| Johne's disease is a chronic granulomatous enteritis caused by Mycobacterium paratuberculosis in ruminants. Survival and multiplication of M. paratuberculosis in bovine mononuclear phagocytes play a role in the pathogenesis of Johne's disease. ATP is known to be released from various cells by nonlytic and lytic mechanisms, and to be cytotoxic to macrophages and induce intracellular killing of mycobacteria in human macrophages. It is possible that ATP release in M. paratuberculosis infected foci might affect intracellular survival of M. paratuberculosis. In this study, we investigated the intracellular fate of M. paratuberculosis in bovine mononuclear phagocytes in vitro, and how this was affected by ATP.;The number of viable intracellular bacilli in bovine monocytes increased between day 0 and day 4, and then declined at day 8 after infection. However, the number of acid-fast bacilli visible microscopically increased steadily during an 8-day incubation period. Using live/dead staining, the percentage of live bacilli decreased during the same time period. These data suggest that bovine monocytes both permit the intracellular multiplication and kill M. paratuberculosis. M. paratuberculosis might survive in bovine monocytes in part because of low production of ROI and RNI, and poor phagosome-lysosome fusion following ingestion of bacilli.;We hypothesized that ATP might induce intracellular killing of M. paratuberculosis in bovine monocytes. We observed that bovine monocytes constitutively released ATP. Removal of secreted ATP by apyrase increased monocyte survival, and increased intracellular killing of M. paratuberculosis. Conversely, addition of ATP decreased monocyte viability and increased intracellular survival of M. paratuberculosis during an 8-day incubation period. In subsequent experiments, ATP (5 mM) or BzATP caused substantial monocyte death at 24 hrs incubation. However, neither ATP nor Bz-ATP treatment (24 hr) induced killing of intracellular M. paratuberculosis in bovine monocyte-derived macrophages, or in the murine macrophage cell lines J774 and RAW.264.3.;The results of this study suggest that ATP is cytotoxic to bovine mononuclear phagocytes, but does not induce intracellular killing of M. paratuberculosis . Inhibition or killing of intracellular M. paratuberculosis is presumed to be regulated by other factors. |
