Neutralization of gamma interferon and tumor necrosis factor alpha blocks in vivo synthesis of nitrogen oxides from L-arginine and protection against Francisella tularensis infection in Mycobacterium bovis BCG-treated mice

Peritoneal cells from Mycobacterium bovis BCG-infected C3H/HeN mice produced nitrite (NO₂^-, an oxidative end product of nitric oxide [NO] synthesis) and inhibited the growth of Francisella tularensis, a facultative intracellular bacterium. Both NO₂^- production and inhibition of bacterial growth were suppressed by N(G)-monomethyl-L-arginine, a substrate inhibitor of nitrogen oxidation of L-arginine, and monoclonal antibodies (MAbs) to gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Intraperitoneal injection of mice with BCG increased urinary nitrate (NO₃^- ) excretion coincident with development of activated macrophages capable of secreting nitrogen oxides and inhibiting F. tularensis growth in vitro. Eight days after BCG inoculation, mice survived a normally lethal intraperitoneal challenge with F. tularensis. Treatment of these BCG-infected mice with MAbs to IFN-γ or TNF-α at the time of BCG inoculation reduced urinary NO₃^- levels to those found in normal uninfected mice for up to 14 days. The same anticytokine antibody treatment abolished BCG-mediated protection against F. tularensis: mice died within 4 to 6 days. Intraperitoneal administration of anti-IFN-γ or anti-TNF-α antibody 8 days after BCG infection also reduced urinary NO₃^- and abolished protection against F. tularensis. Isotype control (immunoglobulin G) or anti-interleukin 4 MAbs had little effect on these parameters at any time of treatment. IFN-γ and TNF-α were clearly involved in the regulation of macrophage activation by BCG in vivo. Protection against F. tularensis challenge by BCG depended upon the physiological generation of reactive nitrogen oxides induced by these cytokines.