Role of Adenosine in Pulmonary Diseases

The acute effect of adenosine on bronchoconstriction is well established by now. Adenosine administration by inhalation was shown to elicit concentrationdependent bronchoconstriction in subjects with asthma whereas the nucleoside had no discernable effect on airway calibre of normal individuals. Since this initial observation, a considerable effort has been directed at revealing the cellular and molecular mechanisms of adenosine-induced bronchoconstriction. One of the proposed mechanisms involves an interaction between adenosine and activated airway mast cells with subsequent release of pre-formed and newly formed mediators. In addition to mast cells, other cells might also play a role in adenosinemediated bronchial hyperresponsiveness (BHR). The observation that adenosine-mediated BHR is reduced but persistent in mast-cell-deficient mice supports the existence of a mast-cell-independent mechanism.

Consistent with the hypothesis of adenosine playing a critical role in the pathogenesis of chronic inflammatory disorders, mice deficient in adenosine deaminase (ADA) develop features of severe pulmonary inflammation and airway remodelling in association with increases in adenosine concentrations in the lung. Features of the pulmonary phenotype noted include the following:

• the accumulation of eosinophils and activated macrophages in the airways;
• mast cell degranulation;
• mucus metaplasia in the bronchial airways; and
• emphysema-like injury of the lung parenchyma.

Although the histological observation in ADAdeficient mice does not completely resemble that of human asthma due to the lack of epithelial shedding, sub-epithelial fibrosis, or muscle/submucosal gland hypertrophy, the ADA-deficient mouse model is a useful tool to study the pathogenic role of adenosine in chronic airway inflammation. The central role of adenosine in chronic lung inflammation is also supported by studies carried out in mice that have increased levels of interleukin (IL)-13 in the lung. These mice develop inflammation, fibrosis and alveolar destruction with concurrent increases in adenosine concentrations in the lung. Treatment with polyethylene glycol (PEG)-ADA to prevent the increases in adenosine concentration results in a marked decrease in the pulmonary phenotypes, suggesting that adenosine mediates IL-13-induced inflammation and tissue remodelling in these models.

An important clinical development in this research area is the use of an adenosine (or adenosine monophosphate, AMP) inhalation challenge as a diagnostic test for asthma and Chronic Obstructive Pulmonary Disease (COPD). Unlike BHR to methacholine, which is related to the changes in airway calibre, BHR to inhaled AMP seems to be more sensitive to treatment with inhaled corticosteroids. In addition, AMP provocation also increases the release of serum neutrophil chemotactic factor and induces sputum eosinophilia. Moreover, inhalation challenge with AMP appears to be useful at establishing the appropriate dose of inhaled corticosteroids (ICS) needed to control airway inflammation, or at predicting safe dose reductions of ICS in patients with mild to moderate asthma. The growing body of evidence supports the hypothesis that BHR to inhaled AMP may reflect the inflammatory status of allergic patients and could be useful in evaluating the effectiveness of different treatment regimens with ICS and monitoring corticosteroid requirements and dose selection in asthma treatment.

Targeting the A_2B Adenosine Receptor Subtype

Extracellular adenosine elicits its biological effects by interacting with four cell surface G-protein coupled receptors designated as A₁, A_2A, A_2B, and A₃ adenosine receptors. All four adenosine receptor subtypes are expressed in the lung and in inflammatory cells involved in asthma, and selective agonists or antagonists to these receptor subtypes are being exploited by the pharmaceutical industry in an attempt to generate novel therapies for asthma and COPD. The A_2B adenosine receptor seems to play a more prominent role in mediating the bronchoconstriction and proinflammatory effect of adenosine in the lung.

The initial evidence for the role of A_2B receptors in asthma came from pharmacological studies of enprofylline, a methylxanthine structurally closely related to theophylline. It was shown that enprofylline is a selective antagonist for the A2B receptors, whereas theophylline has similar binding affinities for A₁, A_2A and A_2B receptors. Importantly, the therapeutic concentrations of theophylline and enprofylline are in the range of their affinities for A_2B receptors. Thus, it was proposed that A_2B receptor might be the therapeutic target for the long-term clinical benefit achieved with relatively low doses of theophylline and enprofylline.

Recently, A_2B receptors have been shown to mediate several pro-inflammatory effects of adenosine in cultured mast cells and lung structural cells. Due to the difficulty in isolating and culturing human lung mast cells, most of the evidence supporting a role of A_2B receptors in mast cells has been obtained using HMC-1, a human mastocytosis-derived cell line. Activation of A_2B receptors in HMC-1 cells increases the release of IL-8 and vascular endothelial growth factor (VEGF), suggesting a role for A_2B receptors in promoting angiogenesis. In addition, activation of A_2B receptors in HMC-1 cells increases the release of Thelper cell (Th)-2 cytokines including IL-4 and IL-13, which in turn promotes the synthesis of immunoglobulin (Ig)E by B lymphocytes. Functional A_2B receptors have been also been identified in bronchial smooth muscle cells, lung fibroblasts and bronchial epithelium. In airway smooth muscle cells, activation of A_2B receptors leads to the release of IL-6 and MCP-1. Similarly, in lung fibroblasts, activation of A_2B receptors increases the release of IL-6, which in turn promotes the differentiations of fibroblasts into myofibroblasts. In bronchial epithelial cells, activation of A_2B receptors increases the release of IL-19, which leads to the release of tumour necrosis factor (TNF)-α from a monocytic cell line. TNF-α is critical to the pathogenesis of asthma as it is known to have a critical role in the induction, maintenance and progression of airway inflammation through enhanced release of proinflammatory/ chemotactic mediators, upregulation of adhesion molecules, and proliferation and activation of sub-epithelial myofibroblasts/fibroblasts. These findings support the hypothesis that A_2B receptors might play a role in the amplification of the allergic inflammatory responses associated with asthma.

Numerous animal models have been used to assess the contribution of adenosine and A_2B receptors in the development of pulmonary diseases. The most commonly used models are allergic animal models, the bleomycin-induced pulmonary fibrosis model, and genetically modified mouse models. In the allergic mouse model, an A_2B antagonist, CVT-6883, inhibits the AMP-induced airway reactivity. In the allergic sheep model, an A_2B antagonist, AS-16, blocks the bronchospasm induced by adenosine. It partially inhibits the early phase of airway response induced by allergen and almost completely blocks the late-phase airway response. In bleomycin-induced pulmonary fibrosis models, treatment with an A_2B antagonist reduces pulmonary inflammation and fibrosis. Mice with genetic deletion of the ADA gene in the lung develop features of pulmonary inflammation and airway remodelling with concurrent increases in tissue levels of adenosine in the lung. In ADA-deficient mice, treatment of CVT-6883 inhibits pulmonary inflammation as determined by the number of inflammatory cells in bronchoalveolar lavage fluid (BALF) as well as the expression of pro-inflammatory cytokines and chemokines. In addition, A_2B antagonism inhibited pulmonary fibrosis revealed by reductions of collagen deposition and accumulation of myofibroblasts in the lung of ADA-deficient mice. Altogether, these findings provide strong support that A_2B antagonists might be promising therapeutic agents in the treatment of pulmonary diseases such as asthma, COPD and pulmonary fibrosis.

Conclusion

It has been 20 years since the first demonstration that adenosine is a bronchoconstrictor in asthmatics. Since then, a large body of literature has supported the hypothesis that adenosine plays an important role in airway hyperresponsiveness. In addition, BHR to adenosine has been shown to correlate well with the inflammatory status of the lungs of asthmatic patients. While all four adenosine receptors have been found in lung tissues and modulated during diseases, a critical role of A_2B antagonists in pulmonary inflammation, fibrosis and airway remodelling has been confirmed in several animal models. It is worthwhile to point out that A_2B receptors are widely distributed in different organs and may participate in the pathogenesis of inflammatory diseases in other organs. This offers opportunities to develop A_2B antagonists for multiple indications as well as challenges to develop A_2B antagonists that are devoid of side effects due to the possible action of A2B receptors in other organ systems. Proof of the efficacy and safety of A_2B antagonists in clinical asthma and other pulmonary diseases is eagerly awaited.