CD16A-Ig competes with cell surface FcγRs and blocks immune-complex binding and phagocytosis by mouse macrophage cells. (A) CD16A-Ig blocked the binding of soluble IC to the mouse macrophage cell line P388D1 in a dose dependent manner. P388D1 cells were incubated with FITC-IC (soluble IC) in the presence and absence of various concentrations of CD16A-Ig and the cells were analyzed for binding of FITC-IC using flow cytometry. (B) Specificity of blocking by CD16A-Ig. P388D1 cells were incubated with soluble IC in the presence of the following reagents: anti-CD16A/32B mAb (2.4G2), CD16A-Ig and CD32A-Ig or mIgG2a. The FITC-IC binding to P388D1 cells was analyzed using standard flow cytometry. (C) CD16A-Ig blocked the binding of particulate IC to the mouse macrophage cells. P388D1 cells (50 μl of 5 × 106/mL) in binding buffer (PBS/5 mM EDTA/1% BSA) were incubated with PKH labeled EA (50 μL of 1.5 × 108) for 2 hours at 4°C. Binding assays were performed in the absence or presence of CD16A-Ig or CD32A-Ig, or 10 μg/mL blocking mAbs for mouse CD16A/CD32B. Monomeric mIgG2a (50 μg/mL) was used to block CD64. Blocking mAbs (2.4G2), CD16A-Ig, and CD32A-Ig were preincubated for 30 minutes at 4°C and then continuously present during their incubation with EA. EA bound to the cells were analyzed by flow cytometry. Cells incubated with PKH-labeled unopsonized-E were used as a background control. The EA binding in presence of medium was taken as 100% to calculate the percentage EA binding. Data shown are the average (mean ± SD) of 3 individual experiments. (D) CD16A-Ig blocked the phagocytosis of opsonized cells by macrophages. Phagocytosis of Alexa-EA was carried out using P388D1 cells. P388D1 cells were incubated with Alexa-EA in the absence or presence of the following blocking reagents: 2.4G2 (10 μg/mL) and CD16A-Ig (25, 50, and 75 μg/mL). The cells were then fixed and the fluorescence of phagocytosed Alexa-EA was measured using flow cytometry. Cells incubated with EA at 4°C served as the background control. The phagocytic index (PI) was calculated using the formula PI = %P × MF/100.59 The experiment was repeated twice. Data shown are the average of 3 individual experiments. *P < .01, **P < .001.
