Hydrogen peroxide release from mouse peritoneal macrophages: dependence on sequential activation and triggering.

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Abstract
Using a specific and sensitive fluorometric assay, the H2O2 release from as few as 2 .times. 105 mouse peritoneal macrophages [PM] could be detected continuously and quantitated. It is emphasized that the assay measured H2O2 release, not production. Induction of H2O2 release required sequential application of 2 stimuli: the administration of an activating agent to the mice from 4 days-10 wk before cell harvest and the exposure of the cells in vitro to a triggering agent. BCG was most effective as an activating agent, resulting in PM which could be triggered to release H2O2 almost as copiously (8 nmol/106 macrophages per 5 min) as mouse peritoneal PMN [polymorphonuclear neutrophils] (9 nmol/106 PMN per 5 min). Casein and Corynebacterium parvum could also serve as activators, but thioglycollate and FCS [fetal calf serum] were ineffective after single injections. PMA [phorbol myristate acetate] was a potent triggering agent, resulting in a maximal rate of H2O2 release after a latency of about 40 s for cells in suspension. Other triggering agents included the ionophore A23187, concanavalin A in the presence of cytochalasin B and phagocytosis. H2O2 release could be attributed to PM and PMN in peritoneal cell suspensions or in preparations of adherent peritoneal cells, but not to lymphocytes. The H2O2 detected was probably formed from superoxide anion. H2O2 may be important in macrophage antimicrobial and antitumor mechanisms.