Adenosine in the airways: Implications and applications

European Journal of Pharmacology 533 (2006) 77 – 88 www.elsevier.com/locate/ejphar

Review

Adenosine in the airways: Implications and applications Lucia Spicuzza ⁎, Giuseppe Di Maria, Riccardo Polosa Dipartimento di Medicina Interna e Medicina Specialistica Sezione Malattie Respiratorie-Università di Catania, Italy Accepted 13 December 2005 Available online 3 February 2006

Abstract Adenosine in a signaling nucleoside eliciting many physiological responses. Elevated levels of adenosine have been found in bronchoalveolar lavage, blood and exhaled breath condensate of patients with asthma a condition characterized by chronic airway inflammation. In addition, inhaled adenosine-5′-monophosphate induces bronchoconstriction in asthmatics but not in normal subjects. Studies on animals and humans have shown that bronchoconstriction is most likely due to the release of inflammatory mediators from mast cells. However a number of evidences suggest that adenosine modulates the function of many other cells involved in airway inflammation such as neutrophils, eosinophils, lymphocytes and macrophages. Although this clear pro-inflammatory role in the airways, adenosine may activate also protective mechanisms particularly against lung injury. For many years this dual role of adenosine in the respiratory system has represented an enigma, and only recently it has become clear that biological functions of adenosine are mediated by four distinct subtypes of receptors (A1, A2A, A2B, and A3) and that biological responses are determined by the different pattern of receptors distribution in specific cells. Therefore, pharmacological modulation of adenosine receptors, particularly A2B, may represent a novel therapeutic approach for inflammatory diseases. Moreover, as bronchial response to adenosine strictly reflects airway inflammation in asthma, bronchial challenge with adenosine is considered a valuable clinical tool to monitor airway inflammation, to follow the response to anti-inflammatory treatments and to help in the diagnostic discrimination between asthma and chronic obstructive lung disease. © 2005 Published by Elsevier B.V. Keywords: Adenosine; Asthma; AMP; Mast cells; Airway inflammation

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adenosine endogenous production and metabolic pathway . . . . . . . Adenosine receptors in the airways. . . . . . . . . . . . . . . . . . . A1 adenosine receptor . . . . . . . . . . . . . . . . . . . . . . . . . A2A adenosine receptors . . . . . . . . . . . . . . . . . . . . . . . . A2B adenosine receptor . . . . . . . . . . . . . . . . . . . . . . . . . A3 adenosine receptor . . . . . . . . . . . . . . . . . . . . . . . . . Should we target adenosine receptors to treat airway inflammation? . . Bronchial response to exogenous adenosine . . . . . . . . . . . . . . Mechanisms of bronchoconstriction . . . . . . . . . . . . . . . . . . 10.1. Mast cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2. Neural mechanisms. . . . . . . . . . . . . . . . . . . . . . . Adenosine and airway inflammation . . . . . . . . . . . . . . . . . . Bronchial challenge with adenosine is a useful clinical tool in asthma.

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⁎ Corresponding author. Dipartimento di Medicina Interna e Medicina Specialistica Sezione Malattie Respiratorie, Via Passo Gravina 187 95125 Catania, Italy. E-mail address: [email protected] (L. Spicuzza). 0014-2999/$ - see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.ejphar.2005.12.056

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13. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Adenosine is an ubiquitous purine nucleoside, playing a pivotal role in many biological processes such as energy generation and proteins metabolism. In the last two decades it has become clear that adenosine is a pro-inflammatory mediator involved in the pathogenesis of asthma and other lung inflammatory disorders. This notion is based on the observation that adenosine induces bronchoconstriction in animal models and in patients with inflammatory airway diseases such as asthma and chronic obstructive pulmonary diseases (COPD) and on the fact that adenosine receptors are present in many cell types involved in airway inflammation (Spicuzza et al., 2003). It is now clear that mediators release from immunologically primed mast cells is the main mechanism responsible for the bronchoconstriction that follows adenosine inhalation although, there is some evidence for neural pathways activation (Polosa et al., 2002). The link between adenosine and airway inflammation is definitively confirmed by the increased levels of adenosine found in biological fluids, such as bronchoalveolar lavage and exhaled breath condensate of patients with asthma (Driver et al., 1993; Huszar et al., 2002). Biological responses to adenosine are mediated by four distinct G-coupled receptors (A1, A2A, A2B, A3) and stimulation of each of these receptors induces a distinct functional response (Polosa et al., 2002). Although the bio-availability of adenosine is an important determinant of its biological functions, the pattern of expression and distribution of these receptors in the anatomical– structural sites of the respiratory system and in immune/ inflammatory cells, accounts for the observation that adenosine may exert either deleterious or protective roles in the lung. In mammalians adenosine can be released as the result of hypoxia, lung injury and chronic inflammation (Blackburn et al., 2003). On the other side, some anti-inflammatory effects of adenosine and protection of tissues have been described in lung (Linden, 2001), heart (Lasley et al., 1990) and brain (Rudolphi et al., 1992). This review will focus on the biological role of adenosine in the respiratory system and on the possibility to modulate airway inflammatory processes by targeting specific adenosine receptors. 2. Adenosine endogenous production and metabolic pathway Adenosine is a nucleoside consisting of the purine base adenine in glycosidic linkage with ribose. Endogenous adenosine derives from dephosphorylation of the nucleotide adenosinemonophosphate (AMP) to adenosine by the enzyme ecto5′-nucleotidase (ectonucleotidases family) known to be present in the cell membrane. The ectonucleotidases include ectonucleoside triphosphate diphosphohydrolases, ectonucleotide pyrophosphatase/phosphodiesterases, alkaline phosphatases and 5′-nucleotidases (Zimmermann et al., 2002). In physiological conditions most adenosine is derived from intracellular AMP, as this diffuses down its concentration gradient out of the cell and thereby encounters the cell membrane ecto-5′-nucleotidase.

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Intracellular AMP is derived from the cleavage of adenosine diphosphate (ADP) and adenosine triphosphate (ATP) during the cycle of energy generation and therefore dephosphorylation of AMP to adenosine is considered the last step of the enzymatic chain (Fredholm et al., 2001). This metabolic pathway lasts few hundred milliseconds and dephosphorylation of AMP to adenosine seems to be the rate-limiting step (Dunwiddie et al., 1997). During hypoxia, or whenever the energy demand is greater, adenosine is also degradated to inosine and hypoxanthine by the enzyme adenosine deaminase (Trams and Lauter, 1974). Adenosine can also be formed in the intracellular environment due to the activity of intracellular 5′-nucleotidases of which two isoforms, cN-I and cN-II, have been cloned (Fredholm et al., 2001). Once formed extracellular adenosine is transported inside the cell by nucleoside transporters which are able to maintain high level of adenosine against a concentration gradient (Fredholm et al., 2001). An increase in extracellular active levels of adenosine can be induced by drugs decreasing the activity of these transporters. Adenosine transporters have been cloned and named as ENT1 and ENT2 (equilibrative transport) and CNT1 and CNT2 (concentrative transport). Inside the cell adenosine is phosphorylated to AMP (which in turn is reconverted to ADP and ATP, as a part of the energy cycle) by adenosine kinase and this process keeps low the intracellular level of adenosine in physiological conditions. Under conditions of high energy demand and/or hypoxia, intracellular AMP is metabolised to adenosine (Bodansky and Schwartz, 1968; Mentzer et al., 1975). An example of this high energy demand is an inflammatory environment where a large number of inflammatory cells compete for a limited oxygen supply. Ischemia, hypoxia and electrical stimulation can also increase the level of adenosine (Zetterstrom et al., 1982; Berne and Rubio, 1974) and the increase can be up to 100-fold during ischemia (Rudolphi et al., 1992). Adenosine release is also affected by neurotransmitters such as NMDA, dopamine and nitric oxide (Fredholm et al., 2001). 3. Adenosine receptors in the airways Biological functions of adenosine are mediated by Gcoupled receptors which have been cloned and pharmacologically identified. Four distinct adenosine receptors, named A1, A2A, A2B and A3, have been cloned in rodents, dog, sheep, cow and humans (Table 1) (Fredholm et al., 2001; Polosa et al., 2002). These receptors, presenting a similar sequence among subtypes, are asparagine-linked glycoproteins with sites for palmitoylation in the proximity of the carboxyl terminus and represent an integral part of membrane proteins (Linden, 2001). Adenosine A1 and A3 receptors are coupled to Gi/0 whereas A2A and A2B to Gs (Fredholm et al., 2001); it is also possible that, particularly after transfection, one adenosine receptor can be coupled to more than one G protein. Adenosine A2B receptors on HEK 293 cells, human and canine mast cells are coupled to

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Table 1 Expression and putative functional role of adenosine receptors subtypes in the human respiratory system A1 Site of expression

Neutrophils, monocytes, airway smooth muscle. Intracellular signal ↓cAMP, ↑IP3 transduction pathway Functional effects Activates neutrophils with chemotaxis and adherence to endothelial cells

A2A

A2B

A3

Mast cells, neutrophils, monocytes, macrophages ↑ cAMP

Bronchial epithelium, smooth muscle, mast cells, monocytes, fibroblasts ↑ cAMP, ↑IP3

Neutrophils, monocytes, eosinophils ↓cAMP, ↑IP3

Inhibits neutrophils degranulation and adherence to surfaces and endothelium. Reduces the release of pro-inflammatory cytokines from macrophages. Reduces histamine release from mast cells

Induces mast cells degranulation and Inhibits neutrophils increases the release of pro-inflammatory and eosinophils cytokines. degranulation Promotes IgE synthesis by lymphocytes B. Induces differentiation of fibroblasts into myofibroblasts

Gs and Gq (Linden et al., 1999; Auchampach et al., 1997). Moreover coupling of adenosine A2A receptors to different G proteins depends on the area where these receptors are expressed. It has been known for long time that intracellular signals activated by adenosine receptors include either stimulation or inhibition of adenyl cyclase (van Calker et al., 1978; Londos et al., 1980). Adenosine A1 and A3 receptors decrease whereas A2A and A2B increase adenylyl cyclase (Sullivan et al., 2001). In addition adenosine A1 and A3 receptors can activate K+ channels and Ca2+-channels and also for A2A and A2B cAMP-independent intracellular pathways have been described (Cronstein et al., 1994). There are numerous evidences that adenosine receptors may interact with other different receptors. Some authors consider this a rule rather than an exception (Fredholm et al., 2001) and this may have important consequences in the expected response (e.g. if two receptors act in synergy, then blockade of either receptors will block the response). Besides, more than one adenosine receptor can be expressed in a single cell (see for example mast cells) and this may some time result in an atypical pharmacological response. In the respiratory system of various species defining the involvement of each adenosine receptor subtype in physiological processes and in the pathogenesis of inflammation has been a challenging task, particularly in the past for the poor selectivity of the adenosine receptors ligands. In addition, although animal models have become recently available, it is now clear that the subtype of receptor involved in adenosine-induced bronchoconstriction and inflammatory responses varies among species. Thus adenosine-induced bronchoconstriction is mediated by adenosine A1 and A2B receptors in rat and mouse, A3 in rat, guinea-pig and mouse (Nyce and Metzger, 1997; Pauwels and Van der Straeten, 1987; Hannon et al., 2002; Thorne et al., 1996), A2B in man (Feoktistov et al., 1998b). To further complicate this scenario recently it has been suggested that an atypical receptor mechanism mediates contraction of lung parenchymal strips from sensitised allergen-challenged Brown Norway rats (Wolber and Fozard, 2005). Confusion may be also generated by evidences that different types of adenosine receptors are expressed in inflammatory/immune cells (mast cells, macrophages, lymphocytes, fibroblasts, eosinophils and neu-

trophils) recruited in the site of inflammation as well as in some structural components of lungs, such as airway smooth muscle, epithelium and secretive cells (Polosa et al., 2002). 4. A1 adenosine receptor A1 adenosine receptors have high affinity for adenosine and are widely distributed in mammals, particularly in nerve terminals. High expression of adenosine A1 receptors mRNA has been found in brain, dorsal horn of spinal cord eye and adrenal gland, intermediate levels in skeletal muscles and in the gastro-enteric tract, whereas low expression has been found in lungs and bronchi (Fredholm et al., 2001). A1 adenosine receptors are likely to be involved in bronchoconstriction and inflammatory responses induced by adenosine in some species. Rabbits immunised at birth with allergen develop airway hyperreactivity to adenosine through a mechanism involving adenosine A1 receptors (Ali et al., 1994; el-Hashim et al., 1996). The role of adenosine A1 receptors in adenosine-induced bronchoconstriction has been also shown in rabbits engineered with an antisense oligo-deoxynucleotide against adenosine A1 receptor (initiation codon of receptor mRNA). In fact sensitised animals lacking of adenosine A1 receptors manifested a reduced bronchoconstrictor response to allergen challenge (Nyce and Metzger, 1997). Adenosine A1 receptors have been found in human neutrophils, where their activation induces chemotaxis and adherence to endothelial cells and human monocytes (Cronstein et al., 1990, 1991), but the presence and the role of A1 adenosine receptors in human airways remain unclear. An early study showed that in the airways of non-asthmatic subjects there was no evidence of A1 adenosine receptors (Joad and Kott, 1993). Conversely, another study has shown that in isolated bronchi from asthmatic patients inhibition of the contraction induced by leukotriene and histamine antagonists was mediated by adenosine A1 receptors (Bjorck et al., 1992). More recently studies with radiobinding ligands have shown the presence of A1 adenosine receptors on cultured human airway smooth muscle cells of nonasthmatics and evidence has been produced that in these cells over-expression of A1 may affect cAMP release in various conditions. Zhong et al., recently found low expression of adenosine A1 receptor in cultured human airway smooth muscle

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cells (Zhong et al., 2004). Recently, experiments in a mice model have raised the interest toward adenosine A1 receptors and their putative role in asthma. In mice lungs all four adenosine receptors have been found with adenosine A1 receptor transcript being the most abundant (Chun et al., 2001). More recently the same authors have shown in a model of amino deaminase-deficient mice elevated transcript levels for adenosine A1 receptor, particularly in alveolar macrophages; genetic removal of these receptors resulted in an increased lung inflammation and injury with mucus metaplasia and alveolar destruction, associated with increased release of Th2 cytokines (Sun et al., 2005). These findings suggest that adenosine A1 receptors might influence the severity of pulmonary inflammation and remodelling in chronic lung diseases. It is noteworthy that previously, studies had shown that adenosine A1 receptor antagonists attenuated ischemic riperfusion and endotoxin-induced lung injury (Neely and Keith, 1995; Neely et al., 1997). From these evidences adenosine A1 receptors seem to play a dual role in the lung probably due to pattern of distribution. Even though, adenosine A1 receptors selective antagonists are currently under development. Very recently it has been shown that the selective adenosine A1 adenosine receptor antagonist L-97-1 induces a significant reduction of histamine- and adenosine-induced airway hyperreactivity in rabbits sensitised to house dust mite (Obiefuna et al., 2005). In the same study a protection against late bronchial response was also observed. The respiratory anti-sense oligonucleotide (EPI2010), targeting adenosine A1 receptors, is the only compound introduced in clinical trials (phase I/II) for patients with asthma, however although this is a safe and well tolerated drug, the efficacy shown so far is poor (Sandrasagra et al., 2002; Ball et al., 2004). 5. A2A adenosine receptors From early studies it was clear that inflammatory mediators release induced by adenosine in humans and rodents mast cells occurred through activation of adenosine A2 receptors. Successively it became clear that two different subtypes of adenosine A2 receptors exist, coupled to different intracellular signalling pathways, and this observation helped to explain some discrepancies found in airway response to adenosine (Holgate, 2005). Adenosine A2A receptors display high affinity for adenosine and a number of selective antagonists have provided a useful tool for their pharmacological identification. These receptors are widely distributed in the body structures and high expression of adenosine A2A mRNA has been found in leukocytes and platelets and in some areas of the central nervous system, while intermediate levels have been found in lung and heart (Fredholm et al., 2001). Several evidences suggest that in the lung adenosine A2A receptors activate a protective mechanism playing a critical role in the down-regulation of inflammation and tissue damage in different models (Ohta and Sitkovsky, 2001; Thiel et al., 2005). In a model of mice lacking of adenosine A2A receptors subthreshold doses of an anti-inflammatory stimulus causing minimal inflammation and tissue damage in the wild-type animal, induced extensive damage, associated to higher levels of inflammatory cytokines, and eventually animal death (Ohta and

Sitkovsky, 2001). Recently it has been suggested that iatrogenic exacerbation of acute lung injury, upon oxygen administration, is due to the oxygen-associated elimination of the adenosine A2 receptor-linked protecting pathway (Thiel et al., 2005). In an in vivo model of lung transplantation activation of adenosine A2A receptors reduces inflammation and preserves pulmonary function (Reece et al., 2005). Activation of adenosine A2A receptors expressed on lymphoid cells inhibits inflammatory responses by increasing cAMP intracellular levels (Cronstein et al., 1994). At least in part the anti-inflammatory effect mediated by adenosine A2A receptors is due to their expression and functional role on neutrophils (Cronstein et al., 1994; Lappas et al., 2005). A first observation to support this notion was that stimulation of adenosine A2A receptors reduced neutrophils adherence to surfaces and endothelium, without affecting degranulation (Cronstein et al., 1992a). It was therefore suggested that activation of adenosine A2A receptors on neutrophils “uncouples” chemoattractant receptors from their stimulus-transduction proteins (Cronstein et al., 1992b). Successively it has been observed that stimulation of adenosine A2A receptors, no only inhibits cellular recruitment and adhesion, but also inhibits degranulation of activated neutrophils and monocytes (Fredholm et al., 1996; Bouma et al., 1994). Another study has shown that elastase and superoxide production by activated human neutrophils is inhibited by the selective adenosine A2A receptor agonist CGS21680 in a dose-dependent fashion, probably, via cAMPmediated sequestration of cytosolic Ca2+ (Visser et al., 2000;). In human and murine monocytes and macrophages adenosine modulates the production of tumor necrosis factor (TNF)-ά, interleukin (IL)-10 and IL-12 particularly via adenosine A2A receptors, reducing the pro-inflammatory cytokine IL-12 and enhancing the protective cytokine IL-10 (Le Vraux et al., 1993; Hasko et al., 1996, 2000). Adenosine A2A receptors are coexpressed with A2B in human mast cells found in bronchoalveolar lavage (Feoktistov et al., 1998; Suzuki et al., 1998). Differently from adenosine A2B receptors, activation of A2A receptors reduces the release of histamine and tryptase from these cells (Hughes et al., 1984; Suzuki et al., 1998). Recently it has been shown that adenosine A2A receptor activation is predominantly responsible for the inhibitory effect of adenosine receptor agonists on TNF-α production from lipopolysaccharide-stimulated monocytes (Zhang et al., 2005). Taken together these data strongly suggest that activation of adenosine A2A receptors affects multiple aspects of the inflammatory process, modulating neutrophils activation and degranulation, oxidative species production, adhesion molecules expression, cytokines release and mast cells degranulation (Lappas et al., 2005), therefore it is not surprising that selective adenosine A2A receptors agonists may hold some potential in the pharmacological control of airway inflammation and in neutrophils–monocytes-mediated lung tissue injury. A first evidence of the potential antiinflammatory role of adenosine A2A receptors derives from a study in an in vivo model of airway inflammation in which inhibition of leukocytes accumulation induced by methrotrexate, a potent non-steroidal anti-inflammatory agent, is blocked by the selective adenosine A2 receptor antagonist DMPX (Cronstein et al., 1993). More recently it has been shown

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that, in sensitised Brown Norway rats, the inflammation induced by ovalbumin (consisting in an increase in leukocytes, protein content and eosinophils peroxidase activity in bronchoalveolar fluid) was inhibited dose-dependently by the adenosine A2A receptor agonist CGS21680, and this effect was quaitatively similar to that obtained with the glucocorticoid budesonide (Fozard and McCarthy, 2002). Unfortunately, a significant decrease in blood pressure, due to the vasodilator effect of this agonist, was also observed and this represents a major limitation for the introduction of this compound in clinical trials. 6. A2B adenosine receptor Adenosine A2B receptors have been cloned in rat hypothalamus, human hippocampus and mouse mast cells (Feoktistov and Biaggioni, 1997). The pharmacological identification indicates a widespread distribution of these receptors in the body: functional A2B receptors are present in brain fibroblasts, hematopoietic cells, mast cells, myocardial cells and muscle cells. Adenosine A2B receptors display a lower affinity for adenosine and agonists as compared to adenosine A2A receptors. Although similarities in the structure and in the ability to increase intracellular cAMP have been shown, the functional role of the two receptors is quite different, probably due to the fact that adenosine A2B receptors can activate other intracellular pathways in addition to cAMP. In fact in contrast to adenosine A2A receptors activation of adenosine A2B receptors can increase phospholipase C in human mast cells and in mouse bone marrow-derived mast cells (Feoktistov and Biaggioni, 1995; Marquardt et al., 1994). The discovery of adenosine A2B receptors in the airways has raised great interest, as the presence of these receptors has helped to explain some of the physiological effects of adenosine, that remained previously unexplained (Polosa et al., 2002; Holgate, 2005). Expression of adenosine A2B receptors has been found in bronchial epithelium (Clancy et al., 1999), in cultured human airway smooth muscle (Mundell et al., 2001), in human mast cells (Marquardt et al., 1994), monocytes (Zhang et al., 2005) and fibroblasts (Zhong et al., 2005). Increasing evidences suggest that in rodents and man activation of adenosine A2B receptors modulates mast cells function. Early studies on mast cells from mouse bone marrow showed that although a co-expression of adenosine A2A and A2B receptors, the ability of adenosine to cause mast cell degranulation was not affected by the selective adenosine A2A receptor agonist CGS 21680 (Marquardt et al., 1994). In human mast cell line HMC-1 activation of adenosine A2B receptors, but not adenosine A2A receptors, increases the release of IL-8 (Feoktistov et al., 2001). In human lung fibroblasts activation of adenosine A2B adenosine receptors increases the release of IL-6 and induces differentiation into myofibroblasts thus suggesting that adenosine, via A2B receptors participates in the remodelling process occurring in chronic inflammatory lung diseases (Zhong et al., 2005). Adenosine, via A2B receptors, increases the release of IL-6 and monocyte chemotactic protein-1 from bronchial smooth muscle cells (Zhong et al., 2004). Recently the pro-inflammatory role of adenosine A2B receptors has been confirmed by a study showing that activation of these receptors up-regulates Th2

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cytokines (IL-3, IL-4, IL-8, IL-13) in mast cells and promotes IgE synthesis by lymphocytes B (Ryzhov et al., 2004a,b). The observation that human B lymphocytes co-cultured with NECAstimulated mast cells produced high level of IgE, as compared with B lymphocytes co-cultured with non-stimulated mast cells, suggests a more specific role for these receptors in the allergic inflammation occurring in asthma (Ryzhov et al., 2004a,b). Taken together these evidences suggest that adenosine A2B receptor are deeply involved in the mechanisms underlying mediators release by mast cells, the major mechanism by which adenosine induces bronchoconstriction and airway inflammation in asthma. In light of this observation, and considering that blocking adenosine A1 and A2A receptors would inhibit the anti-inflammatory pathways activated by adenosine, the development of adenosine A2B antagonists with high selectivity hold therapeutic potential. Available antagonists include IPDX (Feoktistov et al., 2001), 8-SPT, CGS15493 (Fozard et al., 2003) and the recent available CVT5440 which shows high affinity and good selectivity for adenosine A2B receptors (Zablocki et al., 2005). 7. A3 adenosine receptor A3 adenosine receptors are the most recently discovered among adenosine receptors. High expression of their mRNA has been found in rat mast cells (Fredholm et al., 2001). In rats and guinea-pigs activation of adenosine A3 receptors induces mast cells degranulation in response to stimulation with allergens (Thorne et al., 1996; Fozard et al., 1996). In mice inhaled adenosine induces recruitment of mast cells and neutrophils in wild-type animals, but not in A3-deficient animals (Tilley et al., 2003). Unfortunately, the distribution of adenosine A3 receptors in rodents does not reflects distribution in humans, thus decreasing the relevance of these animal models. In man no expression of adenosine A3 receptor protein has been found in mast cells, while high density of adenosine A3 receptors has been found in eosinophils both in the blood (Knight et al., 1997; Kohno et al., 1996) and in the airways (Walker et al., 1997). When activated, adenosine A3 receptors on human eosinophils mediate inhibition of degranulation and superoxide anion release (Ezeamuzie and Philips, 1999). Adenosine A3 receptors are also expressed in human neutrophils (Bouma et al., 1994; Gessi et al., 2002) and their activation inhibits neutrophils degranulation induced by endotoxin (Gessi et al., 2002). Adenosine A3 receptors have also been implicated in the suppression of TNF-α release induced by endotoxin from human monocytes (Le Vraux et al., 1993). It has been shown that mRNA and protein of adenosine A3 receptor are increased in lung from asthmatics (Walker et al., 1997). Nevertheless, the functional role of these receptors in the asthmatic lung is unclear. As adenosine A3 receptors inhibit degranulation of eosinophils it has been speculated that specific adenosine A3 receptor ligands might be useful in eosinophil-dependent allergic diseases such as asthma and rhinitis (Walker et al., 1997). In amino deaminase-deficient mice expression of adenosine A3 receptors has been found in mucus-producing cells in the airways and the selective A3 antagonist MRS 1523 inhibited mucus production and prevented eosinophilia (Young et al., 2004). Finally it is

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possible that adenosine A3 receptors may also modulate the immune response in the airways as recently it has been shown that human lymphocytes express adenosine receptors with pharmacological and biochemical profile typical of the human adenosine A3 receptor subtype (Gessi et al., 2004). Recently a new orally bioavailable dual adenosine A2B/A3 receptor antagonist has become available (Press et al., 2005). Although studies are trying to address this point currently available data are conflicting and we are far from understanding whether or not there is a rationale to develop specific adenosine A3 receptor ligands for therapeutic purpose. 8. Should we target adenosine receptors to treat airway inflammation? A great amount of work has been done in the last decade to characterize adenosine receptors and to understand their physiological role. Indeed evidences suggest their functional role in the airways and their involvement in some aspects of allergic inflammation, such as recruitment of inflammatory cells, release of inflammatory mediators, airway remodelling and mucus secretion. Therefore it has been suggested that targeting adenosine receptors might be a possible approach for the development of anti-inflammatory treatments in diseases characterized by chronic airway inflammation such as asthma and COPD. Although some selective agonist/antagonist are currently under development since the four distinct receptor subtypes are associated to different functional roles, activating either a protective or an inflammatory pathway, one major problem is to establish which receptor/s should be targeted. The balance of the four known adenosine receptor subtypes appears to be controlled by several inflammatory cytokines (Khoa et al., 2001; Xaus et al., 1999). Therefore, distinct pro- and anti-inflammatory functions of adenosine are likely to be dependent on the dynamic regulation of specific adenosine receptors on specific cell types in a given inflammatory environment. Since stimulation of adenosine A2B receptors expressed on human lung mast cell is likely to be the main trigger for adenosine-induced bronchospasm, currently there is agreement that development of selective adenosine A2B receptor antagonists might be the most appealing approach (Polosa et al., 2002; Holgate, 2005). Another problem in translating available data in therapeutic tools is that adenosine receptors are widely spread in human body, particularly in the central nervous system and in the vascular system, therefore a selective targeting of the site of action should be granted to avoid significant side effects. These compounds should be lipophobic to avoid penetration into the blood-brain barrier and lack of side effects on the cardiocirculatory system.

after adenosine was administered intravenously (Drake et al., 1994). Ten years later bronchoconstriction to inhaled 5-AMP was also observed in patients with COPD particularly in smokers (Oosterhoff et al., 1993). Mechanisms underlying bronchoconstriction have been explored by a number of in vitro and in vivo studies but results were often conflicting due mainly to differences in bronchial response among species and to differences in the basal tone of airway smooth muscle (Polosa et al., 2002). In vitro, adenosine weakly contracts human and guinea-pig airways maintained at basal tone but relaxes guinea-pig airways pre-contracted with carbachol (Advenier et al., 1982; Finney et al., 1985). The observation that contraction induced by adenosine is more marked in isolated airway from asthmatics than non-asthmatics (Bjorck et al., 1992) is in agreement with the observation that inhaled AMP induces bronchoconstriction in asthmatics, either allergic or non-allergic, but not in healthy subjects. 10. Mechanisms of bronchoconstriction 10.1. Mast cells Since the description of the bronchoconstrictor effect of adenosine in man, efforts have been made to elucidate the underlying mechanisms (Fig. 1). Although no adenosine antagonist has acceptance to be used in human, alternative approaches have suggested that the bronchial response is not a direct effect of adenosine but most likely is due to degranulation of mast cells. A first correlation between adenosine receptors and mast cells have been observed already in 1978 when Marquardt reported that adenosine, although ineffective alone, potentiated histamine release induced by anti-immunoglobulin E concanavalin A, compound 40/80, and by the calcium ionophore A23187 in isolated rat mast cells (Marquardt et al., 1978). Given the role of mast

9. Bronchial response to exogenous adenosine In 1984 Cushley and co-workers for the first time, administered inhaled adenosine-5′-monophosphate (5-AMP) in groups of asthmatics and normal subjects and found that while a dose-related bronchoconstriction was observed in asthmatics no effect occurred in normal subjects (Cushley et al., 1984). Bronchoconstriction on asthmatics was also confirmed

Fig. 1. Mechanism of bronchoconstriction induced by adenosine in man.

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cells in airway inflammation in asthma the involvement of adenosine receptors in mast cells degranulation has raised great interest and has been widely explored. In vitro studies indicate that adenosine enhances the release of histamine and other preformed mediators from immunologically primed rodents mast cells (Marquardt and Wasserman, 1982). Potentiation of histamine release from rat peritoneal mast cells is insensitive to methylxanthines thus suggesting that this process might be mediated by a receptor with low affinity for xanthines such as the adenosine A3 receptor (Zhou et al., 1992). It is important that biochemical characteristics of mast cells and response to pharmacological agents can vary among species and among anatomical sites and there is recent evidence that adenosine A2B receptors mediate the release of inflammatory mediators. Adenosine is able to increase mediators release in human mast cells obtained with mechanical dispersion or enzymatic dispersion of lung, with leukotrieneC4 being one of these mediators (Peachell et al., 1988). Same results have been shown on mast cells obtained from bronchoalveolar lavage (Forsythe et al., 1999). In parenchymal human lung mast cells, obtained from surgical specimens, adenosine does not induce the release of histamine directly, but only when mast cells are immunologically activated (Peachell et al., 1991) Evidence for a role of mast cells in bronchoconstriction induced by adenosine is also available in vivo. Inhalation of adenosine-5′-monophosphate (5-AMP) in asthmatics increases circulating levels of histamine (Phillips et al., 1990). Agents blocking mast cells degranulation such as sodium cromoglycate and nedocromil sodium inhibit bronchoconstriction induced by 5-AMP in asthmatics (Richards et al., 1988; Phillips et al., 1989a,b). In addition, pre-medication with the H1-histamine receptor antagonists terfenadine and astemizole inhibits bronchoconstriction induced by 5-AMP in asthma and COPD (Rafferty et al., 1987; Phillips et al., 1989a,b) and instillation of 5-AMP in asthmatic bronchi or in the nose of patients with allergic rhinitis increases concentration of histamine and tryptase in the lavage fluid (Polosa et al., 1995, 1999). Inhaled heparin, via inhibition of mast cells activation, attenuates airway response to 5-AMP (Polosa and Holgate, 1997) and the response induced by nasal provocation with 5-AMP (Zeng et al., 2004). It has been shown that the anti-inflammatory agent tetrazolylbenzamido inhibits 5-AMP-induced bronchoconstriction in mild asthma (Persiani et al., 2001). Adenosine also stimulates the release of other mediators from mast cells. These include prostanoids, since bronchoconstriction induced by 5-AMP is attenuated by indomethacin and flurbiprofen (Phillips et al., 1989a,b) and lysine aspirin (Crimi et al., 1995). Increased concentration of prostaglandin D2 has also been found in bronchoalveolar lavage after instillation of adenosine (Polosa et al., 1995). A role for cysteinylleukotrienes in 5-AMP-induced bronchoconstriction has also been suggested since montelukast, a leukotriene receptor antagonist, attenuates acute 5-AMP-induced bronchoconstriction (Rorke et al., 2002). 10.2. Neural mechanisms Cholinergic and peptidergic neural pathways may also be involved in the bronchoconstrictor effect of adenosine. The in-

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volvement of cholinergic reflexes has been suspected since in rat and in man inhaled ipratropium bromide attenuates bronchoconstriction induced by 5-AMP (Polosa et al., 1991). The neural contribution of 5-AMP-induced bronchoconstriction is also suggested by the observation that inhaled frusemide and bumetanide, loop diuretics modulating sensory nerve responses in the airways, inhibit bronchial response to 5-AMP (O'Connor et al., 1991; Polosa et al., 1993). In guinea-pigs in vivo bronchoconstriction induced by adenosine is attenuated by capsaicin (Manzini and Ballati, 1990). In rat pulmonary circulation adenosine induces vasoconstriction by activation of neuropeptidesproducing nerves (Meade et al., 1996). The role of neuropeptides in adenosine-induced bronchoconstriction seems supported by the observation that repeated challenges with inhaled bradykinin (a model of neuropeptides depletion in human airways) attenuate the bronchial response to adenosine (Polosa et al., 1992). Recently it has been speculated that, since both bradykinin B2 and adenosine receptors have been identified on mast cells and peptidergic nerves, adenosine and bradykinin may share a common activation pathway through the release of neuropeptides known to activate mast cells (Rajakulasingam et al., 1994; Holgate, 2005). Besides, inhibition of neutral endopeptidase by phosphoramidon does not affect bronchial response to 5-AMP (Polosa and Holgate, 1997). In addition, the reduction in bronchial response to 5-AMP, induced by the inhalation of ipratropium bromide, has not been observed in patients with COPD. As we have previously speculated perhaps in asthmatics 5-AMP stimulates mast cells release of histamine which in turn stimulates vagal nerve endings, while the role of mast cells in COPD is only marginal (Polosa et al., 2002). 11. Adenosine and airway inflammation Endogenous released adenosine is a potent regulator of airway inflammation, particularly of allergic inflammation. Human lung in vitro releases adenosine upon stimulation with allergens in the presence of inhibitors of adenosine deaminase (Konnaris et al., 1996). In atopic asthmatics it has been shown that bronchial challenge with allergens increases blood levels of adenosine (Mann et al., 1986) and exercise-induced bronchoconstriction also increases circulating adenosine levels (Vizi et al., 2002). Increase in adenosine concentration has also been found in the bronchoalveolar lavage of patients with asthma. In addition to increase mast cells activation adenosine promotes release of inflammatory cytokines from human monocytes (Le Vraux et al., 1993) and smooth muscle, leukocytes chemotaxis via adenosine A2B receptors and eosinophils recruitment and activation via adenosine A3 receptors (Young, 2004). All these aspects are basic mechanisms leading to airway inflammation in asthma. The development of non-invasive techniques to assess airway inflammation, such as evaluation of soluble mediators in exhaled breath, has further confirmed the involvement of adenosine in airway inflammation. In fact, increased levels of adenosine have been found in exhaled breath condensate of steroids-naive asthmatics as compared to healthy subjects and steroids treated patients (Huszar et al., 2002). Adenosine levels also increase in exhaled breath condensate during exercise-induced bronchoconstriction in

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asthmatics, but not in healthy subjects (Csoma et al., 2005). Although the precise source of adenosine release (mast cells, smooth muscle, epithelial cells) remains uncertain it is likely that adenosine may contribute to the bronchoconstriction induced by other stimuli such as allergens (Huszar et al., 1998). It is well known that adenosine potentiates the release of inflammatory mediators when human mast cells are immunologically primed (Peachell et al., 1991) and therefore it has been emphasized the link between airway response to adenosine and the state of atopy. A number of studies indicate that atopic asthmatics are significantly more responsive to inhaled AMP than non-atopic asthmatic (Spicuzza et al., 2003). In patients with allergic rhinitis, nasal provocation with AMP induces a significant histamine release indicating that this response to adenosine provocation reflects mast cells priming in vivo and possibly allergic inflammation (Ludviksdottir et al., 2000). In accordance with these findings, van Daele et al. compared histamine and AMP bronchial challenges in preschool children with recurrent wheeze in order to identify atopic mechanisms for their wheezing and found that in all non-atopic wheezing children the adenosine provocation test was negative (van Daele et al., 2001). Indeed the notion that inflammatory cytokines can regulate adenosine receptors expression (Khoa et al., 2001; Xaus et al., 1999) further confirms a role for adenosine in the inflammatory environment. For example in transgenic animals the severity of airway inflammation and lung remodelling is amplified by the release of adenosine (Blackburn et al., 2003). In a recent elegant commentary Tilley and Boucher have depicted a possible model of adenosine receptors and cell types mediating the pro-inflammatory and anti-inflammatory effect of adenosine in the lung (Tilley and Boucher, 2005) on the basis of an amino deaminase deficient model of mice. According to this model inflammatory effects of adenosine are mediated by the adenosine A3 receptor which is involved in mast-cell dependent increase in vascular permeability, adenosine-induced mast cells degranulation, antigen-induced mast cells degranulation, mucus secretion and recruitment of inflammatory cells in the lung. Adenosine A2B receptors also contribute to mast cells degranulation. In this model adenosine A1 and A3 receptors present on macrophages account for the anti-inflammatory effect of adenosine occurring through an increase in the release of anti-inflammatory mediators (IL-10 and PGE2) and inhibition in the release of pro-inflammatory mediators (TFN-α and matrix metalloproteinase). Of course this is an animal model and in light of the current knowledge it is difficult to establish whether or not it can be extrapolated from mice to human. For example a limitation of this model is that adenosine A3 receptors are widely distributed in the airway of rodents, while very little is known on their distribution on human airways. One interesting aspect of this model is the involvement of adenosine in airway mucus secretion, as altered mucus secretion is an important feature of asthma and COPD. It is noteworthy that recently it has been shown that in airway epithelial cells adenosine up-regulates the mucin gene MUC 2 in asthma (McNamara et al., 2004). Understanding why levels of adenosine are elevated in chronic airway inflammation and why asthmatic airways respond so intensely to adenosine is difficult. There is opinion that

accumulation of adenosine in the lung is not only a by-product of lung inflammation and damage, but can directly affect signaling pathway that lead to features of chronic lung disease (Blackburn, 2003). Some authors have speculated that the pro- and antiinflammatory property of adenosine may be dictated by its level in the lung (Blackburn, 2003; Polosa et al., 2000). Lung inflammation determines a hypoxic environment in which adenosine is generated. In the initial stage low levels of adenosine activate high affinity receptors, such as adenosine A2A receptors, and this triggers a protective pathway; however, when lung inflammation is severe higher levels of adenosine are released, and these, activating the low affinity adenosine A2B receptors, may trigger deleterious signaling pathways that further exacerbate inflammation (Blackburn, 2003). 12. Bronchial challenge with adenosine is a useful clinical tool in asthma As asthma is characterized by chronic airway inflammation, finding non-invasive markers of airway inflammation has represented a major challenge for researchers. Recent evidences suggest that bronchial challenge with inhaled AMP may be a useful tool to asses and monitor airway inflammation and other airway inflammatory diseases. Bronchial hyperresponsiveness to AMP correlates better than bronchial hyperresponsiveness to histamine or methacholine (the two most commonly used agents to assess bronchial hyperresponsiveness) with markers of airway inflammation such as sputum, blood and bronchial tissue eosinophilia and exhaled nitric oxide (eNO) (van den Berge et al., 2004; De Meer et al., 2002; van den Toorn et al., 2001). In addition, bronchial hyperresponsiveness to inhaled AMP appears to reflect the inflammatory component of the disease as shown by the correlation with sputum eosinophils and the number of CD8+ cells in bronchial biopsies of COPD patients (Rutgers et al., 2000). Control of airway inflammation is a major target of the pharmacological treatment in asthma. If changes in bronchial response to AMP reflect fine changes in airway inflammation, bronchial challenge with AMP would provide an excellent tool to follow these changes and to assess the efficacy of the anti-inflammatory treatment. In fact early studies have shown that regular treatment with inhaled steroids reduces airway response to AMP as compared to methacholine (O'Connor et al., 1992; Wilson and Lipworth, 2000). More recently we have shown that in asthmatics bronchial response to inhaled AMP promptly detected inflammatory changes of the airways as early as by the first week of treatment with the inhaled steroid budesonide, whereas changes in bronchial response to methacholine and in percentage eosinophil and epithelial cell counts in the sputum could be observed only by the fourth week of treatment (Prosperini et al., 2002). As atopy is an important determinant of airway response to AMP and studies have cleared that there is a different response to AMP in patients with COPD in which allergic factors are of no significance (van den Berge et al., 2004), therefore test with inhaled adenosine could be exploited as a useful tool for a better discrimination between asthma and COPD (Spicuzza et al., 2003).

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13. Conclusions A substantial amount of work has cleared that adenosine may play an active role in developing airway chronic inflammation. This seems confirmed by the observation that adenosine receptors are present in many cell types involved in airway inflammation and that asthmatic airways are highly responsive to adenosine. Therefore it has been raised the possibility that modulation of adenosine receptors with specific agonists/antagonists may be a valuable approach to control airway inflammation. This picture is very much complicated by the observation that some cells express different types of adenosine receptors, eliciting often opposite effects upon stimulation. Mast cells are likely to play a major role in the bronchoconstriction induced by adenosine and therefore these cells appear to be the most attractive cellular target. In particular A2B adenosine receptors, expressed on the surface human mast cells, trigger mediators release from mast cells and bronchospasm. Therefore the development of potent selective A2B receptor antagonist/s to use in humans might be a particularly beneficial approach. As adenosine receptors are widely distributed in the human body it is important for these compounds to be highly site-selective, to be lipophobic (avoid penetration in the bloodbrain barrier), and to lack of effects on the cardiocirculatory system. Another key point is that asthmatic airways promptly respond to inhaled adenosine and the degree of this response has been well correlated with the degree of airway inflammation, particularly in the state of atopy. There are now several evidences that bronchial challenge with exogenous adenosine may provide a useful clinical tool to assess non-invasively airway inflammation, to monitor the response to anti-inflammatory treatments, and to make a diagnostic discrimination between asthma and COPD. References Advenier, C., Bidet, D., Floch-Saint-Aubin, A., Renier, A., 1982. Contribution of prostaglandins and thromboxanes to the adenosine and ATP-induced contraction of guinea-pig isolated trachea. Br. J. Pharmacol. 77, 39–44. Ali, S., Mustafa, S.J., Metzger, W.J., 1994. Adenosine receptor-mediated bronchoconstriction and bronchial hyperresponsiveness in allergic rabbit model. Am. J. Physiol. l266, L271–L277. Auchampach, J.A., Jin, X., Wan, T.C., Caughey, G.H., Linden, J., 1997. Canine mast cell adenosine receptors: cloning and expression of the A3 receptor and evidence that degranulation is mediated by the A2B receptor. Mol. Pharmacol. 52, 846–860. Ball, H.A., Van Scott, M.R., Robinson, C.B., 2004. Sense and antisense: therapeutic potential of oligonucleotides and interference RNA in asthma and allergic disorders. Clin. Rev. Allergy Immunol. 27, 207–217. Berne, R.M., Rubio, R., 1974. Adenine nucleotide metabolism in the heart. Circ. Res. 35, 109–120. Bjorck, T., Gustafsson, L.E., Dahlen, S.E., 1992. Isolated bronchi from asthmatics are hyperresponsive to adenosine, which apparently acts indirectly by liberation of leukotrienes and histamine. Am. Rev. Respir. Dis. 145, 1087–1091. Blackburn, M.R., 2003. Too much of a good thing: adenosine overload in adenosine-deaminase-deficient mice. Trends Pharmacol. Sci. 24, 66–70. Blackburn, M.R., Lee, C.G., Young, H.W., Zhu, Z., Chunn, J.L., Kang, M.J., Banerjee, S.K., Elias, J.A., 2003. Adenosine mediates IL-13-induced inflammation and remodeling in the lung and interacts in an IL-13adenosine amplification pathway. J. Clin. Invest. 112, 332–344. Bodansky, O., Schwartz, M.K., 1968. 5′-Nucleotidase. Adv. Clin. Chem. 11, 277–328.

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