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* Department of Biochemistry and Molecular Biology, University of Texas-Houston Medical School, Houston, TX 77030;
Department of Neuroscience, Merck Research Laboratories, West Point, PA 19486; and
Department of Medicine, University of North Carolina, Chapel Hill, NC 27599
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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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Mast cells are principal effector cells in allergic diseases, including asthma (18), and have been implicated to play an important role in the exacerbation of certain forms of COPD (19). These cells can release mediators, such as histamine, tryptase, leukotrienes, and cytokines, that have both immediate and chronic effects on airway constriction and inflammation. Substantial evidence suggests that adenosine can modulate mast cell degranulation. Adenosine and adenosine analogs in vitro can enhance mediator release from mast cells in response to challenge with a variety of stimuli (12, 20, 21, 22). In contrast, adenosine can directly initiate mast cell degranulation in the absence of additional stimuli in vivo (23, 24). These observations are supported by recent studies in adenosine deaminase (ADA)-deficient mice in which elevations in endogenous adenosine were shown to lead to the degranulation of lung mast cells (25). The mechanisms through which adenosine elicits these effects are not known; however, these studies demonstrated that ADA-deficient mice can serve as valuable in vivo models to study adenosine signaling in lung mast cells.
Most physiological effects of adenosine are mediated through adenosine receptors. Four subtypes of adenosine receptor, A1, A2A, A2B, and A3, have been identified. Each receptor has unique tissue distribution, ligand affinity, and signal transduction pathways (1). Most studies suggest that the A2B or A3 adenosine receptors are involved in mediating adenosine’s effects on mast cells. The A2B receptor can evoke IL-8 secretion in human HMC-1 mast cells (26), and the A3 receptor is responsible for enhanced histamine release from mouse bone marrow-derived mast cells (mBMMCs) and cutaneous mast cells through a Gi protein and phosphoinositide 3-kinase (PI3K) 
-dependent pathway (27, 28). However, due to the heterogeneity of tissue mast cells, little is known about how adenosine affects mast cells in lung tissue. Understanding the receptor interactions and downstream signaling mechanisms of adenosine’s effects on lung mast cell degranulation, which is a major pathogenic component of asthma and COPD, will help guide new therapies for the treatment of these widespread diseases.
In the current study we examined the expression and function of adenosine receptors on murine lung mast cells. A2A, A2B, and A3 adenosine receptors were found to be expressed on murine primary lung mast cells (mPLMCs), and studies using selective adenosine receptor agonists and antagonists suggested that activation of A3 receptors could induce mPLMC mediator release. Furthermore, this mediator release was associated with increases in intracellular Ca2+ that were mediated through Gi protein- and PI3K-dependent pathways. In addition, a nebulized A3 receptor agonist directly induced lung mast cell degranulation in wild-type mice while having no effect in A3 receptor knockout mice. Finally, lung mast cell degranulation in response to endogenously elevated adenosine was shown to act through A3 receptors. These results demonstrate that the A3 adenosine receptor plays an important role in adenosine-mediated murine lung mast cell degranulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ADA-deficient mice and A3 receptor-deficient (A3-/-) mice were generated and genotyped as previously described (29, 30, 31). ADA-deficient mice were on a mixed background of 129/Sv, C57BL/6, and FVB/N stains. A3-/- mice were inbred on the C57BL/6 strain, as were the wild-type mice used for the generation of mPLMCs.
Cell culture
The mPLMC cultures were established as previously described (32). Briefly, lung mast cells were isolated from the upper airways of wild-type and A3-/- mice by cutting the tissue into 
3-mm3 pieces, which were then incubated in DMEM containing 10 ng/ml murine stem cell factor and IL-3 (Sigma-Aldrich, St. Louis, MO). Culture medium was changed every other day for the first week. At the end of 1 wk nonadherent cells were collected and transferred to fresh medium. These cultures were passaged in the same manner for 2–3 wk more, after which a nonadherent population of granular cells had grown out. The homogeneity of the mast cells was determined by acid toluidine blue staining and immunofluorescence for c-Kit. Cells that had been in culture for 4–8 wk were used in the experiments. C2C12 cells were obtained from American Type Culture Collection (Manassas, VA), and RBL-2H3 cells were a gift from Dr. B. Dickey (Baylor College of Medicine, Houston, TX). Both cell types were cultured according to protocols provided by American Type Culture Collection.
Acid toluidine blue staining and immunofluorescence for c-Kit and adenosine receptors
The mPLMCs were cytospun onto microscope slides, C2C12 and RBL-2H3 cells were grown on Falcon culture slides overnight, and mPLMCs were stained with acid toluidine blue (pH 1.0) according to established protocols (33). For immunofluorescence, cells were fixed in 1% formalin in PBS for 10 min, and permeabilized in 0.2% Triton X-100 for 5 min on ice. For c-Kit immunofluorescence, cells were blocked with 10% normal goat serum for 20 min and incubated with 10 µg/ml polyclonal rabbit c-Kit Ab at 4°C overnight. After washing, cells were incubated with 5 µg/ml goat anti-rabbit IgG-FITC for 1 h. For adenosine receptor immunofluorescence, cells were blocked with 10% normal donkey serum for 20 min and incubated with 20 µg/ml polyclonal goat anti-rat adenosine receptor Abs at 4°C overnight. After washing, cells were incubated with 5 µg/ml donkey anti-goat IgG-FITC, then washed, counterstained with 1 µg/ml bisbenzimide (Sigma-Aldrich) for 2 min to visualize nuclei, and coverslipped. All Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Quantitative real-time RT-PCR
Quantitative real-time RT-PCR was performed using a 7700 Sequence Detector (PE Applied Biosystems, Foster City, CA). Specific quantitative assays for adenosine receptors were developed using Primer Express software (PE Applied Biosystems) following the recommended guidelines based on sequences from GenBank (34). Total RNA was isolated from mPLMCs using an RNeasy Mini Kit (Qiagen, Valencia, CA), followed by DNase treatment to eliminate potential genomic DNA contamination. This was followed by cDNA synthesis and real-time PCR using established protocols (35). The resulting data were analyzed using SDS software (PE Applied Biosystems) with TAMRA as the reference dye. The final data were normalized to 
-actin and are presented as molecules of transcript/molecules of -actin x 100 (% -actin).
Activation of mast cells by adenosine receptor agonists
Wild-type or A3-/- mPLMCs were stimulated with adenosine or adenosine receptor agonists and antagonists at 5 x 105 cells/ml in medium without murine stem cell factor. Adenosine (100 µM), 100 nM CGS21680, 10 µM NECA, 100 nM IB-MECA, 25 µM enprofylline, and 5 µM MRS-1523 (all from Sigma-Aldrich) were used to activate or antagonize adenosine receptors. As a positive control, cells were incubated overnight at 37°C with 100 ng/ml of monoclonal anti-DNP IgE (Sigma-Aldrich). Cells were stimulated by the addition of 10 ng/ml DNP-albumin (Sigma-Aldrich) at 37°C. Reactions were terminated after 20 min by centrifugation at 2000 x g for 5 min. Histamine concentrations were then measured in supernatant and lysed cell pellets using an enzyme immunoassay (Immunotech, Marseilles, France). Data are presented as the percent histamine released or as absolute values released into the supernatant. The percent histamine released was determined by dividing the concentration of histamine in the supernatant by total histamine levels (supernatant plus pellet). For some experiments cells were preincubated with 100 ng/ml pertussis toxin (Sigma-Aldrich) for 2 h or with 25 µM LY294002 (Calbiochem, San Diego, CA) for 20 min before stimulation with IB-MECA.
Intracellular calcium measurements
The mPLMC were plated on 20% Matrigel-coated, 35-mm, glass-bottom microwell dishes (Mattek, Ashland, MA) and then loaded at room temperature with 5 µM fura 2-AM (Molecular Probes, Eugene, OR) for 40 min in buffer (pH 7.4) containing 145 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 0.5 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, and 5 mM glucose. Changes in fura 2 fluorescence in individual cells were measured at 340 and 380 nm excitation and 510 nm emission wavelengths using an InCyt2 Im2 imaging system (Intracellular Imaging, Cincinnati, OH). Fluorescence ratios were monitored using Im2 software to approximate intracellular calcium concentrations. Data are presented as representative tracings from 30–40 individual cells on experiments performed on two different dishes on separate days.
IB-MECA treatment and bronchial alveolar lavages
Wild-type and A3-/- mice were treated with nebulized saline or 1 mM IB-MECA for 5 min. Mice were anesthetized with avertin, and a blunted 21-gauge needle was secured in the trachea. Lungs were lavaged three times with 0.4 ml of PBS, and 1–1.2 ml of pooled lavage fluid was collected. Samples were centrifuged at 1200 rpm for 5 min, and supernatant from these spins was collected for the analysis of histamine levels. Lung tissues from saline- or IB-MECA-exposed animals were then collected for toluidine blue staining to assess mast cell status.
Toluidine blue staining and counting of lung tissue mast cells
Mice were sacrificed, and lungs were processed, embedded in paraffin, and sectioned as previously described (25). Toluidine blue staining was accomplished by immersing hydrated sections in 0.1% toluidine blue in saline for 1 min, followed by rinsing in water. Toluidine blue-positive mast cell numbers in lung tissues were determined by counting the number of toluidine blue-stained cells in longitudinal sections through one mainstream bronchus. Multiple sections from each lung were analyzed to ensure that the entire length of the bronchus was examined.
ADA enzyme therapy and MRS-1523 treatment
Polyethylene glycol-modified ADA (PEG-ADA), also known as ADAGEN, was obtained from Enzon (Piscataway, NJ). Mice were injected i.m. with doses of PEG-ADA designed to deliver 100–500 U of PEG-ADA/kg of body weight (36). Injections were started on postnatal day 1 and were given every 4 days up to postnatal day 17. MRS-1523 was given to mice at a daily dose of 100 µg/kg of body weight using osmotic pumps (Alzet, Cupertino, CA). MRS-1523 was dissolved in DMSO and diluted in saline. Pumps were filled with MRS-1523 or saline and implanted s.c. into mice 2 days after the last PEG-ADA treatment. Mice were sacrificed, and lung tissues were analyzed 13–15 days after stopping PEG-ADA treatment.
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Results |
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Although the expression of adenosine receptors has been demonstrated in mBMMCs (20), there is little information available on the expression of these receptors in the mouse lung. To determine directly the expression of adenosine receptors on murine lung mast cells, mast cells were isolated from the large airways of mice and expanded in tissue culture. After 4 wk in culture the mast cell population appeared >98% homogeneous as determined by acid toluidine blue staining (Fig. 1A) and c-Kit immunofluorescence (Fig. 1B). Real-time RT-PCR was used to quantify adenosine receptor transcript levels in mPLMCs (Fig. 2). Transcripts for the A1 adenosine receptor were not found, while transcripts for the A2A, A2B, and A3 adenosine receptors were detectable. Among these, the A3 receptor had the highest transcript levels in mPLMCs. Immunofluorescence for the adenosine receptors was performed to verify expression at the protein level (Fig. 3). Consistent with what was found at the RNA level, no fluorescent signal for the A1 receptor was found on the surface of mPLMCs, while immunofluorescence for the A2A, A2B, and A3 receptors was detected. These results suggest that A2A, A2B, and A3 adenosine receptors are expressed on mPLMCs.
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To determine whether mPLMCs could be stimulated to release histamine, cells were incubated overnight with anti-DNP-IgE and were then exposed to DNP-albumin. Basal histamine release was 8.2%, whereas Ag stimulation resulted in >50% histamine release (Table I). To determine whether engagement of adenosine receptors could directly mediate murine lung mast cell degranulation, we challenged wild-type mPLMCs with adenosine at a concentration capable of engaging all the adenosine receptors (100 µM). Adenosine alone was capable of stimulating the release of histamine from mPLMCs (Table I; Fig. 4A). Various adenosine receptor agonists and antagonists were then used to determine which of the adenosine receptors were responsible for this release of histamine (Table I; Fig. 4A). The A2A receptor agonist CGS21680 at 100 nM had no effect on histamine release, suggesting that this receptor was not involved. Since no selective A2B receptor agonist was available, we used the nonselective agonist NECA together with the A2B receptor antagonist enprofylline to assess the role of the A2B receptor. Pretreatment with enprofylline had no effect on NECA-stimulated histamine release, suggesting that the A2B receptor was not involved in this process. In contrast, the A3 receptor agonist IB-MECA was able to stimulate histamine release at a concentration of 100 nM. Furthermore, IB-MECA-stimulated histamine release was completely blocked by pretreatment with the A3-selective antagonist MRS-1523 (5 µM; Fig. 4A). To confirm the role of the A3 receptor on mast cell histamine release, A3-/- mPLMCs were stimulated with IB-MECA or NECA. Neither IB-MECA nor NECA was able to stimulate histamine release from A3-/- mPLMCs (Fig. 4B). These findings suggest that engagement of the A3 adenosine receptor can directly mediate the degranulation of mPLMCs.
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Mast cell degranulation is commonly associated with elevations in intracellular Ca2+ (37). To determine whether engagement of the A3 adenosine receptor could increase intracellular Ca2+ levels in mPLMCs, we exposed mast cells to the A3 agonist IB-MECA and monitored intracellular Ca2+ levels. IB-MECA was able to rapidly induce a rise in intracellular Ca2+. Pretreatment of mPLMCs with the A3 receptor antagonist MRS-1523 completely blocked this response. In contrast, the A2A receptor agonist CGS21680 at 100 nM failed to induce a Ca2+ response (Fig. 5A). In addition, IB-MECA failed to induce a Ca2+ response in A3-/- mPLMCs (Fig. 5B). These results suggest that adenosine mediates mPLMC degranulation by increasing intracellular Ca2+ levels following engagement of the A3 adenosine receptor.
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Recent studies have shown that the cooperative effects of Ag exposure and adenosine on mBMMC degranulation are dependent on A3 receptor activation of PI3K through the Gi pathway (28). To determine whether the Gi pathway or PI3K was involved in A3 receptor-mediated lung mast cell degranulation, mPLMCs were preincubated with the Gi inactivator pertussis toxin (100 ng/ml) for 2 h or with the PI3K inhibitor LY294002 (25 µM) for 20 min at 37°C and then stimulated with IB-MECA (100 nM). Compared with cells treated with IB-MECA alone, cells preincubated with pertussis toxin or LY294002 released much less histamine (Fig. 6A). In addition, intracellular Ca2+ levels were monitored during IB-MECA treatment in untreated cells or in cells pretreated with pertussis toxin or LY 294002. Both pertussis toxin and LY294002 incubation completely abolished the Ca2+ response caused by IB-MECA (Fig. 6B). These data suggest that adenosine-induced mPLMC degranulation is mediated by A3 receptor signaling through the Gi and PI3K pathways.
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As described above, treatment of mPLMCs with the A3 receptor agonist IB-MECA can cause degranulation. To determine whether engagement of A3 receptors on lung mast cells could directly lead to degranulation in vivo, wild-type mice were exposed to nebulized IB-MECA for 5 min and then examined for evidence of mast cell degranulation and histamine release into the airways. IB-MECA caused a pronounced mast cell degranulation in the lungs of wild-type animals; however, IB-MECA was unable to promote degranulation in the airways of A3-/- mice (Fig. 7, A–D). Furthermore, IB-MECA-treated wild-type mice showed a pronounced increase in bronchial alveolar lavage fluid (BALF) histamine levels compared with saline-treated mice, while IB-MECA failed to elevate histamine levels in A3-/- BALF (Fig. 7E). These findings suggest that engagement of the A3 adenosine receptor can directly mediate lung mast cell degranulation in vivo.
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Elevated adenosine levels are associated with lung mast cell degranulation in ADA-deficient mice (25). To determine whether the A3 receptor was involved in mast cell degranulation mediated by endogenous adenosine in ADA-deficient lungs, MRS-1523 was administered to ADA-deficient mice. Control and ADA-deficient mice were maintained on ADA enzyme therapy from birth to prevent adenosine accumulation and mast cell degranulation (25). ADA enzyme therapy was discontinued on day 17, and Alzet implants containing concentrations of MRS-1523 designed to sustain a dose of 100 µg/kg/day, were implanted. Lung mast cells were quantified in saline- or MRS-1523-treated control and ADA-deficient mice at 13–15 days after the cessation of ADA enzyme therapy. As expected, there were no toluidine blue-positive mast cells in the lungs of ADA-deficient mice containing saline-filled Alzet pumps (Fig. 8), a feature consistent with mast cell degranulation in this model (25). However, mast cells were consistently found in the lungs of ADA-deficient mice treated with MRS-1523. These experiments demonstrate that treatment with an A3 receptor antagonist can prevent mast cell degranulation in ADA-deficient lungs, suggesting that the A3 receptor mediates degranulation of lung mast cells in response to endogenous elevations in lung adenosine.
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Discussion |
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Modulation of mast cell function by adenosine has been extensively investigated. In vitro studies in rodents using transformed mast cells, such as RBL-2H3 cells, or cells differentiated in culture, such as mBMMCs, have shown that adenosine can only enhance mediator release from stimulated mast cells, but cannot initiate mast cell degranulation alone (20, 21). Our studies using mPLMCs and studies using human bronchial alveolar lavage mast cells (38) demonstrate that adenosine can directly stimulate mediator release in the absence of other stimuli. Consistent with these observations are whole animal studies demonstrating that adenosine can directly initiate mast cell degranulation in vivo (23, 24). These observations suggest that transformed or in vitro differentiated mast cells may have different adenosine signaling pathways compared with tissue mast cells that develop in vivo. Therefore, examining tissue-derived mast cells will be important in deciphering specific pathways involved in mast cell degranulation.
Isolating and amplifying mPLMCs cells allowed us to examine for the first time the expression profile of adenosine receptors on mast cells isolated from the murine lung (Fig. 2). The A3 receptor had the highest transcript levels, while A2A and A2B adenosine receptors were much lower, and the A1 receptor was not detected. This pattern of adenosine receptor expression was similar to that shown for mBMMCs and RBL-2H3 cells (39); however, the relative expression levels of the adenosine receptors in these cells were not clear. The abundance of A3 receptor transcripts in mPLMCs may reflect its importance in adenosine-mediated mast cell degranulation; however, the function of the A2A and A2B receptors on these cells is not clear. Our data suggest that these receptors do not mediate lung mast cell degranulation in the mouse (Fig. 4). They might serve to promote mast cell survival or the production of cytokines, as has been demonstrated in human transformed mast cells (26). The ability to purify lung mast cells and manipulate them in culture will provide a means to assess the role of these receptors using both genetic and pharmacological approaches.
Previous studies have demonstrated that the A3 receptor is responsible for potentiating Ag-induced degranulation of mBMMCs in vitro (31) and directly initiating cutaneous mast cell degranulation in vivo (27). Our studies were consistent with these findings, in that activation of the A3 adenosine receptor could cause histamine release from lung mast cells both in vitro (Fig. 4) and in vivo (Fig. 7). The A3 receptor agonist IB-MECA induced histamine release from mPLMCs in a dose-dependent manner (data not shown); however, IB-MECA failed to cause histamine release from A3-/- mPLMCs or A3 wild-type cells pretreated with an A3 receptor antagonist. Furthermore, the nonselective agonist NECA failed to induce histamine release from A3-/- mPLMCs, suggesting that A2A or A2B activation does not evoke degranulation of these cells. The function of A3 receptors in vivo was tested by exposing mice to the A3 receptor agonist, IB-MECA. Nebulized IB-MECA directly induced lung mast cell degranulation in wild-type mice, but had no effect in A3-/- mice. Previous studies have shown that mast cell numbers are normal in A3-/- mice, and that expression of the other adenosine receptors is intact (27). Therefore, the failure of IB-MECA-induced mast cell degranulation in A3-/- animals is due to the absence of A3 adenosine receptor function rather than abnormal mast cell numbers or adenosine receptor dysregulation. Therefore, the A3 receptor plays a major role in adenosine-mediated murine lung mast cell degranulation.
Elevations in lung adenosine levels have been demonstrated in asthmatics (8); however, the role of such endogenous adenosine elevations has not been thoroughly examined. Our laboratory has developed a model with which to examine the affects of endogenous adenosine elevations (40). Mice lacking the purine catabolic enzyme ADA accumulate adenosine in their lungs and develop severe lung inflammation. A major feature seen in these mice is adenosine-dependent lung mast cell degranulation (25). Since exogenous A3 agonist could cause mast cell mediator release in vivo, we proposed that elevated endogenous adenosine could also lead to mast cell degranulation by acting on the A3 adenosine receptor. This was confirmed in our experiments using ADA-deficient mice, in which treatment with an A3 receptor antagonist partially prevented mast cell degranulation in response to elevated lung adenosine levels (Fig. 8). The complete absence of Toludine Blue-stained mast cells in untreated ADA-deficient lungs is associated with the loss of mast cell granules, in that these cells can still be found in the lungs using c-Kit immunostaining (25). Increases in the levels of cytokines and chemokines are also found in the lungs of ADA-deficient mice (40, 41). These mediators can lead to mast cell degranulation (42). Therefore, the partial protection of mast cell degranulation by A3 antagonism in these mice might represent the influence of other uncharacterized stimulators of mast cell degranulation in this model.
Activation of recombinant A3 adenosine receptors expressed in HEK-293 cells can increase intracellular Ca2+ via a pertussis toxin-sensitive pathway (43). These findings suggest that Ca2+ responses to A3 activation are probably not Gq mediated, but could be mediated by the subunit of the Gi proteins. In support of this, many basic mast cell secretogues induce exocytosis through the subunit of Gi2 and Gi3 proteins (44), and activation of the A3 receptor on mBMMCs enhance IgE-mediated degranulation via the Gi pathway (28). These findings are consistent with our observations that IB-MECA stimulates mPLMC histamine release and Ca2+ responses through the A3 receptor by a pertussis toxin-sensitive Gi pathway (Fig. 6), and it is likely that this pathway might involve the activation of PI3K through its interaction with the subunit (45). A role of PI3K in mast cell degranulation has been demonstrated using the PI3K inhibitor wortmannin, which completely blocked IgE-mediated mast cell degranulation (46). Our finding that the specific PI3K inhibitor LY294002 completely blocks IB-MECA-induced histamine release and the Ca2+ response (Fig. 6) is consistent with this hypothesis. It is not clear which isoform of PI3K is important in adenosine-mediated mPLMC degranulation due to the lack of isoform selectivity of LY294002. However, PI3K was demonstrated to be the sole isoform downstream of G protein-coupled receptors in neutrophils and macrophages (47), and recent studies using PI3K-/- mice showed that adenosine failed to induce cutaneous mast cell degranulation in these animals (28). Therefore, it is likely that PI3K is activated by the subunit of Gi proteins following A3 receptor activation in murine lung mast cells. Further investigations using isoform-selective PI3K inhibitory reagents are needed to clarify this issue.
Ca2+ influx has been implicated to play a central role in mast cell degranulation induced by Ag or other stimuli (37). Activation of PI3K results in local accumulation of phosphatidylinositol-3,4,5-triphosphate at the plasma membrane. Phosphatidylinositol-3,4,5-triphosphate acts as a signaling messenger that can cause increases in intracellular Ca2+ either by activation of phospholipase C to generate inositol-1,4,5-triphosphate, which releases Ca2+ from intracellular stores, or by directly acting on plasma membrane channels to cause Ca2+ influx (48). Ca2+ responses in mPLMCs induced by IB-MECA were completely quenched by extracellular EGTA (data not shown), suggesting that the Ca2+ elevation in these cells produced by activation of the A3 receptor is mediated though plasma membrane Ca2+ channels. In turn, elevations in intracellular Ca2+ probably regulate the direct effects of A3 receptor engagement on murine lung mast cell degranulation.
Numerous studies have demonstrated that inhaled adenosine or its precursor AMP have potent and specific effects on the asthmatic airway (49) and in the airways of certain COPD patients (11). Most studies suggest that adenosine-induced bronchoconstriction is not due to primary effects on airway smooth muscle or nerves, but involve the promotion or enhancement of mast cell degranulation (50). Evidence to support this includes the observations that pretreatment with mast cell-stabilizing agents (51), histamine blockers (52), or inhibitors of leukotriene signaling (53) can attenuate adenosine-induced bronchoconstriction. In addition, theophyline, a broad-spectrum adenosine receptor antagonist, can attenuate adenosine-induced bronchoconstriction (10), suggesting that signaling through adenosine receptors is involved. However, the specific receptors and the downstream signaling pathways involved in adenosine’s effects on mast cells in the asthmatic lung are not fully understood. Recent studies by Tilley and colleagues (54) demonstrate that the A3 receptor plays an important role in mast cell-mediated bronchoconstriction in the mouse. Taken together with our findings, these studies provide strong support for an A3 receptor signaling pathway in the mediation of adenosine’s effects on airway physiology in the mouse. Whether this pathway is present in the asthmatic lung is not clear at this time and awaits further investigation.
The effects of adenosine on mast cell degranulation vary between species. Our studies and many others suggest that the A3 adenosine receptor is important in mast cell degranulation in rodents (21, 27, 31), whereas other studies demonstrate that the A2B adenosine receptor is important in mast cell degranulation in dogs (55). The specific adenosine receptors involved in the degranulation of human mast cells is not clear; however, A2B activity has been demonstrated in a human tumor mast cell line (26, 43). Therefore, additional studies are needed to definitively characterize the expression of adenosine receptors on human lung mast cells as a first step toward determining which pathways are active in human lung mast cells. Doing so will help guide the development of potential adenosine-based therapeutics for the treatment of asthma and COPD.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael R. Blackburn, Department of Biochemistry and Molecular Biology, University of Texas-Houston Medical School, 6431 Fannin, Houston, TX 77030. E-mail address: michael.r.blackburn{at}uth.tmc.edu
3 Abbreviations used in this paper: COPD, chronic obstructive pulmonary disease; ADA, adenosine deaminase; BALF, bronchial alveolar lavage fluid; mBMMCs, mouse bone marrow-derived mast cells; mPLMCs, murine primary lung mast cells; PEG-ADA, polyethylene glycol-modified ADA; PI3K, phosphoinositide 3-kinase.
Received for publication November 19, 2002. Accepted for publication April 18, 2003.
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) or MRS-1523 (
) was continuously delivered to mice at a daily dose of 100 µg/kg of body weight using Alzet osmotic pumps. Toluidine blue-positive mast cells were quantified in the lungs of control and ADA-deficient mice 13–15 days after the cessation of PEG-ADA enzyme therapy. nd, not detected. n ≥ 5 for each. Error bars indicate the SEM. *, p ≤ 0.05 (by Student’s t test).