Abstracts of ECCO Congress, Innsbruck, Austria, 1—3 March 2007 51 epithelial cells with IL-21 resulted in enhanced phosphorylation of ERK1/2 and p38 and increased synthesis of MIP-3alpha, a T cell chemoattractant. Inhibition of ERK1/2 but not p38 suppressed IL-21-induced MIP-3alpha pro- duction. IL-21-treated cell culture supernatants enhanced in vitro lympho- cyte migration, and this effect was inhibited by anti-MIP-3alpha antibody. Treatment of IBD explants with anti-IL-21 reduced MIP-3alpha production. Conclusions: Data show that intestinal epithelial cells are a target of IL-21 and that IL-21 is involved in the cross-talk between epithelial and immune cells in the gut. P184 NF-KB CONTROLS THE EXPRESSION OF FLIP, AN INHIBITOR OF FAS-MEDIATED APOPTOSIS, IN CROHN'S DISEASE LYMPHOCYTES F. Caprioli 1 , D. Fina 1 , R. Caruso 1 , I. Peluso 1 , M.C. Fantini 1 , C. Stolfi 1 , G. Sica 2 , A. Rizzo 1 , S. Janssen 1 , L. Biancone 1 , F. Pallone 1 , G. Monteleone 1 . 1 University of Tor Vergata of Rome; 2 Department of Surgery, University of Tor Vergata of Rome Background and aims: The marked accumulation of T cells in the gut of Crohn's disease (CD) patients is supposed to rely partly on the resistance of such cells to Fas-mediated apoptosis. We previously showed that CD lamina propria lymphocytes (LPL) contain elevated levels of Flip, an inhibitor of Fas- mediated apoptosis, and that inhibition of Flip by antisense oligonucleotides reverted the resistance of CD LPL to Fas-induced apoptosis. How Flip is reg- ulated in CD remains unclear. We here examined the molecular mechanisms that control Flip in CD. Methods: Paired colonic biopsy samples of patients with CD, patients with ulcerative colitis (UC) and healthy controls (HC) were analysed for Flip RNA and protein by real-time PCR and Western blotting (WB) respectively. Sam- ples were also evaluated for Stat3, Akt and NF-kB/p65 activation by WB. Additionally, Flip was evaluated in biopsies of CD and HC cultured with TPCK (NF-kB inhibitor), or AG490 (JAK2/Stat3 inhibitor), or wortmannin (pAkt in- hibitor). Finally, CD and HC CD4+ LPL were treated with TPCK per 24 hours and then with Fas-ligand for further 24 hours. Apoptotic cells were eventu- ally evaluated by FACS. Results: A more pronounced expression of Flip protein was seen in CD as compared to UC and HC. Additionally, Flip RNA levels were significantly in- creased in CD in comparison to UC and HC, indicating that Flip is controlled at the transcriptional level in CD. Notably, the same CD samples contained high levels of active Stat3, Akt and NF-kB, all of which have been reported to enhance Flip in other systems. However, inhibition of NF-kB but neither AkT nor Stat3 reduced Flip in CD biopsies and LPL. Consistently, TPCK restored the susceptibility of CD LPL to FAS-mediated apoptosis. Conclusions: Data show that, in CD, Flip is transcriptionally controlled by NF- kB, thus elucidating an additional mechanism by which NF-kB helps sustain the ongoing inflammation in CD. P185 INCREASED GUT PLASMACYTOID DENDRITIC CELLS IN ACUTE ULCERATIVE COLITIS - KEY MEDIATORS OF IMMUNITY AND INFLAMMATION? S.C. Ng 1 , S. Plamondon 1 , M.A. Kamm 2 , S.C. Knight 1 , A.J. Stagg 1 . 1 Antigen Presentation Research Group, St Mark' s Hospital, London, UK; 2 Gastroenterology, St Mark' s Hospital, London, UK Background & Aim: Breakdown of tolerance against the commensal mi- croflora is believed to be a key factor in the pathogenesis of inflammatory bowel disease (IBD). Dendritic cells (DC) have been implicated in mediating this process in animal models, but data in the human disease are limited. We aimed to identify changes in DC associated with acute flare-up of ulcerative colitis (UC). Methods: Rectal biopsies were obtained from patients with active UC, symp- tomatic for < 4 weeks (Sigmoidoscopy score >2; n=20) and from macro- scopically and histologically normal tissue from controls (referred for rectal bleeding; n=7). DCs were identified by multi-colour flow cytometry as HLA- DR+ lin-/dim cells in freshly isolated lamina propria mononuclear cells. The proportion and number of CD11c+ myeloid DC and CD11c- putative plasma- cytoid DC were assessed. Surface expression of activation markers CD40, CD86, and Toll-like receptors (TLR), TLR-2 and TLR-4 was assessed on each cell population. Results: During acute flare-up, UC patients had significantly more intestinal DC compared with controls (456 versus 108 per mg; p<0.05). The majority of additional DC present in inflamed tissue were CD11c- plasmatocytoid DC (403 versus 51 per mg; p<0.005). In contrast, the number of CD11c+ DC did not differ significantly between UC patients and controls. Unlike CD11c+ myeloid DC, CD11c- DC from UC patients did not express TLR-2 and TLR-4, and only a few expressed CD40 (22% CD11c-DC versus 60% CD11c+DC) and CD86 (11% CD11c-DC versus 48% CD11c+ DC). Preliminary data also suggested that these CD11c- cells lacked expression of blood plasmacytoid DC markers (CD123, BDCA-2 and BDCA-4). Conclusion: Acute exacerbation of UC is associated with an increased num- ber of intestinal DC, the majority of which are CD11c- cells. Recruitment of this specific group of cells in the gut probably contributes to local inflamma- tion and tissue damage. Further characterisation of these cells will provide new insights into their role as a therapeutic target in active UC. P186 INTESTINAL CYTOKINE PRODUCTION BY DENDRITIC CELLS IN ACUTE ULCERATIVE COLITIS - AN IMMUNOREGULATORY ROLE? S.C. Ng 1 , S. Plamondon 1 , M.A. Kamm 2 , S.C. Knight 1 , A.J. Stagg 1 . 1 Antigen Presentation Research Group, St Mark' s Hospital, London, UK; 2 Gastroenterology, St Mark' s Hospital, London, UK Background & Aim: Intestinal dendritic cells (DC) interact with the luminal flora and play a key role in maintaining immune homeostasis. Altered DC function and an unbalanced cytokine production are likely to contribute to the dysregulated recognition of bacteria that drives inflammation. Cytokine profile in chronic inflammation may be different from those in the early stage of active disease. The roles of individual cytokines in acute intestinal inflammation remain unclear. We therefore aimed to assess spontaneous in- tracellular cytokine production by colonic DC and lymphocytes in patients with an acute exacerbation of ulcerative colitis. Methods: Colonic biopsies were obtained from inflamed tissue of patients with active ulcerative colitis, symptomatic for less than four weeks (n=18; sigmoidoscopy score > 2) and controls (n=7; macroscopically and histologi- cally normal intestine of patients referred for rectal bleeding). DC subsets (CD11c+ HLA-DR+ lineage- myeloid DCs; CD11c- HLA-DR+ lineage- plasmacy- toid DCs) and lymphocytes were identified by multi-colour flow cytometry of cells extracted from collagenase digested intestinal tissue. Spontaneous in- terleukin [IL]-10, IL-12, IL-6, and IL-13 production by lamina propria DC and lymphocytes was measured by intracellular staining of permeabilised cells in the absence of exogenous stimulation. Results: In patients with acute ulcerative colitis, a significantly greater pro- portion of CD11c+ DC and lymphocytes produced IL-10 than cells from control tissue (p<0.05). In contrast, production of IL-12p40, IL-6 and IL-13 by DC in ulcerative colitis patients was present but this did not differ from that of control DC. In cells from healthy colonic mucosa from controls, there was no detectable DC production of IL-12p40, IL-6 or IL-13. Compared with CD11c+ DC and lymphocytes, intestinal CD11c- DC were poor spontaneous cytokine producers in ulcerative colitis patients. Conclusion: Acute ulcerative colitis is associated with increased IL-10 pro- duction by DC which may represent an attempt to antagonise inflammation and re-establish homeostasis during the initial phase of inflammation. This observation suggests that local modulation of DC cytokine contributes to the intestinal immune homeostasis. P187 EXPRESSION PROFILE OF PERIPHERAL REGULATORY T CELLS IN IBD ON THE SELF-DEVELOPED HUMAN Treg CHIP J. Maul 1 , S. Pförtner 2 , J. Buer 2,3 , R. Geffers 2 , R. Duchmann 1 . 1 Charité- Universitätsmedizin Berlin, Campus Benjamin-Franklin; 2 Helmholtz Centre for Infection Research, Braunschweig; 3 Institute of Medical Microbiology, Hanover Medical School Background: Previous findings show that regulatory CD4+CD25+ T cells (Treg) are numerically deficient in patients with active inflammatory bowel disease (IBD), but display normal suppressive function to allogeneic antigens in vitro. To further elucidate their pathobiology and therapeutic capacity, the present study investigates potential differences in Treg gene expression in active and inactive IBD patients and healthy controls. Materials and Methods: Treg and naive T cells were isolated from peripheral blood of patients with active Crohn's disease (aCD; n=3), inactive CD (iCD; n=3), inactive ulcerative colitis (UC; n=3) and healthy controls (HC; n=11) using MACS. Hybridisation to a self-developed microarray (Human TReg Chip) enabling simultaneous measurement of expression levels of 350 genes se- lected for their potential implication in Treg biology was performed. First, gene expression level of Treg versus autologous CD4+CD25- T cells was com- pared. Then, significantly differentially expressed genes were further anal- ysed comparing Treg specific gene signatures in HC and IBD patients. Regula- tion of interesting candidate genes was confirmed by quantitative real-time PCR. Results: 94 genes are significantly differentially expressed when comparing Treg specific gene signatures in IBD (inactive and active) and HC. None of these was regulated inversely, but some were expresses at marked differ- ent levels. Regulated genes are involved in apoptosis, proliferation, signal Downloaded from https://academic.oup.com/ecco-jccs/article-abstract/1/1/51/2394128 by guest on 31 May 2020