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.