We have compared the oxidative metabolism of human eosinophils (80%-90% purity) to that of neutrophils. Hexose monophosphate (HMP) shunt activity of eosinophils was higher than that of neutrophils under either resting or phagocytizing conditions. Eosinophil HMP shunt activity also was stimulated by phorbol myristate acetate, a membrane- active agent. Eosinophils showed a marked incorporation of 125I into trichloroacetic acid-insoluble material under resting conditions, which increased markedly during phagocytosis. Eosinophils likewise showed a greater reduction of nitroblue tetrazolium dye during phagocytosis than did neutrophils. Measurement of other parameters of oxidative metabolism indicated that eosinophils generated superoxide anion following phagocytosis and also elicited a burst of chemiluminescence similar to that observed during phagocytosis by neutrophils. Measurement of NADPH oxidase activity demonstrated that this enzyme was 3–6 times more active in fractions isolated from eosinophils than in corresponding fractions isolated from neutrophils; this was observed over a range of substrate concentrations. The eosinophil enzyme sedimented differently than the neutrophil enzyme with differential centrifugation; neither showed sedimentation characteristics of peroxidase. These data indicate that eosinophils possess a similar, although in some ways more potent, oxidative burst than neutrophils and are consistent with a role for NADPH oxidase in the initiation of that burst.

Low doses of aliphatic alcohols produce divergent effects on the function of chemoattractant receptors on human polymorphonuclear leukocytes (PMNs) since they enhance chemotaxis but inhibit stimulation of superoxide production by chemoattractants. As such, alcohols can provide useful pharmacologic tools to probe the mechanisms of stimulus- response coupling in leukocytes. A role for protein kinase C has been implicated in the activation of the respiratory burst in PMNs. Although the vast majority of this enzyme activity is located in the cytosolic fraction of unactivated PMNs, protein kinase C activity appears in the particulate fraction of the cells when they are stimulated to produce superoxide by either chemoattractants or by phorbol myristate acetate (PMA). Doses of the alcohols that selectively inhibited stimulation of superoxide production by chemoattractants also inhibited the appearance of protein kinase C activity as well as an undefined protein kinase activity in the particulate fraction of the cells. In contrast, the alcohols did not affect either the ability of PMA to stimulate the production of superoxide in PMNs nor the appearance of protein kinase activity in the cells' particulate fraction. PMA is known to bind and activate protein kinase C directly, thus bypassing receptor-mediated events. These data suggest that alcohols inhibit the stimulation of the respiratory burst by chemoattractants in PMNs by blocking the ability of receptor occupancy to induce the appearance of protein kinase activity in particulate fractions. These results moreover suggest that the appearance of protein kinase activity in the particulate fraction may be required for activation of the respiratory burst in PMNs.

Cyanide has been shown to stimulate both oxygen uptake and hexose monophosphate shunt activity in phagocytizing human polymorphonuclear leukocytes. It also stimulates the oxidation of NADPH by a particulate fraction derived from phagocytizing cells. This stimulation of NADPH oxidase is not observed in the presence of exogenous Mn2+. Studies with purified enzymes have shown that CN- also stimulates NADPH oxidation by horseradish peroxidase or lactoperoxidase, suggesting that the respiratory burst might be initiated by activation of a peroxidase-like enzyme in the human polymorphonuclear leukocyte. Based on studies of others, however, it does not appear as though the enzyme is identical to myeloperoxidase. The mechanism of the CN- stimulation appears to involve an oxidatic chain reaction, since it stimulates markedly NADPH oxidation in the presence of an artificial superoxide-generating system.