The myeloperoxidase-hydrogen peroxide-halide system as effector of neutrophil-mediated tumor cell cytotoxicity

Human neutrophils activated by exposure to phorbol myristate acetate were highly effective mediators of tumor cell cytotoxicity. Target cell injury was documented by a ⁵¹Cr release assay and by the loss of oncogenicity for test mice. The spectrum of susceptible tumor cells included the murine lines LSTRA, L1210, EL-4, YAC-1, and MPC-11. The potency of the human neutrophil as a killer cell was indicated by the low number of cells required (effector cell:target cell ratios as low as 1:2), the rapidity of target cell lysis (e.g., 1 hr or less), and the degree of ⁵¹Cr release (>59% under most conditions). The involvement of neutrophil myeloperoxidase was demonstrated by the inhibition of cytotoxicity by azide and cyanide, by the markedly impaired activity of neutrophils from a patient with hereditary myeloperoxidase deficiency, and by the specific correction of this defect on addition of purified myeloperoxidase. The participation of H₂O₂ was indicated by the inhibition of cytotoxicity by catalase, but not by heated catalase or superoxide dismutase, by the absence of activity of neutrophils from patients with chronic granulomatous disease, and by the specific correction of this defect on addition of H₂O₂ or a peroxide-generating enzyme system (glucose oxidase). Target cell destruction required the presence of a halide cofactor-chloride, iodide, or bromide. In the chloride system, substantial enhancement of cytotoxicity was seen on addition of glucose. Partial inhibition of cytotoxicity was observed on addition of protein in the form of serum or albumin, an effect that was in part nullified by the addition of glucose and an increased number of neutrophil effector cells. Cytotoxicity was markedly inhibited by certain oxygen radical scavengers, including ascorbic acid, 2-mercaptoethanol, and methionine, but not oxidized methionine. Exposure of human neutrophils to phorbol myristate acetate resulted in protein iodination as well as tumor cell cytotoxicity. Iodination and cytotoxicity displayed remarkably similar properties with respect to inhibitors, oxygen radical scavengers, and phorbol ester dose-response characteristics. Structural specificity for various phorbol derivative was also similar for iodination and cytotoxicity and matched that previously reported for tumor promotion and inflammatory properties of these compounds. The predominant substrate for iodination was extracellular protein, indicating the secretion of myeloperoxidase and H₂O₂.