CYCLOSPORINE-A INHIBITS NITRIC-OXIDE PRODUCTION BY L929 CELLS IN RESPONSE TO TUMOR-NECROSIS-FACTOR AND INTERFERON-GAMMA

We recently reported that tumor necrosis factor (TNF) induced the production of nitric oxide (NO) by TNF-sensitive, but not-resistant, tumor cells. Paradoxically, NO thus produced does not appear to be involved in the mechanism of TNF-mediated cytotoxicity as inhibitors of NO production and NO scavengers did not block cytotoxicity. Because the immunosuppressive drug cyclosporin A (CsA) inhibits several types of immune-mediated killing, we were interested in what effect CsA would have on TNF-mediated cytotoxicity as well as NO production. Treatment with CsA had no effect on the sensitivity L929 cells to TNF-mediated cytotoxicity, either in the presence or absence of interferon-gamma (IFN-gamma). In the presence of IFN-gamma alone, 1,929 cells were slightly less sensitive to the cytotoxic effects of TNF. In contrast to the effect on TNF-mediated cytotoxicity, CsA treatment had a profound effect on the ability of these cells to produce NO in response to TNF and IFN-gamma. Cells treated with CsA produced 75% less NO than did their untreated controls. Inhibition of calmodulin-dependent calcineurin-like phosphatases is one mechanism by which CsA may exert its effects. Therefore, we tested the effect of EGTA, which inhibits calcineurin by chelating calcium, on NO production and found that EGTA treatment resulted in a 15% decrease in the amount of NO produced. In addition, cells treated with the calmodulin antagonist W-13 produced 79% less NO than their untreated controls. Therefore, these results provide further evidence that NO produced by TNF-sensitive cells is not involved in the mechanism of TNF-mediated cytotoxicity because reduction of NO production by CsA has no effect on TNF-mediated killing of these same cells.