Characterisation of the P2X7 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 revealed that ATP-mediated cell death and associated mycobacterial killing could be functionally uncoupled. Inhibitors of phospholipase D (PLD) (wortmannin and butan-1-ol), phospholipase A(2) (PLA(2)) (GW 624A and GW 625A) and the mitogen-activated protein kinase (MAPK) pathway (SB203580 and PD98059) all antagonised the ATP-mediated killing of intracellular mycobacteria, but had no effect on ATP-induced macrophage death. Inhibitors of protein kinase C (PKC), protein tyrosine kinases (PTKs), and protein serine/threonine phosphatases (PSTPs) were ineffective against either ATP-stimulated cell death or bacterial killing. The mycobactericidal effects of ATP were not associated with the induction of apoptosis per se as the broad spectrum caspase inhibitor, ZVAD-fmk, failed to block killing of mycobacteria within human macrophages. Inhibitors of protein synthesis (cycloheximide) and transcription (actinomycin D) were also ineffective against the cytotoxic and bactericidal responses of ATP, suggesting that the terminal effector mediator(s) involved is preformed and does not require de novo synthesis. Immunoelectron microscopy studies using intracellular probes to identify lysosomes revealed that ATP treatment of BCG-infected macrophages induced the progressive colocalisation of mycobacteria-containing phagosomes with lysosomes. The data suggests that ATP-induced killing of intracellular mycobacteria involves the cellular activation of PLD, PLA2, and MAP kinase, and these signalling events lead to the maturation of the BCG-containing phagosome culminating in the demise of the invading mycobacteria. (C) 2001 Wiley-Liss, Inc.