HYDROGEN-PEROXIDE FORMATION OF POLYMORPHONUCLEAR LEUKOCYTES STIMULATED WITH CYTOCHALASIN-D

Possible cellular sites to form superoxide (O2-) and H2O2 were studied in [guinea-pig] polymorphonuclear leukocytes stimulated with either cytochalasin D (CD) or bacteria. When the cells were stimulated with cytochalasin, the ratio of O2- to H2O2 appearing in the medium was about 2 (determined separately) and the appearance of H2O2 in the medium was inhibited by addition of cytochrome c, a scavenger of O2-. The inhibition was reversed by the addition of superoxide dismutase. The cells phagocytosing bacteria released O2- at the beginning but the release subsided corresponding to the increase in the direct release of H2O2. p-Chloromercuribenzenesulfonate, a non-permeant reagent, inhibited H2O2 formation by the cells stimulated with CD; it did not inhibit the formation by the phagocytosing cells. Cells stimulated with the CD may release O2- from the cell surface, whereas phagocytosing cells release H2O2 from an intracellular site, probably the phagosomes formed by the invagination of the plasma membrane. EM showed that vacuole-like structures found in cytochalasin-treated cells had openings to outside. The particle-bound NADPH-oxidase activity of the cytochalasin-treated cells was several times higher than that of the resting cells and activation was apparently parallel with O2--releasing activity of the cells. No activation of the NADH-oxidase was observed.