Hundt M, Zielinska-Skowronek M, Schmidt R E
Department of Clinical Immunology, Hannover Medical School, Hannover, Germany.
Eur J Immunol. 2001 Dec;31(12):3475-83. doi: 10.1002/1521-4141(200112)31:12<3475::aid-immu3475>3.0.co;2-1.
The classic acute-phase reactant C-reactive protein (CRP) plays an important role in innate immunity. Specific CRP receptors have been described on white blood cells and were further characterized as Fcgamma receptors I and II. Here, we used biotinylated, highly purified natural CRP and recombinant human CRP from E. coli to investigate binding to white blood cells. The structural integrity of recombinant CRP was demonstrated by proof of pentamer assembly using non-denaturing gel electrophoresis. Furthermore, the functional capability was confirmed by calcium-dependent ligand binding (phosphorylcholine-coupled BSA and nuclear constituents), and by complement activation (C3 deposition). The monocytic cell line U937 expresses FcgammaRI and FcgammaRII--the proposed CRP receptors--in high density. Binding of biotinylated CRP was only detected by flow cytometry using a partially purified CRP preparation, that contained additional proteins, e.g. IgG as demonstrated by immunoblotting. Highly purified and recombinant CRP, free of IgG, were not bound. To exclude blocking of binding epitopes by labeling on recombinant CRP, biotinylation was performed at various biotin to protein ratios. In addition, competition assays demonstrated that binding of biotinylated, partially purified CRP was only inhibited by partially purified CRP and IgG, but not by highly purified and recombinant CRP. Recombinant CRP bound to U937 cells only after contamination with 0.5 microg IgG per 100 microg CRP before biotinylation. Therefore, we conclude that CRP itself is not bound to white blood cells and strongly suggest a reassessment of previous data.
经典急性期反应物C反应蛋白（CRP）在固有免疫中发挥重要作用。已在白细胞上描述了特异性CRP受体，并进一步鉴定为Fcγ受体I和II。在此，我们使用生物素化的、高度纯化的天然CRP和来自大肠杆菌的重组人CRP来研究与白细胞的结合。使用非变性凝胶电泳证明五聚体组装，证实了重组CRP的结构完整性。此外，通过钙依赖性配体结合（磷酸胆碱偶联的牛血清白蛋白和核成分）以及补体激活（C3沉积）证实了其功能能力。单核细胞系U937高密度表达FcγRI和FcγRII（即推测的CRP受体）。仅使用含有额外蛋白质（如通过免疫印迹证明的IgG）的部分纯化的CRP制剂，通过流式细胞术检测到生物素化CRP的结合。不含IgG的高度纯化和重组CRP未结合。为了排除重组CRP上的标记对结合表位的阻断，以各种生物素与蛋白质的比例进行生物素化。此外，竞争试验表明，生物素化的部分纯化CRP的结合仅被部分纯化的CRP和IgG抑制，而不被高度纯化和重组CRP抑制。重组CRP仅在生物素化前每100μg CRP被0.5μg IgG污染后才与U937细胞结合。因此，我们得出结论，CRP本身不与白细胞结合，并强烈建议重新评估以前的数据。
