357

The Role of Human Intestinal Paneth Cells Expressing Toll-like Receptors in Innate Host Defense Toklyoshi Ayabe, Atsuo Maemoto, Yasuko Miynshi, Toshifumi Ashida, Yusuke Saitoh, Tom Kono, Yoshiaki Ehisawa, Andre J. Ouellette, Yutaka Kohgo

Background&Aim: Small intestinal Paneth cells contnbute to mucosal innate immunity by sensing bactena and secreting microbicidaI peptides, particularly a-defensins (Nature Immu- nol l: 113, 2000). Our aims were to investigate the expression and function of Toll-like receptor (TLR)s and related molecules in crypts of human small intestine. Methods: Ileal mucosa were obtained from surgical specimens resected from patients with colon cancer with normal ileum under informed consent. Intact crypts were isolated by standard EDTA dissociation, and further collagenase dissociation was conducted to prepare crypt single cell s0spensions. RT-PCR experiments were performed on individual crypts or isolated Paneth cells to assay for TLRI-10, CD14, MD2 and MyD88 mRNAs. Specific TLRs were immunoco- localized in isolated crypts and in Paneth cells with a-defensin HD5 using anti-TLR-2 and TLR-4 antibodies and confocal microscopic analysis. Isolated intact crypts were exposed to I x l03 S. typhimurium CFU/crypt to induce Paneth cell secretion. Secretions were assayed for bactericidal activity against defensin sensitive S. typhimurium phoP- and for HD5 secretion by western blot analysis. Inhibition assay was performed using anti-TLR2 neutralizing anti- body in the secretion assay system Results: TLR1-8, TLR10, CD14 and MyD88, but not M02 mRNAs were detected in single isolated crypts and isolated Paneth cells. TLR2 and ]-LR4 expression was restricted to Paneth cells in the isolated crypts. TLR4 staining in single Paneth cells was localized at the membrane surface and also near secretory granules, a- Defensin secretion by Paneth cells in response to bacteria was inhibited by TLR2 antibody pre-treatment as judged by the diminished bactencidal activity and reduced secretion of HD5 in western blots. Conclusion: Paneth cells in human small intestinal crypts express Toll-like receptors that appear to be implicated in ex vivo responses to bacterial exposure.

358

[ipopolysaccharide Induces a Toll-Like 4 Independent Cross-Tolerance to Proinflammatory Cytokines in Human Intestinal Epithelial Cells Tor Savldge, Paul Newman, Wei-Hna Pan, Beth McCormick, W. Allan Walker, Andrea Quaroni

Intestinal epithelial cells (IEC) are constantly exposed to bacterial cell wall components such as LPS, without tnggering proinflammatory immune responses. This tolerance to LPS is due to the absence of a functional toll-like receptor (TLR) 4/MD-2 signaling complex in human IEC under normal conditions. We demonstrate here that chronic exposure of human-derived IEC (Caco-2, H4 and temperature-sensitive SV40-transformed FHI cells) to purified, protein- tree LPS induces a cross-tolerance to IDlb activated 1D8 production that occurs indepen- dently of TLR-4/MD-2 signaling in these cells. IL-8 production in response to activation by unrelated TNF-a and phorbol mynstate acetate (PMA) signaling pathways is also inhibited, indicating a broad-spanning cross-tolerance. Quantitative rtPCR and IL-8 promoter-luciferase assays demonstrate that cross-tolerance is regulated at the transcriptional level and occurs independently of IEC cytodifferentiation. By contrast, LPS does not significantly alter proin- flammatory signaling cascades in IEC that operate independently of IL-8 production, for e~mple the neutrophil transepithelial migration response induced by invasive Salmonella t),phimurium 1EC have therefore developed novel LPS-induced signaling cascades that promote a hyporesponsiveness to proinflammatory cytokines as well as to common bacterial components in the gastrointestinal tract, while on-the-other hand LPS exposure does not compromise the ability of IEC to mount proinflammatory immune responses to invasive enteropathogens. Supported by the Crohns and Colitis Foundation, and the National Insti- tutes of Health (DK33506; DK4-0561)

359

Regulated Production of CCL28 by Human Intestinal Epithelial Cells Hiroyuki Ogawa, Lars Eckmann, Martin F. Kagnoff

Human intestinal epithelial cells (IECs) secrete chemokines that regulate the trafficking of neutrophils and monocytes important in innate mucosal immunity. The CC chemokine CCL28, also termed mucosal epithelial chemokine (MEC), is a ligand for T cells and eosinophils that express the chemokine receptors CCR10 and CCR3, respectively. Human colon epithelial cells constitutively express CCL28, but little is known about its regulated expression under different conditions. The aim of these studies was to examine regulated CCL28 expression by human IECs. Methods. CCL28 mRNA expression in IEC lines (HCA- 7, Caco-2, HT-29, T84, 1-407, HCT-8) and in mature human colon and small intestinal xenografts transplanted in SCID mice was assayed by real-time RT-PCR. CCL28 protein expression in human intestinal biopsies and in xenografts was assessed by immunnstaining. CC/-28 production by cell lines was assayed by ELISA, ELISPOT assay, and immunostaming. BAY 11-7082 and ALLN were used to inhibit NF-KB activation. Results. IEC lines constitu- In'ely expressed CCL28 mRNA. CCL28 mRNA levels were upregulated 5-10 fold in IECs stimulated with lL-la and 20-25 fold in IECs infected with Salmonella dublin. Sodium buts'rate upregnLated CCL28 mRNA levels in IECs by -6-8 fold, and stimulation of IECs ~ath butytate and lL-la resulted in a synergistic ~100 fold increase in CCL28 mRNA levels. Increased agomst-stimuLated CCL28 mRNA levels in IECs were paralleled by increased CC1.28 protein production and secretion. Pharmacologic inhibitors of NF-KB activation almost completely inhibited IL-la-stimuLated upregulation of CCL2B, suggesting CCL28 hnctions as a NF-KB target gene. CCL28 mRNA was upregulated in human colon, but not in small intestinal xenografts, in response to 1L-la injection, whereas IL-8 expression was t~pregulated in both. This was accompanied by increased CCL28 immunostaining of IECs in colon, but not in small intestinal xenografts. Consistent with the studies in cell lines and xenografts, CCL28 was markedly increased in epithelial cells from inflamed compared to normal human colon, whereas little to no CCL28 was detected in normal or inflamed small intestine. Conclusion. The constitutive expression of CCL28 by human colon epithelial cells, but not by human small intestinal epithelium, and its upregnlated expression in response

to proinflammatory mediators, suggest a unique role for this chemokine in region-specific modulation of innate and adaptive immune responses in the intestinal tract. Supported by NIH grants DK35108 and DK58960.

360

A20 Is Required for The Negative Regulation of Lps-lndueed Nf-Kappab in Macrophages David L. Boone, Eric Lee, Sophia Chai, Paula Gibson, Jonathan Tam, Shon Libby, Justin Chang, Faye Chan, Marcia Chien, Averil Ma

The importance of innate immune cells, like macrophages, in the pathology of IBD has recently been highlighted by the discovery of NOD2 mutations associated with IBD. NOD2 regulates LPS signaling and is predominantly expressed in monocyte/macrophages. Thus, studies examining the regulation of LPS responses in macrophages are germane to understand- ing the pathology of IBD. We have previously reported that A20 is critical for the negative regulation of TNF-induced NF-kappaB in fibroblasts and T cells. In addition we have shown that A20-/- mice develop 1BD and die prematurely, and that this IBD can occur in A20-/- RAG-/- mice that have no lymphocytes. These results suggest that A20 plays critical roles in non-lymphocyte cells to prevent 1BD. IBD in A20-/- mice is characterized by markedly increased numbers of activated macrophages in the mucosa. We therefore examined whether A20 regulates LPS signaling in murine macrophages. Western analysis showed that bone marrow derived macrophages (BMDM) express A20 protein and that A20 is induced in BMDM following LPS treatment. In vivo, macrophages from ,420-/- mice had more LPS- induced TNF and IL-6 than wild-type (wt) macrophages. In response to LPS in vitro, A20-/- BMDM produced dramatically higher levels of nitric oxide, TNF and IL-6 than WT BMDM. Further, A20-/- macrophages had prolonged activation of NF-kappaB in response to LPS. Finally, l-kappaB kinase (IKK) activity was prolonged in LPS treated A20-/- macrophages. These results demonstrate that A20 is critical for the normal regulation of LFS-induced NF- kappaB in macrophages. These studies also suggest that one possible mechanism of IBD in A20-A mice involves the disrupted regulation of LPS responses in macrophages; a process that has been implicated in human IBD.

361

Multiple Colorectal Adenomas, Familial Adenomatous Polyposis and Germline Mutations in MYH Lara Lipton VI, Ohver M. Sieber, Michael Crabtree, Karl Heinimann, Paulo Fidalgo, Robin K. S. Phillips, Marie-Luise Bisgaard, Torben F. Ornoft, Lauri A. Aaltonen, Shirley u Hodgson, Ian P. M. Tomlinson, Huw J. W. Thomas

Background: Germline mutations in the base excision repair gene MYH have been associated with recessive inheritance ofmuhiple colorectal adenomas. Tumours from affected individuals displayed excess somatic G:C->T:A trarlsversions in the APC gene. Methods: We screened for germline MYH mutations in 152 patients with multiple (3 to 100) colorectal adenomas and 107 APC mutation-negative probands with classical familial adennmatous polyposis (>100 adenomas). Subsets were analysed for changes in the related genes, MTH1 and OGG1.. Adennmas were tested for somatic APC mutations. Results: Six multiple adenoma patients and eight polyposis patients harboured bi-allelic germline MYH variants. Missense and protein-truncating mutations were found, of which Y165C and G382D were the most frequent. Mutation spectra were very similar in the multiple adenoma and polyposis patient groups. In the tumours of bi-allefic mutation carriers, all somatic APC mutations were G:C- >T:A transversions. In the multiple adenoma group, about one third of patients with more than 15 adenomas had bi-allelic MYH mutations. In the polyposis patient group, no individual with bi-allelic MYH mutations had severe disease (>1000 adenomas), but three had extra- colonic disease (including upper-gastrointestinal tumours and congenital hypertrophy of the retinal pigment epithelium). No clearly pathogenic MTH1 or OGG1 mutations were identi- bed. Conclusions: Germlina MYH mutations predispose to a recessive, multiple adenorna or polyposis coli phenotype. For patients with about 15 or more colorectal adenomas - especially if no germline APC mutation has been identihed and family history is compatible with recessive inheritance - MYH genetic testing is indicated for diagnosis and calculating risks in relatives.

362

Prevalence & Risk Factors for Anal Carcinoma : Results of a Prospective Cohort Study Iradj Sobhani, Francine Walker, Dominique Heinn, Laurent Abramowitz, Thomas Aparido, Jean C. Soule

Background fir Aim. The incidence of anal cancer is increasing likely due to the augmentation of sexually-transmitted diseases (STD). Among these human papilloma vires (HPV) responsi- ble for anal condyloma, a pre-cancerons benign lesion and HW responsible for systemic and tissue immune cell alteration are considered as risk factors for anal carcinoma. In the present prospective cohort study we determined the prevalence of anal cancer in patients with anal canal HPV infection. Patients fir Methods. From Jan. 93 up to Dec. 02, 228 consecutive patients (164 HIV-positive, 64 Hlu with anal canal condyloma, were included after lesions were cured. Patients were then followed every 3-6 months. During follow up relapsed condylomas, it any, were similarly cured. At baseline and at relapses following parameters were charactenzed: Dendritic cells (DC), tissue lymphocytes and viruses (EBV, CMV, HSV1, various HPV types) in the anal mucosa, and LyCD4, HIV load in the serum. Anova, X2 and logistic regression tests were performed. Results. 60 (29%) patients were lost for follow up (less than 2 visits). The overall rate of invasive carcinoma (6 HIV + patients) was 2.6%. HGD was detected during the follow up in 15.3~ (35 patients of whom 29 HIV+). Condyloma relapses occurred more often (p<0.01) in HIV+ (1.4/year/ patient) than in HIV- (0.4/year/patient). H1V, 1-IGD and anal DC < 15/ram (normal cut off) at baseline were independent predictive risk factors for anal HGD and cancer. Among HIV infected patients, HIV load>10000 copies/ml in the serum was the main risk factor for

A-45 AGA Abstracts