Lack of specific receptors for C-reactive protein on white blood cells.
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.
Hundt
M
M
Department of Clinical Immunology, Hannover Medical School, Hannover, Germany. Hundt.Matthias@MH-Hannover.de
Zielinska-Skowronek
M
M
Schmidt
R E
RE
D001665:Binding Sites; D019921:Biotinylation; D002097:C-Reactive Protein / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D019169:Jurkat Cells; D007962:Leukocytes / Q000737:chemistry*; D017452:Receptors, IgG / Q000378:metabolism; D011971:Receptors, Immunologic / Q000097:blood*; D011994:Recombinant Proteins / Q000737:chemistry; D020298:U937 Cells
C071581:CRP protein, human; D007074:Immunoglobulin G; D017452:Receptors, IgG; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D002097:C-Reactive Protein