the biologic relevance is not clear. To investigate the role of PSGL-1 in recruitment of T cells to the colon, naive, transgenic DOll .10 CD 4 + T cells were adoptwely transferred to recipient Balb/C mice. The transferred cells were activated in vivo by priming recipients with i.p. OVA323-329/CFA. Accumulation of activated cells in tissues was measured in primed mice and in primed mice that had been treated with anti-P-selectin. Cells were isolated 7 days after transfer. Results are expressed as the percentage of DO 11.10 cells over total CD 4 + T cells. Transgenic, activated cells preferentially migrated to the colon LP (11.9%) versus the spleen (2.99%), mesenteric lymph node (MLN) (2.25%), Peyer's patch (PP) (3.43%), and small bowel LP (3.74%). Furthermore, anti-P-selectin abrogated recruit- ment to the colon as nearly 100% of migration was blocked (0.2% of CD4+ cells were transgenic). These data suggest that P-selectin mediated pathways dominate activated T cell recruitment to the colon. This implies that that the colon is selectively recruiting Th-1 helper effector cells. Therefore, PSGL-1 likely plays a role in recruitment of T cells to the colon to mediate effector response to colonic pathogens, as well as in IBD.

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T Ceil-lnduced Clearance of Enteropathogenic E. Coli (EPEC) in Murine Intestine Navdha Mittal, Gall Hecht, Ramanarao Dirisina, Neda Bajalo, Suzana Savkovic, Terrence A. Barrett

It is postulated that production of antimicrobial peptides by Paneth cells impedes survival of pathogenic enteric organisms. To examine this question we utilized a novel in vivo mutant of enteropathogenic E. coli (EPEC) where mutation of the bundle forming ptlus(Abfp)gene has been shown to enhances enterocyte attachment in mice. We have found that mice infected with Abfp EPEC develop transient diarrhea and have increased tissue levels of myeloperoxidase (MPO) suggesting that the mutant induces pathogenic responses in murine intestine. Previously, we reported that activation of T cells with T cell receptor-crosslinking anti-CD3 mAb induces more than 3-fold induction in Paneth cell size due to increased production of granules containing active cryptdin peptides. In the current studies we assessed whether production and release of Paneth cell granules influence survival of EPEC. Mice were orally inoculated with Abfp EPEC and colony forming units (CFUs) assessed in small bowel (SB) and colon after treatment with control or anti-CD3 mAb. Mice were examined on days 4 and 5 after infection with Abfp EPEC. Anti-CD3 mAb was administered 24 hr prior to sacrifice (when T cell-induced cryptdin peptide release is greatest). Results indicate that T cell activation reduced SB EPEC CFUs from 104 (given control mAb) to 101 (anti- CD3 mAb) and colon EPEC CFU from 106 to <101. No effects on CFUs of Gram +ve or Gram -ve commensal organisms were detected although further speciation is pending. These findings are consistent with the notion that T cefl-induced Paneth cell release of anumicrobial pepttdes is a natural host defense mechanism that reduces survival of enteric pathogens. These results may be relevant to the development of novel therapies aimed at preventing and treating enteritis caused by pathogenic organisms such as EPEC.

SI091

Mucosal Macrophages Make Substance P (SP) in Murine Thl -Type Colitis Razvan Arsenescu, Arthur Blum, Ahmed Metwafi, Tommy Setiawan, Elliott E. David, Joel V. Weinstock

IL10-A mice given a NSAID develop chronic Thl colitis. Substance P (SP) is a pro-inflamma- tory molecule that drives this inflammation. SP stimulates IFN~/and ILl2 production, and inhibits TGF~ secretion. While nerves make SP, evidence suggests that leukocytes make it also. We determined if intestinal lamina propria mononuclear cells (LPMC) make SP in IL10-A colitis and studied possible mechanisms of regulation. Five-wk-old ILl0-/- C57BL/ 6 mice received a NSAID for 2 wks to induce colitis. All mice had severe colitis 2 wks after stopping NSAID administration. LPMC were isolated using collagenase dispersal and Percoll gradients. The isolated cells were fractionated into T cell and non-T cell subsets using magnetic bead capture to study SP mRNA content. Using RT-PCR, SP transcripts were detected only in the non-T fraction (Figure). Dual color immunohistochemistry on freshly isolated LPMC localized SP production to F4/80 + macrophages. Since SP is a Thl cytokine, experiments determined if the STAT4/6 pathway of activation affected macrophage SP production. Inflammatory macrophages were isolated from WT, STAT4-/- (ILl2 pathway impaired) and STAT6-/- (IL4 pathway impaired) animals to measure SP mRNA content using quantitative PCR. Compared to WT control cells, STAT6-/- macrophages had 10-fold more SP mRNA, while SP transcripts were substantially diminished in STAT4-/- macrophages. Subsequent experiments showed that ILl2 induces SP production. Conclusions: 1) Murine LP macrophages make SP in ILl0 colitis. 2) STAT4/6 pathways of cell activation regulate macrophage SP production. 3) IL 12, a Thl cytokine, induces SP production that in turn amplifies Thl inflammation. Thus, in intestinal inflammation, leukocytes and nerves both may be important sources of mucosal neuropeptides (DK38327, 58755, 02428, 25295, AI49382, CCFA.)

Preprotachykinin mRNA in LPMC Depleted of T Cells

MW T Non-T T Non-T T Non-T T Non-T

1 2 3 4 4 separate experiments

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The Role of Interleukin (IL)-4 in the Early Control of Murine Cryptosporidiosis Stuart McDonald, John O'Grady, Frank Bromhacher, James Alexander, Vincent McDonald

Introduction: The clearance of Cryptosporidium parvum (Cp.) infection from mice is depen- dent on the presence of IFN~. We have previously shown that BALB/c IL-4 -/- mice were more susceptible to C.p. in the acute phase of infection compared with wild type mice. Here we evaluate the role IL-4 plays in the control of C.p. infection in normal, SCID and 1FN~ knockout (-/-) BALB/c mice, in particular its role during the early stages of infection. Materials and Methods: Seven-day-old mice were infected 2.5x104 C.p. oocysts orally. Infection was quantified using an acid-fast stain of faecal smears and intestinal tissue was snap frozen at varying time points post-infection (p.i.). RT-PCR was used to evaluate cytokine expression. Rat anti- IL-4 monoclonal antibody lIBII and recombinant IL-4 were injected subcutaneously (s.c.). Results: Injecting 100~g anti-IL-4 when infection was initiated resulted in heavier parasite burden at 7 days p.i. compared with PBS-injected controls. However, if the anti- IL-4 was injected 4 days p.i. there was no difference in the oocyst counts compared with the control group. Injections of l~g IL-4 when infection was initiated, but not 4 days p.i, results in fewer oocysts being shed at day 7. These results suggest that IL-4 has a protective effect at an early point of infection. Anti- IL-4 treatment of 1FN'y -/- mice infected with Cp. oocysts, however, did not result in a sigmficant increase in C.p. burden when compared with wild type controls. This suggests that IL-4 may play a role in promoting Thl-mediated clearance of C.p. Similarly, anti-IL-4 had no effect on C.p. infection in SCID mice suggesting that any effect of IL-4 on infection is dependent on the presence of T cells. IL-4 mRNA was not detected in the early stages of infection in normal mice (8-48 hours p.i.) whereas IFN3, can be detected from 30 hours.. Conclusion: These data suggest that IL-4 contributes to the limiting of severity of C.p. infection in the presence of IFN'y and that IL-4 may be stored in cells that rapidly infiltrate the intestine (e.g. mast cells) upon infection and release 1L- 4 locally.

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In Vivo Demonstration of Significant Role of C-C Chemokine mLARC in T Lymphocyte Adhesion to the Microvessels of Inflamed Small Intestinal and Colonic Mucosa Chikako Watanabe, Soichiro Miura, Naoki Hosoe, Yoshikazu Tsuzuki, Ken Teramoto, Tokushige Oyama, Yoichi Fujiyama, Ryota Hokari, Hiroshi Nagata, Hiromasa Ishii

Background: Chemokines are fundamentally important in determining the trafficking proper- ties of specific lymphocyte subsets. MIP-3c~ is known to be specifically expressed in the skin and intestine, and its liganfl CCR6 is expressed in immature dendritic cells and memory T cells, especially ~t4~7-expressing cells. The expression patterns of CCR6 and MIP-3a suggest that these molecules might mediate migration of lymphocytes during mucosal immune responses. However, their roles in intestinal inflammation have not been clearly demonstrated. The aim of this study was to determine the role of mLARC, the orthologue of human MIP-3ct in mice, in the integrin-dependent adhesion of T lymphocytes to the microvessels of inflamed intestinal mucosa induced by TNF-c~ or LPS. Methods: T lympho- cytes were isolated from intestinal lamina propria(LPLs). Lymphocytes were labeled with fluorochrome carboxyfluorescein diacetate succinimidyl ester(CFSE) and administered from jugular vein of the recipient mice. Adhesion of fluorescence labeled LPLs to microvessels of small intestinal and colonic mucosa was observed under an intravita] fluorescence micro- scope. In some experiments, TNF-~t(25~g/g) or LPS(100~g/body) was injected intraperitone- ally to mice 5 hours before lymphocyte injection. In another sets of experiments, T lympho- cytes were desensitized with excess amount of mLARC and changes in cell kinetics was examined. Expression of mLARC mRNA was examined by RT-PCR, and its localization was determined by in situ demonstration with Cy-3-1abeled anti-mLARC. Results: Intravital observation demonstrated that TNF-~t or LPS treatment induced a significant increase in LPL accumulation at both small intestine and colonic mucosa. Desensitization of CCR6 with mLARC significantly inhibited the TNF-ct or LPS-indnced LPL accumulation in the inflamed small intestine as well as in the inflamed colonic mucosa. Increased mLARC mRNA expression was induced by TNF-c~ at both small and large intestine, and Cy-3-1abeled anti-mLARC showed that the increase mainly occurs in the lamina propria. Conclusions: Inflammation induced increase in adhesion of T lymphocytes to the microvessels was demonstrated in the small and large intestinal mucosa after administration of TNF-cx or LPS. Combination of mLARC and CCR6 may play significant roles in the enhanced migration of LPLs in these inflamed sites.

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EXPRESSION OF THE FRACTALKINE RECEPTOR, CX3CR1, CHARACTERIZES A SUBPOPULATION OF INTESTINAL ANTIGEN SAMPLING DENDRITIC CELLS Jan H. Niess, Stephan Brand, Xiubin Gu, Steffen Jung, Dan R. Littman, Hans-Christian Reinecker

Background: Fractalkine is a membrane bound chemokine, which is constitutively expressed by intestinal epithelial cells (IEC). In order to identify the cellular targets of IEC derived fractalkine, we utilized mice with a target deletion of CX3CR1 by green fluorescence protein (GFP) insertion. This approach allowed the generation of a mutant CX3CR1 locus but also the examination of the CX3CR1 expression pattern and migration of cells that normally express this receptor. Methods: The distribution of CX3CR1 expressing cells in the gastroin- testinal tract was assessed by confocal microscopy in living tissues. For phenotypic character- ization of CX3CR1 expressing cells we developed a novel technique for the isolation of dendritic cells (DCs) from the small intestine. Results: Three-dimensional reconstruction of confocal image series revealed CX3CR1 positive cells localized in close proximity of IECs within small intestinal villi. In the colon CX3CR1 positive cells were localized beneath the surface epithelium. In the small and large intestine the CX3CR1 positive DCs were interconnected with long dendrites. Restricted to the very distal ileum, CX3CRI expressing DCs extended dendrites across the basal membrane into the villus epithelium. In the colon

AGA Abstracts A-152