Reactive nitrogen intermediates implicated in the inhibition of Encephalitozoon cuniculi (phylum microspora) replication in murine peritoneal macrophages

Abstract

Encephalitozoon cuniculi (phylum microspora) is a protozoan parasite that can replicate within parasitophorous vacuoles in macrophages. Thioglycollate-elicited BALB/c peritoneal macrophages treated with murine recombinant interferon-gamma (rIFN-gamma; 100 7/ml) in combination with lipopolysaccharide (LPS; 10 ng/ml) for 24 h killed E. cuniculi as determined by significant reductions in the number of parasites and percent of infected macrophages 48 h later compared with cultures treated with medium only. Treatment of the elicited macrophages with murine rIFN-gamma (10 u/ml or 100 u/ml) only, resulted in microbistatic activity. Significantly higher levels of nitrite (NO2) were detected in supernatants from macrophage cultures treated with rIFN-gamma (10 u/ml or 100 u/ml) which induced microbistatic macrophage activity as well as from macrophage cultures treated with LPS + rIFN- when compared with levels of nitrite detected in supernatants of infected macrophages treated with medium only. Addition of the L-arginine analogue, N3 monomethyl-L-arginine (NMMA) at concentrations of 50, 100 or 250 uM significantly inhibited nitrite synthesis and prevented microsporidia killing. Addition of exogenous L-arginine at concentrations of 5 mM or 10 mM reversed the NMMA-induced inhibition of parasite killing. These results indicate that reactive nitrogen intermediates contribute to the killing of E. cuniculi by LPS + rIFN-gamma-activated murine peritoneal macrophages in vitro.