In recent years, a great deal of attention has been devoted to elucidating the mechanisms of release of the pro-inflammatory leaderless cytokine IL-1 from monocytes and macrophages. Originally produced as 31-kDa precursors, the two IL-1 isoforms, pro-IL-1α and pro-IL-1β, are subsequently cleaved by interleukin-converting enzyme (ICE; also known as caspase-1 20) to produce the mature 17-kDa forms 21. IL-1α and IL-1β are thought to have identical biological actions, although IL-1β, unlike IL-1α, is inactive in its immature form 21. The mechanism of IL-1β release has been extensively studied in vitro, although there are only a limited number of molecules capable of inducing controlled release, and whether these processes reflect the in vivo situation remains unclear. Upon release, IL-1β is known to elicit diverse responses, including the activation of macrophages, T-cells and signalling cascades, as well as the induction of cyclooxygenase type 2 (COX-2) and fever 22. IL-1 has been shown to be important in many diseases including rheumatoid arthritis 23, multiple sclerosis 24, asthma 25 and chronic obstructive pulmonary disease 26. It is therefore clear that IL-1β is of particular importance in the initiation and propagation of an inflammatory response, with its functions and therapeutic potential extensively reviewed 2227.

Originally, cell death by apoptosis was reported to stimulate the production and release of mature IL-1β, although the mechanism was not identified 28. The release of mature IL-1β appeared to require two consecutive stimuli 29, with LPS stimulation in monocytes only producing pro-ICE and pro-IL-1β 30. The latter authors reported that ATP-stimulated K⁺ efflux was important for the release of mature IL-1β 30, with Ferrari and colleagues subsequently suggesting that it was P2X₇R-mediated, and independent of apoptosis 31. This was latterly confirmed in pharmacological 32 studies and those using P2X₇R knockout mice 3334, with the activation of the P2X₇R by ATP producing a fall in cytoplasmic K⁺ concentration which in turn stimulates processing of pro-ICE to ICE, and thereby inducing release of mature IL-1β (Figure 1; 35). Indeed, in an elegant series of studies Surprenant and colleagues have subsequently demonstrated that ATP-induced activation of the P2X₇R results in the shedding of microvesicles which contain mature IL-1β 36 and more recently IL-1RA 37. With high concentrations (0.5–5 mM) of ATP required for optimal activation of P2X₇R-mediated IL-1β release in vitro 38, alternative endogenous agonists that could produce significant P2X₇R stimulation have been sought. Interestingly, several cationic host defence peptides (CHDP; also known as antimicrobial peptides) have recently been shown to mediate post-translational processing of IL-1β in LPS-primed monocytes. Although the mechanisms of action of the porcine CHDP protegrin-1 and -3 have been shown to be P2X₇R-independent 39, three studies have now proposed that P2X₇R activation underlies some of the immunomodulatory effects of the human CHDP, LL-37 384041. LL-37 is the major active cleavage product of the only human cathelicidin hCAP18, is upregulated in infection and inflammation 4243, and in addition to broad-spectrum antimicrobial activity and direct anti-endotoxic effects, LL-37 has a number of immunomodulatory roles 44. LL-37 has now been shown to induce caspase-1 activation and secretion of mature IL-1β in LPS-primed monocytes, in the absence of cytotoxicity, through P2X₇R activation 38. Furthermore, recent studies have demonstrated that concentrations of LL-37 as low as 250 ng/ml, and well within the physiological range, can inhibit apoptosis in human neutrophils, in a P2X₇R-dependent manner involving the PI3-kinase pathway 4041. Such studies indicate that in addition to extracellular ATP, the endogenous, inducible CHDP, LL-37 may activate the P2X₇R on key innate immune effector cells to modulate cytokine release. Finally, as compounds such as Tenidap, which is being evaluated for its anti-inflammatory and anti-arthritic properties also appear to inhibit the release of IL-1β 45, whilst sensitising the P2X₇R on macrophages to the cytotoxic effects of ATP 46, future studies may show that the P2X₇R could be regulated by a range of ligands.

The importance of, and the mechanisms through which the P2X₇R regulates the production of the pro-inflammatory cytokines IL-1β and IL-18, and potentially the innate immune response, was recently and beautifully described by Mariathasan and colleagues 47. These authors showed that the P2X₇R is up-stream of the inflammasome, an important complex of cytosolic proteins that are known to regulate caspase-1 activation and ultimately the processing of IL-1β and IL-18. With inflammasome dysregulation known to produce inflammatory disorders such as Muckle-Wells syndrome and neonatal onset multisystem inflammatory disease, it is clear that inhibiting inflammasome activation with P2X₇R antagonists could affect the outcome of a range of inflammatory disorders 47. However, one must remember that the P2X₇R may not be the only purinergic receptor involved in IL-1β release. A recent study has shown ATP-dependent Ca²⁺ release from intracellular stores (endoplasmic reticulum) is also involved in the secretion of pro-IL-1β, although it was not independently capable of releasing mature IL-1β 48. As discussed above, K⁺ efflux was also reported to be necessary for the release of mature IL-1β, with Brough and colleagues (2003) proposing that ATP may stimulate both P2X and P2Y receptors 48. The importance of P2Y receptor stimulation and Ca²⁺ release from intracellular stores remains to be determined.

P2X₇R-mediated regulation of IL-1β has also been demonstrated within the central nervous system where microglia are the resident monocytic cells. In a seminal study in 1997, Ferrari et al 49 reported that ATP induced IL-1β production in cultured microglial cells through the activation of the P2X₇R. Subsequent studies showing that cultured microglia from P2X₇R knockout mice do not release IL-1β following exposure to LPS and ATP 50 support the role for P2X₇R in IL-1β production, albeit in vitro. P2X₇R up-regulation has been observed in response to a variety of inflammatory brain insults, underpinning the view that P2X₇R antagonists may be of therapeutic use for the treatment of several disorders including stroke, traumatic brain injury (TBI), multiple sclerosis and Alzheimer's disease 3515253. Since IL-1β has been reported to induce COX-2 in various tissues including glia, it has been proposed that a vicious cycle occurs whereby ATP release (from cell death for example) leads to P2X₇R activation, IL-1β release, COX-2 induction and further cell death with consequent ATP release; this type of self-perpetuating cycle may underlie lesion expansion particularly in stroke and TBI. Once selective P2X₇R antagonists become commercially available it will be possible to test the importance of this receptor in these processes. However, it is interesting to note that non-specific antagonism of P2X receptors by PPADS, and the inhibition of IL-1β, and COX-2, have all been reported to be effective in animal models of stroke and other neurodegenerative disorders 5154. Intriguingly, another function attributed to the P2X₇R that is important in neuropathology is microglial production of superoxide anion 55. The significance of P2X₇R regulation of superoxides was underlined by the observation that P2X₇R expression was up-regulated around β-amyloid plaques in a mouse model of Alzheimer's disease 55. It was also subsequently shown that in human microglia, β-amyloid-induced cytokine release (e.g. IL-1β) was found to be modulated by ATP, probably via the P2X₇R 56.

Understandably, polymorphisms in the genes encoding IL-1, its receptor, and IL-1RA have been found to be associated with a range of diseases including rheumatoid arthritis, systemic lupus erythematosus, atherosclerosis and tuberculosis 57. As a result of the importance of the P2X₇R in IL-1β processing and release, polymorphisms in this unique ion channel have been investigated and to date, in excess of 260 polymorphisms have been identified for the P2X₇R 5859. One such polymorphism is the single nucleotide substitution at position 1513 of the P2X₇R gene which changes a glutamic acid to an alanine at amino acid position 496 (Glu⁴⁹⁶Ala), and leads to loss of function of the receptor 60. It is interesting to note that this polymorphism decreased the ATP-induced K⁺ efflux subsequently delaying the ATP-induced release of IL-1β. The fact that IL-1β release was delayed rather than abrogated indicates that there are compensatory or redundant mechanisms present 61. However there is now evidence from P2X₇R polymorphism studies, that those associated with a loss of function mutation have a reduced sensitivity to inflammation 62.

In the absence of commercially available potent and selective P2X₇R antagonists, P2X₇R knockout mice have provided new insights into the in vivo role of this receptor. Labasi and colleagues 34 reported that peritoneal macrophages from P2X₇R deficient mice were unable to produce mature IL-1β in response to LPS, or ATP application, or with a combination of both stimuli. This study also compared the induction of monoclonal anti-collagen-induced arthritis in P2X₇R-deficient mice and wild-type littermates, with the former group demonstrating reduced susceptibility to, and severity of disease 34. It was therefore suggested that, in normal mice, endogenous ATP is present in sufficient concentrations at sites of inflammation to activate the P2X₇R 34, (an area that has attracted some scepticism based on in vitro work with the addition of exogenous ATP 38). However, as described earlier, care must now be taken in interpreting results observed in vivo, as although ATP was originally thought to be the only endogenous agonist of the P2X₇R, recently other physiological agents such as LL37 (see above) and NAD 63 have been reported to activate the P2X₇R at lower concentrations. New studies in P2X₇R knockout mice continue to indicate that this receptor plays a role in a number of conditions in addition to arthritis and include multiple sclerosis, hepatitis and pain 34646566.

In addition to IL-1β secretion, the P2X₇R has been implicated in the synthesis and release of the related leaderless cytokine IL-18 (interferon-γ-inducing factor), which is also produced through cleavage of pro-IL-18 by ICE 475967, although it has not yet been extensively studied. In contrast to IL-1β, secretion of IL-18 was found to be less dependent on LPS-priming 68, although conflicting data was presented by Mehta et al who found IL-18 production to be LPS-dependent 69. Indeed it has been shown that individuals expressing the Glu⁴⁹⁶Ala P2X₇R polymorphism produce significantly less IL-18 when their monocytes are stimulated by ATP 61. We have also shown that in LPS primed, BzATP stimulated, human monocytic THP-1 cells, both IL-1β and IL-18 release is inhibited by P2X₇R antagonists (Finlayson et al., unpublished observations). The importance of IL-18 in general inflammatory processes, and its suitability as a therapeutic target have been extensively discussed 70, however the simultaneous inhibition of both IL-1β and IL-18 by P2X₇R antagonism has its obvious attractions.

In general, TNF-α is regarded as a pro-inflammatory cytokine that is produced in response to injury, exerting a number of important roles in the immune system and during inflammatory responses. It is of particular interest in neuropathology where this dual role is most clear, with TNF-α having both neurotoxic and neuroprotective effects 71727374. It appears that microglia, the principal immune cells of the central nervous system, have enhanced P2X₇R expression following inflammatory insults (see above) 375. However, as mentioned previously, ATP may act as a 'danger' signal, which recruits microglia to damaged areas of the brain through P2Y rather than P2X receptors 76. In a rat model of neuronal injury, stimulation of the P2X₇R by ATP has been shown to protect neurones by releasing TNF-α 77. In contrast to TNF-α release in rat microglia, Kucher and Neary reported that the P2X₇R was probably responsible for the inhibition of TNF-α release in rat LPS-stimulated astrocytes 78. Indeed, these authors proposed that this could be a mechanism to sense the severity of damage and alter the inflammatory response appropriately. There are also some reports by Perregaux et al 68 that show ATP alters TNF-α production in human monocytes. As the effects of TNF-α in the CNS will be dependent upon the circumstances of its release, and may differ during the acute response to injury versus the long-term recovery from injury 79, it is vital to understand these effects to facilitate the development of novel therapeutic agents.

In addition to the effects that P2X₇R polymorphisms have on IL-1β production, it has also been noted that individuals harbouring such polymorphisms have reduced plasma TNF-α levels (but higher levels of the anti-inflammatory cytokine IL-10) relative to normal subjects 62. Results from this study suggested that during infectious perturbations, 15% of healthy individuals exhibited anti-inflammatory mediator responses, which was correlated with the level of P2X₇R pore activity. While normal pore activity appeared to increase microbial clearance, reduced pore activity may provide some protection from autoimmune disorders as those with an anti-inflammatory cytokine profile are less likely to mount an adaptive immune response to self tissues 62). Since the P2X₇R is important in the production of both TNF-α and IL-1β and as inhibitors of both are in clinical use for the treatment of rheumatoid arthritis 80 and other inflammatory conditions, such observations possibly underlie why AstraZeneca, Pfizer and Abbot amongst others are currently developing P2X₇R antagonists.

In rheumatoid arthritis ATP is found in the synovial fluid where a number of P2X₇R-expressing cells including macrophages are present 8182. In joint diseases such as rheumatoid arthritis and in other conditions such as atherosclerosis the P2X₇R has also been implicated in the secretion of the pro-inflammatory cytokine IL-6 from fibroblasts 83. In atherosclerosis fibroblasts are likely to be exposed to increased concentrations of ATP because of its secretion from platelets and at sites of chronic inflammation 84. In a more recent study the same authors have shown that fibroblasts from type-2 diabetic patients have increased sensitivity to ATP, which is likely to contribute to diabetic vascular disease 85. Furthermore, although mast cells have received little attention with regard to the P2X₇R, it has been known for some time that these cells express this unique receptor (originally described as the P2Z receptor) along with several other P2X and P2Y receptors 86. In addition to inducing cell death, ATP-stimulation of the P2X₇R on murine mast cells has been shown to increase the expression of several pro-inflammatory cytokines, including IL-6 and TNF-α 87. Considering the role of mast cells, especially in allergic inflammation, it would appear pertinent to re-examine the role of the P2X₇R given its therapeutic potential in this area. Finally, new in vivo evidence has been presented supporting the use of P2X₇R antagonists as anti-inflammatory and antipyretic agents (where excessive pro-inflammatory cytokine production or high fever is harmful to the host 88). These authors provided important new insights into LPS-induced febrile response in rats, and showed that the ATP released from activated immune cells stimulated cytokine release which then initiated the febrile response 88. These authors suggested that the P2X₇R plays a central role 88, which is perhaps unsurprising given that the cytokines IL-6, IL-1β and TNF-α all act as endogenous pyrogens 89.

The process of cell death is fundamental to many aspects of physiology and pathophysiology, and of great importance to the regulation of inflammation. Apoptosis is a process of controlled cell death in which cells undergo well characterised morphological changes, including the classical features of chromatin condensation, cell shrinkage, and the formation of apoptotic bodies 93. In contrast to necrotic cell death, apoptotic cell death is a predominantly non-inflammatory process in which the membranes of cells remain intact. This allows the cytotoxic granule contents of cells such as PMN to remain enclosed within the cytoplasmic membrane while the cell is phagocytosed, thereby minimising tissue damage. Furthermore, phagocytosis of apoptotic cells, unlike other particles, has been shown to inhibit the release of pro-inflammatory mediators including IL-1β, IL-8 and TNF-α 94. However, failure of rapid phagocytosis can result in secondary necrosis of the apoptotic cell leading to tissue damage and inflammatory infiltrate (Figure 2). Thus, regulation of innate immune effector cell apoptosis, in particular that of short-lived granulocytes, is critical to the induction, maintenance and resolution of inflammatory processes 95. Apoptosis is regulated at a cellular level by the expression and activation of the Bcl-2 family of proteins and the components of the caspase pathways, which dictate the lifespan and mode of cell death in such cells 96. Importantly, recent studies indicate that P2X₇R activation may modulate a number of cell death processes through effects upon these key regulators of apoptosis.

As described above, the human cathelicidin LL-37 inhibited PMN apoptosis in a P2X₇R-dependent manner 4041. Stimulation of PMN with LL-37 was shown to upregulate expression of the Bcl-2 family protein Mcl-1, a key rapid response component which promotes PMN survival 97, and to inhibit the cleavage and activation of the critical apoptotic regulator pro-caspase-3 9899. Interestingly, whereas lower levels of LL-37 acted primarily as a neutrophil survival factor, higher levels appeared to promote necrotic cell death while inhibiting apoptosis 41. Thus stimulation of the P2X₇R has the capacity to exert a potent effect upon neutrophil survival. These data indicate that PMN express functional P2X₇R, but the cellular localisation of these receptors in this cell type remains unclear. P2X₇R expression on human cells has been demonstrated on PMN, HL-60 promyelocytes and granulocytic differentiated cells, and is reported to increase with granulocytic differentiation 100. However, one report has suggested that human PMN have an intracellular pool of P2X₇R, with little or no surface expression 101. Irrespective, these studies suggest that P2X₇R activation might extend the lifespan of PMN at sites of infection and inflammation, and modulate the mechanism of cell death in these cells.

In contrast to the effects observed in neutrophils, prolonged P2X₇R activation with extracellular ATP has been shown to induce apoptosis in other cell types, including mast cells and epithelial cells 9102103. In addition, murine whole blood exposed to ATP demonstrated a near complete loss of monocytes, and a decrease in lymphocytes, but no change in PMN numbers 34. This effect was not seen in P2X₇R-deficient mice, indicating a P2X₇R-mediated induction of cell death in these cells 34. This induction of apoptosis has been proposed to involve the opening of cation-selective membrane pores, and to be a calcium-independent, ROCK-1-dependent pathway 9. Whereas prolonged or excessive P2X₇R activation with ATP induces apoptosis, transient activation induces a state of pseudoapoptosis in epithelial cells 9. Under these conditions, P2X₇R activation results in a series of very rapid and reversible effects, including calcium-dependent translocation of plasma membrane phosphatidylserine, loss of mitochondrial membrane potential (without cytochrome c release), disruption of the actin filament/microtubule network and membrane blebbing. These data suggest that the P2X₇R can be associated with two different pathways, inducing pseudoapoptosis or apoptosis in epithelial cells. These effects on cell death, assuming the physiological ligand is ATP, are most likely to occur at sites of tissue damage where ATP is released in considerable quantities 104. Interestingly, LL-37 has also been shown to induce eukaryotic membrane permeability 38 and been implicated in the induction of apoptosis in epithelial cells 105. Thus, although the possible role for P2X₇R in mediating these latter effects remains to be determined, it is tempting to speculate that alternative agonists such as LL-37 could induce P2X₇R-dependent apoptosis, and the safe removal of infected cells in an inflammatory environment, even in the absence of high concentrations of ATP.

Thus, an intriguing contrast exists between the effects of P2X₇R stimulation on cell death pathways in different host innate immune effector cells. Nevertheless, the consequences in each case may enhance the inflammatory response and the clearance of infection in acute infection, but have potentially deleterious effects in chronic inflammatory conditions. Indeed, Chen and Brosnan have shown P2X₇R knockout mice to be more susceptible to autoimmune encephalomyelitis (a model for multiple sclerosis), attributing this susceptibility to reduced apoptotic activity in lymphocytes 64. A further understanding of these processes is anticipated to facilitate the development of novel therapeutic agents capable of modulating inflammation via P2X₇R-mediated effects on cell death pathways.

Ferrari et al 106 were the first to show that the P2X₇R was present on eosinophils, with Mohanty et al 107 showing one year later that this expression was dependent upon stimulation by interferon-γ (IFN-γ). This stimulation-dependent expression contrasts with a more recent study which showed functional P2X₇R were expressed endogenously on eosinophils and that inhibition of the P2X₇R, abrogated agonist (BzATP) induced IL-8 release from eosinophils 108. This is interesting in light of the observation that asthmatics secrete more IL-8 from their peripheral blood eosinophils than normal individuals 109. Furthermore, as IL-8 is chemotatic for neutrophils 110 and CD16+ natural killer cells 111 this suggests a role for IL-8 in the initiation and propagation of the inflammatory response 108. As ATP can be released upon tissue damage 104 and in response to inflammatory stimuli 49 (both of which may be present in asthma) it is possible that the P2X₇R would be activated, resulting in IL-8 production and propagation of the immune response (Figure 3). This simplified description of part of the interplay between inflammatory cells and the mediators released, again suggests that the P2X₇R may be a potential target for therapeutic intervention: however, these complex interactions are not yet fully understood. A better understanding of the basic pathophysiology of the initiation of inflammation will allow us to determine whether more specific therapies such as P2X₇R regulation would prevent excessive inflammatory reactions, suppress acute inflammatory reactions and possibly augment the healing process following tissue damage 112.