Receptor 3 Adenosine a Activation of Murine Lung Mast Cells By

Activation of Murine Lung Mast Cells by the Adenosine A 3 Receptor Hongyan Zhong, Sergiy G. Shlykov, Jose G. Molina, Barbara M. Sanborn, Marlene A. Jacobson, Stephen L. Tilley This information is current as and Michael R. Blackburn of September 27, 2021. J Immunol 2003; 171:338-345; ; doi: 10.4049/jimmunol.171.1.338 http://www.jimmunol.org/content/171/1/338 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Activation of Murine Lung Mast Cells by the Adenosine A3 Receptor1

Hongyan Zhong,* Sergiy G. Shlykov,* Jose G. Molina,* Barbara M. Sanborn,* Marlene A. Jacobson,† Stephen L. Tilley,‡ and Michael R. Blackburn2*

Adenosine has been implicated to play a role in asthma in part through its ability to influence mediator release from mast cells. Most physiological roles of adenosine are mediated through adenosine receptors; however, the mechanisms by which adenosine influences mediator release from lung mast cells are not understood. We established primary murine lung mast cell cultures and used real-time RT-PCR and immunofluorescence to demonstrate that the A2A,A2B, and A3 adenosine receptors are expressed on murine lung mast cells. Studies using selective adenosine receptor agonists and antagonists suggested that activation of A3 re- ceptors could induce mast cell histamine release in association with increases in intracellular Ca2؉ that were mediated through Downloaded from Gi and phosphoinositide 3-kinase signaling pathways. 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 while having no effect in A3 receptor knockout mice. Furthermore, studies using adenosine deaminase knockout mice suggested that elevated endogenous adenosine induced lung mast cell degranulation by engaging A3 receptors. These results demonstrate that the A3 adenosine receptor plays an important role in adenosine-mediated murine lung mast cell degranulation. The Journal of Immu- nology, 2003, 170: 338–345. http://www.jimmunol.org/

denosine is an endogenous nucleoside that can be re- (14, 15), epithelial (16), and smooth muscle cells (17). Efforts to leased from metabolically active cells or generated via understand the mechanisms involved in these processes will help A the degradation of extracellular ATP. It is a potent bio- us understand the role of adenosine signaling in the pathogeneses logical signaling molecule that elicits many of its physiological of asthma and COPD. effects by engaging G protein-coupled receptors on target cells (1). Mast cells are principal effector cells in allergic diseases, in- Adenosine signaling plays important roles in the cardiovascular cluding asthma (18), and have been implicated to play an impor- (2), neurological (3), renal (4), and immune systems (5). In addi- tant role in the exacerbation of certain forms of COPD (19). These tion, substantial evidence suggests that adenosine signaling might cells can release mediators, such as histamine, tryptase, leukotri- by guest on September 27, 2021 contribute to the exacerbation of inflammatory lung diseases, such enes, and cytokines, that have both immediate and chronic effects as asthma and chronic obstructive pulmonary disease (COPD)3 (6, on airway constriction and inflammation. Substantial evidence 7). These findings include the observations that asthmatics have suggests that adenosine can modulate mast cell degranulation. elevated lung adenosine concentrations (8), and adenosine receptor Adenosine and adenosine analogs in vitro can enhance mediator transcripts are increased in inflamed lungs (9). In addition, inhaled release from mast cells in response to challenge with a variety of adenosine can provoke bronchoconstriction in asthmatics and COPD patients while having little effect on normal individuals (10, stimuli (12, 20Ð22). In contrast, adenosine can directly initiate 11). Adenosine is also able to influence the function of cells in- mast cell degranulation in the absence of additional stimuli in vivo volved in the exacerbation of asthma, including mast cells (12), (23, 24). These observations are supported by recent studies in lymphocytes (5), eosinophils (9), neutrophils (13), macrophages 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 *Department of Biochemistry and Molecular Biology, University of Texas-Houston elicits these effects are not known; however, these studies demon- Medical School, Houston, TX 77030; †Department of Neuroscience, Merck Research strated that ADA-deficient mice can serve as valuable in vivo mod- Laboratories, West Point, PA 19486; and ‡Department of Medicine, University of North Carolina, Chapel Hill, NC 27599 els to study adenosine signaling in lung mast cells. Received for publication November 19, 2002. Accepted for publication April Most physiological effects of adenosine are mediated through 18, 2003. adenosine receptors. Four subtypes of adenosine receptor, A1,

The costs of publication of this article were defrayed in part by the payment of page A2A,A2B, and A3, have been identified. Each receptor has unique charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tissue distribution, ligand affinity, and signal transduction path- ways (1). Most studies suggest that the A or A adenosine re- 1 This work was supported by a Schissler Foundation Fellowship (to H.Z.), National 2B 3 Institutes of Health Grants AI43572 and HL61888 (to M.R.B.), and a Junior Inves- ceptors are involved in mediating adenosine’s effects on mast cells. tigator Award from the Sandler Family Supporting Foundation (to M.R.B.). The A2B receptor can evoke IL-8 secretion in human HMC-1 mast 2 Address correspondence and reprint requests to Dr. Michael R. Blackburn, Depart- cells (26), and the A receptor is responsible for enhanced hista- ment of Biochemistry and Molecular Biology, University of Texas-Houston Medical 3 School, 6431 Fannin, Houston, TX 77030. E-mail address: michael.r.blackburn@ mine release from mouse bone marrow-derived mast cells (mBM- uth.tmc.edu MCs) and cutaneous mast cells through a Gi protein and phospho- 3 Abbreviations used in this paper: COPD, chronic obstructive pulmonary disease; inositide 3-kinase (PI3K) ␥-dependent pathway (27, 28). However, ADA, adenosine deaminase; BALF, bronchial alveolar lavage fluid; mBMMCs, mouse bone marrow-derived mast cells; mPLMCs, murine primary lung mast cells; due to the heterogeneity of tissue mast cells, little is known about PEG-ADA, polyethylene glycol-modified ADA; PI3K, phosphoinositide 3-kinase. how adenosine affects mast cells in lung tissue. Understanding the

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 339 receptor interactions and downstream signaling mechanisms of ad- was followed by cDNA synthesis and real-time PCR using established enosine’s effects on lung mast cell degranulation, which is a major protocols (35). The resulting data were analyzed using SDS software (PE pathogenic component of asthma and COPD, will help guide new Applied Biosystems) with TAMRA as the reference dye. The final data were normalized to ␤-actin and are presented as molecules of transcript/ therapies for the treatment of these widespread diseases. molecules of ␤-actin ϫ 100 (% ␤-actin). In the current study we examined the expression and function of adenosine receptors on murine lung mast cells. A ,A , and A 2A 2B 3 Activation of mast cells by adenosine receptor agonists adenosine receptors were found to be expressed on murine primary Ϫ/Ϫ lung mast cells (mPLMCs), and studies using selective adenosine Wild-type or A3 mPLMCs were stimulated with adenosine or adeno- ϫ 5 receptor agonists and antagonists suggested that activation of A sine receptor agonists and antagonists at 5 10 cells/ml in medium with- 3 out murine stem cell factor. Adenosine (100 ␮M), 100 nM CGS21680, 10 receptors could induce mPLMC mediator release. Furthermore, ␮M NECA, 100 nM IB-MECA, 25 ␮M enprofylline, and 5 ␮M MRS-1523 this mediator release was associated with increases in intracellular (all from Sigma-Aldrich) were used to activate or antagonize adenosine 2ϩ Ca that were mediated through Gi protein- and PI3K-dependent 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 pathways. In addition, a nebulized A3 receptor agonist directly induced lung mast cell degranulation in wild-type mice while hav- 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 ϫ ing no effect in A3 receptor knockout mice. Finally, lung mast cell g for 5 min. Histamine concentrations were then measured in supernatant degranulation in response to endogenously elevated adenosine was and lysed cell pellets using an enzyme immunoassay (Immunotech, Mar- seilles, France). Data are presented as the percent histamine released or as shown to act through A3 receptors. These results demonstrate that the A adenosine receptor plays an important role in adenosine- absolute values released into the supernatant. The percent histamine re- 3 leased was determined by dividing the concentration of histamine in the Downloaded from mediated murine lung mast cell degranulation. supernatant by total histamine levels (supernatant plus pellet). For some experiments cells were preincubated with 100 ng/ml pertussis toxin (Sig- Materials and Methods ma-Aldrich) for2horwith 25 ␮M LY294002 (Calbiochem, San Diego, Transgenic mice CA) for 20 min before stimulation with IB-MECA.

Ϫ/Ϫ ADA-deficient mice and A3 receptor-deficient (A3 ) mice were gener- ated and genotyped as previously described (29Ð31). ADA-deficient mice Intracellular calcium measurements were on a mixed background of 129/Sv, C57BL/6, and FVB/N stains. http://www.jimmunol.org/ Ϫ Ϫ The mPLMC were plated on 20% Matrigel-coated, 35-mm, glass-bottom A / mice were inbred on the C57BL/6 strain, as were the wild-type mice 3 microwell dishes (Mattek, Ashland, MA) and then loaded at room temper- used for the generation of mPLMCs. ature with 5 ␮M fura 2-AM (Molecular Probes, Eugene, OR) for 40 min in Cell culture 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 The mPLMC cultures were established as previously described (32). fura 2 fluorescence in individual cells were measured at 340 and 380 nm Briefly, lung mast cells were isolated from the upper airways of wild-type excitation and 510 nm emission wavelengths using an InCyt2 Im2 imaging Ϫ/Ϫ ϳ 3 and A3 mice by cutting the tissue into 3-mm pieces, which were then system (Intracellular Imaging, Cincinnati, OH). Fluorescence ratios were incubated in DMEM containing 10 ng/ml murine stem cell factor and IL-3 monitored using Im2 software to approximate intracellular calcium con- (Sigma-Aldrich, St. Louis, MO). Culture medium was changed every other centrations. Data are presented as representative tracings from 30Ð40 in-

day for the first week. At the end of 1 wk nonadherent cells were collected dividual cells on experiments performed on two different dishes on by guest on September 27, 2021 and transferred to fresh medium. These cultures were passaged in the same separate days. manner for 2Ð3 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 IB-MECA treatment and bronchial alveolar lavages had been in culture for 4Ð8 wk were used in the experiments. C2C12 cells Ϫ/Ϫ Wild-type and A3 mice were treated with nebulized saline or 1 mM were obtained from American Type Culture Collection (Manassas, VA), IB-MECA for 5 min. Mice were anesthetized with avertin, and a blunted and RBL-2H3 cells were a gift from Dr. B. Dickey (Baylor College of 21-gauge needle was secured in the trachea. Lungs were lavaged three Medicine, Houston, TX). Both cell types were cultured according to pro- times with 0.4 ml of PBS, and 1Ð1.2 ml of pooled lavage fluid was col- tocols provided by American Type Culture Collection. lected. Samples were centrifuged at 1200 rpm for 5 min, and supernatant Acid toluidine blue staining and immunofluorescence for c-Kit from these spins was collected for the analysis of histamine levels. Lung tissues from saline- or IB-MECA-exposed animals were then collected for and adenosine receptors toluidine blue staining to assess mast cell status. The mPLMCs were cytospun onto microscope slides, C2C12 and RBL- 2H3 cells were grown on Falcon culture slides overnight, and mPLMCs Toluidine blue staining and counting of lung tissue mast cells were stained with acid toluidine blue (pH 1.0) according to established protocols (33). For immunofluorescence, cells were fixed in 1% formalin in Mice were sacrificed, and lungs were processed, embedded in paraffin, and PBS for 10 min, and permeabilized in 0.2% Triton X-100 for 5 min on ice. sectioned as previously described (25). Toluidine blue staining was accom- For c-Kit immunofluorescence, cells were blocked with 10% normal goat plished by immersing hydrated sections in 0.1% toluidine blue in saline for serum for 20 min and incubated with 10 ␮g/ml polyclonal rabbit c-Kit Ab 1 min, followed by rinsing in water. Toluidine blue-positive mast cell num- at 4¡C overnight. After washing, cells were incubated with 5 ␮g/ml goat bers in lung tissues were determined by counting the number of toluidine anti-rabbit IgG-FITC for 1 h. For adenosine receptor immunofluorescence, blue-stained cells in longitudinal sections through one mainstream bron- cells were blocked with 10% normal donkey serum for 20 min and incu- chus. Multiple sections from each lung were analyzed to ensure that the bated with 20 ␮g/ml polyclonal goat anti-rat adenosine receptor Abs at 4¡C entire length of the bronchus was examined. overnight. After washing, cells were incubated with 5 ␮g/ml donkey anti- goat IgG-FITC, then washed, counterstained with 1 ␮g/ml bisbenzimide (Sigma-Aldrich) for 2 min to visualize nuclei, and coverslipped. All Abs ADA enzyme therapy and MRS-1523 treatment were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyethylene glycol-modified ADA (PEG-ADA), also known as ADA- Quantitative real-time RT-PCR GEN, was obtained from Enzon (Piscataway, NJ). Mice were injected i.m. with doses of PEG-ADA designed to deliver 100Ð500 U of PEG-ADA/kg Quantitative real-time RT-PCR was performed using a 7700 Sequence De- of body weight (36). Injections were started on postnatal day 1 and were tector (PE Applied Biosystems, Foster City, CA). Specific quantitative as- given every 4 days up to postnatal day 17. MRS-1523 was given to mice says for adenosine receptors were developed using Primer Express soft- at a daily dose of 100 ␮g/kg of body weight using osmotic pumps (Alzet, ware (PE Applied Biosystems) following the recommended guidelines Cupertino, CA). MRS-1523 was dissolved in DMSO and diluted in saline. based on sequences from GenBank (34). Total RNA was isolated from Pumps were filled with MRS-1523 or saline and implanted s.c. into mice mPLMCs using an RNeasy Mini Kit (Qiagen, Valencia, CA), followed by 2 days after the last PEG-ADA treatment. Mice were sacrificed, and lung DNase treatment to eliminate potential genomic DNA contamination. This tissues were analyzed 13Ð15 days after stopping PEG-ADA treatment. 340 ADENOSINE RECEPTOR-MEDIATED LUNG MAST CELL DEGRANULATION

Results

A2A,A2B, and A3 adenosine receptors are expressed on mPLMCs Although the expression of adenosine receptors has been demon- strated 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 Ͼ98% 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 re- FIGURE 2. Real-time RT-PCR analysis of adenosine receptor tran- scripts in mPLMCs. Total RNA isolated from mPLMCs was subjected to ceptor transcript levels in mPLMCs (Fig. 2). Transcripts for the A1 real-time RT-PCR analysis. The relative adenosine receptor mRNA tran- adenosine receptor were not found, while transcripts for the A2A, A , and A adenosine receptors were detectable. Among these, script levels were calculated by dividing the adenosine receptor levels by 2B 3 ␤ ϭ the A receptor had the highest transcript levels in mPLMCs. Im- the -actin levels measured in the same RNA preparations. n 4. Error 3 bars indicate the SEM. nd, not detected. munofluorescence for the adenosine receptors was performed to verify expression at the protein level (Fig. 3). Consistent with what Downloaded from

was found at the RNA level, no fluorescent signal for the A1 re- ceptor was found on the surface of mPLMCs, while immunoflu- tors could directly mediate murine lung mast cell degranulation, orescence for the A2A,A2B, and A3 receptors was detected. These results suggest that A ,A , and A adenosine receptors are ex- we challenged wild-type mPLMCs with adenosine at a concentra- 2A 2B 3 ␮ pressed on mPLMCs. tion capable of engaging all the adenosine receptors (100 M). Adenosine alone was capable of stimulating the release of hista- http://www.jimmunol.org/ Activation of the A3 adenosine receptor leads to mediator mine from mPLMCs (Table I; Fig. 4A). Various adenosine recep- release from primary lung mast cells tor agonists and antagonists were then used to determine which of To determine whether mPLMCs could be stimulated to release the adenosine receptors were responsible for this release of hista- histamine, cells were incubated overnight with anti-DNP-IgE and mine (Table I; Fig. 4A). The A2A receptor agonist CGS21680 at were then exposed to DNP-albumin. Basal histamine release was 100 nM had no effect on histamine release, suggesting that this

8.2%, whereas Ag stimulation resulted in Ͼ50% histamine release receptor was not involved. Since no selective A2B receptor agonist (Table I). To determine whether engagement of adenosine recep- 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 by guest on September 27, 2021

NECA-stimulated histamine release, suggesting that the A2B re-

ceptor 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 his- tamine release was completely blocked by pretreatment with the ␮ A3-selective antagonist MRS-1523 (5 M; Fig. 4A). To confirm Ϫ/Ϫ 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 Ϫ/Ϫ A3 mPLMCs (Fig. 4B). These findings suggest that engage-

ment of the A3 adenosine receptor can directly mediate the de- granulation of mPLMCs.

A3 receptor-mediated mast cell degranulation is associated with elevations in intracellular Ca2ϩ Mast cell degranulation is commonly associated with elevations in intracellular Ca2ϩ (37). To determine whether engagement of the 2ϩ A3 adenosine receptor could increase intracellular Ca levels in

mPLMCs, we exposed mast cells to the A3 agonist IB-MECA and monitored intracellular Ca2ϩ levels. IB-MECA was able to rapidly induce a rise in intracellular Ca2ϩ. Pretreatment of mPLMCs with

the A3 receptor antagonist MRS-1523 completely blocked this re-

sponse. In contrast, the A2A receptor agonist CGS21680 at 100 nM failed to induce a Ca2ϩ response (Fig. 5A). In addition, IB-MECA FIGURE 1. Cytospun mPLMCs were subjected to acid toluidine blue failed to induce a Ca2ϩ response in A Ϫ/Ϫ mPLMCs (Fig. 5B). staining (A) and immunofluorescence for c-Kit (B). To control for Ab spec- 3 ificity, cells were stained with c-Kit Ab preincubated with a 5-fold excess These results suggest that adenosine mediates mPLMC degranu- 2ϩ of the peptide used to generate the Ab (C). Scale bars in A and C ϭ 10 ␮m lation by increasing intracellular Ca levels following engage-

and also apply to B. ment of the A3 adenosine receptor. The Journal of Immunology 341

FIGURE 3. Immunofluorescence for adenosine receptors. Immunofluorescence for the A1,A2A, A2B, and A3 adenosine receptors was performed on mPLMCs (C and D). C2C12 cells were used as a positive control for the A1 receptor, and RBL-2H3 cells were used as positive controls for the A2A,A2B, and A3 receptors (A and B). To control for Ab spec- ificity, cells were stained with adenosine receptor Abs preincubated with a 5-fold excess of the peptide used to generate the Abs (B and D). Scale bar in D ϭ 10 ␮m and applies to AÐD Downloaded from http://www.jimmunol.org/

Inactivation of Gi and inhibition of PI3K prevent histamine engagement of A3 receptors on lung mast cells could directly lead 2ϩ release and the Ca response caused by A3 receptor to degranulation in vivo, wild-type mice were exposed to nebu- engagement lized IB-MECA for 5 min and then examined for evidence of mast Recent studies have shown that the cooperative effects of Ag ex- cell degranulation and histamine release into the airways. IB- posure and adenosine on mBMMC degranulation are dependent on MECA caused a pronounced mast cell degranulation in the lungs of wild-type animals; however, IB-MECA was unable to promote A3 receptor activation of PI3K through the Gi pathway (28). To Ϫ/Ϫ degranulation in the airways of A3 mice (Fig. 7, AÐD). Fur- determine whether the Gi pathway or PI3K was involved in A3 receptor-mediated lung mast cell degranulation, mPLMCs were thermore, IB-MECA-treated wild-type mice showed a pronounced increase in bronchial alveolar lavage fluid (BALF) histamine lev- by guest on September 27, 2021 preincubated with the Gi inactivator pertussis toxin (100 ng/ml) for ␮ els compared with saline-treated mice, while IB-MECA failed to 2 h or with the PI3K inhibitor LY294002 (25 M) for 20 min at Ϫ/Ϫ 37¡C and then stimulated with IB-MECA (100 nM). Compared elevate histamine levels in A3 BALF (Fig. 7E). These findings with cells treated with IB-MECA alone, cells preincubated with suggest that engagement of the A3 adenosine receptor can directly pertussis toxin or LY294002 released much less histamine (Fig. mediate lung mast cell degranulation in vivo. 6A). In addition, intracellular Ca2ϩ levels were monitored during IB-MECA treatment in untreated cells or in cells pretreated with Mast cell degranulation in ADA-deficient lungs is mediated pertussis toxin or LY 294002. Both pertussis toxin and LY294002 through A receptors incubation completely abolished the Ca2ϩ response caused by IB- 3 MECA (Fig. 6B). These data suggest that adenosine-induced Elevated adenosine levels are associated with lung mast cell de- granulation in ADA-deficient mice (25). To determine whether the mPLMC degranulation is mediated by A3 receptor signaling A3 receptor was involved in mast cell degranulation mediated by through the Gi and PI3K pathways. endogenous adenosine in ADA-deficient lungs, MRS-1523 was

Nebulized A3 receptor agonist can directly stimulate lung tissue administered to ADA-deficient mice. Control and ADA-deficient mast cell degranulation mice were maintained on ADA enzyme therapy from birth to pre- vent adenosine accumulation and mast cell degranulation (25). As described above, treatment of mPLMCs with the A3 receptor agonist IB-MECA can cause degranulation. To determine whether ADA enzyme therapy was discontinued on day 17, and Alzet im- plants containing concentrations of MRS-1523 designed to sustain a dose of 100 ␮g/kg/day, were implanted. Lung mast cells were Table I. Histamine content and release from primary murine lung quantified in saline- or MRS-1523-treated control and ADA-defi- mast cellsa cient mice at 13Ð15 days after the cessation of ADA enzyme ther- apy. As expected, there were no toluidine blue-positive mast cells Treatment Conditions in the lungs of ADA-deficient mice containing saline-filled Alzet pumps (Fig. 8), a feature consistent with mast cell degranulation in Control IgE-DNP Adenosine NECA this model (25). However, mast cells were consistently found in Supernatant (nM) 7.2 Ϯ 1.2 46.3 Ϯ 1.0 22.2 Ϯ 2.0 25.0 Ϯ 1.8 the lungs of ADA-deficient mice treated with MRS-1523. These Ϯ Ϯ Ϯ Ϯ Pellet (nM) 80.4 2.3 44.2 4.0 66.4 0.9 63.5 1.7 experiments demonstrate that treatment with an A receptor an- % Release 8.2 51.2 25.1 28.3 3 tagonist can prevent mast cell degranulation in ADA-deficient a Wild-type primary murine lung mast cells were exposed to medium alone (con- lungs, suggesting that the A3 receptor mediates degranulation of trol) or various treatment conditions for 20 min. Cell supernatants and pellets were assayed for histamine concentrations, and values are presented as the mean nanomolar lung mast cells in response to endogenous elevations in lung concentration Ϯ SEM (n ϭ 3 for each). adenosine. 342 ADENOSINE RECEPTOR-MEDIATED LUNG MAST CELL DEGRANULATION Downloaded from http://www.jimmunol.org/ FIGURE 4. Histamine release from mPLMCs stimulated with adenosine FIGURE 6. Histamine release and changes in intracellular Ca2ϩ con- or adenosine receptor agonists. mPLMCs from wild-type mice were incubated centrations in mast cells preincubated with pertussis toxin (p. toxin) or ␮ ϩ with 100 M adenosine (Ado) to activate all adenosine receptors, 100 nM LY294002. Histamine levels in the medium (A) and intracellular Ca2 ␮ CGS21680 (CGS) to activate A2A receptors, 10 M NECA to activate all concentrations (B) were measured in the mPLMCs preincubated with 100 receptors including A2B, 100 nM IB-MECA (MECA) to activate A3 receptors, ng/ml pertussis toxin for2htoinactivate the G pathway or 25 ␮M ␮ ␮ i 10 M NECA together with 25 M enprofylline (ENP), and 100 nM IB- LY294002 for 20 min to inhibit PI3K. Cells incubated with medium only ␮ ,ء .MECA together with 5 M MRS-1523 (MRS) for 20 min (A). All compounds were used as the control. n ϭ 4 for each. Error bars indicate the SEM were dissolved in DMSO and diluted in DMEM. There was no difference in -p Յ 0.05 compared with IB-MECA ,ءء ;p Յ 0.05, compared with control histamine release from cells incubated with 1% DMSO and cells incubated treated mice (by Student’s t test). IB-MECA was added as indicated by the with medium only (data not shown). Therefore, cells incubated with medium by guest on September 27, 2021 Ϫ/Ϫ arrow in B. only were used as controls. mPLMCs from A3 mice were incubated with ␮ Ϫ/Ϫ 100 nM IB-MECA or 10 M NECA. As positive controls, A3 cells were preincubated with 100 ng/ml anti DNP IgE overnight and stimulated with 10 logically active compounds. Adenosine has been shown to stimu- ng/ml DNP-albumin (B). Histamine levels in the medium were assessed by late or enhance mast cell degranulation, but few studies have p Յ ,ء .enzyme immunoassay. n ϭ 4 for each. Error bars indicate the SEM p Յ 0.05 compared with agonist-treated mice investigated the mechanisms of adenosine’s effects on lung tissue ,ءء ;compared with control 0.05 (by Student’s t test). mast cells. In the current study we demonstrate that adenosine can directly stimulate murine lung mast cell degranulation both in vitro

and in vivo by activating the A3 adenosine receptor. This response Discussion to adenosine on lung mast cells occurred in the absence of Ag Mast cells play a central role in allergic and inflammatory reac- stimulation and involved intracellular Ca2ϩ elevations that were

tions involved in asthma through the release of a variety of bio- mediated through Gi and PI3K signaling pathways.

FIGURE 5. Changes in intracellular Ca2ϩ concentrations in mast cells stimulated with adenosine receptor agonists. Changes in intracellular Ca2ϩ ϩϩ concentrations ([Ca ]i) were determined when wild-type mPLMCs were stimulated with 100 nM IB-MECA (MECA) to activate A3 receptors or 100 nM ␮ CGS21680 (CGS) to activate A2A receptors or were pretreated with 5 M MRS-1523 (MRS) to antagonize A3 receptors and then stimulated with 100 nM 2ϩ Ϫ/Ϫ IB-MECA (A). In addition, intracellular Ca levels were measured in wild-type and A3 primary lung mast cells while 100 nM IB-MECA (MECA) was added (B). Compounds were added as indicated by the arrows. The Journal of Immunology 343 Downloaded from

Ϫ/Ϫ FIGURE 7. Phenotype of mast cells and histamine levels in BALF in IB-MECA-treated mice. Wild-type and A3 mice were exposed to nebulized saline or 1 mM IB-MECA for 5 min. Sections through bronchi were stained with toluidine blue for the detection of mast cells (arrows). A, Wild-type mice Ϫ/Ϫ Ϫ/Ϫ treated with saline. B, Wild-type mice treated with IB-MECA. C,A3 mice treated with saline. D,A3 mice treated with IB-MECA. Inset in B and D demonstrates airway mast cells at a higher magnification. Both scale bars in D ϭ 20 ␮m and apply to AÐD. Histamine levels in BALF collected from Յ ء ϩ/ϩ ϭ these same animals were measured using enzyme immunoassay (E), wild-type (A3 ). n 4 for each. Error bars indicate the SEM. , p 0.05 (by Student’s t test). http://www.jimmunol.org/

Modulation of mast cell function by adenosine has been exten- portant in deciphering specific pathways involved in mast cell sively investigated. In vitro studies in rodents using transformed mast degranulation. cells, such as RBL-2H3 cells, or cells differentiated in culture, such as Isolating and amplifying mPLMCs cells allowed us to examine mBMMCs, have shown that adenosine can only enhance mediator for the first time the expression profile of adenosine receptors on release from stimulated mast cells, but cannot initiate mast cell de- mast cells isolated from the murine lung (Fig. 2). The A3 receptor granulation alone (20, 21). Our studies using mPLMCs and studies had the highest transcript levels, while A2A and A2B adenosine using human bronchial alveolar lavage mast cells (38) demonstrate by guest on September 27, 2021 receptors were much lower, and the A1 receptor was not detected. that adenosine can directly stimulate mediator release in the absence This pattern of adenosine receptor expression was similar to that of other stimuli. Consistent with these observations are whole animal shown for mBMMCs and RBL-2H3 cells (39); however, the rel- studies demonstrating that adenosine can directly initiate mast cell ative expression levels of the adenosine receptors in these cells degranulation in vivo (23, 24). These observations suggest that trans- were not clear. The abundance of A3 receptor transcripts in formed or in vitro differentiated mast cells may have different aden- mPLMCs may reflect its importance in adenosine-mediated mast osine signaling pathways compared with tissue mast cells that develop cell degranulation; however, the function of the A2A and A2B re- in vivo. Therefore, examining tissue-derived mast cells will be im- ceptors 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 phar- macological approaches.

Previous studies have demonstrated that the A3 receptor is re- sponsible for potentiating Ag-induced degranulation of mBMMCs in vitro (31) and directly initiating cutaneous mast cell degranu- lation in vivo (27). Our studies were consistent with these findings,

in that activation of the A3 adenosine receptor could cause hista- mine release from lung mast cells both in vitro (Fig. 4) and in vivo

FIGURE 8. Toluidine blue-positive mast cell numbers in ADA-defi- (Fig. 7). The A3 receptor agonist IB-MECA induced histamine cient mice treated with the A3 adenosine receptor antagonist MRS-1523. release from mPLMCs in a dose-dependent manner (data not Control and ADA-deficient mice were maintained on PEG-ADA enzyme shown); however, IB-MECA failed to cause histamine release therapy until postnatal day 17. Two days following the last PEG-ADA from A Ϫ/Ϫ mPLMCs or A wild-type cells pretreated with an A treatment, saline (Ⅺ) or MRS-1523 (f) was continuously delivered to mice 3 3 3 ␮ receptor antagonist. Furthermore, the nonselective agonist NECA at a daily dose of 100 g/kg of body weight using Alzet osmotic pumps. Ϫ/Ϫ Toluidine blue-positive mast cells were quantified in the lungs of control failed to induce histamine release from A3 mPLMCs, suggest- and ADA-deficient mice 13Ð15 days after the cessation of PEG-ADA en- ing that A2A or A2B activation does not evoke degranulation of zyme therapy. nd, not detected. n Ն 5 for each. Error bars indicate the these cells. The function of A3 receptors in vivo was tested by Յ ء SEM. , p 0.05 (by Student’s t test). exposing mice to the A3 receptor agonist, IB-MECA. Nebulized 344 ADENOSINE RECEPTOR-MEDIATED LUNG MAST CELL DEGRANULATION

IB-MECA directly induced lung mast cell degranulation in wild- Ca2ϩ influx has been implicated to play a central role in mast Ϫ/Ϫ type mice, but had no effect in A3 mice. Previous studies have cell degranulation induced by Ag or other stimuli (37). Activation Ϫ/Ϫ shown that mast cell numbers are normal in A3 mice, and that of PI3K results in local accumulation of phosphatidylinositol- expression of the other adenosine receptors is intact (27). There- 3,4,5-triphosphate at the plasma membrane. Phosphatidylinositol- fore, the failure of IB-MECA-induced mast cell degranulation in 3,4,5-triphosphate acts as a signaling messenger that can cause Ϫ/Ϫ 2ϩ A3 animals is due to the absence of A3 adenosine receptor increases in intracellular Ca either by activation of phospho- function rather than abnormal mast cell numbers or adenosine re- lipase C␥ to generate inositol-1,4,5-triphosphate, which releases 2ϩ ceptor dysregulation. Therefore, the A3 receptor plays a major role Ca from intracellular stores, or by directly acting on plasma ϩ ϩ in adenosine-mediated murine lung mast cell degranulation. membrane channels to cause Ca2 influx (48). Ca2 responses in Elevations in lung adenosine levels have been demonstrated in mPLMCs induced by IB-MECA were completely quenched by 2ϩ asthmatics (8); however, the role of such endogenous adenosine extracellular EGTA (data not shown), suggesting that the Ca elevations has not been thoroughly examined. Our laboratory has elevation in these cells produced by activation of the A3 receptor 2ϩ developed a model with which to examine the affects of endoge- is mediated though plasma membrane Ca channels. In turn, eleva- 2ϩ nous adenosine elevations (40). Mice lacking the purine catabolic tions in intracellular Ca probably regulate the direct effects of A3 enzyme ADA accumulate adenosine in their lungs and develop receptor engagement on murine lung mast cell degranulation. severe lung inflammation. A major feature seen in these mice is Numerous studies have demonstrated that inhaled adenosine or adenosine-dependent lung mast cell degranulation (25). Since ex- its precursor AMP have potent and specific effects on the asthmatic airway (49) and in the airways of certain COPD patients (11). Most ogenous A3 agonist could cause mast cell mediator release in vivo, we proposed that elevated endogenous adenosine could also lead studies suggest that adenosine-induced bronchoconstriction is not Downloaded from due to primary effects on airway smooth muscle or nerves, but to mast cell degranulation by acting on the A3 adenosine receptor. This was confirmed in our experiments using ADA-deficient mice, involve the promotion or enhancement of mast cell degranulation (50). Evidence to support this includes the observations that pre- in which treatment with an A3 receptor antagonist partially pre- vented mast cell degranulation in response to elevated lung aden- treatment with mast cell-stabilizing agents (51), histamine blockers osine levels (Fig. 8). The complete absence of Toludine Blue- (52), or inhibitors of leukotriene signaling (53) can attenuate ad- stained mast cells in untreated ADA-deficient lungs is associated enosine-induced bronchoconstriction. In addition, theophyline, a http://www.jimmunol.org/ broad-spectrum adenosine receptor antagonist, can attenuate ade- with the loss of mast cell granules, in that these cells can still be nosine-induced bronchoconstriction (10), suggesting that signaling found in the lungs using c-Kit immunostaining (25). Increases in through adenosine receptors is involved. However, the specific re- the levels of cytokines and chemokines are also found in the lungs ceptors and the downstream signaling pathways involved in ade- of ADA-deficient mice (40, 41). These mediators can lead to mast nosine’s effects on mast cells in the asthmatic lung are not fully cell degranulation (42). Therefore, the partial protection of mast understood. Recent studies by Tilley and colleagues (54) demon- cell degranulation by A antagonism in these mice might represent 3 strate that the A receptor plays an important role in mast cell- the influence of other uncharacterized stimulators of mast cell de- 3 mediated bronchoconstriction in the mouse. Taken together with granulation in this model. by guest on September 27, 2021 our findings, these studies provide strong support for an A recep- Activation of recombinant A adenosine receptors expressed in 3 3 tor signaling pathway in the mediation of adenosine’s effects on HEK-293 cells can increase intracellular Ca2ϩ via a pertussis tox- airway physiology in the mouse. Whether this pathway is present in-sensitive pathway (43). These findings suggest that Ca2ϩ re- in the asthmatic lung is not clear at this time and awaits further sponses to A activation are probably not G mediated, but could 3 q investigation. be mediated by the ␤␥ subunit of the G proteins. In support of this, i The effects of adenosine on mast cell degranulation vary be- many basic mast cell secretogues induce exocytosis through the ␤␥ tween species. Our studies and many others suggest that the A3 subunit of Gi2 and Gi3 proteins (44), and activation of the A3 adenosine receptor is important in mast cell degranulation in ro- receptor on mBMMCs enhance IgE-mediated degranulation via dents (21, 27, 31), whereas other studies demonstrate that the A2B the Gi pathway (28). These findings are consistent with our obser- adenosine receptor is important in mast cell degranulation in dogs vations that IB-MECA stimulates mPLMC histamine release and (55). The specific adenosine receptors involved in the degranula- 2ϩ Ca responses through the A3 receptor by a pertussis toxin-sen- tion of human mast cells is not clear; however, A2B activity has sitive Gi pathway (Fig. 6), and it is likely that this pathway might been demonstrated in a human tumor mast cell line (26, 43). ␤␥ involve the activation of PI3K through its interaction with the Therefore, additional studies are needed to definitively character- subunit (45). A role of PI3K in mast cell degranulation has been ize the expression of adenosine receptors on human lung mast cells demonstrated using the PI3K inhibitor wortmannin, which com- as a first step toward determining which pathways are active in pletely blocked IgE-mediated mast cell degranulation (46). Our human lung mast cells. Doing so will help guide the development finding that the specific PI3K inhibitor LY294002 completely of potential adenosine-based therapeutics for the treatment of 2ϩ blocks IB-MECA-induced histamine release and the Ca re- asthma and COPD. sponse (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 Acknowledgments LY294002. 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