Suppression of nitric oxide generation by antioxidants in alveolar macrophage-derived cell line MH-S

Abstract

Preincubation of alveolar macrophages (AM) with bacterial endotoxin &I’S) enhances macrophage resistance to intracellular replication of pathogens and potentiates the activating effect of interferon-gamma (IFN-gamma). Such immunologic activation also induces the expression of nitric oxide synthase (iNOS) to generate reactive nitric oxide (NO) which contributes to the antimicrobial activity against pathogens. With the use of MH-S cell line derived from murine AM, we have examined the stimulatory effects of LPS (0.001 mg/ml) and IFN-gamma (100 U/ml) on extracellular NO production. The NO level in the culture medium was l&fold higher than basal value in 18 hours. In the p-ce of N(G)-methyl-L-arginine (L-NMMA, 1 mM), the amount of NO generated was suppressed to 19.3%, indicating that the in- in NO was the consequence of expression of iNOS. S-Methylisothiourea (SMT), a highly selective inhibitor of iNOS, was found to be very effective with an IC, of 0.01 mM concentration. Pyrrolidine dithiocarbamate (PDTC) and diethyldithiocarbamate (DETC) were both strong inhibitors of inducible NO production with IC, of 0.04 and 0.05 mM, respectively. Other antioxidank, e.g. N-acetylcysteine, also exhibit inhibitory effects. but comparatively weaker (IC, = 4 mM). To investigate the role of poly-ADP-ribosylation in iNO! induction, poly_(ADP-ribose) poly-merase (PARP) inhibitors, e.g. 3_aminobenzamide, were examined for their effects on NO production. It was found that all PARP inhibitors are weak inhibitors with IC,, in the range of 10-25 mM. In conclusion, the established transformed murine AM MH-S cell line can be useful in the study of cytokines and oxidative stress in microbicidal defenses of alveolar macrophages.