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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 References This article cites 52 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/171/1/338.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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, 20–22). 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.
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