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 techni ques have been developed for quantitation of oxidative burst activity at th e single cell level using 2',7'-dichlorofluorescin (DCFH). However, the spe cific reactive oxidant species being measured using this method are not cle arly defined. Isolated human PMN were loaded with DCFH diacetate, stimulate d with phorbol myristate acetate (PMA) in the presence or absence of specif ic reagents, and analyzed using flow cytometry. Addition of PMA resulted in a 90-fold increase in the fluorescence intensity of DCFH-loaded neutrophil s (p < .01). Inhibition of NADPH oxidase activity using a calmodulin antago nist (W-13) decreased PMA-induced DCFH oxidation by 70% (p < .05). Inhibiti on 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 sugges t that hydrogen peroxide, and not NO, is primarily responsible for the PMA- induced oxidation of DCFH in human PMN under these conditions.