with omeprazole (400 umol/kg/day i.p) for 2 months. Gastric epithelial and neuroendocrine cells were quantified by morphometry and flow cytometry. Exclusion of Helicobactercolonization in mice was made by quantitative cultures and biochemistry. Results: G - / - BIO mice showed significant inflammation and atrophy compared to the SPF animals. Both morphometdc and flow cytometric analysis of BIO mice showed a 3- and 2-fold increase in the number of G and parietal cells respectively. BIO G - / - animals also had elevated T cells. After 20 days of antibiotic treatment or G-17 infusion, elevated G cells decreased, and inflammation resolved. G+/+ mice showed no inflammation regardless of SPF or BID conditions. When G+/+ mice were treated with omeprazole, the lymphocyte population increased 3-fold over untreated mice. Significant inflammation was resolved after antibiotic therapy. Morphometdc analysis revealed a significant increase in G and parietal cell numbers, with a reciprocal decrease in D cells. Conclusions: In the absence of gastric acid, non-Helicobacterpyloriorganisms colonize the stomach (see abstract Rieder G.) causing inflammation and metaplasia. Therefore, gastritis is not specific for Helicobacter spp., but rather is the natural response of the stomach to bacterial infection.

954

Gene Chip Analysis of Gastric Gene Expression in Gastrin-deficient mice Lennart Friis-Hansen, Philip Bennett, Klaus Bendtzen, Klaus Rieneck, Rigshospitalet, Copenhagen Denmark

Aim Gestrin knockout mice have reduced acid secretion and maturation of parietal and ECL cells. In order to understand the molecular basis for these changes we wanted to characterize the gene expression profiles from gastrin knockout mice, wild type mice and gastrin knockout mice that have been given gastdn infusion. Method Stomachs were isolated from 10 female mice (8 weeks old) from each of the following groups: 1) Gastrin knockout, 2) wild type, and 3) gastrin knockouts that had been given continuous gastrin infusion (6 pmol/g/h) by subcutaneous implantation of osmotic minipumps. RNA was subsequently extracted from each stomach and equal amounts were pooled in groups of five. Expression profiling was done using the mouse expression Gene Chips MU74 subset A, from Affymetrix. Genes that were scored as increased/decreased and had an average fold change greater than 2.3 from the wildtype were scored as differentially expressed and selected for further analysis. Differential expression of key genes was confirmed using real time PCR, immunohistochemistry, western blotting or radioimmunoassay. Results The plasma gastrin concentration was O, 60-+10 and 810 _ 440 pmol/I in gastrin KO, wild type and treated gastdn KO mice, respectively. Gastdn mRNA was absent in both gastrin KO groups. The differential expression of Chromogranin A and histidine decarboxylase both known to he regulated by gastrin was confirmed. Further- more we found downregulation of two cytoskelstal proteins (ankyrin 3 and dystrobrevin), five protein involved in signal transduction and two transport proteins. However, none of the enzymes directly responsible for acid secretion were downregulated. The expression of HB- EGF, TGFalpha and the REG proteins unaffected by lack of gastrin or gastrin re-infusion. However, the expression of gastric EGF was reduced to one-sixth and the expression of NOV (a member CCH gene-famUy) was reduced to a third in the gastrin knockout, but reached wildtype levels after gastrin infusion. Conclusion Despite the profound changes in acid secretion and cellular function of the cells involved in acid secretion in the gastrin knockout mouse only few genes were found to he down regulated in the gastdn knockout mice. The regulation of EGF and NOV expression by gastdn suggests that they rather than TGFalpha, HB-EGF or the REG proteins may be involved in promoting growth of the gastric mucosa during hypergas- trinemia.

955

Overexpression of CCK2/Gastrin Receptors in the Murine Pancreas Results in Growth of the Pancreas, Transdifferentiation of Acinar Cells end Neoplasia Pascal Clerc, Stephaue Leung-Theung-long, Michete Bouisson, IHSERM U531, Toulouse France; Timothy C. Wang, Univ of MA Medical Ctr, Worcester, MA; Graham J. Dockray, Univ of Liverpool, Liverpool United Kingdom; Nicole Vaysse, Lucien Pradayrol, Daniel Fourmy, Marlene Dufresne, INSERM U531, Toulouse France

In order to explore the pancreatic physiopathology of CCK2/gastrin receptor, we created a transgenic mice strain (ElasCCK2) in which the human receptor is expressed in the exocrine pancreas. The transgenic CCK2/gastrin receptor was demonstrated to mediate enzyme release and more efficiently, protein synthesis. We now report results of phenotypic and long-term studies. The growth rate of the pancreas of ElasCCK2 mice was increased by 40 % after birth resulting in a mean 40 % weight increase between 40 and 110 days of age that was correlated to significant increases of 20 to nearly 50 % of the area of exocrine tissue. Abnormalities of the ElasCCK2 pancreatic exocrine tissue were obvious from the postnatal day 50: eosinophilic acinar cells loci that affected entire Iobules at 9 months, anisocaryosis, cellular pleiomorphism and ductal metaplasia. Crossing with INS-GAS mice that express gastrin in pancreatic fl cells resulted in development of morphological changes in youngeranimals in relation with enhanced activation of the CCK2/gastrin receptor. Malignant transformation occurred in two of twenty homozygous ElasCCK2 mice at the age of 18 months, lmmunohistological characterization was performed to characterize the phenotype of these tumors. Tumor 1 was an osteoclast- like giant cell tumor. Duct-like cells present in the tumor were positive for cytokeratins and partially for amylase. Treatment with an anti-CCK2/gastrin receptor antibody showed intense staining of acinar cells in the peritumoral pancreatic region, decrease of the staining in the transitional region where acini undergoing ductal metaplasia and duct-like structures were faintly stained. There was no CCK2/gastrin receptor in the tumor. Tumor 2 had a different phenotype. Nodules, composed of a stroma of pleiomorphic cells surrounding areas of acinar tissue, developped at the periphery of the pancreas that was infiltrated with connective tissue. Many tubular complexes, partially stained with the CCK2/gastdn receptor antibody, were present at the periphery of the tumor and their lumen was filled with mucus. We did not find K-ras mutations at codon 12. The present study clearly support a key role of the CCK2/gastrin receptor in the development of pre- and neoplastic lesions of the pancreas and is in favor of an acinar-ductal carcinoma sequence.

956

CD47 Binds to SIRP alpha -1 and Facilitates Neutrophil Transepithelial Migration Yuan Liu, Emory Univ Sch of Medicine, Atlanta, GA; Hans-Joerg Buehring, Univ of Tuebingen, Tuebingen Germany; Charles A. Parkos, Emory Univ Sch of Medicine, Atlanta, 6A

Background: Neutrophil (PMN) transepithelial migration is a prominent feature of the symptom- atic phase of inflammatory bowel disease (IBD). Studies on PMN transepithelial migration have implicated CD47 as a crucial component of the transmigration response. While the mechanism of CD47 function is not known, a recently identified putative ligand for CD47 is SIRP alpha-l, member of a family of cell surface proteins that signal through interaction with protein tyrosine phosphatase. The goal of this study was to better understand the role of CD47, and its interaction with SIRP in PMN transmigration. Methods: The effects of mAbs against CD47 and SIRP on the time course of fMLP-stimulated PMN transepithelial migration were studied using T84 intestinal epithelial cell monolayers cultured on permeable supports. Transmigration results were correlated with PMN cell surface CD47 and SIRP expression by flow cytometry and immunoprecipitation after cell surface biotinylation. Protein binding was assessed using recombinant CD47 and SIRPs consisting of the extracellular domains linked to alkaline phosphatase (AP) and GST, respectively. Results: Anti-CD47 mAbs caused a significant delay in fMLP-driven PMN migration across T84 monolayers compared to non- inhibitory control mAbs (migration commencing at 2hr vs 1-1.5hr, respectively). However, the total number of PMN that eventually migrated in both conditions were the same. SIRP mAbs also inhibited transmigration, but to a lesser extent (20-40% inhibition). Cell surface labeling and immunoprecipitation experiments demonstrated an increase of cell surface CD47 and SIRP on PMN after stimulation with fMLP and after transmigration. In-vitro binding assays demonstrated specific interaction of CD47 with SIRP alpha-l, but not SIRP beta-1 despite the fact that the extracellular domains of these two proteins are 90% identical. SIRP-CD47 association was also confirmed by co-immunoprecipitation from PMN cell lysates. Conclusions: i) Inhibition of CD47 results in a delay in the rate hut not the amount of PMN transmigration, it) SIRP mAbs partially inhibit PMN transmigration, iii) Both CD47 and SIRP are up-regulated to the PMN cell surface during fMLP-driven chemotaxis, iv} CD47 specifically binds to one particular member of the SIRP family, SIRP alpha-l. These results suggest that CD47- SIRP interactions play an important role in regulating PMN transepithelial migration. Understanding these events may provide ideas for new anti-inflammatory targets in IBD.

957

CCR6 Is A Functional Signal Transducing Receptor On Human Intestinal Epithelial Celts. Hiroyuki Ogawa, Michael B. Dwinell, Martin F. Kagfloff, Univ of CA, San Diego, La Jolla, CA

Background: CCR6, the sole chemokine receptor for the chemokine macrophage inflammatory protein-3~ (MIP-3~) (CCL20) and a putative receptor for human/3 defensin-2, is expressed on immature dendritic cells and CD45RO ÷ memory T cells. We have recently shown that CCR6 is also expressed by human intestinal epithelial cell lines and by human colonic epithelium in vivo. The present study asked whether CCR6 acts as a functional receptor on human intestinal epithelial cells, whether it is G-protein coupled as in myeloid cells, and whether expression of this receptor is strictly constitutive or can be regulated. Methods:These studies used the T84 and HT-29 human colon epithelial cell lines, cAMP was measured by enzyme immunoassay and protein tyrosine phosphorylation was assessed by immunoblot analysis. mRNA expression was assessed by RT-PCR. Results: CCR6 mRNA expression in T84 and HT-29 cells was upregulated by the cytokines interferon-~/or GM-CSF and, in contrast to regulation of it~ ligand MIP-3a, was not upregulated by the proinflammatory cytokines TNFc~ or IL-1. Chemokine receptors in myeloid cells often are coupled to G~-proteins, stimulation of which inhibits adenylyl cyclase production of the second messenger cAMP. To test if epithelial cell CCR6 was also linked to Ga,-proteins, T84 cells were stimulated with forskolin in the presence and absence of MIP-3a. Activation of CCR6 by MIP-3a resulted in marked inhibition of forskolin stimulated cAMP formation in a dose dependent manner. MIP-3a stimulated inhibition of forskolin induced cAMP production was prevented by CCR6 densensiti- zation and was sensitive to pertussis toxin, an inhibitor of signaling through Ga, proteins. Consistent with its role as a functional signaling receptor, CCR6 activation by MIP-3a resulted in altered tyrosine phosphorylation of intracellular proteins and the increased expression of ICAM-1 mRNA in T84 cells. Conc/usions: CCR6, the sole receptor for the proinflammatory chemokine MIP-3~, acts as a functional signal transducing receptor in colon epithelial cells and delivers signals to those cells through G proteins. Supported by N11-1 grants DK58960 and DK35108 and the CCFA.

958

Differential Requirement For Transtorming Growth Factor/~-Activated Kinase 1 (TAK1) And Protein Kinase B (AKT) In LPS, IL-1,8 And TNF-Mediated NF-KB Activation In Intestinal Epithelial Cells (IEC). Maria Pia Russo, Ryan Balfour Sartor, Christian Jobin, Univ of North Carolina, Chapel Hill, NC

Introduction: Cytokines and bacterial products utilize the IKB/NF-KB pathway to trigger an inflammatory response in multiple cell systems. The role of the kinases AKT and TAK1 in cytokine and LPS signaling in IEC is currently unknown. Aims: To determine the role of TAK1 and AKT in IL-1K, TNF and LPS signaling leading to NF-KB activation and proinflammatory gene expression in IEC. Methods: Dominant negative I KKK, TAK1 and AKT genes were delivered in IEC-6 and HT-29 cells by adenoviral vectors. Cells were infected with the various adenoviral vector (Ad5) and then stimulated for different times with IL-11~ and TNF (both at 2ng/ml) or LPS (5/~g/ml). The effect of IL-1/~, LPS and TNF on the IKB/NF-KB system was evaluated by measuring phosphorylated and unphosphorylated IKBaprotein levels by Western blot. NF- KB activation was determined by RelA immunofluorescence. ICAM-1, IL-6 and IL-8 gene expression was analyzed by RT-PCR and ELISA. Results: RelA nuclear localization was slower (30 min) in LPS than in IL-1 or TNF-stimulated IEC-6 cells. LPS-mediated RelA nuclear translocation was inhibited in Ad5dnAKT and Ad5dnTAKl-infected IEC-6 cells. Also, TNF and

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