Neutrophils in lung inflammation: Which reactive oxygen species are being measured?

The oxidative burst in circulating polymorphonuclear leukocytes (PMN) plays a fundamental role in pulmonary defense and injury. Flow cytometric techniques have been developed for quantitation of oxidative burst activity at the single cell level using 2',7'-dichlorofluorescin (DCFH). However, the specific reactive oxidant species being measured using this method are not clearly defined. Isolated human PMN were loaded with DCFH diacetate, stimulated with phorbol myristate acetate (PMA) in the presence or absence of specific reagents, and analyzed using flow cytometry. Addition of PMA resulted in a 90-fold increase in the fluorescence intensity of DCFH-loaded neutrophils (p <.01). Inhibition of NADPH oxidase activity using a calmodulin antagonist (W-13) decreased PMA-induced DCFH oxidation by 70% (p <.05). Inhibition of nitric oxide synthase using N(G)-monomethyl-L-arginine (NMMA) did not significantly reduce DCFH oxidation, and did not alter the action of W-13. Addition of superoxide dismutase (SOD) had no effect, but catalase, with or without SOD, suppressed DCFH oxidation by 90% (p <.01). These data suggest that hydrogen peroxide, and not NO, is primarily responsible for the PMA-induced oxidation of DCFH in human PMN under these conditions.