Effect of neutrophil activation on binding of soluble immune complexes by CD32A.
125I-Transferrin/rabbit antihuman transferrin IgG immune complex (125I-IC) was prepared as described in “Materials and Methods.” The molecular size of the IC was determined by gel filtration. fMLP (1 μmol/L) activation of neutrophils was performed as described in the legend to Figure 5. After washing the cells with RPMI 1640/2% IgG-free fetal bovine serum,125I-IC binding was carried out as described in “Materials and Methods.” Total binding (A) of IC to unactivated (open circle) and activated (closed circle) neutrophils was performed in the presence of a binding buffer. CD32A-dependent binding (C) was performed in the presence of Fab fragments of anti-CD16 mAb, CLBFcgran-1. Scatchard plot analysis of total binding (B) and CD32A-dependent binding (D) was done after converting the specific binding to bound/free IC as functions of the bound IC. Binding in the presence of a 50-fold excess of Fab fragments of mAbs against CD16B and CD32A was taken as nonspecific binding. The IC bound to neutrophils in binding buffer was taken as 100%. Experiments were performed in triplicate.
