Stober C B, Lammas D A, Li C M, Kumararatne D S, Lightman S L, McArdle C A
Medical Research Council Center for Immune Regulation, Division of Immunity and Infection, University of Birmingham, Birmingham, United Kingdom.
J Immunol. 2001 May 15;166(10):6276-86. doi: 10.4049/jimmunol.166.10.6276.
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.
我们之前证明，细胞外ATP可刺激牛分枝杆菌卡介苗（BCG）感染的人巨噬细胞发生死亡并杀伤分枝杆菌。ATP通过G蛋白偶联的P2Y受体动员细胞内Ca(2+)，或通过P2X嘌呤受体促进细胞外Ca(2+)内流，从而增加巨噬细胞胞质Ca(2+)浓度。研究了这些受体和Ca(2+)来源对ATP刺激的巨噬细胞死亡和分枝杆菌杀伤的相对贡献。我们证明：1）ATP可在UTP脱敏的巨噬细胞（在无Ca(2+)培养基中）中动员Ca(2+)；2）UTP而非ATP无法耗尽细胞内Ca(2+)储存，这表明ATP和UTP的药理学特性不同，且除P2Y(2)亚型外，人巨噬细胞上还表达一种可动员Ca(2+)的P2Y嘌呤受体。ATP和Ca(2+)离子载体离子霉素可促进巨噬细胞死亡和BCG杀伤，但在无Ca(2+)培养基中，离子霉素介导的巨噬细胞死亡受到抑制，而BCG杀伤在很大程度上得以保留。用毒胡萝卜素（耗尽肌醇(1,4,5)-三磷酸可动员的细胞内储存）或1,2-双-(2-氨基苯氧基)乙烷-N,N,N',N'-四乙酸乙酰甲酯（一种细胞内Ca(2+)螯合剂）预处理细胞，未能抑制ATP刺激的巨噬细胞死亡，但阻断了分枝杆菌杀伤。使用亲酸性分子探针3-(2,4-二硝基苯胺)-3'-氨基-N-甲基二丙胺发现，ATP刺激可促进人巨噬细胞内含有BCG的吞噬体酸化，且这种效应同样依赖于从细胞内储存中动员Ca(2+)。我们得出结论，ATP的细胞毒性和杀菌作用可以分离，且BCG杀伤并非宿主巨噬细胞死亡的必然结果。
Zotero 插件