SllmmsryWe have examined the effect of killing of host monocytes infected with bacillus Calmette-Gu6rin (BCG) on the viability of the intracellular mycobacteria. Peripheral blood monocytes were infected in vitro with a single bacillus per cell and maintained in culture for 6-8 d to allow the bacilli to replicate. Replicating viable BCG were found singly in perinuclear vacuoles bounded by tightly apposed lipid bilayers. Monocytes were then exposed to toxic mediators that induced killing of cells as evaluated by StCr release into the culture medium. Both hydrogen peroxide (H202) (an inducer of cell necrosis) and adenosine triphosphate (ATP 4-) (an inducer of cell apoptosis) treatment killed infected monocytes. H202-induced killing had no effect on BCG viability. ATPinduced cell death was accompanied by DNA fragmentation and nuclear condensation. Apoptosis was associated with a swelling of the phagocytic vacuoles which became multibacillary and with a reduction of BCG viability as enumerated by colony-forming units.

Host defense against mycobacterial infection requires the participation of monocytes and T cells. Both CD4+ and CD8+ T cells have been shown to be important in resistance to mycobacterial infection in vivo. The main contribution of CD4+ T cells to the protective antituberculosis response involves the production of Th1-type cytokines, including interleukin-2 (IL-2) and gamma interferon (IFN-gamma). CD8+ T cells have been considered to be responsible primarily for cytotoxicity mediated by toxic molecules, including perforin. CD8+ T cells may also elaborate Th1-type cytokines, such as IFN-gamma, in response to the infection. To elucidate the contribution of perforin-mediated target cell death to the control of mycobacterial infection in vivo, mice with a disruption in the perforin gene (P-/-) were infected with Mycobacterium bovis BCG or M. tuberculosis Erdman for 5 and 13 weeks, respectively. At 1, 3, 5, and 13 weeks postinfection, the number of viable mycobacteria in the lungs, spleens, and livers of mice were determined by CFU assay. The infected tissues were examined histologically, and cytokine mRNA levels in the spleens of these mice were determined. Similar studies were carried out in Fas receptor-defective (CBA/lpr(cg)) mice to evaluate the contribution of this alternative cytotoxic pathway to the control of mycobacterial infection. The absence of either perforin gene function or Fas receptor gene function did not modify the course of experimental mycobacterial infection in these mice. In addition, both P-/- and Fas receptor-defective mice appeared to have a compensatory activation of cytokine genes, even in the absence of the experimental infection. P-/- mice had a mean 3.4- to 5-fold increase in mRNA levels for IL-10, IL-12p35, IL-6, and IFN-gamma. Similarly, Fas receptor-defective mice had a mean 3- to 3.6-fold increase in mRNA levels for IFN-gamma, IL-12p35, and IL-10. Our results indicate that both perforin-mediated cytotoxicity and Fas-mediated cytotoxicity do not appear to be necessary for the early control of mycobacterial infection in vivo.

The relative virulence and avirulence of Mycobacterium tuberculosis strains H37Rv and H37Ra were previously defined using animal infection models. To investigate host species' specificity of mycobacterial virulence, growth of the 2 M. tuberculosis strains in human monocyte-derived macrophages in vitro was studied. Mycobacterial growth was evaluated by acid-fast staining, electron microscopy, and colony-forming units (cfu) assay. As expected, the 2 strains demonstrated significantly different growth rates in mouse macrophages in vitro (53 h for H37Rv, 370 h for H37Ra). In marked contrast, in human macrophages the average division times of the strains were nearly equal (80 h for H37Rv and 76 h for H37Ra by cfu measurement, and 96 h for H37Rv and 104 h for H37Ra by acid-fast staining). These findings indicate that observations of mycobacterial virulence in murine systems may not necessarily translate to the human system, in which different mechanisms to control mycobacterial growth may be expressed.

No abstract

Mycobacterium avium-M. intracellulare, an intracellular parasite of mononuclear phagocytes, rarely causes disease in immunocompetent individuals. In contrast, in human immunodeficiency virus type 1-infected patients, M. avium-M. intracellulare can infect almost every tissue and organ. This suggests that immunocompetent individuals have a protective mechanism to control or prevent the infection. How mycobacteria may be killed by the host immune response is unclear. We have recently reported that induction of apoptosis of Mycobacterium bovis BCG-infected macrophages with ATP 4؊ was associated with killing of the intracellular mycobacteria. In the present study, a long-term culture of M. avium-M. intracellulare-infected monocytes was used to further evaluate the interaction between M. avium-M. intracellulare and primary human monocytes. In our system, M. avium-M. intracellulare parasitized the human monocytes and appeared to replicate slowly over 14 days within the host cells. To examine the role of apoptotic mechanisms in survival or death of intracellular mycobacteria, M. avium-M. intracellulare-infected human monocytes were treated with a monoclonal antibody to Fas receptor (APO-1/CD95) or with various concentrations of H 2 O 2 . Although both of these exogenous agents induced monocyte apoptosis, optimal killing (65% reduction in CFU) of intracellular M. avium-M. intracellulare was observed only when M. avium-M. intracellulare-infected cells were treated with 10 mM H 2 O 2 . Fas-induced apoptosis did not affect M. avium-M. intracellulare viability. Our results suggest that not all stimuli of monocyte apoptosis induce killing of intracellular M. avium-M. intracellulare. Since release of H 2 O 2 following phagocytosis of mycobacteria has been documented, H 2 O 2 -induced apoptotic death of M. avium-M. intracellulare-infected monocytes and its association with killing of the intracellular bacilli may be a physiological mechanism of host defense against M. avium-M. intracellulare.