An easily performed assay to identify the C3b and Fc receptor on human polymorphonuclear leukocytes (PMN) was developed. Salmonella typhi were directly fluoresceinated and then incubated in non-immune fresh human serum which led to C3b fixation via activation of the alternative pathway. Similarly, Type II pneumococci were fluoresceinated and opsonized with type specific rabbit anti-serum. PMN bearing C3b and Fc receptors formed rosettes with the respective bacteria which were easily readable because of their bright fluorescence. Studies employing various surface agents were employed to provide information on the physical properties of PMN receptors. Incubation of PMN at 37° C with C3-coated bacteria generated 54±5% C3b rosettes whereas PMN incubated with IgG coated bacteria yielded 78±8% rosettes. Heat inactivation of the fresh human serum at 56° C for 30 minutes completely inhibited the formation of the C3b and addition of goat anti-rabbit IgG inhibited the formation of the Fc rosettes. Pre-incubation of PMN with 1 mg/ml Trypsin, 1 mM N-ethyl maleimide, xanthine-xanthine oxidase to generate 20 nm superoxide anion and hydrogen peroxide (H2O2), 10 mM H2O2, and 8×10−4 M hydrocortisone reduced the number of C3b rosettes to values of 25±6%, 7±6%, 22±4%, 36±2%, and 24±5%, respectively, but had no significant effect on the number of Fc rosettes. Thus, this study provides evidence for selective receptors for C3b and Fc on PMN membranes.
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Boxer, L., Richardso, S. & Baehner, R. EFFECTS OF SURFACE ACTIVE AGENTS ON NEUTROPHIL RECEPTORS. Pediatr Res 11, 485 (1977). https://doi.org/10.1203/00006450-197704000-00690