The effect of
Mycobacterium tuberculosis infection on the viability of healthy (control) human alveolar macrophages was evaluated by staining with
ethidium homodimer and
calcein to discriminate live from dead cells.
Infection with M.
tuberculosis H37Ra or H37Rv increased macrophage mortality at 6 days from the control level of 3.8% +/- 0.7% to 28.7% +/- 6.9% or 12.6% +/- 3.1%, respectively (P < 0.001 for comparisons of all conditions). A role for
tumor necrosis factor alpha (
TNF-alpha) in the M.
tuberculosis-induced cytolysis of alveolar macrophages was demonstrated by increased cytotoxicity following the addition of exogenous
TNF-alpha to the cultures and by enhancement of macrophage survival when M.
tuberculosis-infected alveolar macrophages were treated with
pentoxifylline or anti-
TNF-alpha antibody. The cytolytic mechanism was determined to be apoptosis by the demonstration of a characteristic internucleosomal ladder of genomic
DNA by
agarose gel electrophoresis, by finding nuclear fragmentation and condensation by electron microscopy, and by in situ terminal
transferase-mediated nick end labeling of fragmented
DNA in alveolar macrophages infected with M.
tuberculosis in vitro. The latter technique was employed to reveal extensive apoptosis within caseating
granulomas from lung tissue samples from clinical
tuberculosis cases. The induction of apoptosis in alveolar macrophages by M.
tuberculosis may play a role in the macrophage-pathogen interaction of
tuberculosis in vivo.