- Email: [email protected]
CD47 binds to SIRP alpha-1 and facillitates neutrophil transepithelial migration
with omeprazole(400 umol/kg/dayi.p) for 2 months. Gastricepithelialand neuroendocrinecells were quantified by morphometry and flow cytometry. Exclusionof Helicobactercolonizationin mice was made by quantitativecultures 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 elevatedT cells. After 20 days of antibiotic treatment or G-17 infusion, elevatedG 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 increased3-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 absenceof gastric acid, non-Helicobacterpyloriorganismscolonize the stomach (seeabstract RiederG.) causing inflammationand metaplasia.Therefore,gastritis is not specific for Helicobacter spp., but rather is the natural response of the stomach to bacterial infection.
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) transepithelialmigration is a prominentfeature of the symptomatic 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 CD47and SIRP on the time course of fMLP-stimulated PMN transepithelial migration were studied using T84 intestinal epithelial cell monolayers cultured on permeablesupports. 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 noninhibitory 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 labelingand immunoprecipitationexperimentsdemonstratedan increaseof 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 associationwas also confirmed by co-immunoprecipitationfrom 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 CD47and 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.
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 understandthe molecular basis for these changeswe wantedto characterize the gene expressionprofiles 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. Genesthat were scored as increased/decreasedand had an averagefold changegreaterthan 2.3 from the wildtype were scored as differentially expressedand selectedfor further analysis. Differential expressionof key genes was confirmed using real time PCR, immunohistochemistry,western blotting or radioimmunoassay.ResultsThe 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 decarboxylaseboth known to he regulatedby gastrin was confirmed. Furthermore we found downregulationof 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 HBEGF, TGFalphaand the REG proteins unaffected by lack of gastrin or gastrin re-infusion. However, the expressionof gastric EGFwas reducedto one-sixth and the expressionof NOV (a member CCH gene-famUy) was reduced to a third in the gastrin knockout, but reached wildtype levelsafter gastrin infusion. Conclusion Despitethe profound changesin 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 regulatedin the gastdn knockout mice. The regulation of EGF and NOV expression by gastdn suggests that they rather than TGFalpha, HB-EGFor the REG proteins may be involved in promoting growth of the gastric mucosaduring hypergastrinemia.
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 chemokinereceptorfor the chemokinemacrophageinflammatory 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 CCR6is also expressedby human intestinalepithelialcell 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 expressionof 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 CCR6was 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 inducedcAMP production was preventedby CCR6densensitization and was sensitive to pertussis toxin, an inhibitor of signaling through Ga, proteins. Consistent with its role as a functional signaling receptor, CCR6activation 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 N11-1grants DK58960 and DK35108 and the CCFA.
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/gastrinreceptor was demonstratedto mediateenzyme release and more efficiently, protein synthesis. We now report results of phenotypic and long-term studies. The growth rate of the pancreasof ElasCCK2mice was increasedby 40 % after birth resulting in a mean40 % weight increasebetween40 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 ElasCCK2pancreaticexocrinetissue were obvious from the postnatalday 50: eosinophilic acinar cells loci that affected entire Iobulesat 9 months, anisocaryosis,cellular pleiomorphism and ductal metaplasia.Crossing with INS-GASmice that express gastrin in pancreaticfl cells resulted in developmentof morphologicalchangesin youngeranimals in relationwith enhanced activation of the CCK2/gastrin receptor. Malignant transformation occurred in two of twenty homozygous ElasCCK2mice at the age of 18 months, lmmunohistological characterization was performed to characterizethe phenotypeof these tumors. Tumor 1 was an osteoclastlike giant cell tumor. Duct-like cells present in the tumor were positive for cytokeratins and partially for amylase.Treatmentwith an anti-CCK2/gastrin receptor antibody showed intense staining of acinar cells in the peritumoral pancreatic region, decreaseof the staining in the transitional region where acini undergoing ductal metaplasiaand 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 areasof acinar tissue, developpedat the periphery of the pancreasthat was infiltrated with connectivetissue. 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 presentstudy clearly support a key role of the CCK2/gastrin receptor in the developmentof pre- and neoplastic lesions of the pancreasand is in favor of an acinar-ductal carcinoma sequence.
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 determinethe role of TAK1 and AKT in IL-1K, TNF and LPS signaling leading to NF-KB activation and proinflammatory geneexpressionin IEC.Methods: Dominantnegative IKKK,TAK1 and AKTgeneswere delivered in IEC-6and HT-29 cells by adenoviralvectors. Cellswere 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 phosphorylatedand unphosphorylatedIKBaprotein levels by Western blot. NFKB activation was determined by RelA immunofluorescence. ICAM-1, IL-6 and IL-8 gene expressionwas analyzedby RT-PCRand ELISA. Results: RelA nuclear localizationwas slower (30 min) in LPS than in IL-1 or TNF-stimulated IEC-6 cells. LPS-mediated RelA nuclear translocation was inhibited in Ad5dnAKTand Ad5dnTAKl-infected IEC-6 cells. Also, TNF and
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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) transepithelialmigration is a prominentfeature of the symptomatic 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 CD47and SIRP on the time course of fMLP-stimulated PMN transepithelial migration were studied using T84 intestinal epithelial cell monolayers cultured on permeablesupports. 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 noninhibitory 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 labelingand immunoprecipitationexperimentsdemonstratedan increaseof 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 associationwas also confirmed by co-immunoprecipitationfrom 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 CD47and 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.
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 understandthe molecular basis for these changeswe wantedto characterize the gene expressionprofiles 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. Genesthat were scored as increased/decreasedand had an averagefold changegreaterthan 2.3 from the wildtype were scored as differentially expressedand selectedfor further analysis. Differential expressionof key genes was confirmed using real time PCR, immunohistochemistry,western blotting or radioimmunoassay.ResultsThe 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 decarboxylaseboth known to he regulatedby gastrin was confirmed. Furthermore we found downregulationof 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 HBEGF, TGFalphaand the REG proteins unaffected by lack of gastrin or gastrin re-infusion. However, the expressionof gastric EGFwas reducedto one-sixth and the expressionof NOV (a member CCH gene-famUy) was reduced to a third in the gastrin knockout, but reached wildtype levelsafter gastrin infusion. Conclusion Despitethe profound changesin 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 regulatedin the gastdn knockout mice. The regulation of EGF and NOV expression by gastdn suggests that they rather than TGFalpha, HB-EGFor the REG proteins may be involved in promoting growth of the gastric mucosaduring hypergastrinemia.
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 chemokinereceptorfor the chemokinemacrophageinflammatory 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 CCR6is also expressedby human intestinalepithelialcell 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 expressionof 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 CCR6was 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 inducedcAMP production was preventedby CCR6densensitization and was sensitive to pertussis toxin, an inhibitor of signaling through Ga, proteins. Consistent with its role as a functional signaling receptor, CCR6activation 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 N11-1grants DK58960 and DK35108 and the CCFA.
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/gastrinreceptor was demonstratedto mediateenzyme release and more efficiently, protein synthesis. We now report results of phenotypic and long-term studies. The growth rate of the pancreasof ElasCCK2mice was increasedby 40 % after birth resulting in a mean40 % weight increasebetween40 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 ElasCCK2pancreaticexocrinetissue were obvious from the postnatalday 50: eosinophilic acinar cells loci that affected entire Iobulesat 9 months, anisocaryosis,cellular pleiomorphism and ductal metaplasia.Crossing with INS-GASmice that express gastrin in pancreaticfl cells resulted in developmentof morphologicalchangesin youngeranimals in relationwith enhanced activation of the CCK2/gastrin receptor. Malignant transformation occurred in two of twenty homozygous ElasCCK2mice at the age of 18 months, lmmunohistological characterization was performed to characterizethe phenotypeof these tumors. Tumor 1 was an osteoclastlike giant cell tumor. Duct-like cells present in the tumor were positive for cytokeratins and partially for amylase.Treatmentwith an anti-CCK2/gastrin receptor antibody showed intense staining of acinar cells in the peritumoral pancreatic region, decreaseof the staining in the transitional region where acini undergoing ductal metaplasiaand 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 areasof acinar tissue, developpedat the periphery of the pancreasthat was infiltrated with connectivetissue. 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 presentstudy clearly support a key role of the CCK2/gastrin receptor in the developmentof pre- and neoplastic lesions of the pancreasand is in favor of an acinar-ductal carcinoma sequence.
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 determinethe role of TAK1 and AKT in IL-1K, TNF and LPS signaling leading to NF-KB activation and proinflammatory geneexpressionin IEC.Methods: Dominantnegative IKKK,TAK1 and AKTgeneswere delivered in IEC-6and HT-29 cells by adenoviralvectors. Cellswere 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 phosphorylatedand unphosphorylatedIKBaprotein levels by Western blot. NFKB activation was determined by RelA immunofluorescence. ICAM-1, IL-6 and IL-8 gene expressionwas analyzedby RT-PCRand ELISA. Results: RelA nuclear localizationwas slower (30 min) in LPS than in IL-1 or TNF-stimulated IEC-6 cells. LPS-mediated RelA nuclear translocation was inhibited in Ad5dnAKTand Ad5dnTAKl-infected IEC-6 cells. Also, TNF and
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