Adenosine receptors and asthma

Abstract

The accumulation of evidence implicating a role for adenosine in the pathogenesis of asthma has led to investigations into all adenosine receptor subtypes as potential therapeutic targets for the treatment of asthma. Selective A₁ receptor antagonists are currently in preclinical development since adenosine has been shown experimentally to mediate various features of asthma through this receptor such as bronchoconstriction, mucus secretion and inflammation. The A_2A receptor is expressed on most inflammatory cells implicated in asthma, and as A_2A stimulation activates adenylate cyclase and consequently elevates cAMP, selective A_2A receptor agonists have now reached clinical development. However, initial reports concerning their efficacy are inconclusive. A_2B receptor antagonists are also under investigation based on the rationale that inhibiting the effects of adenosine on mast cells would be beneficial, in addition to other reported pro-inflammatory effects mediated by the A_2B receptor on cells such as airway smooth muscle, epithelial cells and fibroblasts. Whilst the effects in pre-clinical models are promising, their efficacy in the clinical setting has also yet to be reported. Finally, adenosine A₃ receptor stimulation has been demonstrated to mediate inhibitory effects on eosinophils since it also elevates cAMP. However, some experimental reports suggest that A₃ antagonists mediate anti-inflammatory effects, thus the rationale for A₃ receptor ligands as therapeutic agents remains to be determined. In conclusion, establishing the precise role of adenosine in the pathogenesis of asthma and developing appropriate subtype selective agonists/antagonists represents an exciting opportunity for the development of novel therapeutics for the treatment of asthma.

Introduction

This article provides an introduction to the role of adenosine receptors in asthma following the symposium ‘New insights into the anti-inflammatory effects of A_2A adenosine receptor agonists' presented at the Life Sciences Meeting in Glasgow on 11th July, 2007. The potential role of A_2A agonists as a novel treatment for asthma is discussed in more detail by Dr Palmer and Dr Trevethick in this journal in a review entitled ‘New insights into the anti-inflammatory effects of A_2A adenosine receptor agonists and antagonists: an introduction'. Furthermore, this article also provides an interesting comparison with adenosine receptors and cardiac inflammation as reviewed in this journal by Professor Joel Linden (University of Virginia, USA) in ‘A_2A adenosine receptors in tissue reperfusion injury'.

Inhaled adenosine has long been recognized as a potent bronchoprovocant when administered to asthmatic but not healthy subjects, as first demonstrated by Cushley et al. (1983). However, although the effects of inhaled adenosine are well characterized, comparatively less is understood about the physiological role of endogenous extracellular adenosine in the airways and the pathological significance of an increase in the levels of this mediator in the airways. There is also a lack of information concerning the precise location and relative distribution of adenosine receptors in both healthy and asthmatic airways, although as virtually all cells express one or more of the adenosine receptor subtypes, there is a considerable amount of data pertaining to the function of adenosine receptors on a variety of cell types relevant to asthma. Consequently, a number of these subtype-specific cellular effects are currently being exploited to provide novel therapeutic opportunities for the treatment of asthma.

The aim of this review is to therefore discuss the current understanding of the role of endogenous adenosine in the pathophysiology of asthma, and thus the therapeutic basis of inhibiting certain receptor subtype-specific effects of adenosine, in particular the A₁ and A_2B receptors. Conversely, the rationale for activating subtype-specific adenosine receptor pathways, namely the A_2A pathway, as an alternative therapeutic approach will also be reviewed.

Adenosine receptors

The biological action of adenosine is mediated predominantly through specific cell surface receptors of which four subtypes (A₁, A_2A, A_2B and A₃) have been described (reviewed by Ralevic and Burnstock, 1998). Adenosine receptors are G-protein coupled 7-transmembrane receptors, with the A₁ and A₃ subtypes predominantly coupled to G_i/o and the A_2A and A_2B to G_s, thus lowering and elevating the level of intracellular cAMP, respectively (Table 1). Binding studies revealed that the A₁ and A_2A subtypes have the highest affinity for adenosine, whereas the A_2B and A₃ receptors have a significantly lower affinity (Fredholm et al., 2001). Adenosine receptors are ubiquitously expressed throughout the body, with virtually all cells expressing one or more adenosine receptor subtype. The location and identified functions of adenosine receptors on cells important in asthma are described in Table 2. With respect to the lung, little is known about the relative expression of adenosine receptor subtypes; however, binding studies in healthy peripheral lung tissue have suggested that A₂ receptor subtypes are much more abundant than the A₁ and A₃ receptor subtypes (Joad, 1990).

Table 1. G-protein coupling and signal transduction mechanisms of adenosine receptor subtypes.

	A1	A2A	A2B	A3
G-protein	Gi/Go	Gs/Golf	Gs/Gq/11	Gi/Gq/11
Signal transduction	↓cAMP ↑IP3/DAG	↑cAMP	↑cAMP ↑IP3/DAG	↓cAMP ↑IP3/DAG
Affinity for adenosine	High	High	Low	Low

Table 2. Expression and putative functions of adenosine receptor subtypes on cells involved in asthma.

	A1	A2A	A2B	A3	Putative functions
Neutrophils	✓	✓		✓	A1 induces degranulation, respiratory burst, chemotaxis, endothelial adherence A2A is linked to inhibition of degranulation, respiratory burst, endothelial adhesion A3 inhibits degranulation
Eosinophils	✓	✓		✓	A1 is linked to eosinophil activation A2A is linked to inhibition of respiratory burst A3 is linked to inhibition of degranulation, respiratory burst, chemotaxis
Basophils		✓			A2A inhibits leukotriene and histamine release
Mast cells		✓	✓		A2A inhibits degranulation A2B induces/promotes degranulation, cytokine secretion, IgE synthesis
T lymphocytes		✓	✓		A2A inhibits activation and expansion A2B inhibits activation
B lymphocytes		✓			Adenosine indirectly promotes IgE synthesis through mast cell activation
Monocytes	✓	✓	✓	✓	A1 promotes phagocytosis A2A inhibits IL-12 and TNF-α secretion, induces IL-10 and VEGF secretion
Platelets		✓			A2A inhibits aggregation
Macrophages		✓	✓	✓	A2A inhibits IL-12 and TNF-α secretion, NO production and induces IL-10 secretion A3 inhibits respiratory burst
Dendritic cells	✓	✓		✓	A1 and A3 promote chemotaxis (immature cells) A2A inhibits IL-12 secretion (mature cells)
Bronchial epithelial cells	✓	✓	✓		A1 induces mucin secretion A2A stimulates wound healing A2B induces IL-19 secretion
Bronchial smooth muscle	✓		✓		A1 inhibits contraction A2B induces IL-6 and MCP-1 secretion
Fibroblasts			✓		A2B induces IL-6 secretion and differentiation into myofibroblasts
Endothelial cells	✓	✓	✓		A2A inhibits IL-6 and IL-8 secretion and E-selectin and VCAM-1 expression from LPS-stimulated HUVECS; stimulates neo-vascularisation A2B induces VEGF and IL-8 secretion

Source and physiological role of endogenous adenosine

The nucleotide ATP has numerous biological functions, but it is best known as the primary source of free energy for metabolic processes within all living cells. ATP consists of a purine base (adenine) attached to the 1′ carbon atom of ribose and three phosphate groups attached at the 5′ carbon atom of the ribose (Figure 1). The liberation of the terminal phosphate group by hydrolysis is a highly exergonic reaction yielding ADP and inorganic phosphate. Under certain conditions such as high energy demand, ADP is also hydrolysed to AMP and subsequently the nucleoside adenosine through the action of intracellular 5′-nucleotidase (reviewed by Fredholm et al., 2001). As many cells express bidirectional nucleoside transporters (Cass et al., 1998), adenosine is able to cross the membrane and enter the extracellular space. Indeed, when the intracellular concentration of adenosine is very high, it can also passively diffuse out of the cell. ATP also functions as a neurotransmitter in both the central and peripheral nervous system, thus generating an extracellular source of adenosine following exposure to ectonucleotidases (reviewed by Burnstock, 2006).

Figure 1.

The structure of ATP and ATP derivatives.

Many of the diverse effects of extracellular adenosine upon physiological processes have now been characterized and include neurotransmission, vascular smooth muscle dilation and modulation of cardiac, renal, immune and inflammatory functions. Although the constitutively low extracellular concentration of adenosine is normally strictly regulated various metabolically stressful conditions such as hypoxia, ischaemia and inflammation can all cause the level to dramatically increase as a result of increased intracellular energy demand (Fredholm et al., 2001). An acutely elevated level of extracellular adenosine is considered to mediate anti-inflammatory and protective effects, and for this reason, adenosine has been labelled a ‘retaliatory metabolite' (Newby, 1984). Probably the best known example of this is in the ischaemic hypoxic heart, whereby the increase in extracellular adenosine is cardioprotective by virtue of its vasodilatory and negative inotropic effects (Mubagwa et al., 1996). However, in a similar manner to many biological processes intended to be protective only in the short term, prolonged activity frequently generates detrimental consequences. Thus, with respect to asthma, chronic accumulation of adenosine in the airways is now becoming closely associated with various pathological aspects of the disease (Spicuzza et al., 2006).

Endogenous and exogenous adenosine in asthma

In asthmatic subjects, it has been demonstrated that adenosine levels in bronchoalveolar lavage fluid and exhaled breath condensate are significantly higher than those present in healthy subjects (Driver et al., 1993; Huszar et al., 2002). The study by Driver et al. (1993) estimated the concentration of adenosine in epithelial periciliary fluid to be approximately 60 μM in healthy subjects and 200 μM in asthmatic subjects. Furthermore, it has been observed that plasma adenosine levels rapidly increase following allergen challenge in asthmatic subjects (Mann et al., 1986) and are also increased in both plasma and exhaled breath condensate during exercise-induced bronchoconstriction (Vizi et al., 2002; Csoma et al., 2005).

The effects of exogenous inhaled AMP upon lung function in asthmatic subjects are very well characterized, and in addition some pro-inflammatory consequences of AMP challenge have been reported (AMP is used in preference to adenosine for solubility reasons and is rapidly hydrolysed to adenosine by 5′-ectonucleotidase in the lung). This knowledge therefore provides valuable insight into the significance of endogenous adenosine accumulation in asthma and the reported increase associated with an exacerbation. Thus, by considering what is currently understood about the effects of exogenously administered adenosine in asthmatic subjects, in addition to the experimental effects of adenosine upon cells in vitro and in animal models of allergic airway inflammation, we can begin to delineate the contribution of the endogenous accumulation of adenosine to the pathology of asthma.

Bronchoconstrictive effects of exogenous adenosine in asthmatic subjects

Firstly, the inhalation of AMP elicits a concentration-dependent bronchoconstriction. The first study to demonstrate this also showed that the chemically similar nucleoside guanosine had no effect (Cushley et al., 1983), nor did the deaminated adenosine metabolite inosine (Mann et al., 1986), suggesting that this was a specific adenosine receptor-mediated effect. The fall in forced expiratory volume in one second (FEV₁) is similar in duration (but somewhat less potent in molar terms) to that observed with methacholine and histamine, which all induce bronchoconstriction significantly faster than that which occurs following allergen challenge. As the response to AMP correlates more closely with the airway inflammation than methacholine or histamine challenge, AMP challenge is increasingly used as a non-invasive tool to assess disease severity and the response to anti-inflammatory therapy (Spicuzza and Polosa, 2003). Although inhalation of AMP does not generate a ‘late response' as observed with allergen challenge, it has been suggested that adenosine induces mast cell degranulation via activation of the adenosine A_2B receptor subtype, rather than causing bronchoconstriction by acting directly on the airway smooth muscle (reviewed by Holgate, 2005). For this reason, adenosine has been classed as an ‘indirect bronchoprovocant'. However, the mast cell/A_2B receptor theory has not been unequivocally proven due to the technical difficulties in obtaining a pure population of asthmatic airway mast cells, and the conclusions have thus been drawn from largely circumstantial evidence, which is therefore subject to alternative interpretations.

Nevertheless, the strongest evidence that has led to the conclusion of mast cell degranulation occurring in response to administration of adenosine was that obtained from pharmacological modulation of the asthmatic response to inhaled AMP. Pre-treatment with inhibitors of various bronchoconstrictive mast cell products including second-generation selective histamine H₁ receptor antagonists (Phillips et al., 1987; Rafferty et al., 1987a, 1987b, Phillips and Holgate, 1989), cysteinyl leukotriene receptor antagonists (Rorke et al., 2002) and cyclooxygenase inhibitors (Crimi et al., 1989; Phillips and Holgate, 1989), all attenuated AMP-induced bronchoconstriction, as determined by an increased AMP PC₂₀ (provocative concentration required to reduce baseline forced expiratory volume in one second by 20%) value in the presence of these drugs. In addition, pre-treatment with the so-called ‘mast cell stabilizers' nedocromil sodium and disodium cromoglycate also inhibited AMP-induced bronchoconstriction (Crimi et al., 1988; Phillips et al., 1989; Summers et al., 1990). In support of these studies, Polosa et al. (1995) demonstrated the release of mast cell-derived mediators following endobronchial adenosine challenge in asthmatic subjects.

However, these observations can be interpreted in several other ways. Firstly, adenosine receptors are ubiquitously expressed and, given the inflammatory milieu in asthmatic airways, it is possible that adenosine is stimulating the release of inflammatory mediators from other cells, which then promote degranulation of tissue mast cells. Furthermore, the absence of a ‘late response' suggests that the mast cells are not degranulating in the same manner as that which occurs following allergen challenge, and also mast cells are not an exclusive cellular source of histamine, leukotrienes and arachidonic acid derivatives. Secondly, Bjorck et al. (1992) investigated the effects of adenosine on isolated human bronchial strips and demonstrated a variable but overall small contractile response in tissue from healthy subjects, which was significantly increased in tissue from asthmatic subjects closely reflecting the clinical situation. The adenosine-induced contraction observed with tissue from asthmatic subjects was significantly inhibited by the A₁ receptor antagonist 2-thio-((1,3-dipropyl)-8-cyclopentyl)-xanthine, suggesting it was A₁ receptor mediated, an adenosine receptor subtype that mast cells are not considered to express, and therefore the contractile response in tissue from subjects with asthma did not involve the A_2B receptor. However, the same study also showed that the response could be attenuated significantly by pre-incubation with an H₁ receptor antagonist and leukotriene synthesis inhibitor. A subsequent investigation by Ellis and Undem (1994) demonstrated that isolated human bronchial tissue has a high degree of intrinsic tone and that histamine and leukotrienes constitute a major part of this basal tone, inhibition of which potently relaxes the tissue, suggesting further that the results of Bjorck were probably not mediated via mast cell degranulation. Furthermore, it has been suggested that histamine and leukotrienes contribute to bronchial tone in subjects with asthma, as it is recognized that a single-dose administration of both H₁ receptor antagonists and leukotriene receptor antagonists can increase baseline FEV₁ (Reiss et al., 1997; Dekhuijzen and Koopmans, 2002; Nelson, 2003). Thus, it is difficult to ascertain precisely up to what degree the inhibition of adenosine-induced contractile responses by the H₁ receptor antagonist and leukotriene synthesis inhibitor in both the in vitro and clinical setting can be attributed to the direct inhibition of mast cell products released by adenosine or to an altered intrinsic tone. The possibility that inhibiting mast cell products in asthmatic subjects increases the PC₂₀ to inhaled AMP partly through physiological antagonism should therefore be considered. Although the studies described above investigating pharmacological modulation of the AMP response did not observe significant changes in PC₂₀ values to the other bronchoprovocants concurrently tested (with the exception of the corresponding agonist), it is possible that the bronchoconstrictive effects of AMP could be significantly more sensitive to the presence of pharmacological agents acting as a physiological antagonist as demonstrated with β₂ agonists (Phillips et al., 1990; Nightingale et al., 1999). In support of this, the first-generation H₁ receptor antagonist chlorpromazine had no effect upon AMP challenge (Crimi et al., 1986), and the direct bronchodilatory effect of a single dose of first-generation H₁ receptor antagonists was subsequently demonstrated to be less efficacious than that of second-generation H₁ receptor antagonists (Wood-Baker and Holgate, 1993). Furthermore, second-generation H₁ receptor antagonists have been reported to also possess anti-inflammatory effects unrelated to histamine inhibition (Lever et al., 2007; reviewed by Walsh, 2002), which may also affect the response to AMP. Likewise, a number of non-mast cell effects of nedocromil sodium and disodium cromoglycate have also been described, particularly effects on airway sensory nerves (Barnes, 2006).

A very recent study (Liesker et al., 2007) should also be taken into consideration, as this does not support the involvement of mast cell degranulation in response to AMP challenge. This study attempted to compare mast cell density within smooth muscle regions in bronchial biopsy material obtained from healthy, asthmatic and chronic obstructive pulmonary disease (COPD) subjects and to investigate whether the number of mast cells correlated with AMP responsiveness. No differences in mast cell numbers were observed between the three subject groups, leading the authors to conclude that mast cells are not important in the bronchoconstrictor response to AMP.

Nevertheless, it was suggested that bronchoconstriction to adenosine was mediated through the A_2B receptor subtype (reviewed by Holgate, 2005), as the methylxanthine enprofylline, which has a weak but selective antagonistic effect at A_2B receptors, attenuated the effects of AMP challenge (Clarke et al., 1989). However, at the same dose, enprofylline also inhibited the response to histamine challenge to a very similar extent, suggesting a shared inhibitory mechanism, most likely physiological antagonism resulting from phosphodiesterase enzyme inhibition. Interestingly, in the same study, it was demonstrated that theophylline, which is an antagonist at all adenosine receptor subtypes, inhibited the response to histamine in a similar manner to enprofylline, yet was significantly more potent at inhibiting the effects of AMP than histamine, suggesting the presence of an additional inhibitory mechanism. It can be concluded from this study, therefore, that antagonism of adenosine receptors other than the A_2B receptor was more efficacious at inhibiting the AMP-induced bronchoconstriction. Furthermore, a very recent study demonstrated a total lack of effect of a selective dual A_2B/A₃ receptor antagonist upon AMP PC₂₀ in asthmatic subjects (Pascoe et al., 2007), suggesting that A_2B or indeed the A₃ receptors are not involved in this response.

The in vitro evidence implicating mast cell degranulation in response to adenosine in addition to suggesting that the A_2B receptor mediates this effect is also ambiguous. The most convincing evidence was reported by Forsythe et al. (1999), who demonstrated the release of histamine from human bronchoalveolar lavage mast cells following treatment with adenosine. The caveat to this study is that the mast cells were not isolated from the other bronchoalveolar lavage cells, and so it is possible that adenosine was inducing inflammatory mediator release from different cells, which then promoted mast cell degranulation. An earlier study with primary human mast cells demonstrated that adenosine can potentiate mast cell degranulation induced by other stimuli such as IgE and calcium ionophore, but was not able to act alone (Peachell et al., 1988) and indeed was also shown to inhibit degranulation (Hughes et al., 1984).

Other experimental approaches have focused upon the human mastocytoma-1 (HMC-1) cell line, which was derived from a patient with mast cell leukaemia (Butterfield et al., 1988). In summary, these studies have demonstrated that HMC-1 cells express the A_2A, A_2B and A₃ receptors and that adenosine can stimulate interleukin (IL)-8 secretion (Feoktistov and Biaggioni, 1995), in addition to IL-1β, IL-3, IL-4 and IL-13 secretion (Ryzhov et al., 2004), all via the A_2B receptor. Potentiation of inflammatory mediator secretion was also demonstrated with rat peritoneal mast cells (Marquardt et al., 1978) and murine bone marrow-derived mast cells (Marquardt et al., 1984). Thus, although these studies demonstrate that adenosine can certainly exert pro-inflammatory effects upon mast cells in the presence of other stimuli, most likely through activation of the A_2B receptor, none were able to demonstrate adenosine-induced degranulation per se.

In summary, the evidence to date suggests that it is not possible to definitely conclude that inhaled adenosine induces bronchoconstriction through mast cell degranulation, and alternative mechanisms should not be disregarded. The ability of adenosine to mediate bronchoconstriction through direct effects on airway smooth muscle as well as through neuronal mechanisms are two such other possibilities that ought to be considered.

In support of adenosine possessing direct bronchoconstrictor effects, it has been demonstrated that expression of the adenosine A₁ receptor is significantly elevated on airway smooth muscle obtained from asthmatic subjects in comparison to healthy subjects (Brown et al., 2008). Furthermore, culture of human airway smooth muscle cells in asthmatic serum was shown to upregulate A₁ receptor expression (Hakonarson et al., 1998). An increase in airway A₁ receptor expression was also observed in a rabbit model of allergic airway inflammation when compared to healthy naïve animals (Nyce and Metzger, 1997) and in adenosine-deaminase knockout mice, which have elevated extracellular adenosine levels (Chunn et al., 2001; Zhong et al., 2001). Moreover, the A₁ receptor was upregulated on airway smooth muscle in an allergic rabbit model, and when this expression was inhibited with the use of an antisense oligonucleotide directed against the A₁ receptor, the bronchoconstrictive response to inhaled adenosine was inhibited (Nyce and Metzger, 1997; Abebe and Mustafa, 1998). In addition, this was also demonstrated recently with a selective adenosine A₁ receptor antagonist (Obiefuna et al., 2005). Thus, as the A₁ receptor is G_i-protein coupled and therefore lowers cAMP following activation, it is likely that adenosine can directly contract airway smooth muscle, and in support of this, Ethier and Madison (2006) demonstrated that activation of the A₁ receptor on human airway smooth muscle cells in vitro induced calcium mobilization. The study described earlier by Bjorck et al. (1992) also demonstrated involvement of the A₁ receptor in mediating contraction of asthmatic bronchial tissue in vitro. By considering the possibility that adenosine acts directly on the muscle, the pharmacological modulation of AMP PC₂₀ with histamine and leukotriene receptor antagonists described earlier could be attributed to an inhibition of the ability of histamine and leukotrienes to prime airway smooth muscle and thereby increasing the reactivity of muscle cells to various spasmogens (Lee et al., 1984; Jacques et al., 1991, 1992; Carbajal et al., 2005). Although various animal studies provide support for a role of adenosine A₁ receptors in mediating airway obstruction, it is acknowledged that species and methological differences account for the demonstration of a role for atypical (Hannon et al., 1998) A_2B and A₃ receptors in this response (Fan et al., 2003; Hua et al., 2007). Nevertheless, human data support a role for adenosine A₁ receptors in mediating airway smooth muscle contraction.

Evidence implicating a role for nerves in the asthmatic response to adenosine has also been described. One of the first clinical studies to suggest this resulted from observing the inhibitory effects of ipratropium bromide on AMP challenge, indicating a possible involvement of cholinergic neural pathways (Polosa et al., 1991). Furthermore, investigations into the effects of inhaled loop diuretics frusemide and bumetanide on AMP challenge revealed that they could attenuate the effects of AMP, which is thought to result from modifying sensory nerve activity (O'Connor et al., 1991; Polosa et al., 1993). Polosa et al. (1992) demonstrated that previous repeated challenges with inhaled bradykinin, which depletes neuropeptides from sensory nerve endings, significantly attenuated the response to AMP challenge. However, a subsequent study did not support this observation, as previous treatment of asthmatic subjects with phosphoramidon, a neutral endopeptidase inhibitor, failed to enhance bronchoconstrictor sensitivity to AMP (Polosa et al., 1997), which suggested that certain neuropeptides including neurokinin A and substance P may not play a significant role in the response to AMP. Some in vivo evidence does, however, suggest an involvement of sensory nerves; for example, the inhibition of AMP- and the selective A₁ receptor agonist cyclopentyl adenosine-induced airway obstruction by atropine, capsaicin and bilateral vagotomy in allergic guinea pigs suggests a neuronal-dependent mechanism with the particular involvement of capsaicin-sensitive nerves (Keir et al., 2006). In addition, a very recent study investigating the airway effects of adenosine in four strains of naïve mice, each lacking one of the four adenosine receptors, demonstrated that adenosine induces bronchoconstriction through the A₁ receptor, and that this was likely to be mediated through sensory nerves (Hua et al., 2007).

Pro-inflammatory effects of exogenous adenosine in asthmatic subjects

In addition to exogenous adenosine affecting airway tone, inhalation of AMP in asthmatic subjects has been shown to increase airway eosinophilia within 1 h of AMP challenge (van den Berge et al., 2004), supporting earlier observations in an allergic guinea pig model (Spruntulis and Broadley, 2001), which was also accompanied by an increase in macrophage number and inhibited by an A₃ receptor antagonist. Incidentally, studies investigating adenosine receptors expressed on human eosinophils identified A₁, A_2A and A₃ subtypes, with the A₁ receptor linked to activation of the eosinophil and the A_2A and unexpectedly the A₃ receptor inhibiting eosinophil activation (Ezeamuzie and Philips, 1999, 2003) and chemotaxis (Knight et al., 1997). Indeed, the A₃ was found to be atypically positively coupled to adenylate cyclase, which explained these effects (Ezeamuzie and Philips, 2003). Furthermore, expression of A₃ receptors was upregulated in asthmatic lung tissue, where it was located predominantly to eosinophils (Walker et al., 1997). Thus, although the mechanism by which adenosine can increase airway eosinophilia in asthmatic subjects remains to be determined, these data suggest that adenosine may promote inflammatory leukocyte recruitment to the lung in subjects with asthma.

Given the evidence described above, the effects of exogenously administered adenosine in asthmatic subjects are all associated with recognized features of asthma. It is clear that although the precise mechanisms through which exogenous adenosine is able to induce bronchoconstriction remain to be fully elucidated, it can be speculated that the increase in extracellular adenosine during an asthmatic exacerbation contributes to the bronchoconstriction response to some extent; however, how significant a contribution this may be remains to be established. In addition, the evidence suggests that endogenous adenosine may promote inflammatory cell infiltration. Thus, taken together, these data advocate a role for extracellular endogenous adenosine in the pathophysiology in asthma. In support of this hypothesis, a number of experimental in vitro and in vivo studies provide further evidence advocating a role for adenosine in asthma, and will subsequently be described.

Putative effects of elevated endogenous adenosine in asthmatic subjects

Firstly, mucus hypersecretion is an important feature of asthma and adenosine has been shown to upregulate expression of the mucin gene, MUC2, in human tracheal epithelial cells in vitro through activation of the adenosine A₁ receptor (McNamara et al., 2004). In support of adenosine possessing secretagogue properties, it has been demonstrated that adenosine was able to induce canine tracheal mucus secretion in vivo through an A₁ receptor-dependent mechanism (Johnson and McNee, 1985).

Secondly, neutrophils are considered to play an important role in the pathophysiology of moderate-to-severe asthma (Jatakanon et al., 1999), and through activating the A₁ receptor, adenosine has been shown to promote human neutrophil adherence to endothelial cells (Cronstein et al., 1992) and chemotaxis (Cronstein et al., 1990), upregulate CD11b/CD18 expression (Wollner et al., 1993) and trigger the respiratory burst (Cronstein et al., 1985, 1987) and degranulation (Bouma et al., 1997). In addition, adenosine also promotes monocyte phagocytosis through the A₁ receptor (Salmon et al., 1993) and increases endothelial cell permeability (Wilson and Batra, 2002). Adenosine has been shown to induce IL-6 and MCP-1 secretion from human airway smooth muscle cells (Zhong et al., 2004), IL-19 from human bronchial epithelial cells (Zhong et al., 2006) and IL-6 from fibroblasts (Zhong et al., 2005), all through activation of the A_2B receptor, in addition to inducing the differentiation of fibroblasts into myofibroblasts. It should be noted, however, that many inflammatory cells express adenosine A_2A receptors, and activation of these will lead to suppression of cell functions (reviewed by Bours et al., 2006), as highlighted in Table 2. As inflammatory cells usually express more than one adenosine receptor subtype with varying affinity for adenosine, adenosine is capable of inducing signalling pathways that result in opposing cellular effects directly dependent upon the concentration. The overall effect, therefore, of adenosine in asthmatic airways upon local inflammatory cells remains to be determined.

Further to these studies, an important study unequivocally demonstrating the pathological respiratory effects of an increased extracellular adenosine level has been described in mice with a genetically engineered partial adenosine-deaminase deficiency, which reduces their ability to metabolize adenosine to inosine (Chunn et al., 2001). Consequently, these mice accumulate high concentrations of adenosine extracellularly and exhibit a marked pulmonary inflammation. Some of the pathological features reported in this model were consistent with those observed in asthma such as mucus hypersecretion, but the pulmonary inflammatory cell profile differed considerably and consisted mainly of macrophages, with no increase in eosinophil numbers. However, transcript levels for the A₁, A_2B and A₃ receptors were found to be significantly increased, suggesting that an increase in extracellular adenosine promotes adenosine receptor signalling. Mice totally deficient in adenosine deaminase displayed more severe pulmonary inflammation and died at 3 weeks of age from respiratory distress (Blackburn et al., 2000). Additional inflammatory features observed included pulmonary and peripheral blood eosinophilia, extensive mast cell degranulation and an increase in serum IgE (Zhong et al., 2001). Exogenous administration of adenosine deaminase could both prevent and reverse the lung inflammation and damage observed in these genetically altered animals, suggesting that the increased extracellular adenosine was mediating these effects. Interestingly, concurrent knockout of the adenosine A₁ receptor was reported to exacerbate the inflammation further, suggesting a protective role of the A₁ receptor in this model (Sun et al., 2005). However, the levels of eosinophils in this model were of a magnitude unlikely to result in alterations in airway responsiveness, and therefore the relevance of these findings to asthma should be interpreted with caution (less than 2% of total cells, compared with approximately 40% of total in a murine model of allergic inflammation (Riffo-Vasquez et al., 2004)).

Interestingly, a subset of patients with severe combined immunodeficiency disease is deficient in adenosine deaminase, and consequently plasma adenosine levels are elevated. An increased incidence of various respiratory disorders including asthma have been observed in a significant number of these patients, in addition to features associated with asthma such as peripheral blood eosinophilia and elevated serum IgE (reviewed by Blackburn and Kellems, 2005). Furthermore, subjects with COPD also have an increased extracellular level of adenosine in the airways, which provides further evidence of the association between increased adenosine and pulmonary disease (Sullivan et al., 2005). Inhaled AMP can also induce bronchoconstriction in COPD patients, although not to the same extent as in asthmatic subjects. Interestingly however, it has very recently been demonstrated that subjects with COPD have an increased density of A₁, A_2A and A₃ receptors in the lung parenchyma when compared to age-matched smoking controls (Varani et al., 2006), although the associated functions remain to be determined.

In conclusion, the clinical and experimental evidence described above suggests that the chronic accumulation of extracellular adenosine in asthmatic airways may contribute towards the pathogenesis and/or pathophysiology of asthma. Consequently, adenosine receptors have become attractive therapeutic targets for the treatment of asthma. Furthermore, as many cells express more than one subtype of adenosine receptor, activation of which can have opposing cellular effects, developing selective agonists that induce a specific effect on a particular cell type are also under investigation.

Adenosine receptors as targets for the treatment of asthma

The A₁, A_2A and A_2B receptors are the target of various drugs in development for the treatment of asthma. Pre-clinical evidence supports the rationale for the use of antagonists at the A₁ and A_2B receptors and an agonist at the A_2A receptor. The role of the A₃ receptor in asthma has yet to be clarified. Thus, it remains to be determined whether it is of greater therapeutic benefit to inhibit the effects of extracellular adenosine in asthma with selective antagonists against the A₁ and A_2B receptors, or to exploit the inhibitory cellular events downstream of the A_2A receptor with an A_2A selective agonist.

Adenosine A₁ receptor

The rationale for targeting the adenosine A₁ receptor is based on the evidence described above obtained from in vitro and in vivo studies in addition to clinical findings. In summary, by activating the A₁ receptor, adenosine has been shown to mediate bronchoconstriction, mucin production, promote pro-inflammatory functions of neutrophils and monocytes and increase endothelial cell permeability. Thus, to inhibit adenosine A₁ receptor-mediated pathways, two distinct approaches have been taken.

The first approach was to develop an inhaled antisense oligonucleotide against the A₁ receptor, as the therapeutic efficacy of this approach was demonstrated in the pivotal study by Nyce and Metzger (1997), in a rabbit model of allergic airway inflammation. Antisense oligonucleotides block mRNA and therefore protein expression of the targeted gene. The oligonucleotide used in this study, EPI-2010 (EpiGenesis Pharmaceuticals, New Jersey, USA), was also shown to significantly increase AMP PC₂₀ in a primate model of asthma (Nyce, 1999). The main advantages of inhaled antisense oligonucleotides over small-molecule antagonists are a lower incidence of systemic side effects, which is particularly important given the physiological functions of A₁ receptors in other organs, in addition to a longer half-life (once-weekly dosing) and greater specificity for the desired target. Thus, given the pre-clinical data, the antisense oligonucleotide against A₁ receptor mRNA EPI-2010 was investigated as an inhaled therapy for the treatment of asthma in humans. Although well tolerated in Phase I, the Phase IIa trial demonstrated that treatment with EPI-2010 yielded only modest improvements in asthmatic subjects, which were not deemed sufficient to justify continuing the clinical development of this therapy (reviewed by Ball et al., 2004).

The second approach reverted to more traditional methods and has involved the development of a small-molecule A₁ receptor antagonist, L-97-1, for oral administration (Obiefuna et al., 2005). This has proved successful in pre-clinical models at attenuating both adenosine and allergen-induced bronchoconstriction, in addition to airway inflammation (Nadeem et al., 2006). As it is based very closely upon the methylxanthine bamiphylline, which has been approved for the treatment of respiratory diseases for a number of years, serious adverse effects are not anticipated.

Adenosine A_2A receptor

In comparison to the A₁ receptor, there has been significantly more interest in developing selective A_2A receptor agonists for the treatment of respiratory diseases and other chronic inflammatory conditions. Most inflammatory cells express the A_2A receptor, and being G_s-protein coupled, activation almost always elicits inhibitory effects, as described in Table 2 (reviewed by Lappas et al., 2005). To summarize the observations most relevant to asthma, selective activation of the A_2A receptor inhibits histamine and tryptase release from mast cells (Hughes et al., 1984; Peachell et al., 1991; Suzuki et al., 1998), neutrophil adherence to the endothelium (Cronstein et al., 1992; Felsch et al., 1995), integrin upregulation (Wollner et al., 1993) and the respiratory burst (Fredholm et al., 1996; Hannon et al., 1998) and degranulation (Bouma et al., 1997). In addition, activation of the A_2A receptor inhibits IL-12 and TNF-α release from activated monocytes (Bouma et al., 1994; Zhang et al., 2005) and enhances IL-10 and vascular endothelial growth factor secretion (reviewed by Hasko et al., 2007), in addition to inhibiting IL-12 secretion from mature dendritic cells (Panther et al., 2001). Furthermore, the A_2A receptor has been demonstrated to suppress the activation and expansion of T lymphocytes (Huang et al., 1997). In bronchial epithelial cells, the A_2A receptor stimulated wound healing (Allen-Gipson et al., 2006). In Brown-Norway rats sensitized to ovalbumin, administration of an A_2A agonist CGS-21680 attenuated the airway inflammation induced by ovalbumin challenge with a magnitude similar to that achieved by budesonide (Fozard et al., 2002). Finally, in a murine model of allergic airway inflammation, genetic deletion of the A_2A receptor augmented the airway inflammation and hyper-responsiveness (Nadeem et al., 2007), further supporting the role of the A_2A receptor as mediating anti-inflammatory functions.

Thus, by exerting inhibitory effects on multiple inflammatory cell types, it has been predicted that a selective A_2A receptor agonist may have broad-spectrum anti-inflammatory effects similar to glucocorticosteroids. Several pharmaceutical companies now have A_2A agonists in clinical development for other inflammatory conditions such as COPD, but it is expected that they will be investigated for their efficacy in treating asthma, for example, UK-432097 (Pfizer, Sandwich, UK). A GlaxoSmithKline A_2A agonist reached Phase II for the treatment of asthma (GW328267), but was discontinued, possibly due to a lack of efficacy (Luijk et al., 2003). Whether this was due to the pharmacokinetic profile of the compound, or simply that A_2A agonists are not as beneficial as predicated, remains to be established.

Adenosine A_2B receptor

The therapeutic potential of an antagonist at the A_2B receptor is based largely on the cellular effects downstream of this receptor in mast cells, as described earlier, which enhance the release of inflammatory mediators in addition to pro-inflammatory effects on airway smooth muscle cells, epithelial cells and fibroblasts. Data obtained from various animal models of allergic airway inflammation suggest that inhibition of A_2B-mediated effects may be of therapeutic benefit in asthma (Fozard et al., 2003). A very recent study using a murine model by Mustafa et al. (2007) demonstrated that the selective A_2B receptor antagonist CVT-6883 (CV Therapeutics, Palo Alto, USA) attenuated the bronchoconstriction induced by inhaled AMP and allergen challenge in addition to the inflammatory cell infiltration.

Thus, CVT-6883 along with various other compounds, for example, CGS15493 (Novartis, Basel, Switzerland), WO-00125210 (Bayer Shering Pharma), IPDX (Vanderbilt University, Nashville, USA), ATL-907 (Adenosine Therapeutics, Charlottesville, USA), are being investigated for the treatment of asthma, some of which are now in Phase I and Phase II trials. A dual A_2B/A₃ antagonist QAF805 (Novartis, Basel, Switzerland) recently reached Phase II clinical trial, although it demonstrated no effect on AMP PC₂₀ in mild asthmatic subjects (Pascoe et al., 2007).

Adenosine A₃ receptor

The role of the A₃ receptor in the human lung and indeed in asthma remains to be clarified. What is clear, however, is that the expression of the A₃ receptor in asthmatic airways is predominantly located in eosinophils, and in vitro studies have demonstrated that the A₃ receptor mediates inhibition of eosinophil chemotaxis and activation. Thus, the elevated level of adenosine in asthmatic airways may be protective through activating the A₃ receptor. Enhancing the effects, therefore, of endogenous adenosine with a selective A₃ receptor agonist may be beneficial in the treatment of asthma. Conversely, it has been suggested that an A₃ receptor antagonist may be useful in the treatment of asthma, as data obtained from animal models demonstrate that the A₃ receptor can mediate mast cell degranulation, bronchoconstriction, mucus secretion and eosinophilia (Ramkumar et al., 1993; Fan et al., 2003; Tilley et al., 2003; Young et al., 2004). However, these conflicting data only serve to highlight the necessity of further investigations concerning the role of the A₃ receptor in asthma, before considering the therapeutic potential of targeting this receptor in the treatment of asthma although with an agonist or antagonist.

Conclusion

Asthma remains a disease with unmet therapeutic needs. The evidence to date strongly suggests that adenosine contributes to the pathophysiology of asthma, although the precise extent remains to be determined. Pre-clinical evidence, however, certainly warrants the current investigations into the adenosine A₁ and A_2B receptor subtypes as novel therapeutic targets for the treatment of asthma. In addition, stimulating A_2A receptor-mediated cellular pathways remains an exciting prospect, given the potential broad-spectrum activity such a drug may yield. Furthermore, the combination of both therapeutic approaches may provide an even better way of modifying adenosine receptors as a treatment for asthma.

Glossary

COPD

chronic obstructive pulmonary disease

HMC

human mastocytoma

IL

interleukin

PC

provocative concentration