help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on June 15, 2006, doi:10.1165/rcmb.2005-0476OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0476OCv1
35/5/587    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhong, H.
Right arrow Articles by Zeng, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhong, H.
Right arrow Articles by Zeng, D.
American Journal of Respiratory Cell and Molecular Biology. Vol. 35, pp. 587-592, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0476OC

A2B Adenosine Receptors Induce IL-19 from Bronchial Epithelial Cells, Resulting in TNF-{alpha} Increase

Hongyan Zhong, Yuzhi Wu, Luiz Belardinelli and Dewan Zeng

Department of Drug Research and Pharmacological Sciences, CV Therapeutics, Inc., Palo Alto, California

Correspondence and requests for reprints should be addressed to Hongyan Zhong, Ph.D., CV Therapeutics, Inc., 3172 Porter Drive, Palo Alto, CA 94304. E-mail: hongyan.zhong{at}cvt.com


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Adenosine is a signaling nucleoside that has been proposed to contribute to the pathogenesis of asthma and chronic obstructive pulmonary disease. Previous studies suggest that adenosine might play an important role in modulating levels of inflammatory mediators in the lung. Because airway epithelium is an important cellular source of inflammatory mediators, the objective of the present study was to determine whether adenosine affects the expression and release of inflammatory cytokines from human bronchial epithelial cells (HBECs). Among the four subtypes of adenosine receptors, the A2B receptor was expressed at the highest level. 5'-(N-ethylcarboxamido)-adenosine (NECA), a stable analog of adenosine, increased the release of IL-19 by 4.6- ± 1.1-fold. A selective antagonist of the A2B receptor, CVT-6694, attenuated this effect of NECA. The amount of IL-19 released from HBEC was sufficient to activate a human monocytic cell line (THP-1) and increase the release of TNF-{alpha}. Furthermore, TNF-{alpha} was found to upregulate A2B receptor expression in HBECs by 3.1- ± 0.3-fold. Hence, these data indicate that NECA increases the release of IL-19 from HBECs via activation of A2B receptors, and IL-19 in turn activates human monocytes to release TNF-{alpha}, which upregulates A2B receptor expression in HBECs. The results of this study suggest that there is a novel pathway whereby adenosine can initiate and amplify an inflammatory response which might be important in pathogenesis of inflammatory lung diseases.

Key Words: adenosine • TNF-{alpha} • bronchial epithelial cells • IL-19


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Airway epithelium is known to play an important role in airway defense mechanism via a mucociliary system and as a mechanical barrier. Recent studies further indicate that the epithelium is not only a barrier but can also actively generate a range of molecules, including lipid mediators, growth factors, and a variety of cytokines/chemokines, that are important in the inflammatory and remodeling responses that occur in the lungs (1). In asthma, the epithelium seems more sensitive and responds abnormally to various stimuli (2). In addition, airway epithelial cells are able to interact with immune and inflammatory cells via direct adhesion as well as by releasing mediators including cytokines (1). Thus, the epithelium is actively involved as regulator of airway inflammatory responses important in the pathogenesis of airway disorders.

Adenosine is a nucleoside that can elicit many physiologic and pathophysiologic responses by activating one or more of its four subtypes of G protein–coupled receptors (A1, A2A, A2B, and A3) on target cells. Adenosine has been proposed to contribute to the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD) (3). This hypothesis is based on the findings that the interstitial concentration of adenosine is elevated in the lungs of individuals with asthma (4) and inhaled adenosine causes bronchoconstriction in patients with asthma (5). The bronchoconstrictive effect of adenosine has been attributed to the activation of lung mast cells (6, 7). In addition to the mast cells (8, 9), adenosine has been found to modulate the functions of other inflammatory cells such as lymphocytes (10), eosinophils (11, 12), neutrophils (13), and macrophages (14), and lung cells such as bronchial smooth muscle cells (15, 16) and fibroblasts (17). The physiologic effects of adenosine on lung epithelial cells have also been investigated. Adenosine is able to modulate the activity of ion channels (18–20), and induce fibronectin (21) and mucin gene expression (22). However, it is unknown whether and how adenosine affects the release of inflammatory cytokines from epithelial cells and epithelial cell–inflammatory cell communication.

In this study, we used the primary cultured human bronchial epithelial cells (HBECs) as the model system. The objectives of this study were to determine (1) whether adenosine affects the release of inflammatory cytokines from bronchial epithelial cells, (2) what the potential effects of these cytokines on lung inflammation might be, and (3) which adenosine receptor subtype is responsible for the effect of adenosine.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Materials
A selective antagonist to the A2B receptor (CVT-6694) was synthesized by the Department of Bio-Organic Chemistry at CV Therapeutics Inc. (Palo Alto, CA), and was described in our previous publication (15). All other reagents, such as rolipram, forskolin, 5'-(N-ethylcarboxamido)-adenosine (NECA), and adenosine deaminase (ADA), were purchased from Sigma (St. Louis, MO) unless otherwise stated.

Cell Culture
Primary cultured normal HBECs were obtained from Clonetics (San Diego, CA) and cultured using bronchial epithelial cell growth medium (Clonetics). HBECs were routinely grown in a humidified incubator with 5% CO2 at 37°C. Cells from three different donors and from passages 2–3 were used in the following studies. THP-1 cells were purchased from ATCC (Manassas, VA) and cultured according to ATCC's instructions.

Stimulation of HBECs
HBECs were seeded into 12-well tissue culture plates at a density of 1 x 105 cells/well and allowed to adhere overnight and reach ~ 90% confluence. Cells were washed twice in HEPES-buffered saline, and cultured in bronchial epithelial cell basal medium (Clonetics) containing various agonists or antagonists of AdoRs for 1 or 24 h.

RNA Extraction and Real-Time RT-PCR
Total RNA was extracted from HBECs using the Stratagene Absolutely RNA RT-PCR Miniprep Kit (Stratagene Corp., La Jolla, CA) followed by DNase treatment to eliminate potential genomic DNA contamination. Real-time RT-RCR for adenosine receptors was performed as previously described (15). Specific primers for IL-19 (forward: 5'-AAACAATCTCCCCAAGGTGGAT-3'; reverse: 5'-AGGAAATGCTGTCAAGGTTTGC-3'), GRO-beta (forward: 5'-TTTCTTCGTGATGACATATCACATGT-3'; reverse: 5'-TCCTCAGCCTCTATCACAGTGG-3'), and GRO-{gamma} (forward: 5'-TGCTTGTAGGGCATAATGCCT-3'; reverse: 5'-GGGAAAGAGAAACGCTGCAG-3') were designed using Primer Express 2.0 (Applied Biosystems, Foster City, CA) following the recommended guidelines based on sequences from GenBank. At the end of the PCR cycle, a dissociation curve was generated to ensure 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 beta-actin in the same sample, and presented as percentages of beta-actin mRNA.

Measurement of cAMP Accumulation
Cells were harvested using 0.0025% trypsin and 2 mM EDTA in PBS, washed and resuspended in phenol-free DMEM to a concentration of 5 x 105 cells/ml, and then incubated with 1 U/ml of ADA for 30 min at room temperature. Cells were then treated with AdoR agonists, antagonists, and forskolin in the presence of 50 µM of the phosphodiesterase IV inhibitor, rolipram. 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.

cDNA Array Analysis
Expression of inflammatory cytokines was determined using a cDNA array kit (Cat. No. HS-033; SuperArray, Frederick, MD). The assay was performed according to manufacturer's instructions. Biotinylated cDNA probes were generated from 1–2 µg of total RNA using GEArray RT-labeling enzyme kit (SuperArray). The labeled cDNA probes were then hybridized with gene-specific cDNA fragments spotted on nylon membrane. The signals were detected with the chemiluminescent detection reagents. The relative expression level of each gene was analyzed using GEArray analysis software (SuperArray).

Measurement of IL-19 and TNF-{alpha}
ELISA for IL-19 was developed according to manufacturer's protocol (R&D Systems, Minneapolis, MN). Plates (96-well) were pre-coated with 50 ng/well anti-human IL-19 Ab (R&D Systems). IL-19 was detected using 100 ng/ml of biotinylated polyclonal IL-19 Ab (R&D systems), streptavidin-HRP, and TMB (tetramethylbenzidine) chromogen (Biosource, Camarillo, CA). Serial dilutions of recombinant human IL-19 (rhIL-19; PeproTech, Rocky Hill, NJ) was used as standard. The detection limit of rhIL-19 was 0.2 ng/ml. The concentrations of TNF-{alpha} in the cell medium were determined using ELISA kits obtained from Biosource according to the manufacturer's instructions. The minimal detection level of TNF-{alpha} with these kits was 1.7 pg/ml.

Statistical Analysis
Data are presented as mean ± SEM of at least three separate experiments. The statistical analysis was performed using ANOVA followed by Bonferroni test. A P value of < 0.05 was considered significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Expression of AdoR Subtypes in HBECs
Real-time RT-PCR was performed to quantify the levels of transcripts for AdoRs. Among the four subtypes, the A2B receptor had the highest transcript level (0.49 ± 0.04% of beta-actin expression) (Figure 1). Lower levels of A1 and A2A receptor transcripts were also detected (0.0012 ± 0.0002% and 0.0058 ± 0.0003% of beta-actin, respectively), whereas the transcript for A3 receptors was below the detection level. Hence, the rank order of AdoR mRNA levels was A2B > > A2A > A1 > > A3.


Figure 1
View larger version (6K):
[in this window]
[in a new window]
  		Figure 1. The mRNA levels of AdoR subtypes in HBECs. Total RNA isolated from HBECs was subjected to real-time RT-PCR analysis. The relative levels of the AdoR transcripts are presented as percentages of the beta-actin transcript. Data shown are averages ± SEM (n = 6). nd denotes not detected.

 
In many cell types, activation of A2A or A2B receptors leads to increases in cellular cAMP accumulation, whereas activation of A1 or A3 receptors decreases cellular cAMP accumulation caused by the adenylate cyclase activator, forskolin. To identify the AdoR subtype(s) that are functionally expressed in HBECs, the effects of a nonselective agonist NECA and several other selective agonists on cellular cAMP accumulation were determined. NECA is a stable analog of adenosine, and it activates all four AdoR subtypes including A2B receptors. As shown in Figure 2A, NECA increased cellular cAMP accumulation in a concentration-dependent manner, with potency (EC50 value) of 8.8 ± 1.3 µM. In contrast, the A2A-selective agonist CGS-21680 (<= 10 µM) did not cause a significant increase in cellular cAMP concentration. In addition, the A1-selective agonist, CPA (1 µM), and the A3-selective agonist, IB-MECA (1 µM), failed to inhibit the cellular cAMP accumulation caused by forskolin (10 µM, Figure 2B). Because there is no selective agonist for A2B receptors, the effect of a selective antagonist to A2B receptors, CVT-6694, on NECA-induced cellular cAMP accumulation was determined. CVT-6694 has a high affinity for the A2B receptor (Ki = 7 nM) and very low affinity for three other AdoR subtypes (Ki values are > 5 µM for A1, A2A, and A3 receptors) (15, 17). As shown in Figure 2A, CVT-6694 (1 µM) significantly attenuated NECA-induced cellular cAMP accumulation. Thus, using cellular cAMP concentration as readout for the functional expression of AdoRs, the results indicate that A2B receptors are functionally expressed in HBECs, whereas A1, A2A, or A3 receptors are not.


Figure 2
View larger version (13K):
[in this window]
[in a new window]
  		Figure 2. Effects of AdoR agonists and antagonist on cellular cAMP accumulation in HBECs. (A) Concentration–response curves of CGS-21680 (CGS, circles) and NECA in the absence (squares) or presence (triangles) of the A2B receptor antagonist CVT-6694 (1 µM). (B) Lack of effect of CPA (1 µM) and IB-MECA (IM, 1 µM) on forskolin (Fsk, 10 µM)-induced cellular cAMP accumulation. Data shown are averages ± SEM (n = 3). *P < 0.05, compared with control; #P < 0.05, compared with NECA-treated cells in A.

 
Effects of NECA on Expression of Inflammatory Cytokines
The effect of NECA on the gene expression of the inflammatory cytokines was determined using a cDNA array containing 85 inflammatory genes involved in asthma. Among the 85 genes on the microarray, GRO-beta, GRO-{gamma}, and IL-19 genes were increased above 2-fold by NECA (data not shown).

To confirm and quantify NECA-induced expression of GRO-beta, GRO-{gamma}, and IL-19, gene-specific real-time RT-PCR was performed on HBECs treated with NECA (10 µM) for 1 h. As shown in Figure 3, NECA increased mRNA expression of IL-19, GRO-beta, and GRO-{gamma} up to 4.3- ± 0.9-, 5.6- ± 0.6-, and 4.3- ± 0.6-fold above control levels, respectively.


Figure 3
View larger version (8K):
[in this window]
[in a new window]
  		Figure 3. Effects of NECA on the mRNA levels of GRO-beta, GRO-{gamma}, and IL-19 determined using real-time RT-PCR. HBECs were incubated with NECA (10 µM) for 1 h. Cells incubated with vehicle were used as control. The expression levels of the cytokines were normalized to that of beta-actin and are presented as the ratios of the expression level in NECA-treated cells versus that in vehicle-treated cells. The expression levels of GRO-beta, GRO-{gamma}, and IL-19 in control cells are 0.020 ± 0.003%, 0.008 ± 0.002%, and 0.168 ± 0.025% of beta-actin, respectively. Data shown are averages ± SEM (n = 3).

 
Activation of the A2B Receptor Increased the Release of IL-19 from HBECs
Recent publications suggesting a potential role of IL-19 in lung inflammation prompted us to determine the effect of NECA on IL-19 release. IL-19 concentrations in the culture media from cells treated with NECA were measured using ELISA. The levels of IL-19 in the media from vehicle- and NECA-treated cells for 24 h were 19.2 ± 6.3 ng/ml and 89.2 ± 20.2 ng/ml, respectively (Figure 4). Hence, NECA (10 µM) caused 4.6- ± 1.1-fold increase of IL-19 release compared with vehicle-treated cells. To determine the role of A2B receptors in NECA-induced IL-19 production, cells were incubated with CVT-6694 (1 µM) together with NECA. The A2B receptor antagonist CVT-6694 (1 µM) reduced the NECA-increased IL-19 release by 88.9 ± 0.5%. These results confirmed that NECA-induced IL-19 release is mediated by the A2B receptor subtype.


Figure 4
View larger version (7K):
[in this window]
[in a new window]
  		Figure 4. Effect of NECA on the release of IL-19 by HBECs. Cells were treated with vehicle, NECA in the absence or presence of CVT-6694 for 24 h. Media from treated cells were collected, and the concentrations of IL-19 were determined using ELISA. CVT-6994 alone did not change the release of IL-19 by HBECs (data not shown). Data shown are the averages ± SEM (n = 3). *P < 0.05, compared with control; #P < 0.05, compared with NECA-treated cells.

 
Effects of IL-19 Released from HBEC on Activation of a Human Monocytic Cell Line (THP-1)
To determine whether the amount of IL-19 released from HBEC can activate inflammatory cells, the effect of IL-19 alone or the conditional medium from NECA-treated HBEC on the release of TNF-{alpha} from a monocytic cell line (THP-1) was determined. The basal level of TNF-{alpha} in the media from vehicle-treated (24 h) THP-1 cells was 7.5 ± 0.5 pg/ml. IL-19 (100 ng/ml) increased the concentrations of TNF-{alpha} in the media by 4.3- ± 0.4-fold (Figure 5A). In contrast, GRO-beta (100 ng/ml), GRO-{gamma} (100 ng/ml), or NECA (10 µM) alone had no significant effect on TNF-{alpha} release from THP-1 cells (Figure 5A). These data demonstrate that IL-19, but not NECA or GRO-beta/{gamma}, was able to activate THP-1 cells. Furthermore, the conditional media collected from HBECs treated with vehicle (control-M) or NECA (NECA-M) for 24 h were used to incubate THP-1 cells for an additional 24 h. NECA-M caused a 4.6- ± 1.0-fold increase in TNF-{alpha} release from THP-1 cells compared with control-M. This effect was completely abolished by an IL-19–neutralizing Ab (Figure 5B). These results suggest that IL-19 released from NECA-stimulated HBECs is able to and sufficient to activate THP-1 cells.


Figure 5
View larger version (11K):
[in this window]
[in a new window]
  		Figure 5. Effects of cytokines and conditional medium from HBECs on TNF-{alpha} release from THP-1 cells. (A) THP-1 cells were incubated with vehicle (control), GRO-beta (100 ng/ml), GRO-{gamma} (100 ng/ml), IL-19 (100 ng/ml), or NECA (10 µM) for 24 h. (B) Conditional medium collected from HBECs that were treated with vehicle (control-M) or NECA (NECA-M) for 24 h was used to incubate THP-1 cells in the absence or presence of polyclonal IL-19 Ab (1 µg/ml) for an additional 24 h. The concentration of TNF-{alpha} in media was measured using ELISA. Data shown are the averages ± SEM (n = 4). *P < 0.05, compared with control (A) or control-M (B); #P < 0.05, compared with NECA-M in B.

 
Effects of TNF-{alpha} on Expression of AdoRs in HBECs
To further explore the interaction between epithelial cells and monocytes, the effect of TNF-{alpha} on AdoR expression in HBECs was determined using real-time RT-PCR. TNF-{alpha} caused a significant time-dependent increase in mRNA expression of A2B receptors (Figure 6). After 24 h incubation with TNF-{alpha}, mRNA level of A2B receptors was increased to 3.1 ± 0.3 fold above basal levels (Figure 6). Incubation of TNF-{alpha} for 5 and 24 h also significantly upregulated the mRNA levels of A1 and A2A receptors up to 2.0- ± 0.3- and 1.6- ± 0.1-fold above basal levels, although the expression of both subtypes of receptors was still very low (below 0.01% of beta-actin mRNA level). The mRNA expression of the A3 receptors remained below the detection level after TNF-{alpha} treatment (Figure 6).


Figure 6
View larger version (15K):
[in this window]
[in a new window]
  		Figure 6. Effect of TNF-{alpha} on the mRNA levels of AdoR subtypes in HBECs. HBECs were incubated with TNF-{alpha} (5 ng/ml) for 0, 1, 5, and 24 h. The relative levels of the AdoR transcripts are presented as percentages of the beta-actin transcript. Data shown are averages ± SEM (n = 3). nd denotes not detected. *P < 0.05, compared with 0 h.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The main objective of this study was to determine the effect of adenosine on the inflammatory responses mediated by epithelial cells. The main findings of our study are: (1) A2B receptors are the predominant subtype of AdoRs expressed in primary HBECs; (2) activation of A2B receptors on HBECs increases the release of IL-19, which is able to and sufficient to induce TNF-{alpha} production from human monocytes; and (3) TNF-{alpha} can upregulate the expression of A2B receptors in HBECs. These findings support the hypothesis that adenosine can initiate and amplify inflammatory responses which might be important in pathogenesis of inflammatory lung diseases.

Among the four subtypes of AdoRs, the A2B receptor has the lowest affinity for the natural ligand, adenosine, requiring at least micromolar concentrations of adenosine for its activation (23). Hence, under normal physiologic conditions, adenosine levels might be too low to activate the A2B receptor. However, higher levels of adenosine have been detected during hypoxia, ischemia, and inflammation (24–26). In addition, results of recent studies (16, 17, 26, 27) suggest the A2B receptor expression can markedly increase during inflammation and hypoxia. The molecular mechanism of how A2B receptor expression is modulated during inflammation is largely unknown. The results of the current study demonstrate that TNF-{alpha}, a well-known inflammatory marker and mediator, upregulates the expression of A1, A2A, and A2B receptors in HBECs. This is consistent with an earlier report suggesting that TNF-{alpha} regulates the expression of A2A and A2B receptors in microvascular endothelial cells (28).

It should be noted that the expression of A2B receptors are upregulated in the lung of ragweed-challenged mice (27) and IL-13–transgenic mice (26). More strikingly, increased expression of A2B receptors in the lung of IL-13–transgenic mice has been localized in bronchial epithelium. On the other hand, results of a recent study suggested that the density of A2B receptor, determined using saturation binding assays with an antagonist radioligand, was significantly decreased in the lungs of patients with COPD compared with the control group, whereas the radioligand affinity for A2B receptors was not altered (29). However, it is not entirely clear which cells contribute to the decrease of A2B receptors in COPD, how the expression of A2B receptor is regulated in allergic diseases versus COPD, and the precise role of A2B receptor in these diseases. Interestingly, in the same study, the expression level of the A2B receptor was found to be highest in mast cells and macrophages. Regardless, the authors concluded that the A2B receptor might play an important role in the pathogenesis of COPD. Therefore, future studies to determine the effect of A2B antagonist in COPD would be of interest.

Functional expression of the A2B receptor in airway epithelial cells has been reported. This receptor has been shown to mediate adenosine-modulated ion transport (18, 19); however, the function of A2B receptor in mediating cytokine release is not known. The results of our study showed that activation of A2B receptors upregulates the expression of proinflammatory cytokines such as IL-19, GRO-beta, and GRO-{gamma}. GRO-beta (CXCL2) and GRO-{gamma} (CXCL3) are CXC chemokines. The functions of these two chemokines are similar to IL-8 (CXCL8), which belongs to the same chemokine family. GRO-beta and GRO-{gamma} chemoattract and activate neutrophils (30), and promote angiogenesis by activating the CXCR2 receptor on endothelial cells (31). Because neutrophils have been implicated in the pathogenesis of inflammatory lung diseases including asthma and COPD, and because CXC chemokine–mediated angiogenesis is associated with idiopathic pulmonary fibrosis (32), CXCR2 is a potential therapeutic target for these diseases. A more important finding of the present study is that NECA increases the release of IL-19 from HBECs. IL-19 belongs to the IL-10 family, which includes IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26 (33). In contrast to the pleiotropic role of IL-10, IL-19 has been shown to have mainly proinflammatory roles. IL-19 stimulates T cells to produce IL-4 and IL-13 (34) and alters the balance of Th1/Th2 in favor of Th2. IL-19 also induces production of IL-6, TNF-{alpha}, and reactive oxygen species from mouse monocytes (35). IL-19 level is elevated in the serum of patients with asthma compared with healthy control subjects, and its transcript is also increased in the lung of an allergen-challenged mouse model (34). Because adenosine is elevated in the asthmatic lung to hundred-micromolar range (4), and at this level it can increase the release of IL-19 from HBECs (data not shown), it is plausible to postulate that elevated adenosine in the asthmatic lung may contribute to the elevation of IL-19. Interestingly, recent studies have shown that elevated adenosine play a key role in the upregulation of IL-13 and several other cytokines in mouse models of pulmonary diseases (26).

Consistent with earlier reports on the proinflammatory roles of IL-19, the results of our study demonstrate that IL-19 released by bronchial epithelial cells can activate human monocytes. In the present study, conditional medium of NECA-stimulated HBECs was able to activate THP-1 cells. This effect is not due to the activation of AdoRs on THP-1 cells, because this effect was completely abolished by the IL-19–neutralizing Ab and NECA alone did not stimulate TNF-{alpha} production from THP-1 cells. Furthermore, the results in this study suggest that elevated TNF-{alpha} could have a positive feedback on the expression of the adenosine receptors on the epithelial cells. Hence, adenosine may play an important role in the interaction between epithelial cells and inflammatory cells in the airway.

Activation of the A2B receptor increases cAMP, which is generally believed to elicit anti-inflammatory responses. However, proinflammatory roles of this receptor have been reported (36). For example, in human mast cells (HMC-1), activation of A2B receptors increase cAMP (37) and also increase the release of IL-4, IL-8, and IL-13 (8). A possible explanation is that A2B receptor can couple to other cell signaling pathways, such as Ca/PLC, and MAP kinases (38), and these pathways are likely to mediate the proinflammatory effect of adenosine or adenosine analog via activation of A2B receptors. However, it remains to be established which signaling pathway is responsible for the A2B-mediated upregulation of IL-19. It should be noted that anti-inflammatory roles of the A2B receptor have also been reported in other in vitro studies (39–41). Therefore, further studies using animal models or patients will be helpful to determine the role of A2B receptors in the lung.

In summary, adenosine, IL-19, and TNF-{alpha} are important proinflammatory mediators in lung inflammation. These mediators may interact with one another to cause the alleviation or exacerbation of the disease. The results of the present study demonstrated that the A2B receptor subtype is the predominant AdoR expressed in HBECs. Activation of this AdoR subtype increased the release of IL-19. IL-19 released from these cells, in turn, stimulated the human monocytes to release TNF-{alpha}. TNF-{alpha} is able to upregulate the expression of A2B receptors in HBECs (Figure 7). Thus, our findings provide a novel mechanism that could explain the interactions among adenosine, IL-19, and TNF-{alpha} in the initiation, maintenance, and amplification of inflammatory responses. In addition, our findings suggest that the A2B receptor might be a novel therapeutic target for inflammatory lung diseases.


Figure 7
View larger version (13K):
[in this window]
[in a new window]
  		Figure 7. Schematic representation of an inflammatory response initiated and amplified by adenosine involving HBECs and monocytes. Adenosine, via activation of the A2B receptors, increases the release of IL-19 from HBECs. IL-19, in turn, stimulates human monocytes to release TNF-{alpha}. TNF-{alpha} upregulates A2B receptor expression in HBECs.

 

   Footnotes
 
An abstract (less than 250 words) related to this study has been published for the American Thoracic Society International Conference in 2005: Zhong H, Wu Y, Belardinelli L, Zeng D. Adenosine indirectly activates monocytes by releasing IL-19 from human bronchial epithelial cells. Proc Am Thorac Soc 2005;2:A110.

Originally Published in Press as DOI: 10.1165/rcmb.2005-0476OC on June 15, 2006

Conflict of Interest Statement: H.Z., Y.W., L.B., and D.Z. are employees of CV Therapeutics, Inc. (CVT) and own stock and stock options in this company. CVT has patented numerous A2B antagonists, and one such antagonist is currently being developed for the treatment of asthma.

Received in original form December 21, 2005

Accepted in final form June 1, 2006


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Takizawa H. Airway epithelial cells as regulators of airway inflammation [review]. Int J Mol Med 1998;1:367–378.[Medline]
  2. Holgate ST. Epithelial damage and response. Clin Exp Allergy 2000;30:37–41.[CrossRef][Medline]
  3. Holgate ST, Church MK, Polosa R. Adenosine: a positive modulator of airway inflammation in asthma. Ann N Y Acad Sci 1991;629:227–236.[Medline]
  4. Driver AG, Kukoly CA, Ali S, Mustafa SJ. Adenosine in bronchoalveolar lavage fluid in asthma. Am Rev Respir Dis 1993;148:91–97.[Medline]
  5. Cushley MJ, Tattersfield AE, Holgate ST. Adenosine-induced bronchoconstriction in asthma: antagonism by inhaled theophylline. Am Rev Respir Dis 1984;129:380–384.[Medline]
  6. Cushley MJ, Holgate ST. Adenosine-induced bronchoconstriction in asthma: role of mast cell-mediator release. J Allergy Clin Immunol 1985;75:272–278.[CrossRef][Medline]
  7. Polosa R, Ng WH, Crimi N, Vancheri C, Holgate ST, Church MK, Mistretta A. Release of mast-cell-derived mediators after endobronchial adenosine challenge in asthma. Am J Respir Crit Care Med 1995;151:624–629.[Abstract]
  8. Ryzhov S, Goldstein AE, Matafonov A, Zeng D, Biaggioni I, Feoktistov I. Adenosine-activated mast cells induce IgE synthesis by B lymphocytes: an A2B-mediated process involving Th2 cytokines IL-4 and IL-13 with implications for asthma. J Immunol 2004;172:7726–7733.[Abstract/Free Full Text]
  9. Zhong H, Shlykov SG, Molina JG, Sanborn BM, Jacobson MA, Tilley SL, Blackburn MR. Activation of murine lung mast cells by the adenosine A3 receptor. J Immunol 2003;171:338–345.[Abstract/Free Full Text]
  10. Huang S, Apasov S, Koshiba M, Sitkovsky M. Role of A2a extracellular adenosine receptor-mediated signaling in adenosine-mediated inhibition of T-cell activation and expansion. Blood 1997;90:1600–1610.[Abstract/Free Full Text]
  11. Walker BA, Jacobson MA, Knight DA, Salvatore CA, Weir T, Zhou D, Bai TR. Adenosine A3 receptor expression and function in eosinophils. Am J Respir Cell Mol Biol 1997;16:531–537.[Abstract]
  12. Young HW, Molina JG, Dimina D, Zhong H, Jacobson M, Chan LN, Chan TS, Lee JJ, Blackburn MR. A3 adenosine receptor signaling contributes to airway inflammation and mucus production in adenosine deaminase-deficient mice. J Immunol 2004;173:1380–1389.[Abstract/Free Full Text]
  13. Cronstein BN. Adenosine regulation of neutrophil function and inhibition of inflammation via adenosine receptors. In: Jacobson KA, Jarvis MF, editors. Purinergic approaches in experimental therapeutics. New York: Wiley-Liss; 1997. pp. 285–299.
  14. Hasko G, Szabo C, Nemeth ZH, Kvetan V, Pastores SM, Vizi ES. Adenosine receptor agonists differentially regulate IL-10, TNF-alpha, and nitric oxide production in RAW 264.7 macrophages and in endotoxemic mice. J Immunol 1996;157:4634–4640.[Abstract]
  15. Zhong H, Belardinelli L, Maa T, Feoktistov I, Biaggioni I, Zeng D. A2B adenosine receptors increase cytokine release by bronchial smooth muscle cells. Am J Respir Cell Mol Biol 2004;30:118–125.[Abstract/Free Full Text]
  16. Feoktistov I, Ryzhov S, Zhong H, Goldstein AE, Matafonov A, Zeng D, Biaggioni I. Hypoxia modulates adenosine receptors in human endothelial and smooth muscle cells toward an A2B angiogenic phenotype. Hypertension 2004;44:649–654.[Abstract/Free Full Text]
  17. Zhong H, Belardinelli L, Maa T, Zeng D. Synergy between A2B adenosine receptors and hypoxia in activating human lung fibroblasts. Am J Respir Cell Mol Biol 2005;32:2–8.[Abstract/Free Full Text]
  18. Huang P, Lazarowski ER, Tarran R, Milgram SL, Boucher RC, Stutts MJ. From the Cover: Compartmentalized autocrine signaling to cystic fibrosis transmembrane conductance regulator at the apical membrane of airway epithelial cells. Proc Natl Acad Sci USA 2001;98:14120–14125.[Abstract/Free Full Text]
  19. Kornerup KN, Page CP, Moffatt JD. Pharmacological characterisation of the adenosine receptor mediating increased ion transport in the mouse isolated trachea and the effect of allergen challenge. Br J Pharmacol 2005;144:1011–1016.[CrossRef][Medline]
  20. Szkotak AJ, Ng AM, Sawicka J, Baldwin SA, Man SF, Cass CE, Young JD, Duszyk M. Regulation of K(+) current in human airway epithelial cells by exogenous and autocrine adenosine. Am J Physiol Cell Physiol 2001;281:C1991–C2002.[Abstract/Free Full Text]
  21. Roman J, Rivera HN, Roser-Page S, Sitaraman SV, Ritzenthaler JD. Adenosine induces fibronectin expression in lung epithelial cells: implications for airway remodeling. Am J Physiol Lung Cell Mol Physiol 2006;290:L317–L325.[Abstract/Free Full Text]
  22. McNamara N, Gallup M, Khong A, Sucher A, Maltseva I, Fahy JV, Basbaum C. Adenosine up-regulation of the mucin gene, MU2, in asthma. FASEB J 2004;18:1770–1772.[Abstract/Free Full Text]
  23. Fredholm BB, Irenius E, Kull B, Schulte G. Comparison of the potency of adenosine as an agonist at human adenosine receptors expressed in Chinese hamster ovary cells. Biochem Pharmacol 2001;61:443–448.[CrossRef][Medline]
  24. Mentzer RM Jr, Rubio R, Berne RM. Release of adenosine by hypoxic canine lung tissue and its possible role in pulmonary circulation. Am J Physiol 1975;229:1625–1631.[Abstract/Free Full Text]
  25. Hagberg H, Andersson P, Lacarewicz J, Jacobson I, Butcher S, Sandberg M. Extracellular adenosine, inosine, hypoxanthine, and xanthine in relation to tissue nucleotides and purines in rat striatum during transient ischemia. J Neurochem 1987;49:227–231.[CrossRef][Medline]
  26. Blackburn MR, Lee CG, Young HW, Zhu Z, Chunn JL, Kang MJ, Banerjee SK, Elias JA. Adenosine mediates IL-13-induced inflammation and remodeling in the lung and interacts in an IL-13-adenosine amplification pathway. J Clin Invest 2003;112:332–344.[CrossRef][Medline]
  27. Fan M, Qin W, Mustafa SJ. Characterization of adenosine receptor(s) involved in adenosine-induced bronchoconstriction in an allergic mouse model. Am J Physiol Lung Cell Mol Physiol 2003;284:L1012–L1019.[Abstract/Free Full Text]
  28. Khoa ND, Montesinos MC, Reiss AB, Delano D, Awadallah N, Cronstein BN. Inflammatory cytokines regulate function and expression of adenosine A(2A) receptors in human monocytic THP-1 cells. J Immunol 2001;167:4026–4032.[Abstract/Free Full Text]
  29. Varani K, Caramori G, Vincenzi F, Adcock I, Casolari P, Leung E, MacLennan S, Gessi S, Morello S, Barnes PJ, et al. Alteration of Adenosine Receptors in Patients with Chronic Obstructive Pulmonary Disease. Am J Respir Crit Care Med 2006;173:398–406.[Abstract/Free Full Text]
  30. Geiser T, Dewald B, Ehrengruber M, Clark-Lewis I, Baggiolini M. The interleukin-8-related chemotactic cytokines GRO alpha, GRO beta, and GRO gamma activate human neutrophil and basophil leukocytes. J Biol Chem 1993;268:15419–15424.[Abstract/Free Full Text]
  31. Romagnani P, Lasagni L, Annunziato F, Serio M, Romagnani S. CXC chemokines: the regulatory link between inflammation and angiogenesis. Trends Immunol 2004;25:201–209.[CrossRef][Medline]
  32. Strieter RM, Belperio JA, Keane MP. CXC chemokines in angiogenesis related to pulmonary fibrosis. Chest 2002;122:298S–301S.[Medline]
  33. Kempuraj D, Frydas S, Kandere K, Madhappan B, Letourneau R, Christodoulou S, Boucher W, Riccioni G, Conti P, Theoharides TC. Interleukin-19 (IL-19) network revisited. Int J Immunopathol Pharmacol 2003; 16:95–97.[Medline]
  34. Liao SC, Cheng YC, Wang YC, Wang CW, Yang SM, Yu CK, Shieh CC, Cheng KC, Lee MF, Chiang SR, et al. IL-19 induced Th2 cytokines and was up-regulated in asthma patients. J Immunol 2004;173:6712–6718.[Abstract/Free Full Text]
  35. Liao YC, Liang WG, Chen FW, Hsu JH, Yang JJ, Chang MS. IL-19 induces production of IL-6 and TNF-alpha and results in cell apoptosis through TNF-alpha. J Immunol 2002;169:4288–4297.[Abstract/Free Full Text]
  36. Caruso M, Holgate ST, Polosa R. Adenosine signalling in airways. Curr Opin Pharmacol 2006;6:251–256.[CrossRef][Medline]
  37. Feoktistov I, Biaggioni I. Adenosine A2b receptors evoke interleukin-8 secretion in human mast cells: an enprofylline-sensitive mechanism with implications for asthma. J Clin Invest 1995;96:1979–1986.[Medline]
  38. Fredholm BB, Jzerman API, Jacobson KA, Klotz K-N, Linden J. International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev 2001;53:527–552.[Abstract/Free Full Text]
  39. Kreckler LM, Wan TC, Ge Z-D, Auchampach JA. Adenosine inhibits tumor necrosis factor-{alpha} release from mouse peritoneal macrophages via A2A and A2B but not the A3 adenosine receptor. J Pharmacol Exp Ther 2006;317:172–180.[Abstract/Free Full Text]
  40. Pelletier S, Dube J, Villeneuve A, Gobeil F Jr, Bernier SG, Battistini B, Guillemette G, Sirois P. Adenosine induces cyclic-AMP formation and inhibits endothelin-1 production/secretion in guinea-pig tracheal epithelial cells through A2B adenosine receptors. Br J Pharmacol 2000; 129:243–250.[CrossRef][Medline]
  41. Xaus J, Valledor AF, Cardo M, Marques L, Beleta J, Palacios JM, Celada A. Adenosine inhibits macrophage colony-stimulating factor-dependent proliferation of macrophages through the induction of p27kip-1 expression. J Immunol 1999;163:4140–4149.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
G. Hasko and P. Pacher
A2A receptors in inflammation and injury: lessons learned from transgenic animals
J. Leukoc. Biol., March 1, 2008; 83(3): 447 - 455.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
A. Elizur, T. L. Adair-Kirk, D. G. Kelley, G. L. Griffin, D. E. deMello, and R. M. Senior
Clara cells impact the pulmonary innate immune response to LPS
Am J Physiol Lung Cell Mol Physiol, August 1, 2007; 293(2): L383 - L392.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
S. J. Mustafa, A. Nadeem, M. Fan, H. Zhong, L. Belardinelli, and D. Zeng
Effect of a Specific and Selective A2B Adenosine Receptor Antagonist on Adenosine Agonist AMP and Allergen-Induced Airway Responsiveness and Cellular Influx in a Mouse Model of Asthma
J. Pharmacol. Exp. Ther., March 1, 2007; 320(3): 1246 - 1251.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0476OCv1
35/5/587    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhong, H.
Right arrow Articles by Zeng, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhong, H.
Right arrow Articles by Zeng, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2006 American Thoracic Society.