Abstract
Adenosine (Ado) has been suggested to play a role in inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease. The goal of this study was to determine the effect of Ado and its receptor subtypes on cytokine release by bronchial smooth muscle cells. The A2B Ado receptor (AdoR) was expressed at the highest level among the four AdoR subtypes. Activation of the A2B AdoR by an Ado analog, 5′-(N-ethylcarboxamido)-adenosine (NECA), increased cAMP accumulation with potency (EC50 value) of 21.2 ± 0.2 μM. The effect of NECA on the expression of the inflammatory cytokines was determined using a cDNA array consisting of 23 cytokine genes and confirmed using real-time reverse transcription–polymerase chain reaction and enzyme-linked immunosorbent assay. NECA increased the release of interleukin-6 and monocyte chemotactic protein-1 proteins with EC50 values of 1.26 ± 0.25 μM and 0.40 ± 0.08 μM, respectively, and the maximal folds of induction were 20.8 ± 1.7– and 6.4 ± 0.7–fold, respectively. Selective agonists for the A1, A2A, and A3 AdoR subtypes had no effect on cytokine release. The effects of NECA were attenuated by selective antagonists of the A2B AdoR. Thus, Ado increases the release of interleukin-6 and monocyte chemotactic protein-1 from bronchial smooth muscle cells via activation of the A2B AdoR. Our findings provide a novel mechanism whereby Ado acts as a proinflammatory mediator in the airway.
Asthma is a chronic disease of the conducting airways characterized by airway inflammation, reversible airway obstruction, and hyperresponsiveness (1). The bronchial smooth muscle cell (BSMC) contributes to the pathophysiologic changes that occur in asthma. The well-characterized functions of the BSMC include its contractile properties, which produce immediate airway narrowing, and its ability to proliferate during airway remodeling (2). Recent studies have suggested a further role of BSMCs in lung inflammation as a source of cytokines and chemokines. Upon stimulation with cytokines, BSMCs can release proinflammatory molecules, including interleukin (IL)-1β, IL-6, granulocyte macrophage colony-stimulating factor (GM-CSF), transforming growth factor (TGF)-β, and eotaxin (3–5).
Adenosine (Ado) is a potent signaling nucleoside that can elicit many physiologic responses by activating G-protein–coupled receptors on the target cells. Inhaled Ado caused bronchoconstriction in patients with asthma (6), and the interstitial concentration of Ado was elevated in the lungs of individuals with asthma (7). Thus, adenosine has been implicated in asthma and chronic obstructive pulmonary disease (COPD) (8). The mechanism of the Ado-mediated bronchoconstriction is most likely due to its effect on mediator release from mast cells (9–12). In addition, Ado modulates the functions of many inflammatory cells such as lymphocytes (13), eosinophils (14), neutrophils (15), and macrophages (16). All of these inflammatory cells are potentially involved in the exacerbation of asthma. As discussed above, increasing evidence indicates that BSMCs play an active role in the inflammatory process by secreting cytokines and chemokines. However, the effect of Ado on inflammatory cytokine release by BSMCs has not been determined.
The goal of the current study was to determine the effects of Ado and its receptor subtypes on cytokine expression and release by BSMCs. We first identified that the A2B Ado receptor (AdoR) is the predominant AdoR subtype in BSMCs. Using a cytokine array, we showed that 5′-(N-ethylcarboxamido)-adenosine (NECA) increased the expression and release of IL-6 and monocyte chemotactic protein (MCP)-1, two important proinflammatory cytokines. In contrast to NECA, selective agonists for the A1, A2A, and A3 AdoR subtypes had no effect on the cytokine expression and release. The selective antagonists of the A2B AdoR attenuated all the effects of NECA. Furthermore, we evaluated the potential involvement of cAMP pathway in NECA-induced IL-6 and MCP-1 production. Our findings indicate that Ado increases the release of IL-6 and MCP-1 by BSMCs via activation of the A2B AdoR and further support that the A2B AdoR antagonist has potential therapeutic utility for the treatment of asthma and COPD.
Dibutyryl cAMP (DBcAMP) and H-89 were purchased from Calbiochem (San Diego, CA). CVT-6694 and 3-isobutyl-8-pyrrolidinoxanthine (IPDX) were made at CV Therapeutics (Palo Alto, CA). All other chemicals (such as forskolin, rolipram, adenosine, NECA, N6-cyclopentyladenosine [CPA], CGS21680, and N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide [IB-MECA]) were from Sigma (St. Louis, MO).
Normal human BSMCs were obtained from Clonetics (San Diego, CA) and cultured using smooth muscle cell growth medium supplemented with 5% fetal bovine serum, 0.5 ng/ml epidermal growth factor, 5 μg/ml insulin, 2 ng/ml fibroblast growth factor, 50 μg/ml gentamicin, and 50 ng/ml amphotericin B. BSMCs were grown under a humidified 5% CO2 atmosphere at 37°C. Immunofluorescence staining of BSMCs using a monoclonal anti-human smooth muscle α-actin antibody (Sigma) demonstrated that the cultures were essentially (> 99%) free of other contaminating cell types. BSMCs from passages 2–5 were used in the following studies.
BSMCs were seeded into 6-well tissue culture plates at a density of 3 × 105 cells/well and allowed to adhere overnight and reach ∼ 80% confluence. Cells were washed twice in HBSS, and cultured in serum-free basal medium (Clonetics) containing antibiotics and various agonists or antagonists of AdoRs. For some experiments, cells were preincubated with 20 μM H-89 for 20 min before stimulation with NECA.
Total RNA was extracted from BSMCs using the Stratagene Absolutely RNA reverse transcription–polymerase chain reaction (RT-PCR) Miniprep Kit followed by DNase treatment to eliminate potential genomic DNA contamination. cDNA was synthesized from 2 μg of total RNA using TaqMan Reverse Transcription Reagents from PE Applied Biosystems (Foster City, CA). TaqMan real-time PCR analysis was applied using 2 μl cDNA per reaction and YBR Green PCR Core Reagents on ABI Prism Sequence Detection System 5,700 (PE Applied Biosystems) according to the manufacturer's instructions. The primers for AdoRs and β-actin were designed as previously described (17). Specific primers for IL-6 (forward: 5′CCGGGAACGAAAGAGAAGCT3′; reverse: 5′CGCTTGTGGAGAAGGAGTTCA3′) and MCP-1 (forward: 5′CGCCTCCAGCATGAAAGTCT3′; reverse: 5′GGAATGAAGGTGGCTGCTATG3′) were designed using Primer Express (PE Applied Biosystems) following the recommended guidelines based on sequences from Genbank. At the end of the PCR cycle, a dissociation curve was generated to ensure that the amplification of a single product, and the threshold cycle times (Ct values) for each gene were determined. Relative mRNA levels were calculated based on the Ct values, normalized to β-actin in the same sample, and presented as percentages of β-actin mRNA.
Expression of cytokines was determined using human inflammation response cytokines via the GEArray kit (SuperArray, Inc., Bethesda, MA). The assay was performed according to manufacturer's instructions. [32P]-labeled cDNA probes were generated from 5–10 μg of total RNA. The cDNA probes were then hybridized with gene-specific cDNA fragments spotted on nylon membrane. The relative expression level of each gene was analyzed using a phosphorimager and Image Quant software (Molecular Dynamics, Sunnyvale, CA).
Cells were harvested using 0.0025% trypsin and 2 mM EDTA in phosphate-buffered saline, washed, and resuspended in phenol-free Dulbecco's modified Eagle's medium to a concentration of 1 × 106 cells/ml, and then incubated with 1 U/ml of adenosine deaminase (Sigma) for 30 min at room temperature. A final concentration of 50 μM of the phosphodiesterase IV inhibitor, rolipram, was added to the cells immediately before addition of adenosine receptor agonists, antagonists, and forskolin. After incubating for 15 min at 37°C, cells were lysed and cAMP concentrations were determined using cAMP-Screen Direct System (Applied Biosystems) according to the manufacturer's instructions.
The concentrations of IL-6 and MCP-1 in the cell medium were determined using enzyme-linked immunosorbent assay (ELISA) kits obtained from R&D Systems (Minneapolis, MN) according to the manufacturer's instructions. The minimal detection levels of IL-6 and MCP-1 with these kits were 0.7 and 5 pg/ml, respectively.
BSMCs were seeded into 6-well tissue culture plates at a density of 2 × 105 cells/well, and allowed to adhere overnight and reach ∼ 50% confluence. Transfection of BSMCs was performed using Fugene 6 transfection reagent (Roche, Indianapolis, IN) according to the manufacturer's instructions. Cells in each well were transfected with 0.5 μg of pAP-1-Luc, pCRE-Luc, pNFAT-Luc, or pNF-κB-Luc (all from Clontech, Palo Alto, CA), and together with 0.5 μg of pSV-β-galactosidase (Promega, Madison, WI) to normalize transfection efficiencies. Forty-eight hours after transfection, cells were washed, cultured in serum-free basal medium, and treated with NECA for 4 h. Cells were then harvested, and luciferase and β-galactosidase activities were assessed using Promega luciferase and β-galactosidase assay systems.
Data were presented as mean ± SEM. Statistical analysis was performed by using a two-tailed, paired Student's t test. Values of P < 0.05 were considered significant.
Real-time RT-PCR was performed to quantify the levels of AdoR mRNA in BSMCs. Among the four subtypes of AdoRs, the A2B AdoR had the highest transcript levels (0.51 ± 0.05% β-actin) (shown in Figure 1)
Figure 1. Real-time RT-PCR analysis of AdoR transcripts in BSMCs. Total RNA isolated from BSMCs was subjected to real-time RT-PCR analysis. The relative AdoR mRNA transcript levels were presented as percentages of the expression level of β-actin mRNA. Data shown are averages ± SEM from four independent experiments. nd, not detected.
[More] [Minimize]Activation of the A2A and A2B AdoRs increased cAMP accumulation via coupling to Gs proteins, whereas activation of the A1 and A3 AdoRs decreased cAMP accumulation induced by forskolin. To identify the AdoR subtypes that are functionally expressed in BSMCs, the effects of a stable adenosine analog NECA (which is a nonselective AdoR agonist that activates all four AdoR subtypes including A2B AdoRs) and several selective AdoR agonists on cAMP accumulations were determined. As shown in Figure 2A
Figure 2. Effects of AdoR agonists and antagonist on cAMP accumulations in BSMCs. (A) Cells were pretreated with ADA (1 U/ml) for 30 min at room temperature to eliminate the endogenous adenosine and then incubated with NECA in the presence (open circle) or absence (open square) of 1 μM A2B adenosine receptor antagonist CVT-6694 or CGS-21680 (open triangle). (B) Cells were incubated with 10 μM forskolin (Fsk) with or without 1 μM CPA and 1 μM IB-MECA. Data shown are averages ± SEM from four independent experiments. *P < 0.05 compared with control; **P < 0.05 compared with NECA-treated cells in A.
[More] [Minimize]The effect of NECA on the gene expression of the inflammatory cytokines was determined using a cDNA array containing 23 inflammatory cytokine genes. BSMCs were incubated with 100 μM NECA or vehicle for 1 h or 6 h, and total RNA was isolated. [32P]-labeled cDNA probes were generated from 5–10 μg of total RNA, and these cDNA probes were then hybridized to nylon membranes that were spotted with gene-specific cDNA fragments from 23 inflammatory cytokine genes. The expression levels of cytokine genes were normalized to the expression level of β-actin gene. Among 23 cytokine genes (listed in Table 1)
|
Gene/Protein Name |
Genbank Accession No. |
|---|---|
| CSF 3 (granulocyte) | X03438 |
| CSF 2 (granulocyte-macrophage) | M11734 |
| IL-1α | M28983 |
| IL-1β | M15330 |
| IL-2 | U25676 |
| IL-4 | M13982 |
| IL-5 | X04688 |
| IL-6 | M14584 |
| IL-8 | M17017 |
| IL-10 | M57627 |
| IL-12A, p35 | M65271 |
| IL-12B, p40 | M65272 |
| IL-16 | M90391 |
| IL-17 | U32659 |
| IL-18 | NM001562 |
| Lymphotoxine α | D12614 |
| Lymphotoxine β | NM_002341 |
| MCP-1 | X14768 |
| Macrophage migration inhibitory factor | NM_002415 |
| TGF-β1 | X02812 |
| TGF-β2 | M19154 |
| TGF-β3 | NM_003239 |
| TNF-α
|
X01394
|
Figure 3. Effect of NECA on expression of IL-6 and MCP-1 determined using cDNA array analysis. BSMCs were incubated with 0.1% DMSO (control) or 100 μM NECA for 1 h or 6 h. The expression levels of IL-6 and MCP-1 were normalized to that of β-actin. Data shown are average ± SEM from three independent experiments. nd, not detected.
[More] [Minimize]To confirm and quantify NECA-induced expression of IL-6 and MCP-1, gene-specific real-time RT-PCR was performed on BSMCs treated with NECA for 1 h. As shown in Figure 4
Figure 4. Effects of AdoR agonist and antagonists on expression of IL-6 and MCP-1 determined using real-time RT-PCR. (A and C) Concentration response curves for the effect of NECA on expression of IL-6 (A) and MCP-1 (C). (B and D) Effect of selective AdoR agonists and antagonists on the expression of IL-6 (B) and MCP-1 (D). BSMCs were incubated with NECA (N, 10 μM), with or without CVT-6694 (6694, 1 μM) or IPDX (10 μM), CPA (1 μM), CGS21680 (CGS, 1 μM), or IB-MECA (1 μM) for 1 h. Cells incubated with 0.1% DMSO vehicle were used as control. The expression levels of IL-6 and MCP-1 were normalized to that of β-actin. Data shown are the averages ± SEM from three independent experiments. *P < 0.05 compared with control; **P < 0.05 compared with NECA-treated cells in B and D.
[More] [Minimize]To further confirm the role of adenosine and NECA in cytokine protein production, IL-6 and MCP-1 concentrations in the culture media from cells treated with adenosine or NECA were measured using ELISA. NECA (10 μM) increased IL-6 and MCP-1 release in a time-dependent manner (Figure 5
Figure 5. Effects of AdoR agonist and antagonists on the release of IL-6 and MCP-1 by BSMCs. (A and B) Time course of the effect of NECA (10 μM) on release of IL-6 (A) and MCP-1 (B). BSMCs were incubated with vehicle (filled bars) or NECA (open bars) for 1, 6, or 24 h. (C and D) Concentration response curves of NECA (squares) and adenosine (triangles). Cells were stimulated with NECA or adenosine for 24 h. (E and F) Effects of selective AdoR agonists and antagonists on the release of IL-6 (E) and MCP-1 (F). BSMCs were incubated with NECA (N, 10 μM) with or without CVT-6694 (6694, 1 μM), or IPDX (10 μM), CPA (1 μM), CGS21680 (CGS, 1 μM), or IB-MECA (1 μM) for 24 h. Media from treated cells were collected, and the concentrations of IL-6 (A, C, E) and MCP-1 (B, D, F) were determined using ELISA. Data shown are averages ± SEM from three independent experiments. *P < 0.05 compared with control; **P < 0.05 compared with NECA-treated cells in E and F.
[More] [Minimize]Activation of A2B AdoRs in BSMCs by NECA increased cAMP accumulation and expression of IL-6 and MCP-1. We determined the potential role of the cAMP pathway in NECA-induced cytokine expression and release in BSMCs. The adenylyl cyclase activator forskolin, a cAMP analog DBcAMP, and a cAMP-dependent kinase inhibitor H-89, were used in this study. DBcAMP (500 μM) and forskolin (10 μM) increased expression and release of IL-6 (Figures 6A and 6C)
Figure 6. Influence of cAMP pathway on NECA-induced mRNA expression and release of IL-6 and MCP-1. BSMCs were incubated with DBcAMP (DBA, 500 μM), forskolin (Fsk, 10 μM), and NECA (10 μM) in the presence or absence of H-89 (20 μM) for 1 h (A and B) or 24 h (C and D). Cells incubated with 0.1% DMSO were used as control. Data shown are averages ± SEM from three independent experiments. *P < 0.05 compared with control; **P < 0.05 compared with NECA-treated cells.
[More] [Minimize]To further determine the effect of NECA on the activities of transcription factors, activator protein-1 (AP-1), cAMP responsive element-binding protein (CREB), nuclear factor of activated T cells (NFAT), and nuclear factor-κB (NF-κB), known to mediate cytokine expression, BSMCs were transfected with pAP-1-Luc, pCRE-Luc, pNFAT-Luc, or pNF-κB-Luc, and then stimulated with NECA. CREB-mediated luciferase activity increased to 12.39 ± 1.03 fold of control after treatment with NECA (100 μM) for 4 h. However, AP-1–, NFAT-, and NF-κB–mediated luciferase activity was not significantly altered by NECA treatment (Figure 7A)
Figure 7. Effect of NECA on specific promoter-mediated transcription. (A) BSMCs transfected with pAP-1-Luc, pCRE-Luc, pNFAT-Luc or pNF-κB-Luc were incubated with NECA (100 μM) for 4 h. (B) BSMCs transfected with pCRE-Luc were incubated with 0.01–100 μM NECA for 4 h. Cells incubated with 0.1% DMSO were used as control. Cells were then harvested, and luciferase and β-galactosidase activities were assessed. Data shown are average ± SEM from 4–8 replicates. *P < 0.05 compared with control.
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The novel findings of this study are that Ado and its stable Ado analog NECA increase the expression and release of IL-6 and MCP-1 by BSMCs, and that this effect of NECA is mediated by the A2B AdoR subtype. To our knowledge, this is the first report on the effect of adenosine and its receptor subtype on inflammatory cytokine releases by BSMCs, and it represents a novel mechanism for the role of Ado in asthma and COPD.
Several reports demonstrated the presence of AdoRs in airway smooth muscle cells from different species. In rat airway smooth muscle cells, transcripts of A1, A2B, and A3 AdoR were detected (19). In contrast, our study demonstrates that in human BSMCs, A2B transcript was highest among the four subtypes of AdoRs; lower levels of A1 and A2A transcripts were also detected, but A3 AdoR transcripts were below detection limit. This difference in AdoR expression between rat and human airway smooth muscle cells is not entirely surprising because differences in AdoR functions in airway smooth muscles from different species have been previously described (20). In addition to gene expression, using cAMP accumulation as a functional readout, we confirmed the presence of functional A2B AdoRs in BSMCs, whereas the presence of functional A1, A2A, and A3 AdoRs were not detected. This result is consistent with another report on the predominant role of the A2B AdoR in mediating cAMP accumulation in human tracheal smooth muscle cells (21).
Adenosine has been suggested to participate in the pathogenesis of allergic airway disease (22) through activation of AdoRs on the inflammatory cells in the airway. However, the role of adenosine and its receptor subtypes on cytokine expression and release by BSMCs had not been previously reported. In this study, we examined the effect of the adenosine analog NECA on cytokine gene expression using an inflammatory cytokine cDNA array, which contains most cytokines implicated in asthma and COPD. Among these 23 human inflammatory cytokines, IL-6 and MCP-1 genes showed the greatest induction by NECA. Expression of some other cytokine genes including macrophage migration inhibitory factor, TGF-β1, and TGF-β2, TGF-β3, and lymphotoxin-α, was detected in both control and NECA-treated BSMCs using the microarray technology, but NECA has no or minimal effects on the expression of these genes. The effect of NECA on the expression of these five genes was also investigated using real-time RT-PCR. The results from RT-PCR confirmed that NECA did not significantly change the expression of these genes (data not shown). The expression of the other 16 genes was below the detection limit by the array technique. IL-6 and MCP-1 are well-characterized inflammatory cytokines. The elevated concentrations of IL-6 and MCP-1 in bronchoalveolar lavage fluid are characteristic features in humans with asthma (23, 24). IL-6, a pleiotropic cytokine, is considered to be a proinflammatory mediator in airway wall inflammation. The effects of IL-6 include promoting mucus hypersecretion (25), stimulating hyperplasia and hypertrophy of cultured guinea-pig airway smooth muscle (26), and inducing subepithelial fibrosis and myofibroblast hyperplasia (27). MCP-1, a C-C class chemokine, is a mediator of both acute and chronic lung inflammation. The functions of MCP-1 include mediating leukocyte infiltration and activation (28), T cell differentiation (29), and airway hyperresponsiveness (30). Our finding that adenosine increases the release of IL-6 and MCP-1 from BSMCs elucidates a novel mechanism for the effect of adenosine in lung inflammation.
Many physiologic roles of Ado are mediated through cell surface AdoRs. In this study, we provided evidence that the A2B AdoR subtype mediates the effect of the Ado analog NECA on IL-6 and MCP-1 release. Our results show the following: (i) The nonselective agonist NECA increased the expression and release of IL-6 and MCP-1, whereas selective agonists for A1, A2A, and A3 AdoRs, such as CPA, CGS-21680, and IB-MECA, had no effect. These agonists are very potent and, at concentrations that range from 0.1–1 μM, they fully activate their cognate receptors without significant activation of the A2B AdoR; at concentrations higher than 1 μM, they may activate A2B AdoRs. This was the rationale for determining the effect of these agonists at a concentration of 1 μM. (ii) The effects of NECA on cytokine release were attenuated by two selective antagonists of the A2B AdoR subtype with the expected rank order of potency. Collectively, these findings provide strong evidence for the role of the A2B AdoR in upregulating the expression and release of IL-6 and MCP-1 caused by NECA.
Transcriptional regulation of IL-6 has been studied intensively in recent years. The promoter of the human IL-6 gene contains the binding sites for AP-1, CREB, NFAT, and NF-κB (31). The MCP-1 promoter contains a variety of transcription factor binding sites, including AP-1– and NF-κB–binding sites (32). In this study, we investigated the potential role of cAMP pathway in NECA-induced expression and release of IL-6 and MCP-1 by BSMCs. Several findings support the role of cAMP pathway: (i) NECA increased cAMP accumulation and expressions of IL-6 and MCP-1, whereas the selective antagonists to A2B AdoR attenuated these effects of NECA; (ii) the adenylyl cyclase activator, forskolin, and the cAMP analog, DB-cAMP, increased cAMP accumulation and expressions of IL-6 and MCP-1; (iii) an inhibitor to cAMP-dependent kinase H-89 attenuated the effect of NECA on expression of IL-6 and MCP-1; and (iv) using various promoter–luciferase constructs transiently transfected into BSMCs, we showed that NECA increased CRE-luciferase activity but had no effect on AP-1–, NFAT-, or NFκB-mediated transcription in BSMCs. Our findings are in agreement with an early report that demonstrated the CREB binding site, but not the NFκB binding site, was critical for adenosine induced IL-6 release by COS 7 cells (33).
Closer examination of the agonist concentration-response curves revealed that NECA was more potent at increasing cytokine expression and release than at increasing cAMP accumulation. The EC50 value of NECA to increase cAMP accumulation was 21 μM, whereas the EC50 values of NECA to increase expression of IL-6 and MCP-1 were 1.3 μM (∼ 16-fold more potent) and 0.2 μM (∼ 100-fold more potent), respectively. It is difficult to reconcile these differences in EC50 values of a given agonist. One plausible explanation is that activation of adenylyl cyclase is proximal to receptor activation and gene expression is distal to receptor activation, and there is progressive amplification of signal along each step of the transduction pathway. Hence, a minor change in cAMP accumulation could result in a major increase in cytokine expression. Regardless of the mechanism that leads to the differences in EC50 values, our study highlighted the importance of determining the agonist potency using the appropriate cellular and physiologic end points rather than using the accumulation of intracellular second messengers.
In summary, the A2B AdoR subtype is the predominant AdoR expressed in BSMCs. Activation of this receptor increased expression and release of IL-6 and MCP-1 in time- and concentration-dependent manners. This process is at least in part mediated via the cAMP pathway. Our findings provide a novel mechanism whereby adenosine acts as a proinflammatory mediator in the airway. Furthermore, these findings suggest that the A2B AdoR antagonist has potential therapeutic utility for the treatment of asthma and COPD.
The authors thank Drs. Jeff Zablocki, Venkata Palla, Rao Kalla, Elfatih Elzein, Ms. Thao Perry, and Ms. Xiaofen Li for their contribution to the discovery and chemical synthesis of CVT-6694, and Dr. Jack Wells for chemical synthesis of IPDX.
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