A microscopic study of Fc gamma RIII-mediated respiratory burst in neutrophils

We investigated the relationship between the respiratory burst in neutrophils and the membrane distribution of the IgG receptor, Fc gamma RIII. Fc gamma RIII receptors were labeled with a fluoresceinated antibody that does not block binding of immune complexes. The respiratory burst was detected using covalently bound rosamine stain previously described for flow cytometric applications. This method allows visualization of intracellular oxidant production in fixed cells using attenuated illumination with a laser. Strong cytosolic oxidation of rosamine was observed only in those cells that displayed prominent receptor endocytosis upon interaction with insoluble immune complexes. Soluble immune complexes or insoluble complexes in the presence of cytochalasin B did not stimulate endocytosis of Fc gamma RIII and induced no rosamine oxidation. Extracellular superoxide production measured by the cytochrome c test did not correlate with intracellular rosamine oxidation: it was maximal in cytochalasin-treated cells and did not require any visible receptor rearrangement. Our results demonstrate the utility of the rosamine stain as an intracellular marker of the oxidative burst, support the role of Fc gamma RIII in neutrophil activation and emphasize the compartmental regulation of the oxidative burst.