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

Bharadwaj D, Stein MP, Volzer M, Mold C, Du Clos TW - J. Exp. Med. (1999)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of New Mexico School of Medicine, Albuquerque, New Mexico 87108, USA.

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.

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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.
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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.

Mentions: In an attempt to identify proteins reacting with CRP on FcR-bearing cells, receptor precipitation studies were performed. THP-1 cells express both FcγRI and FcγRII. Attempts to immunoprecipitate surface-labeled proteins with immobilized CRP showed weak and nonspecific bands. However, it had previously been determined that the interaction of IgG with FcγRII could be enhanced by pronase treatment of cells 18. We recently determined that pronase treatment of FcγRII-bearing cells also markedly increases the binding of CRP (Stein, M.-P., J.C. Edberg, R.P. Kimberly, E.K. Mangan, D. Bharadwaj, C. Mold, and T.W. Du Clos, manuscript submitted for publication). When pronase-treated cells were radiolabeled and precipitated with CRP, distinct binding of two major bands was seen. These bands with Mr of 70 and 42 kD corresponded to the bands precipitated by mAb to CD64 and CD32 (Fig. 5).


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

Bharadwaj D, Stein MP, Volzer M, Mold C, Du Clos TW - J. Exp. Med. (1999)

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.
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Related In: Results  -  Collection

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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.
Mentions: In an attempt to identify proteins reacting with CRP on FcR-bearing cells, receptor precipitation studies were performed. THP-1 cells express both FcγRI and FcγRII. Attempts to immunoprecipitate surface-labeled proteins with immobilized CRP showed weak and nonspecific bands. However, it had previously been determined that the interaction of IgG with FcγRII could be enhanced by pronase treatment of cells 18. We recently determined that pronase treatment of FcγRII-bearing cells also markedly increases the binding of CRP (Stein, M.-P., J.C. Edberg, R.P. Kimberly, E.K. Mangan, D. Bharadwaj, C. Mold, and T.W. Du Clos, manuscript submitted for publication). When pronase-treated cells were radiolabeled and precipitated with CRP, distinct binding of two major bands was seen. These bands with Mr of 70 and 42 kD corresponded to the bands precipitated by mAb to CD64 and CD32 (Fig. 5).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of New Mexico School of Medicine, Albuquerque, New Mexico 87108, USA.

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.

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