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C5a modulates cytokine production critical for intestinal inflammation in inflammatory bowel disease
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Abstracts / Molecular Immunology 48 (2011) 1666–1733
O12 Diminished neutrophil chemotaxis to C5a in vitamin D binding protein deficient mice R. Kew ∗ , G.T. Trujillo, D.H. Habiel, L.G. Ge, M.R. Ramadass Stony Brook University, Stony brook, NY, United States Introduction: Complement activation peptides C5a and C5a des Arg are potent leukocyte chemoattractants. Previously, we have shown that the vitamin D binding protein (DBP) functions as a chemotactic cofactor for C5a/C5a des Arg in vitro, significantly enhancing their chemotactic activity. However, this cofactor effect of DBP has not been demonstrated in vivo. Methods: The DBP null mouse (DBP−/−) and its wild-type C57BL/6 parental strain (DBP+/+) were utilized to investigate whether DBP is required for optimal C5a-induced chemotaxis in vivo. Purified mouse C5a was instilled into lungs using the oropharyngeal aspiration method. At various times after onset of injury, lungs were lavaged and the number and type of cells recruited were evaluated. Results: DBP−/− mice had significantly fewer cells in the bronchoalveolar lavage fluid at 4, 6 and 24 h after C5a instillation. Flow cytometric analysis revealed that a decrease in GR1+ neutrophils largely accounted for this difference. Whole lung homogenates analyzed after lavaging also showed diminished number of neutrophils in the DBP−/− group. This disparity in neutrophil recruitment into the lungs was not due to variations in total number of neutrophils (blood and bone marrow) or their C5a receptor expression levels between the DBP−/− and++ groups. The diminished capacity of DBP−/− mice to recruit neutrophils was largely corrected by instilling C-activated DBP+/+ serum into the lung. Conclusion: These results provide the first evidence that DBP functions as a significant C5a chemotactic cofactor in vivo, signifying that the regulation of C5a chemotactic activity may be more complex than previously recognized. doi:10.1016/j.molimm.2011.06.232 O13 C5a modulates cytokine production critical for intestinal inflammation in inflammatory bowel disease A.T. Reinicke a,∗ , E.S. Reis b , J. Köhl b a b
Universitätsklinikum Schleswig-Holstein, Lübeck, Germany University of Lübeck, Lübeck, Germany
Increased complement activity correlates with inflammation in experimental and clinical inflammatory bowel disease (IBD). Surprisingly, the role of complement in intestinal inflammation remains ill defined. Antigen presenting cells (APCs) exert a key role in maintaining mucosal tolerance as well as in mounting potent immune responses to face off pathogens. We and others have previously shown that cross talk between Toll-like receptors (TLR) and anaphylatoxin receptor signaling pathways regulate APC functions in lung and spleen by promoting differentiation of naive Th cells towards Th1, Th2, Th17, or Treg phenotypes. Intestinal inflammation in IBD generates C3a and C5a, however, their role in modulating APC functions and subsequent T cell responses in the intestine is undefined. To assess such modulatory functions of the anaphylatoxins, we isolated APCs from Lamina propria of small intestine and defined plasmacytoid DC, conventional DC (cDC), and macrophage populations through surface and costimulatory marker expression. We found that FACS-sorted cDCs and macrophages express mRNA for C3aR and C5aR as well as for TLR4, Dectin-1, and NOD2. IL10 and IL-12p40 cytokine production was upregulated in response
to TLR2 stimulation in cDCs and macrophages. Importantly, we found that C5aR expression modulated TLR2-induced cytokine production. IL-12p40 secretion from wt cDCs was significantly lower than from C5aR−/− DCs. In contrast, IL-10 production was significantly higher from wt as compared with C5aR−/− macrophages. We propose novel cross talk signaling between C5aR and TLR2 that differentially modulates the production of cytokines critical for the balance between maladaptive immunity and tolerance in the intestinal mucosa. doi:10.1016/j.molimm.2011.06.233 O14 Independent roles for complement C3a and C5a receptors and the membrane attack complex in the pathogenesis of collagen antibody-induced arthritis in mice: Potential therapeutic implications N.K. Banda a,∗ , S. Hyatt a , M. Glogowska a , K. Takahashi b , T.J. Merkel c , G.L. Stahl d , B. Lu e , C. Gerard e , R.A. Wetsel f , W.P. Arend a , V.M. Holers a a
University of Colorado at Denver, Aurora, United States Massachusetts General Hospital for Children, Boston, United States c CBER, FDA, Bethesda, MD, United States d Brigham and Women’s Hospital, Boston, MA, United States e Children’s Hospital, Harvard Medical School, Boston, United States f University of Texas Medical School, Houston, TX, United States b
The main goal of this study was to explore the comparative role of receptors for C3a and C5a as well as C6, a complement protein that is required for the formation of the membrane attack complex (MAC), in the pathogenesis of CAIA in mice. Clinical disease activity (CDA), histopathological scores and C3 deposition in knee joints were examined in C3aR−/− , C5aR−/− , C6−/− and wild type (WT) mice. The CDA in C3aR−/− , C5aR−/− , and C6−/− mice was significantly (p < 0.05) decreased by 51%, 95%, and 65%, respectively, compared to WT mice. Scores for histopathology and C3 deposition in vivo were consistent with the CDA in all experimental mice. Despite the decrease in C3 deposition in vivo, C3 activation in vitro induced by adherent anti-collagen mAb was indistinguishable using sera from WT, C3aR−/− , C5aR−/− , and C6−/− mice. Macrophage and neutrophil infiltration was decreased into the synovium of C3aR−/− and C5aR−/− mice, whereas only neutrophils were decreased in C6−/− mice. A significant (p < 0.05) decrease in the mRNA levels of TNF-a and IL-1- was observed in the knee joints of C5aR−/− and C6−/− mice with CAIA. Our data indicate that the lack of C3aR, C5aR or MAC formation each increases resistance to CAIA. Multiple mechanisms are likely to be responsible for the effects of C3aR, C5aR, and MAC on the induction of CAIA. However, a consistent feature is a decrease in C3 activation in the target organ site, likely due to an effector pathway mediated enhancement of C3 convertase activation. doi:10.1016/j.molimm.2011.06.234 O15 Alkaline protease of Pseudomonas aeruginosa evades innate immunity by blocking activation of complement C2 and Tolllike receptor 5 B.W. Bardoel ∗ , A.J. Laarman, M. Ruyken, K.P.M. Van Kessel, S.H.M. Rooijakkers, J.A.G. Van Strijp UMC Utrecht, Utrecht, Netherlands Introduction: The innate immune system recognizes bacteria via conserved molecular patterns. The Toll-like receptor (TLR) family activates inflammatory cells and initiates the production of
Abstracts / Molecular Immunology 48 (2011) 1666–1733
O12 Diminished neutrophil chemotaxis to C5a in vitamin D binding protein deficient mice R. Kew ∗ , G.T. Trujillo, D.H. Habiel, L.G. Ge, M.R. Ramadass Stony Brook University, Stony brook, NY, United States Introduction: Complement activation peptides C5a and C5a des Arg are potent leukocyte chemoattractants. Previously, we have shown that the vitamin D binding protein (DBP) functions as a chemotactic cofactor for C5a/C5a des Arg in vitro, significantly enhancing their chemotactic activity. However, this cofactor effect of DBP has not been demonstrated in vivo. Methods: The DBP null mouse (DBP−/−) and its wild-type C57BL/6 parental strain (DBP+/+) were utilized to investigate whether DBP is required for optimal C5a-induced chemotaxis in vivo. Purified mouse C5a was instilled into lungs using the oropharyngeal aspiration method. At various times after onset of injury, lungs were lavaged and the number and type of cells recruited were evaluated. Results: DBP−/− mice had significantly fewer cells in the bronchoalveolar lavage fluid at 4, 6 and 24 h after C5a instillation. Flow cytometric analysis revealed that a decrease in GR1+ neutrophils largely accounted for this difference. Whole lung homogenates analyzed after lavaging also showed diminished number of neutrophils in the DBP−/− group. This disparity in neutrophil recruitment into the lungs was not due to variations in total number of neutrophils (blood and bone marrow) or their C5a receptor expression levels between the DBP−/− and++ groups. The diminished capacity of DBP−/− mice to recruit neutrophils was largely corrected by instilling C-activated DBP+/+ serum into the lung. Conclusion: These results provide the first evidence that DBP functions as a significant C5a chemotactic cofactor in vivo, signifying that the regulation of C5a chemotactic activity may be more complex than previously recognized. doi:10.1016/j.molimm.2011.06.232 O13 C5a modulates cytokine production critical for intestinal inflammation in inflammatory bowel disease A.T. Reinicke a,∗ , E.S. Reis b , J. Köhl b a b
Universitätsklinikum Schleswig-Holstein, Lübeck, Germany University of Lübeck, Lübeck, Germany
Increased complement activity correlates with inflammation in experimental and clinical inflammatory bowel disease (IBD). Surprisingly, the role of complement in intestinal inflammation remains ill defined. Antigen presenting cells (APCs) exert a key role in maintaining mucosal tolerance as well as in mounting potent immune responses to face off pathogens. We and others have previously shown that cross talk between Toll-like receptors (TLR) and anaphylatoxin receptor signaling pathways regulate APC functions in lung and spleen by promoting differentiation of naive Th cells towards Th1, Th2, Th17, or Treg phenotypes. Intestinal inflammation in IBD generates C3a and C5a, however, their role in modulating APC functions and subsequent T cell responses in the intestine is undefined. To assess such modulatory functions of the anaphylatoxins, we isolated APCs from Lamina propria of small intestine and defined plasmacytoid DC, conventional DC (cDC), and macrophage populations through surface and costimulatory marker expression. We found that FACS-sorted cDCs and macrophages express mRNA for C3aR and C5aR as well as for TLR4, Dectin-1, and NOD2. IL10 and IL-12p40 cytokine production was upregulated in response
to TLR2 stimulation in cDCs and macrophages. Importantly, we found that C5aR expression modulated TLR2-induced cytokine production. IL-12p40 secretion from wt cDCs was significantly lower than from C5aR−/− DCs. In contrast, IL-10 production was significantly higher from wt as compared with C5aR−/− macrophages. We propose novel cross talk signaling between C5aR and TLR2 that differentially modulates the production of cytokines critical for the balance between maladaptive immunity and tolerance in the intestinal mucosa. doi:10.1016/j.molimm.2011.06.233 O14 Independent roles for complement C3a and C5a receptors and the membrane attack complex in the pathogenesis of collagen antibody-induced arthritis in mice: Potential therapeutic implications N.K. Banda a,∗ , S. Hyatt a , M. Glogowska a , K. Takahashi b , T.J. Merkel c , G.L. Stahl d , B. Lu e , C. Gerard e , R.A. Wetsel f , W.P. Arend a , V.M. Holers a a
University of Colorado at Denver, Aurora, United States Massachusetts General Hospital for Children, Boston, United States c CBER, FDA, Bethesda, MD, United States d Brigham and Women’s Hospital, Boston, MA, United States e Children’s Hospital, Harvard Medical School, Boston, United States f University of Texas Medical School, Houston, TX, United States b
The main goal of this study was to explore the comparative role of receptors for C3a and C5a as well as C6, a complement protein that is required for the formation of the membrane attack complex (MAC), in the pathogenesis of CAIA in mice. Clinical disease activity (CDA), histopathological scores and C3 deposition in knee joints were examined in C3aR−/− , C5aR−/− , C6−/− and wild type (WT) mice. The CDA in C3aR−/− , C5aR−/− , and C6−/− mice was significantly (p < 0.05) decreased by 51%, 95%, and 65%, respectively, compared to WT mice. Scores for histopathology and C3 deposition in vivo were consistent with the CDA in all experimental mice. Despite the decrease in C3 deposition in vivo, C3 activation in vitro induced by adherent anti-collagen mAb was indistinguishable using sera from WT, C3aR−/− , C5aR−/− , and C6−/− mice. Macrophage and neutrophil infiltration was decreased into the synovium of C3aR−/− and C5aR−/− mice, whereas only neutrophils were decreased in C6−/− mice. A significant (p < 0.05) decrease in the mRNA levels of TNF-a and IL-1- was observed in the knee joints of C5aR−/− and C6−/− mice with CAIA. Our data indicate that the lack of C3aR, C5aR or MAC formation each increases resistance to CAIA. Multiple mechanisms are likely to be responsible for the effects of C3aR, C5aR, and MAC on the induction of CAIA. However, a consistent feature is a decrease in C3 activation in the target organ site, likely due to an effector pathway mediated enhancement of C3 convertase activation. doi:10.1016/j.molimm.2011.06.234 O15 Alkaline protease of Pseudomonas aeruginosa evades innate immunity by blocking activation of complement C2 and Tolllike receptor 5 B.W. Bardoel ∗ , A.J. Laarman, M. Ruyken, K.P.M. Van Kessel, S.H.M. Rooijakkers, J.A.G. Van Strijp UMC Utrecht, Utrecht, Netherlands Introduction: The innate immune system recognizes bacteria via conserved molecular patterns. The Toll-like receptor (TLR) family activates inflammatory cells and initiates the production of