C-reactive protein inhibits plasmacytoid dendritic cell interferon responses to autoantibody immune complexes.

OBJECTIVE: C-reactive protein (CRP) is a serum pattern recognition molecule that binds to apoptotic cells and nucleoprotein autoantigens and Fcγ receptors (FcγR). In systemic lupus erythematosus (SLE), immune complexes (ICs) containing nucleoprotein autoantigens activate plasmacytoid dendritic cells (PDCs) to produce type I interferon (IFN), which contributes to disease pathogenesis. Autoantibody ICs are taken up by PDCs through FcγR type IIa into endosomes, where the nucleic acid components activate Toll-like receptor 7 (TLR-7) or TLR-9. The objective of this study was to investigate the effect of CRP on PDC and monocyte responses to nucleoprotein autoantigens and ICs. METHODS: Peripheral blood mononuclear cells (PBMCs), purified monocytes, and PDCs were isolated from healthy volunteers and stimulated with autoantibody ICs containing apoptotic cells, small nuclear RNPs (snRNPs), or DNA, or directly with TLR-7 and TLR-9 agonists. Supernatants were analyzed for IFNα and cytokine levels by enzyme-linked immunosorbent assay and multiplex assay. Small nuclear RNPs were fluorescence-labeled, and the effect of CRP on binding, uptake, and intracellular localization of autoantibody snRNP complexes was measured by flow cytometry and confocal microscopy. RESULTS: CRP bound to autoantigen did not induce IFNα in PBMCs or PDCs, whereas complexes formed with autoantibody did. Significantly, CRP inhibited the IFNα response to both anti-U1 RNP-snRNP complexes and anti-DNA-DNA complexes, but not to other TLR-7 and TLR-9 agonists. CRP directly inhibited PDC IFNα release, promoted PDC differentiation, and increased late endosome localization of autoantigen in PDCs and monocytes. CONCLUSION: CRP is a regulator of the type I IFN response to SLE ICs. CRP increased the intracellular processing of ICs in late endosomes, which is associated with decreased synthesis of type I IFN after intracellular TLR activation.

Analysis of binding sites in human C-reactive protein for Fc{gamma}RI, Fc{gamma}RIIA, and C1q by site-directed mutagenesis.

Human C-reactive protein (CRP) is a classical, acute phase serum protein synthesized by the liver in response to infection, inflammation, or trauma. CRP binds to microbial antigens and damaged cells, opsonizes particles for phagocytosis and regulates the inflammatory response by the induction of cytokine synthesis. These activities of CRP depend on its ability to activate complement and to bind to Fcgamma receptors (FcgammaR). The goal of this study was to elucidate amino acid residues important for the interaction of CRP with human FcgammaRI (CD64) and FcgammaRIIa (CD32). Several mutations of the CRP structure were studied based on the published crystal structure of CRP. Mutant and wild-type recombinant CRP molecules were expressed in the baculovirus system and their interactions with FcgammaR and C1q were determined. A previous study by our laboratory identified an amino acid position, Leu(176), critical for CRP binding to FcgammaRI and work by others (Agrawal, A., Shrive, A. K., Greenhough, T. J., and Volanakis, J. E. (2001) J. Immunol. 166, 3998-4004) determined several residues important for C1q binding. The amino acid residues important to CRP binding to FcgammaRIIa were previously unknown. This study newly identifies residues Thr(173) and Asn(186) as important for the binding of CRP to FcgammaRIIa and FcgammaRI. Lys(114), like Leu(176), was implicated in binding to FcgammaRI, but not FcgammaRIIa. Single mutations at amino acid positions Lys(114), Asp(169), Thr(173), Tyr(175), and Leu(176) affected C1q binding to CRP. These results further identify amino acids involved in the binding sites on CRP for FcgammaRI, FcgammaRIIa, and C1q and indicate that these sites are overlapping.