- Email: [email protected]
Alternative pathway mediated generation of C3a regulates the allostimulatory function of dendritic cells
226
Abstracts / Molecular Immunology 44 (2007) 147–266
to sense different ligands which collectively provide strong “eat me” signals. These observations shed new light on the role of C1q as a major bridging molecule in apoptotic cell recognition and clearance. doi:10.1016/j.molimm.2006.07.183 179 Inhibitory Fc␥ receptors negatively regulate C5aRmediated effector functions in immune complex diseases Manoj K. Pandey, Gabriele K¨ohl, J¨org K¨ohl Division of Molecular Immunology, Cincinnati Children’s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH 45229,USA Immune complexes (ICs) are integral to the pathogenesis of several autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus. While ligation of activating Fc␥R is indispensable for IC-mediated inflammation, we and others have previously shown that C5aR signaling sets the threshold for Fc␥R activation by positively regulating the balance between activating Fc␥Rs and inhibitory Fc␥RII. Here, we demonstrate that the interaction between C5aR and Fc␥R signaling is bidirectional. Mice lacking activating Fc␥Rs (FcR ␥-chain−/− mice) are protected from inflammation in IC-mediated peritonitis. Surprisingly, ablation of Fc␥RII signaling in FcR ␥-chain−/− mice using an anti-Fc␥RII-specific mAb restored the inflammatory response, suggesting inhibitory properties of Fc␥RII beyond counterbalancing activating Fc␥Rs. This inflammatory response was substantially reduced following C5aR blockade pointing towards a regulatory impact of Fc␥RII on C5aR signaling pathways. Importantly, the in vivo data were reconciled by in vitro studies. Ligation of Fc␥RII on bone marrow (BM)-derived neutrophils from FcR ␥-chain−/− mice suppressed C5aR-induced chemotaxis and upregulation/clustering of complement receptor 3 (CR3; CD11b/CD18). Intriguingly, a similar effect was found for CXCL2 (MIP-2)-induced chemotaxis. In search for mechanisms that may account for the negative regulatory effect of Fc␥RII, we first determined the C5aR signaling pathways that contribute to C5a-mediated neutrophil chemotaxis. Using specific inhibitors of phosphoinositide-3 kinase, p38 mitogenactivated protein kinase (MAPK), and MAP/extracellularrelated kinase (ERK), we found that ERK1/2 is crucial to C5ainduced neutrophil chemotaxis. Importantly, ligation of Fc␥RII substantially suppressed C5a-induced ERK1/2 phosphorylation in BM-derived neutrophils from FcR ␥-chain−/− mice. Collectively, our findings strongly suggest that Fc␥RII signaling in neutrophils negatively regulates effector functions of chemoattractant G-protein-coupled receptors. As IL-4 and the regulatory cytokines TGF-/IL-10 increase the expression of Fc␥RII, this novel regulatory pathway is likely to affect inflammatory responses in atopy and autoimmune diseases. doi:10.1016/j.molimm.2006.07.184
180 Regulation of MAC complex in rainbow trout: A molecular view A.D. Papanastasiou, A. Londou, E. Georgaka, D. Marioli, I.K. Zarkadis Department of Biology, School of Medicine, University of Patras, 26 500 Rion Patras, Greece The survival of host cells requires their protection from autologous complement attack. Mammalian CD59, clusterin and vitronectin proteins regulate the synthesis of membrane attack complex (MAC) inhibiting cytolysis of the host cells. CD59, a GPI-linked glycoprotein, regulates complement activation cascade at the final step, inhibiting formation of MAC. Clusterin and vitronectin are two secreted glycoproteins which act on the soluble hydrophobic complex C5b-9, preventing its stabilization on the target cell surface and inhibiting the formation of MAC. Although the terminal complement component domains (TSP/LDLa/MACPF/EGF/CCP/FIMAC) were established very early in deuterostome lineage, teleost is the most ancient species where the MAC complex has been microscopically observed as small pores in the target cell surface. Very little is known regarding the MAC regulation in low vertebrates. Here we report the cloning, nucleotide sequence, tissue expression profile, exon–intron organization and phylogenetic analysis of CD59, clusterin and vitronectin genes from rainbow trout (Oncorhynchus mykiss). Two isoforms for each of these complement regulatory genes have so far been identified in the trout genome. The tissue expression profiles of trout CD59, clusterin and vitronectin genes differ in relation to their mammalian orthologs. Partial data from analysis of genomic clones show an agreement with their mammalian counterparts in the exon–intron gene organization. Acknowledgement: Supported by “K. Karatheodori” (University of Patras) and Empeirikeio Foundation. doi:10.1016/j.molimm.2006.07.185 181 Alternative pathway mediated generation of C3a regulates the allostimulatory function of dendritic cells Qi Peng, Ke Li, Steven H. Sacks, Wuding Zhou Department of Nephrology and Transplantation, King’s College London School of Medicine at Guy’s, King’s College and St. Thomas’ Hospitals, Guy’s Hospital, London SE1 9RT, UK Our previous studies have shown that production of C3 by dendritic cells (DCs) has a significant impact on DC effector functions such as inducing TH 1-cell responses and influencing regulatory T cell development. However, the precise mechanism remains unclear. The aim of this study is to address how complement and DCs interact. To understand how locally synthesised complement could affect DC effector functions, first, we investigated the synthesis
Abstracts / Molecular Immunology 44 (2007) 147–266
of complement components, regulators and receptors by murine bone marrow DCs using Gene array and RT-PCR; then using a MHC fully mismatched mouse strain combination (H-2b and H-2d) we investigated the role of C3aR pathway in modulating DC function. We also investigated the involvement of main three pathways in modulating DC’s capacity to stimulate alloreactive T cells. Our data showed: (1) C1q, C3, factor B, Crry, CR3, CR4, C3aR and C5aR are produced in mature DCs, but not C2, C4 and terminal pathway components; (2) DCs treated with antagonist for C3aR (by adding the antagonist in DC culture medium during DC culturing) are demonstrated to produce significantly less IL-12 and more IL-10 than untreated cells; when na¨ıve alloreactive CD4+ T cells are exposed to antagonist-treated DCs they produce significantly lower level of IFN-␥ and are less proliferative; (3) DCs prepared from factor B−/− mice, but not C4−/− and C6−/− mice are shown to have significantly reduced T cell stimulatory capacity, which was similar to that observed with C3−/− DCs previously. Thus, our findings demonstrate that local complement activation through the alternative pathway is involved in modulating DC function, and C3aR signalling pathway is essential for DC activation and effector functions, suggesting a key mechanism for local production of complement by DCs in modulating DC functions thus regulating alloreactive T cell response.
227
lization of combined dose of corticosteroid and anti-C5 mAb had enhanced efficacy than monotherapy. In contrast to blocking the engagement of C5a receptor with anti-C5aR mAb as recently reported by Kohl et al. [Kohl, J., Baelder, R., Lewkowich, I.P., Pandey, M.K., Hawlisch, H., Wang, L., Best, J., Herman, N.S., Sproles, A.A., Zwirner, J., Whitsett, J.A., Gerard, C., Sfyroera, G., Lambris, J.D., Wills-Karp, M., 2006. A regulatory role for the C5a anaphylatoxin in type 2 immunity in asthma. J. Clin. Invest. 116, 783–796], repeated chronic aerosol administrations of anti-mouse C5 mAb, BB5.1, did not induce intrapulmonary Th2 cytokine production in normal BALB/c mice. Histology examination showed normal airways without detectable airway inflammation after 10 weeks of intrapulmonary C5 inhibition. These data demonstrated that intrapulmonary activation of C5, as an innocuous by-product of innate immune responses to allergens or infections, plays a critical role in the pathogenesis of asthma. Blocking intrapulmonary activation of C5 is a potential clinical approach for treating patients with asthma. Finally, our data suggests that blocking the engagement of C5a receptor with anti-C5aR mAb is significant different from that of blocking the generation of C5a and C5b-9 with anti-C5 mAb. doi:10.1016/j.molimm.2006.07.187
doi:10.1016/j.molimm.2006.07.186
183 Spontaneous haemolytic uraemic syndrome (HUS) in factor H-deficient mice transgenic for murine factor H protein lacking the five C-terminal domains (FH16–20)
182 Effect of blocking intrapulmonary activation of C5 on the development of airway hyperresponsiveness (AHR) and airway inflammation
M.C. Pickering a,1 , E. Goicoechea de Jorge c,1 , K.L. Rose a , J. Moss c , H.T. Cook b , S. Rodriguez de Cordoba c,1 , M. Botto a,1 a
Alexion Pharmaceuticals Inc., Cheshire, CT 06410, United States
Molecular Genetics and Rheumatology Section & Department of Histopathology, Imperial College School of Medicine, London, UK; b Imperial College School of Medicine, London, UK; c Departamento de Inmunologia, Centro de Investigaciones Biologicas (CSIC), Madrid, Spain
This study is aimed at testing the hypothesis that intrapulmonary activation of C5 plays a critical role in the pathogenesis of asthma. BALB/c mice developed airway inflammation after repeated systemic and aerosol sensitizations with OVA. Significant elevated level of intrapulmonary C5 activity can be detected after aerosol challenge sensitized mice with OVA. Animals developed severe airway inflammation and AHR. Intrapulmonary C5 inhibition can be achieved by nebulization administration of anti-mouse C5 mAb, BB5.1. Anti-mouse C5 mAb as well as a humanized anti-human C5 mAb can be successfully nebulized with common Jet-Air nebulizers. Both antibodies retained the activity and integrity after nebulization with a mass median aerodynamic diameter less than 2.6 m, suitable for aerosol delivery to lower airways. Aerosol delivery of antimouse C5 mAb via nebulization achieved intrapulmonary C5 inhibition without inhibiting serum C5 activity. A single nebulization of either anti-mouse C5 mAb or dexamethasone significantly blocked aerosol methacholine induced AHR in animals that had established airway inflammation. Interestingly, nebu-
Atypical haemolytic uraemic syndrome (aHUS) is frequently associated with mutations in the C-terminal region of complement factor H. To develop a murine model of aHUS we generated factor H-deficient (Cfh−/− ) mice transgenic for a mutant mouse factor H protein lacking the C-terminal five SCR domains (FH16–20). In transgenic Cfh−/− .FH16–20 mice plasma levels of FH16–20 were approximately 64% of wildtype factor H levels. The median plasma C3 level in these mice (77.6 mg/l) was 45% of the median level (171.6 mg/l) in littermate Cfh+/− .FH16–20 mice demonstrating that FH16–20 efficiently controls C3 activation in vivo. In contrast, FH16–20 showed markedly impaired binding to HUVEC. Spontaneous aHUS developed in Cfh−/− .FH16–20 mice aged between 6 and 12 weeks. Glomerular histology showed features of thrombotic microangiopathy including glomerular thrombosis, mesangiolysis and microaneurysm formation. Ultrastructural abnormalities included endothelial cell swelling and accumulation of floccular material under the damaged endothelium. Deposition of C3, in the absence of immunoglobulin, was evi-
Tao Peng, Xiao Su, Karen Kumor, Susan Liu-Chen, Helen Zhou, Dino Miano, Yamin Shen, Yi Wang
Abstracts / Molecular Immunology 44 (2007) 147–266
to sense different ligands which collectively provide strong “eat me” signals. These observations shed new light on the role of C1q as a major bridging molecule in apoptotic cell recognition and clearance. doi:10.1016/j.molimm.2006.07.183 179 Inhibitory Fc␥ receptors negatively regulate C5aRmediated effector functions in immune complex diseases Manoj K. Pandey, Gabriele K¨ohl, J¨org K¨ohl Division of Molecular Immunology, Cincinnati Children’s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH 45229,USA Immune complexes (ICs) are integral to the pathogenesis of several autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus. While ligation of activating Fc␥R is indispensable for IC-mediated inflammation, we and others have previously shown that C5aR signaling sets the threshold for Fc␥R activation by positively regulating the balance between activating Fc␥Rs and inhibitory Fc␥RII. Here, we demonstrate that the interaction between C5aR and Fc␥R signaling is bidirectional. Mice lacking activating Fc␥Rs (FcR ␥-chain−/− mice) are protected from inflammation in IC-mediated peritonitis. Surprisingly, ablation of Fc␥RII signaling in FcR ␥-chain−/− mice using an anti-Fc␥RII-specific mAb restored the inflammatory response, suggesting inhibitory properties of Fc␥RII beyond counterbalancing activating Fc␥Rs. This inflammatory response was substantially reduced following C5aR blockade pointing towards a regulatory impact of Fc␥RII on C5aR signaling pathways. Importantly, the in vivo data were reconciled by in vitro studies. Ligation of Fc␥RII on bone marrow (BM)-derived neutrophils from FcR ␥-chain−/− mice suppressed C5aR-induced chemotaxis and upregulation/clustering of complement receptor 3 (CR3; CD11b/CD18). Intriguingly, a similar effect was found for CXCL2 (MIP-2)-induced chemotaxis. In search for mechanisms that may account for the negative regulatory effect of Fc␥RII, we first determined the C5aR signaling pathways that contribute to C5a-mediated neutrophil chemotaxis. Using specific inhibitors of phosphoinositide-3 kinase, p38 mitogenactivated protein kinase (MAPK), and MAP/extracellularrelated kinase (ERK), we found that ERK1/2 is crucial to C5ainduced neutrophil chemotaxis. Importantly, ligation of Fc␥RII substantially suppressed C5a-induced ERK1/2 phosphorylation in BM-derived neutrophils from FcR ␥-chain−/− mice. Collectively, our findings strongly suggest that Fc␥RII signaling in neutrophils negatively regulates effector functions of chemoattractant G-protein-coupled receptors. As IL-4 and the regulatory cytokines TGF-/IL-10 increase the expression of Fc␥RII, this novel regulatory pathway is likely to affect inflammatory responses in atopy and autoimmune diseases. doi:10.1016/j.molimm.2006.07.184
180 Regulation of MAC complex in rainbow trout: A molecular view A.D. Papanastasiou, A. Londou, E. Georgaka, D. Marioli, I.K. Zarkadis Department of Biology, School of Medicine, University of Patras, 26 500 Rion Patras, Greece The survival of host cells requires their protection from autologous complement attack. Mammalian CD59, clusterin and vitronectin proteins regulate the synthesis of membrane attack complex (MAC) inhibiting cytolysis of the host cells. CD59, a GPI-linked glycoprotein, regulates complement activation cascade at the final step, inhibiting formation of MAC. Clusterin and vitronectin are two secreted glycoproteins which act on the soluble hydrophobic complex C5b-9, preventing its stabilization on the target cell surface and inhibiting the formation of MAC. Although the terminal complement component domains (TSP/LDLa/MACPF/EGF/CCP/FIMAC) were established very early in deuterostome lineage, teleost is the most ancient species where the MAC complex has been microscopically observed as small pores in the target cell surface. Very little is known regarding the MAC regulation in low vertebrates. Here we report the cloning, nucleotide sequence, tissue expression profile, exon–intron organization and phylogenetic analysis of CD59, clusterin and vitronectin genes from rainbow trout (Oncorhynchus mykiss). Two isoforms for each of these complement regulatory genes have so far been identified in the trout genome. The tissue expression profiles of trout CD59, clusterin and vitronectin genes differ in relation to their mammalian orthologs. Partial data from analysis of genomic clones show an agreement with their mammalian counterparts in the exon–intron gene organization. Acknowledgement: Supported by “K. Karatheodori” (University of Patras) and Empeirikeio Foundation. doi:10.1016/j.molimm.2006.07.185 181 Alternative pathway mediated generation of C3a regulates the allostimulatory function of dendritic cells Qi Peng, Ke Li, Steven H. Sacks, Wuding Zhou Department of Nephrology and Transplantation, King’s College London School of Medicine at Guy’s, King’s College and St. Thomas’ Hospitals, Guy’s Hospital, London SE1 9RT, UK Our previous studies have shown that production of C3 by dendritic cells (DCs) has a significant impact on DC effector functions such as inducing TH 1-cell responses and influencing regulatory T cell development. However, the precise mechanism remains unclear. The aim of this study is to address how complement and DCs interact. To understand how locally synthesised complement could affect DC effector functions, first, we investigated the synthesis
Abstracts / Molecular Immunology 44 (2007) 147–266
of complement components, regulators and receptors by murine bone marrow DCs using Gene array and RT-PCR; then using a MHC fully mismatched mouse strain combination (H-2b and H-2d) we investigated the role of C3aR pathway in modulating DC function. We also investigated the involvement of main three pathways in modulating DC’s capacity to stimulate alloreactive T cells. Our data showed: (1) C1q, C3, factor B, Crry, CR3, CR4, C3aR and C5aR are produced in mature DCs, but not C2, C4 and terminal pathway components; (2) DCs treated with antagonist for C3aR (by adding the antagonist in DC culture medium during DC culturing) are demonstrated to produce significantly less IL-12 and more IL-10 than untreated cells; when na¨ıve alloreactive CD4+ T cells are exposed to antagonist-treated DCs they produce significantly lower level of IFN-␥ and are less proliferative; (3) DCs prepared from factor B−/− mice, but not C4−/− and C6−/− mice are shown to have significantly reduced T cell stimulatory capacity, which was similar to that observed with C3−/− DCs previously. Thus, our findings demonstrate that local complement activation through the alternative pathway is involved in modulating DC function, and C3aR signalling pathway is essential for DC activation and effector functions, suggesting a key mechanism for local production of complement by DCs in modulating DC functions thus regulating alloreactive T cell response.
227
lization of combined dose of corticosteroid and anti-C5 mAb had enhanced efficacy than monotherapy. In contrast to blocking the engagement of C5a receptor with anti-C5aR mAb as recently reported by Kohl et al. [Kohl, J., Baelder, R., Lewkowich, I.P., Pandey, M.K., Hawlisch, H., Wang, L., Best, J., Herman, N.S., Sproles, A.A., Zwirner, J., Whitsett, J.A., Gerard, C., Sfyroera, G., Lambris, J.D., Wills-Karp, M., 2006. A regulatory role for the C5a anaphylatoxin in type 2 immunity in asthma. J. Clin. Invest. 116, 783–796], repeated chronic aerosol administrations of anti-mouse C5 mAb, BB5.1, did not induce intrapulmonary Th2 cytokine production in normal BALB/c mice. Histology examination showed normal airways without detectable airway inflammation after 10 weeks of intrapulmonary C5 inhibition. These data demonstrated that intrapulmonary activation of C5, as an innocuous by-product of innate immune responses to allergens or infections, plays a critical role in the pathogenesis of asthma. Blocking intrapulmonary activation of C5 is a potential clinical approach for treating patients with asthma. Finally, our data suggests that blocking the engagement of C5a receptor with anti-C5aR mAb is significant different from that of blocking the generation of C5a and C5b-9 with anti-C5 mAb. doi:10.1016/j.molimm.2006.07.187
doi:10.1016/j.molimm.2006.07.186
183 Spontaneous haemolytic uraemic syndrome (HUS) in factor H-deficient mice transgenic for murine factor H protein lacking the five C-terminal domains (FH16–20)
182 Effect of blocking intrapulmonary activation of C5 on the development of airway hyperresponsiveness (AHR) and airway inflammation
M.C. Pickering a,1 , E. Goicoechea de Jorge c,1 , K.L. Rose a , J. Moss c , H.T. Cook b , S. Rodriguez de Cordoba c,1 , M. Botto a,1 a
Alexion Pharmaceuticals Inc., Cheshire, CT 06410, United States
Molecular Genetics and Rheumatology Section & Department of Histopathology, Imperial College School of Medicine, London, UK; b Imperial College School of Medicine, London, UK; c Departamento de Inmunologia, Centro de Investigaciones Biologicas (CSIC), Madrid, Spain
This study is aimed at testing the hypothesis that intrapulmonary activation of C5 plays a critical role in the pathogenesis of asthma. BALB/c mice developed airway inflammation after repeated systemic and aerosol sensitizations with OVA. Significant elevated level of intrapulmonary C5 activity can be detected after aerosol challenge sensitized mice with OVA. Animals developed severe airway inflammation and AHR. Intrapulmonary C5 inhibition can be achieved by nebulization administration of anti-mouse C5 mAb, BB5.1. Anti-mouse C5 mAb as well as a humanized anti-human C5 mAb can be successfully nebulized with common Jet-Air nebulizers. Both antibodies retained the activity and integrity after nebulization with a mass median aerodynamic diameter less than 2.6 m, suitable for aerosol delivery to lower airways. Aerosol delivery of antimouse C5 mAb via nebulization achieved intrapulmonary C5 inhibition without inhibiting serum C5 activity. A single nebulization of either anti-mouse C5 mAb or dexamethasone significantly blocked aerosol methacholine induced AHR in animals that had established airway inflammation. Interestingly, nebu-
Atypical haemolytic uraemic syndrome (aHUS) is frequently associated with mutations in the C-terminal region of complement factor H. To develop a murine model of aHUS we generated factor H-deficient (Cfh−/− ) mice transgenic for a mutant mouse factor H protein lacking the C-terminal five SCR domains (FH16–20). In transgenic Cfh−/− .FH16–20 mice plasma levels of FH16–20 were approximately 64% of wildtype factor H levels. The median plasma C3 level in these mice (77.6 mg/l) was 45% of the median level (171.6 mg/l) in littermate Cfh+/− .FH16–20 mice demonstrating that FH16–20 efficiently controls C3 activation in vivo. In contrast, FH16–20 showed markedly impaired binding to HUVEC. Spontaneous aHUS developed in Cfh−/− .FH16–20 mice aged between 6 and 12 weeks. Glomerular histology showed features of thrombotic microangiopathy including glomerular thrombosis, mesangiolysis and microaneurysm formation. Ultrastructural abnormalities included endothelial cell swelling and accumulation of floccular material under the damaged endothelium. Deposition of C3, in the absence of immunoglobulin, was evi-
Tao Peng, Xiao Su, Karen Kumor, Susan Liu-Chen, Helen Zhou, Dino Miano, Yamin Shen, Yi Wang