W1154 Macrophage Mannose Receptor Deficient Mice Are More Susceptible to Helicobacter Hepaticus Induced Colitis

AGA Abstracts

seen after 6 months. Our objectives were to assess patient acceptance of Home Automated Telemanagement in UC (UC HAT). Methods: UC HAT consists of a laptop connected to a scale, a decision support server and a web-based portal. Patients respond to a series of questions about UC symptoms, side effects, and adherence using the laptop. Body weight is assessed and an educational curriculum is delivered. Providers use the web portal to individualize alerts and action plans. Individualized action plans are set up at a designated web site and uploaded to a patient unit. Alerts are generated if pre-setup clinical conditions are met and then processed by a coordinator. Ten adult patients with UC were trained to use UC HAT at a 30 minute session. Patients returned one week later to complete selftesting without assistance; attitudinal surveys and qualitative interviews were performed. Results: All patients reported use of the computer was not complicated. 90% reported the symptom diary and side effect questions were not difficult to answer. All patients reported the training session was adequate and 70% reported that testing took little time. 70% would feel safer using the system. 90% would use UC HAT in the future. Analysis of the qualitative interviews revealed other positive aspects of UC HAT. Patients felt the provider would be able to monitor the disease more effectively as they would be aware of flares “right away” and UC HAT would provide “constant communication”. UC HAT seemed to empower patients “…because by being monitored, I would be aware if I needed to change behavior or meds”. Patients liked the constant feedback the system provided them. UC HAT resulted in “increasing awareness of what is going on... If you are doing well it is positive, and if you are in a flare it may make me feel more safe”. Improved safety was cited as an important benefit of UC HAT. UC HAT may “catch something I might not recognize” and help me “respond quickly to a threat”. Discussion: Improved monitoring is needed for UC. Patients with UC can be easily trained to use UC HAT, and patient acceptance of UC HAT is high. UC HAT has potential in improving clinical outcomes and patient satisfaction in UC.

H., Fullekrug, J., Stremmel, W., Griffiths, G., and Ehehalt, R. (2007) J Biol Chem 282, 27155-64 W1151 A Mechanism of Action for Cyclosporine a in Ulcerative Colitis: the Role of the Tec Kinase Itk in Disease Pathogenesis Raja Narayana Atreya, Benno Weigmann, Brigitte Bartsch, Peter R. Galle, Markus F. Neurath Introduction: The well established immunosuppressant Cyclosporine A (CsA) mediates rapid anti-inflammatory effects in the treatment of acute steroid-refractory ulcerative colitis (UC), inducing impetuous remission. In the treatment of Crohn's disease (CD) however, CsA does possess little if any therapeutic potential. The mechanism of action by which CsA exerts its therapeutic effect in UC has not yet been identified. The aim of our study was therefore to elucidate the molecular mechanism of CsA in UC. Methods: Lamina propria mononuclear cells (LPMC) were isolated from biopsies taken from inflammatory bowel disease (IBD) and control patients. The rate of apoptosis induction upon treatment with CsA was assessed via flow cytometric analysis of annexin V/propidium iodide staining. Cytokine concentration (IFN-γ, IL-13, IL-2) in the supernatant of cell cultures were analyzed using ELISA. The itk expression rate in LPMCs was assessed via flow cytometry. Immunfluorescence staining of cytospins of LPMCs for itk were performed. Oxazolone colitis was induced in itk deficient mice and controls. Disease activity was evaluated by means of body weight, histological and endoscopic score of inflammatory activity. Results: Upon administration of CsA there is significant induction of apoptosis in LPMCs from patients with UC but not in CD or corresponding controls. Analysis of cytokine concentrations in UC revealed a significant inhibition of IFN-γ and IL-13 production while IL-2 was not suppressed after CsA treatment. Histological analysis of gut cytospins showed a significant increase of itk expression in UC compared to CD and controls. Importantly in the oxazolone induced colitis model, itk deficient mice were significantly protected against the development of intestinal inflammation compared to control mice. Conclusion: Our results indicate for the first time, that the rapid therapeutic effect of CsA in UC is due to its capability to induce apoptosis in LPMCs. The augmented intestinal itk expression in UC and the protection of itk deficient mice in an experimental model of colitis, demonstrate a pivotal role of the T cell associated tec kinase itk in disease pathogenesis. These data show that in UC an itk mediated pathway leads to the augmented resistance of LPMCs to apoptosis and that the interaction of CsA with itk leads to the induction of apoptosis explaining its clinical efficacy. This pathway offers the basis for the development of novel therapeutic strategies in the treatment of UC.

W1149 Role of Fecal Polymerase Chain Reaction in the Differentiation of Intestinal Tuberculosis from Crohn's Disease Sucharita P. Chittaranjan, Ramadass Balamurugan, Balakrishnan S. Ramakrishna Background: The differentiation of intestinal tuberculosis (ITB) and Crohn's disease (CD) is often difficult in clinical practice. Culture of intestinal biopsy specimens requires 3-8 weeks with a yield of 20-30%. PCR on biopsies has shown variable results. This study examined the ability of fecal PCR for Mycobacterium tuberculosis (MTB) DNA to reliably distinguish patients with ITB from CD. Subjects: Fecal samples were collected from 75 patients, 27 with untreated ITB and 48 with CD. ITB was diagnosed using Paustian's criteria and response to therapy, while the diagnosis of CD was based on clinical, endoscopic and histological findings characteristic of the disease with response to aminosalicylates, steroids and /or immunosuppressants. Methods: DNA was extracted from feces and subjected to PCR using primers that amplified a 123 bp fragment of the IS 6110 sequence that is specific to the MTB genome. The sequences of the primers were MTB 1: 5'CCT GCG AGC GTA GGC GTC GG 3'and MTB 2: 5' CTC GTC CAG CGC CGC TTC GG 3'. The amplicon contained an internal endonuclease site that allowed confirmation of the product by digestion with SalI. We have earlier confirmed the sensitivity and specificity of these primers (J Clin Microbiol 2006). Results: Fecal PCR was positive in 21 of 27 patients with untreated ITB, compared to 7 of 48 patients with CD. Sensitivity, specificity, positive predictive value and negative predictive value of the test were 75%, 87%, 77%, and 85% respectively. Conclusion: Fecal PCR for the IS 6110 sequence of MTB is a simple non-invasive test with high sensitivity and specificity for the differentiation of ITB from CD.

W1153 Anti-TNF Agents Target Mucosal Intercellular Signaling in Inflammatory Bowel Diseases: A Common Molecular Mechanism of Action of Clinically Effective Anti-TNF Agents Raja Narayana Atreya, Brigitte Bartsch, Peter R. Galle, Markus F. Neurath Introduction The anti-TNF antibodies infliximab and adalimumab and the PEGylated Fab' certolizumab pegol have proven clinical efficacy in the treatment of Crohn's disease (CD). The role apoptosis induction plays in the action of these agents is still unclear; certolizumab pegol, in contrast to the other anti-TNF agents, does not appear to induce apoptosis in peripheral blood mononuclear cells (PBMCs). These results contradict the current paradigm of a direct apoptosis-inducing modality explaining clinical efficacy; hence other mechanisms must exist that result in anti-TNF-mediated suppression of gut inflammation in inflammatory bowel disease (IBD). Aim To identify a common mechanism of action for TNF agents in IBD. Methods Antigen-presenting cells (APCs) and CD4+ cells were isolated from PBMCs and lamina propria mononuclear cells (LPMCs) of gut specimens from patients with IBD (CD or ulcerative colitis) and controls. Immunofluorescence staining of cryosections of gut specimens was performed for membrane TNF (mTNF), TRAF2, and TNFR2. CD4+, CD11b+, and CD14+ cells of PBMCs or LPMCs were cultured and treated with each anti-TNF agent. Apoptosis induction was determined by flow cytometric analysis. Results There was no difference in mTNF expression between IBD and control group PBMCs or between cell subtypes in the two groups. Importantly, there was no significant induction of apoptosis in monocultured or co-cultured PBMC subtypes treated with anti-TNF agents in the IBD and control groups. Histologic analysis of gut cryosections showed a significant increase of mTNF expression in lamina propria APCs compared with CD4+ cells in IBD samples. TNFR2 expression was elevated in CD4+ T cells of IBD samples. In Vitro application of the antiTNF agents to cultured LPMCs from IBD samples resulted in a significant induction of apoptosis in co-cultured CD4+/CD14+ cells; this effect was not apparent when these cells were cultured alone. Discussion Our data show for the first time that all clinically effective anti-TNF agents are able to induce apoptosis In Vitro in gut CD4+ cells when co-cultured with CD14+ cells from IBD samples. Augmented mTNF expression in gut APCs and elevated TNFR2 expression in gut CD4+ cells in IBD indicate that this pathway mediates the resistance of T cells to apoptosis, contributing to the perpetuation of inflammation. Specific targeting of this pathway by anti-TNF agents results in binding to mTNF on CD14+ cells, causing indirect rather than direct apoptosis of gut CD4+ cells. These data concerning the mechanism of action of anti-TNF agents have important implications for the development of new therapeutic strategies in IBD.

W1150 Phosphatidylcholine Delays TNF-α-Induced Pro-Inflammatory Signalling and Interferes with the Compartementalization of TNF-α Receptors to Sphingolipid/ Cholesterol Enriched Microdomains (Lipid Rafts) At the Plasma Membrane Irina Treede, Petia Jeliaskova, Annika Braun, Thomas Giese, Joachim Fuellekrug, Wolfgang Stremmel, Gareth Griffiths, Robert Ehehalt Background & Aims: Phosphatidylcholine (PC) is a major lipid of the gastrointestinal mucus layer. We recently showed that mucus from patients suffering from ulcerative colitis has low levels of PC (1). Interestingly, clinical studies reveal that addition of PC to the colonic mucus using slow release preparations is therapeutically beneficial (2,3). The positive role of PC in this disease is elusive. However recent data showed that PC has an instrinsic antiinflammatory property. Exogenous application of PC inhibits membrane dependent actin assembly In Vitro and In Vivo as well as the TNF-α induced NF-κB activation (4,5). Methods: We now further investigated the hypothesis that exogenous application of PC has antiinflammatory properties. Different PC species were applied to human Caco-2 cells treated with TNF-α to induce a pro-inflammatory response. TNF-α-induced NF-κB-activation was analyzed by transient expression of a NF-κB-luciferase reporter system, pro-inflammatory gene transcription by quantitative RT-PCR analysis. Binding of TNF-α to its receptor was analyses by FACS. Lipid rafts were analysed by isolation of detergent resistant membranes (DRMs). Results: Exogenous addition of all PC species tested strongly inhibited TNF-α induced pro-inflammatory signalling via NF-κB. The expression level of selected genes involved in inflammation was significantly delayed after PC-pretreatment for at least 2 hours. This was not due to a reduced binding of TNF-α to its receptor or a decreased surface expression of TNF-α receptors. PC treatment changed the compartementalization of TNFR1 and TNF-R2 to DRMs. Conclusion: PC induces a prolonged inhibition of TNF-α induced pro-inflammatory signalling. This might be caused by a shift of the TNF-α receptors at the surface to lipid rafts. Our results provide a molecular explanation for the clinical studies showing a beneficial effect of PC therapy in ulcerative colitis. 1. Ehehalt, R., Wagenblast, J., Erben, G., Lehmann, W. D., Hinz, U., Merle, U., and Stremmel, W. (2004) Scand J Gastroenterol 39, 737-42 2. Stremmel, W., Ehehalt, R., Autschbach, F., and Karner, M. (2007) Ann Intern Med 147, 603-10 3. Stremmel, W., Merle, U., Zahn, A., Autschbach, F., Hinz, U., and Ehehalt, R. (2005) Gut 54, 966-71 4. Anes, E., Kuhnel, M. P., Bos, E., Moniz-Pereira, J., Habermann, A., and Griffiths, G. (2003) Nat Cell Biol 5, 793-802 5. Treede, I., Braun, A., Sparla, R., Kuhnel, M., Giese, T., Turner, J. R., Anes, E., Kulaksiz,

AGA Abstracts

W1154 Macrophage Mannose Receptor Deficient Mice Are More Susceptible to Helicobacter Hepaticus Induced Colitis. Sigrid Heinsbroek, Kevin J. Maloy, Philip Ahern, Mark Asquith, Sofia M. Buonocore, Fiebo J. ten Kate, Wouter de Jonge, Guy E. Boeckxstaens, Siamon Gordon

Background and Aim: The macrophage mannose receptor (MR) is a recycling receptor expressed by macrophages and selected dendritic cell populations. MR can recognise a large range of endogenous ligands such as lysosomal hydrolases and myeloperoxidase, mediating

A-644

W1155 IL-21 Receptor Signaling Enhances NK Cell Cytolytic Activity and Induces Proinflammatory Cytokine Production in Inflammatory Bowel Disease Zhanju Liu, Jinxia Jiu, Huixia Zhang Aims: IL-21 is a CD4+ T cell-derived cytokine, which is involved in innate and adaptive immune response, and induces T and NK cell activation and effector response. The aim of this study was to analyze IL-21 receptor (IL-21R) expression in inflamed mucosa of inflammatory bowel disease (IBD), and evaluate its role in the induction of proinflammatory cytokine production and NK cell cytotoxicity and activation. Materials and Methods: Expression of IL-21R was performed by immunohistochemistry and flow cytometry. NK cell cytotoxic activity was detected by a standard 51Cr-release assay. Cytokine levels were determined by ELISA. Results: IL-21R-positive cells were found to be significantly increased in inflamed mucosa of IBD patients compared with those in controls by immunohistochemistry. Flow cytometric analysis confirmed that IL-21R was mainly expressed in freshly isolated PB- and LP-CD4+, CD8+ T, B and NK cells. IL-21R was found to be decreased gradually in PB-T cells 24h after anti-CD3 stimulation In Vitro. PB-NK cells from IBD patients, when stimulated with human IgG and IL-21, produced higher levels of IFN-γ and TNF than controls (P < 0.05). Importantly, IL-21-primed IBD NK cells showed a potent antitumour cytotoxicity to NK-sensitive K562 cells than controls. Moreover, freshly isolated PB-T cells and LPMC from IBD patients, when stimulated with IL-21 and anti-CD3 mAb, secreted large amounts of proinflammatory cytokines (e.g., TNF, IFN-γ, IL-2) than controls. Conclusion: IL-21R is highly expressed in the inflamed mucosa of IBD patients. IL-21 triggers IBD T cells to produce proinflammatory cytokine secretion and enhances NK cell cytotoxic response, suggesting that strategies aimed at blocking IL-21R signaling may have a therapeutic potential in IBD.

W1158 Azathioprine Modulates Human Monocyte Function Via the Pak-2 Signaling Pathway Lu Zhou, Klaas Nico Faber, Maikel P. Peppelenbosch, Gerard Dijkstra Introduction: Azathioprine is the main treatment option for Crohn's disease. Recent studies showed that the azathioprine metabolite 6-Thioguanine (6-TG) causes immunosuppression by blocking Rac1 GTPase activation in T lymphocytes[1]. However, the mechanisms explaining why such inhibition is clinically useful remain poorly understood. Here, we studied the action of 6-TG on human monocytes, the main innate mediators of immunity in Crohn's disease. Materials and Methods: Peripheral blood monocytes of Crohn's disease patients and healthy controls were isolated by gradient centrifugation followed by adherence. For evaluating the activation status of Rac1 we used p21-activated kinase 2 (phospho-PAK2), the downstream target of Rac1, as a surrogate marker[2]. Western blotting was used to assess the phospho-PAK-2 levels in unstimulated, peptidoglycan (PGN)-, or lipopolysaccharide (LPS)-stimulated monocytes in the presence or absence of 6-TG. Since PAK-2 is implicated in cytoskeletal remodelling, we studied monocyte phagocytosis in the absence or presence of various concentrations 6-TG by quantifying incorporation of FITC-labelled E. coli by monocytes using fluorescence microscopy. Results: A 30 minute incubation with 30 µM 6TG strongly reduced phospho-PAK2 levels in monocytes from 2 healthy controls, as well as in 2 patients with Crohn's disease. Moreover, PGN (30 µg/ml)-induced Toll-like receptor (TLR)-2-mediated induction of phospho-PAK-2 is inhibited by 6-TG (analyzed for 3 healthy controls and 5 Crohn's disease patients). The TLR-4 ligand LPS (2 µg/ml) did not increase phospho-PAK2 levels in monocytes. Treatment of monocytes from a healthy donor with 6TG led to a dose (0-30 uM 6-TG)-dependent increase in monocyte phagocytosis as quantified by the number of FITC-E. coli-positive monocytes as well as the total number of FITClabelled bacteria per 100 monocytes. Treatment with higher concentrations of 6-TG (60100 uM) led to a subsequent decrease in monocyte phagocytosis. Conclusion: Azathioprine metabolite 6-TG inhibits monocyte (Rac1-dependent) PAK-2 signaling pathway under both basal and stimulated conditions. This inhibition results in increased phagocytosis at low to intermediate 6-TG concentrations, suggesting that 6-TG may actually stimulate innate immunity. As impaired innate immunity seems a general feature of Crohn's disease[3], the therapeutic effects of Azathioprine in patients may be partially dependent on elevating Racdependent suppression of monocyte function. References: 1. Poppe D, et al. J Immunol. 2006,176:640-51. 2. Renkema GH et al. Mol Cell Biol. 2002, 22:6719-25. 3. Marks DJ, et al. Lancet. 2006,367:668-78.

W1156 IL-23 Is Highly Expressed in Inflammatory Bowel Disease and Induces T Cell Proinflammatory Cytokine Secretion Zhanju Liu, Jinxia Jiu, Huixia Zhang Aims: IL-23 is composed of IL-23p19 and p40 subunit of IL-12, and plays an important role in the induction of Th1 immune response and autoimmune immunity. In this study, we analyzed expression of IL-23p19 in inflamed areas of inflammatory bowel disease (IBD) and its role in the induction of T cell activation and proinflammatory cytokine secretion. Materials and Methods: Expression of IL-23p19 was performed by immunohistochemistry. IL-23R expression in CD4+, CD8+ T and NK cells was analyzed by flow cytometry. Cytokine levels were determined by ELISA. Intracellular expression of IL-17 by peripheral blood (PB)CD4+ T cells after IL-23 stimulation was determined by flow cytometry. Results: Expression of IL-23p19 was significantly increased in inflamed mucosa of CD at both the transcriptional and translational levels compared with that in UC and healthy controls. Double staining confirmed IL-23p19-positive cells were mainly CD68+ macrophages/dendritic cells. IL-23R was mainly expressed in PB- and lamina propria (LP)-CD4+, CD8+ T cells and NK cells. IL-23 strongly induced PB-T cell CD69 expression in IBD patients than in controls In Vitro, and triggered CD PB- and LP-T cells to produce significantly higher levels of IFN-gama, TNF and IL-2 than those from UC and healthy controls (P < 0.05). Moreover, IL-23 potently induced IBD PB-T cells to secrete IL-17 than healthy controls. Conclusion: IL-23p19 is increased expression in IBD, particularly in CD, and may play an important role in the induction of T cell activation and proinflammatory cytokine secretion. Targeted therapy directed against IL-23 may have a therapeutic role in the treatment human IBD. W1157 Intestinal IL-7 Is Not Essential for the Persistence of IL-7-Dependent Chronic Colitis Takayuki Tomita, Takanori Kanai, Yasuhiro Nemoto, Toshimitsu Fujii, Teruji Totsuka, Mamoru Watanabe

W1159

Backgroud & Aims: We previously demonstrated that IL-7 is produced by intestinal goblet cells and is essential for the persistence of colitis. It's well known, however, that goblet cells are depleted in chronically inflamed mucosa of colitis in animal models and human inflammatory bowel diseases. Methods: Thus, we here questioned if intestinal IL-7 is actually required for the persistent of colitis using an adoptive transfer RAG-1/2-/- colitis model induced by adoptive transfer of CD4+ CD45RBhigh T cells in combination with a parabiosis system (Fig.1). Results: Surprisingly, both IL-7-/- x RAG-1-/- (Gr. 3) and IL-7+/+ x RAG-1-/(Gr. 2) host mice developed colitis to a similar extent of the colitic RAG-2-/- donor mice previously transferred with CD4+CD45RBhigh T cells 4 wk after parabiosis. Of note, although the number of CD4+ T cells recovered from the spleen or the BM of the IL-7-/- x RAG-1-/-

Interleukin-22: A Marker of Activity in Crohn's Disease Sabine Thieler, Ulrike Strauch, Gerhard Rogler, Claudia Ott, Jürgen Schölmerich, Florian Obermeier BACKGROUND: IL-22 is a proinflammatory, TH17-cell derived cytokine and part of the IL-10 cytokine family. Its receptor complex is expressed on a number of tissues as skin, liver, lung, pancreas and intestine, but not on immune cells. It has been shown by immunohistochemical staining that IL-22 is not detected in healthy gastrointestinal mucosa but is increased in the inflamed mucosa of patients with inflammatory bowel disease (IBD). The

A-645

AGA Abstracts

AGA Abstracts

(Gr. 3) host mice was significantly decreased compared with that of the IL-7+/+ x RAG-1-/(Gr. 2) host mice, equivalent number of CD4+ T cells was recovered from the lamina propria of both mice, indicating that the expansion of CD4+ T cells in the spleen or in the bone marrow is dependent on IL-7, but is not dependent in the LP. Conclusions: Collectively, systemic IL-7 is essential, but intestinal IL-7 is not essential, for the persistence of colitis, suggesting that therapeutic approaches targeting systemic IL-7/IL-7R signaling pathway may be feasible in the treatment of IBD.

clearance of these molecules by macrophages and limiting tissue damage. Moreover, the mannose receptor is a pattern recognition receptor shown to recognise a variety of microbes. We hypothesized that a lack of mannose receptor influences gut flora recognition thereby inducing susceptibility to inflammatory bowel disease. We assessed the role of MR in H. hepaticus induced colitis using MR deficient mice.

Methods: Wildtype and MR deficient mice were fed with H. hepaticus, known to induce colitis in susceptible strains, on day 0, 2 and 4, and injected with anti-IL10 receptor each week for a period of 4 weeks. After four weeks mice were killed and inflammation was assessed in spleen and colon.

Results: Histochemistry showed a more severe colitis in MR deficient mice compared to wildtype mice, with increase in inflammation, amount of lymphoid follicles, ulceration in the submucosa and spreading to the muscle layer. The colon inflammation score for MR deficient mice and control mice were 2.280 ± 0.6598; N=5 and 6.200 ± 1.463; N=5, respectively. Systemic disease was more severe in MR deficient mice as was shown by a 10% increase of granulocytes in the spleen compared to WT mice.

Conclusions: These data demonstrate that mannose receptor deficiency exacerbates H. hepaticus induced colitis, possibly due to lack of microbe recognition. This suggests that MR might play a role in inflammatory bowel diseases.