April 2000 IL-18 in the gut. In the present study, we investigated if circulating and/or locally produced IL-18 levels correlate with disease severity in IBD and C patients, and the contribution of both IEC and intestinal M0 s to the production of pro- and/or mature IL-18. Whole blood (N =8/grp) and mucosal biopsies (N =8/grp) were collected from patients with active and inactive disease as well as involved and non-involved areas of inflamma- tion, respectively. Enriched human epithelial antigen (HEA)pos. IEC and CD3neg/CDl4pos M0 s from resected gut specimens of the aforemen- tioned patient groups (N= 4/group), as well as the human colonic and M0 cell lines, Caco-2 and U937 respectively, were cultured in the presence or absence of TNF (10, 100 ng/ml) and collected at various time points following stimulation. Processed samples were assayed by ELISA for total IL-18 protein and by Western blot to distinguish IL-18 isoforms. Our results show that plasma IL-18 levels correlate with disease activity in CD (act-34 .3±9.9 vs inact-1 4.4±5 .7 pg/rnl), but not UC (act- 12.4±4.2 vs inact-8.3±3.7 pg/ml) patients, compared to C (14.0±4.6 pg/ml). Interest- ingly, an inverse correlation of total IL-18 protein levels was observed in "patient-matched" inv vs non-inv mucosal samples from CD (avg= inv- 2242 .89± IOO.33 vs non-inv-3851.78±256.21 pg/ml), but not UC (avg =i nv-8207.87± 1076.45 vs non-inv- I0372. 34± 1236.88 pg/ml) pa- tients, with strikingly elevated levels in UC vs CD and C (9852.54± 1274.32 pg/ml) patients. In addition, pro1L-1 8 is solely present in C and prevalent in non-inv areas of both CD and UC (UC>CD) vs "matched" inv areas of inflammation. Conversely, bioactive mature IL-18 was increased in inv areas from CD vs UC tissues. Cultured HEApos as well as Cac02 cells produced only pro1L-18 in response to TNF stimulation which peaked at 48 hr while CD3neg/CDl4pos and U937 produced both pro and mature IL-18. These data demonstrate that while IL-18 plasma levels may be an adequate index of CD disease activity. the balance of mature/pro IL-18 levels may prove to be a superior way to assess disease specificity and severity. 717 DIFFERENTIAL EXPRESSION OF SURFACE THI MARKER (CCRS) AND TH2 MARKER (CRTH2) ON INFILTRATED CELLS IN COLONIC MUCOSA OF ULCERATI VE COLITIS. Koji Matsuzaki, Ryota Hokari, Shingo Kate, Atsuhiro Iwai, Chic Kurihara, Atsushi Kawaguchi. Shigcaki Nagao. Tohru Miyahara, Kazuro Itoh, Soichiro Miura. Kinya Nagata. National Defense Med Coli, Saitarna, Japan; Biomed Lab. Saitarna, Japan. Background: The pathogenesis of ulcerative colitis (UC) is unclear, but abnormal infiltration of T lymphocytes in the colonic mucosa has been implicated in the mucosal tissue damage. Although ThlITh2 imbalance in infiltrating lymphocytes has been proposed in patients with UC. the Thl / Th2 dominance of unstimulated lymphocytes in inflammatory mucosa has not been defined. In this study. we investigated the expression of cherno- kinc receptor 5 (CCR5) as Th I marker and chemoallractant receptor- homologues molecule expressed on Th2 cells (CRTH2, J Immunol 162: 1278,1999) to determine polarized ThlITh2 responses in colonic mucosa of UC patients. Methods: Tissue samples were obtained by colonoscopic biopsies from patients with ulcerative colitis with informed consent. As controls colonic biopsy specimens were obtained from patients with co- lonic polyps and amoebic colitis. The histological scores of haematoxylin- eosin staining sections were assessed using a five point scale. Immunoh is- tochemi cal analysis was performed on PLP fixed serial cryostat sections using LSAB method. Monoclonal antibodies against CD4 (MT31O), CCR5 (RBI83) or CRTH2 (mouse monoclonal IgG) were used for the primary antibodies. These sections were observed under fluorescence microscope or confocal laser scanning microscope. The number of cells expressing CCR5 or CRTH2 per mucosal area was calculated by using image analyzer. Results: The express ion of CRTH2 was already observed in the colonic mucosa of controls and in the uninflamed colonic mucosa of UC patients, Especially significant increase in CRTH2 positive cells were found in the colonic mucosa of patients with amoebic colitis. On the other hand, few CCR5 positive cells were observed in the colonic mucosa of control subjects. The expression of CRTH2 and CCR5 was observed on some pan of CD4 lymphocytes in the colonic mucosa of UC patients. In this case no lymphocyte showed simultaneous expressio n of both CCR5 and CRTH2. The expression of CCR5 was significantly increased in the inflamed mucosa of UC. and the degree of CCR5 appeared to be correlated with the disease activity. Conclusions: In the present study we demonstrated that uninflamed colonic mucosa shows a relatively Th2 dominant condition. The present results also suggested that polarized Th I response occurred in the inflamed colonic mucosa of UC patients. There is a possibility that the Th11Th2 imbalance in colonic mucosa is responsible for the disease pro- gression in Uc. AGAAlll 718 TNFA SYNTHESIS AND RELEASE BY IL-I-PRIMED INTESTI· NAL SUBEPITHELIAL MYOFIBROBLASTS IS ENHANCED BY VARIOUS NSAIDS, BUT NOT S·ASA. Randy C. Mifflin, Jamal I. Saada, John F. DiMari , Don W. Powell, Univ of Texas Med Branch, Galveston, TIC TNFaSynthesis and Release by IL-I-Primed Intestinal Subepithelial Myo- fibroblasts is Enhanced by NSAIDs. but not 5-ASA The efficacy of anti-TNFaantibodies in the treatment of inflammatory bowel disease (IBD) suggests that TNFaplays an important role in the pathophysiology of intestinal inflammation. With the exception of 5-aminosalicylate (5-ASA), NSAIDs cause gastrointestinal inflammation and ulceration and precipitate disease activity in IBD patients.. Intestinal subepithelial myofibroblasts (ISEMF) exist as a syncytium of cells separated from the epithelium by only the basement membrane. These cells play key roles in epithelial barrier function, proliferation, differentiation and repair. We propose that cytokinc release by ISEMF initiates damage induced by NSAlDs . Because TNF ale vels are elevated in patients undergo ing NSAID therapy, we have studied the effect of NSAIDs on the synthesis and release of TNFaby a human ISEMF cell line, 18CO. Confluent monolayers were incubated with various NSAIDs in the presence or absence of IL-Ia . TNFamRNA levels and the amount of TNFarelcased into the culture medium was then determined , Significant accumulati on of TNFain the culture medium was only observed in cells treated with the combination of IL-I plus ibuprofen (ibuprof. 1.0 roM, 240 ± 66 pg/rnl TNF) or IL-I plus aspirin (ASA, 5.0 mM, 735 ± 86 pg/ml TNF) 24 hours after treatment. TNFamRNA fol- lowed a similar pattern of induction. IL-I induced elevated TNFamRNA levels within four hours of treatment and these returned to undetectable by 8 hours. NSAIDs alone did not induce TNFaexpression. However, ibuprof and ASA. when combined with IL-I synergistically prolonged the duration and increased the magnitude of TNFamRNA induction as well as in- creased the amount of TNFaobscrved in the medium after 24 hours. compared to IL-I alone. In contrast, 5-ASA did not significantl y affect IL-l -mediatcd TNFaexpression. These results suggest that the paracrine secretion of TNFain the subepithelial compartment is enhanced by NSAIDs, exclusive of 5-ASA. which plays a role in the exacerbation of IBD by NSAIDs. (Supported by CCFA and NIDDK grants to DWP.) 719 FRACTALKINE IS AN EPITHELIAL AND ENDOTHELIAL CELL DERIVED CHEMOATTRACTANT FOR INTRAEPITHELIAL LYMPHOCYTES IN THE SMALL INTESTINAL MUCOSA. Andreas Muehlhoefer, Lawerence 1. Saubermann, Xuibin Gu, Kerstin Luedtke-Heckenkamp, Ramnik Xavier, Richard S. Blumberg. Daniel K. Podolsky, Richard P. MacDermott, Hans-Christian Reinecker, MA Gen Hosp GI-Unit CSffiD, Boston, MA; Brigham and Women's Hosp Div Gastroenterology, Boston, MA; Brigham & Women 's Hosp, Boston, MA; Albany Med Coli Div of Gastroenterology, Albany, NY. Background: Fractalkine is a unique chemokine, which combines proper- ties of both chemoattractants and adhesion molecules. However, the role of fractalkine in the healthy intestine and during inflammatory mucosal re- sponses is not known. Methods: Fractalkine mRNA expression was as- sessed in small intestinal and colonic tissues as well as intestinal epithelial cell lines by Northern blot analysis. Fractalkine protein expression was determined by immunoblotting, immunoprecipitation, and immunohi sto- chemistry. Membrane compartmentalization of fractalkine was determined after membrane fractioning on sucrose gradients . Expression of CX3CR I was determined by RT-PCR and flow cytometry in freshly isolated and cultured human intestinal IEL (iIEL). Boyden chamber experiments were carried out to determine chemotactic activity of soluble fractalkine. Re- sults: We identified intestinal epithelial cells as a novel source of fracta- lkine. The basal expression of fractalkine mRNA and protein in T-84 cells was under the control of IL-I f3 . Induction of fractalkine expression in intestinal epithelial cells may mediate long lasting effects. Whereas ex- pression of fractalkine mRNA was maximally induced within 4 hours after a single induction by IL- I f3. membrane-bound fractalkine protein levels increased over 12 hours. and the amount of soluble fractalkine increased steadily over 24 hours. Fractalkine localized with caveolin- I in detergent insoluble glycolipid-enriched membrane microdomains in T-84 cells. Hu- man CD8+ ilEL expressed the fractalkine receptor CX3CRI and migrated specifically along fractalkin e gradient s. Immunohi stochem istry demon- strated that fractalkine was basolaterally expres sed in primary intestinal epithelial cells of the human small intestine as well as on endothelial cells. Northern blot analysis demonstrated the marked upregulation of fractalkine mRNA expression in Crohn 's disease mucosa. Conclusion: Fractalkine expression within the intestinal mucosa may have a dual function. Endo- thelial expressed fractalkine may initiate capture of lymphocytes by firm adhesion to the activated endothelium and intestinal epithelial cell derived soluble fractalkinc may direct lymphocytes to the site of inflammation and tissue repair. Membrane-bound fractalkine expressed by intestinal epithe- lial cells may then help to retain ilEL within the regenerating intestinal epithelium.