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M1687 Ecto-Phosphorylation of CD98 Regulates Lymphocytes-Epithelia Interactions
Increased Macromolecular Permeability in Non-Inflamed Colon of Ulcerative Colitis Involves Muscarinic Receptor Signalling Via Subepithelial Eosinophils Conny Wallon, Ann-Charlott Ericson, Ping-Chang Yang, Derek M. McKay, Philip M. Sherman, Mary H. Perdue, Johan D. Soderholm Background and aim: Increased intestinal permeability has been shown in patients with UC In animal studies and studies in human volunteers, uptake of protein antigens can be regulated by cholinergic signalling pathways, eosinophils and mast cells. Our aim was to elucidate macromolecular permeability and ion secretion in macroscopically non-inflamed colon in UC. Materials and methods: Biopsies from fifteen UC patients in remission and fifteen healthy volunteers were studied in Ussing chambers for macromolecular permeability (horseradish peroxidase, HRP, 51Cr-EDTA), and electrophysiology during modulation of muscarinic receptors and mast cell stabilizer. The biopsies were examined by electron and light microscopy for eosinophils and mast cells in relation to cholinergic receptor localization. Results: Permeability to HRP was increased in UC patients (2.28 ± 0.20 pmol/cm2/h) compared to controls (0.99 ± 0.19). The elevated level of HRP uptake in UC was normalized by pretreatment with atropine (0.76 ± 0.26)or the mast cell stabilizer lodoxamide tromethamine (1.16 ± 0.28). Immunohistochemistry showed increased numbers of lamina propria eosinophils expressing muscarinic receptor subtypes M2 and M3 in the UC group, whereas mast cells did not express muscarinic receptors. Electron microscopy showed signs of activation (degranulation) of eosinophils and mast cells upon exposure to the acteylcholine analogue, carbachol. Conclusions: Increased transmucosal uptake of protein antigens in the non-inflamed colon of UC involves cholinergic signalling and activation of mast cells. Subepithelial eosinophils expressing muscarinic receptors may be involved in regulating this process.
M1686 CXCR6 Is Upregulated in Effector Memory CD4+ T Cells Infiltrating Into Chronically Inflamed Colonic Tissue Yasushi Mandai, Koji Hase, Toru Sato, Daisuke Takahashi, Masashi Ebisawa, Tatsuro Katsuno, Yasushi Saito, Hiroshi Ohno Background & Aim. The recently identified CXCL16 is a unique chemokine with dual functions: A transmembrane adhesion molecule and a soluble chemokine. In the present study, we investigated the role of CXCL16 and its receptor CXCR6 in the development of inflammatory bowel disease (IBD). Methods. A murine experimental colitis model was induced by adoptive transfer of CD4+CD45Rbhi T cells from wild-type mice into Rag-1-/mice. CXCL16 mRNA and protein expressions in the colonic tissue of the murine colitis model as well as human IBD patients were analyzed by quantitative RT-PCR and immunohistochemistry (IHC), respectively. Surface markers of T cells prepared from colonic lamina propria were analyzed by flow cytometry. Results. We found a significant increase in CXCL16 expression in inflamed intestinal mucosa of the murine colitis model as well as human patients with active IBD, namely Crohn's disease and ulcerative colitis. IHC analysis revealed that CXCL16 was detected in epithelial cells and a subset of immune cells, possibly activated dendritic cells (DCs). Likewise, treatment of HT-29 human colonic epithelial cell line with proinflammatory cytokines TNF-alpha and IFN-gamma upregulated CXCL16. FACS analysis showed that nearly 80% of colonic laminal propria CD4+ T cells upregulated CXCR6 in the murine colitis model. These CXCR6+CD4+ T cells showed CD62LloCD44hi effector memory phenotype, and highly produced IL-17 and IFN-gamma, whereas the cytokine production by CXCR6- population was minimal. Conclusions. This study suggests that CXCR6 could be a marker of colitogenic effector memory T cells. CXCL16 expressed by epithelium and DCs may play an important role in migration to and retention in the colonic tissue of colitogenic T cells. Our finding raises the possibility that disrupting the CXCL16CXCR6 interaction could serve as a promising therapeutic approach for IBD.
M1684 The Roles of p53 and Death Receptor-5 in Augmenting Activated T-Cell Induced Apoptosis Edwin K. McDonald, Ramanarao Dirisina, Niklas Finnberg, Meghan Barrett, Tatiana Goretsky, Jennet Manjali, Mutazz Darweesh, Jeffrey Brown, Wafik S. El-Deiry, Terrence A. Barrett Background/Aims: Epithelial cell apoptosis is important in the pathogenesis of mucosal injury in T-cell induced inflammatory disorders of the intestine. It has been shown previously that radiation-induced epithelial cell apoptosis requires p53 and Death Receptor-5 (DR5). DR5 is an extrinsic mediator of apoptosis and is induced by p53. Here we address the role of p53 and DR5 in T-cell mediated epithelial cell apoptosis using the anti-CD3 model of systemic T-cell activation. Methods: C57Bl/6, B6/p53-/-, and B6/DR5-/- mice were sacrificed 24 hours after activating T-cells with .2mg anti-CD3 mAb i.p. Alterations in intestinal morphology were assessed and apoptosis was detected by TUNEL and active caspase-3 staining. p53, caspase-3, caspase-8, and caspase-9 levels were assessed by Western blots from 12 hours. p53 was also detected by IHC. Results: Activation of T-cells induced an intestinal enteropathy with morphologic changes including mucosal flattening, villous blunting, and paneth cell hyperplasia in both C57Bl/6 and DR5-/- mice. Villous flattening was more pronounced in the wild type strains. In B6 mice, p53 was induced 3.4-fold in isolated crypt epithelial cells (leukocyte depleted) by Western blot and localized to lower crypt enterocytes by IHC. There was no significant difference in p53 levels between wild type and DR5-/- by IHC. There was a 63-fold increase in TUNEL staining in lower crypt in B6 mice after anti-CD3. TUNEL in lower crypt correlated with an 170-fold and 50-fold induction of cleaved caspase-3 and 9 respectively, but not 8 (Western blot). TUNEL and caspase-3 staining were reduced by 85% and 76% respectively in p53-/- mice and by 41% and 20% in DR5-/- respectively. Conclusions: The reduction of T-cell induced apoptosis in p53-/and DR5-/- mice is consistent with the notion that DR5 contributes to p53-mediated crypt cell apoptosis. The induction of caspase-3 and 9 but not 8 indicates that p53-mediated DR5-induced apoptosis operates through the intrinsic (mitochondrial) pathway. As the largest effects on apoptosis (ie reduction) were seen in cells of the lower crypt in both p53-/and DR5-/- mice, we speculate that these mechanisms operate in progenitor populations of enterocytes responding to a potent immune stimulus. These results also suggest that inhibition of DR5 in colitis may reduce epithelial apoptosis and aide restitution of barrier function following injury.
M1687 Ecto-Phosphorylation of CD98 Regulates Lymphocytes-Epithelia Interactions Hang Thi Thu Nguyen, Yutao Yan, Guillaume Dalmasso, Laetitia Charrier-Hisammudin, Shanthi V. Sitaraman, Didier Merlin Background and Aims: Ecto-phosphorylation has been shown to play an important role in several cellular functions. Many membrane proteins and soluble extracellular protein can be phosphorylated by ecto-protein kinases (ePK) using extracellular ATP as a phophoryl donor. CD98, a type II transmembrane glycoprotein, is a heavy chain of the family of heterodimeric amino acid transporter. Since CD98 contains predicted phosphorylation sites, our aim was to investigate the ecto-phosphorylation of CD98 by ePK and its functional consequences. Results: Recombinant CD98 is phosphorylated by purified casein kinase 2 (CK2) and by ePK prepared from Jurkat T cells. Km and Vmax for the phosphorylation of CD98 by CK2 are 0.84 µM and 8.7e+6 µmol/min, respectively. In human intestinal epithelial Caco2-BBE cells, CD98 is also ecto-phosphorylated by ePK from Jurkat cells. Interestingly, activation of CD98 in CHO cells expressing CD98 enhances the ecto-phosphorylation of CD98 and increases the attachment of Jurkat cells to CHO cells, suggesting that the ectophosphorylation of CD98 is important for cell-cell interactions. To examine the role of CD98 ectodomain phosphorylation in cell-cell interactions, we generated CD98-CD69 and CD69CD98 constructs, as well as CD98 lacking PDZ-binding domain or mutated at predicted serine phosphorylation sites and transfected these constructs into CHO cells. Cell adhesion assays show that mutation at S305-S307-S309 or deletion of extracellular C-terminal or PDZ-binding domain of CD98 significantly decreases the binding of Jurkat cells to CHO cells. Conclusion: CD98 is ecto-phosphorylated by ePK and its phosphorylation affects cellcell interactions.
M1685 Curcumin Inhibits IFN-γ Signaling in Colonic Epithelial Cells Monica T. Midura-Kiela, Claire B. Larmonier, Daniel Laubitz, Fayez K. Ghishan, Pawel R. Kiela In recent years, there has been a growing interest in the use of curcumin in treatment and/ or prevention of Inflammatory Bowel Diseases with multiple reports of benefit in chemically induced rodent models of IBD as well as in a recent clinical trial with UC patients. However, poor systemic bioavailability following oral administration, as well as rapid metabolism by intestinal epithelial cells (IEC), suggest that IEC may be the primary target of the compound's biological activity. IFN-γ is one of the most prominent proinflammatory cytokines with profound effects on colonic epithelial cells. Elevated IFN-γ disrupts the epithelial barrier function by inducing tight junction internalization, prevents epithelial cell migration and wound healing by causing endocytosis of β1-integrin containing focal adhesions and by preventing inter-enterocyte gap junction communication. IFN-γ also primes IEC cells to express MHC class II molecules and to serve as non-professional antigen presenting cells. IEC isolated from IBD patients have been shown to express co-stimulatory molecules (CD58, CD86, B7H) and to potently induce proliferation of CD4+ lymphocytes when co-cultured. In this report we present data demonstrating that curcumin inhibits IFN-γ signaling in colonic epithelial cells. T-84 cells were transfected with a reporter construct driven by a tandem of IFN-γ activation sites (pGAS-Luc). Curcumin significantly inhibited IFN-γ induced Luc activity. IFN-γ (100U/mL) stimulated expression of MHC class II genes, HLA-DRα and HLA-DPα1, by 140,000 times and 40,000 times respectively. Pretreatment with curcumin
M1688 Dysregulation of the Polymeric Immunoglobulin Receptor in Crohn's Disease Razvan I. Arsenescu, Maria Bruno, Willem J. de Villiers, Charlotte Kaetzel Background: Crohn's disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract, in which a dysregulated host immune response to ubiquitous microbes may play a role. Secretory IgA forms the first line of immune defense at mucosal surfaces, and contributes to intestinal homeostasis by neutralization of pro-inflammatory factors. The polymeric immunoglobulin receptor (pIgR) transports IgA into mucosal secretions. We hypothesize that dysregulated expression of pIgR in the intestinal mucosa contributes to tissue inflammation in CD. Methods: Intestinal mucosal biopsies and serum were collected from CD patients (n=43) and healthy controls (n=26). Disease activity was assessed by Harvey-Bradshaw score, endoscopy and histology. Levels of mRNA for pIgR and IL-8 were measured by quantitative reverse transcriptase PCR. Serum IgA levels were measured by ELISA. Immunohistochemistry was performed for pIgR, CD71 and IgA in controls, CD involved and uninvolved mucosa. Results: pIgR mRNA, was higher in normal colon than ileum likely reflecting the increased bacterial load in the former.. Expression of pIgR was significantly decreased in colonic
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AGA Abstracts
AGA Abstracts
at 25 µM significantly reduced this increase, and 75 µM brought it to near background level. Similarly, curcumin in a dose-dependent fashion inhibited IFN-γ stimulated expression of CII TA , a master regulator of MHCII class genes. Gel mobility shift analysis showed curcumin-mediated inhibition of STAT-1 binding to both GAS and ISRE (Interferon Stimulatory Response Element) cis-elements. Immunofluorescence studies demonstrated that curcumin inhibited IFN-γ-stimulated nuclear translocation of Stat-1. IFN-γ-induced and JAKdependent phosphorylation of Stat-1 at Tyr701, a residue critical in Stat-1 dimerization and nuclear translocation, was also inhibited by curcumin in a concentration-dependent fashion. These observations were confirmed in conditionally immortalized mouse colonic epithelial cells (YAMC). In summary, curcumin acts as a potent inhibitor of Jak-Stat signaling pathway in the colonic epithelial cells, a phenomenon which may partially account for the beneficial effects of curcumin in experimental colitis as well as in human IBD. 5R01DK067286
M1683
M1686 CXCR6 Is Upregulated in Effector Memory CD4+ T Cells Infiltrating Into Chronically Inflamed Colonic Tissue Yasushi Mandai, Koji Hase, Toru Sato, Daisuke Takahashi, Masashi Ebisawa, Tatsuro Katsuno, Yasushi Saito, Hiroshi Ohno Background & Aim. The recently identified CXCL16 is a unique chemokine with dual functions: A transmembrane adhesion molecule and a soluble chemokine. In the present study, we investigated the role of CXCL16 and its receptor CXCR6 in the development of inflammatory bowel disease (IBD). Methods. A murine experimental colitis model was induced by adoptive transfer of CD4+CD45Rbhi T cells from wild-type mice into Rag-1-/mice. CXCL16 mRNA and protein expressions in the colonic tissue of the murine colitis model as well as human IBD patients were analyzed by quantitative RT-PCR and immunohistochemistry (IHC), respectively. Surface markers of T cells prepared from colonic lamina propria were analyzed by flow cytometry. Results. We found a significant increase in CXCL16 expression in inflamed intestinal mucosa of the murine colitis model as well as human patients with active IBD, namely Crohn's disease and ulcerative colitis. IHC analysis revealed that CXCL16 was detected in epithelial cells and a subset of immune cells, possibly activated dendritic cells (DCs). Likewise, treatment of HT-29 human colonic epithelial cell line with proinflammatory cytokines TNF-alpha and IFN-gamma upregulated CXCL16. FACS analysis showed that nearly 80% of colonic laminal propria CD4+ T cells upregulated CXCR6 in the murine colitis model. These CXCR6+CD4+ T cells showed CD62LloCD44hi effector memory phenotype, and highly produced IL-17 and IFN-gamma, whereas the cytokine production by CXCR6- population was minimal. Conclusions. This study suggests that CXCR6 could be a marker of colitogenic effector memory T cells. CXCL16 expressed by epithelium and DCs may play an important role in migration to and retention in the colonic tissue of colitogenic T cells. Our finding raises the possibility that disrupting the CXCL16CXCR6 interaction could serve as a promising therapeutic approach for IBD.
M1684 The Roles of p53 and Death Receptor-5 in Augmenting Activated T-Cell Induced Apoptosis Edwin K. McDonald, Ramanarao Dirisina, Niklas Finnberg, Meghan Barrett, Tatiana Goretsky, Jennet Manjali, Mutazz Darweesh, Jeffrey Brown, Wafik S. El-Deiry, Terrence A. Barrett Background/Aims: Epithelial cell apoptosis is important in the pathogenesis of mucosal injury in T-cell induced inflammatory disorders of the intestine. It has been shown previously that radiation-induced epithelial cell apoptosis requires p53 and Death Receptor-5 (DR5). DR5 is an extrinsic mediator of apoptosis and is induced by p53. Here we address the role of p53 and DR5 in T-cell mediated epithelial cell apoptosis using the anti-CD3 model of systemic T-cell activation. Methods: C57Bl/6, B6/p53-/-, and B6/DR5-/- mice were sacrificed 24 hours after activating T-cells with .2mg anti-CD3 mAb i.p. Alterations in intestinal morphology were assessed and apoptosis was detected by TUNEL and active caspase-3 staining. p53, caspase-3, caspase-8, and caspase-9 levels were assessed by Western blots from 12 hours. p53 was also detected by IHC. Results: Activation of T-cells induced an intestinal enteropathy with morphologic changes including mucosal flattening, villous blunting, and paneth cell hyperplasia in both C57Bl/6 and DR5-/- mice. Villous flattening was more pronounced in the wild type strains. In B6 mice, p53 was induced 3.4-fold in isolated crypt epithelial cells (leukocyte depleted) by Western blot and localized to lower crypt enterocytes by IHC. There was no significant difference in p53 levels between wild type and DR5-/- by IHC. There was a 63-fold increase in TUNEL staining in lower crypt in B6 mice after anti-CD3. TUNEL in lower crypt correlated with an 170-fold and 50-fold induction of cleaved caspase-3 and 9 respectively, but not 8 (Western blot). TUNEL and caspase-3 staining were reduced by 85% and 76% respectively in p53-/- mice and by 41% and 20% in DR5-/- respectively. Conclusions: The reduction of T-cell induced apoptosis in p53-/and DR5-/- mice is consistent with the notion that DR5 contributes to p53-mediated crypt cell apoptosis. The induction of caspase-3 and 9 but not 8 indicates that p53-mediated DR5-induced apoptosis operates through the intrinsic (mitochondrial) pathway. As the largest effects on apoptosis (ie reduction) were seen in cells of the lower crypt in both p53-/and DR5-/- mice, we speculate that these mechanisms operate in progenitor populations of enterocytes responding to a potent immune stimulus. These results also suggest that inhibition of DR5 in colitis may reduce epithelial apoptosis and aide restitution of barrier function following injury.
M1687 Ecto-Phosphorylation of CD98 Regulates Lymphocytes-Epithelia Interactions Hang Thi Thu Nguyen, Yutao Yan, Guillaume Dalmasso, Laetitia Charrier-Hisammudin, Shanthi V. Sitaraman, Didier Merlin Background and Aims: Ecto-phosphorylation has been shown to play an important role in several cellular functions. Many membrane proteins and soluble extracellular protein can be phosphorylated by ecto-protein kinases (ePK) using extracellular ATP as a phophoryl donor. CD98, a type II transmembrane glycoprotein, is a heavy chain of the family of heterodimeric amino acid transporter. Since CD98 contains predicted phosphorylation sites, our aim was to investigate the ecto-phosphorylation of CD98 by ePK and its functional consequences. Results: Recombinant CD98 is phosphorylated by purified casein kinase 2 (CK2) and by ePK prepared from Jurkat T cells. Km and Vmax for the phosphorylation of CD98 by CK2 are 0.84 µM and 8.7e+6 µmol/min, respectively. In human intestinal epithelial Caco2-BBE cells, CD98 is also ecto-phosphorylated by ePK from Jurkat cells. Interestingly, activation of CD98 in CHO cells expressing CD98 enhances the ecto-phosphorylation of CD98 and increases the attachment of Jurkat cells to CHO cells, suggesting that the ectophosphorylation of CD98 is important for cell-cell interactions. To examine the role of CD98 ectodomain phosphorylation in cell-cell interactions, we generated CD98-CD69 and CD69CD98 constructs, as well as CD98 lacking PDZ-binding domain or mutated at predicted serine phosphorylation sites and transfected these constructs into CHO cells. Cell adhesion assays show that mutation at S305-S307-S309 or deletion of extracellular C-terminal or PDZ-binding domain of CD98 significantly decreases the binding of Jurkat cells to CHO cells. Conclusion: CD98 is ecto-phosphorylated by ePK and its phosphorylation affects cellcell interactions.
M1685 Curcumin Inhibits IFN-γ Signaling in Colonic Epithelial Cells Monica T. Midura-Kiela, Claire B. Larmonier, Daniel Laubitz, Fayez K. Ghishan, Pawel R. Kiela In recent years, there has been a growing interest in the use of curcumin in treatment and/ or prevention of Inflammatory Bowel Diseases with multiple reports of benefit in chemically induced rodent models of IBD as well as in a recent clinical trial with UC patients. However, poor systemic bioavailability following oral administration, as well as rapid metabolism by intestinal epithelial cells (IEC), suggest that IEC may be the primary target of the compound's biological activity. IFN-γ is one of the most prominent proinflammatory cytokines with profound effects on colonic epithelial cells. Elevated IFN-γ disrupts the epithelial barrier function by inducing tight junction internalization, prevents epithelial cell migration and wound healing by causing endocytosis of β1-integrin containing focal adhesions and by preventing inter-enterocyte gap junction communication. IFN-γ also primes IEC cells to express MHC class II molecules and to serve as non-professional antigen presenting cells. IEC isolated from IBD patients have been shown to express co-stimulatory molecules (CD58, CD86, B7H) and to potently induce proliferation of CD4+ lymphocytes when co-cultured. In this report we present data demonstrating that curcumin inhibits IFN-γ signaling in colonic epithelial cells. T-84 cells were transfected with a reporter construct driven by a tandem of IFN-γ activation sites (pGAS-Luc). Curcumin significantly inhibited IFN-γ induced Luc activity. IFN-γ (100U/mL) stimulated expression of MHC class II genes, HLA-DRα and HLA-DPα1, by 140,000 times and 40,000 times respectively. Pretreatment with curcumin
M1688 Dysregulation of the Polymeric Immunoglobulin Receptor in Crohn's Disease Razvan I. Arsenescu, Maria Bruno, Willem J. de Villiers, Charlotte Kaetzel Background: Crohn's disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract, in which a dysregulated host immune response to ubiquitous microbes may play a role. Secretory IgA forms the first line of immune defense at mucosal surfaces, and contributes to intestinal homeostasis by neutralization of pro-inflammatory factors. The polymeric immunoglobulin receptor (pIgR) transports IgA into mucosal secretions. We hypothesize that dysregulated expression of pIgR in the intestinal mucosa contributes to tissue inflammation in CD. Methods: Intestinal mucosal biopsies and serum were collected from CD patients (n=43) and healthy controls (n=26). Disease activity was assessed by Harvey-Bradshaw score, endoscopy and histology. Levels of mRNA for pIgR and IL-8 were measured by quantitative reverse transcriptase PCR. Serum IgA levels were measured by ELISA. Immunohistochemistry was performed for pIgR, CD71 and IgA in controls, CD involved and uninvolved mucosa. Results: pIgR mRNA, was higher in normal colon than ileum likely reflecting the increased bacterial load in the former.. Expression of pIgR was significantly decreased in colonic
A-397
AGA Abstracts
AGA Abstracts
at 25 µM significantly reduced this increase, and 75 µM brought it to near background level. Similarly, curcumin in a dose-dependent fashion inhibited IFN-γ stimulated expression of CII TA , a master regulator of MHCII class genes. Gel mobility shift analysis showed curcumin-mediated inhibition of STAT-1 binding to both GAS and ISRE (Interferon Stimulatory Response Element) cis-elements. Immunofluorescence studies demonstrated that curcumin inhibited IFN-γ-stimulated nuclear translocation of Stat-1. IFN-γ-induced and JAKdependent phosphorylation of Stat-1 at Tyr701, a residue critical in Stat-1 dimerization and nuclear translocation, was also inhibited by curcumin in a concentration-dependent fashion. These observations were confirmed in conditionally immortalized mouse colonic epithelial cells (YAMC). In summary, curcumin acts as a potent inhibitor of Jak-Stat signaling pathway in the colonic epithelial cells, a phenomenon which may partially account for the beneficial effects of curcumin in experimental colitis as well as in human IBD. 5R01DK067286
M1683