Purinergic receptors are divided into P1 and P2 receptors. The adenosine receptors are referred to as P1 receptors. P2 receptors are the receptors for adenosine triphosphate (ATP) and can be further subdivided into metabotropic P2Y receptors, which are G-protein-coupled receptors and ionotropic P2X receptors, which are cation-selective ion channels. Macrophages express a wide variety of P2X and P2Y receptors; analysis of mouse macrophages using a variety of techniques indicates the presence of P2X₄, P2X₇, P2Y₁, P2Y₂, P2Y₄, and P2Y₆ receptors (3). Additionally, the expression of P2Y₁₂ receptor has been reported in microglia (4) and macrophages (5). Historically, macrophages were also thought to express an additional ATP-sensitive large conductance channel termed P2Z receptor (6). However, this receptor was later shown to be P2X7 by Surprenant et al. (7).

As non-excitable cells, macrophages rely on Ca²⁺-permeable channels that are not gated by voltage. In addition to P2X channels, macrophages express the store-operated Orai channels and some members of the transient receptor potential (TRP) channel superfamily. The regulation and function of these ion channels remains a mystery and is an emerging topic of significance to inflammation (Table 1).

Extracellular ATP induces Ca²⁺ elevations in myeloid cells through the activation of G_q-coupled P2Y receptors and Ca²⁺-permeable P2X channels. P2Y receptors have a higher affinity for ATP and can elicit Ca²⁺ mobilization from the intracellular stores at low micromolar concentrations of extracellular ATP (13). In contrast, the P2X channels open at millimolar concentrations of ATP and mediate the influx of extracellular Ca²⁺ and other cations (43). Notably, the mobilization of Ca²⁺ stored in the endoplasmic reticulum is not sufficient for the activation of caspase-1 and secretion of IL-1β. When cells are stimulated with ATP in extracellular medium that is depleted of Ca²⁺, IL-1β secretion is nearly abolished (19). These observations indicate that activation of caspase-1 requires a sustained and more intense rise in intracellular Ca²⁺, which can be mediated by the activation of P2X₇ channels but not P2Y receptors.

An alternative explanation for differential requirements of Ca²⁺ stores and Ca²⁺-entry in the activation of caspase-1 involves the efflux of K⁺ through the activated P2X₇ channels. In this model, a concomitant efflux of K⁺ is necessary for the activation of caspase-1 and the rise in intracellular Ca²⁺ without K⁺ efflux is insufficient (44). In any case, although the activation of P2Y receptors by low concentrations of ATP is insufficient to activate caspase-1, the resulting Ca²⁺ oscillations have been shown to promote the transcription of proinflammatory cytokines such as IL-6 (45). The relative contributions of P2X and P2Y receptors in nucleotide-induced Ca²⁺-signaling have not been adequately defined but the use of knockout mice has provided useful insights into this complex aspect of inflammation (46). In myeloid cells, Ca²⁺-dependent activation of PKC plays a pivotal role in the NFκB pathway (47) and the cellular outputs at the site of inflammation are thus likely to be shaped by the purinergic microenvironment. Even in the absence of purinergic signals, Ca²⁺ stores can be mobilized by Toll-like receptors through the activation of tyrosine kinases and phospholipase C (48), but the presence of ATP in the microenvironment likely functions as a potent amplifying mechanism for inflammatory processes.

In addition to the regulation of proinflammatory gene expression and cytokine secretion, Ca²⁺-signaling plays a major role in phagosome maturation. This link is exploited by the internalized mycobacterium for immunoevasive block of phagosome maturation (49). Although the role of Ca²⁺ in phagosome maturation is incompletely defined, it is clear that the lysosomal synaptotagmin VII, a Ca²⁺-sensitive protein, is essential for the fusion of lysosomes with phagosomes (50, 51). Ca²⁺-influx is also essential for the engulfment of apoptotic cells and a subsequent anti-inflammatory response (36). Macrophage phenotypic polarization results in significant differences in the execution of phagocytosis and phagosome maturation but the associated differences in the role of purinergic and Ca²⁺-signals between differentially polarized macrophages remain undefined (52). Ca²⁺-influx has also been shown to be essential to maintain the leading-edge structure in migrating macrophages. In this context, Ca²⁺-influx may be necessary for the activity of PKCα, which is preferentially localized at the leading edge (53).

Macrophages and other cells of myeloid lineage respond at three basic levels to extracellular ATP gradients. First, they migrate toward increasing ATP concentrations; second, they use the ATP gradients emanating from dying cells as a “find-me” signal to locate and phagocytose the cell corpse; and third, at high concentrations of ATP, macrophages respond by robust secretion of proinflammatory cytokines.

Chemotactic responses to ATP were first convincingly demonstrated using cultured microglial cells. Extracellular ATP at micromolar concentrations induced pronounced membrane ruffling, chemokinesis, and chemotaxis (14). This aspect of purinergic response was not confined to ATP gradients emanating from a distant site, indicating ATP acted on cells in an autocrine manner. Indeed, migrating human neutrophils release ATP from their leading edges to amplify and steer their migration using an autocrine feedback loop that involves multiple types of purinergic receptors (54). In macrophages, the chemotactic response to C5a also utilizes an “autocrine purinergic loop” that involves the release of ATP at the leading edge and activation of multiple purinergic receptors (5). In asthmatic airway inflammation, ATP-induced chemotaxis appears to play a critical role in eosinophil and dendritic cell infiltration (55).

The analysis of dendritic cell responses to ATP clearly demonstrates that the chemotactic response to ATP is mechanistically dissociated from other cellular effects of ATP such as secretion of proinflammatory cytokines (16). However, the ATP-induced chemotactic response is intricately connected to the role of purinergic signaling in the location and phagocytosis of apoptotic cells. In the central nervous system, damaged neurons release UDP, which triggers the phagocytic response in neighboring microglial cells (15) and similar mechanisms are likely at work in other tissue-resident macrophages. A definitive role for extracellular ATP as a find-me signal for phagocytes was demonstrated by Elliott et al. (9). The release of ATP and UTP is dependent on the activation of caspases and Pannexin 1 during the early stages of apoptosis and the concentration gradient generated by such release is highly efficient at recruiting monocytes and macrophages. Whether different macrophage phenotypes migrate differently in response to ATP gradients has not been explored. Phagocytosis of dying cells that release ATP serves an anti-inflammatory role, and phagocytotic capacity is drastically inhibited in Mox macrophages that accumulate at the sites of oxidative tissue damage (56). In this context, it would be interesting to know whether alternatively activated M2 or M2-like macrophages show any significant specialization in locating dying cells using the ATP gradients.

Sustained exposure to relatively high concentrations of ATP has been shown to be a critical signal for the secretion of proinflammatory cytokines, such as IL-1β and IL-18. The unconventional secretion of these cytokines is accomplished through proteolytic processing by caspase-1. The activation of caspase-1 is regulated by large multimeric complexes called inflammasomes and the activation of one such inflammasome, the NLRP3 inflammasome, is highly sensitive to the presence of extracellular ATP. Early evidence for ATP-induced maturation of IL-1β came from studies involving apoptosis of peritoneal exudate cells when exposed to high concentrations of extracellular ATP. This form of apoptosis, which we now refer to as pyroptosis, was accompanied by proteolytic processing and release of IL-1β (57), and was especially pronounced in LPS-stimulated mouse peritoneal macrophages where exposure to millimolar concentrations of ATP resulted in rapid processing and release of IL-1β. It was further demonstrated that exposure to high concentrations of extracellular K⁺ prevented the processing and release of IL-1β (58), suggesting that depletion of intracellular K⁺ was essential for ATP-induced IL-1β processing. Subsequent studies of this phenomenon were greatly facilitated by the isolation of the human monocytic cell line THP-1, which was shown to be highly sensitive to purinergic stimulation of IL-1β processing. At least in human monocytes, the purinergic activation of IL-1β processing and secretion is also accompanied by release of proteolytically activated caspase-1 and a commitment to cell death (59).

A study conducted by Dixit and colleagues tested the role of channel-mediated ATP release by characterizing Pannexin 1-deficient mice in the context of inflammasome activation (60). Authors show that the activation of caspase-1 and secretion of IL-1β in response to a wide variety of stimuli including ATP is normal in Pannexin-1-deficient macrophages. In contrast, Pannexin 1-deficient thymocytes failed to recruit macrophages after undergoing apoptosis. Overall, these studies indicate that ATP released through Pannexin 1 is sufficient to reach extracellular concentrations that are of functional relevance to chemotaxis. However, the activation of caspase-1 typically requires high concentrations of ATP that are unlikely to be reached when cytosolic ATP is released through Pannexin 1. The role of vesicular release of ATP in the autocrine activation of inflammasome has not yet been ruled out and that may hold the key to reconcile these studies. A key component of vesicular ATP release is the vesicular nucleotide transporter VNUT (also known as SLC17A9), which is responsible for the accumulation of ATP into secretory vesicles (61). The human monocytic cell line THP-1 has been shown to express VNUT, which mediates the rapid secretion of ATP in response to LPS treatment (62). The function of vesicular ATP secretion in mouse macrophages and its physiological significance have not been reported yet. It is also not clear whether M1 and M2 macrophages exhibit mechanistic and functional differences in ATP release mechanisms.

The mechanisms through which activated caspase-1 and IL-1β are secreted have remained unclear but there is evidence for the involvement of Ca²⁺-influx elicited by ATP (19). Interestingly, although high concentrations of ATP are required for caspase-1 activation, low concentrations of ATP (10 μM) or UTP (10 μM) are sufficient to induce oscillations in intracellular Ca²⁺ and increased transcription of the proinflammatory cytokine IL-6 (45). An autocrine role for ATP-induced activation of inflammasome has also been suggested in the case of primary human monocytes that are stimulated by danger-associated uric acid. According to this model, the activation of inflammasome is dependent upon the initial release of ATP, which then acts on purinergic receptors in an autocrine manner. Whether such secretion results in local ATP concentrations that are high enough to activate the inflammasomes is not clear (11). In addition to the regulation of IL-1β and IL-18 secretion, ATP has also been shown to regulate the secretion of lysosomal cathepsins (27). These proteases are involved in the degradation of extracellular matrix and can result in auto-inflammatory tissue damage. Recently, ATP was shown to potentiate the release of IFNβ in LPS-stimulated macrophages (30).

With respect to macrophage phenotypes, a recent study provided evidence that inflammatory M1 macrophages are more sensitive to ATP and more efficient at ATP-induced IL-1β release when compared to M2 macrophages (31). While the physiological significance of these findings remains to be elucidated, this study provides the initial exploratory foray into these outstanding questions pertaining to how macrophage polarization fine-tunes the sensitivity to the purinergic microenvironment.

Many cells express membrane-bound ectonucleotidases that convert the extracellular ATP and ADP to adenosine. A common pathway involves the conversion of ATP and ADP to AMP by CD39 and subsequent conversion of AMP to adenosine by CD73. These enzymatic biochemical conversions have potent implications for the termination of inflammatory response due to the reduction in ATP levels. More significantly, adenosine serves as a ligand for G-protein coupled adenosine receptors or P1 receptors on myeloid cells (Table 1). The four P1 adenosine receptors (A₁, A_2A, A_2B, and A₃) transmit a “calm down” signal that may orchestrate the resolution of inflammation, a process conceptually different from anti-inflammatory signals that restrain the initiation of inflammatory process by preventing the recruitment and activation of immunocytes. Pertinently, extracellular adenosine has been shown to augment the polarization of macrophages toward the M2 phenotype (63).

Early studies showed that adenosine inhibited the secretion of TNFα, IL-6, and IL-8 by LPS-activated human monocytes (64). Subsequently, it was shown that the adenosine receptor A₃ plays a major role in downregulating the synthesis of proinflammatory cytokines in monocytes and macrophages in response to extracellular adenosine (65, 66). Synthetic agonists of A₃ adenosine receptor have been shown to have potent therapeutic effects in various mouse models of rheumatoid arthritis (67). A similar role for the A_2A adenosine receptor has also been reported. Mice deficient in the A_2A receptor show extensive tissue damage and prolonged inflammation to sub-threshold doses of inflammatory stimuli in three different disease models (68). In the context of infectious diseases and polymicrobial sepsis, A_2A receptors are required for the control of IL-10 production by alternatively activated macrophages (69). The A_2A receptors also play an anti-inflammatory role in neuroinflammation. The ligation of A_2A receptors on the activated microglia has been shown to retract their processes and initiate the resolution of inflammation in the brain (70).

In the case of endotoxin-induced lung injury, the pharmacological inhibition or genetic deletion of A_2B receptors greatly exacerbates lung injury. In contrast, the A_2B receptor agonist attenuates endotoxin-induced lung inflammation (71). The role of A_2B receptors in dampening endotoxin-induced inflammation is evident in mouse models of polymicrobial sepsis. Deletion of A_2B receptors greatly increases the mortality of mice from cecal ligation and puncture-induced sepsis (72). The A_2B-deficient mice showed increased levels of proinflammatory cytokines and chemokines in the serum, coincident with augmented activation of NFκB and p38 in the spleen.

In summary, the adenosine receptors provide crucial information to monocytes and macrophages and calibrate their response to the complex mix of purinergic stimuli in the inflammatory microenvironment (73). In this context, the ratio of ATP and adenosine may provide the crucial cues necessary for the polarization of macrophages toward the M2 phenotype induced by IL-4. The role of A_2A and A_2B receptors to augment this process of M2 polarization has been illuminated (63) but surprisingly, a recent study shows that adenosine may control this process independent of IL-4 (74). Similarly, a recent study provides evidence suggesting a role for adenosine in IL-10-induced STAT3 activation in alternatively activated phenotype termed M2c (75). Manipulation of ATP and adenosine levels in the tissue microenvironment is thus likely to emerge as a potent mechanism to guide the plasticity of macrophages and holds clinical potential for therapeutic intervention. This concept may find traction in a large variety of diseases where inflammation plays a major pathological role. A recent preclinical study exemplifies the application of this strategy in the treatment of osteolysis; the authors show that activation of A_2A receptors prevents wear-induced osteolysis (76).

Some of the earliest evidence indicating a role for extracellular ATP in monocyte and macrophage function came from the findings that the cells of the myeloid lineage exhibit rapid elevations in intracellular Ca²⁺ when treated with micromolar amounts of ATP (13). The in vivo significance of this finding was further highlighted by the studies of Bertics and colleagues who showed that LPS-induced activation of macrophages was greatly enhanced by extracellular ATP (77) and that mice treated with the adenine nucleotide analog 2-methylthio-ATP were protected from endotoxic shock (78). Subsequent studies indicated a potent role for purinergic signals in controlling the inflammatory gene expression in response to LPS stimulation (79). The extensive contribution of purinergic signaling in inflammatory processes has now been established in a wide variety of pathologies (2), some of which are outlined below.

The tissue-resident macrophages of the central nervous system, the microglia, have been shown to be especially sensitive to extracellular ATP. In vitro studies with cultured microglia revealed that extracellular ATP and ADP stimulate chemotaxis and morphological changes (14). Local trauma in the brain, which results in cell death, is thought to increase the extracellular levels of ATP significantly and elegant studies using multiphoton imaging have demonstrated that microglia respond rapidly to local injury through dynamic changes in their morphology. The convergence of microglial processes at the injury site could also be stimulated by local injection of ATP and this response was demonstrated to be highly sensitive to the presence of ATP-hydrolyzing enzymes and blockers of purinergic receptors (80).

In the lungs of asthmatic patients, allergic challenges cause rapid accumulation of ATP and this has been modeled successfully in mice using experimentally induced asthma (55). Interestingly, hydrolysis of ATP in the airways through the application of apyrase greatly reduces eosinophil infiltration, production of Th2 cytokines, and bronchial hyper-reactivity. Corollary experiments show that exogenous ATP potentiates airway inflammation (55) and similar findings have demonstrated a role for purinergic signals in cigarette smoke-induced inflammation and emphysema (25). The significance of purinergic signals in allergic reactions is also evident in the skin. The accumulation of ATP in response to contact allergans is critical for the production of inflammatory cytokines by the myeloid cells and the subsequent sensitization process (23). Accumulation of ATP has also been observed in the ascites of patients and mice undergoing GVHD. In mice, the severity of GVHD is greatly reduced by neutralizing the ATP or by blocking purinergic signaling (24). Furthermore, recent studies have also implicated extracellular ATP in the development of intestinal inflammation in patients with Crohn’s disease. In corresponding mouse models, blocking purinergic signaling greatly reduces the activation of intestinal mast cells and thereby blocks the subsequent rise in proinflammatory cytokines and leukotrienes (26).

In summary, studies in human beings and mice provide conclusive evidence that purinergic signals play a major role in inflammation and tissue injury. Purinergic receptors are expressed ubiquitously and a comprehensive understanding of how purinergic signals influence the physiology and pathology is still in rudimentary stages. Purinergic control of macrophage function promises to play a central role in these processes and understanding the effects of purinergic signals on macrophage function provides an immediate window toward therapeutic intervention.

Our understanding of the fundamental role played by purinergic and Ca²⁺ signaling in macrophage activity is increasing rapidly but the signaling mechanisms that drive specific cellular outputs still remain largely enigmatic. The close coupling of purinergic stimulation and Ca²⁺ influx suggests that the purinergic receptors, Orai channels, and TRP channels function in a co-ordinated network that responds rapidly to the changes in the inflammatory microenvironment. By virtue of being excellent drug targets, purinergic G-protein coupled receptors and ion channels offer an enticing pharmacological path to shape the plasticity of macrophage function in various diseases. To make this a reality, we will need to develop experimental models where the influence of the purinergic microenvironment and the resulting Ca²⁺-dynamics in macrophages can be interrogated in situ. All indications are that we have only just scratched the surface in this exciting area of innate immunity.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.