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

(1) 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. (2) H₂O₂ production increased with the duration of exposure to LK and was evident at high dilutions of supernatant (1/64). (3) 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. (4) The marked increase in H₂O₂ production was associated with only a moderate increment in superoxide (O₂^-) liberation and this was not specific for LK treated cells. (5) 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. (6) H₂O₂ is, apparently, derived by non-enzymatic dismutation of O₂^- taking place within the cell; (7) LK treatment of MPs also resulted in a significant reduction in catalase activity.