Nitric oxide regulates interleukin 1 bioactivity released from murine macrophages

The bioactivity of interleukin-1 (IL-1), a major proinflammatory cytokine, can be modulated by a variety of factors including inhibitors of IL-1 production and release and receptor blockade by IL-1 receptor antagonist and by binding to nonsignaling soluble receptors. This study demonstrates that the free radical nitric oxide (NO) is also a regulator of IL-1 bioactivity. Lipopolysaccharide-activated murine macrophage RAW264.7 cells, and lipopolysaccharide plus interferon-gamma-activated murine peritoneal macrophages release IL-1 bioactivity, which is increased 10-fold over control levels by 24 h. NG-Monomethyl -arginine (NMMA), a nitric oxide synthase (NOS) inhibitor, almost completely inhibits the release of IL-1 bioactivity from activated macrophages in a time- and concentration-dependent manner with an IC50 of 50 microM. IL-1 activity was determined by thymocyte proliferation bioassay and by a new spectrophotometric bioassay based on IL-1-specific induction of NOS and NO production by an insulinoma cell line, RINm5F. Neither NO nor NOS inhibitors present in the macrophage supernatant interfere with the bioassays. Aminoguanidine and iodonium diphenyl, mechanistically unrelated NOS inhibitors, also prevent the release of IL-1 activity from RAW 264.7 cells. The addition of the NO donor S-nitroso-acetylpenicillamine reconstituted the release of IL-1 bioactivity inhibited by NMMA in a concentration-dependent manner. NO appears to increase the amount of IL-1 protein released by activated macrophages as determined by enzyme-linked immunosorbent assay, but not by mechanisms involving cell death nor modification of IL-1 precursor processing. A cGMP donor, 8-bromo-cGMP, dose-dependently reverses NMMA inhibition of bioactive IL-1 release, suggesting that NO regulates IL-1 release by a cGMP-dependent mechanism. These observations suggest that NO stimulation of the activity of IL-1, a key mediator of the immune response, may be a potentially important mechanism for control of IL-1 activity in vivo.