1"1051

Intestinal Epithelial Cells Express Functional lnterlenldn-8-Receptors kndreas Sturm, Johanna Harder-D'beureuse, Bertram Vv'iedenmann, Axel U Dignass

Background: IL-8, a member of the c~.cbemokme tamily of chemotactic cytokines, has been demomstrated to be a potent stimulator of nautrophil activation and chemotaxis within the intestinal nmcosa As intestinal epithelial cells have been recently shown to produce IL-8, we were interested, it intestinal epithelial cells may express receptors for IL-8 and if these n!ceptors may mediate fimctional act*yules of IL-8 on intestinal epithelial ceils Methods: The expression of the IL-.8 receptors CXCRI and CXCR2 was assessed by RT-PCR and FACS analysis in human Caco-2 ceils and in rat 1EC-6 cells The effects of 1L-8 on intestinal epithelial proliferation were assessed with color*metric MTI" assays m subcontluent monolayers of IEC-6 and Caco-2 cells and the etfects on epithelial restitution were assessed w~th an in vitro migration model using lEG6 and Cas ceils Resuhs: While the expression of both Ik-8 receptor CXCR1 mRNA and protein could be demonstrated by RT-PCR and FACS analysis, no expression of 1L-8 receptor CXCR2 was observed in Caco~2 and IEC-6 cells. IL-8 did not significaufly modulate cell proliteration in intestinal epithelial cells in vitro over a broad range of concentranons up to supraphysiological concentrations of 100 ng/ml. In contrast, lL-8 si~itkantly enhanced the migration of Caco-2 and IEG6 cells approximately 2 5-fold m a close-dependent manner. Similar rates of cell migration were observed in the presence of replication inh:biting doses of mitomycin C, indicating that cell proliferation was not necessary ~br enhanced restitution No significant enhancement of both latent and bioactive TGFI3 pep~ide levels in cells treated with IL-8 and no modulation of IL-8 effects by immunoneutralizing TGF~-antibodies were observed, suggesting that enhancement of epiIhehal cell restitution by IL-8 was TGF~-independent Conclusion: Our study' demon- strates the presence of tunctic, nal IL-8 receptors in intestinal ep:thefial cells, in addition to its we//<haracterized etfi'cts on nentrophils [k-8 also exerts functional effects on intestinal epithelial cells that may be relevant thr intestinal inflammation and mncosal healing.

T1052

EvaLuation of Gut Mucosal Diamine Oxidase Activity (Dao) in Patients with Food Allergy and Ulcerative Colitis, Idiopathic Ulcerative Colitis and Crohn's Disease Martin RaitheI, Astrid Riedel, Michael Kud'ner, Norbert Donhauser, Eckhart Hahn

An accmuulation of histamine and mast cells is found in IBD and gastrointestinal food allergy, Histamine may exert a broad range of pminflammatory- and immuno-regulatory dfects, particularly if this amine isn't degraded enaynmtically w:thin short time. In this study ~be enzyme acti'~:ty of diamine oxidase (DAO), one of the main histamine catabolizing enzyrnes, was investigated in patients with food allergy and ulcerative colitis, idiopathic ulcerative colitis and Crohn's Disease Tbe enzyme activity was determined in the homogenate of the biopsy using 14C-pntreseine (substmte) during 60 minutes, The 14C labelled organic end product (1-pyrmlin) was extracted in the organm phase and then measured by liquid scintillation counting. DAO activities (cpm/h/mg ww) showed a physiological value of 61,6 + 44 in control persons (n= 67 biopsies) Patients with food allergy' and ulcerative colitis (n=31) had significantly reduced I)AO levels (407 + 46, p=0,001) similarly to chronic idiopathic ulcerative colitis (384 + 39, p = 0001, n=40), Although reduced, DAO levels in Cmhn's D:sease did not ~vach statistical signiflcane (45.6 + :32 @<0.07, n = 47) Interest- ingly, corresponding to the decrease of DAO acti:qty, increased tissue histamine levels were observed in patients with tood allergy and ulcerative colitis and in idiopathic ulcerative colitis (both p<001 ), but not in Cr0hn s Disease. This analysis of the DAO activity demonstrates that the ox:dative capacity ot gut mucosa to rapidly degrade histamine, is impaired in food aIler~/and IBD, suggesting that histamine may hmher enhance mucosal inflammation. Th2 dominated &sease as food allergy" and ulcerative colitis are found to be at a higher risk to loose DAO from epithelial cells during inflammation and to exhibit more elevated tissue histamine levels than Crohns Disease The reduced activity of DAO also suggests to use histamine-poor or -fl'ee diets and antihistamines during acute phases of mucosal inflamma- tion

T1053

Vanilloid Receptor Subtyped Deficient Mice Are Protected from Clostr/dium Difficile Toxin-A-induced Intestinal Edema Sebastian G De La Fuente, Kazunori Fujino l~eodore N Pappas, Christopher R. Mantyh

Background: Vanilloid receptor subtype-I (VR-I) is fbund on sensory neurons and may mediate neurogenic inflammation. Pravlous studies have shown that VR-I antagonists attenu~ ate animal models of intestinal inflammation. The purpose of his study was to evaluate wbe[her mice lacking VR- 1 are protected against intra~uminal admra:stration of Clostridium diffidle toxin-A (TA), a weII-established model of acute intestinal inflammation. Methods: "{A (10gg m 100~d of phosphate-buflered saline, PBS) was infused mw real and colonic loops (4 cm) of anesthetized mice as previously described. Control animals were infused with PBS alone Studies were conducted in VR-I gene-deficient (VR-1 -/-, n = 5) and C57BU 6 wild-type (VR- 1 + / o4, u = 6) mice as confirmed by PCR, Three hours tbllowing administra- tion, animals were euthanized and loops were removed and weighed. Fluid secretion was expressed as the loop weigfit-to-lengtfi ratio (mr/era) Statistical analysis was determined using Student-t test ~,tfi significance assumed to occur at a p < 0 0 5 Results are expressed as mean a: 5EM Resuhs: Infusion of PBS into ileal loops stimulated similar amounts of fluid secretion in both study groups (VR-1 -/- 26,8 • 17 vs. VR-1 + / + 30,8 • 2.1, p = n s ) However, TA administration significantly increased fluid secretiou in VR-1 + / + mice as compared to VR-1 /* ammals (VR-I + / + 50.8 • 6,2 vs VR-1 4- 298 • 3.6, p < 0 05) Preliminary results in colonic segments show corresponding data to that observed in the ileum Conclusions: Animals genetically deficient of VR-1 are protected agan~t TA- induced intlammatory edema These findings underscore the pivotal role of VR-1 in the pathogenesis ot acute intestinal inilamraation,

T1054

The Developing Intestinal Epithelial Response to Tnf-A Is in Part Modulated by Specific Components of The Extracellnlar Matrix (ECM) Cheryl Young, Jae-Snng Ko, W, Allan Walker, N Nanda Nanthakumar

In response to pro-inflammatory stimuli, the human intestinal epithelium recruits nentmphils by secreting lL-8, The undifferentiated crypt epithelial ceils secrete the greatest amount oI IL-8 leading to increased neutrophd infiltrates during inflammation A number of investigators have tested the role of undtftEren~iated cells m this increased response However, ECM underlying the crypt epithelium is different from diflerentiated vilhis epithelium. The difler- ence in the composition o[ ECM plays a critical role in the rate of epithelial proliferation and diflerentiation, Recently,, the specific role of ECM ni recruitment of lymphocyces to the mucoa] surface during inflammation has been demonstrated However, the role of the ECM in the intestinal immune thnetion has not been characterized Since specific components of the matrix play a critical role in the developing gut, we have hypotfiesized that these matrix molecules are partially responsible tbr the enhanced epithehal response to pro-inflaromatory stimuli in the preterm infant gut. Using T-84 cells as a model for mature enterocytes and H4 cells (a primary nonmmhgnant small intestinal fetal cell line) as a model tot immature entemcytes, we have tested this hypothesis Tbe ceils were grown on Matt*Gel, Collagen I, Collagen IV, Laminin, and Fibronectin. Upon reaching confluence, the cells were stimulated with TNF-a and as~yed for ID8, H4 cells grown on Matt*Gel showed a threedold inducuon of intestinal alkaline phosphatase suggesting partial epithelial diflerentiation, Whereas H4 cells remain undifferentiated when grown on other ECM components, When grown on laminin and fibronectin a two and three told ll-8 induction, :~spectively, was observed Whereas no response was observed when grown on Matt*Gel, Collagen-1 or Collagen-iV. T84 cells grown on these specific ECM components showed a non-specific enhancement of IL-8 to TNF-e* stimulation with out any" specificity These data taken together suggest a distinct role of specific ECM components in cellular differentiation and in modulating the immune response in the immature infimt gut There~bre, we conclnde that an age-dependent change in inflammatory responses are also dependent on tie state of cellular differentiation as well as specific composition of various ECM components contiguous with epithelial monolayer These ECM components may play an important role in pathophysiolocial responses during inflammation in infants comlYared to adults

T1055

Intestinal Stromal Cell Products, including TGF-~ Recruit Blood Monocytes and Down-regulate Monocyte CCR5 Expression and HIV-1 Replication: Mechanism by Which Resident Intestinal Macrophages Are Unable to Serve as a Reservoir for HIV-1 Phillip D Smith, Gang Meng, Lesley SmFhies

Background. To understand the role of the intestinal mucosa in the transmission ot HIVe1 and as a reservoir for HIV-l-intec:ed cells, we have shown that intestinal macrophages rarely" express H1V-1 mRNA in vivo (,[ Exp Med 1994:180:1541), that primary intestinal epithelial cells selectively transfer macrophage-tropic (R5), but not lymophocy'~e-tropic (X4), HIV-1 m vitro (Nature Med 2002;6:150), and that intestinal macrophages are phenotypical]y and functionally distinct from blood monocytes (] Immund 2001;167:2651), lack CCR5 (R5 receptor) and CXCR4 (X4 receptor) (Gastroenterology 1999;116:1043), and do not support HIV-1 replication (,l infect Dis 2000;182:785), These findings indicate that macmphage- tropic viruses initially infect intestinal Iymphocytes, not macropbages Aim. Since CCR5 CXCR4- intestinal macmphages are derived from CCR5+CXCR4" blood monocBes, we sought to determine how btood nmnocytes are down-regulated tbr CCR5 expression and HIV-1 permissiveness. Methods. Lamina propria mononuclear cells, epithelial cells, and stroma (mesem'hymal cells) were purified from normal human intestine and used to generate conditioned media (CM), HIV-1 infections were pertbrmed as previously described. Results Stroma-conditioned media (S-CM), but not other intestinal cell CMs, was strongly ebemotac- tic tot blood monoc)r and the chemotactic activity w~as inhibited by anti-TGF-13 anubodies. S-CM induced dose-dependent reduction in blood monocyte, but not lymphocyte, CCR5 and CXCR4 expression, When the monocytes were pre-incubated with S-CM, monocyte permissiveness to HIV-I was inhibited in a dose-dependent maturer In addition, real-time PeR showed that exposure of monocytes already inlected with HIV-1 to S-CM resulted in a dose-dependent down-regulation of HIV-1 replication, These doum-regulatory activities were partially reversed by anti-TGF-13 antibodies. Conclusion These findings suggest that lamina propria stroma produces factors, including TGFq3 which recruit monocytes to the mucosa and then down-regulate monocyte CCR5 and CXCR4 expression as well as monocyte permissiveness to HIVq and H1V-1 replication.

T1056

Lactobacillus casei DN-114 001 and Bacteroides thetaiotaomicron VPl-5482 inhibit rotavirus infection by modulating apical glyeosylation pattern of cultured human intestinal HT29-MTX cells Miguel Freitas*, Emmanuelle Tavan*, Karine Thoreux, Chantal Cayuela, Catherine Sap*n, Germain Tmgnan

The glycosylation pattern of the intestinal cell layer is implicated m the intestinal host ddeuse and is influenced by the gut micmilora The goal of this sntdy was to test whether pmbiotic and commensal bacteria could modulate the glycosylation pattern of HT29-MTX intestmaI epithelial cells in vitro, and consequently influence the intestinal host detense. Such modula- tion was tested on the inhibition of rotavirus infection, as clinical trials provide evidences that certain fermented milks decrease the incidence of acute infant:le rota~urna diarrhea. Difterentiated ttT29-MTX cells grown for 5 days with 20% of spent culture supernatant (SCS) either fi'om Lactobacillus case: DN-114 00 i (Lease* DN-114 001) or from Bacteroides thetoiotaomicron VPl-5482 (It.thetaiotaomicron), displayed a strong increased [3-galactose- specific lectin binding, compared to cells incubated with control media Quantitative analysis by fluorimetry revealed a 160% and 150% increase (P<0,05) between contrd ceils and those incubated with SCS fi'om Lcasei DN-114 001 and B.thetaiotaomiceo~: respectively, The

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