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Published ahead of print on October 7, 2004, doi:10.1165/rcmb.2004-0103OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 2-8, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0103OC

Synergy between A2B Adenosine Receptors and Hypoxia in Activating Human Lung Fibroblasts

Hongyan Zhong, Luiz Belardinelli, Tenning Maa 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 Dewan Zeng, Ph.D., CV Therapeutics, Inc., 3172 Porter Drive, Palo Alto, CA 94304. E-mail: dewan.zeng{at}cvt.com


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Chronic inflammatory airway diseases, such as asthma, chronic obstructive pulmonary disease and pulmonary fibrosis, are associated with subepithelial fibroblast activation, myofibroblast hyperplasia, hypoxia, and increase in interstitial adenosine concentrations. The goal of this study was to determine the effect of adenosine and its receptors on activation of human lung fibroblasts under normoxia (21% O2) and hypoxia (5% O2). Under the normoxic condition, adenosine and its stable analog, 5'-(N-ethylcarboxamido)-adenosine, via activation of A2B adenosine receptors, increased the release of interleukin (IL)-6 by 14-fold and induced the differentiation of human lung fibroblasts to myofibroblasts. This latter effect of 5'-(N-ethylcarboxamido)-adenosine was abolished by an IL-6–neutralizing antibody. Hypoxia increased the release of IL-6 by 2.8-fold, and there was a synergy between hypoxia and activation of A2B adenosine receptors to increase the release of IL-6 and to induce differentiation of fibroblasts into myofibroblasts. Hypoxia increased the expression of A2B adenosine receptors by 3.4-fold. Altogether, these data suggest that hypoxia amplifies the effect of adenosine on the release of IL-6 and cell differentiation by upregulating the expression of A2B adenosine receptors. Our findings provide a novel mechanism whereby adenosine participates in the remodeling process of inflammatory lung diseases.

Key Words: adenosine • interleukin-6 • hypoxia • myofibroblast


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Chronic inflammatory airway diseases, such as asthma, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis, are associated with activation of subepithelial fibroblasts (1). The activated fibroblasts play a key role in airway inflammation and remodeling by producing extracellular matrix components, expressing surface molecules, and by releasing proinflammatory cytokines and chemokines (2, 3). In addition, in the fibrotic lesions and in several other pathologic conditions present in asthma and pulmonary fibrosis, fibroblasts are often differentiated into myofibroblasts (4). A key marker of this differentiation is the expression of {alpha}-smooth muscle actin ({alpha}-SMA). Myofibroblasts are particularly important in airway remodeling because they are the main source of the extracellular matrix and they also alter the compliance of the lung (4).

Adenosine is a potent signaling nucleoside that can elicit many physiologic responses by activating its receptors on the target cells. Adenosine has been proposed to contribute to the pathogenesis of asthma and COPD (5). This hypothesis is based on the findings that the interstitial concentration of adenosine is elevated in the lungs of individuals with asthma (6), and inhaled adenosine causes bronchoconstriction in patients with asthma (7). The effect of adenosine on bronchoconstriction appears to be mainly due to the activation of lung mast cells (8–11). In addition to the acute bronchoconstriction effect, adenosine has been suggested to play a role in modulating the functions of other inflammatory cells such as lymphocytes (12), eosinophils (13), neutrophils (14), and macrophages (15). However, it is unknown whether adenosine plays a role in the airway remodeling.

The objectives of this study were to determine (1) whether adenosine affects the release of inflammatory cytokines and promotes the differentiation of fibroblasts to myofibroblasts, and (2) which adenosine receptor subtype is responsible for the effect of adenosine. We used the primary cultured human lung fibroblasts (HLFs) as a model system. In addition, because chronic inflammatory airway diseases are usually associated with hypoxia (16), which has been shown to be a powerful stimulus for gene expression and cell differentiation (17), another objective of this study was to compare the effects of adenosine on the functions of HLFs under normoxic and hypoxic conditions.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Materials
A selective antagonist to the A2B adenosine receptor (CVT-6694) was synthesized by the Department of Bio-Organic Chemistry at CV Therapeutics Inc. (Palo Alto, CA), and was described in our earlier publication (18). All other compounds, such as rolipram, forskolin, adenosine, 5'-(N-ethylcarboxamido)-adenosine (NECA), cyclopentyladenosine (CPA), 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine (CGS-21680), N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA), and adenosine deaminase (ADA), were purchased from Sigma (St. Louis, MO).

Culture of Primary Human Lung Fibroblasts
Three different batches of primary cultured normal HLFs were obtained from Clonetics (San Diego, CA) and cultured using fibroblast basal medium supplemented with 2% fetal bovine serum, 5 µg/ml insulin, 1 ng/ml fibroblast growth factor, 50 µg/ml gentamicin, and 50 ng/ml amphotericin B (all from Clonetics). HLFs were routinely grown under the normoxic condition in a humidified incubator with 5% CO2 at 37°C. Cells from passages 2 to 8 were used in the following studies.

Hypoxia
Cells were incubated in the hypoxic chamber (Billups-Rothenberg, Inc., Del Mar, CA). The hypoxic condition (5% O2) was created by flushing the chamber with a gas mixture of 95% N2 and 5% CO2 at the flow rate of 25 liters per min for 5 min, according to the manufacturer's instruction. The chamber with cells inside was then sealed and incubated at 37°C for 1 or 24 h.

Stimulation of HLFs
HLFs were seeded into 12-well tissue culture plates at a density of 2.5 x 104 cells/well and allowed to adhere overnight and reach ~ 80% confluence. Cells were washed twice in HEPES buffered saline, and cultured in serum-free fibroblast basal medium (Clonetics) containing antibiotics and various agonists or antagonists of adenosine receptors for 24 h.

RNA Extraction and Real-Time RT-PCR
Total RNA was extracted from HLFs using the Stratagene Absolutely RNA RT-PCR Miniprep Kit followed by DNase treatment to eliminate potential genomic DNA contamination. The cDNA was synthesized from 2 µg of total RNA using TaqMan Reverse Transcription reagents from 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 5700 (Applied Biosystems) according to the manufacturer's instructions. The primers for adenosine receptors and ß-actin were designed as previously described (18). 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 ß-actin in the same sample, and presented as percentages of ß-actin mRNA.

Measurement of cAMP Accumulation
Cells were harvested using 0.0025% trypsin and 2 mM EDTA in phosphate-buffered saline (PBS), washed, and resuspended in phenol-free Dulbecco's modified Eagle's Medium to a concentration of 106 cells/ml, and then incubated with 1 U/ml of ADA for 30 min at room temperature. Cells were then treated with adenosine receptor 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.

Immunofluorescence Staining for the A2B Adenosine Receptor and {alpha}-Smooth Muscle Actin
HLFs were grown on culture slides overnight and allowed to reach ~ 80% confluence. For the immunofluorescence study on the expression of the A2B adenosine receptor, cells were fixed in 4% paraformaldehyde in PBS for 10 min, blocked with 5% normal goat serum for 30 min, and incubated with 5 µg/ml polyclonal rabbit anti-human A2B antibody (Ab; Alpha Diagnostic, San Antonio, TX) at 4°C overnight. After washing, cells were incubated with 10 µg/ml Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, Eugene, OR) for 1 h, washed with PBS, and mounted using VectaShield mounting medium with DAPI (Vector Laboratories, Burlingame, CA).

For the immunofluorescence study on the expression of the {alpha}-smooth muscle actin ({alpha}-SMA), cells were fixed, permeabilized with 0.2% Triton-100 in PBS for 10 min, blocked with 5% normal goat serum for 30 min, and incubated for 1 h with monoclonal mouse anti-human {alpha}-SMA Ab (Sigma) diluted 1:50 in 2% goat serum in PBS. After washing, cells were incubated with 10 µg/ml Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes) for 1 h, washed with PBS, and mounted using VectaShield mounting medium with DAPI.

To quantify the changes in {alpha}-SMA expression, numbers of {alpha}-SMA–positive cells in each field were counted and normalized to the total numbers of cells (positively stained by DAPI). The {alpha}-SMA–positive cell was defined as a cell whose nucleus was completely covered by {alpha}-SMA staining. This counting was done in a blind fashion, i.e., the individual who performed counting was not aware of conditions of cell treatment.

Measurement of IL-6
The concentrations of IL-6 in the cell medium were determined using ELISA kits obtained from Biosource (Camarillo, CA) according to the manufacturer's instructions. The minimal detection levels of IL-6 with these kits were 2 pg/ml.

Statistical Analysis
Data were presented as mean ± SEM of at least three independent experiments. Statistical analysis was performed by using a two-tailed, paired Student's t test. A P value of < 0.05 was considered significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Expression of Adenosine Receptor Subtypes in HLFs
Real-time RT-PCR was performed to quantify the levels of transcripts for adenosine receptors. Among the four subtypes, the A2B adenosine receptor had the highest transcript level (0.89 ± 0.11% of ß-actin expression) (Figure 1A). Lower levels of A1 and A2A adenosine receptor transcripts were also detected (0.025 ± 0.010% and 0.058 ± 0.024% of ß-actin, respectively), whereas the transcript for A3 adenosine receptors was below the detection level. Hence, the rank order of adenosine receptor mRNA levels was A2B > > A2A > A1 > > A3. In addition, as shown in Figure 1B, immunofluorescence study was performed to confirm the expression of the A2B adenosine receptor at the protein level.



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  		Figure 1. Expression of adenosine receptor subtypes in HLFs. (A) Real-time RT-PCR analysis of the adenosine receptor transcripts. The relative levels of the adenosine receptor transcripts were presented as percentages of the ß-actin transcript. Data shown are averages ± SEM from four independent experiments done in triplicate. "nd" denotes "not detected". (B) Immunofluorescence staining of HLFs with anti-human A2B adenosine receptor antibodies. (C) Immunofluorescence staining of HLFs with anti-human A2B adenosine receptor antibodies that were pre-incubated with 5-fold excess of the peptide used to generate the antibody. HLFs were coverslipped using mounting medium with DAPI to visualize nuclei (B and C).

 
Activation of the A2A or A2B adenosine receptors increases cellular cAMP accumulation, whereas activation of the A1 or A3 adenosine receptors decreases cellular cAMP accumulation induced by forskolin. To identify the adenosine receptor subtype(s) that are functionally expressed in HLFs, the effects of NECA and several other agonists on cellular cAMP accumulation were determined. NECA is a stable analog of adenosine, and it activates all four adenosine receptor subtypes including A2B adenosine receptors. As shown in Figure 2A, NECA increased cellular cAMP accumulation in a concentration-dependent manner, with a potency (EC50 value) of 8.4 ± 1.4 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 the A2B adenosine receptors, the effect of a selective antagonist to A2B receptors, CVT-6694, on NECA-induced increase in cellular cAMP accumulation was determined. As shown in Figure 2A, CVT-6694 (1 µM) significantly attenuated NECA-induced cellular cAMP accumulation. Collectively, using cellular cAMP concentration as readout for the functional expression of adenosine receptors, our results indicate that A2B adenosine receptors are functionally expressed in HLFs whereas A1, A2A, or A3 adenosine receptors are not.



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  		Figure 2. Effects of adenosine receptor agonists and antagonist on cellular cAMP accumulation in HLFs. (A) Concentration-response curves of CGS-21680 (CGS, circles) and NECA in the absence (squares) or presence (triangles) of A2B adenosine receptor antagonist CVT-6694 (1 µM). (B) Lack of effect of CPA (1 µM) and IB-MECA (1 µM) on the forskolin (Fsk, 10 µM)-induced increase in cellular cAMP accumulation. Data shown are averages ± SEM from three independent experiments done in duplicate. *P < 0.05, compared with control; **P < 0.05, compared with NECA-treated cells in A.

 
Activation of A2B Adenosine Receptors Increased the Release of IL-6 from HLFs
Our previous study demonstrated that activation of A2B adenosine receptors increased the expression and the release of IL-6 by human bronchial smooth muscle cells (18). To determine the role of adenosine in the release of IL-6 from HLFs, HLFs were treated for 24 h with adenosine, NECA or vehicle, and the concentrations of IL-6 in the culture media from treated cells were measured. Adenosine and NECA increased the release of IL-6 in a concentration-dependent manner, with potencies (EC50 values) of 20.5 ± 3.7 µM and 1.8 ± 0.9 µM, respectively (Figure 3A). The fold induction by adenosine (100 µM) and NECA (10 µM) were similar, 14.3 ± 0.7- and 14.4 ± 3.7-fold, respectively. The basal level of IL-6 in the media from vehicle-treated cells was 28.6 ± 2.5 pg/ml.



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  		Figure 3. Effects of adenosine receptor agonists and antagonist on the release of IL-6 by HLFs. (A) Concentration-response curves of adenosine (triangles) and NECA (squares) on the release of IL-6. Cells were treated with adenosine or NECA for 24 h. (B) Effects of selective adenosine receptor agonists and antagonist on the release of IL-6. HLFs were incubated with vehicle (control), NECA (10 µM), NECA (10 µM) plus CVT-6694 (6694, 1 µM), CPA (1 µM), CGS-21680 (CGS, 1 µM), or IB-MECA (1 µM) for 24 h. Media from treated cells were collected, and the concentrations of IL-6 were determined using ELISA. Data shown are averages ± SEM from three (A) and five (B) independent experiments done in duplicate. *P < 0.05 compared with control; **P < 0.05 compared with NECA-treated cells in B.

 
To determine which of the adenosine receptor subtypes mediate the NECA-induced release of IL-6, cells were incubated for 24 h with selective adenosine receptor agonists or antagonists. Unlike NECA, the selective A1 agonist CPA (1 µM), the A2A agonist CGS21680(1 µM), and the A3 agonist IB-MECA (1 µM) failed to increase the release of IL-6. The selective A2B antagonist, CVT-6694 (1 µM), significantly reduced NECA (10 µM)-induced release of IL-6 (90.3 ± 5.6% inhibition) (Figure 3B). These results demonstrate that NECA-induced release of IL-6 is mediated by the A2B adenosine receptor subtype.

NECA-Induced Differentiation of Fibroblasts into Myofibroblasts Is Mediated by IL-6
To determine the effect of NECA on the differentiation of fibroblasts into myofibroblasts, HLFs were incubated with NECA (10 µM) for 72 h, and the differentiation of HLFs to myofibroblasts was determined using immunofluorescence staining of {alpha}-SMA. NECA (10 µM) markedly increased the expression of {alpha}-SMA (Figures 4C and 4G, 20.1 ± 1.9% of cells were stained positively) when compared with vehicle-treated cells (Figure 4A and 4G, 6.4 ± 0.7% of cells were stained positively). Similarly, IL-6 (500 pg/ml) also increased the expression of {alpha}-SMA (Figures 4E and 4G, 48.4 ± 5.0% of cells were stained positively). To determine whether the effect of NECA on cell differentiation is dependent on the release of IL-6, the IL-6–neutralizing Ab was added to the cell media during NECA treatment. The IL-6–neutralizing Ab greatly decreased the effect of NECA, and percentage of positive cells in the absence or presence of anti–IL-6 Ab were 20.1 ± 1.9% and 7.2 ± 0.7%, respectively (Figures 4C, 4D, and 4G). As expected, the IL-6–neutralizing Ab also decreased the effect of IL-6 on {alpha}-SMA expression, and the percentages of positive cells in the absence and presence of anti–IL-6 Ab were 48.4 ± 5.0% and 13.6 ± 2.0%, respectively (Figure 4E, 4F, and 4G). An isotype control for the IL-6 Ab had no effect on {alpha}-SMA expression (data not shown). These data suggest that NECA-induced differentiation of HLFs into myofibroblasts is mediated by the release of IL-6.



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  		Figure 4. Immunofluorescence staining of HLFs with anti–{alpha}-SMA antibodies. HLFs were incubated with media alone (A and B), or media containing 10 µM NECA (C and D), or 500 pg/ml IL-6 (E and F), in the absence (A, C, and E, white bars in G) or presence of 5 ng/ml IL-6–neutralizing Ab (B, D, and F, black bars in G) for 72 h. Mouse IgG1 (5 ng/ml), the isotope control for the IL-6–neutralizing Ab, has no significant effect on expression of {alpha}-SMA (data not shown). Similar results were observed in three independent experiments. The {alpha}-SMA–positive cells from six random x400 fields in each treatment group were counted (G). Percentages of {alpha}-SMA–positive cells were calculated by dividing the numbers of {alpha}-SMA–positive cells by the total numbers of cells. Values are mean ± SEM. *P < 0.05 compared with control; **P < 0.05 compared with cells in the absence of anti–IL-6.

 
Synergy between Hypoxia and NECA (via Activation of A2B Adenosine Receptors) to Increase the Release of IL-6 by HLFs
The role of adenosine in the release of IL-6 during a hypoxic condition (5% oxygen) was determined. As shown in Figure 5A, hypoxia (5% oxygen) alone increased the release of IL-6 by 2.8 ± 0.4-fold over normoxia (21% oxygen). Hypoxia-induced release of IL-6 was not affected by ADA (1 U/ml) or the A2B adenosine receptor antagonist, CVT-6694 (1 µM), indicating that hypoxia-induced release of IL-6 was not mediated by endogenous adenosine (Figure 5A). As shown in Figure 3A, NECA (10 µM) alone increased the release of IL-6 from HLFs by 14.4-fold. Combined hypoxia and NECA increased the release of IL-6 by 49.3 ± 4.8-fold above the basal level observed during normoxia. The A2B adenosine receptor antagonist CVT-6694 completely abolished this augmented effect of NECA (Figure 5B), whereas the A1 and A2A adenosine receptor agonists, CPA and CGS-21680 respectively, did not affect the hypoxia-induced release of IL-6 (Figure 5A). These data indicate that a synergy exists between hypoxia and the activation of the A2B adenosine receptors in increasing the release of IL-6.



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  		Figure 5. Effects of hypoxia on the release of IL-6 by HLFs. (A) HLFs were incubated under normoxia (21% oxygen) or hypoxia (5% oxygen) for 24 h in the absence or presence of ADA (1 U/ml), CVT-6694 (6694, 1 µM), CPA (1 µM) or CGS-21680 (CGS, 1 µM). (B) HLFs were incubated under normoxia or hypoxia in media containing vehicle (white bars), NECA (10 µM) alone (black bars) or NECA (10 µM) plus CVT-6694 (1 µM) (gray bars) for 24 h. The concentration of IL-6 in media from cells treated with vehicle under normoxia was used as control. The control value was 28.6 ± 0.4 pg/ml. Data shown are averages ± SEM from three independent experiments done in duplicate. *P < 0.05 compared with control; **P < 0.05, compared with NECA-treated cells in B.

 
The effect of hypoxia on adenosine receptor expression was assessed using real-time RT-PCR. Hypoxia significantly (P < 0.05) upregulated the mRNA level of A2B adenosine receptors up to 3.4 ± 0.2-fold of the expression level observed during normoxia. Hypoxia had no significant effect on the mRNA expression of the other three subtypes (A1, A2A, or A3) of adenosine receptors (Figure 6).



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  		Figure 6. Effect of hypoxia (5% oxygen) on the expression of adenosine receptor subtypes in HLFs. HLFs were incubated under normoxia (21% oxygen) or hypoxia (5% oxygen) for 1 h. Total RNA isolated from these cells was subjected to real-time RT-PCR analysis. The relative levels of adenosine receptor mRNA transcripts were normalized to the expression level of ß-actin mRNA, and are presented as the ratios of the expression level of a given receptor in hypoxia verses that in normoxia. Data shown are averages ± SEM from three independent experiments done in triplicate. The expression of the A3 adenosine receptor is undetectable under both normoxic and hypoxic conditions.

 
Synergy between Hypoxia and NECA (via Activation of A2B Adenosine Receptors) to Mediate the Differentiation of HLFs into Myofibroblasts
To determine the interaction between hypoxia and NECA in mediating fibroblast differentiation, HLFs were incubated under a hypoxic condition (5% oxygen) for 24 h in the absence or presence of NECA and then placed under the normoxic condition (21% oxygen) for another 48 h. Immunofluorescence staining for {alpha}-SMA expression was performed. Under this culture condition, hypoxia alone did not significantly change {alpha}-SMA expression compared with normoxia (percentage of positively stained cells were 9.0 ± 1.2% and 6.9 ± 0.9%, respectively, Figures 7B and 7A), whereas hypoxia and NECA together greatly increased the expression of {alpha}-SMA (Figure 7C) and this combined effect of hypoxia and NECA was significantly attenuated by the IL-6–neutralizing Ab (Figure 7D) with percentage of positively stained cells in the absence and presence of anti–IL-6 Ab being 74.8 ± 6.2% and 22.5 ± 2.8%, respectively. Thus, under our experimental condition, hypoxia alone is insufficient to induce the differentiation of HLFs into myofibroblasts, whereas NECA and hypoxia synergistically induce the differentiation of HLFs via IL-6.



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  		Figure 7. Effects of hypoxia (5% oxygen) on the expression of {alpha}-SMA in HLFs. HLFs were incubated under the normoxic condition (21% oxygen) in medium alone (A) or under the hypoxic condition (B, C, and D) in medium alone (B), media containing NECA (10 µM) (C), or media containing NECA (10 µM) plus IL-6–neutralizing Ab (D) for 24 h. All cells were then incubated under the normoxic condition for additional 48 h, cells were then stained with anti–{alpha}-SMA antibodies. Similar results were observed in three independent experiments. The {alpha}-SMA–positive cells from six random x400 fields in each treatment group were counted (E). Percentage of {alpha}-SMA–positive cells was calculated by dividing the number of {alpha}-SMA–positive cells by the total number of cells. Values are mean ± SEM. *P < 0.05 compared with control; **P < 0.05 compared with cells in the absence of anti–IL-6.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The following are the novel findings of this study: (1) Activation of A2B adenosine receptors increases the release of IL-6 and induces differentiation of HLFs into myofibroblasts. (2) Hypoxia and activation of A2B adenosine receptors act synergistically to promote the release of IL-6 and cell differentiation. (3) Hypoxia amplifies effects of A2B adenosine receptors via upregulation of the expression of A2B receptors.

Adenosine has been suggested to play a role in asthma and COPD via activation of mast cells. However, limited information is available regarding the role of adenosine and its receptor subtypes in the airway remodeling. Because fibroblasts and myofibroblasts are important in airway remodeling and chronic lung inflammation, we determined the effects of adenosine on these cells. The results of our study showed that activation of A2B adenosine receptors increased the release of IL-6 and induced differentiation of HLFs into myofibroblasts. IL-6 is a proinflammatory cytokine that mediates the inflammation of airway walls, and its concentration is elevated in the lung of individuals with asthma (19). The effects of IL-6 in the airway include promoting mucus secretion by human airway epithelial cells (20), stimulating hyperplasia and hypertrophy of cultured guinea pig airway smooth muscle (21), and inducing subepithelial fibrosis and myofibroblast hyperplasia in mouse lungs (22). The findings that adenosine increases the release of IL-6, and this cytokine, in turn, induces differentiation of fibroblasts into myofibroblasts suggest a novel mechanism whereby adenosine could participate in the remodeling process of inflammatory lung diseases.

Adenosine levels are elevated in the chronic inflammatory diseases. For example, adenosine levels are elevated in the bronchoalveolar lavage fluid of patients with asthma and COPD compared with control subjects (6), and in the exhaled breath condensate of individuals with atopic asthma versus nonatopic control subjects (23). More recently, levels of adenosine in the lung were shown to be elevated in several animal models of lung inflammation (24). Similarly, chronic lung diseases are often associated with inflammation and the inflamed microenvironment is generally known to be hypoxic (25). Hypoxia has been shown to serve as a powerful stimulus for increased formation of adenosine (26), gene expression, and cell differentiation (17). Our study demonstrated that both hypoxia and adenosine induced IL-6 release from HLFs, but the maximal fold induction of IL-6 by hypoxia (2.8-fold) is much smaller than that caused by adenosine or NECA (14-fold). The measured adenosine concentrations in the conditioned media were similar in normoxia and hypoxia (~ 50 nM, data not shown) and below the threshold for activation of A2B adenosine receptors. This is supported by the findings that ADA, an enzyme that degrades adenosine, and the A2B adenosine receptor antagonist, CVT-6694, had no effect on hypoxia-induced IL-6 release. These findings are consistent with the results from a previous study that demonstrated that adventitial fibroblasts are a poor source of ATP compared with endothelial cells (27). As mentioned earlier, the interstitial concentration of adenosine is elevated in the lungs of individuals with asthma and patients with COPD (6) and in the hypoxic canine lung tissues (26). However, the cellular source of the adenosine is unknown. Regardless, we hypothesize that adenosine released by other cells in proximity to fibroblasts acts synergistically with hypoxia to activate HLFs in airway inflammatory diseases.

Many physiologic roles of adenosine are mediated through cell surface adenosine receptors. In this study, we provided evidence that the A2B adenosine receptor subtype mediates the effect of the adenosine on the release of IL-6 and differentiation of fibroblasts to myofibroblasts. Our results show the following: (1) The nonselective agonist NECA increased the release of IL-6, whereas selective agonists for A1, A2A, and A3 adenosine receptors, such as CPA, CGS-21680, and IB-MECA, had no effect. These selective agonists are very potent, and, in concentration range of 0.1–1 µM, they can 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 selective agonists at a concentration of 1 µM. (2) The effect of NECA on the release of IL-6 was attenuated by CVT-6694 (1 µM). As recently reported (18), CVT-6694 has a high affinity for the A2B adenosine receptors (Ki value = 7 nM) and very low affinities for the three other adenosine receptors (that is, Ki values are more than 5 µM for A1, A2A, and A3 receptors); thus, 1 µM of CVT-6694 should inhibit only the effects mediated by A2B adenosine receptors. Collectively, these findings provide strong evidence that activation of A2B adenosine receptors increases the release of IL-6 and induces the differentiation of fibroblasts into myofibroblasts.

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 activator protein 1, cAMP response element binding protein (CREB), nuclear factor of activated T cells, nuclear factor (NF)–IL-6, and NF-{kappa}B. We and others have shown that the CREB binding site is critical for adenosine induced IL-6 expression in epithelial cells and smooth muscle cells (18, 28). The signaling pathway that mediates hypoxia-induced IL-6 expression has also been investigated. Hypoxia has been shown to increase IL-6 expression via activation of either NF-{kappa}B (cardiac myocytes) or NF–IL-6 (29, 30). Thus, it is likely that adenosine and hypoxia increases IL-6 expression via different pathways in either additive or synergistic fashions. In addition, our data showed that hypoxia increases the expression of A2B adenosine receptors and this could also contribute to the synergistic effect of adenosine and hypoxia on IL-6 release.

Induction of {alpha}-SMA expression is an essential feature during the differentiation of fibroblasts into myofibroblasts. In the current study, we showed that, in normoxia, adenosine increased the percentage of cells that expressed {alpha}-SMA and anti–IL-6 antibody blocks this increase completely, suggesting that the effect of adenosine on {alpha}-SMA expression in normoxia is mainly mediated by IL-6. In comparison, under hypoxia, the effect of adenosine on {alpha}-SMA expression was not completely blocked by anti–IL-6. It is possible that other factors, such as platelet-activating factor (PAF) and platelet-derived growth factor (PDGF), also contribute to the synergistic effect of adenosine and hypoxia on {alpha}-SMA. Besides {alpha}-SMA, collagen production has also been recognized as a key marker for fibrotic responses. Under our culture condition, adenosine did not affect the collagen production from HLF (data not shown). Although the mechanism for these differential effects of adenosine on the expression of {alpha}-SMA and collagen is unknown, we postulate that it is likely that adenosine alone is not sufficient to induce complete fibrotic responses and it requires the presence of other growth factors. In addition, one limitation of our study is that the disease histories of the tissue donors are unknown. As shown in a recent publication (31), IL-6 inhibited the proliferation of normal fibroblasts and induced proliferation of IPF fibroblasts. In our future studies, we hope to use human lung tissues with known disease history to explore the interaction of adenosine with other critical growth factors in fibrosis. Interestingly, a recent publication by Blackburn and colleagues demonstrated that adenosine mediates lung inflammation and remodeling including fibrosis in IL-13 transgenic mice (24). Although the cellular mechanism of action of adenosine in mice is not fully characterized, these data support that adenosine may play a critical role in the initiation of fibroblast differentiation into the myofibroblast, leading to the fibrotic phenotypes.

In summary, the A2B adenosine receptor subtype is the predominant adenosine receptor expressed in HLFs. Activation of this adenosine receptor subtype increases the release of IL-6 in a concentration-dependent manner. IL-6 released from these cells, in turn, induced the differentiation of fibroblasts into myofibroblasts. Hypoxia alone increased IL-6 release and it synergistically augments NECA-induced IL-6 release and differentiation of HLFs by a mechanism that most likely involves the upregulation of the expression of A2B adenosine receptors. Our findings provide a novel mechanism whereby adenosine participates in the remodeling process of inflammatory lung diseases.


   Acknowledgments
 
The authors thank Drs. Jeff Zablocki, Rao Kalla, Elfatih Elzein, Venkata Palla, Ms. Thao Perry, and Ms. Xiaofen Li for their contributions to the discovery and chemical synthesis of CVT-6694. They also thank Drs. Italo Biaggioni, Igor Feokistov, and Michael Blackburn for critical review of this manuscript.


   Footnotes
 
Conflict of Interest Statement: H.Z., L.B., T.M., and D.Z. are employees of CV Therapeutics, Inc., and own stock and stock options in this company, which has an interest in the subject matter discussed in this manuscript.

Received in original form March 26, 2004

Received in final form September 16, 2004


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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