NITRITE PRODUCTION BY MACROPHAGES DERIVED FROM BCG-RESISTANT AND BCG-SUSCEPTIBLE CONGENIC MOUSE STRAINS IN RESPONSE TO IFN-GAMMA AND INFECTION WITH BCG

Reactive nitrogen intermediates (RNI) have been implicated in the inte rferon-gamma (IFN-gamma)-induced anti-microbial action of macrophages against a wide variety of pathogens. We have been studying the product ion of NO2- by macrophage lines derived from the bone marrow of either B10.A (Bcg(s)) strain mice (B10S cell lines), or their congenic BCG-r esistant partners of the B10A.Bcg(r) (Bcg(r)) strain (B10R cell lines) . We have discovered that there is a significant difference in the pro duction of NO2- of B10S compared with B10R macrophages in response to IFN-gamma. By 48 hr following treatment with 10 U/ml IFN-gamma, B10R m acrophages had produced an approximately threefold higher level of NO2 - than B10S macrophages. Similar results were obtained when experiment s were performed with total splenic cells harvested from the spleens o f B10.A.Bcg(r) and B10.A strain mice. The bacteriostatic activity, as assessed by the [H-3]uracil incorporation by Mycobacterium bovis BCG, was higher in B10R macrophages compared to B10S macrophages. The bacte riostatic activity of B10R and B10S macrophages correlated with the am ount of nitric oxide produced by the macrophages. The anti-mycobacteri al activity was inhibited by N(g)MMLA, a specific inhibitor of nitrite and nitrate synthesis from L-arginine. Addition of L-arginine to IFN- gamma-stimulated macrophages in the presence of N(g)MMLA restored nitr ite production and bacteriostatic activity of macrophages. Northern bl ot analysis of macrophage nitric oxide synthase (iNOS) revealed that t he difference in NO2- production by IFN-gamma-treated B10S and B10R li nes was reflective of the difference in iNOS mRNA expression.