Macrophage-mediated cytolysis of erythrocytes in the guinea pig: II. Role of oxidative burst products in macrophage cytotoxicity activated by lectins and phorbol ester derivatives

Abstract

Guinea pig peritoneal macrophages (GPPM) exhibited enhanced production of O 2 − and H 2O 2, and cytolytic activity toward erythrocytes, in response to reagents such as 12- O-tetradecanoyl-phorbol-13-acetate (TPA), its methylated derivative 4- O-MeTPA, Con A, wheat germ agglutinin (WGA), and opsonized zymosan. In order to examine the possible role of oxidative burst products such as O 2 − and H 2O 2 in the cytolytic process, we used reagents and enzymes which influence the balance of O 2 − and H 2O 2 outside and inside the GPPM cells. Macrophage-mediated cytolysis (MMC) of erythrocytes in the presence of the activators and modulators was assessed by 51Cr release assay. MMC activated by TPA and 4- O-MeTPA was inhibited by scavengers of H 2O 2 such as catalase and α-tocopherol, and was augmented by the catalase inhibitor 3-amino-1,2,4-triazole, and by horseradish peroxidase. TPA- and 4- O-MeTPA-activated MMC was only partially inhibited by the O 2 − scavenger cytochrome c and the enzyme superoxide dismutase and unaffected by cytochalasin D (an inhibitor of phagocytosis). MMC activated by the lectins Con A and WGA was unaffected by the scavengers and enzymes used, but markedly inhibited by cytochalasin D. Activation of MMC by TPA, WGA, and phagocytosis of opsonized zymosan, as well as O 2 − and H 2O 2 generation triggered by these reagents, were markedly inhibited by chlorpromazine. The results indicate that GPPM-mediated cytolysis activated by lectins, phorbol ester derivatives, and phagocytosis of opsonized zymosan, is dependent on the generation of oxidative burst products, mainly H 2O 2. TPA- or 4- O-MeTPA-activated MMC is mainly an extracellular event, while lectin-activated MMC may take place within the macrophages.