April 2000

644

REGULATION OF CHEMO/CYTOKINES BY SOMATOSTATININ A HUMAN COLONIC EPITHELIAL CELL LINE.Taral Patel, Ping Jiang, Sheharyar Ali, Mark W. Babyatsky, Mount SinaiSch of Medicine, New York, NY; Saint Barnabas Med Ctr, West Orange,NJ.Somatostatin (SST) inhibits a variety of immune functions and may beimportant in regulating mucosal inflammation. We have previously shownthat SST inhibits TNF-ain cell lines and isolated cells (Gastroenterology1997;lI2:Al1l0). Recently, the intestinal epithelial has been shown to bean important regulator of multiple mucosal immune responses. We studiedwhether SST regulates two important pro-inflammatory chemokines, in­terleukin(IL)-S and monocyte chemoattractant protein(MCP)-I, and anti­inflammatory IL-IO, to test whether SST regulates epithelial immuneresponses. Methods: Col0205, HT-29, LoVo, LSl74T and Caco-2 cellswere grown to near confluence. Isolated mRNA from lysed cells weretested for SST receptor subtype 2 (SSTR2), IL-S, MCP-I, and IL-10mRNA expression by RT-PCR using specific primers. Col0205 cells,which expressed all of these factors abundantly, were treated with IL­I(3(2ng/ml)with or without SST at 10-5 to 10-11Min media for various timepoints. Extracted RNA underwent semi-quantitative RT-PCR using prim­ers specific for IL-S, MCP-l, IL-lO, and (3-actin (loading control). Results:All of the tested colonic epithelial cell lines expressed RT-PCR; onlyCol0205 cells expressed all three mediators. IL-ll3stimulated IL-S, MCP-l ,and IL-IO mRNA (maximal at 4 hours). SST, in a dose-dependent manner,inhibited IL-II3-stimulated IL-S and MCP-I expression at all time pointstested. SST stimulated IL- 10 expression in an additive manner to IL-l {3.Conclusions: J. All human epithelial cell lines tested express SSTR2. 2.SST inhibits stimulated MCP-l and IL-S mRNA expression in a dose­dependent manner in a colonic epithelial cell line. 3. SST's stimulation ofIL-IO expression in epithelial cells may be part of the mechanism by whichSST suppresses inflammation.

645

INTERACTION OF II-CS ON GASTRIC EPITHELIAL CELLSWITH CD44 ON LOCAL T CELLS: IMPLICATIONS IN THE IM­MUNOPATHOGENESIS OF H. PYLORnNFECTION.Carlos A. Barrera, Xuejun Fan, Gang Ye, Rosario Espejo, Tian Chang,Sheila E. Crowe, Victor E. Reyes, Univ of Texas Med Branch, Galveston,TX; Univeersity of Texas Med Branch. Galveston, TX.

Background!Aim: Helicobacter pylorus an important gastrointestinalpathogen that is implicated in chronic gastritis, peptic ulceration and gastriccancer. The infected tissue has a substantial increase of CD4+ T cells thatexpress markers of activation, including CD44. However, it is not knownhow those T cells become activated. An isoform of the class II associatedinvariant chain (Ii) known as the chondroitin sulfate form of Ii (Ii-CS) actsas a co-receptor for CD44 on T cells. The interaction between CDM andIi-CS leads to enhanced CD4+ T cell proliferation. The present study wasperformed to establish that during infection with H. pylori, the binding ofIi-CS on gastric epithelial cells to CDM on T cells plays a role in theimmunopathogenesis of gastric disease. Methods: Flow cytometry, meta­bolic radiolabeling, immunoprecipitation and SDS-PAGE were used tocharacterize the expression of Ii-CS by human gastric epithelial cell lines.Blocking of CDM with specific antibodies was used to demonstrate thespecificity of li-CS binding and the role of this interaction in T cellactivation as determined by eH] thymidine uptake. Results: Kato IIIgastric epithelial cells were noted to express Ii-CS by immunoprecipitationfrom [35S0 4] metabolic radiolabeled cells. The specific binding of recom­binant fusion protein (CD44H-Ig) bound to purified radiolabeled li-CSfrom KATO III cells was confirmed by competition with unlabeled li-CSand with anti-CD44 antibody. We correlated the binding of [5 1Cr]

CD4+CD44+Jurkat Tcells to plates coated with Ii-CS. Finally, the bindingof CDM and Ii-CS was abrogated following enzymatic removal of CSfrom Ii. Conclusion: Results strongly suggest that the interaction of Ii-CSand CDM playa role in activation of gastric T cells. These studies enhanceour understanding of cell-mediated immunity in the gastric mucosa inresponse to H. pyloriinfection.

AGAA93

646

CYTOTOXIC LYMPHOCYTES OF PATIENTS WITH INFLAM­MATORY BOWEL DISEASE REVEAL INCREASED INDUCTIONCAPACITY OF INTRACELLULAR IFN-r CO-CULTURED WITHINTESTINAL EPITHELIAL CELLS - A MHC-CLASS IRE·STRICTED PATHWAY?Guido Bisping, Norbert Luegering, Stefan Luetke-Brintrup, Hans G.Pauels, Wolfram Domschke, Torsten Kucharzik, Dept of Medicine B,Munster, Germany; Inst of Immulnology, Munster, Germany.

Background: Intestinal epithelial cells are known as central players forregulation of natural and acquired immune responses especially duringinflammatory bowel disease (IBD). In the following co-culture model weinvestigated the influence of intestinal epithelial cells on cytokine expres­sion of T-cytotoxic and T-helper cells from patients with IBD and healthycontrols. Methods: Purified PBMC's were co-incubated with epithelialcells in direct contact as well as separated by transwell filters. We usedCaco-2 layers as well as freshly isolated, purified colonic crypt epitheliaobtained from surgical specimens. MHC class I restricted interactionsbetween cytotoxic lymphocytes and epithelial cells were blocked by anti­MHC class I antibody. Three-colour flow cytometry was performed afterstaining of PBMC's with anti-CD4, anti-CDS, anti-IFN--y and anti·IL-4.Patients with IBD (CD, n=12; DC, n=16) and healthy controls (n=lO)were included in the study. Results: After 24 hrs of direct co-incubationwith Caco-2 cells we found a significant increase of IFN--y.producingCDS+ lymphocytes in patients with IBD (CD: 20.1% ± 5.6 IFN--y+/CDS+ in transwell samples up to 24.0% ± 5.8 in direct co-cultures(p=0.0l2), compared to 15,5% in monocultures; DC: 19.7% ± 3.5 IFN­-y+/CDS+ in transwell samples up to 26.4% ± 5.S in direct co-cultures(p=0.005S), compared to 14.6% in monocultures, mean ± SEM). Incontrast, healthy controls did not respond to the epithelial stimulus. Nosignificant differences could be found between Crohn' s disease and ulcer­ative colitis. A significant increase of IFN.-y+/CDS+ lymphocytes inpatients with ulcerative colitis was also seen after direct co-incubation withprimary cultures of colonic crypt cells (22.97% ± 2.2 IFN--y+/CDS+ indirect co-cultures compared to 12.27% ± 0.4 in monocultures, mean ±SEM). The observed epithelial-lymphocyte interactions could be blockedby anti MHC class I antibody. No significant epithelial cell mediatedeffects on cytokine expression were detected in the PBMC CD4+ subsets.Conclusion: Patients with IBD - even in an inactive state of disease - exertan increased capacity for IFN--yinduction in CD8+ lymphocytes mediatedby intestinal epithelial cells. This mechanism may be important duringchronic intestinal inflammation as in case of altered mucosal barrier func­tion epithelial cells may become targets for cytotoxic lymphocytes.

647

HLA·E IS EXPRESSED BY INTESTINAL EPITHELIAL CELLSAND IS REGULATED BY r -INTERFERON.Kelly Evans, Bana Jabri, Lloyd Mayer, Mount Sinai Med Ctr, New York,NY.

Several studies have supported a unique role for intestinal epithelial cells(IEC), that of antigen presenting cell to local lymphoid populations. Recentdata suggest that despite the expression of classical restriction elements,class I and II, on the lEC surface, nonclassical pathways may play adominant role in lEC:T cell interactions. Specifically CDld appears toserve as a restriction element regulating T cell activation by lEe. HLA-Eis a class Ib molecule whose ligand includes the NK receptor (NKR)complex CD94INKG2A, a killer inhibitory receptor which down regulatesNK cell activity. Intraepitheliallymphocytes (up to 30%) have been shownto express this NKR complex. In order to determine whether this NKRcomplex might be functional on lELs, we first investigated whether IECsexpress HLA-E. The colonic IEC line HT29 or the gastric epithelial cellline KATO 3 were cultured in the presence or absence of -yIFN (100 u/ml),TNFa(lO ng/ml), ILl5 (15 ng/ml) or ILIO (100 ng/ml) for 24h, followedby staining with the anti-HLA-E mAb, 3D12. kindly provided by Dr.Geraghty. There was constitutive expression ofHLA·E on KATO cells butnot on HT29. -yIFN induced expression of HLA-E by HT29 and augmentedexpression on KATO cells (change in mean channel ftuorescence[cellsurface density] from 32 to 212). IL15, an activator ofNK cells was unableto enhance expression of the NKR ligand. Similarly, neither TNFanor IL I0had any effect on HLA-E expression in these lines. The expression of yetanother class Ib molecule by lEC supports the concept that Ag presentationby lEC is unique and regulated by distinct cell surface interactions.