Human neutrophils produce free radicals from the cell-zymosan interface during phagocytosis and from the whole plasma membrane when stimulated with calcium ionophore A23187

Abstract

The production of free radicals, superoxide anions (O 2 −, and hydrogen peroxide (H 2O 2) was histochemically investigated in human neutrophils that were stimulated by either phagocytosis or the calcium ionophore A23187. To demonstrate O 2 −, peripheral neutrophils from healthy donors were incubated at 37 °C in a medium containing nitroblue tetrazolium and glucose in the presence of either opsonized zymosan A and/or A23187. To demonstrate H 2O 2, neutrophils pretreated with a stimulant for 10 min were washed and incubated in a cerium medium containing CeCl 3 and glucose in a Tris-maleate buffer. In cells engaged in phagocytosis, diformazan (for O 2 −) and cerium perhydroxide deposits (for H 2O 2) 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 O 2 − and H 2O 2 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.