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Identification of the gC1qR binding sites for HIV-1 gp41 and HCV core protein
Abstracts / Immunobiology 217 (2012) 1129–1222
136 Identification of the gC1qR binding sites for HIV-1 gp41 and HCV core protein Lina Pednekar 1 , Nithin Tumma 2 , Christopher Valentino 2 , Kinga K. Hosszu 2 , Alisa Valentino 2 , Ellinor I.B. Peerschke 3 , Uday Kishore 1 , Berhane Ghebrehiwet 2 1 Center for Infection, Immunity and Disease Mechanisms, Brunel University, Uxbridge, UK 2 The Department of Medicine, Sony Brook University, Stony Brook, NY, USA 3 The Department of Laboratory Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA
A substantial body of evidence accumulated over the past 20 years supports the concept that gC1qR is a major pathogenassociated pattern recognition receptor. This conclusion is based on the fact that, a wide range of bacterial and viral ligands are able to exploit gC1qR to either suppress the host’s immune response and thus enhance their survival, or to gain access into cells to initiate disease. Of the extensive array of viral ligands that have affinity for gC1qR, the HIV-1 envelope glycoprotein gp41, and the core protein of hepatitis C virus (HCV) are of major interest as they are known to contribute to the high morbidity and mortality caused by these pathogens. While the HCV core protein binds gC1qR and suppresses T cell proliferation resulting in a significantly diminished immune response, the gp41 employs gC1qR to induce the surface expression of the NK cell ligand, NKp44L, on uninfected CD4+ T cells thus rendering them susceptible to autologous destruction by NKp44 expressing NK cells. Because of the potential for the design of peptide-based or antibody-based therapeutic options, the present studies were undertaken to define the gC1qR interaction sites for these pathogen-associated molecular ligands. Employing a solid phase microplate-binding assay, we examined the binding of each viral ligand to wild type gC1qR and 10 gC1qR deletion mutants. Results obtained from these studies conclusively identify a major HCV core protein site on a domain of gC1qR comprising of residues 196–202. This domain is located in the last half of the larger gC1qR segment encoded by exons IV–VI (residues 159–282), which was proposed previously to contain the site for HCV core protein (Kittlesen et al. 2000). The gC1qR site for gp41 on the other hand, was found to be in a highly conserved region encoded by exon IV and comprises of residues 174–180. Interestingly, gC1qR residues 174–180 also constitute the cell surface-binding site for soluble gC1qR (sgC1qR), which can bind to the cell surface in an autocrine/paracrine manner via a fibrinogen-like molecule. http://dx.doi.org/10.1016/j.imbio.2012.08.138 137 IgG2a immune complexes enhance C5a-mediated inflammation Marina Wagner 1 , Christian M. Karsten 1 , Jörg Köhl 1,2 1
Institute for Systemic Inflammation Research, UKSH, Lübeck, Germany 2 Division of Cellular and Molecular Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA C5a promotes many pro-inflammatory effects in immune complex (IC)-mediated autoimmune diseases including rheumatoid arthritis or systemic lupus erythematosus. The onset of these diseases is also Fc gamma receptor (FcgR)-dependent, since the inflammatory response is reduced in the absence of activating FcgRs and more severe, when the inhibitory FcgRIIB is lacking. Nevertheless, despite the critical roles of C5a and FcgRs in
1177
autoimmune diseases, our knowledge about the cross-talk between C5aR and FcgRs is limited. Previously, we found that highly galactosyalated IgG1 IC induce an FcgRIIB-dependent signaling pathway that blocks C5aR-mediated extracellular signal-regulated kinases (ERK) 1/2 phosphorylation and effector functions, including neutrophil chemotaxis in vivo and in vivo, upregulation of CD11b and increase of intracellular calcium. Here we determined the effects of the IgG2a isotype on several C5a-mediated effector functions. We performed in vitro assays using bone marrow derived neutrophils. In a chemotaxis assay, we found that neutrophils pretreated with IgG2a IC showed increased migration towards C5a. This effect was dependent on the presence of FcgRIV, since pharmacological targeting by an FcgRIV-specific monoclonal antibody or using cells from FcgRIV knock out mice abrogated the inhibitory effect. Investigating the signaling pathways driving the enhancing effect of IgG2a, we found that the effect is spleen tyrosine kinase (SYK)dependent, since we observed no enhancing IgG2a IC effect in cells treated with SYK inhibitors or in cells from SYK−/− mice. Surprisingly, the effect was limited to the chemotactic response. We found no changes in CD11b expression or in intracellular calcium following IgG2a treatment. In summary, we describe a novel pathway by which IgG2a IC enhance the chemotactic potency of C5a. This effect may enhance complement-mediated bacterial defense and tumor killing but could be detrimental in Th1-associated autoimmune diseases. http://dx.doi.org/10.1016/j.imbio.2012.08.139 138 IgM-mediated enhancement of immune responses Zhoujie Ding, Anna Bergman, Christian Rutemark, Birgitta Heyman Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden IgM administered with its specific antigen induces a higher antibody response than antigen administered alone. Data suggest that this requires complement activation. In line with this, animals lacking C1q, C2, C3, C4, and complement receptors 1 and 2 (CR1/2) have impaired antibody responses. We have elucidated three aspects of IgM-mediated enhancement: (i) its dependence of complement activation, (ii) its dependence of expression of CR1/2 on B cells and/or follicular dendritic cells, and (iii) its ability to enhance CD4+T cell responses. A novel knock-in mouse strain with a point mutation in the gene encoding the third constant domain of the mu heavy chain was used. These mice produce IgM where proline in position 436 is substituted with serine, a mutation previously shown to abrogate the ability of mouse IgM to activate C. IgM specific for SRBC or KLH were obtained from these animals as well as from WT controls. These antibodies were administered to mice together with SRBC or KLH respectively. Whereas WT IgM enhanced the specific antibody response, the same amount of mutant IgM failed to do so. Four groups of bone marrow chimeric mice (expressing CR1/2 on both B cells and FDC, on neither cell type, or on either B cells or FDC) were generated. Upon immunization with IgM antiSRBC and SRBC, it was evident that expression of CR1/2 on FDC was most important for a robust antibody response. However, in mice where also B cells expressed CR1/2, the responses were further enhanced. To analyze whether IgM could enhance the proliferation of CD4+T cells, OVA-specific transgenic T cells from DO11.10 mice were transferred to WT recipients. These animals were immunized with IgM and SRBC-OVA or SRBC-OVA alone. Whereas IgM enhanced the antibody response, no effect on CD4+T cell responses was seen. In conclusion, IgM enhances the antibody but not the T
136 Identification of the gC1qR binding sites for HIV-1 gp41 and HCV core protein Lina Pednekar 1 , Nithin Tumma 2 , Christopher Valentino 2 , Kinga K. Hosszu 2 , Alisa Valentino 2 , Ellinor I.B. Peerschke 3 , Uday Kishore 1 , Berhane Ghebrehiwet 2 1 Center for Infection, Immunity and Disease Mechanisms, Brunel University, Uxbridge, UK 2 The Department of Medicine, Sony Brook University, Stony Brook, NY, USA 3 The Department of Laboratory Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA
A substantial body of evidence accumulated over the past 20 years supports the concept that gC1qR is a major pathogenassociated pattern recognition receptor. This conclusion is based on the fact that, a wide range of bacterial and viral ligands are able to exploit gC1qR to either suppress the host’s immune response and thus enhance their survival, or to gain access into cells to initiate disease. Of the extensive array of viral ligands that have affinity for gC1qR, the HIV-1 envelope glycoprotein gp41, and the core protein of hepatitis C virus (HCV) are of major interest as they are known to contribute to the high morbidity and mortality caused by these pathogens. While the HCV core protein binds gC1qR and suppresses T cell proliferation resulting in a significantly diminished immune response, the gp41 employs gC1qR to induce the surface expression of the NK cell ligand, NKp44L, on uninfected CD4+ T cells thus rendering them susceptible to autologous destruction by NKp44 expressing NK cells. Because of the potential for the design of peptide-based or antibody-based therapeutic options, the present studies were undertaken to define the gC1qR interaction sites for these pathogen-associated molecular ligands. Employing a solid phase microplate-binding assay, we examined the binding of each viral ligand to wild type gC1qR and 10 gC1qR deletion mutants. Results obtained from these studies conclusively identify a major HCV core protein site on a domain of gC1qR comprising of residues 196–202. This domain is located in the last half of the larger gC1qR segment encoded by exons IV–VI (residues 159–282), which was proposed previously to contain the site for HCV core protein (Kittlesen et al. 2000). The gC1qR site for gp41 on the other hand, was found to be in a highly conserved region encoded by exon IV and comprises of residues 174–180. Interestingly, gC1qR residues 174–180 also constitute the cell surface-binding site for soluble gC1qR (sgC1qR), which can bind to the cell surface in an autocrine/paracrine manner via a fibrinogen-like molecule. http://dx.doi.org/10.1016/j.imbio.2012.08.138 137 IgG2a immune complexes enhance C5a-mediated inflammation Marina Wagner 1 , Christian M. Karsten 1 , Jörg Köhl 1,2 1
Institute for Systemic Inflammation Research, UKSH, Lübeck, Germany 2 Division of Cellular and Molecular Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA C5a promotes many pro-inflammatory effects in immune complex (IC)-mediated autoimmune diseases including rheumatoid arthritis or systemic lupus erythematosus. The onset of these diseases is also Fc gamma receptor (FcgR)-dependent, since the inflammatory response is reduced in the absence of activating FcgRs and more severe, when the inhibitory FcgRIIB is lacking. Nevertheless, despite the critical roles of C5a and FcgRs in
1177
autoimmune diseases, our knowledge about the cross-talk between C5aR and FcgRs is limited. Previously, we found that highly galactosyalated IgG1 IC induce an FcgRIIB-dependent signaling pathway that blocks C5aR-mediated extracellular signal-regulated kinases (ERK) 1/2 phosphorylation and effector functions, including neutrophil chemotaxis in vivo and in vivo, upregulation of CD11b and increase of intracellular calcium. Here we determined the effects of the IgG2a isotype on several C5a-mediated effector functions. We performed in vitro assays using bone marrow derived neutrophils. In a chemotaxis assay, we found that neutrophils pretreated with IgG2a IC showed increased migration towards C5a. This effect was dependent on the presence of FcgRIV, since pharmacological targeting by an FcgRIV-specific monoclonal antibody or using cells from FcgRIV knock out mice abrogated the inhibitory effect. Investigating the signaling pathways driving the enhancing effect of IgG2a, we found that the effect is spleen tyrosine kinase (SYK)dependent, since we observed no enhancing IgG2a IC effect in cells treated with SYK inhibitors or in cells from SYK−/− mice. Surprisingly, the effect was limited to the chemotactic response. We found no changes in CD11b expression or in intracellular calcium following IgG2a treatment. In summary, we describe a novel pathway by which IgG2a IC enhance the chemotactic potency of C5a. This effect may enhance complement-mediated bacterial defense and tumor killing but could be detrimental in Th1-associated autoimmune diseases. http://dx.doi.org/10.1016/j.imbio.2012.08.139 138 IgM-mediated enhancement of immune responses Zhoujie Ding, Anna Bergman, Christian Rutemark, Birgitta Heyman Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden IgM administered with its specific antigen induces a higher antibody response than antigen administered alone. Data suggest that this requires complement activation. In line with this, animals lacking C1q, C2, C3, C4, and complement receptors 1 and 2 (CR1/2) have impaired antibody responses. We have elucidated three aspects of IgM-mediated enhancement: (i) its dependence of complement activation, (ii) its dependence of expression of CR1/2 on B cells and/or follicular dendritic cells, and (iii) its ability to enhance CD4+T cell responses. A novel knock-in mouse strain with a point mutation in the gene encoding the third constant domain of the mu heavy chain was used. These mice produce IgM where proline in position 436 is substituted with serine, a mutation previously shown to abrogate the ability of mouse IgM to activate C. IgM specific for SRBC or KLH were obtained from these animals as well as from WT controls. These antibodies were administered to mice together with SRBC or KLH respectively. Whereas WT IgM enhanced the specific antibody response, the same amount of mutant IgM failed to do so. Four groups of bone marrow chimeric mice (expressing CR1/2 on both B cells and FDC, on neither cell type, or on either B cells or FDC) were generated. Upon immunization with IgM antiSRBC and SRBC, it was evident that expression of CR1/2 on FDC was most important for a robust antibody response. However, in mice where also B cells expressed CR1/2, the responses were further enhanced. To analyze whether IgM could enhance the proliferation of CD4+T cells, OVA-specific transgenic T cells from DO11.10 mice were transferred to WT recipients. These animals were immunized with IgM and SRBC-OVA or SRBC-OVA alone. Whereas IgM enhanced the antibody response, no effect on CD4+T cell responses was seen. In conclusion, IgM enhances the antibody but not the T