Augmentation of spontaneous macrophage-mediated cytolysis by eosinophil peroxidase

Abstract

Eosinophil peroxidase (EPO), a cationic protein purified from horse blood, adhered to four different types of tumor cells, markedly potentiating their lysis by preformed or enzymatically generated H2O2 (up to 76-fold, as assayed in serum-containing tissue culture medium without supplemental halide). Similarly, compared with uncoated tumor cells, EPO-coated tumor cells were up to 32 times more sensitive to lysis when incubated with macrophages or granulocytes whose respiratory burst was triggered by PMA. However, EPO-coated tumor cells were also readily lysed by bacillus Calmette-Guerin-activated macrophages in the absence of exogenous triggering agents. This spontaneous cytolysis was rapid (50% at 2 h) and potent (50% lysis at macrophage/tumor cell ratios of 1.5 to 4.6), and was observed with both a peroxide-sensitive tumor (TLX9) and a peroxide-resistant tumor (NK lymphoma). Under the conditions used, neither EPO alone nor macrophages alone were spontaneously cytolytic. Neither EPO nor EPO-coated tumor cells triggered a detectable increment in H2O2 release from macrophages. Nonetheless, spontaneous macrophage-mediated cytolysis of EPO-coated tumor cells was completely inhibitable by catalase (50% inhibition, 23 U/ml), although not by heated catalase, indicating a requirement for H2O2. Cytolysis was also completely inhibitable by azide (50% inhibition, 2.5 x 10-5 M), indicating a requirement for enzymatic activity of EPO. Thus, a cytophilic peroxidase from eosinophils and H2O2 spontaneously released from activated macrophages interacted synergistically in a physiologic medium to destroy tumor cells.