Two distinct cytolytic mechanisms of macrophages and monocytes activated by phorbol myristate acetate

Pulmonary alveolar macrophages (PAM) and peripheral blood monocytes (PBMO) of the miniature swine can be converted to cytolytically active effector cells by treating with phorbol myristate acetate (PMA) as determined by enhancement of cytotoxicity to various target cells. Kinetics of the PMA-activated PAM and PBMO in cytotoxicity show that the effective PMA concentration ranges from 10 to 1,000 ng/ml. Induction of cytotoxic macrophages and monocytes occurred as early as 30 min and to their maximum cytotoxicity after 1 hr exposure to PMA and the enhanced cytotoxic activity persisted up to 24 to 40 hr when PMA was removed by washing after 1 hr exposure, but prolonged exposure to PMA for more than 6 hr resulted in a drastic decrease of cytolytic activities suggesting the prolonged exposure to PMA causes macrophages and monocytes to become refractory to PMA stimulation. Target cells displayed varying degrees of cytotoxic sensitivity to the PMA-activated PAM and PBMO; PRBC, SRBC, and K562 were sensitive, WEHI-164 and U937 were relatively sensitive, and SB was very resistant to these activated effector cells. The mechanisms of PMA-induced cytotoxicity could largely be divided into two categories. One was the H2O2 mediated killing as shown by complete reduction of cytotoxicity after adding catalase in the assay. The other was the proteases mediated cytolysis, which could be blocked by protease inhibitors, Phenyl methyl sulfonyl fluoride (PMSF), and N- alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK). H2O2 was the only mediator produced in large enough quantities from PBMO to kill target cells, whereas PAM could produce both mediators (H2O2 and proteases). PRBC, SRBC, and K562 appeared to be killed by H2O2 produced by PAM and PBMO. In contrast, U937 and WEHI-164 appeared to be killed by proteases in PAM mediated cytolysis but by H2O2 in PBMO-mediated cytolysis. These results suggest that the observed cytolytic mechanisms can be differed by type of target cells as well as the source of mononuclear phagocytes within the individual animal.