Adenosine and Adenosine Receptors in the Pathogenesis and Treatment of Rheumatic Diseases

Abstract

Adenosine, a nucleoside derived primarily from the extracellular hydrolysis of adenine nucleotides, is a potent regulator of inflammation. Adenosine mediates its effects on inflammatory cells by engaging one or more cell-surface receptors. The expression and function of adenosine receptors on different cell types change during the course of rheumatic diseases, such as rheumatoid arthritis. Targeting adenosine receptors directly for the treatment of rheumatic diseases is currently under study; however, indirect targeting of adenosine receptors by enhancing adenosine levels at inflamed sites accounts for most of the anti-inflammatory effects of methotrexate, the anchor drug for the treatment of rheumatoid arthritis. In this Review, we discuss the regulation of extracellular adenosine levels and the role of adenosine in regulating the inflammatory and immune responses in rheumatic diseases such as Rheumatoid Arthritis, psoriasis and other types of inflammatory arthritis. In addition, adenosine and its receptors are involved in promoting fibrous matrix production in the skin and other organs and the role of adenosine in fibrosis and fibrosing diseases will also be discussed.

Introduction

Many different intercellular signals maintain homeostasis in tissues and organs. One of the first of these signals identified in the regulation of a large number of physiological and pathological processes was adenosine. First identified as a potent vasodilator in 1929 by Drury and Szent-Gyorgi¹, adenosine was subsequently shown to mediate its effects on cells via engagement of specific receptors (A₁, A_2a, A_2b and A₃ ^2,3). Indeed, adenosine has been described as a ‘retaliatory metabolite’ ⁴ that is released from cells in response to hypoxia, metabolic stress or injury and promotes the processes required to alleviate these noxious stimuli. Among its other functions, adenosine is an endogenous regulator of inflammation that mediates the transition from inflammation to healing. Pathological changes in, or pharmacological manipulation of, adenosine metabolism or adenosine receptor expression and/or function might have a role in both the pathogenesis and therapy of rheumatic diseases. We review the metabolic changes that regulate adenosine levels in inflamed tissue, the receptors that mediate the pharmacological and pathological effects of adenosine and their role in rheumatic diseases, as well as the potential role for therapeutic targeting of adenosine and its receptors.

Adenosine in inflammation and fibrosis

Inflammation of the joints, connective tissue, muscle and bone are the most common manifestations of the rheumatic diseases; both the innate and adaptive immune systems can contribute to the inflammation observed in diseases such as rheumatoid arthritis (RA) and psoriatic arthritis. In most inflammatory arthritides neutrophils are the most abundant cells in synovial fluids whereas T cells, B cells and macrophages predominate in the synovial tissues. At inflamed sites a large number of intercellular messengers and effector molecules secreted by the various cells are present, ranging in size from low molecular weight products (such as nitric oxide and prostaglandins among many others) to proteins (cytokines, growth factors, proteolytic enzymes and others). The cells (for example, synovial fibroblast-like cells and vascular endothelial cells) of the tissues that comprise the joint also contribute to injury and destruction of the structures of the joint; osteoclasts are critical to bone destruction, and chondrocytes secrete enzymes that destroy cartilage in inflamed joints. Adenosine, acting at its receptors, regulates the activation of all of these cell types and their secretion of intercellular messengers and effector molecules of all classes.

A common end point of inflammation is fibrosis or scarring, which can disrupt the appropriate functioning of the organ affected. Thus, joint contractures are also characteristic of inflammatory arthritis, and fibrosis characterizes organs affected in extra-articular RA, such as in the lung. The major manifestations of systemic sclerosis (SSc) are fibrosis of the skin and internal organs. Adenosine and its receptors also have a role in the pathogenesis of fibrosis in many different organs and might have a role in SSc as well.

Sources of adenosine

Intracellular ATP, the most abundant molecule in the cell, serves as the reservoir for the production of adenosine; however, most adenosine is formed in the extracellular space as a result of the sequential dephosphorylation of adenine nucleotides to adenosine (Figure 1). A number of transporters are involved in the export of ATP, including the proteins connexin-43 (also known as gap junction alpha-1 protein) ⁵, progressive ankylosis protein homolog (ANK) ⁶, pannexin-1 and pannexin-3 ⁷, and probably others as well, including P2×7⁸. Under resting conditions some ATP is dephosphorylated to adenosine but injury, hypoxia or other metabolic assault can trigger increased rates of intracellular conversion of ATP to adenosine or, more commonly, stimulate the release of adenine nucleotides into the extracellular space where they are dephosphorylated to adenosine by ectoenzymes at the cell surface (ecto-5’nucleotidase [CD73] and ecto-nucleoside triphosphate phosphohydrolase [CD39]) and by enzymes in blood or other extracellular fluids (for example, alkaline or acid phosphatases, including tissue nonspecific alkaline phosphatase [TNAP]). Once formed or released into the extracellular space adenosine can be deaminated to inosine and, in humans, ultimately to uric acid or taken up directly by cells by specific nucleoside transporters (ENT1 and ENT2) ⁹ and re-phosphorylated to ATP (Figure 1) ^6,9-16.

Figure 1. Cellular adenosine production in response to hypoxia and medications.

Adenosine is produced extracellularly from the hydrolysis of adenine nucleotides (ATP, ADP and AMP) by the ectoenzymes nucleoside triphosphate phosphohydrolase (NTPP or CD39) and ecto- 5’nucleotidase (CD73) whereas adenosine levels in the extracellular space are reduced by cellular uptake via the extracellular nucleoside transporter (ent1) and breakdown extracellularly to inosine by adenosine deaminase. Both methotrexate and sulfasalazine diminish the activity of aminoimidazolecarboxamido ribonucleotide (AICAR) transformylase leading to accumulation of AICAR and reduction of its metabolite formyl AICAR (FAICAR). Intracellular accumulation of AICAR leads to increased ATP release into the extracellular space.

Inflammation in the joint has long been known to lead to hypoxia due, in part, to pressure from synovial exudates, the disordered vasculature that develops in chronically inflamed synovium and the marked influx of inflammatory cells ^17-19. Although acute hypoxia leads to increased adenosine release that helps to ameliorate tissue injury following ischemia–reperfusion (reviewed elsewhere²⁰), in settings of chronic hypoxia and inflammation the ongoing cellular injury results in mitochondrial dysfunction²¹. In addition to release of inflammatory reactants from dysfunctional mitochondria, reduced ATP production probably leads to a reduction of ATP and/or adenosine levels in extracellular fluid (Corciulo and Cronstein, unpublished observations. Despite upregulation of adenosine receptors at inflamed sites (see below) the diminished adenosine level present in chronically inflamed joints probably permits further activation of inflammatory cells in the synovium ²¹. Hypoxia contributes to both acute and chronic inflammation by other mechanisms as well ²¹.

Adenosine receptors

Four subtypes of adenosine receptor exist, named in order of discovery: A₁, A_2a, A_2b and A₃. These receptors are all members of the G-protein-coupled family of 7-transmembrane spanning receptors ³ (FIGURE 2). A₁ and A_2a are high affinity receptors with activity in the low to mid-nanomolar range whereas A_2b has a substantially lower affinity for adenosine (micromolar). Considerable species-dependent variability in A₃ exists²². Adenosine receptors are widespread in their expression and are important regulators of many different types of physiological and pathological processes. A₁ and A₃ signal via Gα_i signalling proteins, which downregulate cAMP expression, whereas A_2a and A_2b are linked to Gα_S proteins, which trigger increases in cellular cAMP content. A_2b also signals through Gq proteins²³.

Figure 2. Adenosine receptors.

Adenosine receptors are all G protein-coupled receptors whose signal transduction is mediated by activation of intracellular G proteins. ADORA1 has the highest affinity for adenosine and is activated by high picomolar to low nanomolar adenosine concentrations; ADORA2A is activated by concentrations of adenosine in the mid-nanomolar range. By contrast, ADORA2B and ADORA3 are activated by adenosine concentrations in the micromolar range.

Expression of adenosine receptors is regulated by a number of stimuli, including inflammatory stimuli. In particular, A_2a is upregulated by agents that stimulate activation of NFκB (a central transcriptional regulator in the inflammatory process), such as TNF, IL-1 and endotoxin, ^24-28 and acts, as described below, as a feedback inhibitor of inflammation. Evidence from patients with RA confirms these observations: increased expression of A_2a on peripheral white blood cells in these patients is reduced by treatment with anti-TNF agents^29,30. In addition to regulation of A_2a expression, TNF and other proinflammatory cytokines increase the function of these receptors by preventing receptor desensitization³¹, further downregulating inflammation. By contrast, interferon-γ (IFNγ) downregulates both the expression and function of A_2a ^32-34. This downregulation (which occurs following the increase in both extracellular adenosine levels and A_2a expression and function after cellular injury or necrosis) indicates the potent role of adenosine and its receptors as feedback regulators of inflammation and innate immune responses.

Similarly, A₃ adenosine receptor expression is increased on peripheral blood white cells in patients with RA, and treatment with anti-TNF antibodies reduces adenosine receptor overexpression^29,35-37.

Adenosine receptors and innate immunity

Neutrophils

The anti-inflammatory effects of adenosine were first suggested in 1983 when the capacity of extracellular adenosine to inhibit stimulated neutrophil superoxide anion generation was demonstrated ³⁸, effects subsequently confirmed and expanded upon by many others ^39-42. Later studies revealed that these effects were mediated by adnesoine receptor A_2a ^43-45. Thus, A_2a engagement inhibits phagocytosis.

Before neutrophils can destroy invading bacteria or clear debris from a wound they must be recruited from the vasculature into the extravascular space, a process mediated by a series of adhesive interactions among neutrophils, endothelial cells and matrix. Numerous studies revealed that engagement of adenosine receptor A_2a and A_2b inhibited adhesion of neutrophils to both endothelial cells and other surfaces ^46-50 by inhibiting both selectin and integrin-mediated adhesive interactions. Interestingly, in vitro studies demonstrated that A₁ enhanced neutrophil adhesion to endothelial cells via α4 integrins ⁴⁹. This same group had reported, based on studies with pharmacological inhibitors, that stimulation of A₁ enhanced neutrophil chemotaxis; ^43,51 however, later studies using knockout mice and their neutrophils demonstrated that, in fact, it was A₃ that mediated enhanced chemotaxis ^52,53.

One of the mechanisms by which inflammatory cell activation is terminated or downregulated is by engulfment of apoptotic cells, such as occurs at sites of crystal-induced inflammation. A_2a engagement promotes engulfment-mediated neutrophil downregulation, whereas A₃ engagement diminishes engulfment-mediated downregulation of proinflammatory actions (TABLE 1) ^54,55.

TABLE 1.

Adenosine receptors regulate the function of inflammatory cells

Inflammatory cell	Adenosine receptor
	ADORA1	ADORA2A	ADORA2B	ADORA3
Neutrophil	Increases adhesion to matrix49	Inhibits superoxide anion generation43-45 Inhibits adhesion and recruitment Increases engulfment-mediated downregulation of neutrophil function46-50	Unknown	Chemotaxis52,53
Macrophage	Increases giant cell formation91 Increases osteoclast differentiation92,93	Promotes M1 to M2 transition62,71 Inhibits cytokine expression61-72 Inhibits osteoclast formation94-96	Inhibits osteoclast formation94-96 Promotes M1 to M2 transition75-77	Inhibits cytokine expression70,78-86
T cell	Unknown	Inhibits TCR-triggered activation103,104 Inhibits activation-induced cell death112 Inhibits Fas/FasL-mediated cell death112	Stimulates TH17 differentiation by increasing dendritic cell IL-6 production87-89	Unknown
Endothelial cell	Unknown	Increases angiogenesis123 Increases barrier integrity (prevents oedema) 123,125	Increases angiogenesis123 Increases oedema formation in arthritis90 Promotes clearance of pulmonary oedema126	Unknown
Fibroblast	Unknown	Stimulates fibroblast production of collagen I and III130-133 Promotes skin, lung and hepatic fibrosis130-144	Stimulates collagen production130-133	Unknown

It has often been noted that imitation is the most sincere form of flattery and this adage is no less true for imitation of critical biological mechanisms by pathogens. Although most of the adenosine that mediates suppression of inflammation is produced endogenously some is produced by pathogens as a virulence factor. Invasive forms of Candida albicans, Staphylococcus aureus and Streptococcus suis have all been reported to produce adenosine as a virulence factor to protect against phagocyte-mediated injury ^56-58. The hijacking of adenosine-mediated immunosuppression by pathogens provides strong evidence for the importance of this biological mechanism.

Monocytes and macrophages

Macrophages have critical roles in initiating the innate immune response against microbes and in eliminating debris at sites of injury. In addition to destroying most microbes, macrophages produce a variety of cytokines and mediators involved in regulating inflammatory responses and present antigens to T cells in order to orient the adaptive immune response. Macrophages are antigen presenting cells that promote the selective expansion and differentiation of lymphocytes specific for invaders or cancer cells and, in the rheumatic diseases, direct immune response to self-antigens. Two types of macrophages have been identified: M1 or classical macrophages and M2 or alternatively activated macrophages ^59,60. In diseases such as RA, M1 macrophages have an important role by releasing a variety of proinflammatory cytokines (including TNF, IFNγ and IL-12 among many others), as well as oxidants, nitric oxide and other small-molecule mediators of tissue injury. M2 macrophages have a role in terminating inflammation after ingestion of apoptotic cells and promote wound healing by producing angiogenic and profibrotic cytokines.

Adenosine, acting at A_2a, suppresses production of proinflammatory cytokines and stimulates expression of anti-inflammatory mediators such as IL-10 and vascular endothelial growth factor (VEGF) ^61-72. More generally, A_2a stimulation induces a switch from an M1 to a modified M2 phenotype ^62,71. One mechanism by which A_2a ligation alters macrophage function is via stimulating expression of the orphan nuclear receptor NR4A, which diminishes activation of NFκB-dependent gene expression ^73,74. A_2b also stimulates the switch from an M1 to an M2 phenotype ^75-77. The role of A₃ in regulation of inflammatory macrophage function is less clear; A₃ stimulation clearly suppresses cytokine expression and release by macrophages and diminishes inflammation in mouse models of arthritis and inflammatory bowel disease ^70,78-86. Indeed, the relatively poor anti-infective function of macrophages from human neonates is thought to reflect the dominance of A₃ expressed by these cells ⁸⁰. By contrast, studies have indicated that A₃ ligation diminishes the suppression of inflammation induced by engulfment of apoptotic cells ⁵⁴. In patients with RA, A₃ expression is a marker of disease severity and can predict the efficacy of treatment of symptoms with a selective adenosine receptor A₃ ligand (TABLE 1) ³⁵.

The effect of A_2b activation on the overall immune response remains ambiguous. As noted above, A_2b promotes a phenotypical switch from M1 to M2 macrophages, ^75-77 but also stimulates the differentiation of dendritic cells, which can promote type 17 T helper cells (T_H17) immune responses via production of IL-6 ^87-89. Indeed, in in vivo studies high concentrations of adenosine exacerbated arthritis and tissue destruction in rats via an A_2b mechanism ⁹⁰.

Multinucleated giant cells, formed from the fusion of macrophages, are characteristic accompaniments of inflammation in response to foreign bodies or certain pathogens (most notably tuberculosis) and their presence in inflamed vessels gives Giant Cell Arteritis its name.. Adenosine receptors have a critical role in the formation of these syncytia as A₁ stimulation is essential for formation of giant cells in vitro ⁹¹. Osteoclasts, a specialized form of multinucleated giant cell involved in bone remodelling and turnover, also depend on A₁ to complete differentiation by a mechanism involving the signalling molecule TRAF6 ^92,93. By contrast, A_2a and A_2b stimulation inhibits osteoclast differentiation; mice lacking these receptors have osteopaenia ^94-96 due, in part, to increased osteoclast-mediated bone resorption. Owing to its effects on osteoclasts and macrophages and production of proinflammatory cytokines, A_2a also inhibits inflammatory bone damage induced by prosthetic wear particles⁹⁷. Interestingly, both A_2a and A_2b stimulate bone formation in vivo ⁹⁸ although only the direct stimulatory effects of A_2b on osteoblast function have been well characterized ^99,100.

The adaptive immune system

Regulation of T cells by A_2a

Effector functions of T cells in RA synovium are triggered by the T cell receptor (TCR) recognition event followed by transmembrane signalling of the TCR–CD3 complex and by multiple interconnected intracellular biochemical pathways; therefore, it is important to explore the therapeutic opportunities of targeting T cells in both hypoxic and adenosine-rich microenvironments and normoxic and adenosine-poor tissue microenvironments of inflamed joints in RA. Studies of adenosine receptors on T cells were largely motivated by the need to understand their role in anti-pathogen and anti-tumour immunity, with a primary focus, until recently, on A_2a ^101,102. The high-affinity A_2a, but not A₁ or A₃, was identified as the predominant adenosine receptor on the surface of mouse T cells^103,104. Expression and functional coupling of A_2a in mouse peripheral T cells and B cells correlated with adenosine-induced cAMP accumulation in lymphocytes and with adenosine-induced inhibition of TCR-triggered T cell activation^103,104. The capacity of A_2a stimulation to inhibit effector functions of different subsets of mouse T cells was subsequently confirmed¹⁰⁵ and in vivo genetic and pharmacological evidence was provided showing that A_2a is critical and non-redundant in controlling the extent of T-cell-mediated immune responses, inflammation and tissue damage in different models of antipathogen immune responses and antitumour immune responses ^101,106. Non-redundance of A_2a in T cells and myeloid cells was demonstrated by the observation that none of the other cAMP-elevating G-protein-coupled receptors on immune cells could replace A_2a and compensate for the A_2a-mediated attenuation of T-cell-driven immune responses and inflammatory damage in models of autoimmune and viral hepatitis¹⁰¹.

ADORA2A gene–dose effect

Naive T cells express low levels of A_2a ^103,104 but following TCR-triggered activation of T cells ADORA2A mRNA and A_2a-mediated signalling rapidly increase¹⁰⁵. Other studies have demonstrated a clear gene–dose effect for extracellular adenosine signalling at A_2a and that no receptor reserve exists on these cells ¹⁰⁷. The lack of ‘spare’ immunosuppressive A_2a on T cells points toward the importance of every individual A_2a molecule on these cells in maintaining the balance between immunosuppression and immunostimulation and the need for the precise regulation of numbers of A_2a molecules to avoid autoimmunity without tolerization of T cells. Extracellular adenosine is short-lived in vivo but its immunosuppressive effects on T cells are prolonged due to the intracellular biochemical ‘memory’ of exposure of T cells to adenosine¹⁰⁴.

In view of the direct evidence for the effect of A_2a on T cells and the role of T_H17 and T regulatory (T_reg) cells in the pathogenesis of RA it would be of significant interest to investigate the genetic differences in ADORA2A between individual patients with RA and to search for correlations between the number of A_2a molecules per immune cell and severity of the disease. ADORA2A gene polymorphisms, as well as the number of A_2a molecules per immune cell, might also predispose a patient to developing RA or, equally importantly, to respond to adenosine-dependent therapies such as methotrexate (see below). An interesting experimental approach to testing this hypothesis was presented in studies of functional changes caused by genetic variation of ADORA2A that used personalized lymphoblastoid cell lines from twins as a model system¹⁰⁸. This study demonstrated that there was altered potency of a partial A_2A agonist for A_2A receptor activation in cells expressing a receptor in which there was a previously described polymorphism associated with caffeine-induced sleep disorders.

A_2a and subsets of human T cells

Further characterization of which T cell subsets express A_2a and whether and how their functions are regulated is required, although early studies clearly demonstrated differential expression and function of this receptor on T cell subsets^109,110. CD4⁺ T helper cells express A_2a which, when stimulated by adenosine or an A_2A agonist, inhibits the proinflammatory functions of these cells^110,111. Similar to mouse T cells, human T helper cells increased ADORA2A mRNA levels as soon as 4 hours after activation, and A_2a was the predominant subtype of adenosine receptor expressed by these cells. Activation of A_2a on T cells is of great consequence not only to T cell function but also to the fate of T cells, memory T cells and T-cell homeostasis as A_2a counteracts processes leading to activation-induced cell death and Fas–Fas-ligand-mediated cell death in CD4+ T lymphocytes¹¹². (TABLE 1) These original studies were carried out on mouse CD4+ hybridomas and human Jurkat cells (a transformed T cell line and were subsequently expanded to in vivo studies of naive T cell development and peripheral maintenance, which is the overall outcome of extracellular signalling to T cells in different tissue microenvironments¹¹³. The possibility of A_2a involvement in T-cell selection processes was also the implication of early studies of immature T cells¹¹⁴.

A_2a signalling in T cells

A_2a is a Gs-linked receptor that stimulates an increase in intracellular cAMP levels, which in turn activates protein kinase A (PKA). cAMP–PKA signalling directly inhibits all known TCR-triggered effector functions^104,106 because cAMP–PKA is the ‘high fidelity’ immunosuppressor that intercepts both early and late events in the TCR-triggered intracellular T cell activation pathway ^115,116. Moreover, signalling mediated by A_2a, cAMP, PKA, cAMP response element (CRE) and cAMP response element binding (CREB) protein can redirect proinflammatory T cell responses toward an immunosuppressive phenotype. This change in phenotype also occurs because the extracellular adenosine accumulation driven by hypoxia and hypoxia-inducible factor (HIF)-1α results in hypoxia response element (HRE) and CRE-dependent redirection of CD8 and CD4 and myeloid-cell-mediated immune responses toward production of immunosuppressive cytokines and molecules such as TGFβ, IL-10 and CTLA-4, and promotes the development of T_reg cells and their inhibition of effector T cells^117,118. Taken together, any interpretation of the effects of adenosine on T cells should consider A_2a signalling regulating T cell development, maintenance and effector functions.

Regulation of T cells by A_2b

Compared with adenosine receptor A_2a, much less is known about the low affinity A_2b on T cells, but—of relevance to RA—evidence indicates that activation of A_2b by synthetic agonists could have T_H17-promoting effects by redirecting the differentiation of bone marrow cells to a CD11c⁺GR1⁺ dendritic cell subset^89,119. (TABLE 1) Moreover, A_2b stimulation might also promote T_H17 differentiation by stimulating IL-6 expression in a cAMP-independent manner^88,119. Others have reported that A_2b promotes T_reg-cell differentiation and limits inflammatory injury¹²⁰. A_2b signals by multiple pathways; not only does A_2b increase cAMP via coupling to G_s proteins but they might also signal via G_q and G_i proteins, thereby explaining their cAMP-independent effects on generation of IL-6¹²¹.

ADORA1 and ADORA3 in T cells

The expression and function of A₁ and A₃ on T cells and their subsets is not fully established. A report indicates that A₃ is expressed on Jurkat cells but its expression and function in primary cells has not been established¹²².

Other cell types in inflammation

New blood vessel formation is a characteristic of the inflamed synovium, and hypoxia is a major driving force for neovascularization. Adenosine and its receptors have direct and indirect roles in stimulating new vessel formation. Acting at both A_2a and A_2b, adenosine stimulates both angiogenesis and production of such proangiogenic mediators as VEGF¹²³. Moreover, both A_2a and A_2b directly stimulate endothelial cells to form new vessels¹²³. (TABLE 1)

The effects of adenosine receptor stimulation on the formation and resolution of vascular oedema seems to vary with the tissue studied. A_2a has been reported to promote vascular leakage in healing wounds and inflamed sites as a result of the increase in new blood vessels that form at these sites¹²⁴. By contrast, the barrier integrity of human umbilical vein endothelial cells is enhanced by A_2a stimulation¹²⁵. Interestingly, A_2b stimulation promotes clearance of pulmonary oedema¹²⁶. A_2b stimulation might have opposing effects in other vascular beds: excess adenosine generation, acting via A_2b, can increase inflammation and inflammatory oedema in rat models of arthritis¹²⁷. Thus, targeting adenosine receptors in the prevention of angiogenesis and vascular oedema in inflammation is complex.

Similar to the effects on oedema formation and angiogenesis, the effects of adenosine and adenosine receptors on collagen and matrix production by fibroblasts and fibroblast-like cells varies from tissue to tissue. Adenosine, acting at A_2a, directly stimulates dermal fibroblasts to produce collagen and also stimulates the production of factors, such as IL-13 and connective tissue growth factor, that are capable of further amplifying collagen production ¹²⁸. By contrast, A_2b stimulation inhibits collagen production by some tissue fibroblasts (cardiac fibroblasts) but stimulates collagen production by fibroblasts in other tissues ¹²⁸.

Adenosine and resolution of inflammation

The inflammatory response is critical for clearance of debris and protection from invading microorganisms; inflammation sets the stage for tissue repair. As with suppression of inflammation, adenosine, acting at its receptors, promotes tissue repair and regeneration. As noted above, A_2a stimulation promotes the transformation of macrophages to an M2 phenotype, which is associated with lower levels of production of TNF, IL-12 and other proinflammatory mediators than the M1 phenotype, and with increased production of IL-10, IL-13 and VEGF,^59,60 which promote angiogenesis and fibrosis. Moreover, A_2a and A_2b stimulation promotes angiogenesis and generation of matrix by directly stimulating endothelial cells, endothelial cell precursors and fibroblasts ^123,129.

Adenosine receptors and fibrosis

Adenosine mediates the transition from inflammation to wound healing but an excess of wound healing might result in fibrosis, which can disrupt organ architecture and function. Fibrous contractions of the joints and fibrosis in the lungs and other tissues are common complications of RA and other rheumatic diseases, and fibrosis characterizes SSc. Results from animal models of scarring or diffuse fibrosis, a manifestation of SSc, indicate a role for adenosine and its receptors in fibrosis ^130-133. Moreover, mice lacking adenosine deaminase have high levels of adenosine in the skin and spontaneously develop diffuse dermal fibrosis ¹³¹. Elevated adenosine levels in the tissue of mice lacking adenosine deaminase also lead to pulmonary fibrosis by engaging both A_2a and A_2b ^134-141. In the liver, extracellular adenosine production with A_2b stimulation mediates hepatic fibrosis in mouse models, a phenomenon that is inhibited by caffeine, a nonselective adenosine receptor antagonist^142-144. This latter finding might account for the well-documented observation that coffee drinking and caffeine ingestion reduces deaths from liver disease ^145-149. Peritoneal fibrosis also depends on A_2a stimulation in animal models¹⁵⁰. In the setting of priapism, in which the chronically engorged penis becomes hypoxic, adenosine release leads to both vasodilation and fibrosis via activation of A_2b ^151,152. Thus, although adenosine and its receptors generally have a beneficial role in terminating inflammation, these same receptors, if persistently stimulated, lead to enhanced fibrosis with resulting tissue and organ dysfunction.

Regulation of extracellular adenosine

Under some pathological conditions tissue adenosine levels are markedly increased. Perhaps the best studied condition under which adenosine levels increase is hypoxia. As noted above, adenosine was first described as a coronary vasodilator in 1929¹ and in subsequent studies it became increasingly clear that the hyperaemic response to hypoxia was attributable, in large part, to adenosine release¹⁵³. Presumably the breakdown of ATP and release of adenine nucleotides into the extracellular space has a role in the generation of increased extracellular adenosine; HIF-1α and adenosine kinase were shown to have an important role in this pathway¹⁵⁴. In addition, ENT1-mediated uptake of adenosine is downregulated by increases in intracellular cAMP¹⁵⁵, and increases in extracellular adenosine drive further increases in adenosine levels by diminishing ENT1 function via adenosine-receptor-mediated increases in cAMP ¹⁵⁶. It was recognized quite early that inflamed joints tend to be chronically hypoxic as a result of the increased cellular bulk, tissue oedema (with resulting compression of the microvasculature) and the pressure of increased synovial fluid^{17-19,157,158}. Nonetheless, estimating the actual level of adenosine in synovial fluid is difficult as cells present in synovial fluid might undergo lysis, thus releasing ATP (which is converted to adenosine during processing); furthermore, adenosine is hydrolyzed by the adenosine deaminase present in synovial fluid^159-161.

Extracellular adenosine levels can also fluctuate in other settings. A number of drugs can regulate adenosine levels in the extracellular fluid. Most notable among these are dipyridamole and ticagrelor, which are agents that inhibit ENT1-mediated adenosine uptake ^156,162,163. Ethanol, which is metabolized as a lipid and requires ATP hydrolysis in order to be metabolized to acetyl-CoA, also causes adenosine release as a result of ATP breakdown^156,164-167. More recently, it has been appreciated that extracellular adenosine levels might reach extremely high concentrations in the setting of rapid cellular breakdown and hypoxia, such as occurs in tumours, leading to consequences such as immunosuppression by tumours^101,102,168.

Therapeutic targeting of adenosine

Although no drugs have been developed to treat rheumatic diseases that directly target adenosine and its receptors, adenosine-based targeting has been reported for a number of commonly used drugs. Anti-inflammatory and antirheumatic drugs that mediate at least some of their anti-inflammatory effects via targeting adenosine metabolism include methotrexate, sulfasalazine and high-dose aspirin.

Methotrexate

Weekly, low-dose methotrexate remains the gold standard therapy for patients with rheumatic diseases. Although its mechanism of action has not been fully elucidated (Box 1), it seems that enhancing adenosine release at inflamed sites has a major role in the anti-inflammatory actions of low-dose methotrexate. The enzyme inhibited best by intracellular methotrexate polyglutamates is aminoimidazolecarboxamidoribonucleotide (AICAR) transformylase; intracellular accumulation of AICAR leads to adenosine release¹⁶⁹ (Figure 1). In 1991, the first results were published suggesting that methotrexate promotes adenosine release, which diminishes inflammation in vitro¹⁷⁰; it was subsequently demonstrated that adenosine mediates the anti-inflammatory effects of methotrexate in an in vivo model in which increased intracellular AICAR levels at an inflamed site were documented¹⁷¹. Adenosine mediates the anti-inflammatory effects of methotrexate via occupancy of A_2a and A₃¹⁶⁹. Moreover, adenosine receptor antagonists, including caffeine, block the anti-inflammatory effects of methotrexate treatment in an animal model of arthritis¹⁷² and in patients with Rheumatoid Arthritis. in prospective studies^173,174. A large retrospective study of patients taking methotrexate did not support the contention that caffeine ingestion reversed the anti-inflammatory effects of weekly low-dose methotrexate; however, given that patients in this study had been successfully treated for RA for a number of years, those who had not responded to methotrexate were excluded from the analysis¹⁷⁵. Nonetheless, other studies provide further evidence for the role of adenosine in the mechanism of action of methotrexate; for example, the expression of CD39, an ectoenzyme that catalyzes the dephosphorylation of ATP and ADP to AMP (which is critical for extracellular adenosine production), is a biomarker for methotrexate efficacy in patients with RA¹⁷⁶. Other mechanisms of action have been described for the anti-inflammatory actions of methotrexate in the treatment of Rheumatoid Arthritis and other rheumatic conditions that do not include a role for adenosine^169,177-179.

BOX 1.

Methotrexate was originally developed as a folic acid antagonist to prevent the de novo synthesis of purines and pyrimidines in the treatment of cancer and leukaemia, which remain indications for use of this drug. Although high-dose aminopterin, an analogue of methotrexate, was reported to be useful in the treatment of rheumatoid arthritis (RA) in the early 1950s¹⁹⁰, methotrexate was not widely used to treat inflammatory arthritis until anecdotal reports and uncontrolled trials were carried out in the 1970s followed by placebo-controlled trials in the 1980s and approval for the treatment of RA in 1989¹⁹¹. Although initially thought to block lymphocyte proliferation via inhibition of purine and pyrimidine synthesis, the inability of folic acid supplementation to reverse the efficacy of methotrexate treatment while preventing folate-dependent toxicities did not support this effect for methotrexate used at doses up to 1/100^th of the dose given to treat malignancy^192-194. In the treatment of Rheumatoid Arthritis, psoriasis and other inflammatory conditions methotrexate is administered, in low doses (5-25mg) once per week and has a half-life that is relatively short. It is undetectable in the circulation after 18 h but is widely distributed and present in tissues as long-lived polyglutamates, which remain potent inhibitors of a number of enzymes involved in the synthesis of purines and pyrimidines. The enzyme inhibited most potently by methotrexate polyglutamates is aminoimidazole carboxamidoribonucleotide (AICAR) transferase (AICART); inhibition of this enzyme leads to intracellular AICAR accumulation (see Figure 1). Increased intracellular AICAR promotes adenosine release, and blockade or deletion of adenosine receptors reverses the anti-inflammatory effects of methotrexate in animal models and patients with Rheumatoid Arthritis ¹⁶⁹.

Sulfasalazine

Sulfasalazine was first developed as a specific therapy for RA in the 1940s and remains in use today. Sulfasalazine is broken down by gut bacteria to 5-aminosalicylate and sulfapyridine; sulfasalazine itself and 5-aminosalicylate are poorly absorbed from the gut and sulfapyridine acts as an antifolate once absorbed. Indeed, folate deficiency is a recognized side effect of sulfasalazine¹⁸⁰. Among its other effects sulfasalazine promotes accumulation of intracellular AICAR and release of adenine nucleotides into the extracellular space where they are dephosphorylated to adenosine; blockade or loss of ecto-5’nucleotidase prevents adenosine release induced by sulfasalazine both in vitro and in vivo and reverses the adenosine-receptor-dependent anti-inflammatory effects of the drug in a mouse model of inflammation^181,182 (Figure 1). These results suggest that, like methotrexate, adenosine mediates at least some of the anti-inflammatory effects of sulfasalazine.

High-dose aspirin

Although not commonly used anymore for the treatment of RA and other rheumatic diseases, high-dose aspirin was a mainstay of antirheumatic therapy until the development of less-toxic NSAIDs in the 1970s. Before the recognition that aspirin and other NSAIDs mediated many of their actions by inhibiting cyclooxygenases, it was thought that one mechanism by which high-dose aspirin mediated its therapeutic and toxic effects was by uncoupling oxidative phosphorylation and loss of intracellular ATP ^183,184. Results of studies in human neutrophils indicated that some of the effects of high-dose salicylates on stimulated neutrophil function resulted from ATP loss and extracellular conversion to adenosine ¹⁸⁵. Subsequent studies in mice confirmed that adenosine mediates the anti-inflammatory effects of high-dose aspirin in vivo as well ¹⁸⁶. The doses of aspirin required verge on the toxic and are almost never used at present.

Antirheumatic drugs in development

On the basis of their anti-inflammatory activity in a variety of animal models [¹⁸⁷, A₃ agonists are currently under development for the treatment of RA. Indeed, a phase II clinical trial of CF101, an A₃ agonist, reduced RA activity although the improvement did not achieve statistical significance¹⁸⁸. Interestingly, an increased clinical response to CF101 was seen in patients in whom A₃ was overexpressed on peripheral blood mononuclear cells ¹⁸⁸. CF101 has also been administered to patients for the treatment of psoriasis and an early phase study demonstrates clear evidence of efficacy for this indication ¹⁸⁹.

Conclusions

The role of adenosine and its receptors in regulating inflammation and immune responses has been well established. Adenosine, released by hypoxic or injured tissues or following treatment with drugs such as methotrexate, suppresses inflammation and immune responses via stimulation of its receptors on inflammatory and immune cells. Although this mechanism of action is useful for the treatment of diseases such as RA, in the setting of tumours the immunosuppression that occurs might be deleterious. The conditional benefits of modulating inflammation and immunity dictate that targeting adenosine receptors with agonists or antagonists will require individual tailoring.

Key Points.

• Adenosine, generated from the extracellular hydrolysis of ATP, is a potent endogenous regulator of inflammation and immune reactions via interaction with one or more cell surface receptors.

• The principal adenosine receptor involved in regulation of adaptive T cell responses is ADORA2A.

• ADORA2A, ADORA2B and ADORA3 downregulate macrophage-mediated inflammatory actions, although ADORA2B might indirectly stimulate type 17 T helper cell immune responses via increased IL-6 production.

• Adenosine mediates the anti-inflammatory effects of low-dose methotrexate treatment as well as some of the anti-inflammatory effects of sulfasalazine.

Biography

Bruce Cronstein is the Paul R. Esserman Professor of Medicine at NYU School of Medicine where he directs the Clinical and Translational Science Institute. Trained as a physician and Rheumatologist, he first described the role of adenosine and its receptors in inflammation and demonstrated that adenosine mediates the anti-inflammatory effects of low-dose methotrexate in the treatment of rheumatic diseases. In other studies he has demonstrated the role of adenosine and its receptors in bone and liver physiology and the pathologic role of adenosine in fibrosis of the skin and liver.

Michail Sitkovsky is the E.W. Black Professor of Immunophysiology and Director of the New England Inflammation and Tissue Protection Institute at Northeastern University, Boston, Massachusetts, USA. Trained as a biophysicist and immunologist, he uncovered the non-redundant and domineering role of A_2A adenosine receptor and hypoxia-inducible factor 1α in pathophysiological immunosuppression and redirection of immune response. These findings led to the conceptually novel repurposing of several classes of drugs in order to weaken the immunosuppressive hypoxia–A_2A adenosine receptor axis and thereby enable the use of immunotherapies for cancer and infectious diseases.