Another part of the removed intestine wasfixed for 12 h at 4 C in periodatelysineparaformaldehyde(PLP). Subsequently, these specimens were washed and de-hydrated for 12 h with PBS containing 10, 15, and 20%sucrose. After fixation, they were embedded in Tissue-TekO.C.T. compound (Sakura Fineteck Inc., USA) and frozen inliquid nitrogen. Cryostat sections of frozen tissue were cutat 7m. Immunohistochemistry was done with the labeledstreptavidin biotin technique (LSAB). Primary antibodiesused in the immunostaining were monoclonal antibodies thathad reacted to CD4 (RM4-5, rat IgG2a PharMingen, SanDiego, CA), and CD8 (53-6.7, rat IgG2a PharMingen, SanDiego, CA). The tissue sections were incubated with ad-equately diluted primary antibodies overnight at 4 C, andtreated with subclass- and host-matched biotinylated anti-bodies for 1 h at room temperature. They were visualized bystreptavidinFITC. Rinsing with PBS containing 1% bovineserum albumin was performed after each step. A cover slipwas applied using glycerol jelly. These sections were ob-served under an fluorescence microscope (BX60 Olympus,Tokyo). The infiltrated cells were expressed as the num-ber of CD4 and CD8 positive cells per mm in muscularismucosa.

2.3. Measurement of translocation of inert particlesand bacteria

To investigate the translocation of inert particlesfrom the gut lumen, groups of mice were given 1mfluorescein-labeled latex beads (Polysciences, Warrington,PA) in drinking water (1 108/ml) for 7 days before theywere to be sacrificed. After the mice were sacrificed, mesen-teric lymph nodes (MLNs) were removed, fixed in O.C.T.compound and frozen, then sectioned at a thickness of10m. The latex beads in sections of MLNs were observedthrough a fluorescent microscope. Six sections per MLNwere examined, and the mean number of beads per sectionwas calculated. The control mice were also given beadswith the same method for 7 days, and the mean number ofbeads per section of MLNs was calculated.

To measure bacterial translocation, organs were har-vested aseptically and cultured for bacteria as previouslydescribed by Spaecth et al. [16]. Briefly, the MLNs, spleenand liver were removed, weighed, and homogenized insterile glass tissue grinders. An equal volume of tissuehomogenate (10l) from various experimental groups wascultured separately on blood agar plates to culture totally

T. Inamura et al. / Immunology Letters 90 (2003) 311 5

aerobic bacteria and on McConkey agar plates to culturegram-negative enteric bacilli. The agar plates were incu-bated for 24 and 48 h at 37 C. Bacterial colony-formingunits (CFUs) were counted. If the agar plates did not showany bacterial growth up to 48 h, the organ was considerednegative for the presence of bacteria.

2.4. Isolation and flow cytometry of IELs

Intraepithelial lymphocytes were isolated by using themodified procedures described previously by Ishikawa et al.[17]. Briefly, the inverted intestine was cut into four seg-ments and the segments were transferred to a 50 ml conicaltube containing 45 ml of 5% fetal calf serum in Ca2+- andMg2+-free Hanks balanced salt solution (HBSS; Gibco Lab-oratories, Grand Island, NY). The tube was shaken horizon-tally in an orbital shaker for 45 min at 37 C. Cell suspen-sions were collected and passed through a glasswool col-umn to remove cell debris and adhering cells. The cells werethen suspended in 30% (w/v) Percoll (Pharmacia Biotech,Uppsala, Sweden) and centrifuged for 20 min at 1800 g;those at the bottom of the solution were subjected to Percolldiscontinuous-gradient centrifugation and IELs were recov-ered at the interface of 44 and 70% Percoll. The purity ofIELs was confirmed by flowcytometry using a FACSort ma-chine (Becton-Dickinson immunocytometry systems). Thepurity of CD3-positive cells was proven to be at least 96%and IELs exhibited a strong expression of E-integrin ontheir surface. The cells were washed, resuspended in RPMI(pH 7.4) with 5% fetal calf serum, and stored on ice untilthey were used.

To determine subpopulation, antibodies were obtainedfrom PharMingen, San Diego, CA and these consisted of:PE-conjugated anti-mouse CD4, CD8 and FITC-conjugatedanti-mouse TCR, TCR. Isotype-matched, irrelevantantibodies were used as negative controls. Flow cytome-try was done using standard techniques. Acquisition andanalysis were undertaken on a Becton-Dickinson FACSort(Becton-Dickinson, Mountainview, CA) using a Macin-tosh Power PC (Apple Computer Inc., Cupertino, CA) andCellQuest (Becton-Dickinson) software.

2.5. IFN- production assay from IELs

IELs were stimulated in vitro with plate-coated anti-CD3mAb, or IL-12/IL-18. IELs (4105 cells per well) were cul-tured in triplicate in microtiter wells coated with anti-CD3(145-2C11, 10g/ml, PharMingen) that were kept at 37 Cin an incubator filled with humidified air containing 5% CO2.The cells were cultured in 0.2 ml of RPMI medium supple-mented with 5% of FCS, 2 mM of glutamine, 100 U/ml ofpenicillin, 100 U/ml of streptomycin, and 20g/ml of gen-tamycin sulfate. In some experiments, they were stimulatedwith IL-12 (100 pg/ml, PharMingen) and IL-18 (500 pg/ml,MBL) which were added to the cultures. Supernatants werecollected on day 3 and the IFN- in the supernatants was

measured with ELISA kits (TECHNE corporation, Min-neapolis, MN).

2.6. Cell proliferation assay of IELs

To measure IEL proliferation, IELs were resuspendedin RPMI supplemented with l-glutamine (2 mM), 2-mercaptoethanol (50M), HEPES (10 mM), gentamycin(50 mg/ml), and FCS (10%) at a density of 5 106 cells/ml.One hundred microliters of cell suspensions were addedto the wells of a 96-well plate. The cells were culturedat 37 C in 5% CO2 in the presence or absence of phy-tohemagglutinin (PHA) (15g/ml) (DIFCO, Detroit, MI)or plate-coated anti-CD3 mAb. After 66 h of culturing,0.5Ci of 3H-thymidine (ICN Biomedicals, Costa Mesa,CA; 6.7 Ci/mM) were added to each well containing thecells. After an additional 6 h of incubation, cells wereharvested on glass filter papers (LKB, Gaitherburg, MD).Radioactivity was determined using a Skatron cell platecounter (LKB), and data was expressed as cpm.

2.7. Statistical analysis

All results were expressed as meansS.E.M. The differ-ences between groups were evaluated by one-way analysisof variance (ANOVA) and Fishers post hoc test. Statisticalsignificance was set at P < 0.05.

3. Results

3.1. Histologic and immunohistochemical evaluation

There was a marked increase in collagen deposition in theCCl4 exposed mouse livers, revealing a cirrhotic appearancein the paraffin-embedded liver sections stained with Azan.By 12-week exposure to CCl4, about 72% of mice survivedand all of them developed cirrhosis (n = 86). Conversely,in mouse livers with chronic alcohol exposure there was nosignificant deposition of collagen. Fig. 1 shows morphome-tric analysis of intestinal morphology determined by lightmicroscopy in the jejunum and ileum of the small intestine.The mucosal architecture in mice exposed to alcohol and/orCCl4 was not significantly different from that of the controls,and epithelial shedding was absent. However, there was asignificant decrease in the villus height of the jejunum andileum mucosa in both the alcohol and cirrhosis groups. Con-versely, there was an increase in the crypt depth comparedwith the control animals in both groups. In particular, therewas a significant increase in the crypt depth in the ileum ofthe cirrhosis groups.

Fig. 2 compares the number of CD4 and CD8 positiveIELs (A) and LPLs (B) per mm in the muscularis mucosa ofthe 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 mucosalarchitecture was compared in three different groups (control, alcohol, and cirrhosis). () P < 0.05. Values are means S.E.M. of six animals.

the alcohol and cirrhosis groups compared with the controlgroup in the jejunum, and the increase was more significantin the cirrhosis group. However, in the ileum, only the cir-rhosis group had a significant increase in the CD8 IEL pop-ulation compared with the control animals. There was onlya small number of CD4 positive cells in the IELs, and thesewere not altered after alcohol and/or CCl4 treatment. Thissignificant increase in CD8 cells was also observed in theLPL populations of the alcohol and cirrhosis groups com-pared with the control group (Fig. 2B), although it was more

striking in the cirrhosis group than in alcohol group. Whenthe number of CD4 positive LPLs was determined in thedifferent groups, there were significant increases in the alco-hol and cirrhosis groups compared with the control animals,and the increase was more significant in the cirrhotic group.

3.2. Flow cytometric analysis of IELs

IELs were isolated from the small intestines of differ-ent groups and the yield and changes in subpopulation


Fig. 2. Lymphocyte subpopulations in the intestinal mucosa determined by immunohistochemistry. Data compares the number of CD4 and CD8 positiveIELs (A) and LPLs (B) per mm muscularis mucosa in the jejunum and ileum of the small intestine in alcohol and cirrhosis groups compared with thecontrol group. () P < 0.05. Values are means S.E.M. of six animals.

were determined by flow cytometry. We obtained a largeramount of IELs from the intestines of the cirrhosis groups(2.38 0.50 105/cm intestine) compared with the al-cohol (1.25 0.21 105/cm intestine) or control groups(0.79 0.12 105/cm intestine) (P < 0.05). The results offlow cytometric analysis are summarized in Table 1. Therewas a preferential increase in the TCR+CD8+ populationwith a relative decrease in the other subpopulations in thealcohol group. Despite the larger number of IELs in theintestinal mucosa of the cirrhosis group, the subpopulationdetermined by flow cytometry did not change significantly

Table 1Flow cytometric analysis on subpopulations of small intestinal (ileal) IELs

Controlgroup (%)

Alcoholgroup (%)

Cirrhosisgroup (%)

TCR+CD4+ (n = 3) 9.5 4.0 6.8 2.4 10.3 2.2TCR+CD8+ (n = 3) 16.4 2.1 15.0 4.6 16.8 0.8TCR+CD8+ (n = 3) 39.6 2.1 55.7 6.6 38.1 5.1Values are expressed as Mean S.E.M.


Table 2Determination of bacterial translocation

Experimental groups Liver (colony-formingunits (CFUs) in samples)

Spleen (colony-formingunits (CFUs) in samples)

Mesenteric lymph nodes (colony-forming units (CFUs) in samples)

Control group (n = 6) None None NoneAlcohol group (n = 6) None None 3.0 0.6Cirrhosis group (n = 6) 5.4 0.8 5.2 1.0 31.0 5.0Values are expressed as mean S.E.M. None: not detected.

compared with the control group. These suggest the possi-bility that there was an increase in the number of IELs inthe cirrhosis groups, while maintaining the same proportionof subpopulations as the control groups.

3.3. Measurement of translocation of inert particles andbacteria

Fig. 3A is a representative micrograph of the transloca-tion of fluorescein-labeled latex beads taken up in the MLNsof the cirrhosis groups (upper panel) and controls (lowerpanel). As we can see from Fig. 3B there were few particlestaken up in the MLNs of the control groups, and a smallnumber of latex beads were translocated to the MLNs inthe alcohol groups. However, a large number of latex beadswere found to be transferred in the MLNs in the cirrhoticgroup. Table 2 compares the detection of bacterial translo-cation in the splanchnic organs (MLNs, spleen and liver) inthe three groups. All cultures were negative in the controlgroups, and there were no colony forming units (CFUs) inany samples. However, a small number of CFUs were notedin the MLNs of the alcohol groups, although these numberswere small. There was a significant number of CFUs notedin all cultures in the cirrhotic group, with a remarkable in-crease in the MLNs.

3.4. Cell proliferation assay of IELsTo determine whether the increased number of IELs in the

small intestine of the cirrhosis group was due to increasedproliferation, we evaluated the mitogen (PHA)-stimulated orTCR-mediated (anti-CD3) IEL proliferative responses of thethree groups. As we can see from Fig. 4A, the IELs obtainedfrom the control groups responded to PHA stimulation atlevels different from the alcohol groups. Furthermore, theIELs obtained from the cirrhosis groups did not proliferate asmuch as the controls. Similarly, the proliferative responsesto anti-CD3 were significantly suppressed in the cirrhosisand alcohol groups (Fig. 4B).

3.5. IFN- production from IELs stimulated with anti-CD3or with combination of IL-12 and IL-18

Fig. 5A has the IFN- concentration in the culture mediaafter the cells were treated with anti-CD3. IFN- could notbe detected in the culture media after 3 days when the cellshad not been stimulated, but after stimulation with anti-CD3the culture media of IELs from the control group contained

10 1 ng/ml IFN-. There was no significant change inanti-CD3 induced IFN- production in the alcohol groups;in contrast, the IFN- production from IELs was markedlydiminished in the cirrhosis group.

Fig. 5B depicts the IFN- concentration in culture mediaafter the cells had been treated through a combination ofIL-12 and IL-18. The culture media of the IELs from thecontrol group contained 252 ng/ml IFN- after stimulationwith IL-12 and IL-18. IL-12 and IL-18 together increasedIFN- concentration to levels greater than those produced byanti-CD3 treatment, consistent with the previous results [18].The IFN- production from the IELs was slightly decreasedin the alcohol group. However, it was remarkably suppressedin the cirrhosis group.

4. Discussion

This study demonstrated that experimental cirrhosisinduced through the administration of chronic doses ofethanol and CCl4 resulted in a several-fold increase inthe translocation of latex particles and bacteria. We foundthat the gastrointestinal tract is affected during cirrhosisand that there are mucosal abnormalities secondary to por-tal hypertension [5]. The reduction in the height of villifound in cirrhotic mice in our study is consistent withthe morphologic changes discovered in other studies [19].The decreased number of cells in the villus region duringcirrhosis may be related to alterations in actin cytoskele-tal protein, which interfere with normal cellular migrationalong the crypt-villus axis [19,20]. The exact physiologicaland biochemical mechanisms involved in intestinal barrierdysfunction during cirrhosis have not been clearly under-stood. However, the structural changes in the epithelial celllayer may be an important factor in facilitating intestinalpermeability, leading to bacterial translocation. A distendedinterenterocyte space together with shorter microvilli hasbeen observed in the duodenal mucosa of cirrhotic patients[21], and changes in brush border membrane composition,which include decreased structural proteins and microvil-lus enzymes have been reported in cirrhotic rats [2224].Recently, Ramachandran et al. focused on oxygen-free rad-icals and showed that cirrhosis results in oxidative stressin the intestine, and this may affect mitochondrial functionand increased lipid peroxidation in enterocytes [19].

The other factors contributing to inducing barrier dys-function in the gut, leading to increased bacterial transloca-tion, include altered gut motility and bacterial overgrowth,


Fig. 3. Measurement of translocation of inert particles into mesentericlymph nodes 7 days after administration. (A) A representative micrographof translocation of fluorescein-labeled latex beads taken up in MLNs ofcirrhosis groups (upper panel) and controls (lower panel). Large numberof latex beads are taken up in MLNs in cirrhotic group, while there are fewparticles in control groups. (B) The number of latex beads determined in a10m section of MLNs through a fluorescence microscope. Six sectionsper MLN were examined, and the mean number of beads per section wascalculated. () P < 0.05. Values are means S.E.M. of six animals.

which have been discovered in patients with liver cirrho-sis or experimental cirrhosis [3,25,26]. However, anotherintriguing and possible etiologic factor is inappropriate im-mune activity, especially at the level of the intestinal mu-cosa, since the intestinal mucosal immune system functionsas the first line of defense against enteric bacteria. The lym-phoid tissues associated with the intestine are continuouslyexposed to antigens in the lumen of the gut. Under healthyconditions, a few indigenous bacteria are known to continu-ously translocate to the mesenteric lymph node, but becauseimmune defenses are intact, these bacteria do not survive.However, it is known that injuries, caused by such factorsas alcohol and burns [27,28], as well as specific conditions,such as obstructive jaundice [29,30] or total parenteral nu-trition [31,32], frequently disrupt effective mucosal defenseto intraluminal microorganisms. When these conditions oc-cur, there is a suppression of intestinal immune responseagainst injury [27] or heightened immunologic activity andactivation within intramucosal lymphoid tissue [29]. In thepresent study, we found that there was a marked impairmentin IFN- production in each IEL from cirrhotic intestinescompared with the controls or alcohol groups. The exactmechanism for reduced IFN- production from IELs is notknown in cirrhostic mice, but this may not be due to the directtoxic effect of CCl4 because our preliminary experimentsrevealed that short term injection (2 weeks) of CCl4 did notsignificantly affect IFN- production. Since it has been re-ported that IL-12/IL-18 induction is not overridden by theTCR pathway [33], one can speculate that by cirrhotic con-dition the downstream common transcriptional pathway wasinhibited. Anyhow, such decreased IFN- production duringcirrhosis may affect the phagocytic ability of macrophagesand phagocytic cells and thus allow bacteria to multiply andtransfer to extraintestinal sites. IFN- produced by intesti-nal T cell helps in the resolution of Yersinia enterocolitiainfection [34]. In another study [35], IFN- was shown toconfer protection against the S. typhimurium invasion of ep-ithelial cells and fibroblasts. We also found that the prolifer-ating response of IELs to mitogenic stimuli was significantlysuppressed, suggesting an impaired response of IELs in theintestinal mucosa of cirrhotic condition. This finding is inaccordance with the suppression in T cell mitogenesis thathas been recorded after exposure to alcohol [28]. In our pre-vious study using cirrhotic rats, induced by CCl4, we alsodemonstrated a remarkable suppression in the appearance ofanti-cholera-toxin containing cells in the intestinal mucosaand mesenteric lymph nodes [10]. Such combinatorial de-creases in IFN- production, mitogenic ability and the pro-duction of antigen-specific antibodies will potentially resultin disrupting the hosts complex defense network, resultingin immune dysfunctions against microorganisms.

Despite the significant inhibition of proliferating re-sponses in lymphocytes, we found that there was a signif-icant increase in the number of lymphocytes both in theintraepithelial region as well as in the lamina proprialregions in cirrhotic animals, without a change in the


Fig. 4. Proliferative response of IELs from small intestine induced by PHA (A) and anti-CD3 stimulation (B). Sixty-six hours after starting to cultureIELs in PHA (5g/ml)-treated or anti-CD3 coated wells, the wells were pulsed with 0.5Ci of 3H-thymidine and 6 h later radioactivity of harvestedcells was evaluated. IELs from alcohol and cirrhosis groups are compared with control group. () P < 0.05 compared with controls. Mean S.E.M. offour 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 (10g/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.


subpopulations of mucosal lymphocytes. The exact reasonfor the increased number of mucosal lymphocytes is notknown, but we speculate that there might be an increasedmigration of these lymphocytes toward the intestinal epithe-lium in cirrhosis. We previously demonstrated in situ thatIELs are able to selectively adhere to the villus microves-sels of the small intestine [36]. It is therefore reasonableto assume that the accumulation of lymphocytes in theintestinal epithelium occurs in cirrhosis to compensate forthe immunological dysfunction against enhanced intralu-minal stress, although functional abilities like the cytokineproduction of each IEL is significantly inhibited.

However, we also noted that there was a slight but constantincrease in the translocation of latex particles, with a signif-icant decrease in cytokine production from IELs even in thealcohol drinking group. It was also interesting to note thatalcohol imbibition by the mice directly induced a significantincrease in TCR+CD8+ population. These results sug-gest that chronic exposure to ethanol may elicit a predisposi-tion to more significant intestinal mucosal changes by otherstressor such as CCl4. Napolitano et al. demonstrated thatalthough chronic alcohol exposure itself resulted in damageto both gut villi and the submucosal region [27], they andother groups also found that mice receiving ethanol intakeprior to burn injuries exhibited significant impairment to in-testinal reparative responses, increased bacterial transloca-tion rates, and increased susceptibility to infection [27,28].These results suggest chronic oral ethanol intake may havea possible enhancer effect on significant intestinal morphol-ogy as well as contributing to immunological alterations aswe discovered in our present study.

To the best of our knowledge, this is the first direct demon-stration that experimental liver cirrhosis induces a significantreduction in IFN- production from IELs, which may beclosely correlated with the disruption of effective mucosaldefense against intraluminal microorganisms in cirrhosis.

Acknowledgements

This study was supported in part by Grants-in-Aid forScientific Research from the Japanese Ministry of Educa-tion, Science and Culture of Japan and by grants from KeioUniversitys School of Medicine, and from the National De-fense Medical College.

References

[1] T.P. Almdal, P. Skinhoj, Scand. J. Gastroenterol. 22 (1987) 295300.[2] M. Andreu, R. Sola, A. Sitges-Serra, C. Alia, M. Gallen, M.C. Vila,

S. Coll, M.I. Oliver, Gastroenterology 104 (1993) 11331138.[3] C.S. Chang, G.H. Chen, H.C. Lien, H.Z. Yeh, Hepatology 28 (1998)

11871190.[4] E.A. Deitch, Ann. Surg. 216 (1992) 117134.[5] A. Ramachandran, K.A. Balasubramanian, J. Gastroenterol. Hepatol.

16 (2001) 607612.

[6] J.W. Alexander, S.T. Boyce, G.F. Babcock, L. Gianotti, M.D. Peck,D.L. Dunn, T. Pyles, C.P. Childress, S.K. Ash, Ann. Surg. 212 (1990)496510.

[7] H. Kita, I.R. Mackay, J. Van De Water, M.E. Gershwin, Gastroen-terology 120 (2001) 14851501.

[8] T. Nakamura, T. Morizane, T. Watanabe, K. Tsuchimoto, Y. Inagaki,N. Kumagai, M. Tsuchiya, Int. J. Cancer 32 (1983) 573575.

[9] S. Miura, M. Tsuchiya, in: K. Okuda, J.-P. Benhamou (Eds.), PortalHypertension, SpringerVerlag, Tokyo, 1991, pp. 6384.

[10] S. Miura, H. Serizawa, Y. Hamada, S. Tanaka, M. Yoshioka, T. Hibi,M. Tsuchiya, J. Gastroenterol. Hepatol. 1 (4 Suppl.) (1989) 7174.

[11] M. Salmi, S. Jalkanen, Gastroenterol. Clin. North Am. 20 (1991)495510.

[12] G. Pulverer, H. Lioe Ko, J. Beuth, Scand. J. Gastroenterol.222 (Suppl.) (1997) 107111.

[13] G.-K. Sim, Adv. Immunol. 58 (1995) 297343.[14] H. Komano, Y. Fujiura, M. Kawaguchi, S. Matsumoto, Y. Hashimoto,

S. Oana, P. Mombaert, S. Tonegawa, H. Yamamoto, S. Itohara, M.Nanno, H. Ishikawa, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 61476151.

[15] G. Alpini, I. Elias, S.S. Glaser, R.E. Rodgers, J.L. Phinizy, W.E.Robertson, H. Francis, J. Lasater, M. Richards, G.D. LeSage, J.Hepatol. 27 (1997) 371380.

[16] G. Spaecth, R.D. Berg, R.D. Specian, Surgery 108 (1990) 240247.[17] H. Ishikawa, Y. Li, A. Abeliovich, S. Yamamoto, S.H.F. Kaufmann,

S. Tonegawa, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 82048208.[18] Y. Hara, S. Miura, S. Komoto, T. Inamura, S. Koseki, C. Watanabe,

R. Hokari, Y. Tsuzuki, T. Ogino, H. Nagata, S. Hachimura, S.Kaminogawa, H. Ishii, Immun. Lett. 86 (2003) 139148.

[19] A. Ramachandran, R. Prabhu, S. Thomas, J.B. Reddy, A. Pulimood,K.A. Balasubramanian, Hepatology 35 (2002) 622629.

[20] T.M. Albers, I. Lomakina, R.P. Moore, Cell Biol. Int. 20 (1996)821830.

[21] J. Such, J.V. Guardiola, J. de Juan, J.A. Casellas, S. Pascual, J.R.Aparicio, J. Sola-Vera, M. Perez-Mateo, Eur. J. Gastroenterol. Hep-atol. 14 (2002) 371376.

[22] B. Manevska, Eksp. Med. Morfol. 15 (1976) 107111 (Bulgarianwith English abstract).

[23] I. Castilla-Cortazar, J. Prieto, E. Urdaneta, M. Pascual, M. Nunez,E. Zudaire, M. Garcia, J. Quiroga, S. Santidrian, Gastroenterology113 (1997) 11801187.

[24] J.P. Buts, N. De Keyser, E. Collette, M. Bonsignore, L. Lambotte,J.F. Desjeux, E.M. Sokal, Pediatr. Res. 40 (1996) 533541.

[25] A.M. Madrid, F. Cumsille, C. Defilippi, Dig. Dis. Sci. 42 (1997)738742.

[26] C. Guarner, B.A. Runyon, S. Young, M. Heck, M.Y. Sheikh, J.Hepatol. 26 (1997) 13721378.

[27] L.M. Napolitano, M.J. Koruda, K. Zimmerman, K. McCowan, J.Chang, A.A. Meyer, J. Trauma 38 (1995) 198207.

[28] M.A. Choudhry, N. Fazal, M. Goto, R.L. Gamelli, M.M. Sayeed,Am. J. Physiol. Gastrointest. Liver Physiol. 282 (2002) G937G947.

[29] F.K.S. Welsh, C.W. Ramsden, K. MacLennan, M.B. Sheridan, G.R.Barclay, P.J. Guillou, J.V. Reynolds, Ann. Surg. 227 (1998) 205212.

[30] R.W. Parks, C.H. Stuart Cameron, C.D. Gannon, C. Pope, T. Dia-mond, B.J. Rowlands, J. Pathol. 192 (2000) 526532.

[31] J. Li, K.A. Kudsk, M. Hamidian, B.L. Gocinski, Arch. Surg. 130(1995) 11641169.

[32] I. Kiristioglu, D.H. Teitelbaum, J. Surg. Res. 79 (1998) 9196.[33] J. Yang, T.L. Murphy, W. Ouyang, K.M. Murphy, Eur. J. Immunol.

29 (1999) 548555.[34] V.A. Kempf, E. Bohn, A. Noll, C. Bielfeldt, I.B. Autenrieth, Clin.

Exp. Immunol. 113 (1998) 429437.[35] S. Bao, K.W. Beagley, M.P. France, J. Shen, A.J. Husband, Im-

munology 99 (2000) 464472.[36] S. Koseki, S. Miura, H. Fujimori, R. Hokari, S. Komoto, Y. Hara,

T. Ogino, H. Nagata, M. Goto, S. Hachimura, S. Kaminogawa, H.Ishii, Int. Immunol. 13 (2001) 11651174.

Alteration of intestinal intraepithelial lymphocytes and increased bacterial translocation in a murine model of cirrhosisIntroductionMaterials and methodsInduction of cirrhosis in mice (animal model)Histologic evaluation and immunohistochemistryMeasurement of translocation of inert particles and bacteriaIsolation and flow cytometry of IELsIFN-gamma production assay from IELsCell proliferation assay of IELsStatistical analysis

ResultsHistologic and immunohistochemical evaluationFlow cytometric analysis of IELsMeasurement of translocation of inert particles and bacteriaCell proliferation assay of IELsIFN-gamma production from IELs stimulated with anti-CD3 or with combination of IL-12 and IL-18

DiscussionAcknowledgementsReferences

Alteration of Intestinal Intraepithelial Lymphocytes and Increased

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