The mechanism of action of lymphokines. VIII. Lymphokine-enhanced spontaneous hydrogen peroxide production by macrophages.

Oil-elicited guinea-pig peritoneal macrophages (MPs) cultured for 2-3 days in medium containing supernatant of concanavalin A-activated lymphocytes (lymphokine, LK) generated large amounts of hydrogen peroxide (H2O2), as detected by the horseradish peroxidase (HRP)-dependent oxidation of phenol red, in the absence of further stimulation. H2O2 production increased with the duration of exposure to LK and was evident at high dilutions of supernatant (1/64). Parallel cultures of MPs in medium or a supernatant of non-activated lymphocytes also increased their H2O2 production during culture but levels at all time intervals were significantly lower than those measured in LK treated cultures. The marked increase in H2O2 production was associated with only a moderate increment in superoxide (O-2) liberation and this was not specific for LK treated cells. Detection of LK-dependent H2O2 production was dependent on ongoing pinocytosis during the assay. This and other arguments suggest that the HRP-phenol red assay, as applied here, detects H2O2 generation occurring at the level of intracellular vesicles and it is concluded that LK elicits H2O2 production that is limited to the intracellular compartment. H2O2 is, apparently, derived by non-enzymatic dismutation of O-2 taking place within the cell; LK treatment of MPs also resulted in a significant reduction in catalase activity.