84 At the end of their infection cycle, each of these intracellular parasites is released from the host cell, triggering macrophage death and inducing local inflam- mation, accompanied by the possible release of ATP, as shown for macrophages infected with Mycobacterium tuberculosis [11]. As extracellular ATP (ATPe) can be used by neighbouring macrophages as ammunition to inhibit infection (described below), many intracellular pathogens— such as Mycobacterium bovis BCG, Vibrio cholerae, M. tuberculosis, T. cruzi and Leishmania—also secrete or express on their outer surface enzymes that degrade or synthesise nucleotides [12–15]; and microbial enzymes that consume or produce ATP are considered as virulence factors for M. tuberculosis, Leishmania amazonensis and T. cruzi [14, 16–18]. Macrophages activate microbicidal pathways and con- tribute to inflammation after the ligation of purinergic P2 receptors by ATPe. This review will, therefore, describe some of the P2-dependent mechanisms used by macro- phages to eliminate infection by intracellular bacteria and protozoan parasites, and, whenever possible, will correlate these findings with host susceptibility and resistance to infection. Effect of ATPe on macrophage infection by M. tuberculosis The first evidence for an involvement of ATPe in the control of intracellular infections came from the laboratory of Kaplan, who demonstrated in 1994, that ATPe-mediated apoptosis in Mycobacterium tuberculosis-infected macro- phages is associated with the inhibition of mycobacterial infection [19]. This early study also showed that ATPe- induced macrophage apoptosis, but not H2O2-induced necrosis, is associated with the killing of the intracellular mycobacteria. These findings were confirmed by Lammas et al., who proposed that the ATPe-induced elimination of BCG-infected human macrophages are mediated by the purinergic receptor, P2X7, through a mechanism indepen- dent of both RNIs and ROIs [20]. In comparison with other ligands that can trigger the lysis of macrophages, including complement-mediated cytolysis, Fas ligation and CD69 activation, only ATPe treatment could stimulate the death of both host macrophages and intracellular mycobacteria. Subsequently, several laboratories confirmed that the P2X7 receptor plays a role in limiting infection in murine and bovine macrophages infected with mycobacteria, and in human macrophages infected with BCG bacillus and virulent strains of M. tuberculosis [11, 21–23]. In addition to the P2X7 receptor, other P2 receptor subtypes, possibly P2Y, are apparently involved in ATPe- mediated bactericidal activity in macrophages [11, 21, 23]. Purinergic Signalling (2007) 3:83–90 Experiments using macrophages derived from P2X7-defi- cient mice revealed that ATPe stimulates the production of reactive species such as RNIs equally well in both wildtype and P2X7-deficient macrophages [11]. Moreover, it was found that ATPe induces bactericidal effects in the macro- phages better than BzATP (the most potent known agonist for the P2X7 receptor), suggesting that P2X7 receptors are necessary, but not sufficient, for maximal ATPe-dependent killing of intracellular M. tuberculosis by human and bovine macrophages [21, 22]. Lammas et al. have further observed that the ATPe activity is potentiated by extracel- lular Zn2+ [20]. This effect was initially ascribed to the P2X7 receptor, but now, P2X7 activity is known to be blocked by extracellular Zn2+, while the activity of another purinergic receptor, P2X4, is potentiated by Zn2+ [24]. Since macrophages express functional P2X4 receptors [25] and inflammatory mediators can upregulate this receptor on macrophages [26], it is likely that both P2X7 and P2X4 are involved in the ATPe-induced killing of M. tuberculosis in macrophages. More recent reports have also confirmed the predomi- nant role of the P2X7 receptor in mycobacterial clearance, extending these results to show that loss-of-function poly- morphisms in human P2X7 receptors lead not only to reduced ATPe-induced apoptosis, but also to impaired ATPe-induced killing of intracellular mycobacteria (BCG) by macrophages [27–29]. Nonetheless, more experiments will be required to elucidate the role that P2X7, possibly in conjunction with other P2 receptors, may play in the killing of intracellular mycobacteria in vivo, since P2X7-deficient mice control lung infection as well as wildtype mice after low-dose aerosol infection with virulent M. tuberculosis [30]. Cellular mechanisms of ATPe-induced mycobacterial killing ATPe ligation of P2X7 on macrophages results in a variety of different cellular effects, including the activation of phospholipase D (PLD), maturation and release of inter- leukin-1β (IL-1β), generation of macrophage polykarions, modulation of lipopolysaccharide (LPS) induced macro- phage activation through modulation of iNOS expression and NO production, and the induction of macrophage death by necrosis and/or apoptosis [31–40]. The original findings from the Kaplan laboratory suggested that the apoptosis of macrophages is necessary for ATPe-mediated killing of intracellular bacteria [19], but it was later established that the ATPe-induced killing of mycobacteria in human and mice macrophages can occur without macrophage death, through a pathway requiring PLD activation, the acidifica- tion of phagosomes and phagosome–lysosome fusion [21]