Nitric Oxide-Mediated Modulation of Interleukin-8 Production by a Human Glioblastoma Cell Line,T98G,Cocultured with Myeloid and Monocytic Cell Lines

Abstract

Coculture of T98G glioblastoma cells with the myeloid and monocytic cell lines, HL-60, and THP-1 produced minimal amounts of interleukin-8 (IL-8). Pretreatment of HL-60 or THP-1 cells with phorbol myristate acetate (PMA) enhanced their capacity to induce IL-8 production by T98G cells. In contrast, the murine macrophage cell lines J774 A.1 and RAW 264.7 induced high levels of IL-8 production by T98G cells without PMA activation. To determine the molecules responsible for the induction of IL-8 by T98G cells, we carried out coculture experiments with a membrane fraction prepared from RAW cells and indicated that membrane-associated and free forms of murine IL-1α acted on human T98G cells to produce IL-8. RAW cells were unique in that increasing the number of RAW cells relative to the number of T98G cells (RAW/T98G ratio > 4:1) significantly suppressed IL-8 production by T98G cells. Because RAW cells produce large amounts of nitric oxide (NO), we assumed that the suppression of IL-8 production was ascribable to the NO produced by the RAW cells. This was supported by the inverse relationship between increasing concentrations of NO and IL-8 production seen in this coculture system. The involvement of NO in the suppression of IL-8 production was confirmed by the finding that N-monomethyl-l-arginine (NMMA), which inhibits NO production, reversed this suppression, whereas S-nitroso-N-acetyl-d,l-penicillamine (SNAP), a strong NO generator, suppressed IL-8 production. Our results indicate that high levels of NO suppress IL-8 production by T98G cells, and murine IL-1α plays a major role in the induction of IL-8 production by T98G cells. It is, therefore, possible that excessive production of NO during the interaction of glioma cells with macrophages may play a regulatory role in chemokine production, thus mitigating inflammatory responses.