Biological Defense Mechanisms THE EFFECT OF BACTERIA AND SERUM ON SUPEROXIDE PRODUCTION BY GRANULOCYTES

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Abstract
We previously reported that granulocytes are able to produce superoxide (O2-), a highly reactive compound formed by the one-electron reduction of oxygen. The demonstration of O2- production was based on the observation that the reduction of extra-cellular cytochrome c by granulocytes was greatly diminished by superoxide dismutase, an enzyme catalyzing the conversion of O2- to hydrogen peroxide and oxygen. In the present report, studies concerning the effect of bacteria and serum on O2--dependent cytochrome c reduction by granulocytes are described. In the absence of bacteria, the O2--dependent reduction of extracellular cytochrome c by granulocytes under optimal assay conditions amounted to 9.2±2.8 SD nmol/3 × 106 cells/20 min. When bacteria (100 organisms/cell) were present, the O2--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 by cells killed by heat abolished O2--dependent cytochrome c reduction. Bacteria killed by autoclaving were almost as effective as live bacteria in stimulating granulocyte O2- production. Measurements of particle uptake and O2 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 O2- by this enzyme. Stimulation of O2- production by bacteria was strongly dependent on the presence of serum in the incubation mixture. Serum heated to 56°C for 45 min was as effective as unheated serum in stimulating O2- production in the presence of bacteria, but boiled serum had no effect. Other experiments suggested that incubation of bacteria with serum resulted in the release of a nonparticulate heat-labile substance capable of stimulating O2- production in the absence of bacteria. Certain characteristics of the O2--dependent cytochrome c reduction by granulocytes were studied, including the dependence of this process on granulocyte, cytochrome c, and bacterial concentrations. In addition, O2--dependent cytochrome c reduction was followed as a function of time. A constant rate was found with resting granulocytes. With bacteria the time course was more complex. A well-defined lag was followed by a fairly brief period of extremely vigorous cytochrome c reduction. During this period, the maximum rate of cytochrome c reduction exceeded the rate observed in the absence of bacteria by a factor of 12. The rate then decreased until by 40 min, it had slowed to the rate observed in the absence of bacteria. From the above results, it was concluded that the exposure of the granulocyte to bacteria plus serum initiates a process in which a defined quantity of O2- is formed in a rapid burst lasting 20-30 min. It is conceivable that the O2- generated by this process may be involved in the killing of bacteria by the granulocytes.