Analysis of the factor(s) responsible for macrophage killing of Mycobacterium tuberculosis.
thesisposted on 19.11.2015, 09:10 by Seamus. O'Brien
The development of a suitable model of macrophage killing of M. tuberculosis, in vitro, using alveolar macrophages from guinea-pigs that had been vaccinated, boosted and then intravenously challenged (VBC) with Mycobacterium bovis BCG, enabled the mechanisms responsible for tuberculocidal activity to be examined. Alveolar macrophages from the lungs of unvaccinated and VBC guinea-pigs released hydrogen peroxide after stimulation with tubercle bacilli. There was no correlation between the susceptibility of tubercle bacilli to intracellular killing by macrophages from VBC guinea-pigs, in vitro, and triggering of H2O2 release in these macrophages in vitro. The ability of nine strains of tubercle bacilli, representing a spectrum of virulence in the guinea-pig, to trigger H2O2 release in alveolar macrophages from normal and VBC guinea-pigs was equal. There was no correlation between the virulence of M. tuberculosis, represented by the root-index of virulence (RIV), and the extent of H2O2 release triggered. However, the association of these strains of M. tuberculosis, with monolayers of alveolar macrophages from VBC guinea-pigs, was significantly (P 0.05) inversely correlated with virulence. Furthermore, the association of M. tuberculosis with monolayers of macrophages from VBC guinea-pigs was significantly (P 0.001) less than association with monolayers of macrophages from normal guinea-pigs. Alveolar macrophages fron VBC guinea-pigs, killed both a H2O2 resistant strain and hydrogen peroxide sensitive strain of M. tuberculosis, to the same extent, in vitro. Pretreatment of alveolar macrophages in vitro, with catalase and mannitol, agents which remove H2O2 and the hydroxyl radical, respectively, did not decrease the extent of killing of either strain. In contrast, pretreatment of alveolar macrophages with catalase reversed the inhibition of growth of L. monocytogenes. Murine fibroblasts, cells deficient in then respiratory burst, inhibited the replication of M. tuberculosis over 24 hours, after stimulation with IFN-?. However IFN-? stimulated fibroblasts had no effect on the replication of L. monocytogenes, over 4 hours. Lysosomes are a possible site for oxygen-independent anti-mycobacterial factors. A heterogenous population of lysosomes was identified in alveolar macrophages from normal guinea-pigs. The effect of BCG vaccination was to reduce the heterogeneity of lysosomes in alveolar macrophages. BCG vaccination had a variable effect on lysosomal enzyme activity. Further evidence for oxygen-independent tuberculocidal activity came from experiments with cell-free preparations of alveolar macrophages from VBC guinea-pigs. Soluble and membrane extracts of homogenates of alveolar macrophages, from VBC guinea-pigs, displayed heat-labile and heat stable tuberculocidal activity, at an acidic pH. The heat-labile tuberculocidal activity, at acidic pH, was enhanced in alveolar macrophage homogenates enriched for lysosomes. A preliminary investigation suggested that tryptophan degradation was not responsible for the tuberculocidal activity of guinea-pig alveolar macrophages. The addition of exogenous tryptophan had no effect on the killing of M. tuberculosis by guinea-pig alveolar macrophages.