Regulation of complement activation by C-reactive protein: Targeting of the inhibitory activity of C4b-binding protein

Abstracts / Molecular Immunology 44 (2007) 147–266

216 Regulation of complement activation by C-reactive protein: Targeting of the inhibitory activity of C4b-binding protein Andreas P. Sj¨oberg a , Leendert A. Trouw a , Fabian McGrath b , C. Erik Hack b,c , Anna Blom a a

Lund University, Department of Laboratory Medicine, Section of Clinical Chemistry, The Wallenberg Laboratory, University Hospital Malm¨o, Malm¨o, Sweden; b Sanquin Research at the CLB, and Laboratory for Experimental and Clinical Immunology, Academic Medical Center, Amsterdam, The Netherlands; c Department of Clinical Chemistry, VU Medical Center, Amsterdam, The Netherlands C-reactive protein (CRP) is the major acute phase protein in humans. It has been shown that CRP interacts with factor H, an inhibitor of the alternative pathway of complement, and now we demonstrate binding of CRP to the fluid-phase inhibitor of the classical pathway, C4b-binding protein (C4BP). C4BP bound both to directly immobilized recombinant CRP as well as CRP attached to phosphorylcholine (PC). The binding was sensitive to ionic strength and was enhanced in the presence of calcium. C4BP lacking beta-chain and protein S (PS), which is a form of C4BP increasing upon inflammation bound CRP with higher affinity than the C4BP–PS complex. The binding could not be blocked with monoclonal antibodies directed against peripheral parts of the alpha-chains of C4BP while the isolated central core of C4BP obtained by partial proteolytic digestion bound CRP indicating that the binding site for CRP is localized in the central core of the C4BP molecule. Furthermore we found complexes in serum from a patient with elevated CRP-level and trace amounts of CRP were also identified in a plasma derived C4BP preparation. We were also able to detect C4BP–CRP complexes in solution and established that C4BP retains full complement regulatory activity in the presence of CRP. In addition we found that C4BP can compete with C1q for binding to immobilized CRP and that it inhibits complement activation locally. We hypothesize that CRP limits excessive complement activation on targets via its interactions with both factor H and C4BP. doi:10.1016/j.molimm.2006.07.221 217 The extracellular matrix proteoglycan fibromodulin activates the classical pathway of complement by directly binding C1q b , Matthias M¨ ¨ Andreas P. Sj¨oberg a , Patrik Onnerfjord orgelin b , b a Dick Heineg˚ard , Anna Blom a

Departments of Laboratory Medicine, University Hospital Malm¨o, BMC Lund University, Sweden; b Experimental Medical Science, BMC Lund University, Sweden Components that propagate inflammation in joint disease may be derived from cartilage since the inflammation resolves after joint replacement. We found that the cartilage component fibromodulin has the ability to activate an inflammatory cascade-

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complement. Fibromodulin and immunoglobulins cause comparable deposition of C1q, C4b and C3b from human serum. Using C1q and factor B-deficient sera in combination with varying contents of metal ions, we established that fibromodulin activates both the classical and the alternative pathways of complement. Further studies revealed that fibromodulin binds directly to the globular heads of C1q, leading to activation of C1. However, deposition of the membrane attack complex and C5a release were lower in the presence of fibromodulin as compared to human immunoglobulins. This can be explained by the fact that fibromodulin also binds complement inhibitor factor H. Accordingly, fibromodulin induces increased deposition of iC3b. Factor H and C1q bind to non-overlapping sites on fibromodulin, but none of the interactions is mediated by the negatively charged keratan substituents of fibromodulin. Furthermore, fluid phase fibromodulin in normal human serum was able to inhibit complement deposition on IgG. Factor H and C1q bind to non-overlapping sites on fibromodulin while C1q but not factor H binds to an N-terminal fragment of fibromodulin previously reported to be implicated to be affected in cartilage stimulated with the inflammatory cytokine interleukin 1. Taken together our observations indicate fibromodulin as one factor involved in the sustained inflammation of the joint. doi:10.1016/j.molimm.2006.07.222 218 Mutation in factor H (Y402H) associated with age related macular degeneration (AMD) results in reduced binding activities Christine Skerka a , Nadine Lauer a , Andrea Hartmann a , Stefan Heinen a , Ursula Schl¨otzer-Schrehardt b , Andreas c W.A. Weinberger , Claudia Keilhauer d , Juergen Suehnel e , Bernhard H.F. Weber f , Peter F. Zipfel a a

Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, 07745 Jena, Friedrich Schiller University, 07743 Jena, Germany; b Department of Ophthalmology, University of Erlangen–Nuernberg, 91054 Erlangen, Germany; c Department of Ophthalmology, University of Aachen, 52074 Aachen, Germany,; d Department of Ophthalmology, Julius Maximilians University of Wuerzburg, 97080 Wuerzburg, Germany; e Biocomputing Group, Fritz Lipmann Institute for Age Research, 07745 Jena, Germany; f Institute of Human Genetics, University of Regensburg, 93042 Regensburg, Germany Age related macular degeneration is the most frequent cause of irreversible blindness in developed countries. Recently it has been proposed that the variant Y402H in the complement factor H gene increases the risk for the development of soft Drusen and advanced age related macular degeneration (AMD). However, so far it is not clear if this mutation results in functional changes of factor H, and how such changes contribute to the occurrence of Drusen and the progression to AMD. In order to define a molecular role of factor H in AMD we purified factor H from plasma of AMD patients and control persons with the genomic