ATP-mediated killing of Mycobacterium bovis Bacille Calmette-Guerin withinhuman macrophages is calcium dependent and associated with the acidification of mycobacteria-containing phagosomes

We previously demonstrated that extracellular ATP stimulated macrophage dea th and mycobacterial killing within :Mycobacterium bovis Bacille Calmette-G uerin (BCG)-infected human macrophages. ATP increases the cytosolic Ca2+ co ncentration in macrophages by mobilizing intracellular Ca2+ via G protein-c oupled P2Y receptors, or promoting the influx of extracellular Ca2+ via P2X purinoceptors. The relative contribution of these receptors and Ca2+ sourc es to ATP-stimulated macrophage death and mycobacterial killing was investi gated. We demonstrate that 1) ATP mobilizes Ca2+ in UTP-desensitized macrop hages (in Ca2+-free medium) and 2) UTP but not ATP fails to deplete the int racellular Ca2+ store, suggesting that the pharmacological properties of AT P and UTP differ, and that a Ca2+-mobilizing P2Y purinoceptor in addition t o the P2Y(2) subtype is expressed on human macrophages. ATP and the Ca2+ io nophore, ionomycin, promoted macrophage death and BCG killing, but ionomyci n-mediated macrophage death was inhibited whereas BCG killing was largely r etained in Ca2+-free medium. Pretreatment of cells with thapsigargin (which depletes inositol (1,4,5)-trisphosphate-mobilizable intracellular stores) or 1,2-bis-(2-aminophenoxy)ethane-N, N, N',N'-tetraacetic acid acetoxymethy l ester (an intracellular Ca2+ chelator) failed to inhibit ATP-stimulated m acrophage death but blocked mycobacterial killing. Using the acidotropic mo lecular probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyl dipropylamine, it w as revealed that ATP stimulation promoted the acidification of BCG-containi ng phagosomes within human macrophages, and this effect was similarly depen dent upon Ca2+ mobilization from intracellular stores. We conclude that the cytotoxic and bactericidal effects of ATP can be uncoupled and that BCG ki lling is not the inevitable consequence of death of the host macrophage.