NADPH oxidase-derived oxygen metabolites inhibit caspase-3–like activity and promote PS exposure in PMA-activated neutrophils. (A) PMA-stimulated neutrophils were cultured for the indicated times in the presence (▪) or absence (□) of the NADPH oxidase inhibitor DPI (10 μmol/L), and the rate of AMC liberation was determined in a fluorometric assay. Treatment of the cells with DPI did not affect the morphologic changes induced by PMA (data not shown). Solvent alone (ie, DMSO) had no discernible effect on either morphology or DEVD-AMC cleavage (data not shown). Data are presented as the mean and SD of three separate experiments performed in duplicate. (B) PS exposure was determined in neutrophils by flow cytometry after in vitro culture for 3 hours. Panel 1 shows cells treated with PMA alone (200 nmol/L). As seen in panel 2, addition of the NADPH oxidase inhibitor DPI (10 μmol/L) competely blocks PS exposure, whereas the general caspase inhibitor zVAD-cmk (10 μmol/L) has no effect in this system (panel 3). Reproducible results were obtained in three separate donors.