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Regulated expression of Gb3 synthase and the shiga-like toxin (STX) receptor Gb3 in human colon epithelial cells
1685
(p=O.O3B) and of CD163 (p=0.014) and CD68 (p=0.026) in the sublining. Conclusions: Macrnphagesexpressingthe scavengerreceptorCD163 are increasedin synovium and colonic mucosa in SpA, highlighting the relation betweenjoint and gut. The correlation with inflammatory parameters, the HI.A-DR expression, the production of TNF-e but not IL-IO and the reduction by anti-TNF-a therapy support a role for CD163 in SpA. The increase of CD163+ macrophagesin CD and SpA colon but not in UC provides a new argument for the hypothesis that SpA can be considered as a human model to study early changes in the development of CD.
Prolonged Exposure of Lipopolysaccharide Induces Intestinal Epithelial Tolerance to Endotoxin via Downregulation of the TolHike Receptor Signaling Pathway. Elke Carlo, Daniel K. Podolsky, GastrointestinalUnit, MGH, CSIBD, Harvard Medical Sch, Boston, MA Background: Lipopolysaccharide(LPS) is a potent toxin which may elicit several immediate pro- and anti-inflammatory responsesof the innate and adaptive immune system. We have recently demonstratedthat intestinal epithelial cells may respond to singular LPS-stimulation resulting in activation of p42/p44 Mapk and NF-KBvia ToIHike Receptors(TLRs) (J. Immunol. 2000 164:966-972). However, as frontline of the mucosal immune system, the intestinal epithelium is constantly exposed to high amounts of lumenal LPS in-vivo. The aim of this study was to investigate whether IEC may render "tolerant" to sequential LPS stimulation through prolonged exposure leading to attenuation of inflammatory stress responsesvia the TLR-signaling pathway. Material & Methods: LPS-induced activation of the TLR-signaling pathway (TLR2, TLR3, TLR4, p42/p44 Mapk, NF-KB) was assessed by immunoblotting and transfection studies in differentiated and non-differentiated human IEC (Caco-2, HT29, T84). Results: IEC pro-exposedto LPS (lO/~g/ml, 6-24 hours) exhibited reduced activation of p42/ p44 Mapkwhen exposedto secondstimulation with LPSwhich was time- and dose-dependent In addition, activation of NF-KB was not observed in IEC suggesting that IEC may become tolerant by prolonged exposure to LPS. Induction of tolerance was dependent on state of differentiation of IECs. However, suppression of the LPS-signaling pathway in tolerant IEC did not modulate TLR2,3 and 4 expression, suggesting that the mechanism of tolerance induction may be located downstream from TLRs. Conclusions: The present study suggests that the intestinal epithelium may becometolerant to lumenal LPS following prolonged exposure. Further studies are needed to determine whether LPS-tolerant IEC are still able to respond to serious non-LPS mediated bacterial challenges - via intact TLRs.
1688 Glutamine Prevents TNF~-induced Bacterial Translocation by Acting as a Specific Metabolic Fuel, Edwin C. Clark, Sanjay Patel, Univ of Manchester, Manchester United Kingdom; Paul R. Chadwick, Hope Hosp, Salford United Kingdom; Geoff Warhurst, Univ of Manchester, Manchester United Kingdom; Gordon L. Carlson, Hope Hosp, Salford United Kingdom AIMS: Glutamine may reduce septic morbidity in critical illness but the mechanisms are unclear. The aim of this study was to test the hypothesisthat glutamine availability influences bacterial translocation across intestinal epithelia and to provide an understanding of the underlying cellular mechanisms. METHODS:Human derived intestinal epithelial cells (Caco2) were grown as confluent polarised monolayers in a bicameral system Transwell in DMEM containing 2mU glutamine. Cells were then incubated with TNFa (20ng/ml) for 6 hr, with (n = 6) or without (n = 6) prior removal of glutamine. Reversibilityand specificity of glutamine effects were assessed by replacing glutamine in the culture for 2 hr (n=6) or adding an isonitrogenousmixture of non-essentialamino acids (n = 6). Finallycells in glutaminecontaining medium were incubated with and without TNF,~ (20ng/ml) for 6 hr in the presence of 0.CraM aminooxyacetate,an inhibitor of glutamine oxidation (n=B), or 3.2 mM buthionine sulfoximine, an inhibitor of glutamine conversion to glutathione (n=8). In each study the ability of a 108CFU inoculum of E. Coil C25 to translocate across the epithelium from apical to basal chambers after 4 hr incubation was measured by Miles and Misra serial dilution assay. All results are expressedas mean _+ SD and comparison between groups made with Student's t-test. RESULTS: Glutamine deprivation or incubation with TNFc~alone had no significant effect on bacterial translocation vs control, (3.6 -+ 0.4 & 3.7 ± 0.5 vs 3.4 _+ 0.3 log CFU/ml respectively, P = NS). However, glutamine deprivation significantly increased bacterial translocation in the presenceof TNF~ vs control (5.5 -+ 0.4 vs 3.3 +- 0.3 log CFU/ ml, P
1686 Regulated Expression of Gb3 Synthase and the Shiga-like Toxin (STX) Receptor Gb3 in Human Colon Epithelial Cells. Ariette Darfeuille-Michaud, Michael P. Housley, Martin F. Kagnoff, Univ of CA, San Diego, La Jolla, CA
Background: Enterohemorrhagic £ coli(EHEC) 0157:117 is a major cause of hemorrhagic colitis and hemolytic-uremic syndrome. EHECcolonize the human colon and produce Stx's. The Six B subunits bind a globotriaosylceramidereceptor, Gb3, that is mainly expressedby endothelialcells and renalglomerular epithelium whereasthe A subunit inhibits cellular protein synthesis. Little is known about Gb3 expression by human colon epithelial cells and the role Six plays in the pathogenesisof intestinal epithelial cell infection by Stx-producing EHEC. Methods: Caco-2 colon epithelial cells were infected with a wild-type Stx-2-producing E. coil 0157:H7 strain or a nontoxic isogenic mutant with a mutation in the active site of the Stx-2 A subunit. Responseswere studied in control cells or in cells treated with 5 mM sodium butyrate (NAB) or proinflammatory cytokines (TNFa, IL-I~, IFNx) before bacterial infection. Bacterial adherencewas assessed using colony counts. Gb3 synthasemRNA was determined by real time RT-PCR.Cell surface Gb3 was assessedby flow cytometry. Chemokinesecretion was measuredby ELISA.Results:Adherenceof wild-type EHECto Caco-2cells was significantly increased relative to the nontoxic isogenic mutant. Moreover, wild type EHEC induced 18fold more IL-8 production than the mutant. Gb3 mRNA levels, surface Gb3 expression, and bacterial adherencewere markedly increased in Caco-2 cells treated with NaB, an inducer of histone hyeracetylationand cellular differentiation, Stimulation of Caco-2 cells with TNFa, IL1,~ and IFN~,also increased Gb3 synthase mRNA levels and surface Gb3 expression,but did not alter bacterialadhesion. IL-8 responsesafter infection were markedlyincreasedin butyrate treated compared to untreated colon epithelial cells. Conclusion: Short chain fatty acids normally present in human colon and proinflammatory cytokines produced during colon inflammation regulateGb3 synthaseand surface expressionof the Gb3 receptorfor Stx. EHEC that produce functional Stx manifest increasedadhesion to colon epithelial cells and induce increased production of epithelial cell proinflammatory mediators. Supported by NIH grants DK58960 and DKB510B.
1689 Aotin Filament-Dependent Modulation of Translocated RhoA, PKC a and Association with HSP27 in the Padiculate Fraction of Smooth Muscle Cells Isolated from the Rabbit Colon. Khalil N. Bitar, Mercy Pawar, PeedikayilE. Thomas, Univ of Michigan, Ann Arbor, MI BACKGROUND:Actin is a ubiquitous cellular protein essential for the motility of nonmuscle and muscle cells. Disruption of actin filaments by cytochalasins in nonmusclecells attenuates cellular processes. OBJECTIVE:We have hypothesizedthat actin filaments may be involved in receptor-mediatedsignal transduction in colonic smooth muscle cells. We have previously shown that agonist induced contraction is associatedwith translocation of PKCa and of RhoA to the membrane.We haveinvestigated:1) the possible involvementof actin and its association with translocated PKC • and with translocated RhoA in the particulate fraction, and 2) the role of phosphorylated Hsp27 in maintaining and modulating this functional association. METHODS: Smooth muscle cells were isolated from the circular muscle of the rabbit colon. After stimulation with acetylcholine(107M), the particulatefractions were isolatedand immunoprecipitated with monoclonal antibodies to either of the following: PKCa, RhOA,actin and Hsp27. RESULTS:Immunoprecipitation with actin monoclonal antibody followed by immunoblotting with either RhoA antibody or with PKCe antibody indicated that acetylcholine (107M) induced an immunocomplexing of actin-RhoA in the particulate fraction, and an immunocomplexing of actin-PKCa in the particulate fraction. Similarly immunoprecipitation of the particulate fraction with either anti-RhoA antibody or anti-PKC a antibody followed by immunobtothng with anti-actin antibody indicated that acetylcholine (107M) induced an association of the translocated RhoA and of translocated PKC a with actin in the particulate fraction. Preincubating the cells with cytochalasin for 20 rain inhibited this association. Immunoprecipitationwith Hsp27 monoclonalantibody followed by immunoblotting with either RhoA antibody, or with PKCa antibody or with actin antibody indicated that acetylcholine induced an immunocomplexing of Hsp27-RhoA, of Hsp27-PKC a and of Hsp27-actin in the particulate fraction. CONCLUSION: The data indicate that agonist induced contraction is associated with: 1) Transiocation of PKC a and RhoA to the membrane. 2) Association of the translocated and activated PKC a and RhoA with actin in the cytoskeleton,3) Association of activatedand translocated PKC a and RhoA with HSP27 in the particulate fraction and 4) the formation of a dynamic complex in the particulate fraction between HSP27-ACtin- PKC a RhOA.
1687 Macrophages Expressing the Scavenger Receptor CD163: A Link Between Got and Synovial Inflammation in Spondyloarthropathy. Pieter R. Demetter, Pathology, Ghent Univ Hosp, Gent Belgium; Dominique Baeten, Filip De Keyser, Elli Kruithof, Nancy Van Damme, Rheumatology,Ghent Univ Hosp, Gent Belgium; Martine De Vos, Gastroenterology,Ghent Univ Hosp, Gent Belgium; Eric M. Veys, Rheumatology, Ghent Univ Hosp, Gent Belgium; Claude A. Cuvsiier, Pathology, Ghent Univ Hosp, Gent Belgium Objectives:To investigatethe presence,phenotypeand role of synovial and gut macrophages in spondyloarthropathy(SPA), a human model to study early changes in the developmentof Crohn's disease.Methods: Immunohistochemicalstainingsfor macrophagemarkers, including CD14, CD68, CD163 and HI_A-DR,and dendritic cell markers, including CDla, CD80, CD83 and CD 86, were performed on synovial biopsies of 17 SpA and 18 rheumatoid arthritis (RA) patients, on colonic biopsies of 10 SpA, 8 Crohn's disease (CD), 11 ulcerative colitis (UC) patients and 12 healthy controls, and on paired synovial biopsies of 8 SpA patients before and after anti-TBF-~ therapy. Slides were scored semiquantitativelyon a 4-point scale by two independent observers, who were blinded for diagnosis. The CD163+ macrophages were further analyzedby flow cytometry. Results: In the synovial lining CD68 (p=0.048), CD163 (p = 0.014) and HLA-DR (p = 0.009) were increasedin SpA versus RA; in the sublining CD163 (p =0.037) and HI.A-DR (p=O,OO7) were also increased. Interestingly, both CD (p=O.018) and colon of SpA patients (p = 0.005), but not UC, showed increased numbers of activated macrophages, characterizedby the expression of the scavengerreceptor CD163. CD163 and HLA-DR expression in the synovial sublining were significantly correlated with CRP and sedimentation rate. CD163" macrophages expressed high levels of HI_A-DRand produced TNF-~ but not IL-lO. Finally, anti-TNF-a induced a decrease of CD163 in the synovial lining
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(p=O.O3B) and of CD163 (p=0.014) and CD68 (p=0.026) in the sublining. Conclusions: Macrnphagesexpressingthe scavengerreceptorCD163 are increasedin synovium and colonic mucosa in SpA, highlighting the relation betweenjoint and gut. The correlation with inflammatory parameters, the HI.A-DR expression, the production of TNF-e but not IL-IO and the reduction by anti-TNF-a therapy support a role for CD163 in SpA. The increase of CD163+ macrophagesin CD and SpA colon but not in UC provides a new argument for the hypothesis that SpA can be considered as a human model to study early changes in the development of CD.
Prolonged Exposure of Lipopolysaccharide Induces Intestinal Epithelial Tolerance to Endotoxin via Downregulation of the TolHike Receptor Signaling Pathway. Elke Carlo, Daniel K. Podolsky, GastrointestinalUnit, MGH, CSIBD, Harvard Medical Sch, Boston, MA Background: Lipopolysaccharide(LPS) is a potent toxin which may elicit several immediate pro- and anti-inflammatory responsesof the innate and adaptive immune system. We have recently demonstratedthat intestinal epithelial cells may respond to singular LPS-stimulation resulting in activation of p42/p44 Mapk and NF-KBvia ToIHike Receptors(TLRs) (J. Immunol. 2000 164:966-972). However, as frontline of the mucosal immune system, the intestinal epithelium is constantly exposed to high amounts of lumenal LPS in-vivo. The aim of this study was to investigate whether IEC may render "tolerant" to sequential LPS stimulation through prolonged exposure leading to attenuation of inflammatory stress responsesvia the TLR-signaling pathway. Material & Methods: LPS-induced activation of the TLR-signaling pathway (TLR2, TLR3, TLR4, p42/p44 Mapk, NF-KB) was assessed by immunoblotting and transfection studies in differentiated and non-differentiated human IEC (Caco-2, HT29, T84). Results: IEC pro-exposedto LPS (lO/~g/ml, 6-24 hours) exhibited reduced activation of p42/ p44 Mapkwhen exposedto secondstimulation with LPSwhich was time- and dose-dependent In addition, activation of NF-KB was not observed in IEC suggesting that IEC may become tolerant by prolonged exposure to LPS. Induction of tolerance was dependent on state of differentiation of IECs. However, suppression of the LPS-signaling pathway in tolerant IEC did not modulate TLR2,3 and 4 expression, suggesting that the mechanism of tolerance induction may be located downstream from TLRs. Conclusions: The present study suggests that the intestinal epithelium may becometolerant to lumenal LPS following prolonged exposure. Further studies are needed to determine whether LPS-tolerant IEC are still able to respond to serious non-LPS mediated bacterial challenges - via intact TLRs.
1688 Glutamine Prevents TNF~-induced Bacterial Translocation by Acting as a Specific Metabolic Fuel, Edwin C. Clark, Sanjay Patel, Univ of Manchester, Manchester United Kingdom; Paul R. Chadwick, Hope Hosp, Salford United Kingdom; Geoff Warhurst, Univ of Manchester, Manchester United Kingdom; Gordon L. Carlson, Hope Hosp, Salford United Kingdom AIMS: Glutamine may reduce septic morbidity in critical illness but the mechanisms are unclear. The aim of this study was to test the hypothesisthat glutamine availability influences bacterial translocation across intestinal epithelia and to provide an understanding of the underlying cellular mechanisms. METHODS:Human derived intestinal epithelial cells (Caco2) were grown as confluent polarised monolayers in a bicameral system Transwell in DMEM containing 2mU glutamine. Cells were then incubated with TNFa (20ng/ml) for 6 hr, with (n = 6) or without (n = 6) prior removal of glutamine. Reversibilityand specificity of glutamine effects were assessed by replacing glutamine in the culture for 2 hr (n=6) or adding an isonitrogenousmixture of non-essentialamino acids (n = 6). Finallycells in glutaminecontaining medium were incubated with and without TNF,~ (20ng/ml) for 6 hr in the presence of 0.CraM aminooxyacetate,an inhibitor of glutamine oxidation (n=B), or 3.2 mM buthionine sulfoximine, an inhibitor of glutamine conversion to glutathione (n=8). In each study the ability of a 108CFU inoculum of E. Coil C25 to translocate across the epithelium from apical to basal chambers after 4 hr incubation was measured by Miles and Misra serial dilution assay. All results are expressedas mean _+ SD and comparison between groups made with Student's t-test. RESULTS: Glutamine deprivation or incubation with TNFc~alone had no significant effect on bacterial translocation vs control, (3.6 -+ 0.4 & 3.7 ± 0.5 vs 3.4 _+ 0.3 log CFU/ml respectively, P = NS). However, glutamine deprivation significantly increased bacterial translocation in the presenceof TNF~ vs control (5.5 -+ 0.4 vs 3.3 +- 0.3 log CFU/ ml, P
1686 Regulated Expression of Gb3 Synthase and the Shiga-like Toxin (STX) Receptor Gb3 in Human Colon Epithelial Cells. Ariette Darfeuille-Michaud, Michael P. Housley, Martin F. Kagnoff, Univ of CA, San Diego, La Jolla, CA
Background: Enterohemorrhagic £ coli(EHEC) 0157:117 is a major cause of hemorrhagic colitis and hemolytic-uremic syndrome. EHECcolonize the human colon and produce Stx's. The Six B subunits bind a globotriaosylceramidereceptor, Gb3, that is mainly expressedby endothelialcells and renalglomerular epithelium whereasthe A subunit inhibits cellular protein synthesis. Little is known about Gb3 expression by human colon epithelial cells and the role Six plays in the pathogenesisof intestinal epithelial cell infection by Stx-producing EHEC. Methods: Caco-2 colon epithelial cells were infected with a wild-type Stx-2-producing E. coil 0157:H7 strain or a nontoxic isogenic mutant with a mutation in the active site of the Stx-2 A subunit. Responseswere studied in control cells or in cells treated with 5 mM sodium butyrate (NAB) or proinflammatory cytokines (TNFa, IL-I~, IFNx) before bacterial infection. Bacterial adherencewas assessed using colony counts. Gb3 synthasemRNA was determined by real time RT-PCR.Cell surface Gb3 was assessedby flow cytometry. Chemokinesecretion was measuredby ELISA.Results:Adherenceof wild-type EHECto Caco-2cells was significantly increased relative to the nontoxic isogenic mutant. Moreover, wild type EHEC induced 18fold more IL-8 production than the mutant. Gb3 mRNA levels, surface Gb3 expression, and bacterial adherencewere markedly increased in Caco-2 cells treated with NaB, an inducer of histone hyeracetylationand cellular differentiation, Stimulation of Caco-2 cells with TNFa, IL1,~ and IFN~,also increased Gb3 synthase mRNA levels and surface Gb3 expression,but did not alter bacterialadhesion. IL-8 responsesafter infection were markedlyincreasedin butyrate treated compared to untreated colon epithelial cells. Conclusion: Short chain fatty acids normally present in human colon and proinflammatory cytokines produced during colon inflammation regulateGb3 synthaseand surface expressionof the Gb3 receptorfor Stx. EHEC that produce functional Stx manifest increasedadhesion to colon epithelial cells and induce increased production of epithelial cell proinflammatory mediators. Supported by NIH grants DK58960 and DKB510B.
1689 Aotin Filament-Dependent Modulation of Translocated RhoA, PKC a and Association with HSP27 in the Padiculate Fraction of Smooth Muscle Cells Isolated from the Rabbit Colon. Khalil N. Bitar, Mercy Pawar, PeedikayilE. Thomas, Univ of Michigan, Ann Arbor, MI BACKGROUND:Actin is a ubiquitous cellular protein essential for the motility of nonmuscle and muscle cells. Disruption of actin filaments by cytochalasins in nonmusclecells attenuates cellular processes. OBJECTIVE:We have hypothesizedthat actin filaments may be involved in receptor-mediatedsignal transduction in colonic smooth muscle cells. We have previously shown that agonist induced contraction is associatedwith translocation of PKCa and of RhoA to the membrane.We haveinvestigated:1) the possible involvementof actin and its association with translocated PKC • and with translocated RhoA in the particulate fraction, and 2) the role of phosphorylated Hsp27 in maintaining and modulating this functional association. METHODS: Smooth muscle cells were isolated from the circular muscle of the rabbit colon. After stimulation with acetylcholine(107M), the particulatefractions were isolatedand immunoprecipitated with monoclonal antibodies to either of the following: PKCa, RhOA,actin and Hsp27. RESULTS:Immunoprecipitation with actin monoclonal antibody followed by immunoblotting with either RhoA antibody or with PKCe antibody indicated that acetylcholine (107M) induced an immunocomplexing of actin-RhoA in the particulate fraction, and an immunocomplexing of actin-PKCa in the particulate fraction. Similarly immunoprecipitation of the particulate fraction with either anti-RhoA antibody or anti-PKC a antibody followed by immunobtothng with anti-actin antibody indicated that acetylcholine (107M) induced an association of the translocated RhoA and of translocated PKC a with actin in the particulate fraction. Preincubating the cells with cytochalasin for 20 rain inhibited this association. Immunoprecipitationwith Hsp27 monoclonalantibody followed by immunoblotting with either RhoA antibody, or with PKCa antibody or with actin antibody indicated that acetylcholine induced an immunocomplexing of Hsp27-RhoA, of Hsp27-PKC a and of Hsp27-actin in the particulate fraction. CONCLUSION: The data indicate that agonist induced contraction is associated with: 1) Transiocation of PKC a and RhoA to the membrane. 2) Association of the translocated and activated PKC a and RhoA with actin in the cytoskeleton,3) Association of activatedand translocated PKC a and RhoA with HSP27 in the particulate fraction and 4) the formation of a dynamic complex in the particulate fraction between HSP27-ACtin- PKC a RhOA.
1687 Macrophages Expressing the Scavenger Receptor CD163: A Link Between Got and Synovial Inflammation in Spondyloarthropathy. Pieter R. Demetter, Pathology, Ghent Univ Hosp, Gent Belgium; Dominique Baeten, Filip De Keyser, Elli Kruithof, Nancy Van Damme, Rheumatology,Ghent Univ Hosp, Gent Belgium; Martine De Vos, Gastroenterology,Ghent Univ Hosp, Gent Belgium; Eric M. Veys, Rheumatology, Ghent Univ Hosp, Gent Belgium; Claude A. Cuvsiier, Pathology, Ghent Univ Hosp, Gent Belgium Objectives:To investigatethe presence,phenotypeand role of synovial and gut macrophages in spondyloarthropathy(SPA), a human model to study early changes in the developmentof Crohn's disease.Methods: Immunohistochemicalstainingsfor macrophagemarkers, including CD14, CD68, CD163 and HI_A-DR,and dendritic cell markers, including CDla, CD80, CD83 and CD 86, were performed on synovial biopsies of 17 SpA and 18 rheumatoid arthritis (RA) patients, on colonic biopsies of 10 SpA, 8 Crohn's disease (CD), 11 ulcerative colitis (UC) patients and 12 healthy controls, and on paired synovial biopsies of 8 SpA patients before and after anti-TBF-~ therapy. Slides were scored semiquantitativelyon a 4-point scale by two independent observers, who were blinded for diagnosis. The CD163+ macrophages were further analyzedby flow cytometry. Results: In the synovial lining CD68 (p=0.048), CD163 (p = 0.014) and HLA-DR (p = 0.009) were increasedin SpA versus RA; in the sublining CD163 (p =0.037) and HI.A-DR (p=O,OO7) were also increased. Interestingly, both CD (p=O.018) and colon of SpA patients (p = 0.005), but not UC, showed increased numbers of activated macrophages, characterizedby the expression of the scavengerreceptor CD163. CD163 and HLA-DR expression in the synovial sublining were significantly correlated with CRP and sedimentation rate. CD163" macrophages expressed high levels of HI_A-DRand produced TNF-~ but not IL-lO. Finally, anti-TNF-a induced a decrease of CD163 in the synovial lining
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