Many stimuli induce neutrophils to undergo an oxidative burst and generate toxic oxygen metabolites. The major products are O2- and H2O2, the latter being presumed to arise by spontaneous dismutation of the former. If H2O2 were indeed derived exclusively from released O2- according to the equation 2O2- + 2H+----H2O2 + O2, one would expect that relationship to be reflected in the ratio of the two metabolites detectable in the extracellular mileu of stimulated neutrophils. A second corollary is that H2O2 should not form when cytochrome c is present to scavenge O2- before it can dismutate. Although H2O2 cannot be measured directly in the presence of cytochrome c because it is consumed in reoxidizing reduced cytochrome c, its presence can be detected indirectly by the ability of catalase to improve the apparent yield of reduced cytochrome c. We found that the relative amounts of extracellular H2O2 and O2- that could be measured in the environment of stimulated neutrophils varied with the stimulus and that catalase protected reduced cytochrome c from H2O2 oxidation when some stimuli were used but not with others. For example, the ratio of O2- to H2O2 produced by neutrophils exposed to PMA was about 2:1, the expected result if H2O2 were derived from O2-. However when cytochalasin B was added to the cells before the stimulus, the yield of H2O2 was reduced but not the yield of O2-. When cells were allowed to settle and spread on tissue culture plastic they produced equimolar amounts of O2- and H2O2. Coating the plastic with IgG doubled cytochrome c reduction without effecting H2O2. In contrast, coating with albumin reduced H2O2 without effecting cytochrome c reduction. Soluble IgG aggregates induced production of mostly O2- whereas immune complexes resulted in release of both metabolites. FMLP and A23187 were similar to the soluble IgG aggregates in their effects and induced release of proportionately more O2- than H2O2. The addition of catalase to the cytochrome c solution improved the yield of reduced cytochrome c when PMA or IgG was used to stimulate the cells but not when FMLP was used. These and other data suggest that H2O2 release is not a linear function of the amount of O2- generated and that either a variable fraction of O2- spontaneously dismutates to H2O2 or the neutrophil NADPH oxidase, in a manner analogous to xanthine oxidase, is capable, under some circumstances, of producing H2O2 as well as O2-.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract. Phospholipid remodeling resulting in arachidonic acid (AA) release and metabolism in human neutrophils stimulated by calcium ionophore A23187 has been extensively studied, while data obtained using physiologically relevant stimuli is limited. Opsonized zymosan and immune complexes induced stimulus-specific alterations in lipid metabolism that were different from those induced by A23187.[3H]AA release correlated with activation of phospholipase A2 (PLA2) but not with cellular activation as indicated by superoxide generation. The latter correlated more with calcium-dependent phospholipase C (PLC) activation and elevation of cellular diacylglycerol (DAG) levels. When cells that had been allowed to incorporate [3H]AA were stimulated with A23187, large amounts of labeled AA was released, most of which was metabolized to 5-HETE and leukotriene B4. Stimulation with immune complexes also resulted in the release of [3H]AA but this released radiolabeled AA was not metabolized. In contrast, stimulation with opsonized zymosan induced no detectable release of [3H]AA.Analysis of [3H]AA-labeled lipids in resting cells indicated that the greatest amount of label was incorporated into the phosphatidylinositol (PI) pool, followed closely by phosphatidylcholine and phosphatidylserine, while little [3H]AA was detected in the phosphatidylethanolamine pool. During stimulation with A23187, a significant decrease in labeled PI occurred and labeled free fatty acid in the pellet increased. With immune complexes, only a small decrease was seen in labeled PI while the free fatty acid in the pellets was unchanged. In contrast, opsonized zymosan decreased labeled PI, and increased labeled DAG. Phospholipase activity in homogenates from human neutrophils was also assayed. A23187 and immune complexes, but not zymosan, significantly enhanced PLA2 activity in the cell homogenates. On the other hand, PLC activity was enhanced by zymosan and immune complexes. Stimulated increases in PLC activity correlated with enhanced superoxide generation induced by the stimulus.T I V A T I O S of neutrophils is intimately linked to changes in phospholipid metabolism (40,43,44). Released arachidonic acid (AA)' from membrane phospholipids (PLs) provides a substrate for the formation of bioactive lipids that can modulate cell responses (4, 33, 34). Additionally, nonmetabolized AA has been shown to activate the neutrophil NADPH oxidase (2, 15). It is also possible that phospholipid remodeling resulting in the release of AA and/or the formation of lysophospholipids may be a method of altering cell membrane fluidity during signal transduction (36). Crucial cell functions (i.e., phagocytosis, carrier-mediated transport, membrane-bound enzymes) can be altered by fatty acid enrichment (21,22,31,39).1. Abbreviations used in this paper: AA, arachidonic acid; DAG, diacylglycerol; LTB4, leukotriene B4; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PL, phospholipid; PLA2, phospholipase A:; PLase, phospholipase; PLC, phospholipase...

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