E-NTPDases in human airways: Regulation and relevance for chronic lung diseases
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Chronic obstructive lung diseases are characterized by the inability to prevent bacterial infection and a gradual loss of lung function caused by recurrent inflammatory responses. In the past decade, numerous studies have demonstrated the importance of nucleotide-mediated bacterial clearance. Their interaction with P2 receptors on airway epithelia provides a rapid ‘on-and-off’ signal stimulating mucus secretion, cilia beating activity and surface hydration. On the other hand, abnormally high ATP levels resulting from damaged epithelia and bacterial lysis may cause lung edema and exacerbate inflammatory responses. Airway ATP concentrations are regulated by ecto nucleoside triphosphate diphosphohydrolases (E-NTPDases) which are expressed on the mucosal surface and catalyze the sequential dephosphorylation of nucleoside triphosphates to nucleoside monophosphates (ATP → ADP → AMP). The common bacterial product, Pseudomonas aeruginosa lipopolysaccharide (LPS), induces an acute reduction in azide-sensitive E-NTPDase activities, followed by a sustained increase in activity as well as NTPDase 1 and NTPDase 3 expression. Accordingly, chronic lung diseases, including cystic fibrosis (CF) and primary ciliary dyskinesia, are characterized by higher rates of nucleotide elimination, azide-sensitive E-NTPDase activities and expression. This review integrates the biphasic regulation of airway E-NTPDases with the function of purine signaling in lung diseases. During acute insults, a transient reduction in E-NTPDase activities may be beneficial to stimulate ATP-mediated bacterial clearance. In chronic lung diseases, elevating E-NTPDase activities may represent an attempt to prevent P2 receptor desensitization and nucleotide-mediated lung damage.
Keywordsapyrase bacterial clearance CD39 chrosobstructive lung diseases diphosphohydrolase endotoxin purinergic signaling oxidative stress
airway surface liquid
cyclic compressive stress
cystic fibrosis transmembrane regulator
epithelial sodium channel
- NS AP
non-specific alkaline phosphatase
polymerase chain reaction
tumor necrosis factor alpha
Airway epithelia constitute an essential protective barrier against lung infection, coordinating luminal and interstitial responses to inhaled pathogens through signals (growth factors, cytokines and nucleotides) provided by epithelial, inflammatory and immune cells . It is now widely accepted that extracellular nucleotides provide an elaborated cell communication system in mammalian tissues [2, 3] including the airways [4, 5]. Each signaling event constitutes a brief ‘on-and-off’ switch mechanism allowing the target cells to perceive the subsequent signal. The major source of extracellular nucleotides in normal airways is the epithelium, releasing ATP under resting conditions and in response to mechanical or osmotic stress . While basal ATP concentrations maintained under resting conditions are insufficient to activate surface receptors, the levels reached near the site of stimulated release initiate a variety of P2 receptor-mediated responses . Two P2 receptor families have been identified: Fast-acting (ionotropic) P2X ligand-gated cation channels and slow-acting (metabotropic) G protein-coupled P2Y receptors . Although ATP activates members of both families, P2X receptors generally respond to higher concentrations (EC50 = 1–10 µM) than P2Y receptors (EC50 = 0.1–1.0 µM). Each signal is promptly terminated by surface conversion of ATP to adenosine [9, 10] and dispersal into the interstitial fluid. Adenosine also initiates cellular responses through P1 (A1, A2A, A2B or A3) surface receptors . In the airways, nucleotide- and adenosine-mediated communications are involved in wound healing , bacterial clearance  and inflammatory responses [1, 3, 13, 14].
Cell surface nucleotide concentrations are regulated by three families of ectonucleotidases in mammalian tissues: Ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs: ATP → AMP), alkaline phosphatases (APs: ATP → AMP → AMP → adenosine) and ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases: ATP → AMP → AMP) . This review describes the importance of regulating extracellular nucleotide concentrations for airway homeostasis and the impact of chronic lung diseases on E-NTPDases. Few studies have addressed the activity or expression of airway E-NTPDases. In this review, the available information is complemented by original data.
Nucleotide-mediated bacterial clearance
Extracellular nucleotides and nucleosides regulate CFTR and ENAC . The basal activity of CFTR is maintained by adenosine (A2B) receptors, which induce G protein-coupled adenylate cyclase, followed by cAMP-dependent activation of type II protein kinase A . In contrast, ATP stimulates Cl− secretion through Ca2+-activated Cl− channels. This signaling pathway involves P2Y2 receptors, leading to G protein-coupled phospholipase C activation and cytosolic Ca2+ mobilization. Water efflux is also stimulated by P2Y2 receptor-mediated inhibition of Na+ absorption by ENAC using Ca2+-dependent signaling pathways . Members of the P2X receptor family also support luminal Cl−/water efflux in human airway epithelia, as demonstrated in primary cultures and cell lines (Beas2B, 16HBE14o−, Calu-3, (ΣCFTE-29o−) by Ussing chamber and patch-clamp experiments . The receptors identified in airway epithelia by RT-PCR were P2X2, P2X4 and P2X5. These non-selective cation channels allow Ca2+ influx, which then stimulates Ca2+-activated Cl− channels. Interestingly, P2X7 receptors are only detected in CF airway epithelia, primarily in nasal polyps . Because they are known to induce apoptosis in response to high ATP concentrations , P2X7 receptors may contribute to disease-associated epithelial damage.
The central role of ASL nucleotides for MCC was further substantiated by the observation that P2Y2 receptor activation constitutes the most potent stimulus for cilia beating activity [24, 25, 26] and mucin secretion [27, 28]. In cultured CF airway epithelia, P2Y2 receptor activation restores normal PCL height and rotational mucus transport . Based on these findings, aerosolized nucleotides were proposed for the treatment of chronic obstructive lung diseases . However, clinical studies showed that patients with asthma or chronic obstructive pulmonary disease exposed to aerosolized metabolites of ATP (AMP and adenosine) experience coughing and bronchoconstriction [30, 31]. Furthermore, aerosolized ATP (not adenosine) induced similar reactions from healthy subjects [32, 33]. In developing nucleotide-mediated MCC therapies, the equally potent P2Y2 receptor agonist, UTP, was evaluated . Airway clearance in sheep, measured with nebulized technetium-labeled albumin, was transiently increased by UTP . Aerosolized UTP also improved MCC in patients with mild chronic bronchitis , CF [36, 37] and primary ciliary dyskinesia .
A major obstacle to the treatment of chronic lung diseases with aerosolized UTP is the rapid disappearance of therapeutic concentrations (0.1 mM) from the ASL layer (<5 min) [10, 29]. Incidentally, the metabolically stable UTP analogs INS365 (diquafosol) and INS37217 (denufosol) developed by Inspire Pharmaceuticals Inc.  were reported to improve cough induced-sputum clearance in CF patients [40, 41]. However, long-term treatments with these drugs may require dose optimization to minimize P2Y2 receptor desensitization .
P2 receptors and airway inflammation
In vivo studies conducted on the injurious effects of mechanical ventilation demonstrated that endogenous ATP may reach ASL concentrations sufficiently high to trigger inflammatory responses and lung damage in the absence of infection . The bronchoalveolar lavage fluid of rats subjected to positive-pressure mechanical ventilation contains significantly higher protein, ATP and cytokine (IL-6, TNFα) levels than control animals. Instillation of an equivalent ATP concentration increased lung fluid volume, supporting the existence of P2 and/or P1 receptor-mediated lung edema . Analysis of bronchoalveolar lavage fluid nucleotide content by etheno-derivatization revealed an increase in AMP + adenosine/ADP + ATP ratio , suggesting that mechanical ventilation up-regulates airway ectonucleotidases. Real-time PCR on total lung tissue indicated that mechanical ventilation increases A2B, but decreases P2X7, receptor expression. Rats subjected to both mechanical ventilation and positive end-expiratory pressure exhibited normal bronchoalveolar lavage composition and receptor expression. These findings suggest that patients subjected to positive end-expiratory pressure during large-volume ventilation may avoid ATP-mediated lung injuries. On the other hand, the fact that adenosine levels and A2B receptor expression were raised, whereas ATP levels and P2X7 receptor expression were decreased, by mechanical ventilation supports a dynamic role for ectonucleotidases in the regulation of purine signaling in the airways.
The role of P2 receptors in innate defense against bacterial infection was investigated using mice deficient in P2Y1, P2Y2 or both receptors . All these mice exhibited lower survival and lower cytokine levels in lung homogenates than wild-type animals measured 24 h after intranasal instillation of P. aeruginosa. These results suggest a protective role for ATP-induced inflammation . In human airways, P2Y2 receptor activation amplified inflammatory responses to bacterial infection. Primary cultures of bronchial epithelial cells exposed to UTP and sputum from CF patients exhibited an eight-fold increase in IL-8 secretion, compared to two-fold with UTP alone . Furthermore, P2X7 receptors are known to support several processes relevant to inflammation, including LPS neutralization , pathogen killing, nitric oxide production  and cytokine release from mast cells, leukocytes, dendritic cells and neurons [3, 14]. In human blood, LPS-induced IL-1β release is increased 5-fold by P2X7 receptor activation . Altogether, these studies entail that P2 receptor activation may not only induce innate immunity but also amplify pathogen-induced inflammatory responses in chronic infectious lung diseases.
Too much of a good thing..
Excessive inflammatory response to recurrent bacterial infection is a major component in the pathogenesis of chronic lung diseases, irreversibly damaging the airways and leading to bronchiectasis and respiratory failure [49, 50, 51]. With respect to purine signaling, nucleotide-mediated cell communications may be overwhelmed by massive ATP release from cell lysis of bacteria and damaged epithelia, as well as stimulated leukocytes and epithelia . The sensitivity of certain P2 receptors (P2Y2 , P2X1 and P2X3 ) to agonist-induced desensitization may reduce the efficiency of nucleotide-mediated MCC . On the other hand, chronically elevated ATP may recruit signaling pathways normally dormant in healthy lungs. For instance, P2X7 receptor activation only induces cytokine release and cell death in response to high ATP concentrations encountered in damaged tissues . On astrocytes, LPS-induced TNFα secretion is increased by P2Y receptor activation at low micromolar ATP, but reduced by P2X7 receptor activation at high ATP concentrations . The authors propose a beneficial effect for the potentiation of TNFα secretion by P2Y receptors during mild or acute inflammation and a protective role for P2X7 receptors in damaged tissues or chronic infectious diseases. These studies suggest that P2X7 receptor-mediated responses may be tissue-specific and/or influenced by bacterial infections. In the respiratory system, P2Y2 receptors are expressed throughout airway epithelia  while P2X7 receptors have only been reported in nasal polyps of CF patients . The impact of P2X and P2Y receptor activation on inflammatory responses mediated by airway epithelia under normal and pathological conditions remains to be investigated.
E-NTPDases regulate airway ATP
The physiological importance of azide-sensitive E-NTPDases for nucleotide-mediated MCC was recently established using an in vitro model of rhythmic breathing. Airway epithelia are continuously subjected to mechanical stress generated by breathing, coughing or chest movement. Since mechanical stress induces epithelial ATP release , static culture conditions may underestimate the lungs capacity to regulate MCC in vivo. Rhythmic pressure changes mimicking normal tidal breathing were reproduced in vitro by a system applying cyclic compressive stress (CCS) to the mucosal surface of primary bronchial epithelial cultures . Whereas CF cultures under static conditions exhibit a depleted PCL layer and mucostasis, CCS mimicking normal tidal breathing (20 cmH2O; 15 cycles/min) restored normal PCL height and mucus transport through ATP release and P2Y2 receptor activation. These results also suggest that purine signaling may provide an explanation for the beneficial effects of oscillatory therapeutic devices clinically used to stimulate sputum clearance .
Cyclic compressive stress also enhances MCC through a reduction in ASL nucleotide metabolism. We recently demonstrated that CCS decreases the rate of ATP hydrolysis on the mucosal surface of normal and CF bronchial epithelial cultures . More importantly, CCS restored normal ectoATPase activities on CF epithelial surfaces. The inhibitory effect of CCS on ATP metabolism was abrogated by 20 mM azide, indicating that E-NTPDase 1 and/or E-NTPDase 3 are key components in the regulation P2 receptormediated MCC. Perhaps the ability of CF airway epithelia to provide adequate nucleotide-mediated MCC under conditions mimicking normal breathing may contribute to the relatively healthy state of young patients before the establishment of chronic infection .
Chronic lung diseases shorten the signals
Oxidative stress is one of the major causes of epithelial injury in chronic lung diseases . Reactive oxygen and nitrogen species released from airway epithelia or activated leukocytes damage cell membranes by peroxidation of lipids, amino acids and carbohydrates. P aeruginosa LPS was reported to induce their release from airway epithelia . Incidentally, intrahepatic cholestasis induced by intraperitoneal injection of LPS reduced total liver NTPDase 1 activity within 2 h . In a rat model of glomerulopathy, short-term exposure (1 h) to LPS inactivated endothelial NTPDase 1, which was prevented by pre-treatment with the anti-oxidant superoxide dismutase . Oxidative stress induced by membrane depolarization  or ischemia/ reperfusion  also inhibited endothelial NTPDase 1. Furthermore, short exposures (4 h) to the pro-inflammatory cytokine, TNFα, inhibited endothelial NTPDase 1, which was mimicked by hydrogen peroxide but prevented by superoxide dismutase . Altogether, these studies suggest that oxidative stress may be responsible for the early and transient reduction in azide-sensitive E-NTPDase activities detected in LPS-treated airway epithelia. Since the mRNA expression of E-NTPDases is unaffected by acute oxidative stress  or LPS (Fig. 5B), and the inactivation of NTPDase 1 by ischemia/reperfusion was prevented by inhibitors of lipid peroxidation , the early loss of surface activity may involve local peroxidation of membrane lipids.
The long-term effects of LPS on the azide-sensitive E-NTPDases have not been documented in mammalian tissues. On the other hand, a 24 h exposure to endotoxin stimulated NTPDase 2 on astrocytes , as well as NS AP on endothelial, mesengial [82, 83] and bronchial epithelial  cells. Additionally, the mRNA expression of NTPDase 1 in rat forebrain was up-regulated seven days after transient ischemia . P. aeruginosa LPS [85, 86] is well-known to influence gene expression through the activation of the transcription factors: Nuclear factor-kappa B and activator protein-1 . Recent studies have shown that CFTR acts as a pattern recognition molecule for P. aeruginosa LPS . Endocytosis of the complex triggers translocation of these transcription factors to the nucleus, which initiates innate immunity through the expression of numerous mediators recruiting and activating inflammatory cells. These signaling pathways could be involved in the up-regulation of NTPDase 1 and NTPDase 3 expression resulting from long-term exposures to P. aeruginosa LPS in human airways.
Adaptation of purine signaling to airway diseases
The information provided in this review also exposes the complexity of the signaling interactions taking place between different classes of mediators, including bacterial products, cytokines and extracellular nucleotides, as combinations of mediators may induce different responses than when tested individually. Furthermore, communication networks in the airways may evolve with time during the establishment of a chronic infection, as new target cells (bacteria, leukocytes, lymphocytes) agglomerate on either side of the epithelial barrier. It is proposed that nucleotide-mediated airway functions defined in normal tissues or aseptic culture conditions may require reassessment under conditions mimicking chronic lung diseases before their therapeutic potential may be clearly established.
The human nasal and bronchial epithelial cells were provided by the Tissue Culture Core of the Cystic Fibrosis Center (UNC-CH), directed by Dr Scott H. Randell. This work was supported by the Cystic Fibrosis Foundation (Picher 02I0 and Picher 05G0).
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