Murine hypodense eosinophils induce tumour cell apoptosis by a granzyme B-dependent mechanism

Purpose: Eosinophils have been shown to potentiate anti-tumour cytotoxicity in both clinical and animal studies. The mechanism by which eosinophils in duce tumour cell damage, however, has largely been speculative. The purpose of this study was to identify the mechanisms involved in eosinophil-induce d tumour cell cytotoxicity. Methods: To investigate eosinophil cytotoxicity , eosinophils were isolated from the peritoneal cavity of Mesocestoides cor ti-infected BALB/c mice, and were separated into normodense (ND) and hypode nse (HD) populations using discontinuous Percoll density gradient centrifug ation. The tumoricidal activity of ND and HD eosinophils was assessed using the [Cr-51]-release cytotoxicity assay (a measure of cytolytic activity) a nd the JAM assay (a measure of apoptotic activity). Investigation of apopto sis-inducing molecules in HD eosinophils was undertaken by RT-PCR. The calc ium chelator EGTA, serine protease inhibitor aprotinin and a competitive su bstrate for granzyme B were used to assess the role of perforin and granzym e B in HD eosinophil killing. Results: Cytotoxic activity induced by HD eos inophils was significantly greater than that of ND eosinophils, and apoptos is was the principal killing mechanism. RT-PCR analysis revealed that HD eo sinophils express mRNA for perforin, granzyme B and Fas ligand. Furthermore , HD eosinophil killing was markedly inhibited by EGTA, intracellular aprot inin and the granzyme B competitive substrate. Conclusions: These data are consistent with a hypothesis that murine HD eosinophils elicit tumoricidal activity via a granzyme B-dependent mechanism.