Reduced apoptosis and increased inflammatory cytokines in granulomas caused by tuberculous compared to non-tuberculous mycobacteria: role of MPT64 antigen in apoptosis and immune response

Inhibition of apoptosis of infected macrophages by pathogenic mycobacteria is suggested to be an important virulence mechanism, but little is known about the mycobacterial proteins involved in the inhibition of apoptosis. In this study we investigated differences in apoptosis and immune response and their correlation with the expression of Mycobacterium tuberculosis complex-specific secretory protein MPT64 in lesions caused by tuberculous or non-tuberculous mycobacteria by analysing the in situ expression of apoptosis-related proteins (FasL, Fas, Bax, Bcl-2), apoptotic cells, inflammatory cytokines [tumour necrosis factor (TNF)-alpha, interleukin (IL)-10, transforming growth factor (TGF)-beta, interferon (IFN)-gamma] and MPT64 antigen. The discrimination of mycobacteria was made by nested polymerase chain reaction (PCR) amplification of IS6110, which is specific for M. tuberculosis complex organisms. Forty-seven cases of lymphadenitis with necrotic granulomas were evaluated. With nested PCR, 30/47 cases were positive for M. tuberculosis. MPT64 antigen was detected specifically in the PCR-positive cases. Granulomas caused by tuberculous mycobacteria had fewer apoptotic cells, higher numbers of cells expressing TNF-alpha and TGF-beta and less extensive necrosis than granulomas caused by non-tuberculous mycobacteria. There was a significant negative correlation between apoptotic cells and the number of cells expressing MPT64 antigens, suggesting a role for MPT64 protein in the inhibition of apoptosis. Granulomas with higher amounts of MPT64 also showed a greater number of cells expressing TGF-beta than those with lower amounts of MPT64. In conclusion, this study supports the hypothesis that inhibition of apoptosis is a virulence mechanism for tuberculous mycobacteria. Correlation of MPT64 antigen with expression of macrophage deactivating cytokines and reduced apoptosis suggests its role in pathogenesis and bacillary persistence.