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Adenosine is a key endogenous molecule that regulates tissue function by activating four G-protein-coupled adenosine receptors: A1, A2A, A2B and A3. Cells of the immune system express these receptors and are responsive to the modulatory effects of adenosine in an inflammatory environment. Animal models of asthma, ischaemia, arthritis, sepsis, inflammatory bowel disease and wound healing have helped to elucidate the regulatory roles of the various adenosine receptors in dictating the development and progression of disease. This recent heightened awareness of the role of adenosine in the control of immune and inflammatory systems has generated excitement regarding the potential use of adenosine-receptor-based therapies in the treatment of infection, autoimmunity, ischaemia and degenerative diseases.
In 1929, Drury and Szent-Györgyi first reported the concept of adenosine acting as an extracellular signalling molecule1. The authors found that when simple extracts of heart muscle and other tissues were injected intravenously into the whole animal they produced decreased heart rate and increased coronary blood flow. The active constituent of the extracts turned out to be adenosine, and since the 1980s adenosine has been used to slow down the heart of patients suffering from excessively increased heart rate caused by supraventricular tachycardia2. In addition, adenosine has been used as a diagnostic agent, that is, a coronary vasodilator, to assess coronary artery function in conjunction with radionuclide myocardial perfusion imaging3. Following the original discovery of the effects of adenosine on cardiac function, adenosine has been found to have regulatory roles in virtually every organ system studied.
Adenosine accumulates in the extracellular space in response to metabolic stress and cell damage (BOX 1), and elevations in extracellular adenosine are found in conditions of ischaemia, hypoxia, inflammation and trauma4,5. The rapid release of adenosine in response to tissue-disturbing stimuli has a dual role in modulating homeostasis. First, extracellular adenosine represents a pre-eminent alarm molecule that reports tissue injury in an autocrine and paracrine manner to surrounding tissue. Second, extracellular adenosine generates a range of tissue responses that can be generally viewed as organ protective thereby mediating homeostasis. Adenosine elicits its physiological responses by binding to and activating one or more of the four transmembrane adenosine receptors, denoted A1, A2A, A2B and A3 (BOX 1). A prominent body of evidence supports the notion that the ability of adenosine, acting at its receptors, to control the immune and inflammatory systems plays a key role in the modulatory effects of adenosine in both health and disease. There are many promising emerging therapeutic approaches that are centred on the modulation of adenosine in the immune system. These encompass pharmacological compounds that interfere with the breakdown and generation of adenosine, as well as selective agonists and antagonists of various adenosine receptor subtypes (FIG. 1). Some of these compounds are in preclinical investigations, whereas others have already entered clinical trials for various indications. In this Review, we provide a general overview of adenosine receptors, covering aspects of cell biology, molecular biology and pharmacology. Separate sections focus on the multitude of roles that adenosine has in dictating the function of cell types that are considered basic constituents of the innate and adaptive immune systems. Finally, we discuss the therapeutic basis of harnessing the adenosine receptor system in managing patients suffering from inflammatory, autoimmune and ischaemic diseases.
Extracellular adenosine levels increase following the release of adenosine from cells or as a result of extracellular catabolism of released adenine nucleotides. Intracellular adenosine, which can be derived from increased intracellular metabolism of ATP during cellular stress or S-adenosyl homocysteine, is liberated mainly via equilibrative nucleoside transporters. Extracellular ATP and ADP are catabolized by a cascade of ectoenzymes comprising CD39 (ENTPD1; ectonucleoside triphosphate diphosphohydrolase 1) and CD73 (ecto-5′-nucleotidase). CD39 is an enzyme that hydrolyses ATP and ADP to AMP, and CD73, in turn, rapidly dephosphorylates AMP to adenosine126. Owing to the ubiquitous presence of equilibrative nucleoside transporters, adenosine generated from extracellular ATP is rapidly taken up by cells from the extracellular space. Adenosine in the cytosol is then metabolized either by adenosine kinase to form AMP or adenosine deaminase, which deaminates adenosine to inosine6,127. As a result of the rapid uptake and metabolism of adenosine, levels of this mediator are maintained low in unstressed, healthy tissues. However, under pathophysiological conditions adenosine removal can not keep pace with its generation, resulting in markedly increased extracellular adenosine concentrations.
All of the adenosine receptors contain seven transmembrane domains and couple to intracellular GTP-binding proteins (G proteins). Adenosine can activate A1, A2A and A3 receptors with EC50 values that are between 0.01 μM and 1 μM, whereas A2B receptor activation generally requires adenosine levels that exceed 10 μM (EC50 of 24 μM)128. Because physiological adenosine concentrations are lower than 1 μM, physiological levels of adenosine can activate A1, A2A and A3 receptors, whereas A2B receptor activation requires pathophysiological conditions129. Although the cellular responses to adenosine are highly dependent on the adenosine concentrations at the cell surface, several other factors, such as receptor density and the functionality of the intracellular signalling pathways coupled to adenosine receptors, are also key determinants in dictating the nature and magnitude of the effect of adenosine on the cell. For example, A2A receptor activation potently suppresses the production of the T helper 1-inducing cytokine interleukin 12 (IL12) by monocytes that are preincubated with the pro-inflammatory cytokine IL1 or tumour-necrosis factor-α, agents that also upregulate A2A receptors in these cells119. This example highlights the importance of receptor expression in defining the magnitude of the response to receptor stimulation. Furthermore, the effect of adenosine can also depend on the polarized localization of adenosine receptors. For example, A3 adenosine receptors accumulate at the leading edge of migrating neutrophils and have an important role in facilitating the directional movement of cells in response to chemotactic stimuli35. Finally, caution should be exercised in interpreting data assessing adenosine receptor function in different species, because sequence variations in cloned adenosine receptors have been shown to be associated with varying pharmacological responses to selective agonists and antagonists. This is best illustrated by the fact that A3 receptors remained undiscovered until they were cloned130, because the rodent A3 receptor is insensitive to xanthines such as theophylline and caffeine, antagonists that had been extensively used in identifying adenosine receptor-mediated effects.
A large body of evidence supports the view that adenosine receptors govern cell function by coupling to G proteins, although some G-protein-independent effects have also been reported6. Traditionally, adenosine receptor signalling is thought to occur through the inhibition or stimulation of adenylyl cyclase with a concomitant decrease or increase in intracellular cyclic AMP concentrations. Based on their ability to decrease or increase cAMP accumulation, adenosine receptors were initially classified as A1 or A2 receptors, respectively7. Subsequent studies have refined the classification of adenosine receptors and cAMP-increasing A2 receptors have been divided into two groups: high-affinity A2A receptors and low-affinity A2B receptors8. The more recent discovery and characterization of A3 receptors have made it clear that in addition to A1 receptors, A3 receptors dictate certain cellular responses such as rodent mast-cell degranulation, in part by decreasing intracellular cAMP concentrations9. This early picture of adenosine receptor signalling through the adenylyl cyclase–cAMP system has been substantially expanded, and it is now established that adenosine receptors can be linked to various other pathways.
A1 receptor activation had traditionally been linked to Gi-mediated inhibition of adenylyl cyclase. However, it is now known to be also linked to various kinase pathways including protein kinase C (PKC), phosphoinositide 3 (PI3) kinase and mitogen-activated protein (MAP) kinases10. In addition, A1 receptor activation can directly activate K+ channels and inhibit Q-, p- and N-type Ca2+ channels. It is noteworthy that most of these signalling pathways were uncovered in non-immune cell types, and A1 receptor signalling mechanisms in cells of the immune system are not known.
A2A receptors, similar to other Gs-protein-coupled receptors, signal mainly by the adenylate cyclase–cAMP–protein kinase A (PKA) canonical pathway, but they can also signal through the activation of an exchange factor that is directly activated by cAMP (Epac)6. Signalling downstream from PKA proceeds through phosphorylation of the transcription factor CREB on serine residue 133, resulting in its activation11. Activated CREB can mediate gene expression directly by interacting with gene promoters or indirectly by competing with nuclear factor-κB (NF-κB) or other transcription factors for an important cofactor, CBP6. In other cell types A2A receptors have been shown to signal for increased collagen production by activating MAP kinases12 and inhibiting neutrophil superoxide production by activating protein phosphatase13. Furthermore, recent results indicate that CEBPβ is responsible for the stimulatory effect of A2A receptor agonists on interleukin 10 (IL10) production by macrophages14 (FIG. 2). which of the outlined mechanisms mediate A2A receptor signalling in the various immune cell types remains to be defined in more detail.
A2B receptor stimulation can trigger adenylyl cyclase activation via Gs and phospholipase C (PLC) activation via Gq (REF. 15). Cross-talk between these two pathways appears to be essential for the upregulation of IL4 production by human mast cells following A2B receptor activation16. In particular, Gq-mediated activation of PLCβ leads to calcium mobilization and an increase in NFATc1-dependent IL4 transcription, and this response is amplified by the Gs-mediated accumulation of NFATc1 protein.
The classical signalling pathways associated with A3 receptor activation comprise Gi-mediated inhibition of adenylyl cyclase and Gq-mediated stimulation of PLC17. In addition, A3 receptors can utilize the PLD, RhoA, WNT, MAP kinase and PI3 kinase pathways in dictating cell function. In fact, A3 receptor-dependent enhancement of histamine release in sensitized murine mast cells was prevented by inhibition of Gi proteins using pertussis toxin and by pharmacological inhibition of PI3 kinase18.
The effect of adenosine on cytokine production by macrophages has attracted considerable attention, because macrophage-derived cytokines are crucial initiators and orchestrators of immune responses. As tumour necrosis factor-α (TNF-α) was one of the first cytokines to be discovered, a substantial body of information has accumulated regarding the ability of adenosine receptor activation to limit TNF-α production following macrophage activation. Recent studies using adenosine-receptor knockout mice have painted a detailed but still not complete picture of the receptors involved. Several studies agree that the A2A receptor is the primary and dominant adenosine receptor subtype that mediates inhibition of TNF-α19-21. A role for other receptors was postulated based on the observation that adenosine, and the agonists NECA and IB-MECA (CF-101) can each inhibit, albeit to a lesser extent, TNF-α production even in A2A-receptor knockout mice19,20. A study using a combined approach of using A2A-receptor knockout mice and the A2B receptor antagonist MRS 1754 supports a role for A2B receptors as the other inhibitory receptor20. However, it appears that A2B receptors become operational only when their effect is not masked by A2A receptors, because both MRS 1754 and genetic deletion of A2B receptors in the presence of functional A2A receptors fails to affect the suppression of TNF-α production20,21.
A similar picture is emerging regarding how adenosine receptor activation augments IL10 production. A key role for A2A receptors was recently documented when adenosine failed to upregulate Escherchia coli-induced IL10 release by macrophages lacking A2A receptors but not wild-type macrophages14. In the RAW264.7 macrophage cell line, which expresses negligible levels of A2A receptors, adenosine upregulates IL10 production via an A2B-receptor-mediated mechanism22. while A2B receptor activation has a minor impact on TNF-α and IL10 production, it is key to the stimulatory effect of adenosine on IL6 production, as NECA is unable to induce the release of IL6 in A2B-receptor knockout but not wild-type macrophages21.
Finally, it is important to emphasize that the issue of which adenosine receptors regulate cytokine production by human monocytes/macrophages is even more contentious with evidence pointing to the involvement of all four receptors23. These results, however, must be interpreted with caution, because they are based entirely on pharmacological approaches, and several of the ligands used, especially in early studies, are not particularly selective. Nevertheless, the recent observations that human newborn plasma contains elevated concentrations of adenosine when compared with adult plasma, and degrading adenosine with adenosine deaminase or interrupting adenosine signalling by adenosine receptor blockade augments TNF-α production by neonatal but not adult blood, highlight the importance of the inhibitory effect of adenosine on TNF-α production in human immunity24. Based on these results it was speculated that adenosine might protect the fetus from excessive inflammatory responses that drive alloimmune reactions and premature delivery. However, this protective effect would come at a price of increased susceptibility to infections following birth.
Although dendritic cell responses from adenosine-receptor knockout mice have yet to be characterized, available data from human studies support a consistent role for adenosine receptors in orchestrating dendritic cell function. Gi-coupled (A1, A3 or both depending on the experimental system) adenosine receptors are expressed on both immature myeloid25 and plasmocytoid dendritic cells26, and their activation results in the mobilization of intracellular calcium from intracellular stores and reorganization of the actin cytoskeleton. Consistent with these adenosine-induced intracellular responses, immature dendritic cells migrate along different concentration gradients of adenosine, indicating that adenosine receptor activation induces chemotaxis in immature dendritic cells25,26. These stimulatory responses induced by Gi-coupled adenosine receptors can be observed only in immature dendritic cells, as these receptors undergo downregulation during dendritic cell maturation25,26.
Although A2A receptors are present on immature dendritic cells, they are expressed at low levels and appear to be silent, as their activation is unable to elicit downstream signalling events such as accumulation of intracellular cAMP25. However, dendritic cell maturation is accompanied by the emergence of A2A-receptor-mediated signalling responses, owing to both increased expression and coupling of A2A receptors25,26. A2A receptor activation on mature dendritic cells shifts their cytokine profile from a pro-inflammatory to an anti-inflammatory one, with reduced IL12, IL6 and interferon-α (IFN-α) production and augmented IL10 production25-27. It is likely that dendritic cells in the presence of adenosine have a reduced capacity to induce T helper 1 (TH1) cell versus TH2 cell polarization of naive CD4+ cells27. This is due to the adenosine-induced switch in dendritic cell cytokine production away from the TH1-inducing IL12 towards the TH2-inducing IL10.
In summary, the available data support a dual role for adenosine in dictating dendritic cell function. Adenosine promotes the recruitment of immature dendritic cells to sites of inflammation and injury via A1 or A3 receptors. At these sites adenosine produces, via A2A receptors, an anti-inflammatory dendritic cell phenotype driving T-cell responses towards a TH2 profile.
Adenosine is a potent modulator of neutrophil function and it has long been appreciated that adenosine, by activating its receptors, regulates stimulated production of reactive oxygen species by these cells and phagocytosis28-30.
Individual neutrophils do not produce large quantities of cytokines; however, because of the large numbers of accumulated neutrophils the cumulative contribution to pro-inflammatory cytokine levels at a given site is large. Adenosine, acting at A2A receptors, regulates the production of a range of cytokines including TNF-α, macrophage inflammatory protein (MIP)-1α (also known as CCL3), MIP-1β (CCL4), MIP-2α (CXCL2) and MIP-3α (CCL20)31. Neutrophils are recruited to inflammatory sites by the post-capillary venular endothelium, which alters the expression of adhesive molecules on its surface to capture neutrophils from the circulation. Adenosine, via A2A receptors, inhibits the adhesion of neutrophils to the endothelium by decreasing the expressionand stickiness of the adhesion molecules expressed on neutrophils32-34.
By contrast, A1 receptors promote neutrophil adhesion to different adhesive molecules on the endothelium and on other surfaces32. Once in the tissue neutrophils migrate along gradients of chemoattractants. There are many chemoattractants including activated complement components (C5a), bacterial products (formylated peptides) and chemokines, and studies have shown that adenosine promotes directed migration of neutrophils via A1 and A3 receptors35-37. In addition, neutrophils cluster their A3 receptors at the leading edge of the cell and release ATP, which is converted at the cell surface to adenosine, which then acts in an autocrine manner to stimulate migration35. At inflamed sites neutrophils undergo apoptosis and adenosine, acting at A2A receptors, prevents neutrophils from undergoing apoptosis38-40. Thus, virtually every function carried out by neutrophils is regulated by adenosine and its receptors.
The observation that inhaled adenosine provokes bronchoconstriction in individuals suffering from asthma but not in normal volunteers has propelled adenosine and adenosine receptors into the forefront of asthma research41. Although the mechanisms of how adenosine mediates bronchoconstriction are still elusive, there is overwhelming evidence that mast-cell mediators such as cytokines and histamine may have a key role in evoking airway constriction in response to adenosine. Although A2A, A2B and A3 receptors are clearly present on the cell membrane of mast cells42, there is uncertainty regarding which receptor(s) account for the increased mast-cell activation following adenosine receptor activation.
Adenosine was recently documented to stimulate release of the major asthma-promoting cytokine IL13 by mouse mast cells that were obtained from wild-type but not A2B-receptor-knockout mice. This result implies that A2B receptors are the receptors involved in the pro-inflammatory effects of adenosine43. In addition, it is clear from genetic studies with knockout mice that A3 but not A2B receptor agonism can produce histamine release of lung mast cells in the absence of specific antigenic stimulation18. Thus, A2B and A3 receptors subserve different pro-inflammatory roles in naive mouse mast cells. The role of A2B receptors in regulating antigen-stimulated histamine release in mouse mast cells is controversial. Mast cells isolated from wild-type animals display decreased histamine release following stimulation with antigen when compared with mast cells obtained from A2B-receptor knockout mice44. This observation was interpreted as indicating that constitutive activation of A2B receptors on cultured mast cells suppresses the release of histamine. A subsequent study confirmed the increased degranulation of A2B-deficient mast cells, but based on additional approaches the authors questioned this interpretation of A2B-receptor-mediated decrease of degranulation of mast cells43. The reason for the different interpretation in the latter study was that exogenous adenosine maintained its stimulatory effect on histamine release in A2B-receptor knockout mast cells43. In addition, no evidence of constitutive activity of A2B receptors was revealed by studies using inverse agonists and measuring intracellular cAMP levels. Thus, it appears likely that the decreased release of histamine by mast cells isolated from A2B receptor wild-type versus knockout mice reflects an altered responsiveness of A2B receptor wild-type versus knockout cells to antigen. This is possibly from altered development of mast cells in A2B-receptor-knockout mice. Unlike A2B receptors, A3 receptors directly control histamine release by antigen-stimulated mouse mast cells, because the stimulatory effect of exogenous adenosine noted in wild-type mast cells is not observed in A3-receptor-knockout mast cells45.
In canine and human mast cells, degranulation or cytokine release appears to be mediated primarily by A2B receptor activation46,47. A2B receptor stimulation in human mastocytoma HMC-1 cells induces secretion of the TH2 cytokines IL4 and IL13, as well as a number of other pro-inflammatory cytokines such as IL1β and IL8. Although it is not clear whether adenosine receptor activation is able to stimulate the degranulation of human mast cells44, the observation of increased IL4 and IL13 production following adenosine receptor activation strongly implicates human mast-cell adenosine receptors as important players in the pathophysiology of human asthma.
In addition to regulating lymphocyte function indirectly by stimulating adenosine receptors on innate immune cells such as dendritic cells, adenosine can also directly affect lymphocyte responses by binding and activating adenosine receptors on lymphocytes. A number of recent studies using adenosine-receptor-knockout mice have evaluated the effect of adenosine receptor activation on various lymphocyte functions. The consensus emerging from these studies, as well as pharmacological studies, is that A2A receptors are the dominant adenosine receptors in dictating lymphocyte responses.
Studies using A2A-knockout models have shown that A2A receptor activation inhibits IL2 secretion48 by naive CD4+ T cells thereby reducing their proliferation49 following T-cell receptor stimulation. A2A receptor activation also suppresses the production of both IFN-γ and IL4 by both naive CD4+ T cells48,50 and polarized TH1 and TH2 cells51, thus challenging the hypothesis that A2A receptors on lymphocytes might shift TH cell responses towards a TH2 profile. Further immunosuppressive effects of A2A receptor stimulation include the upregulation of the expression of negative co-stimulatory molecules such as cytotoxic T-lymphocyte antigen 4 (CTLA4) and programmed cell death 1 (PD1), and downregulation of the positive co-stimulatory molecule CD-40L49.
Similar to CD4+ cells, adenosine inhibits IL2 production by both polarized type 1 cytotoxic T (TC1) and TC2 CD8+ cells, the effect of which was proposed to proceed through A2A receptors based on pharmacological evidence52. However, the production of neither TC1 (IFN-γ) nor TC2 (IL4 and IL5) cytokines was influenced by A2A receptor activation. In addition, pharmacological A2A receptor activation failed to reduce TC1 or TC2 cell cytolytic function52, which suggests that another subtype, possibly the A3 receptor, may mediate the anti-cytotoxic effect of adenosine noted in prior studies53,54. In contrast to these results with CD8+ cytotoxic cells, recent data with A1-, A2A- and A3-receptor-knockout mice support a primary role for A2A receptors in preventing the cytolytic activity of IL2-activated natural killer (NK) cells55.
Recent studies have revealed an important role for adenosine in mediating the immune suppressive properties of regulatory T (TReg) cells, a cell type that is thought to have a key role in keeping the immune system at bay, thereby preventing excessive tissue injury (FIG. 3). It was found that TReg cells — as defined by their expression of CD4+/CD25+/FoXp3+ and an ability to suppress the proliferation of CD4+/CD25− cells — express high levels of both CD39 (REF. 56) and CD73 (REFs 56,57), cell surface ectoenzymes that convert extracellular ATP and ADP to adenosine (BOX 1, FIG. 3). The finding that TReg cells selectively co-express CD39 and CD73 prompted the authors to propose that CD39 and CD73 could represent new specific markers of TReg cells. The mechanistic link between the transcription factor FOXP3+ and CD39 is suggested by recent studies showing that FOXP3+ actually drives the expression of CD39 (REF. 58).
The mechanisms of the immune suppressive effects of TReg cells have been elusive. In this regard, it was recently discovered that TReg cells can efficiently hydrolyse exogenous ADP to generate immune suppressive adenosine via CD39 and CD73 (REF. 56). Furthermore, the regulatory function of TReg cells depended largely on their ability to produce adenosine, because TReg cells isolated from CD39-knockout mice lost their potential to suppress proliferation of CD4+/CD25− cells, the effect of which was reconstituted by addition of soluble exogenous forms of CD39 (REF. 56). Further support to highlight an important role for adenosine was provided by the observation that A2A-receptor-knockout target cells (CD4+/CD25−) proliferated more than wild-type cells when cultured with wild-type TReg cells. It is noteworthy that CD4+/CD25− cells increased their expression of A2A receptor by day 4 in culture, at a time when TReg-cell-mediated suppression of target cell proliferation is highest, again confirming that adenosine generation is an important component of the TReg-cell armamentum. In addition to target cells, TReg cells also express A2A receptors and the activation of these receptors upregulates FOXP3+ expression in TReg cells59. The functional significance of A2A receptors on TReg cells was demonstrated in a mouse model of colitis48. In this model, adoptively transferred TReg cells lacking A2A receptors were found to be defective in their ability to suppress colitis. Hence, in an adenine-nucleotide-rich environment, TReg cells generate adenosine via ectoenzymes and are responsive to adenosine via A2A receptors.
Conventional T cells are activated via major histocompatability complex (MHC) molecules on antigen-presenting cells that present antigens to T-cell receptors. The subsequent expansion of activated T cells is responsible for adaptive immunity. A subset of T cells known as invariant NKT (iNKT) cells plays an important role in the innate immune response that triggers rapid host responses to infection. These cells express an invariant α,β T-cell receptor (vα14Jα18 in mice) in addition to molecular markers found on NK cells, such as NK1.1 (REF. 60). Unlike conventional T cells that recognize peptides, iNKT cells recognize lipid antigens that are derived from pathogens or injured host tissue. These lipid antigens are presented by antigen presenting cells to iNKT cells by the MHC class I-related molecule CD1d61. A2A receptors are found on iNKT cells and strongly suppress the release of pro-inflammatory cytokines such as INF-γ62. The presence of A2A receptors on iNKT cells suggests that there could be novel therapeutic applications of A2A agonists or antagonists as activation of iNKT cells has been implicated in a number of disease processes including atherosclerosis, type 1 diabetes, arthritis and various allergic diseases63.
In summary, lymphocyte function is potently regulated by A2A receptors, suggesting that the anti-inflammatory effects of A2A receptor agonists in animal models of autoimmunity and ischaemia are mediated, in part, by targeting lymphocytes.
Several lines of clinical and preclinical evidence support the notion that adenosine and its receptors are intimately involved in defining the pathophysiology of asthma and chronic obstructive pulmonary disorder (COPD). Adenosine receptors on immune cells contained in the lung appear to have particularly important roles. Most compelling are the observations that inhaled adenosine can induce bronchoconstriction in patients suffering from asthma or COPD, but not in healthy individuals, and adenosine receptor blockade can prevent this bronchoconstrictive response42. These observations, combined with the demonstration that both adenosine levels and adenosine receptor expression on immune cells in the lung are elevated in patients with asthma and COPD, implicate endogenous adenosine as a prominent signalling molecule in lung diseases. As the bronchoconstrictive response to adenosine can be blocked by mast-cell membrane-stabilizing agents that can prevent the release of injurious mast-cell mediators, and because histamine receptor antagonists are also effective against adenosine, one major hypothesis explaining the mechanism of adenosine-mediated bronchoconstriction centres around a key role played by mast cells42. This general scheme of the injurious role of adenosine receptor activation on mast cells has been reinforced by studies using rodent models of asthma. These studies have also identified A2B (REF. 43) and A3 (REFS 18,45) receptors as the main contributors that mediate the stimulatory effect of adenosine on mast-cell activation. Although there is a paucity of human in vivo studies detailing the role of adenosine receptor subtypes in asthma and COPD, in vitro studies using human mast cells have narrowed down the candidate adenosine receptors to the A2B receptor (FIG. 4). In addition to mast cells, pro-inflammatory effects of A2B stimulation have also been observed with human bronchial smooth-muscle cells64, human bronchial epithelial cells65 and human lung fibroblasts66, which respond to adenosine by increased release of Il6 (REFS 64,66) and IL19 (REF. 65). A2B receptor activation on human lung fibroblasts promotes their differentiation into myofibroblasts that are capable of overproducing extracellular matrix66, which suggests that adenosine may participate in the fibrosis and remodelling of the lung during asthma and COPD.
This substantial evidence documenting the pro-inflammatory effects of selective A2B receptor activation in human and rodent cellular systems combined with the utility of A2B receptor antagonism in preventing disease progression in rodent animal models67,68 suggest that A2B antagonists may be a viable treatment option for the management of asthma and COPD in human patients. Indeed, it is now well established that enprofylline, an anti-asthmatic agent, is a relatively selective (albeit not potent) A2B receptor antagonist69. The selective A2B receptor antagonist CVT-6883 (REFS 67,68), which has shown efficacy in preventing disease in rodent models of asthma and COPD, appeared to be safe and well tolerated in a recent Phase I study, raising hopes that this agent might have utility in the treatment of individuals suffering from asthma and COPD (TABLE 1).
Lung inflammation results in the accumulation of neutrophils and other leukocytes in the interstitial space and in the airway. This is accompanied by an increased microvascular permeability and the release of chemotactic cytokines. A2A agonists have been found to reverse these effects. To determine which pulmonary cells respond to A2A receptor activation, pulmonary inflammation has been assessed in wild-type and A2A-receptor-knockout mice70. To differentiate the role of A2A receptors on haematopoietic and parenchymal cells, chimeric mice were created by transfer of bone marrow between wild-type and A2A receptor knockout mice. In wild-type mice, A2A receptor activation reduces lipopolysaccharide-induced neutrophil recruitment and release of cytokines. Pretreatment, but not post-treatment, also reduces the increase in vascular permeability. A2A receptor activation only reduced lung inflammation when the A2A receptor was present on bone-marrow-derived cells. In addition, using A2A-receptor-knockout mice, the adenosine–A2A receptor–cAMP axis was identified as a potent endogenous anti-inflammatory signalling pathway that reduces airway reactivity and inflammatory-cell infiltration following sensitization with ragweed71. Thus, A2A agonists appear to be effective at curbing inflammatory lung tissue damage. However, in clinical trials, the utility of the A2A agonist GW328267X was limited by cardiovascular side effects72 (TABLE 1).
During reperfusion following ischaemic injury, many tissues have been shown to be protected from reperfusion injury owing to the activation of A2A receptors on bone-marrow-derived cells (FIG. 5). These include liver73, kidney74, heart75, skin76, spinal cord77,78 and lung79. In liver and kidney tissues iNKT cells have been found to be sensitive to A2A receptor activation and to have a crucial role in initiating an inflammatory cascade during reperfusion injury. These cells are rapidly stimulated to produce IFN-γ within 2 hours after the initiation of reperfusion, and the use of antibodies to deplete NK1.1-positive cells (NK and iNKT) or to block CD1d-mediated lipid-antigen presentation to iNKT cells replicates, but is not additive to the protection from reperfusion injury afforded by A2A adenosine receptor activation62,80. Liver reperfusion injury is also reduced in Rag1-knockout mice that lack mature lymphocytes, and can be restored to the level seen in wild-type mice by adoptive transfer of iNKT cells purified from wild-type or A2A receptor knockout mice, but not IFN-γ-knockout mice62. Additionally, animals with transferred A2A-knockout iNKT cells are not protected from hepatic reperfusion injury by A2A receptor activation. In vitro, A2A receptor activation potently inhibits activation of iNKT cells. These findings suggest that reperfusion injury is initiated by CD1d-dependent activation of iNKT cells, and the activation of these cells is inhibited by A2A receptor activation. The activation of iNKT cells results in the recruitment and transactivation of other immune cells such as macrophages and neutrophils that propagate tissue inflammation and injury.
Despite the introduction of a number of effective biological agents for the treatment of rheumatoid arthritis over the past decade methotrexate remains one of the most effective and most commonly used therapies for inflammatory arthritis. Although originally introduced for the treatment of cancer as a folic acid analogue, the mechanism by which methotrexate, at very low dosages (average dosage in the United States, 17.5 mg per week for rheumatoid arthritis versus as much as 5 g per week for cancer), diminishes inflammation differs from the anti-proliferative mechanism of the drug. At low doses methotrexate is taken up by cells and polyglutamated to a long-lasting metabolite81. Methotrexate Polyglutamates have a different spectrum of enzyme inhibition and AICAR transformylase, an enzyme that is part of the de novo purine synthetic pathway, appears to be most sensitive to inhibition by methotrexate polyglutamates82. At low doses of methotrexate, this leads to the intracellular accumulation of AICAR83,84. AICAR is a competitive inhibitor of AMP deaminase leading, ultimately, to enhanced adenosine release from cells83. Although it is difficult to measure adenosine levels in biological fluids owing to the short half-life (2–8 seconds) of adenosine in blood and other fluids85 in patients with rheumatoid arthritis treated with methotrexate there is strong evidence that methotrexate therapy promotes adenosine release86,87. Studies in mice and rats demonstrate that the anti-inflammatory effects of methotrexate are mediated by adenosine and that the anti-inflammatory effect of methotrexate is lost if animals are treated with adenosine receptor antagonists or if their adenosine A2A or A3 receptors have been deleted88-94. Similar loss of methotrexate efficacy has been noted in patients with rheumatoid arthritis who ingest significant quantities of caffeine, an adenosine receptor antagonist95. Thus, by promoting adenosine release at inflamed sites methotrexate diminishes inflammation in patients with rheumatoid arthritis. Building on the observation that methotrexate exerts some of its beneficial effects via A3 receptors, coupled with the finding that the selective A3 receptor agonist IB-MECA ameliorates the course of arthritis in mouse models96-98, IB-MECA was recently tested in a phase II trial in patients with rheumatoid arthritis. IB-MECA was safe and well tolerated, and patients treated with this drug achieved a moderate reduction of symptoms99 (TABLE 1). Methotrexate and IB-MECA have additive anti-inflammatory effects in rat adjuvant arthritis, which are mediated by an upregulation of the expression of A3 receptors on inflammatory cells100.
Preclinical studies using both knockout and pharmacological approaches have provided insights into the role of the various adenosine receptors in the physiological response of the organism to sepsis101. Inactivation of A1 receptors increased mortality in intra-abdominal sepsis provoked by cecal ligation and puncture in mice, an effect that was correlated with heightened inflammation-induced hepatic and renal injury102. These results suggest that A1 receptor activation may have a beneficial effect and additional studies testing the efficacy of A1 receptor agonists are warranted. Similar to results with A1 receptor blockade in cecal ligation and puncture, both knockout and pharmacological antagonism of A3 receptors increased mortality, whereas stimulation of A3 receptors with IB-MECA was protective103. In contrast to observations with blockade of A1 and A3 receptors, A2A receptor inactivation by either gene deletion or administration of ZM241385 prevented cecal ligation and puncture-induced mortality by a mechanism that involved decreased bacterial dissemination that appeared to be secondary to sustained immune system function104. Paradoxically, A2A receptor activation, when combined with antibiotics, has been noted to reduce mortality from sepsis induced by injecting with live E. coli, possibly by suppressing an exaggerated inflammatory response that can result from the rapid drug-induced killing of large numbers of bacteria105. Recent preliminary studies evaluating the role of A2B receptors have demonstrated an anti-inflammatory and protective role of A2B receptor stimulation in cecal ligation and puncture (C. Csoka, G. Hasko, Z. H. Nemeth and p. Pacher, unpublished observations).
Crohn's disease and ulcerative colitis are chronic, relapsing inflammatory bowel diseases (IBDs) that are characterized by a dysfunction of mucosal T cells, imbalanced cytokine production and cellular inflammation leading to damage of the intestinal mucosa. Although the aetiology of IBD remains to be determined, recent studies suggest that disease results from an inappropriately regulated immune response to luminal antigens in a genetically susceptible host106. CD4+CD25+ TReg cells have emerged as master regulators of the inflammatory response of the mucosa in IBD and have also been documented to offer a therapeutic option in severe inflammatory colitis107. Studies have highlighted the protective effects of A2A receptor activation in various animal models of colitis, and these protective effects can be ascribed to two major mechanisms: decrease of inflammatory-cell infiltration and function in the mucosa, and increased activity of TReg cells. The first mechanism is supported by evidence that A2A receptor activation by ATL146e (an A2A receptor agonist) decreases both leukocyte infiltration and the production of inflammatory cytokines by disease-inducing T-effector cells108. The role of A2A receptors on TReg cells was inferred by results showing that co-transfer of CD4+CD25+ TReg cells obtained from A2A receptor wild-type mice was able to prevent disease induction in severe combined immunodeficiency mice that were transferred adoptively with disease-inducing CD45RBhigh CD4+ T cells, a rodent model of IBD, whereas co-administration of CD4+CD25+ TReg cells isolated from A2A-receptor-knockout mice in conjunction with disease-inducing CD45RBhigh CD4+ T cells was inefficient in blocking the development of disease48.
The observation that activation of A2B receptors on intestinal epithelial cells can augment Il6 production by these cells resulting in increased neutrophil activation109 combined with increased detection of A2B receptors in epithelial cells isolated from mouse or human colitis110 prompted recent studies assessing the effect of A2B receptor blockade in IBD. Administering ATL-801, a selective A2B antagonist, to mice suffering from colitis markedly decreased IL6 production and neutrophil infiltration, and reduced the extent of mucosal damage thereby ameliorating the course of disease111. This pro-inflammatory role of A2B receptor activation by endogenous adenosine was recently confirmed using A2B-receptor-knockout mice112, confirming that blockade of A2B receptor represents a potentially advantageous treatment option that selectively targets the gut for patients afflicted with IBD. This proposition is predicated on the findings that A2B receptor expression is highest in the gut when compared with other organs and that levels of endogenous adenosine that appear to perpetuate the disease process through A2B receptors are elevated mostly locally in the gut111.
In the skin, inflammation is part of a continuum in which injured tissue is repaired and replaced resulting in wound healing. Montesinos et al. first reported that topical application of adenosine A2A receptor agonists increases the rate of wound healing in normal mice and in diabetic rats113. Increases in matrix and vessel formation were induced by adenosine receptor agonists and the improvement in the rate of wound healing was significantly greater than that induced by recombinant platelet-derived growth factor113-116. A2A and A2B receptor stimulation increases angiogenesis both directly and indirectly: adenosine receptors directly stimulate microvascular endothelial cell-proliferation as well as the autocrine production of vascular endothelial growth factor (VEGF), a central stimulus for endothelial proliferation and angiogenesis117-119, and inhibit the production of thrombospondin 1, an anti-angiogenic matrix protein120. In addition, adenosine stimulates VEGF production by macrophages116,121,122. Moreover, A2A and A2B receptor stimulation promotes matrix production by skin fibroblasts, an essential step in tissue repair66,123-125. In this context, it is noteworthy that A2A-receptor-deficient mice do not form granulation tissue indicating that endogenous adenosine plays a central role in wound healing114. Sonedenoson (MRE0094), an A2A agonist that regulates the inflammatory response and enhances tissue regeneration, is currently undergoing trials for the treatment of diabetic foot ulcers(TABLE 1).
The adenosine receptor system has evolved as both a rapid sensor of tissue injury and the major ‘first-aid’ machinery of tissues and organs. Adenosine receptor activation thus preserves tissue function and prevents further tissue injury following an acute injurious insult, such as reperfusion injury, actions in which the immune system has a paramount role. This primordial protective function of the adenosine receptor system following acute insults can, however, be overshadowed by its reduced ability to protect against chronic insults. In addition, in certain chronic disease states, such as asthma, the adenosine receptor system can even exacerbate tissue dysfunction.
Recent advances in understanding the role of the distinct adenosine receptors and the complex network of cellular players that determine the adenosine response to tissue injury have helped identify novel pharmacological targets to restore tissue function in various diseases(BOX 2). Despite growing enthusiasm regarding these targets, it is important to emphasize that because most of the data were obtained using animal models, caution should be exercised in translating results to the clinic. potential difficulties in translating results may stem from, in part, species differences and the fact that animal studies generally are done in young, relatively healthy animals over a short time-span. Of particular relevance is species differences in adenosine signalling in asthma studies. In rodents, primed mast-cell degranulation appears to be mediated primarily by A3 receptors, whereas in humans A2B receptors are more important.
Although adenosine receptor agonists have powerful immunomodulatory actions, the wide tissue distribution of adenosine receptors may limit their usefulness in the treatment of inflammatory diseases. Adenosine receptor antagonists, however, represent an ideal target for the therapy of certain immune-related disorders because their action is selectively targeted to the site of injury, where endogenous adenosine is released. Similarly, interfering with the function of enzymes and transporters that are responsible for the accumulation of extracellular adenosine allows local targeting of adenosine receptors, providing an opportunity for interventions with limited side effects. It is clear that a better understanding of adenosine receptor function will be required before the enormous potential of adenosine-based therapies can be realized to ease human suffering.
This work was supported by the National Institutes of Health (NIH) Grant R01GM66189 and the Intramural Research Program of NIH, National Institute on Alcohol Abuse and Alcoholism.
Competing interests statement
The authors declare competing financial interests: see web version for details.
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