ABSTRACT
In earlier studies, it was shown that ex vivo Mycobacterium tuberculosis-infected type II alveolar epithelial cells generate de novo nitric oxide [NO], but the mycobactericidal quantity of NO was released only by stimulation of these cells with proinflammatory cytokines, i.e. IFN-gamma, TNF-alpha and IL-1beta. In the present communication, it was demonstrated that M. tuberculosis-infected/mycobacterial antigens stimulated cells utilize both, JAK-STAT and NF-kappaB pathways for the induction of inducible Nitric Oxide Synthase [iNOS] mRNA and NO production. Alveolar epithelial cell line A549 were either infected with M. tuberculosis or stimulated with M. tuberculosis components. Confocal microscopy, NO estimation and EMSA were performed on the infected/stimulated A549 cells. Nuclear extracts prepared from M. tuberculosis infected A549 cells alone or stimulated with IFN-gamma or a combination of three cytokines [IFN-gamma, TNF-alpha and IL-1beta] formed DNA protein complexes with probes from both -5.2 kb region [specific for binding of STAT-1 protein] and -5.8 kb region [specific for binding of both STAT-1 and NF-kappaB] of the iNOS promoter. However, TNF-alpha or IL-1beta stimulated M. tuberculosis-infected A549 cells showed no protein DNA complexes with construct from -5.2 kb region. This differential response indicated that TNF-alpha/IL-1beta does not allow STAT-1 production or its translocation to nucleus in M. tuberculosis-infected A549 cells in the absence of IFN-gamma. This differential signaling of iNOS induction in M. tuberculosis-infected alveolar epithelial cells by cytokines may be responsible for controlled production of NO intracellularly.