title: ATP-mediated killing of Mycobacterium bovis bacille Calmette-Guérin within human macrophages is calcium dependent and associated with the acidification of mycobacteria-containing phagosomes.
abstract: We previously demonstrated that extracellular ATP stimulated macrophage death and mycobacterial killing within Mycobacterium bovis Bacille Calmette-Guérin (BCG)-infected human macrophages. ATP increases the cytosolic Ca(2+) concentration in macrophages by mobilizing intracellular Ca(2+) via G protein-coupled P2Y receptors, or promoting the influx of extracellular Ca(2+) via P2X purinoceptors. The relative contribution of these receptors and Ca(2+) sources to ATP-stimulated macrophage death and mycobacterial killing was investigated. We demonstrate that 1) ATP mobilizes Ca(2+) in UTP-desensitized macrophages (in Ca(2+)-free medium) and 2) UTP but not ATP fails to deplete the intracellular Ca(2+) store, suggesting that the pharmacological properties of ATP and UTP differ, and that a Ca(2+)-mobilizing P2Y purinoceptor in addition to the P2Y(2) subtype is expressed on human macrophages. ATP and the Ca(2+) ionophore, ionomycin, promoted macrophage death and BCG killing, but ionomycin-mediated macrophage death was inhibited whereas BCG killing was largely retained in Ca(2+)-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 acetoxymethyl ester (an intracellular Ca(2+) chelator) failed to inhibit ATP-stimulated macrophage death but blocked mycobacterial killing. Using the acidotropic molecular probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyl dipropylamine, it was revealed that ATP stimulation promoted the acidification of BCG-containing phagosomes within human macrophages, and this effect was similarly dependent upon Ca(2+) mobilization from intracellular stores. We conclude that the cytotoxic and bactericidal effects of ATP can be uncoupled and that BCG killing is not the inevitable consequence of death of the host macrophage.
authors.affiliation: Medical Research Council Center for Immune Regulation, Division of Immunity and Infection, University of Birmingham, Birmingham, United Kingdom.
authors.forename: C B
authors.initials: CB
authors.lastname: Stober
authors.forename: D A
authors.initials: DA
authors.lastname: Lammas
authors.forename: C M
authors.initials: CM
authors.lastname: Li
authors.forename: D S
authors.initials: DS
authors.lastname: Kumararatne
authors.forename: S L
authors.initials: SL
authors.lastname: Lightman
authors.forename: C A
authors.initials: CA
authors.lastname: McArdle
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology / Q000502:physiology* / Q000633:toxicity; D001433:Bacteriolysis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D016923:Cell Death / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006131:Growth Inhibitors / Q000633:toxicity; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology* / Q000648:ultrastructure; D009163:Mycobacterium bovis / Q000187:drug effects / Q000254:growth & development* / Q000276:immunology; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism* / Q000382:microbiology* / Q000648:ultrastructure; D012333:RNA, Messenger / Q000032:analysis; D018048:Receptors, Purinergic P2 / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D019284:Thapsigargin / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000502:physiology
chemical_list: D006131:Growth Inhibitors; D007476:Ionophores; C109734:P2RY11 protein, human; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D015759:Ionomycin; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D002118:Calcium; D014544:Uridine Triphosphate