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Complement C4d deposition on T lymphocytes: Mechanisms and significance in systemic lupus erythematosus (SLE)
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Abstracts / Molecular Immunology 44 (2007) 147–266
133 Dissecting complement regulatory sites in the smallpox inhibitor of complement enzymes (SPICE) M.K. Liszewski, M.K. Leung, C.J. Fang, T.P. Srokowski, P. Bertram, J.P. Atkinson Division of Rheumatology, Washington University School of Medicine, St. Louis, MO, USA Public health concerns have emerged regarding use of smallpox as a bioterrorist weapon. Clandestine stockpiles exist in rogue nations and most citizens are no longer immune. Poxviruses express virulence factors that down-modulate the host’s complement system. In previous studies, we constructed SPICE from the vaccinia complement control protein (VCP) by substituting the 11 amino acid differences of SPICE into VCP. Both proteins were then expressed in mammalian and bacterial systems. We confirmed that SPICE possessed enhanced activity over VCP. Further, we demonstrated that VCP and SPICE, surprisingly, possessed only classical pathway decay accelerating activity. Interestingly, SPICE and VCP produced disulfide-linked, function-enhancing homodimers, especially in mammalian systems. Our present studies were aimed at dissecting active sites of SPICE. Using VCP-SPICE chimeras, a general observation was that C3b and C4b regulatory activity increased as more SPICE amino acids were substituted into VCP. We also targeted a region in the second complement control module (CCP2) that is a functionally important site in the related human regulators. We identified residues in CCP2 of SPICE that were critical for C3b binding and cofactor activities. Other observations included substitutions in CCP2 that selectively diminish (a) only C4b activity or (b) only C3b/C4b cofactor activity while maintaining ligand binding capability. The electrophoretic differences on SDS-PAG between SPICE and VCP mapped to a single amino acid in CCP3. We have previously determined that SPICE binds heparin. SPICE sequence suggests the presence of three heparin binding sites in CCP1. By substitution mutagenesis analysis, we found a hierarchy of importance for binding to heparin in which site 2 > site 3 > site 1. Our studies extend our knowledge of the complement inhibitory sites within poxviral inhibitors of complement, suggest possible contact points with heparin, and have implications for the rational design of therapeutic agents against smallpox.
134 Complement C4d deposition on T lymphocytes: Mechanisms and significance in systemic lupus erythematosus (SLE) Chau-Ching Liu, Susan Manzi, Joseph M. Ahearn Lupus Center of Excellence, Division of Rheumatology and Clinical Immunology, University of Pittsburgh Schools of the Health Sciences, Pittsburgh, PA, United States SLE, the prototypic autoimmune disease, is characterized by aberrant immune responses including lymphocyte hyperreactivity, autoantibody production, and complement activation. The consequences of complement activation with regard to T cell function in SLE are largely unexplored. We hypothesize that complement activation-derived products may bind to T cells and cause functional dysregulation of these cells, thereby participating in SLE pathogenesis. The current study was performed to characterize C4d deposition on T cells of SLE patients and to investigate its underlying mechanisms and functional consequences. T-C4d and lymphocyte-reactive autoantibodies were determined by flow cytometric analyses of peripheral blood obtained from SLE patients (n = 136), patients with other inflammatory diseases (n = 119), and healthy controls (n = 91). The results demonstrate that highly elevated levels of C4d are present on T cells of SLE patients (mean fluorescence intensity (MFI) = 23, range 1–198), compared with T cells from patients with other diseases (MFI = 5, range 0–54, p < 0.00001) or healthy controls (MFI = 3, range 0–14, p < 0.00001). In a significant fraction of SLE patients, IgM and/or IgG were detected concomitantly with C4d on T cells, suggesting a process triggered by lymphocyte-reactive autoantibodies. In vitro functional studies demonstrate that C4d-positive T cells derived from SLE patients proliferate more profoundly in response to polyclonal T cell stimulation as compared to C4d-negative T cells. Furthermore, C4d-positive T cells exhibit abnormalities in signal transduction as evidenced by aberrant phosphorylation of signaling proteins upon stimulation. Together, these results support that: (1) C4d deposition on T cells results, at least in part, from an autoantibody-mediated mechanism and (2) C4d may participate in T cell dysfunction in SLE by covalent attachment to molecules on the T cell membrane. Future investigations into the molecular basis underlying C4d deposition on T cells may lead to additional clues to SLE pathogenesis and may identify targets for therapeutic intervention.
doi:10.1016/j.molimm.2006.07.138 doi:10.1016/j.molimm.2006.07.139
Abstracts / Molecular Immunology 44 (2007) 147–266
133 Dissecting complement regulatory sites in the smallpox inhibitor of complement enzymes (SPICE) M.K. Liszewski, M.K. Leung, C.J. Fang, T.P. Srokowski, P. Bertram, J.P. Atkinson Division of Rheumatology, Washington University School of Medicine, St. Louis, MO, USA Public health concerns have emerged regarding use of smallpox as a bioterrorist weapon. Clandestine stockpiles exist in rogue nations and most citizens are no longer immune. Poxviruses express virulence factors that down-modulate the host’s complement system. In previous studies, we constructed SPICE from the vaccinia complement control protein (VCP) by substituting the 11 amino acid differences of SPICE into VCP. Both proteins were then expressed in mammalian and bacterial systems. We confirmed that SPICE possessed enhanced activity over VCP. Further, we demonstrated that VCP and SPICE, surprisingly, possessed only classical pathway decay accelerating activity. Interestingly, SPICE and VCP produced disulfide-linked, function-enhancing homodimers, especially in mammalian systems. Our present studies were aimed at dissecting active sites of SPICE. Using VCP-SPICE chimeras, a general observation was that C3b and C4b regulatory activity increased as more SPICE amino acids were substituted into VCP. We also targeted a region in the second complement control module (CCP2) that is a functionally important site in the related human regulators. We identified residues in CCP2 of SPICE that were critical for C3b binding and cofactor activities. Other observations included substitutions in CCP2 that selectively diminish (a) only C4b activity or (b) only C3b/C4b cofactor activity while maintaining ligand binding capability. The electrophoretic differences on SDS-PAG between SPICE and VCP mapped to a single amino acid in CCP3. We have previously determined that SPICE binds heparin. SPICE sequence suggests the presence of three heparin binding sites in CCP1. By substitution mutagenesis analysis, we found a hierarchy of importance for binding to heparin in which site 2 > site 3 > site 1. Our studies extend our knowledge of the complement inhibitory sites within poxviral inhibitors of complement, suggest possible contact points with heparin, and have implications for the rational design of therapeutic agents against smallpox.
134 Complement C4d deposition on T lymphocytes: Mechanisms and significance in systemic lupus erythematosus (SLE) Chau-Ching Liu, Susan Manzi, Joseph M. Ahearn Lupus Center of Excellence, Division of Rheumatology and Clinical Immunology, University of Pittsburgh Schools of the Health Sciences, Pittsburgh, PA, United States SLE, the prototypic autoimmune disease, is characterized by aberrant immune responses including lymphocyte hyperreactivity, autoantibody production, and complement activation. The consequences of complement activation with regard to T cell function in SLE are largely unexplored. We hypothesize that complement activation-derived products may bind to T cells and cause functional dysregulation of these cells, thereby participating in SLE pathogenesis. The current study was performed to characterize C4d deposition on T cells of SLE patients and to investigate its underlying mechanisms and functional consequences. T-C4d and lymphocyte-reactive autoantibodies were determined by flow cytometric analyses of peripheral blood obtained from SLE patients (n = 136), patients with other inflammatory diseases (n = 119), and healthy controls (n = 91). The results demonstrate that highly elevated levels of C4d are present on T cells of SLE patients (mean fluorescence intensity (MFI) = 23, range 1–198), compared with T cells from patients with other diseases (MFI = 5, range 0–54, p < 0.00001) or healthy controls (MFI = 3, range 0–14, p < 0.00001). In a significant fraction of SLE patients, IgM and/or IgG were detected concomitantly with C4d on T cells, suggesting a process triggered by lymphocyte-reactive autoantibodies. In vitro functional studies demonstrate that C4d-positive T cells derived from SLE patients proliferate more profoundly in response to polyclonal T cell stimulation as compared to C4d-negative T cells. Furthermore, C4d-positive T cells exhibit abnormalities in signal transduction as evidenced by aberrant phosphorylation of signaling proteins upon stimulation. Together, these results support that: (1) C4d deposition on T cells results, at least in part, from an autoantibody-mediated mechanism and (2) C4d may participate in T cell dysfunction in SLE by covalent attachment to molecules on the T cell membrane. Future investigations into the molecular basis underlying C4d deposition on T cells may lead to additional clues to SLE pathogenesis and may identify targets for therapeutic intervention.
doi:10.1016/j.molimm.2006.07.138 doi:10.1016/j.molimm.2006.07.139