Extracellular ATP Is Cytotoxic to Mononuclear Phagocytes but Does Not Induce Killing of IntracellularMycobacterium aviumsubsp.paratuberculosis

Mycobacterium aviumsubsp.paratuberculosisis the etiologic agent of Johne's disease, a chronic granulomatous enteritis in ruminants. ATP has been reported to induce cell death of macrophages and killing ofMycobacteriumspecies in human and murine macrophages. In this study we investigated the short-term effect of ATP on the viability ofM. aviumsubsp.paratuberculosis-infected bovine mononuclear phagocytes and the bacilli within them. Addition of 5 mM ATP toM. aviumsubsp.paratuberculosis-infected bovine monocytes resulted in 50% cytotoxicity of bovine monocytes at 24 h. Addition of 2′(3′)-O-(4-benzoylbenzoyl) ATP triethylammonium salt (Bz-ATP), which is a longer-lived ATP homologue and purinergic receptor agonist, significantly increased the uptake of YO-PRO, which is a marker for membrane pore activation by P2X receptors. Addition of Bz-ATP also stimulated lactate dehydrogenase release and caspase-3 activity in infected bovine monocytes. Neither ATP nor Bz-ATP reduced the survival ofM. aviumsubsp.paratuberculosisin bovine mononuclear phagocytes. Likewise, addition of ATP or Bz-ATP was cytotoxic to murine macrophage cell lines (RAW 264.7 and J774A.1 cells) but did not affect the intracellular survival ofM. aviumsubsp.paratuberculosis, nor were the numbers of viableMycobacterium aviumsubsp.aviumorMycobacterium bovisBCG cells altered in bovine mononuclear phagocytes or J774A.1 cells following ATP or Bz-ATP treatment. These data suggest that extracellular ATP does not induce the killing of intracellularM. aviumsubsp.paratuberculosisin bovine mononuclear phagocytes.