Human C-reactive protein does not bind to fcγRIIa on phagocyti cells

C-reactive protein (CRP) is an acute phase protein in humans with an important role in innate immunity. During inflammation it can be upregulated from a concentration of less than 1 μg/ml to as high as 500 μg/ml. CRP opsonizes foreign particles (1), activates complement (2), and can directly interact with phagocytic cells (3–6). The identification of CRP-binding receptors on phagocytic cells has been tedious, but in February 2000, Stein et al. reported in the JCI that CRP binds to FcγRIIa (CD32) and, more specifically, to the R131 polymorphic form of the receptor (6).

Repeating these experiments and using different CRP-detecting antibodies of the same isotype used in that study (a mouse IgG1), we have confirmed that anti-CRP reagents can detect interaction between CRP and leukocytes (Figure 1a). CRP, indeed, bound the H131 form to a much lesser extent in spite of similar levels of FcγRIIa expression on cells (data not shown). Identical results were obtained with two different anti-CRP antibodies and using a number of secondary reagents. Upon biotinylation of anti-CRP antibody, however, binding to cells was abrogated, even though biotinylated antibodies effectively bound CRP in ELISA (data not shown). We then generated F(ab′)₂ fragments of the mIgG1 anti-CRP antibodies by pepsin digestion, removed the Fc portion on a protein A column, and demonstrated purity of F(ab′)₂ fragments by SDS-PAGE. No residual binding of CRP to FcγRIIa-R131 on polymorphonuclear leukocytes or FcγRIIa-transfected IIA1.6 cells could be detected using F(ab′)₂ fragments (in a concentration range of 4–100 μg/ml) (Figure 1, b and d, and data not shown), even though the F(ab′)₂ fragments effectively bound CRP in ELISA. An intact Fc region of anti-CRP antibodies was thus found to be crucial for binding of CRP to FcγRIIa.

Figure 1.

Detection of CRP binding to FcγRIIa depends on Fc region of anti-CRP antibodies. Polymorphonuclear leukocytes (PMNs) were isolated from donors genotyped for FcγRIIa-R131 or H131 polymorphic forms. CRP was isolated from peritoneal fluid (kindly provided by C.E. Hack, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands), and 100 μg/ml CRP was incubated with 3 × 10⁵ PMNs (a and b) or FcγRIIa-transfected IIA1.6 cells (c and d) in PBS with 10% BSA (Roche Nederland BV, Mijdrecht, The Netherlands) and 0.05% sodium azide for 1 hour at 4°C. Cells were washed and incubated with 50 μg/ml of a whole mouse IgG1 anti-CRP antibody (Clone CRP 8; Sigma Chemical Co., St. Louis, Missouri, USA) (a and c) or F(ab′)₂ fragments of anti-CRP (b and d) for 30 minutes, washed again, and further incubated with an FITC-labeled goat F(ab′)₂ anti-mouse κ light chain antiserum (Jackson ImmunoResearch Laboratories Inc., West Grove, Pennsylvania, USA) (a, b, and d) or FITC-labeled goat F(ab′)₂ fragments of anti-mIgG1 (Southern Biotechnology Associates, Birmingham, Alabama, USA) (c) for 30 minutes. An FITC-labeled mIgG1 isotype control (DAKO A/S, Glostrup, Denmark) was included in all experiments, and cells were analyzed by flow cytometry. Data are representative of more than five individual experiments yielding almost identical results.

Our data are in excellent agreement with earlier work, where it has been documented that mIgG1 binds preferentially to the R131 form of the receptor (7, 8). This observation is, furthermore, consistent with other work documenting that CRP binding to phagocytic cells does not require Fc receptors (3, 4). Our present data indicate that FcγRIIa cannot be considered a phagocytic CRP-binding molecule, although they do not exclude the possibility that CRP interacts with other receptors on these cells. We therefore wish to alert other investigators to the dangers of using whole antibodies for detection of CRP binding. Because of interaction with Fc receptors, this approach may significantly affect the outcome of in vitro analyses.