The major receptor for C-reactive protein on leukocytes is fcgamma receptor II

Abstract

C-reactive protein (CRP) is an acute phase serum protein that shares several functions with immunoglobulin (Ig)G including complement activation and binding to receptors on monocytes and neutrophils. The identity of the receptor for CRP has been the target of extensive research. We previously determined that CRP binds to the high affinity receptor for IgG, FcgammaRI (CD64). However, this interaction could not account for the majority of binding of CRP to neutrophils or monocytic cells. We now determine that CRP also interacts with FcgammaRIIa (CD32), the low affinity receptor for IgG on monocytes and neutrophils. COS-7 cells were transfected with a construct containing the human FcgammaRIIA cDNA. CRP binding and the presence of CD32 were detected by mAb and analyzed by two-color flow cytometry. Cells expressing CD32 bound CRP in a dose-dependent and saturable manner consistent with receptor binding. CRP bound to transfectants and K-562 cells with similar kinetics, and in both cases binding was completely inhibited by aggregated IgG. On monocytic cell lines, treatment with Bt(2)cAMP increased FcgammaRII expression and enhanced CRP binding. CRP also specifically precipitated FcgammaRI and FcgammaRII from the monocytic cell line, THP-1. It is suggested that the major receptor for CRP on phagocytic cells is FcgammaRII.

Figure 1

CRP binds to COS–7 cells transfected with the FcγRIIA cDNA, and to K-562 cells with similar kinetics. Cells were incubated with increasing doses of CRP and binding was detected with 2C10 and PE-GAM, as described in Materials and Methods. Results are expressed as the increase in gMCF compared with the secondary antibody controls. (A) COS-7 cells transfected with the FcγRIIA plasmid or pcDSRα296, or mock-transfected. Specific binding was determined by subtracting gMCF of mock-transfected cells from gMCF of transfected cells. (B) K-562 cells.

Figure 2

Analysis of CRP binding to transfected COS-7 cells by two-color flow cytometry. COS-7 cells were transfected with the FcγRIIA plasmid, pcDSRα296. Mock-transfected cells received no DNA. Cells were incubated with 200 μg/ml of CRP and binding was detected with 2C10 and PE-GAM as described in Materials and Methods. Levels of FcγRII were determined by binding of FITC-AT10. A, B, C, and D, transfected; E, F, G, and H, mock-transfected. A and E, staining with 2C10 and PE-GAM; B and F, staining for CD-32 only; C and G, staining for CRP only; D and H, staining for CRP and CD32.

Figure 4

Treatment of U-937 cells with Bt₂cAMP increases CRP binding. U-937 cells were grown for 60 h in medium containing 1 mM Bt₂cAMP before analysis of CRP binding. CRP binding was detected with 2C10 and PE-GAM as described in Materials and Methods. Results are expressed as the increase in gMCF compared with the secondary antibody controls. (Inset) FcγRI and FcγRII levels were measured using mAb 32.2 and PE-F(ab′)₂ GAM IgG and PE-C1KM5, respectively.

Figure 3

CRP binding to FcγRIIa is inhibited by aggIgG. K-562 cells or COS-7 cells transfected with the FcγRIIA plasmid, pcDSRα296, were incubated with 25 μg/ml of CRP in the presence of increasing concentrations of aggIgG. CRP binding was detected with 2C10 and PE-GAM as described in Materials and Methods. Results are expressed as the increase in gMCF compared with the secondary antibody controls. The solid line represents binding of CRP to K-562 cells and the dotted line represents the binding of CRP to transfected COS cells in the absence of aggIgG.

Figure 5

Immobilized CRP precipitates FcγRI and FcγRII from radiolabeled, pronase-treated THP-1 cells. THP-1 cells were treated with 50 μg/ml pronase, radiolabeled with ¹²⁵I, and lysed with 1% NP-40. (A) Membrane extracts (2.5 × 10⁶ cell equivalents) were incubated with CRP beads, anti-CD32 (mAb IV.3) protein A–Sepharose, or anti-CD64 (mAb 32.2) protein A–Sepharose, and then washed. The samples were boiled in Laemmli's sample buffer and analyzed by SDS-PAGE. Autoradiography was performed using a Storm imaging system. MW, mol wt markers: lane 1, Affigel; lane 2, CRP-Affigel; lane 3, protein A–Sepharose; lane 4, anti-CD64–protein A–Sepharose; lane 5, anti-CD32–protein A–Sepharose.