THE EFFECT OF BACTERIA AND SERUM ON SUPEROXIDE PRODUCTION BY GRANULOCYTES

A B S T R A C T We previously reported that granulocytes are able to produce superoxide (02-), a highly reactive compound formed by the one-electron reduction of oxygen. The demonstration of 02- production was based on the observation that the reduction of extracellular cytochrome c by granulocytes was greatly diminished by superoxide dismutase, an enzyme catalyzing the conversion of 02- to hydrogen peroxide and oxygen. In the present report, studies concerning the effect of bacteria and serum on O-dependent cytochrome c reduction by granulocytes are described. In the absence of bacteria, the 02-dependent reduction of extracellular cytochrome c by granulocytes under optimal assay conditions amounted to 9.2±2.8 SD nmol/3 X 106 cells/20 min. When bacteria (100 organisms/cell) were present, the 02-dependent cytochrome c reduction under otherwise similar conditions increased by a factor of nearly four (34.5+9.4). There was no effect of albumin or catalase on cytochrome c reduction, and boiled dismutase had only a small effect. Omission of granulocytes or substitution of live cells by cells killed by heat abolished 02-dependent cytochrome c reduction. Bacteria killed by autoclaving were almost as effective as live bacteria in stimulating granulocyte 02- production. Measurements of particle uptake and 02 uptake by granulocytes indicated that superoxide dismutase did not affect granulocyte metabolism nonspecifically, supporting the conclusion that the diminution of cytochrome c reduction in the presence of dismutase was due to the destruction of 02 by this enzyme. Stimulation of 02- production by bacteria was strongly dependent on the presence of serum in the incubation