rIFN-gamma-activated rat neutrophils induce tumor cell apoptosis by nitric oxide

We have previously shown that 1) neutrophils activated with various cytokines, including rat recombinant interferon gamma (rIFN-gamma), inhibit tumor cell growth and that 2) nitric oxide (NO) is the effector molecule in tumor inhibition by rIFN-gamma-stimulated rat peritoneal exudate neutrophils. In this study, we examined the nature of tumor cell death induced by rat peritoneal neutrophils activated by rIFN-gamma in order to clarify the mechanism of apoptosis in neoplastic tumor cell death. DNA of 3 syngeneic rat tumor cell lines was significantly fragmented within 3 hr of incubation in the presence of rIFN-gamma-activated neutrophils, and this effect was dependent on both the concentration of rIFN-gamma and the number of neutrophils. This DNA fragmentation was inhibited by L-N-(I-iminoethyl)-ornithine (L-NIO), a NO synthase inhibitor, but not by superoxide dismutase (SOD). Tumor cells treated with the activated neutrophils were shown by electron microscopy to be apoptotic, exhibiting necrotic features with a longer incubation. On the other hand, cytolysis of tumor cells, as determined by a [3H]-uridine release assay, was first observed only at 24 hr of incubation with the rIFN- gamma-activated neutrophils. Taken together, our results suggest that tumor cell apoptosis by activated neutrophils is NO-dependent and that apoptotic tumor cells undergo necrosis as a secondary process. We suggest that tumor cell apoptosis induced by activated neutrophils plays an important role in regulation of neoplastic tumor cell growth and death in vivo.