synergy between a 2b adenosine receptors and hypoxia in activating human...

Synergy between A2B Adenosine Receptors and Hypoxiain Activating Human Lung FibroblastsHongyan Zhong, Luiz Belardinelli, Tenning Maa, and Dewan Zeng

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

Chronic inflammatory airway diseases, such as asthma, chronic ob-structive pulmonary disease and pulmonary fibrosis, are associatedwith subepithelial fibroblast activation, myofibroblast hyperplasia,hypoxia, and increase in interstitial adenosine concentrations. Thegoal of this study was to determine the effect of adenosine and itsreceptors 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)-adeno-sine, via activation of A2B adenosine receptors, increased the releaseof interleukin (IL)-6 by 14-fold and induced the differentiation ofhuman lung fibroblasts to myofibroblasts. This latter effect of 5�-(N-ethylcarboxamido)-adenosine was abolished by an IL-6–neutraliz-ing 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 inducedifferentiation of fibroblasts into myofibroblasts. Hypoxia increasedthe expression of A2B adenosine receptors by 3.4-fold. Altogether,these data suggest that hypoxia amplifies the effect of adenosineon the release of IL-6 and cell differentiation by upregulating theexpression of A2B adenosine receptors. Our findings provide a novelmechanism whereby adenosine participates in the remodeling pro-cess of inflammatory lung diseases.

Keywords: adenosine; interleukin-6; hypoxia; myofibroblast

Chronic inflammatory airway diseases, such as asthma, chronicobstructive pulmonary disease (COPD), and pulmonary fibrosis,are associated with activation of subepithelial fibroblasts (1).The activated fibroblasts play a key role in airway inflammationand remodeling by producing extracellular matrix components,expressing surface molecules, and by releasing proinflammatorycytokines and chemokines (2, 3). In addition, in the fibroticlesions and in several other pathologic conditions present inasthma and pulmonary fibrosis, fibroblasts are often differenti-ated into myofibroblasts (4). A key marker of this differentiationis the expression of �-smooth muscle actin (�-SMA). Myofibro-blasts are particularly important in airway remodeling becausethey are the main source of the extracellular matrix and theyalso alter the compliance of the lung (4).

Adenosine is a potent signaling nucleoside that can elicitmany physiologic responses by activating its receptors on thetarget cells. Adenosine has been proposed to contribute to thepathogenesis of asthma and COPD (5). This hypothesis is basedon the findings that the interstitial concentration of adenosineis elevated in the lungs of individuals with asthma (6), and inhaledadenosine causes bronchoconstriction in patients with asthma(7). The effect of adenosine on bronchoconstriction appears tobe mainly due to the activation of lung mast cells (8–11). Inaddition to the acute bronchoconstriction effect, adenosine has

(Received in original form March 26, 2004 and in revised form September 16, 2004)

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: [email protected]

Am J Respir Cell Mol Biol Vol 32. pp 2–8, 2005Originally Published in Press as DOI: 10.1165/rcmb.2004-0103OC on October 7, 2004Internet address: www.atsjournals.org

been suggested to play a role in modulating the functions ofother inflammatory cells such as lymphocytes (12), eosinophils(13), neutrophils (14), and macrophages (15). However, it isunknown whether adenosine plays a role in the airway remod-eling.

The objectives of this study were to determine (1) whetheradenosine affects the release of inflammatory cytokines and pro-motes the differentiation of fibroblasts to myofibroblasts, and(2) which adenosine receptor subtype is responsible for the effectof adenosine. We used the primary cultured human lung fibro-blasts (HLFs) as a model system. In addition, because chronicinflammatory airway diseases are usually associated with hypoxia(16), which has been shown to be a powerful stimulus for geneexpression and cell differentiation (17), another objective of thisstudy was to compare the effects of adenosine on the functionsof HLFs under normoxic and hypoxic conditions.

MATERIALS AND METHODS

Materials

A selective antagonist to the A2B adenosine receptor (CVT-6694) wassynthesized by the Department of Bio-Organic Chemistry at CV Thera-peutics Inc. (Palo Alto, CA), and was described in our earlier publi-cation (18). All other compounds, such as rolipram, forskolin, adeno-sine, 5�-(N-ethylcarboxamido)-adenosine (NECA), cyclopentyladenosine(CPA), 2-p-(2-carboxyethyl)phenethylamino-5�-N-ethylcarboxamidoadenosine (CGS-21680), N6-(3-iodobenzyl)-adenosine-5�-N-methyluro-namide (IB-MECA), and adenosine deaminase (ADA), were purchasedfrom Sigma (St. Louis, MO).

Culture of Primary Human Lung Fibroblasts

Three different batches of primary cultured normal HLFs were obtainedfrom Clonetics (San Diego, CA) and cultured using fibroblast basalmedium supplemented with 2% fetal bovine serum, 5 �g/ml insulin,1 ng/ml fibroblast growth factor, 50 �g/ml gentamicin, and 50 ng/mlamphotericin B (all from Clonetics). HLFs were routinely grown underthe 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 flushingthe chamber with a gas mixture of 95% N2 and 5% CO2 at the flowrate of 25 liters per min for 5 min, according to the manufacturer’sinstruction. The chamber with cells inside was then sealed and incubatedat 37 �C for 1 or 24 h.

Stimulation of HLFs

HLFs were seeded into 12-well tissue culture plates at a density of2.5 � 104 cells/well and allowed to adhere overnight and reach � 80%confluence. Cells were washed twice in HEPES buffered saline, andcultured in serum-free fibroblast basal medium (Clonetics) containingantibiotics and various agonists or antagonists of adenosine receptorsfor 24 h.

RNA Extraction and Real-Time RT-PCR

Total RNA was extracted from HLFs using the Stratagene AbsolutelyRNA RT-PCR Miniprep Kit followed by DNase treatment to eliminatepotential genomic DNA contamination. The cDNA was synthesized

Zhong, Belardinelli, Maa, et al.: Adenosine Induces IL-6 and Cell Differentiation 3

from 2 �g of total RNA using TaqMan Reverse Transcription reagentsfrom Applied Biosystems (Foster City, CA). TaqMan real-time PCRanalysis was applied using 2 �l cDNA per reaction and YBR GreenPCR Core Reagents on ABI Prism Sequence Detection System 5700(Applied Biosystems) according to the manufacturer’s instructions. Theprimers for adenosine receptors and �-actin were designed as previouslydescribed (18). At the end of the PCR cycle, a dissociation curve wasgenerated to ensure that the amplification of a single product, andthe threshold cycle times (Ct values) for each gene were determined.Relative mRNA levels were calculated based on the Ct values, normal-ized to �-actin in the same sample, and presented as percentages of�-actin mRNA.

Measurement of cAMP Accumulation

Cells were harvested using 0.0025% trypsin and 2 mM EDTA in phos-phate-buffered saline (PBS), washed, and resuspended in phenol-freeDulbecco’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 roomtemperature. Cells were then treated with adenosine receptor agonists,antagonists, and forskolin in the presence of 50 �M of the phosphodies-terase IV inhibitor, rolipram. After incubating for 15 min at 37 �C, cellswere lysed and cAMP concentrations were determined using cAMP-Screen Direct System (Applied Biosystems) according to the manufac-turer’s instructions.

Immunofluorescence Staining for the A2B Adenosine Receptorand �-Smooth Muscle Actin

HLFs were grown on culture slides overnight and allowed to reach� 80% confluence. For the immunofluorescence study on the expressionof the A2B adenosine receptor, cells were fixed in 4% paraformaldehydein PBS for 10 min, blocked with 5% normal goat serum for 30 min,and incubated with 5 �g/ml polyclonal rabbit anti-human A2B antibody(Ab; Alpha Diagnostic, San Antonio, TX) at 4 �C overnight. Afterwashing, cells were incubated with 10 �g/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 (VectorLaboratories, Burlingame, CA).

For the immunofluorescence study on the expression of the �-smoothmuscle actin (�-SMA), cells were fixed, permeabilized with 0.2% Tri-ton-100 in PBS for 10 min, blocked with 5% normal goat serum for 30min, and incubated for 1 h with monoclonal mouse anti-human �-SMAAb (Sigma) diluted 1:50 in 2% goat serum in PBS. After washing, cellswere incubated with 10 �g/ml Alexa Fluor 488 goat anti-mouse IgG(Molecular Probes) for 1 h, washed with PBS, and mounted usingVectaShield mounting medium with DAPI.

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

Measurement of IL-6

The concentrations of IL-6 in the cell medium were determined usingELISA kits obtained from Biosource (Camarillo, CA) according to themanufacturer’s instructions. The minimal detection levels of IL-6 withthese kits were 2 pg/ml.

Statistical Analysis

Data were presented as mean � SEM of at least three independentexperiments. Statistical analysis was performed by using a two-tailed,paired Student’s t test. A P value of � 0.05 was considered significant.

RESULTS

Expression of Adenosine Receptor Subtypes in HLFs

Real-time RT-PCR was performed to quantify the levels oftranscripts for adenosine receptors. Among the four subtypes,the A2B adenosine receptor had the highest transcript level

Figure 1. Expression of adenosine receptor subtypes in HLFs. (A ) Real-time RT-PCR analysis of the adenosine receptor transcripts. The relativelevels of the adenosine receptor transcripts were presented as percent-ages of the �-actin transcript. Data shown are averages � SEM fromfour independent experiments done in triplicate. “nd” denotes “notdetected”. (B ) Immunofluorescence staining of HLFs with anti-humanA2B adenosine receptor antibodies. (C ) Immunofluorescence staining ofHLFs with anti-human A2B adenosine receptor antibodies that were pre-incubated with 5-fold excess of the peptide used to generate the anti-body. HLFs were coverslipped using mounting medium with DAPI tovisualize nuclei (B and C ).

(0.89 � 0.11% of �-actin expression) (Figure 1A). Lower levelsof A1 and A2A adenosine receptor transcripts were also detected(0.025 � 0.010% and 0.058 � 0.024% of �-actin, respectively),whereas the transcript for A3 adenosine receptors was below thedetection level. Hence, the rank order of adenosine receptormRNA levels was A2B A2A A1 A3. In addition, asshown in Figure 1B, immunofluorescence study was performedto confirm the expression of the A2B adenosine receptor at theprotein level.

Activation of the A2A or A2B adenosine receptors increasescellular cAMP accumulation, whereas activation of the A1 orA3 adenosine receptors decreases cellular cAMP accumulationinduced by forskolin. To identify the adenosine receptor sub-type(s) that are functionally expressed in HLFs, the effects ofNECA and several other agonists on cellular cAMP accumula-tion were determined. NECA is a stable analog of adenosine,and it activates all four adenosine receptor subtypes includingA2B adenosine receptors. As shown in Figure 2A, NECA in-creased cellular cAMP accumulation in a concentration-depen-dent manner, with a potency (EC50 value) of 8.4 � 1.4 �M. Incontrast, the A2A selective agonist CGS-21680 ( 10 �M) didnot cause a significant increase in cellular cAMP concentration.In addition, the A1 selective agonist, CPA (1 �M), and the A3

selective agonist, IB-MECA (1 �M), failed to inhibit the cellularcAMP accumulation caused by forskolin (10 �M, Figure 2B).Because there is no selective agonist for the A2B adenosine recep-tors, the effect of a selective antagonist to A2B receptors, CVT-6694, on NECA-induced increase in cellular cAMP accumulationwas determined. As shown in Figure 2A, CVT-6694 (1 �M)significantly attenuated NECA-induced cellular cAMP accumu-lation. Collectively, using cellular cAMP concentration as read-out for the functional expression of adenosine receptors, our

4 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 32 2005

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

results indicate that A2B adenosine receptors are functionallyexpressed in HLFs whereas A1, A2A, or A3 adenosine receptorsare not.

Activation of A2B Adenosine Receptors Increased the Releaseof IL-6 from HLFs

Our previous study demonstrated that activation of A2B adeno-sine receptors increased the expression and the release of IL-6by human bronchial smooth muscle cells (18). To determine therole of adenosine in the release of IL-6 from HLFs, HLFs weretreated for 24 h with adenosine, NECA or vehicle, and theconcentrations of IL-6 in the culture media from treated cellswere measured. Adenosine and NECA increased the release ofIL-6 in a concentration-dependent manner, with potencies (EC50

values) of 20.5 � 3.7 �M and 1.8 � 0.9 �M, respectively(Figure 3A). The fold induction by adenosine (100 �M) andNECA (10 �M) 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.

To determine which of the adenosine receptor subtypes medi-ate the NECA-induced release of IL-6, cells were incubated for24 h with selective adenosine receptor agonists or antagonists.Unlike NECA, the selective A1 agonist CPA (1 �M), the A2A

agonist CGS21680 (1 �M), and the A3 agonist IB-MECA (1 �M)failed to increase the release of IL-6. The selective A2B antago-nist, CVT-6694 (1 �M), significantly reduced NECA (10 �M)-induced release of IL-6 (90.3 � 5.6% inhibition) (Figure 3B).These results demonstrate that NECA-induced release of IL-6is mediated by the A2B adenosine receptor subtype.

NECA-Induced Differentiation of Fibroblasts intoMyofibroblasts Is Mediated by IL-6

To determine the effect of NECA on the differentiation of fi-broblasts into myofibroblasts, HLFs were incubated with NECA

Figure 3. Effects of adenosine receptor agonists and antagonist on therelease of IL-6 by HLFs. (A ) Concentration-response curves of adenosine(triangles) and NECA (squares) on the release of IL-6. Cells were treatedwith adenosine or NECA for 24 h. (B ) Effects of selective adenosinereceptor agonists and antagonist on the release of IL-6. HLFs wereincubated with vehicle (control), NECA (10 �M), NECA (10 �M) plusCVT-6694 (6694, 1 �M), CPA (1 �M), CGS-21680 (CGS, 1 �M), or IB-MECA (1 �M) for 24 h. Media from treated cells were collected, andthe concentrations of IL-6 were determined using ELISA. Data shownare averages � SEM from three (A ) and five (B ) independent experi-ments done in duplicate. *P � 0.05 compared with control; **P � 0.05compared with NECA-treated cells in B.

(10 �M) for 72 h, and the differentiation of HLFs to myofi-broblasts was determined using immunofluorescence staining of�-SMA. NECA (10 �M) markedly increased the expression of�-SMA (Figures 4C and 4G, 20.1 � 1.9% of cells were stainedpositively) when compared with vehicle-treated cells (Figure 4Aand 4G, 6.4 � 0.7% of cells were stained positively). Similarly,IL-6 (500 pg/ml) also increased the expression of �-SMA (Fig-ures 4E and 4G, 48.4 � 5.0% of cells were stained positively).To determine whether the effect of NECA on cell differentiationis dependent on the release of IL-6, the IL-6–neutralizing Abwas added to the cell media during NECA treatment. The IL-6–neutralizing Ab greatly decreased the effect of NECA, andpercentage of positive cells in the absence or presence ofanti–IL-6 Ab were 20.1 � 1.9% and 7.2 � 0.7%, respectively(Figures 4C, 4D, and 4G). As expected, the IL-6–neutralizingAb also decreased the effect of IL-6 on �-SMA expression, andthe percentages of positive cells in the absence and presence ofanti–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 Abhad no effect on �-SMA expression (data not shown). Thesedata suggest that NECA-induced differentiation of HLFs intomyofibroblasts is mediated by the release of 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 hypoxiccondition (5% oxygen) was determined. As shown in Figure 5A,hypoxia (5% oxygen) alone increased the release of IL-6 by2.8 � 0.4-fold over normoxia (21% oxygen). Hypoxia-inducedrelease of IL-6 was not affected by ADA (1 U/ml) or the A2B


Figure 4. Immunofluorescence staining of HLFs with anti–�-SMA anti-bodies. HLFs were incubated with media alone (A and B ), or mediacontaining 10 �M NECA (C and D ), or 500 pg/ml IL-6 (E and F ), inthe 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 nosignificant effect on expression of �-SMA (data not shown). Similarresults were observed in three independent experiments. The �-SMA–positive cells from six random �400 fields in each treatment groupwere counted (G ). Percentages of �-SMA–positive cells were calculatedby dividing the numbers of �-SMA–positive cells by the total numbersof cells. Values are mean � SEM. *P � 0.05 compared with control;**P � 0.05 compared with cells in the absence of anti–IL-6.

adenosine receptor antagonist, CVT-6694 (1 �M), indicatingthat hypoxia-induced release of IL-6 was not mediated by endog-enous adenosine (Figure 5A). As shown in Figure 3A, NECA(10 �M) alone increased the release of IL-6 from HLFs by 14.4-fold. Combined hypoxia and NECA increased the release ofIL-6 by 49.3 � 4.8-fold above the basal level observed duringnormoxia. The A2B adenosine receptor antagonist CVT-6694completely abolished this augmented effect of NECA (Figure 5B),whereas the A1 and A2A adenosine receptor agonists, CPA andCGS-21680 respectively, did not affect the hypoxia-induced re-lease of IL-6 (Figure 5A). These data indicate that a synergyexists between hypoxia and the activation of the A2B adenosinereceptors in increasing the release of IL-6.

The effect of hypoxia on adenosine receptor expression wasassessed using real-time RT-PCR. Hypoxia significantly (P �

0.05) upregulated the mRNA level of A2B adenosine receptorsup to 3.4 � 0.2-fold of the expression level observed duringnormoxia. Hypoxia had no significant effect on the mRNA ex-pression of the other three subtypes (A1, A2A, or A3) of adenosinereceptors (Figure 6).

Figure 5. Effects of hypoxia on the release of IL-6 by HLFs. (A ) HLFswere 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 �M), CPA (1 �M) or CGS-21680 (CGS, 1 �M). (B ) HLFs were incubatedunder normoxia or hypoxia in media containing vehicle (white bars),NECA (10 �M) alone (black bars) or NECA (10 �M) plus CVT-6694(1 �M) (gray bars) for 24 h. The concentration of IL-6 in media fromcells treated with vehicle under normoxia was used as control. The controlvalue was 28.6 � 0.4 pg/ml. Data shown are averages � SEM from threeindependent experiments done in duplicate. *P � 0.05 compared withcontrol; **P � 0.05, compared with NECA-treated cells in B.

Synergy between Hypoxia and NECA (via Activation of A2B

Adenosine Receptors) to Mediate the Differentiationof HLFs into Myofibroblasts

To determine the interaction between hypoxia and NECA inmediating fibroblast differentiation, HLFs were incubated undera hypoxic condition (5% oxygen) for 24 h in the absence orpresence of NECA and then placed under the normoxic condi-tion (21% oxygen) for another 48 h. Immunofluorescence stain-ing for �-SMA expression was performed. Under this culturecondition, hypoxia alone did not significantly change �-SMAexpression compared with normoxia (percentage of positivelystained cells were 9.0 � 1.2% and 6.9 � 0.9%, respectively,Figures 7B and 7A), whereas hypoxia and NECA togethergreatly increased the expression of �-SMA (Figure 7C) andthis combined effect of hypoxia and NECA was significantlyattenuated by the IL-6–neutralizing Ab (Figure 7D) with per-centage of positively stained cells in the absence and presenceof anti–IL-6 Ab being 74.8 � 6.2% and 22.5 � 2.8%, respectively.Thus, under our experimental condition, hypoxia alone is insuf-ficient to induce the differentiation of HLFs into myofibroblasts,whereas NECA and hypoxia synergistically induce the differenti-ation of HLFs via IL-6.

DISCUSSION

The following are the novel findings of this study: (1) Activationof A2B adenosine receptors increases the release of IL-6 andinduces differentiation of HLFs into myofibroblasts. (2) Hypoxiaand activation of A2B adenosine receptors act synergistically to


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

promote the release of IL-6 and cell differentiation. (3) Hypoxiaamplifies effects of A2B adenosine receptors via upregulation ofthe expression of A2B receptors.

Adenosine has been suggested to play a role in asthma andCOPD via activation of mast cells. However, limited informationis available regarding the role of adenosine and its receptorsubtypes in the airway remodeling. Because fibroblasts and myo-fibroblasts are important in airway remodeling and chronic lunginflammation, we determined the effects of adenosine on thesecells. The results of our study showed that activation of A2B

adenosine receptors increased the release of IL-6 and induceddifferentiation of HLFs into myofibroblasts. IL-6 is a pro-inflammatory cytokine that mediates the inflammation of airwaywalls, and its concentration is elevated in the lung of individualswith asthma (19). The effects of IL-6 in the airway include pro-moting mucus secretion by human airway epithelial cells (20),stimulating hyperplasia and hypertrophy of cultured guinea pigairway smooth muscle (21), and inducing subepithelial fibrosisand myofibroblast hyperplasia in mouse lungs (22). The findingsthat adenosine increases the release of IL-6, and this cytokine,in turn, induces differentiation of fibroblasts into myofibroblastssuggest a novel mechanism whereby adenosine could participatein the remodeling process of inflammatory lung diseases.

Adenosine levels are elevated in the chronic inflammatorydiseases. For example, adenosine levels are elevated in the bron-choalveolar lavage fluid of patients with asthma and COPDcompared with control subjects (6), and in the exhaled breathcondensate of individuals with atopic asthma versus nonatopiccontrol subjects (23). More recently, levels of adenosine in thelung were shown to be elevated in several animal models of lunginflammation (24). Similarly, chronic lung diseases are oftenassociated with inflammation and the inflamed microenviron-ment is generally known to be hypoxic (25). Hypoxia has beenshown to serve as a powerful stimulus for increased formationof adenosine (26), gene expression, and cell differentiation (17).Our study demonstrated that both hypoxia and adenosine in-duced IL-6 release from HLFs, but the maximal fold inductionof IL-6 by hypoxia (2.8-fold) is much smaller than that causedby adenosine or NECA (14-fold). The measured adenosine con-centrations in the conditioned media were similar in normoxiaand hypoxia (� 50 nM, data not shown) and below the thresholdfor activation of A2B adenosine receptors. This is supported bythe findings that ADA, an enzyme that degrades adenosine, and

Figure 7. Effects of hypoxia (5% oxygen) on the expression of �-SMAin 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 �M) (C ), ormedia containing NECA (10 �M) plus IL-6–neutralizing Ab (D ) for 24 h.All cells were then incubated under the normoxic condition for addi-tional 48 h, cells were then stained with anti–�-SMA antibodies. Similarresults were observed in three independent experiments. The �-SMA–positive cells from six random �400 fields in each treatment groupwere counted (E ). Percentage of �-SMA–positive cells was calculatedby dividing the number of �-SMA–positive cells by the total numberof cells. Values are mean � SEM. *P � 0.05 compared with control;**P � 0.05 compared with cells in the absence of anti–IL-6.

the A2B adenosine receptor antagonist, CVT-6694, had no effecton hypoxia-induced IL-6 release. These findings are consistentwith the results from a previous study that demonstrated thatadventitial fibroblasts are a poor source of ATP compared withendothelial cells (27). As mentioned earlier, the interstitial con-centration of adenosine is elevated in the lungs of individualswith asthma and patients with COPD (6) and in the hypoxiccanine lung tissues (26). However, the cellular source of theadenosine is unknown. Regardless, we hypothesize that adeno-sine released by other cells in proximity to fibroblasts acts syner-gistically with hypoxia to activate HLFs in airway inflammatorydiseases.

Many physiologic roles of adenosine are mediated throughcell surface adenosine receptors. In this study, we provided evi-dence that the A2B adenosine receptor subtype mediates theeffect of the adenosine on the release of IL-6 and differentiationof fibroblasts to myofibroblasts. Our results show the following:(1) The nonselective agonist NECA increased the release ofIL-6, whereas selective agonists for A1, A2A, and A3 adenosinereceptors, such as CPA, CGS-21680, and IB-MECA, had noeffect. These selective agonists are very potent, and, in concen-tration range of 0.1–1 �M, they can fully activate their cognatereceptors without significant activation of the A2B AdoR; at


concentrations higher than 1 �M, they may activate A2B AdoRs.This was the rationale for determining the effect of these selec-tive agonists at a concentration of 1 �M. (2) The effect of NECAon the release of IL-6 was attenuated by CVT-6694 (1 �M). Asrecently reported (18), CVT-6694 has a high affinity for the A2B

adenosine receptors (Ki value � 7 nM) and very low affinitiesfor the three other adenosine receptors (that is, Ki values aremore than 5 �M for A1, A2A, and A3 receptors); thus, 1 �Mof CVT-6694 should inhibit only the effects mediated by A2B

adenosine receptors. Collectively, these findings provide strongevidence that activation of A2B adenosine receptors increasesthe release of IL-6 and induces the differentiation of fibroblastsinto myofibroblasts.

Transcriptional regulation of IL-6 has been studied inten-sively in recent years. The promoter of the human IL-6 genecontains the binding sites for activator protein 1, cAMP responseelement binding protein (CREB), nuclear factor of activated Tcells, nuclear factor (NF)–IL-6, and NF-�B. We and others haveshown that the CREB binding site is critical for adenosine in-duced IL-6 expression in epithelial cells and smooth muscle cells(18, 28). The signaling pathway that mediates hypoxia-inducedIL-6 expression has also been investigated. Hypoxia has beenshown to increase IL-6 expression via activation of either NF-�B(cardiac myocytes) or NF–IL-6 (29, 30). Thus, it is likely thatadenosine and hypoxia increases IL-6 expression via differentpathways 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 syner-gistic effect of adenosine and hypoxia on IL-6 release.

Induction of �-SMA expression is an essential feature duringthe differentiation of fibroblasts into myofibroblasts. In the cur-rent study, we showed that, in normoxia, adenosine increasedthe percentage of cells that expressed �-SMA and anti–IL-6antibody blocks this increase completely, suggesting that theeffect of adenosine on �-SMA expression in normoxia is mainlymediated by IL-6. In comparison, under hypoxia, the effect ofadenosine on �-SMA expression was not completely blockedby 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 hypoxiaon �-SMA. Besides �-SMA, collagen production has also beenrecognized as a key marker for fibrotic responses. Under ourculture condition, adenosine did not affect the collagen produc-tion from HLF (data not shown). Although the mechanism forthese differential effects of adenosine on the expression of �-SMAand collagen is unknown, we postulate that it is likely that adeno-sine alone is not sufficient to induce complete fibrotic responsesand it requires the presence of other growth factors. In addition,one limitation of our study is that the disease histories of thetissue donors are unknown. As shown in a recent publication(31), IL-6 inhibited the proliferation of normal fibroblasts andinduced proliferation of IPF fibroblasts. In our future studies,we hope to use human lung tissues with known disease historyto explore the interaction of adenosine with other critical growthfactors in fibrosis. Interestingly, a recent publication by Black-burn and colleagues demonstrated that adenosine mediates lunginflammation and remodeling including fibrosis in IL-13 trans-genic mice (24). Although the cellular mechanism of action ofadenosine in mice is not fully characterized, these data supportthat adenosine may play a critical role in the initiation of fibro-blast differentiation into the myofibroblast, leading to the fibroticphenotypes.

In summary, the A2B adenosine receptor subtype is the pre-dominant adenosine receptor expressed in HLFs. Activation ofthis adenosine receptor subtype increases the release of IL-6 ina concentration-dependent manner. IL-6 released from these

cells, in turn, induced the differentiation of fibroblasts into myo-fibroblasts. Hypoxia alone increased IL-6 release and it synergis-tically augments NECA-induced IL-6 release and differentiationof HLFs by a mechanism that most likely involves the upregula-tion of the expression of A2B adenosine receptors. Our findingsprovide a novel mechanism whereby adenosine participates inthe remodeling process of inflammatory lung diseases.

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

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

References

1. Roche WR. Fibroblasts and asthma. Clin Exp Allergy 1991;21:545–548.2. Doucet C, Brouty-Boye D, Pottin-Clemenceau C, Jasmin C, Canonica

GW, Azzarone B. IL-4 and IL-13 specifically increase adhesion mole-cule and inflammatory cytokine expression in human lung fibroblasts.Int Immunol 1998;10:1421–1433.

3. Teran LM, Mochizuki M, Bartels J, Valencia EL, Nakajima T, Hirai K,Schroder JM. Th1- and Th2-type cytokines regulate the expressionand production of eotaxin and RANTES by human lung fibroblasts.Am J Respir Cell Mol Biol 1999;20:777–786.

4. Phan SH. The myofibroblast in pulmonary fibrosis. Chest 2002;122:286S–289S.

5. Holgate ST, Church MK, Polosa R. Adenosine: a positive modulator ofairway inflammation in asthma. Ann NY Acad Sci 1991;629:227–236.

6. Driver AG, Kukoly CA, Ali S, Mustafa SJ. Adenosine in bronchoalveolarlavage fluid in asthma. Am Rev Respir Dis 1993;148:91–97.

7. Cushley MJ, Tattersfield AE, Holgate ST. Adenosine-induced broncho-constriction in asthma: antagonism by inhaled theophylline. Am RevRespir Dis 1984;129:380–384.

8. Cushley MJ, Holgate ST. Adenosine-induced bronchoconstriction inasthma: role of mast cell-mediator release. J Allergy Clin Immunol1985;75:272–278.

9. Phillips GD, Polosa R, Holgate ST. The effect of histamine-H1 receptorantagonism with terfenadine on concentration-related AMP-inducedbronchoconstriction in asthma. Clin Exp Allergy 1989;19:405–409.

10. Polosa R, Ng WH, Crimi N, Vancheri C, Holgate ST, Church MK,Mistretta A. Release of mast-cell-derived mediators after endobron-chial adenosine challenge in asthma. Am J Respir Crit Care Med 1995;151:624–629.

11. Spicuzza L, Bonfiglio C, Polosa R. Research applications and implicationsof adenosine in diseased airways. Trends Pharmacol Sci 2003;24:409–413.

12. Huang S, Apasov S, Koshiba M, Sitkovsky M. Role of A2a extracellularadenosine receptor-mediated signaling in adenosine-mediated inhibi-tion of T-cell activation and expansion. Blood 1997;90:1600–1610.

13. Walker BA, Jacobson MA, Knight DA, Salvatore CA, Weir T, Zhou D,Bai TR. Adenosine A3 receptor expression and function in eosino-phils. Am J Respir Cell Mol Biol 1997;16:531–537.

14. Cronstein BN. Adenosine regulation of neutrophil function and inhibi-tion of inflammation via adenosine receptors. In: Jacobson KA, JarvisMF, editors. Purinergic approaches in experimental therapeutics. NewYork: Wiley-Liss; 1997. pp. 285–299.

15. Hasko G, Szabo C, Nemeth ZH, Kvetan V, Pastores SM, Vizi ES. Adeno-sine receptor agonists differentially regulate IL-10, TNF-alpha, andnitric oxide production in RAW 264.7 macrophages and in endotoxe-mic mice. J Immunol 1996;157:4634–4640.

16. Wright JL. Diseases of the small airways. Lung 2001;179:375–396.17. Stenmark KR, Gerasimovskaya E, Nemenoff RA, Das M. Hypoxic acti-

vation of adventitial fibroblasts: role in vascular remodeling. Chest2002;122:326S–334S.

18. Zhong H, Belardinelli L, Maa T, Feoktistov I, Biaggioni I, Zeng D. A2Badenosine receptors increase cytokine release by bronchial smoothmuscle cells. Am J Respir Cell Mol Biol 2004;30:118–125.

19. Broide DH, Lotz M, Cuomo AJ, Coburn DA, Federman EC, WassermanSI. Cytokines in symptomatic asthma airways. J Allergy Clin Immunol1992;89:958–967.

20. Martin LD, Rochelle LG, Fischer BM, Krunkosky TM, Adler KB. Airwayepithelium as an effector of inflammation: molecular regulation ofsecondary mediators. Eur Respir J 1997;10:2139–2146.


21. De S, Zelazny ET, Souhrada JF, Souhrada M. IL-1 beta and IL-6 inducehyperplasia and hypertrophy of cultured guinea pig airway smoothmuscle cells. J Appl Physiol 1995;78:1555–1563.

22. Elias JA, Zhu Z, Chupp G, Homer RJ. Airway remodeling in asthma.J Clin Invest 1999;104:1001–1006.

23. Huszar E, Vass G, Vizi E, Csoma Z, Barat E, Molnar Vilagos G, Herja-vecz I, Horvath I. Adenosine in exhaled breath condensate in healthyvolunteers and in patients with asthma. Eur Respir J 2002;20:1393–1398.

24. Blackburn MR, Lee CG, Young HW, Zhu Z, Chunn JL, Kang MJ,Banerjee SK, Elias JA. Adenosine mediates IL-13-induced inflamma-tion and remodeling in the lung and interacts in an IL-13-adenosineamplification pathway. J Clin Invest 2003;112:332–344.

25. Sitkovsky MV, Lukashev D, Apasov S, Kojima H, Koshiba M, CaldwellC, Ohta A, Thiel M. Physiological control of immune response andinflammatory tissue damage by hypoxia-inducible factors and adeno-sine A2A receptors. Annu Rev Immunol 2004;22:657–682.

26. Mentzer RM Jr, Rubio R, Berne RM. Release of adenosine by hypoxiccanine lung tissue and its possible role in pulmonary circulation. AmJ Physiol 1975;229:1625–1631.

27. Gerasimovskaya EV, Ahmad S, White CW, Jones PL, Carpenter TC,

Stenmark KR. Extracellular ATP is an autocrine/paracrine regulatorof hypoxia-induced adventitial fibroblast growth. Signaling throughextracellular signal-regulated kinase-1/2 and the Egr-1 transcriptionfactor. J Biol Chem 2002;277:44638–44650.

28. Sitaraman SV, Merlin D, Wang L, Wong M, Gewirtz AT, Si-Tahar M,Madara JL. Neutrophil-epithelial crosstalk at the intestinal lumenalsurface mediated by reciprocal secretion of adenosine and IL-6. J ClinInvest 2001;107:861–869.

29. Matsui H, Ihara Y, Fujio Y, Kunisada K, Akira S, Kishimoto T, Yama-uchi-Takihara K. Induction of interleukin (IL)-6 by hypoxia is medi-ated by nuclear factor (NF)-kappa B and NF-IL6 in cardiac myocytes.Cardiovasc Res 1999;42:104–112.

30. Yan SF, Tritto I, Pinsky D, Liao H, Huang J, Fuller G, Brett J, May L,Stern D. Induction of interleukin 6 (IL-6) by hypoxia in vascular cells:central role of the binding site for nuclear factor-IL-6. J Biol Chem1995;270:11463–11471.

31. Moodley YP, Scaffidi AK, Misso NL, Keerthisingam C, McAnulty RJ,Laurent GJ, Mutsaers SE, Thompson PJ, Knight DA. Fibroblastsisolated from normal lungs and those with idiopathic pulmonary fibro-sis differ in interleukin-6/gp130-mediated cell signaling and prolifera-tion. Am J Pathol 2003;163:345–354.

synergy between a 2b adenosine receptors and hypoxia in activating human...

Documents