TNF-α controls intracellular mycobacterial growth by both inducible nitric oxide synthase-dependent and inducible nitric oxide synthase-independent pathways

The role of TNF-a in the control of mycobacterial growth in murine macrophages was studied in vitro. Infection of macrophages from TNF-a gene disrupted (TNF-knockout (KO)) mice with recombinant Mycobacterium bovis bacillus Calmette Guerin (BCG) expressing the vector only (BCG-vector) resulted in logarithmic growth of the intracellular bacilli. Infection with BCG-secreting murine TNF-a (BCG-TNF) led to bacillary killing. Killing of BCG-TNF was associated with rapid accumulation of inducible NO synthase (iNOS) protein and the production of nitrite. The uncontrolled growth of BCG-vector was associated with low iNOS expression but no nitrite production. Thus, iNOS expression appears to be TNF-a independent but iNOS generation of NO requires TNF-a. In cultures of TNF-KO macrophages infected with BCG-TNF, inhibition of iNOS by aminoguanidine (AMG) abolished the killing of the bacilli. However, the growth of the organisms was still inhibited, suggesting an iNOS-independent TNF-alpha -mediated growth inhibition. To confirm this, macrophages from iNOS-KO mice were infected with either BCG-vector or BCG-TNF. As expected, no nitrite was detected in the culture medium. TNF-a was detected only when the cells were infected with BCG-TNF. In the iNOS-KO macrophages, the growth of BCG was inhibited only in the BCG-TNF infection. These results suggest that in the absence of iNOS activity, TNF-a stimulates macrophages to control the growth of intracellular BCG. Thus, there appears to be both a TNF-alpha -dependent-iNOS-dependent killing pathway as well as a TNF-alpha- dependent-iNOS-independent growth inhibitory pathway for the control of intracellular mycobacteria in murine macrophages. The Journal of immunology 2001,166:6728-6734.