Agonist-dependent Superoxide Formation

Human monocytes purified by elutriation were cultured for 3 d in Teflon bags with or without human recombinant interferongamma (rIFN'y). The cells were then collected and used in suspension to determine the rate of stimulus-dependent superoxide or hydrogen peroxide formation as a measure of the NADPH-oxidase. The treatment with IFN7 increased this rate twoto threefold when phorbol myristate acetate (PMA) was used as the stimulus. By contrast, no IFNy-dependent increase in superoxide production was observed when the cells were stimulated with different concentrations of the receptor agonist N-formyl-methionyl-leucyl-phenylalanine (f-Met-LeuPhe) alone or in combination with another receptor agonist, platelet-activating factor (PAF). At optimum concentrations, f-Met-Leu-Phe elicited rates of superoxide formation that could not be exceeded under other stimulatory conditions including PMA after treatment with IFNy. It thus appears that f-Met-Leu-Phe can lead to maximum activation of the NADPH-oxidase, and that this response is not influenced by IFN'y. Treatment with IFNy also failed to affect the affinity of PMAor f-Met-Leu-Phe-stimulated oxidase for NADPH, the K. values being 30 to 40 ;&M under all conditions. IFN-y did not alter the cellular levels of cytochrome bss5, as measured by low-temperature spectroscopy, and protein kinase C, as measured by [3Hjphorbol dibutyrate binding, and did not appreciably influence the stimulus-dependent increase of cytosolic free calcium. These results indicate that activation of human mononuclear phagocytes by IFNy does not affect the level and the kinetic properties of NADPH-oxidase or its activation by receptor agonists. They confirm, however, that IFN-y enhances the respiratory burst response to PMA.