Human neutrophils produce free radicals from the cell-zymosan interface during phagocytosis and from the whole plasma membrane when stimulated with calcium ionophore A23187.
The production of free radicals, superoxide anions (O2-), and hydrogen peroxide (H2O2) was histochemically investigated in human neutrophils that were stimulated by either phagocytosis or the calcium ionophore A23187. To demonstrate O2-, peripheral neutrophils from healthy donors were incubated at 37 degrees C in a medium containing nitroblue tetrazolium and glucose in the presence of either opsonized zymosan A and/or A23187. To demonstrate H2O2, neutrophils pretreated with a stimulant for 10 min were washed and incubated in a cerium medium containing CeCl3 and glucose in a Tris-maleate buffer. In cells engaged in phagocytosis, diformazan (for O2-) and cerium perhydroxide deposits (for H2O2) were restricted to the neutrophil-particle interface and on the inner surface of phagosomes. The remaining free surface of the plasma membrane was devoid of reaction products. In the case of neutrophils stimulated with A23187, the production of O2- and H2O2 was visualized over the whole surface of the plasma membrane. These histochemical reactions were inhibited by p-benzoquinone, superoxide dismutase, ferricytochrome c or catalase, and p-diazobenzenesulfonate (a membrane-impermeable protein denaturant). The results showed that human neutrophils produce free radicals exocellularly and that the site of production varies with different stimuli.