Nitrotyrosine formation after activation of murine macrophages with mycobacteria and mycobacterial lipoarabinomannan

Abstract

Murine peritoneal macrophages, elicited with thioglycollate, were stimulated in vitro with lipopolysaccharide (LPS). The production of nitrite, superoxide anion (SOA), and the accumulation of nitrotyrosine in the cells increased after treatment, and all were inhibitable by the NO synthase inhibitor N(G)-monomethyl-L-arginine monoacetate (L-NMMA). This effect suggests a direct correlation between the accumulation of those metabolites and NO synthase activity. Lipoarabinomannan (LAM) purified from Mycobacterium tuberculosis was added to peritoneal macrophages in the presence of interferongamma (IFN-γ); the cells produced nitrite and SOA, both inhibitable by L-NMMA. There was, as well, accumulation of nitrotyrosine in the macrophage proteins. Strikingly, the amount of nitrotyrosine measured after LAM plus IFN-γ or LAM plus the low molecular weight adjuvant glutamylmuramyl dipeptide (GMDP), increased significantly in the presence of L-NMMA. These results suggest that murine macrophages, upon LAM stimulation, might generate reactive nitrogen metabolites by a route other than NO synthase. Nitrotyrosine accumulation after infection of macrophages in vitro, with either live bacille Calmette-Guerin (BCG) or live M. tuberculosis, in the presence or absence of IFN-γ showed no correlation with nitrite production, suggesting a low superoxide production.