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* Department of Medicine, Division of Pulmonary and Critical Care Medicine, and
Department of Genetics, University of North Carolina, Chapel Hill, NC 27599; and
Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The potential role of adenosine in asthma and the mechanisms by which it might influence airway function in this disorder have been studied intensively (6, 8). In 1983 it was recognized that aerosolized adenosine produced bronchoconstriction in asthmatics, but not in normal volunteers (9). Moreover, airway responsiveness to adenosine has been shown to reflect the degree of underlying airway inflammation in a more sensitive fashion than does responsiveness to other agents that can induce nonspecific airway hyper-reactivity in subjects with asthma, such as methacholine (10). In addition to its capacity to produce bronchoconstriction, adenosine has been shown to amplify the inflammatory response in some animal models of asthma following sensitization and challenge (11, 12). In genetically engineered mice with elevated levels of adenosine due to adenosine deaminase (ADA)3 deficiency, eosinophilic lung inflammation and mucus hypersecretion develop, and these animals die from respiratory failure at 3 wk of age (13). These studies coupled with the observation that adenosine levels are elevated in the lungs of subjects with asthma suggest that endogenously produced adenosine may contribute to the pathophysiology of this disorder (14).
Studies in both humans and animals have suggested that adenosine-induced bronchoconstriction can occur indirectly, in part through mediator release from mast cells (15). However, the relative contributions of the proposed mast cell-dependent and mast cell-independent components of adenosine-induced bronchoconstriction differ among species (16). For example, rabbits are thought to have a large mast cell-independent component, reflecting direct actions of adenosine on airway smooth muscle (ASM) (17), whereas findings in humans (18, 19) and rats (20, 21) indicate that these species may have a large mast cell-dependent component to adenosine responsiveness.
There is also uncertainty about which receptors account for adenosine’s effects on airway function. Adenosine can act through any of four distinct G protein-coupled receptors, and various studies have implicated three of these four receptors (A1, A2b, and A3) in adenosine-induced bronchoconstriction. While studies using pharmacological reagents and antisense DNA suggest that the A1 receptor is responsible for the mast cell-independent component of adenosine-induced bronchoconstriction (17, 22, 23, 24), there has been considerable controversy about the receptor(s) involved in adenosine-induced mast cell degranulation. For example, in vitro pharmacological studies employing a human mast cell line and a dog mastocytoma line have supported a role for the A2b receptor in adenosine-induced mast cell activation (25, 26, 27), whereas a number of in vitro and in vivo studies with rodents suggest that the A3 receptor is involved (28, 29). Several explanations may account for these discrepancies, including incomplete selectivity of the adenosine analogs used, different signaling pathways among mast cells from various tissues (or that have been generated in vitro using different methods), and species differences. However, it remains unclear to what extent the A2b and A3 adenosine receptors contribute to adenosine-induced mast cell degranulation in asthmatics.
A useful approach for assigning specific physiological roles to particular receptors has been to examine responses to adenosine in mouse lines lacking each adenosine receptor. We previously used mice lacking the A3 receptor to identify an important role for that receptor in adenosine-induced degranulation of skin mast cells in vivo and in the ensuing adenosine-induced and mast cell-dependent enhancement of cutaneous vascular permeability (30). Here we use wild-type, A3-deficient, and mast cell-deficient mice to assess the importance of the A3 receptor and mast cells in airway responsiveness to adenosine.
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Materials and Methods |
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All studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals as well as the institutional animal care and use committee guidelines of University of North Carolina and Stanford University. Mast cell-deficient mice (WBB6F1/J-KitW/KitW-v; 8–12 wk old), and their congenic normal littermates (WBB6F1/J-Kit+/+) were purchased from The Jackson Laboratory (Bar Harbor, ME). Compared with the congenic wild-type mice, adult WBB6F1/J-KitW/KitW-v contain <1.0% of the number of skin mast cells and essentially no detectable mature mast cells in the respiratory system (trachea, bronchi, and lungs) and many other anatomical sites (31). Mice deficient in the A3 adenosine receptor (A3-/-) were generated as previously described (32). The A3 null mutation was backcrossed six generations onto the C57BL/6 background, and A3-/- and A3+/+ controls were obtained by intercrossing A3+/- heterozygotes. All animals were genotyped by Southern blot analysis as described previously and were used between 5–9 mo of age (32). All experimental animals were matched for gender and age within each experiment.
Measurement of airway responsiveness in conscious mice
Mice were placed in a whole body plethysmograph (Buxco Electronics, Troy, NY), and baseline measurements of Penh were obtained. Penh is a dimensionless index calculated from inspiratory pressures, expiratory pressures, and expiratory time and has been shown to correlate with direct measures of pulmonary resistance in mechanically ventilated animals (33). Penh was then measured in response to aerosols of PBS vehicle or adenosine.
Histological evaluation of mast cell degranulation and airway inflammation
Four hours following exposure to 5 min of PBS or adenosine (6 mg/ml) aerosol in the whole body plethysmograph, mice were euthanized with an overdose of sodium pentobarbital (150 mg/kg). Lungs were inflated with Karnovsky’s-2 fixative (2% paraformaldehyde, 2.5% glutaraldehyde, 0.1 M sodium cacodylate buffer, and 0.025% CaCl2) (34, 35), the trachea was tied off with suture, and the trachea and lungs were removed en bloc and immediately placed in Karnovsky’s-2 fixative (20/1, v/v) at room temperature for 4–6 h. Samples were then placed at 4°C for 1 h before being transferred into 0.1 M sodium cacodylate buffer and returned to 4°C until processing. Samples were shipped on wet ice to Stanford University, where they were processed into 1-µm, Epon-embedded, Giemsa-stained sections (34, 35, 36). Sections taken through the trachea were coded so that the observer was not aware of the identity of the individual specimens and were examined at x1000 by light microscope. Mast cells were classified as extensively degranulated (>50% of the cytoplasmic granules exhibiting fusion, staining alternations, and/or extrusion from the cell), moderately degranulated (10–50% of the granules exhibiting fusion or discharge), or normal (<10% of the granules exhibiting alterations). The numbers of neutrophils infiltrating in the trachea (in the interstitium, in the epithelium, or near the surface of the epithelium in the lumen) were also quantified and expressed as neutrophils per square millimeter of the trachea (34, 35, 36).
Statistical analysis
Data are presented as the mean ± SEM. Statistical significance was assessed by ANOVA for adenosine dose-response comparison between WBB6F1/J-KitW/KitW-v and WBB6F1/J-Kit+/+ mice and for comparisons of the responses of various groups of mice to PBS or adenosine over time. Student’s t test was used for all other analyses.
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Results |
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To test the contribution of mast cells to adenosine-induced airway responsiveness in mice, we exposed mast cell-deficient (WBB6F1/J-KitW/KitW-v) mice and their congenic normal littermates (WBB6F1/J-Kit+/+ mice) to aerosolized adenosine and measured airway responsiveness by whole body plethysmography. As shown in Fig. 1A, responsiveness to various concentrations of adenosine was observed in both groups of animals, but the response was significantly attenuated (by 
50%) in mast cell-deficient animals (p = 0.0007). When the response to 5-min exposure to a single concentration of adenosine (6 mg/ml) was examined (Fig. 1B), similar results were obtained. Also, we noted that the adenosine-induced enhancement of Penh rapidly diminished after discontinuation of the aerosol challenge with this agent (Fig. 1B). These findings show that there are both mast cell-dependent and mast cell-independent components to adenosine-induced airway responsiveness in WBB6F1/J mice.
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To test the contribution of the A3 receptor to adenosine-induced airway responsiveness, we evaluated the responses of wild-type and A3-/- mice to 5-min aerosols of PBS or adenosine (6 mg/ml). PBS produced no changes in airway responsiveness in A3+/+ (p = 0.11–0.71) or A3-/- (p = 0.22–0.94) mice compared with baseline measurements (Fig. 2A). Adenosine aerosolization resulted in significant increases in Penh in A3+/+ mice, and this response was markedly attenuated in A3-/- animals (Fig. 2B). However, a statistically significant rise in Penh above baseline was still observed in A3-/- mice (p < 0.01) in response to adenosine, suggesting the presence of an A3-independent component to the response (Fig. 2B).
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To assess the extent to which the observed airway responsiveness to adenosine was associated with activation of mast cells, we looked for histological evidence of mast cell activation in the same animals that had been studied physiologically. Four hours following adenosine or PBS exposure, lung tissue was obtained from all animals to quantify mast cell degranulation and neutrophil accumulation (as additional evidence of mast cell activation) (36). In wild-type mice, significantly more degranulated mast cells were observed in the adenosine- vs PBS-treated group (40.5 ± 4.3 vs 16.1 ± 3.7%; p = 0.00005; Figs. 3A and 4A vs Fig. 4B). By contrast, the percentage of degranulated mast cells in adenosine-exposed A3-/- mice was no different from that in PBS-treated A3-/- animals (17.8 ± 3.4 vs 16.8 ± 3.1%; Figs. 3A and 4C vs Fig. 4D). The numbers of mast cells present in the tracheae were not significantly different between groups: adenosine-treated A3+/+, 16.1 ± 2.1/mm2; PBS-treated A3+/+, 18.2 ± 2.6/mm2; adenosine-treated A3-/-, 19.8 ± 2.6/mm2; and PBS-treated A3-/-, 20.2 ± 1.9/mm2.
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Adenosine-induced neutrophil recruitment is mast cell dependent
To confirm that neutrophil recruitment was the result of mast cell activation by adenosine, we exposed mast cell-deficient (WBB6F1/J-KitW/KitW-v) mice and their congenic normal littermates (WBB6F1/J-Kit+/+ mice) to aerosolized adenosine (6 mg/ml) for 5 min and examined neutrophil recruitment to the lung 4 h postexposure. As shown in Fig. 6, more neutrophils were observed in the tracheae of adenosine-treated, wild-type than in adenosine-treated, mast cell-deficient mice (69.67 ± 7.6 vs 38.41 ± 5.32; p = 0.0044)). No differences were observed among wild-type, PBS-treated; mast cell-deficient, PBS-treated; and mast cell-deficient, adenosine-treated groups (32.05 ± 4.23 vs 35.82 ± 6.36 vs 38.41 ± 5.32). These findings show that adenosine-induced neutrophil recruitment to the lung is mast cell dependent and are consistent with a model in which mast cell activation by adenosine, via the A3 receptor, results in neutrophil recruitment via chemoattractant release from stimulated mast cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We observed that the administration of adenosine by aerosol could trigger degranulation of airway mast cells in vivo without additional stimuli of mast cell activation. This finding is consistent with observations by ourselves and others that adenosine alone can induce degranulation of mast cells in other tissues and organs in vivo, including the skin (28, 29, 30). These findings are also consistent with human studies showing adenosine-induced histamine release from bronchoalveolar lavage mast cells (39). However, these observations do contrast with those of some other studies (2, 30, 40, 41), including one of our own studies in which we showed that while adenosine, acting via the A3 receptor, could enhance the degranulation of bone marrow mast cells in response to IgE and specific Ag, adenosine alone was ineffective at mediating bone marrow mast cell degranulation (30). However, in this issue Zhong et al. (42) demonstrate that adenosine can induce degranulation of mast cells that have been derived in vitro from mouse pulmonary mast cells, a result consistent with the findings in our in vivo studies of mouse airway mast cells in situ.
Taken together these observations indicate that the responsiveness of distinct mast cell populations in vitro to adenosine-induced degranulation can vary, perhaps because of differences in the species or anatomical site of origin of the mast cells or as a result of differences in the methods used to isolate the cells and/or to maintain and expand these mast cells in vitro. However, we show that for mouse airway mast cells, adenosine-dependent degranulation can be induced in vivo in the absence of other stimuli of mast cell activation, such as IgE and specific Ag. The capacity of adenosine to evoke mast cell degranulation in vivo without additional stimuli supports an important role for this mediator as an activator of mast cells in asthma and perhaps other disorders.
The observation that mast cell degranulation in response to adenosine is dramatically decreased in mice lacking the A3 receptor is consistent with a model in which adenosine mediates degranulation of these cells directly by activation of the A3 receptor on mast cells. This model is also supported by the observation by Zhong et al. (42) that pharmacological reagents with high specificity for the A3 receptor can induce histamine release by cultured primary mouse lung mast cells. However, we cannot formally rule out the possibility that in vivo adenosine can activate airway mast cells by an indirect mechanism(s) as well.
Nevertheless, we clearly show that adenosine can induce increased airway responsiveness in mice and that this increase is largely dependent on both the presence of mast cells and the expression of the A3 receptor. These findings together with the observed A3-dependent degranulation of mast cells in vivo suggest a model in which the full expression of bronchoconstriction in response to adenosine requires the release of bronchoactive mediators by mast cells upon their activation by an A3 receptor-dependent mechanism(s). Furthermore, this proposed mechanism is consistent with the findings that 1) IgE-dependent mast cell activation can result in mast cell-dependent bronchoconstriction in the mouse (35); and 2) the airway hyper-responsiveness to methacholine that follows anti-IgE challenge in mice is mast cell dependent (43). However, the specific mast cell-derived mediators that may contribute to the effects of adenosine challenge on airway responsiveness in the mouse remain to be defined.
Our findings in mice may have clinical relevance in that there is substantial indirect evidence that mast cells can contribute to adenosine-induced bronchoconstriction in humans. In vitro studies have shown that adenosine can potentiate the release of histamine and leukotrienes from immunologically activated human mast cells obtained from lung parenchyma (41) and that adenosine can independently induce histamine release from human bronchoalveolar lavage mast cells (39). Moreover, adenosine-induced contractions of isolated bronchi from asthmatics can be blocked by leukotriene and histamine antagonists (44). In vivo, adenosine-induced bronchoconstriction can be attenuated by drugs that block mast cell degranulation (nedocromil sodium) (45) and by drugs that block mast cell-derived products (antihistamines) (19) capable of producing bronchoconstriction in humans. Finally, concomitant bronchoconstriction and mast cell mediator release have been shown following the endobronchial installation of AMP in asthmatics (18).
While adenosine produces bronchoconstriction in the asthmatic airway, it has little effect on the airway caliber of normal individuals. A possible explanation for this observation is suggested by recent histological studies showing that the location of mast cells is markedly altered in the asthmatic airway. While in the healthy lung mast cells are found primarily in the submucosa, this study showed that mast cells infiltrate the ASM of asthmatics (46). It is easy to imagine that under these circumstances mast cell degranulation might have a more pronounced effect on airway smooth muscle tone. Alternatively the increased responsiveness of the asthmatic to adenosine might reflect increased expression of the A3 receptors on mast cells in the inflamed airway. Support for this hypothesis comes from studies showing that while in situ expression of the A3 receptor was not detected in normal lung, specific hybridization was present in mast cells in the airway wall in asthmatic lung. (47).
In our studies, unlike the human studies, we found a significant increase in airway responsiveness in naive mice following adenosine exposure. Our ability to measure changes in response to adenosine without induction of an inflammatory response could simply reflect the sensitivity of the system for measuring changes in the airways of mice vs humans. For example, the whole body plethysmograph used in our studies is sensitive to changes in airway caliber not only in the lower airways but also in the nasal passages and larynx, regions rich in mast cells. We would therefore expect that the response to adenosine might be significantly increased in the inflamed mouse airway. Alternatively, the ability to measure this response in the naive mouse may reflect anatomical differences in the distribution of the mast cells and/or differences in the expression levels of adenosine receptors between the two species.
We also show that exposure of the mouse airway to adenosine can lead to increased inflammation, specifically the recruitment of neutrophils, and that this process is dependent on A3 expression. We also show that this neutrophil recruitment is mast cell dependent, supporting the hypothesis that activation of A3 receptors on mast cells by adenosine stimulates the mast cell to produce a number of chemokines, cytokines, and lipid mediators that can contribute to neutrophil chemotaxis (38). While additional work will be required to fully define the mechanisms of adenosine-induced neutrophil recruitment in this setting, our data show that activation of the A3 receptor plays a critical role in this response.
Chronic elevations of adenosine in the ADA-deficient mice are associated with several dramatic histopathological changes in the lung, including eosinophilia, macrophage activation, and goblet cell hyperplasia (13). In contrast, mast cell degranulation and neutrophil influx were the only histological changes observed in our study following acute exposure of the airways to aerosolized adenosine. There are a number possible explanations for these differences. The changes in the ADA-deficient mice might reflect alterations and adaptive changes on the part of the organism to loss of the receptor throughout development. Second, it is possible that inhaled adenosine does not reach the levels in all tissue compartments achieved in animals lacking ADA. Finally, it is possible that continual chronic exposure of the mice to inhaled adenosine (if achievable) would, given enough time, lead to changes similar to those observed in ADA-deficient mice. In this regard it is interesting to speculate that the changes we observed in the acutely exposed animal may represent the initiating events that eventually lead to the dramatic abnormalities observed in ADA-deficient mice, suggesting that the mast cell may have an important initiating role in the development of airway disease. Second, our findings suggest that the early recruitment of neutrophils, an effector cell not typically associated with allergic airway disease, may play an important role early in disease pathogenesis. It is interesting that while the eosinophil is classically associated with mild to moderate asthma, neutrophils are increased in the airways of severe, steroid-dependent asthmatics and are a prominent feature of patients dying from sudden-onset fatal asthma (48, 49).
While our studies support important roles for mast cells and the A3 receptor in the full expression of adenosine-induced changes in airway responsiveness, they also indicate that other cell types and receptors contribute to this response. Statistically significant changes in airway responsiveness were observed in both mast cell-deficient and A3-deficient mice. The cells that account for the mast cell-independent component of adenosine’s ability to induce airway responses (via either direct or indirect mechanisms) remain to be determined, but candidates include ASM, epithelia, and nerves. Studies of human airway tissue suggest that one or several of these cell types could contribute to adenosine-induced bronchoconstriction. While human ASM cultures exposed to adenosine predominantly show elevations of cAMP via A2b, evidence for A1-mediated effects were demonstrated, and it is possible that in the asthmatic lung the differential contribution of A1 vs A2b to ASM function is altered (50). Cultured airway epithelial cells express A2a, A2b, and A3 adenosine receptors (51), and mediators released from these cells upon activation by adenosine could influence airway tone. Finally, it has been demonstrated that adenosine-induced bronchoconstriction in humans can be attenuated by local anesthetics or anticholinergics, suggesting the involvement of neural pathways in adenosine-induced actions on human airway function (52, 53, 54).
Human alveolar macrophages, primary cultured tracheal epithelial cells, and eosinophils and mast cells from asthmatic lung have all been shown to express the A3 receptor, but the contribution of this adenosine receptor subtype to immune cell function or to epithelial biology in humans is largely unknown (51, 55, 56). A functional role for the A2b receptor on human lung mast cells has been suggested by pharmacological studies showing that an A2b agonist can induce IL-8 secretion in a human mast cell line (25). However, high agonist concentrations were used to produce this effect (10 µM), which may have decreased receptor selectivity, and the human mast cell leukemia line used in that study exhibits many differences from normal, nonneoplastic human mast cells (57).
In summary, we show that adenosine can have a profound effect on airway tone and inflammation, and that A3 receptors and mast cells contribute significantly to these processes. The ability of adenosine to activate mast cells in the absence of IgE and Ag has important implications. While basal levels of adenosine produced in the lung are likely to be insufficient to activate the A3 receptor, adenosine levels increase dramatically during inflammation, and the level measured in the asthmatic airway is sufficient to activate the A3 receptor (14). The infiltration of airway smooth muscle cells with mast cells in the asthmatic lung (46) coupled with this elevation in adenosine levels and subsequent activation of these cells via the A3 receptor may play an important role in the pathogenesis of asthma as well as other diseases in which coordinate increases in adenosine and mast cells is observed.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Beverly Koller, 7027 Thurston-Bowles Building, University of North Carolina, Chapel Hill, NC 27599-7248. E-mail address: treawouns{at}aol.com
3 Abbreviations used in this paper: ADA, adenosine deaminase; ASM, airway smooth muscle.
Received for publication November 25, 2002. Accepted for publication March 24, 2003.
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. J. Clin. Invest 87:446.
by human mast cells: increased anti-IgE-dependent secretion after IgE-dependent enhancement of mast cell IgE-binding ability. Lab. Invest. 77:185.[Medline]This article has been cited by other articles:
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M. R. Blackburn A role for neural pathways in adenosine-induced bronchoconstriction Am J Physiol Lung Cell Mol Physiol, July 1, 2007; 293(1): L22 - L24. [Full Text] [PDF]
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, mast cell-deficient mice (WBB6F1/J-KitW/KitW-v); •, wild-type controls (WBB6F1/J-Kit+/+; n = 18/group); B, baseline. *, p < 0.01 (vs baseline value for mice of the same genotype). The dose response of KitW/KitW-v mice was significantly different (p < 0.001) from that of wild-type mice by ANOVA. B, Airway function (Penh) was measured before, during, and after a 5-min aerosolization of adenosine at 6 mg/ml. 
) or PBS (
). Data represent the mean number of extensively degranulated mast cells (A) or the mean number of neutrophils (PMN, polymorphonuclear leukocytes) per square millimeter; B) ± SEM (n = 14/group). *, p ≤ 0.01; **, p < 0.001 (vs either PBS-challenged mice of the same genotype or adenosine-challenged mice of the other genotype).
) or PBS (