The Quintiles Prize Lecture 2004: The identification of the adenosine A_2B receptor as a novel therapeutic target in asthma

Abstract

Adenosine is a powerful bronchoconstrictor of asthmatic, but not normal, airways. In vitro studies on isolated human mast cells and basophils revealed that adenosine and selective analogues augmented inflammatory mediator release from mast cells by stimulating A₂ receptors. Pharmacological blockade of mast cell mediator release in vivo also attenuated adenosine-induced bronchoconstriction, as did theophylline, by adenosine A₂ receptor antagonism. Further in vitro studies revealed that the asthmatic response to adenosine is likely to be mediated via the A_2B subtype which is selectively antagonised by enprofylline. Studies in animal models, especially mice, have shown a close synergistic interaction between adenosine, Th2 and airway remodelling responses. The recent description of A_2B receptors on human airway smooth muscle cells that mediate cytokine and chemokine release and induce differentiation of fibroblasts into myofibroblasts strengthens the view that adenosine maybe more than an inflammatory mediator in asthma but also participates in airway wall remodelling in this disease. These data have provided a firm basis for developing adenosine A_2B receptor antagonists as a new therapeutic approach to this disease.

Early observations

Adenosine is a purine nucleoside that plays a key role in nucleic acid, energy and protein metabolism. As an extracellular autacoid generated by 5′-nucleoside cleavage of adenosine 5′-monophosphate, it is a powerful mediator acting through specific cell surface purinoreceptors. In 1978, while working as a post-doctoral research fellow in Dr Frank Austen's laboratory at Harvard University, I showed that adenosine and related synthetic analogues were potent agents in augmenting IgE-dependent mediator release from isolated rodent mast cells (Holgate et al., 1980). On returning to the UK in 1980, I set about exploring whether adenosine had any role as a mediator of asthma. In 1983, Michael Cushley, a clinical research fellow, demonstrated that inhaled adenosine (but not its metabolite inosine or the unrelated nucleoside guanosine) was a powerful bronchoconstrictor of asthmatic but, importantly, not of normal airways (Cushley et al., 1983b). Further work showed that both allergic and nonallergic asthmatics responded in a similar way and that the effect was also seen with adenosine 5′-monophosphate (AMP) and adenosine 5′-diphosphate (ADP) (Mann et al., 1986b). A detectable, but lesser response of the lower airways was also observed in patients with allergic rhinitis (Phillips et al., 1990). However, when adenosine was injected intradermally into atopic skin, the vasodilator and small wheal response was no different from that observed in nonatopic skin (Djukanovic et al., 1989). Since asthma accompanies rhinitis in ∼80% of patients, the intermediate airway response observed with adenosine challenge in allergic rhinitis was most likely due to concomitant mild asthma (Djukanovic et al., 1992; Doull et al., 1996), but could not be explained by a generalised increased responsiveness of epithelial surfaces in atopic subjects. As AMP was more soluble in an aqueous solvent than adenosine, most of the future inhalation challenge work was conducted using this nucleotide.

These preliminary observations led to the hypothesis that ‘adenosine, which accumulates in inflamed mucosa under conditions of cell stress and hypoxia, contributes as a mediator of bronchoconstriction in both acute and chronic asthma'. To pursue this, we first demonstrated that following inhalation allergen challenge of sensitised asthmatic subjects adenosine was released into the circulation (Mann et al., 1986a) and locally into the airways (Polosa et al., 1995). Adenosine was also shown to be released from antigen-challenged human lung fragments in vitro in the presence of inhibitors of adenosine deaminase and adenosine kinase (Konnaris & Lloyd, 1996). Blockade of adenosine re-uptake by dipyridamole increased the bronchoconstrictor response to inhaled AMP, indicating that accumulation of extracellular adenosine was closely associated with the asthmatic airway response (Cushley et al., 1985). The ability of dipyridamole to enhance another adenosine-mediated effect was later shown in humans on the hypercapnic ventilatory response, thereby confirming its mode of action in vivo of increasing extracellular adenosine levels (Griffiths et al., 1990; 1997). In vitro studies confirmed that adenosine and A₂ receptor analogues (e.g. 5′-N-ethylcarboxamidoadenosine (NECA)) could augment IgE-dependent mediator release from both human mast cells and basophils (Church et al., 1983; Hughes et al., 1983; 1984) and that activated leukocytes were a major source of extracellular adenosine (Mann et al., 1986c). Adenosine also releases histamine directly from human bronchoalveolar lavage mast cells (Forsythe et al., 1999).

Mechanism(s) of adenosine-induced bronchoconstriction

The possibility that adenosine caused bronchoconstriction in asthma indirectly via mast cell activation as suggested by our early in vitro studies was pursued in several ways. Firstly, AMP provocation of asthmatic airways in vivo was accompanied by a rise in circulating histamine levels (Phillips et al., 1990). Secondly, the immediate bronchoconstriction provoked by inhaled AMP was shown to be antagonised by inhibiting the effects of individual mast cell mediators using selective histamine H₁ antagonists (e.g. terfenadine, astemizole) (Holgate, 1987; Phillips et al., 1987; Rafferty et al., 1987a, 1987b; Phillips & Holgate, 1989), cysteinyl leukotriene receptor 1 (cystLT₁) antagonists (e.g. montelukast) (Rorke et al., 2002) and inhibition of cyclooxygenase 1 and 2 (e.g. flurbiprofen and indomethacin) (Crimi et al., 1989; Phillips & Holgate, 1989). Inhibition of cyclooxygenase activity ablates the production of the powerful bronchoconstrictor mediator prostaglandin (PG)D₂ from activated mast cells. Secondly, the mast cell stabilising drugs sodium cromoglicate (Richards et al., 1988; Phillips et al., 1989b; Richards et al., 1989), nedocromil sodium (Phillips et al., 1989b; Richards et al., 1989; Summers et al., 1990; Church & Holgate, 1993) and more recently andolast (Persiani et al., 2001) were shown to be powerful inhibitors of AMP-induced bronchoconstriction in asthma. Thirdly, when administered by inhalation, the loop diuretics frusemide and bumetanide also inhibited adenosine-provoked bronchoconstriction through their known inhibitory effects in ion channels on mast cells to reduce their threshold of activation and mediator secretion (Polosa et al., 1993a; Rajakulasingam et al., 1994; Bradding et al., 2003; Duffy et al., 2004). Heparin, a highly sulphated unbranched glycosaminoglycan, when given by inhalation protects against bronchoconstriction provoked by allergen and exercise (Ahmed et al., 1993; Bowler et al., 1993) is also inhibitory against AMP challenge of the lower (Polosa et al., 1997) and upper (Zang et al., 2004) airways, again through suppression of mast cell mediator release.

In allergic asthmatics, AMP-induced bronchoconstriction with inhaled AMP was more rapid in onset than that observed with inhaled allergen, indicating that airway narrowing was the consequence of rapid mast cell degranulation with release of histamine and generation of newly formed eicosanoids – PGD₂ and LTC₄ (Cushley & Holgate, 1985; Phillips & Holgate, 1988a) rather than the additional induction of newly formed cytokines and chemokines that are considered to underpin the late phase allergen response (Phillips & Holgate, 1988b). The absence of a late-phase response with inhaled AMP provocation highlighted a fundamental difference in the way that adenosine and allergen interacted with airway mast cells for mediator secretion (Holgate et al., 1987; Church & Holgate, 1988; Holgate et al., 1988). Blockade of muscarinic cholinergic receptors using inhaled ipratropium bromide had only minimal effect in antagonising bronchoconstriction provoked by AMP, leading to the conclusion that cholinergic reflexes were of limited importance in mediating bronchoconstriction (Mann et al., 1985; Polosa et al., 1991). By contrast, inhaled β₂-agonists such as salbutamol had a powerful inhibitory effect on AMP-induced bronchoconstriction by serving as a functional antagonist and as a direct inhibitor of human mast cell activation–secretion coupling (Phillips et al., 1990a).

Unusual features of adenosine-induced bronchoconstriction

Several interesting features about the pro-asthmatic effect of adenosine have emerged. Repeated provocation of asthmatic airways with inhaled AMP led to the development of tolerance, which took 6–8 h to recover (Daxun et al., 1989). Of significance was the further finding that, while in this refractory state, the airways were hyperresponsive to allergen inhalation, suggesting that prior adenosine exposure had produced mast cell priming as we had previously demonstrated in vitro (Holgate et al., 1980; Church et al., 1983; Hughes et al., 1983; 1984; Phillips et al., 1989a). Bronchoconstriction provoked by AMP also rendered the airways refractory to exercise and inhaled bradykinin and vice versa, but not to methacholine challenge, suggesting that the former stimuli operated through a common mast cell-mediated mechanism (Finnerty et al., 1990; Polosa et al., 1992). It has long been known that exercise-induced asthma is a mast cell-dependent phenomena (Finnerty & Holgate, 1990; 1993; Roach et al., 1998), but cross-tolerance between AMP and bradykinin was less easy to explain. We had shown that bradykinin caused bronchoconstriction through activation of bradykinin B2 receptors (Polosa & Holgate, 1990) and that repeated challenge with this peptide also rapidly led to the development of tolerance (Polosa et al., 1993b; Rajakulasingam et al., 1993). Both bradykinin B2 and adenosine receptors have been identified on mast cells (Reissmann et al., 2000; Sylvin et al., 2001) and also on peptidergic nerves (Fox et al., 1996; Chung, 2002), raising the possibility that adenosine and bradykinin share some common activation pathways possibly through the release of neuropeptides such as substance P or other neurokinins, which are known to activate mast cells for mediator secretion (Rajakulasingam et al., 1994).

Adenosine receptors mediate the proasthmatic response

Early work on both rodent and human mast cells demonstrated that adenosine was a powerful stimulator of mast cell and basophil adenylate cyclase to increase cellular levels of cyclic 3′5′-AMP operating through the A₂ subtype of purinoceptor (Holgate et al., 1980; Hughes et al., 1983; 1984; Church & Holgate, 1993). Shortly after describing the bronchoconstrictor activity of adenosine, we demonstrated that both inhaled (Cushley et al., 1983a; 1984; Holgate et al., 1984) and oral theophylline were able to selectively antagonise AMP-induced bronchoconstriction beyond their ability to act as functional antagonists (Mann & Holgate, 1985; Church et al., 1986). The fact that this occurred at drug concentrations one order of magnitude lower than that required to inhibit cyclic AMP phosphodiesterase and in the same range as therapeutic plasma concentrations of theophylline (Holgate et al., 1987) opened up the possibility that the known antiasthmatic effect of methylxanthines could, in part, be due to adenosine antagonism. This view was challenged when enprofylline became available because it was shown that this drug was a powerful inhibitor of cyclic AMP phosphodiesterase but, different from theophylline, was devoid of A₂ receoptor antagonism that had been linked to the diuretic and cardiac arrhythmic properties of xanthines (Lunell et al., 1983; Persson et al., 1986). Thus, in the early 1990s, the idea that adenosine was an important mediator of asthma was being seriously eroded.

However, based on current in vitro pharmacology available at the time, it had been assumed that adenosine was active through a single A₂ receptor linked to adenylate cyclase and that was quite distinct from the other purinergic receptors that responded more selectively to ATP and UTP (e.g. P2Yand P2X). However, a paradox that could not be explained was how an agent which increased cyclic AMP within mast cells and basophils could augment rather than inhibit mediator release, as would be expected since increases in cyclic 3′5′-AMP produced by other agonists, for example, with β₂-adrenoceptor agonists (Okayama & Church, 1992) or PG E₂ (Peters et al., 1982) were strongly inhibitory for mediator release. Further clarity came with the discovery that adenosine A₂ receptors existed as two subtypes – A_2A linked to adenylate cyclase and involving G_s coupling, and A_2B linked to both adenylate cyclase and the phosphatidyl trisphosphate (PI₃)-calcium signalling pathway involving both G_s and G_q coupling (Feoktistov & Biaggioni, 1995; Feoktistov et al., 1998). Thus, while exhibiting no antagonist properties against adenosine A_2A receptors, enprofylline was shown to be a highly selective, albeit weak, antagonist of A_2B receptors (Feoktistov & Biaggioni, 1995; Kim et al., 2002; Fan et al., 2003). This critical observation helped explain our finding of a preferential inhibitory effect of intravenous emprofylline on AMP-induced bronchoconstriction (Clarke et al., 1989). The identification of the A_2B receptor subtype revitalised interest in adenosine as a mediator of asthma and becoming a new therapeutic target for this disease (Feoktistov et al., 1998). Although most of the work identifying A_2B receptors on human mast cells was conducted on the HMC-1 mastocytosis derived cell line, recently A_2B receptors mediating enhanced mediator release have also been found on mast cells dispersed from human lung tissue (Zhong H, personal communication). In addition to causing mast cell mediator release, activation of A_2B receptors on HMC-1 cells cultured with human B cells results in Ig isotype, switching to IgE involving costimulation utilising CD40 and enhanced IL-4 and IL-13 secretion (Ryzhov et al., 2004).

With the identification of this new subclass of A₂ receptors, the ease with which repeated exposure to adenosine (and AMP) results in tolerance and cross-tolerance became of the target of further study. The A_2B receptor appears to be regulated differently from many other G-protein-coupled receptors. Mundell and co-workers have shown that agonist activation of A_2B receptors results in arrestin-dependent internalisation of the receptor complex with antisense neutralisation of arrestin, resulting in loss of desensitisation (Mundell et al., 2000; Matharu et al., 2001). Recent work has shown that human A_2B receptors associate with intracellular signalling proteins other than G proteins such as those containing PDZ (PSD-95, Dig 20-1) domains, and more specifically with the PDZ domain-containing protein E3KARP (Sitaraman et al., 2002). This is known to interact with ezrin/radixin/moesin (ERM) proteins which in turn interact with the actin cytoskeleton that control A_2B receptor trafficking. This molecular-based work provides a good explanation for the ease with which A_2B receptor stimulation results in rapid and profound tachyphylaxis, and also for cross-desensitisation between A_2B and other G-protein-coupled receptors (Sitaraman et al., 2000).

The first observation that inhaled corticosteroids were highly active in rapidly suppressing AMP-induced bronchoconstriction (Doull et al., 1997; Holgate et al., 2000) and the recent demonstration that AMP challenge induces eosinophil influx into the airways (van den Berge et al., 2004) further strengthened interest of the role of A_2B receptor in asthma. The rapidity with which this occurs (Wilson et al., 2003) suggests that a unique effect of corticosteroids on the A_2B receptor internalisation mechanisms possibly involving the recently described rapid steroid response receptor (Long et al., 2005).

Observation on the role of adenosine in animal models

Adenosine receptors are also involved in mediating bronchoconstriction in a number of animal models, but between animal species there is heterogeneity of the receptors involved. In the rabbit the airway response is mediated through A₁ receptors (Nyce & Metzger, 1997), in the rat by A₁, A_2B and A₃ receptors (Pauwels & Van der Straeten, 1987) or an atyptical adenosine receptor (Hannon et al., 2002), in the guinea-pig by A₃ receptors (Thorne et al., 1996) and in the mouse by A_2B and A₃ receptors (Fan et al., 2003). It has further been shown that adenosine deaminase (ADA)-deficient mice develop progressive lung inflammation which can be effectively reversed by adenosine deaminase therapy and markedly reduced by treatment with selective adenosine A_2B receptor antagonists (Chunn et al., 2001). Using mice lacking the A_2A receptor and, therefore, the adenylate cyclase signal associated with its activation (Ohta & Sitkovsky, 2001), a key role for endogenously generated adenosine in providing a regulatory feedback mechanism capable of limiting or terminating inflammatory responses has been shown. In a rat ‘model' of allergic asthma, the A_2A agonist CGS 21680 exhibits anti-inflammatory activity similar to that of the corticosteroid, budesonide (Fozard et al., 2002). Most recently, Sun et al. (2005) have shown that the A₁ receptor plays an anti-inflammatory role in the pulmonary phenotype seen in ADA-deficient mice. GlaxoSmithKline are also investigating an inhaled A_2A agonist GW328267X in both asthma and chronic obstructive pulmonary disease (Luijk et al., 2003), but this has recently been dropped from development due to cardiovascular side effects. On the proinflammatory side, the important influence that adenosine has over asthma pathogenesis has recently received additional support from the observation that, in dual transgenic mice, adenosine and the pro-inflammatory and pro-remodelling cytokine interluekin-13 interact synergistically (Blackburn et al., 2003). Since there is now good evidence to support the involvement of A_2B receptor in mast cell activation, promising antagonists for this receptor are being developed, such as IPDX (Feoktistov et al., 2001), 8-SPT, XAC, CGS15493 (Fozard et al., 2003) and CVT 6883. Some of these are now entering clinical trial in the long-term treatment of asthma (Fozard, 2003; Wolber & Fozard, 2005).

Adenosine bronchoprovocation as a diagnostic test

A second development from the adenosine research describes in this brief review is the use of adenosine (or AMP) inhalation challenge as a diagnostic test for asthma where its specificity and sensitivity appear to be superior to that of inhaled histamine and methacholine (Holgate, 2002a; Polosa et al., 2002; Joos et al., 2003). When compared to agents that produce bronchconstriction directly such as methacholine, airway responsiveness to AMP also seems to be more closely associated with airway inflammation (Van den Berge et al., 2001). In addition, AMP responsiveness is also used as a test for distinguishing asthma from COPD (Spicuzza et al., 2003). Since the airway response to AMP is so sensitive to the effect of inhaled corticosteroids and also is a good marker of disease activity, AMP bronchoprovocation has been suggested as useful as a biomarker to assess disease control (Lee et al., 2003; Prieto et al., 2003).

Concluding comments

Over a span of 20 years, the initial observation of the unique pro-asthmatic effects of inhaled adenosine has evolved to provide the basis for a new asthma therapy as well as a possible diagnostic test (Holgate, 2002b; Rorke & Holgate, 2002). Recently, Inbe et al. (2004) has described a second novel receptor P2Y-15 for adenosine and AMP on human mast cells, but this has recently been challenged (Abbracchio et al., 2005). The recent discovery that A_2B receptors are also functionally active on human airway smooth muscle cells to enhance cytokine and chemokine release (Zhong et al., 2004a, 2004b) and on lung fibroblasts where they promote differentiation to a myofibroblast phenotype (Zhong et al., 2004b) adds to the view that this receptor may be involved in airway wall remodelling as well as in inflammation in asthma. The next 5 years will be critical in determining whether targeting the A_2B receptor will translate into clinical efficacy for patients with chronic asthma.

Abbreviations

3′5′-AMP

3′5′-cyclic adenosine monophosphate

AMP

adenosine 5′-monophosphate

ATP

adenosine triphosphate

IPDX

3-isobutyl-8-pyrrolidinoxanlthine

LTC₄

leukotriene C₄

NECA

5′-N-ethylcarboximidoadenosine

PGD₂

prostaglandin D₂

UTP

uridine triphosphate