Electron microscopic identification of hydrogen peroxide detected in fixed human polymorphonuclear leukocytes during phagocytosis.

Polymorphonuclear leukocytes (PMNs) engaged in phagocytosis produce reactive oxygen species (ROS), such as those that occur in an activated NADPH oxidase reaction, to eliminate ingested microorganisms. The translocation of NADPH oxidase components to produce antimicrobial free radicals from the vesicles to the phagosomes may be important. Hydrogen peroxide (H2O2) derived from O2- has been observed by electron microscopy using a cerium method. However, 2'-7'-dichlorofluorescin diacetate can also detect H2O2 through fluorescence. The main objective of the present study was to measure the H2O2-dependent fluorescence of PMNs after opsonized zymosan A (OPZ) phagocytosis using a microplate reader under different fixation conditions, including 0.5, 1, and 10% glutaraldehyde (GA) individually for 1, 5, 10, or 30 min. An additional objective was to visualize, through the use of electron microscopic cytochemistry, the process of H2O2 generation in OPZ phagocytic fixed PMNs. The fixed PMNs showed that the largest fluorescent value was produced by a concentration of 0.5% GA for all fixation times. This suggested that the fixation of PMNs with a high concentration of GA inhibited phagocytosis and produced ROS. In the fixed PMNs, electron microscopic results showed that after 1 min of mixing, some PMNs attached to particles and exhibited mild deposits in their secretory vesicles. When PMNs engulfed particles, free radical-producing vesicles had enhanced reaction deposits 10 min later and fused to the phagosomal membrane, releasing numerous free radicals into the lumen. Time-dependent H2O2 production was enhanced in the secretory vesicles, some of which were fused exactly to the phagosome membranes.