Characterisation of the P2X(7) receptor-mediated signalling pathway associated with rapid killing of intracellular mycobacteria within human macrophages

Investigation of the P2X(7) receptor-associated signalling events following ATP stimulation of Mycobacterium bovis BCG-infected human macrophages reve aled that ATP-mediated cell death and associated mycobacterial killing coul d be functionally uncoupled. Inhibitors of phospholipase D (PLD) (wortmanni n and butan-1-ol), phospholipase A(2) (PLA(2)) (GW 624A and GW 625A) and th e mitogen-activated protein kinase (MAPK) pathway (SB203580 and PD98059) al l antagonised the ATP-mediated killing of intracellular mycobacteria, but h ad no effect on ATP-induced macrophage death. Inhibitors of protein kinase C (PKC), protein tyrosine kinases (PTKs), and protein serine/threonine phos phatases (PSTPs) were ineffective against either ATP-stimulated cell death or bacterial killing. The mycobactericidal effects of ATP were not associat ed with the induction of apoptosis per se as the broad spectrum caspase inh ibitor, ZVAD-fmk, failed to block killing of mycobacteria within human macr ophages. Inhibitors of protein synthesis (cycloheximide) and transcription (actinomycin D) were also ineffective against the cytotoxic and bactericida l responses of ATP, suggesting that the terminal effector mediator(s) invol ved is preformed and does not require de novo synthesis. Immunoelectron mic roscopy studies using intracellular probes to identify lysosomes revealed t hat ATP treatment of BCG-infected macrophages induced the progressive coloc alisation of mycobacteria-containing phagosomes with lysosomes. The data su ggests that ATP-induced killing of intracellular mycobacteria involves the cellular activation of PLD, PLA2, and MAP kinase, and these signalling even ts lead to the maturation of the BCG-containing phagosome culminating in th e demise of the invading mycobacteria. (C) 2001 Wiley-Liss, Inc.