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Am J Physiol Lung Cell Mol Physiol 295: L505-L514, 2008. First published June 27, 2008; doi:10.1152/ajplung.00046.2008
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Effect of β2-adrenoceptor agonists and other cAMP-elevating agents on inflammatory gene expression in human ASM cells: a role for protein kinase A

Manminder Kaur,1,2,3 Neil S. Holden,1 Sylvia M. Wilson,2 Maria B. Sukkar,4 Kian Fan Chung,3 Peter J. Barnes,3 Robert Newton,1 and Mark A. Giembycz2

Departments of 1Cell Biology and Anatomy and 2Pharmacology and Therapeutics, Airway Inflammation Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada; 3Airway Disease Section, National Heart and Lung Institute, Imperial College London, London, United Kingdom; and 4Respiratory Research Group Faculty of Pharmacy, University of Sydney, New South Wales, Australia

Submitted 24 January 2008 ; accepted in final form 23 June 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
In diseases such as asthma, airway smooth muscle (ASM) cells play a synthetic role by secreting inflammatory mediators such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, or IL-8 and by expressing surface adhesion molecules, including ICAM-1. In the present study, PGE2, forskolin, and short-acting (salbutamol) and long-acting (salmeterol and formoterol) β2-adrenoceptor agonists reduced the expression of ICAM-1 and the release of GM-CSF evoked by IL-1β in ASM cells. IL-1β-induced IL-8 release was also repressed by PGE2 and forskolin, whereas the β2-adrenoceptor agonists were ineffective. In each case, repression of these inflammatory indexes was prevented by adenoviral overexpression of PKI{alpha}, a highly selective PKA inhibitor. These data indicate a PKA-dependent mechanism of repression and suggest that agents that elevate intracellular cAMP, and thereby activate PKA, may have a widespread anti-inflammatory effect in ASM cells. Since ICAM-1 and GM-CSF are highly NF-{kappa}B-dependent genes, we used an adenoviral-delivered NF-{kappa}B-dependent luciferase reporter to examine the effects of forskolin and the β2-adrenoceptor agonists on NF-{kappa}B activation. There was no effect on luciferase activity measured in the presence of forskolin or β2-adrenoceptor agonists. This finding is consistent with the observation that IL-1β-induced expression of IL-6, a known NF-{kappa}B-dependent gene in ASM, was also unaffected by β2-adrenoceptor agonists, forskolin, PGE2, 8-bromo-cAMP, or rolipram. Collectively, these results indicate that repression of IL-1β-induced ICAM-1 expression and GM-CSF release by cAMP-elevating agents, including β2-adrenoceptor agonists, may not occur through a generic effect on NF-{kappa}B.

human airway smooth muscle cells; asthma; inflammation; prostaglandin

PRODUCTION AND RELEASE of inflammatory mediators, including histamine, leukotrienes, and kinins, occur after antigen challenge or other inhaled insults and may lead to contraction of the airway smooth muscle (ASM) to elicit bronchoconstriction (4). Thus the mechanical effector responses of ASM are central to the pathogenesis of asthma, and defects or increases in ASM responsiveness, or airway hyperresponsiveness, possibly due to the inflammatory milieu, are features of asthma (14). Furthermore, the phenotypic plasticity, for example, proliferation and hypertrophy, of ASM in asthma may contribute not only to remodeling and airway wall thickening, but also to airway hyperresponsiveness (16, 28).

A more recently appreciated function of ASM cells is their ability to elaborate a host of cytokines, chemokines, and other immunomodulatory factors (22). Thus proinflammatory cytokines acting on ASM cells induce the expression of cyclooxygenase-2 (6, 33), cytokines and chemokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-8 (23, 39), and adhesion molecules, including ICAM-1 (3). This, in turn, promotes airway inflammation by virtue of the enhanced recruitment, attractant, and prosurvival properties of these factors on invading inflammatory cells, such as the eosinophil (17, 22). In this context, classical constrictor stimuli, for example, bradykinin or leukotrienes, may also act on ASM to promote the expression of key inflammatory genes, such as IL-8, to further contribute to asthmatic inflammation (20, 34).

The realization that the biosynthetic properties of ASM contribute to airway inflammation means that this cell type is also an important target of inhaled anti-inflammatory medications, such as glucocorticoids (corticosteroids) (31). Similarly, inhaled β2-adrenoceptor agonists represent the mainstay and most effective treatment option for the acute relief of bronchoconstriction (15). These drugs, whether short- or long-acting β2-adrenoceptor agonists, act via the β2-adrenoceptor and Gs{alpha} to enhance the activity of adenylyl cyclase (15). Classically, this leads to accumulation of intracellular cAMP and activation of cAMP-dependent protein kinase (PKA) to elicit biological responses, including the relaxation of ASM. However, there is considerable evidence in vitro that the elevation of intracellular cAMP may downregulate inflammatory gene expression, possibly via a repressive effect on the acute-phase transcription factor NF-{kappa}B (43), which is critical to the expression of many inflammatory genes in ASM (7). Thus, in the context of IL-1β stimulation, β2-adrenoceptor agonists reduced the expression of RANTES (regulated on activation normal T cell expressed and presumably secreted), eotaxin, GM-CSF, and, to a lesser extent, IL-8 (18). Although GM-CSF release following stimulation of ASM with TNF{alpha} + IL-1β or ICAM-1 expression following TNF{alpha} treatment is also inhibited by β2-adrenoceptor agonists (26, 32), the expression of TNF{alpha}-induced IL-6 is augmented by β2-adrenoceptor agonists, whereas RANTES expression is reduced (1, 2). Similarly, GM-CSF expression in bronchial epithelial cells is also repressed by cAMP-elevating agents, including β2-adrenoceptor agonists (29), whereas recent reports indicate that β2-adrenoceptor agonists may upregulate the expression of IL-6 and IL-8 (12, 37).

In the present study, we have further examined and clarified the effect of various β2-adrenoceptor agonists, as well as other cAMP-elevating agents, on the expression of the inflammatory genes GM-CSF, IL-6, IL-8, and ICAM-1 induced by the proinflammatory cytokine IL-1β in primary human ASM cells. Furthermore, there are numerous pathways, including protein kinase G, exchange factor activated by cAMP, Src, and MAPKs, that, in addition to PKA, may mediate β2-adrenoceptor agonist responses (15). Therefore, the role of PKA in the repression of inflammatory gene expression was also evaluated. However, many of the traditional pharmacological tools for PKA analysis, in particular the ATP binding site inhibitor H-89, are known to be nonselective as kinase inhibitors and, in the case of H-89, also behave as β2-adrenoceptor antagonists (11, 36). Since these properties make such compounds unsuitable as mechanistic probes, we have utilized the overexpression of PKI{alpha} as a highly selective means to evaluate the role of the classical cAMP effector kinase PKA (29).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Materials. Recombinant IL-1β was obtained from R & D Systems (Hornby, ON, Canada). PGE2, forskolin, rolipram, salbutamol, salmeterol, and formoterol (all from Sigma, Oakville, ON, Canada) were dissolved in DMSO, and 8-bromo-cAMP (8-Br-cAMP; Sigma) was dissolved in sterile water. Recombinant adenoviruses were prepared by the Libin Gene Therapy Unit at the University of Calgary. All other reagents were obtained from Sigma unless otherwise stated.

Cell culture and treatments. ASM cells were isolated and cultured from fresh lobar or main bronchus obtained from patients undergoing lung transplantation or resection at the Royal Brompton Hospital with informed patient consent and local ethics approval. After dissection from the surrounding tissue, the epithelium was removed from the bronchi, and the underlying smooth muscle bundles were separated from surrounding tissue. These bundles were washed and incubated in DMEM (supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 1:100 nonessential amino acids, 20 U/l penicillin, 20 µg/ml streptomycin, and 2.5 µg/ml amphotericin B) containing 1 mg/ml collagenase and maintained in a humidified atmosphere at 37°C in 5% CO2-95% air. After 30–45 min, the enzymatically dispersed cell suspension was centrifuged (200 g, 5 min), and the pellet was resuspended in DMEM containing 10% FBS. Cells were incubated at 37°C in 5% CO2-95% air (vol/vol), and the medium was replaced every 3–5 days. Confluent cells were passaged into 175-mm2 flasks. When examined by light microscopy, cultured ASM cells displayed the typical "hill-and-valley" growth pattern. In previous immunofluorescent studies, with use of this method, >95% of the cells were positive for smooth muscle {alpha}-actin, calponin, and smooth muscle myosin heavy chain (40). In all cases, cells were incubated for 24 h in serum-free medium, without supplements, before treatments. Final concentrations of the carrier DMSO did not exceed 0.1% (vol/vol), and this concentration does not affect the measured output responses (data not shown).

Western blot analysis and cytokine measurement. Western blot analysis was carried out according to standard methods using 4–12% Bis-Tris NuPAGE gels (Invitrogen, Burlington, ON, Canada) and electrotransfer to Hybond-ECL membranes (GE Healthcare Bio-Sciences, Baie d'Urfé, QC, Canada). Immunodetections were performed using primary antibodies recognizing ICAM-1 (sc 8439, Santa Cruz Biotechnology, Santa Cruz, CA) and GAPDH (catalog no. 4699-9555, AbD Serotec, Raleigh, NC) and standard ECL reagents (GE Healthcare Bio-Sciences). Quantification of Western blots was performed by densitometry using Total Lab software (Durham, NC), where the exposure of the film was empirically maintained within the linear, nonsaturated, range. The release of GM-CSF, IL-6, and IL-8 into the supernatants of 24-well plates, each with ~60,000 ASM cells, was measured using standard ELISAs performed according to the manufacturer's specifications (R & D Systems, Minneapolis, MN).

PKI{alpha} overexpression in ASM cells. The adenoviral expression vector Ad5.CMV.PKI{alpha}, which efficiently directs overexpression of PKI{alpha}, was previously described (29). Human ASM cells at ~70% confluence were incubated for 24 h with the indicated multiplicities of infection (MOIs) of adenoviral vector. The medium was then changed to serum-free medium, and after a further 24 h the cells were utilized as indicated. The control adenovirus, Ad5.CMV.null, is an empty adenoviral expression vector.

Luciferase reporters, stable transfection, and luciferase assay. As previously described (24), stable transfection by electroporation was used to generate human ASM cells containing the cAMP response element (CRE)-dependent luciferase reporter pADneo2-C6-BGL, which contains six tandem CRE motif repeats upstream of a minimal β-globin (29). Approximately 1 x 106 human ASM cells, in DMEM supplemented with 10% FCS, were electroporated at 200 V and 950 µF for 1 s with 10 µg of plasmid DNA in an electroporator (Gene Pulser II, Bio-Rad, Mississauga, ON, Canada). After electoporation, the cells were seeded into T-75 cell culture flasks containing DMEM supplemented with 10% FCS. After 24 h, the medium was replaced with fresh medium supplemented with 0.4 mg/ml G-418. The medium was replaced every 3 days until foci of stable transfectants appeared. These foci were harvested to create heterogeneous populations of cells in which the site of integration is randomized. For experiments, confluent cells in 24-well plates were incubated in serum-free medium, without G-418, for 24 h before treatments. Cells were harvested 6 h after treatments in 1x reporter lysis buffer (Promega, Madison, WI), and luciferase activity was measured using a Monolight Luminometer (BD Biosciences, San Diego, CA).

NF-{kappa}B-dependent transcription was analyzed with a validated NF-{kappa}B luciferase reporter that was introduced into human ASM cells by adenoviral delivery (7). This viral construct, Ad5-NF-{kappa}B-luc, was added to cells at 1 MOI for 24 h in DMEM supplemented with 10% FCS. After incubation overnight in serum-free medium, the cells were treated with drugs and/or IL-1β. Cells were harvested after 8 h in 1x reporter lysis buffer (Promega), and luciferase activity was assayed using a commercial kit (Promega).

MTT assay. Potential effects on cell proliferation or viability were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as previously described (7). After removal of culture medium, ASM cells were incubated at 37°C with 1 mg/ml MTT solution. After 30 min, the MTT solution was removed, and the cells were dissolved in 200 µl of DMSO before optical density determination at 550 nm.

Statistical analysis. Values are means ± SE of n independent determinations. Comparison between groups of experimental data was performed using one-way analysis of variance with Bonferroni's or Dunnett's post test or a t-test as appropriate.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Effect of cAMP-elevating agents on GM-CSF release from ASM cells. Exposure of ASM cells to IL-1β (1 ng/ml) for 18 h resulted in increased release of GM-CSF into the culture medium (5.8 ± 1.3 and 229.5 ± 55.5 pg/ml in untreated and IL-1β-treated ASM cells, respectively) in 26 determinations from 13 different subjects. Treatment of ASM cells for 30 min with PGE2 (1 µM) or forskolin (10 µM) before stimulation with IL-1β significantly repressed GM-CSF release by ~90% (Fig. 1). A similar significant effect was observed with 8-Br-cAMP (1 mM), but repression was reduced to 45% of the IL-1β-evoked response. Similarly, rolipram repressed GM-CSF release to 56% of the IL-1β-evoked response (Fig. 1). However, this did not reach statistical significance in these experiments. Similarly, treatment of ASM cells with the short-acting β2-adrenoceptor agonist salbutamol (10 µM) or either of the two long-acting β2-adrenoceptor agonists, salmeterol (0.1 µM) or formoterol (10 nM), for 30 min before IL-1β stimulation resulted in a significant repression of GM-CSF release to 25–30% of the IL-1β-evoked response. These results demonstrate clear inhibition of IL-1β-induced GM-CSF release in ASM cells by a number of distinct activators of the cAMP pathway.


Figure 1
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  		Fig. 1. Effect of cAMP-elevating agents on IL-1β-induced granulocyte-macrophage colony-stimulating factor (GM-CSF) release in human airway smooth muscle (ASM) cells. ASM cells were treated with PGE2 (1 µM), forskolin (Forsk, 10 µM), 8-bromo-cAMP (8-Br-cAMP, 1 mM), and rolipram (30 µM; left) or salbutamol (Salb, 10 µM), salmeterol (Salm, 0.1 µM), and formoterol (Form, 10 nM; right) for 30 min before stimulation with IL-1β (1 ng/ml). After 18 h, cells were harvested, and supernatants were collected for analysis of GM-CSF by ELISA. Values are means ± SE [n = 13 (left) and n = 14 (right)], expressed as percentage of response to IL-1β. *P < 0.05. **P < 0.01.

 
One issue that derives from these data is the possibility that endogenous prostaglandins, particularly PGE2, that are produced by the ASM cells may act via autocrine feedback to mask responses generated by other cAMP-elevating agents (25, 26). Despite other studies, which indicate a very low or negligible level of PGE2 production by ASM cells stimulated with IL-1β alone (5, 6, 35), we elected to examine the effect of indomethacin, at a concentration (10 µM) that abolishes prostanoid release, on GM-CSF expression (Table 1). In these experiments, neither basal nor IL-1β-induced production of GM-CSF was significantly affected by indomethacin. Similarly, no change in the repressive ability of exogenous PGE2 was detected in IL-1β-induced GM-CSF release. Taken together with the low level of PGE2 release from IL-1β-treated ASM cells (5, 6, 35), these data indicate that the production of endogenous prostaglandins is not a major confounding factor in this experimental system.


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  		Table 1. Effect of IL-1β and indomethacin on GM-CSF release from ASM cells

 
Effect of cAMP-elevating agents on ICAM-1 expression in ASM cells. Western blot analysis revealed low levels of ICAM-1 protein in untreated ASM cells (Fig. 2). Upon stimulation with IL-1β, ICAM-1 was highly induced (Fig. 2) and pretreatment of cells for 30 min with PGE2 (1 µM) or forskolin (10 µM) significantly repressed ICAM-1 expression by >80%. Furthermore, 8-Br-cAMP also significantly repressed IL-1β-induced ICAM-1 expression by 59%. Similarly, rolipram produced a 38% repressive effect on the expression of ICAM-1, but, as with GM-CSF release, this did not reach statistical significance. Pretreatment of ASM cells with short- and long-acting β2-adrenoceptor agonists also resulted in significant 60–70% repression of IL-1β-induced ICAM-1 expression. Taken together, these data indicate that, as with GM-CSF, IL-1β-induced ICAM-1 expression can be inhibited by mechanistically distinct cAMP-elevating agents.


Figure 2
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  		Fig. 2. Effect of cAMP-elevating agents on IL-1β-induced ICAM-1 expression in human ASM cells. ASM cells were treated with PGE2 (1 µM), forskolin (10 µM), 8-Br-cAMP (1 mM), and rolipram (30 µM; left) or salbutamol (10 µM), salmeterol (0.1 µM), and formoterol (10 nM; right) for 30 min before stimulation with IL-1β (1 ng/ml). After 18 h, cell proteins were harvested and analyzed for ICAM-1 and GAPDH expression by Western blot analysis. Representative blots are shown. Data from densitometric analysis of Western blots are means ± SE (n = 6). *P < 0.05. **P < 0.01.

 
Effect of cAMP-elevating agents on IL-6 and IL-8 release in ASM cells. Exposure of ASM cells to IL-1β (1 ng/ml) for 18 h resulted in considerable increases in the release of IL-6 and IL-8 into the culture medium. In 11 determinations from 6 individuals, IL-6 release was increased from 31.1 ± 6.3 pg/ml in untreated cells to 2,256 ± 386 pg/ml in IL-1β-treated cells. Prior treatment of ASM cells with PGE2, forskolin, 8-Br-cAMP, rolipram, or β2-adrenoceptor agonists revealed no effect on IL-1β-induced IL-6 release (Fig. 3). Similarly, in 18 determinations from 12 individuals, IL-8 release was augmented from 1.5 ± 0.9 ng/ml in untreated cells to 32.3 ± 18.9 ng/ml in IL-1β-treated cells. As described for GM-CSF, pretreatment of cells with PGE2 and forskolin inhibited IL-1β-induced release of IL-8 to 51.2% and 54.3%, respectively (Fig. 3). Overall, 8-Br-cAMP reduced IL-8 to a similar extent (62.5% of the response to IL-1β). However, variability in the absolute levels of IL-8 release prevented this effect from reaching statistical significance. By contrast, rolipram had no effect on this response (Fig. 3). Similarly, salbutamol, salmeterol, or formoterol had no significant effect on IL-1β-induced IL-8 release.


Figure 3
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  		Fig. 3. Effect of cAMP-elevating agents on IL-1β-induced IL-6 and IL-8 expression in human ASM cells. ASM cells were treated with PGE2 (1 µM), forskolin (10 µM), 8-Br-cAMP (1 mM), and rolipram (30 µM; left) or salbutamol (10 µM), salmeterol (0.1 µM), and formoterol (10 nM; right) for 30 min before stimulation with IL-1β (1 ng/ml). After 18 h, cells were harvested, and supernatants were collected for analysis of IL-6 (top) and IL-8 (bottom) by ELISA. Values are means ± SE [n = 6 (top left and top right), n = 5 (bottom left), and n = 13 (bottom right)]. *P < 0.05.

 
Effect of cAMP-elevating agents and mimetics on induction of CRE-dependent transcription. The data presented above reveal some marked differences in the ability of cAMP-elevating compounds to repress inflammatory gene expression. Thus the β2-adrenoceptor agonists and rolipram did not inhibit IL-8 release but partially prevented GM-CSF release and ICAM-1 expression. This contrasts with the effects of PGE2 and forskolin, which were highly effective at inhibiting GM-CSF and ICAM-1 expression and were partially effective at inhibiting IL-8 secretion. To investigate whether these differences were simply due to differential activation of the cAMP-PKA cascade, we tested the ability of each agonist to induce CRE-dependent transcription (Table 2). In these experiments, PGE2 and forskolin produced 4.9- and 3.7-fold enhancements of CRE-dependent transcription. By comparison, the β2-adrenoceptor agonists also yielded similar (3.2- to 4.3-fold) enhancements, suggesting that the superior efficacy of PGE2 and forskolin on inflammatory gene repression was not a simple matter of elevated activation of the cAMP-PKA pathway. This concept was confirmed in experiments with 8-Br-cAMP, which was routinely weaker in terms of inflammatory gene repression; yet it was the most efficacious inducer of CRE-dependent transcription. Conversely, since rolipram only induced CRE-dependent transcription by 2.4-fold and was consistently poor in terms of repression of inflammatory gene expression, it is possible that this effect may be explained by a relatively low efficacy on the cAMP-PKA pathway.


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  		Table 2. Effect of β2-adrenoceptor agonists, PGE2, forskolin, and 8-Br-cAMP, on CRE-dependent transcription

 
In addition, we examined the effect of IL-1β on the induction of CRE-dependent transcription. In each case, there was no significant effect of IL-1β on basal or induced levels of CRE-dependent transcription (Table 2). These data are consistent with findings that PGE2 is not substantially induced by IL-1β alone and that endogenous PGE2 is not contributing to the activation of CRE-dependent transcription in the present model.

Effect of salmeterol and forskolin on CRE-dependent transcription and IL-1β-induced GM-CSF release in ASM cells infected with Ad5.CMV.PKI{alpha}. To examine the potential role of PKA in the repression of ICAM-1, GM-CSF, and IL-8 expression by cAMP-elevating drugs, we used an adenoviral vector that overexpresses PKI{alpha}, a highly selective inhibitor of PKA (29). Initially, expression of PKI{alpha} after infection of ASM cells with Ad5.CMV.PKI{alpha} was examined by Western blot analysis (Fig. 4A). Western blot analysis revealed overexpression of PKI{alpha} in a manner that correlated with increasing MOI of Ad5.CMV.PKI{alpha} and was maximal at 300 MOI.


Figure 4
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  		Fig. 4. Effect of Ad5.CMV.PKI{alpha} on cAMP response element (CRE)-dependent transcription and IL-1β-induced GM-CSF release in human ASM cells. A: ASM cells were infected with 1, 3, 10, 30, 100, and 300 multiplicity of infection (MOI) of Ad5.CMV.PKI{alpha}. Proteins were harvested after 48 h, and PKI{alpha} and GAPDH expression was examined by Western blot analysis. A: blots representative of 2 independent analyses. B: ASM cells stably transfected with a 6x CRE reporter were infected with 1, 3, 10, 30, 100, and 300 MOI of Ad5.CMV.PKI{alpha} (PKI{alpha}) or Ad5.CMV.null (Null). After 24 h, cells were transferred to serum-free medium. After a further 24 h, cells were stimulated with forskolin (10 µM) or salmeterol (0.1 µM) for 6 h and then harvested for determination of luciferase activity. Values are means ± SE (n = 6–7), expressed as fold induction. *P < 0.05. **P < 0.01. C: ASM cells were infected with 0.3, 1, 3, 10, 30, 100, and 300 MOI of Ad5.CMV.PKI{alpha} for 24 h. After incubation for 24 h in serum-free medium, cells were treated with forskolin (10 µM) or salmeterol (0.1 µM) for 30 min before stimulation with IL-1β (1 ng/ml). Supernatants were collected after 18 h for analysis of GM-CSF by ELISA. Values are means ± SE (n = 4). D: ASM cells were infected, or not, with MOI 30, 100, and 300 of Ad5.CMV.PKI{alpha} or Ad5.CMV.null virus. After 24 h, cells were transferred to serum-free medium for a further 24 h. Cells were then treated with IL-1β (1 ng/ml) in the absence or presence of forskolin (10 µM). After 18 h, cells were analyzed for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Values are means ± SE (n = 4). *P < 0.05.

 
To confirm the functionality of the overexpressed PKI{alpha}, the effect of Ad5.CMV.PKI{alpha} was examined on forskolin- and salmeterol-induced CRE-dependent transcription in ASM cells (Fig. 4B). Forskolin (10 µM) induced CRE-dependent transcription by 10.8-fold, whereas salmeterol (0.1 µM) produced a 3.9-fold increase. (The absolute levels of CRE-dependent transcription cannot be compared, since these studies were not performed in parallel and the activity of the reporter can deteriorate over time.) In each case, increasing MOI of Ad5.CMV.PKI{alpha} produced a concentration-dependent inhibition of forskolin- and salmeterol-induced CRE-dependent transcription, such that CRE activity was significantly inhibited to near the basal level at 300 MOI (Fig. 4B). Parallel experiments using equivalent MOIs of the empty vector Ad5.CMV.null revealed no effect on forskolin- or salmeterol-induced CRE-dependent transcription (Fig. 4B). This indicates that inhibition of CRE-dependent transcription is not an effect of the adenoviral vector but, rather, is due to the presence of overexpressed PKI{alpha}. This point is reinforced by our previous data showing that PKI{alpha} overexpression abolishes serine 133 phosphorylation of CRE-binding protein induced by PGE2 and 8-Br-cAMP and that neither Ad5-CMV.PKI{alpha} nor Ad5.CMV.null alone has an effect on CRE-binding protein phosphorylation in unstimulated cells (8, 10). Since forskolin and salmeterol also activate the cAMP-PKA cascade, these data validate the effectiveness of adenoviral-expressed PKI{alpha} in inhibition of this pathway.

To investigate the potential role of PKA in the inhibition of IL-1β-induced GM-CSF by forskolin and salmeterol, ASM cells were infected with various MOIs of Ad5.CMV.PKI{alpha} before treatment with IL-1β in the presence of forskolin or salmeterol (Fig. 4C). As described above, treatment of ASM cells for 30 min with forskolin (10 µM) or salmeterol (0.1 µM) before IL-1β (1 ng/ml) stimulation repressed GM-CSF release to 11.4 ± 7.0% and 35.4 ± 7.4% of the maximum response, respectively. Increasing the MOI of Ad5.CMV.PKI{alpha} resulted in a progressive loss of repression, with complete reversal at 300 MOI (Fig. 4C). These data are consistent with the overexpression of PKI{alpha} and the inhibition of CRE-dependent transcription, which supports a causal link between these events.

To test for possible effects of PKA inhibition on the number of viable cells, ASM cells were treated with various MOIs of Ad5.CMV-PKI{alpha} or Ad5.CMV.null before stimulation with IL-1β in the absence or presence of forskolin (Fig. 4D). IL-1β produced a small, nonsignificant increase in MTT activity (Fig. 4D). The addition of forskolin produced no observable effect, but the prior addition of Ad5.CMV.PKI{alpha} increased MTT activity. This effect was significant at the highest MOI of virus, whereas no effect was observed in cells infected with empty virus, suggesting that the effect was specific to PKA inhibition (Fig. 4D). Although this increase was modest (<20%), it is nonetheless consistent with the findings of Misior et al. (30), who reported elevated ASM proliferation after PKA inhibition by PKI{alpha}. In the context of the present study, these data are important, since increased cell number may contribute to reversal of the cAMP-dependent repression of inflammatory gene expression. However, as shown in Fig. 4C, GM-CSF expression was repressed to 11% and 35% by forskolin and salmeterol, respectively, and was returned to ~100% in the presence of PKI{alpha}. Thus the changes in cell number cannot account for the observed reversal of inhibition, and these data therefore represent genuine effects operating at the level of cytokine expression.

Effect of cAMP-elevating agents on ICAM-1 expression and release of GM-CSF and IL-8 in ASM cells infected with ad5.CMV.PKI{alpha}. The above-described data establish that 300 MOI is required to abolish CRE-dependent transcription induced by forskolin and salmeterol after infection of ASM cells with Ad5.CMV.PKI{alpha}. Therefore, to examine the potential role of PKA in the repression of ICAM-1, GM-CSF, and IL-8 expression by cAMP-elevating agents, we infected ASM cells with Ad5.CMV.PKI{alpha} at 300 MOI. To control for possible effects due to adenoviral vector infection, we also infected cells with the empty vector Ad5.CMV.null at an equivalent MOI.

Initially, we examined the effects of Ad5.CMV.PKI{alpha} and Ad5.CMV.null on the level of IL-1β-induced GM-CSF, ICAM, and IL-8 expression. This analysis showed no significant effect of Ad5.CMV.PKI{alpha} or Ad5.CMV.null on unstimulated or IL-1β-stimulated release of GM-CSF and IL-8 compared with naïve cells (Table 3) and is consistent with our previous data (8, 9). In the case of ICAM-1 protein, visual inspection of the Western blots confirmed that there was no obvious effect of either virus compared with unstimulated and IL-1β-treated cells. Subsequently, the effect of PGE2 and forskolin, as representative cAMP-elevating agents, was examined along with β2-adrenoceptor agonists in the presence of Ad5.CMV.PKI{alpha} and Ad5.CMV.null. As described above for naïve cells (Fig. 2), treatment of ASM cells that had been infected with Ad5.CMV.null with PGE2 or forskolin before IL-1β stimulation significantly reduced ICAM-1 expression to 28.8 ± 5.6% and 26.2 ± 7.0% of the response to IL-1β alone (Fig. 5A). However, in cells infected with Ad5.CMV.PKI{alpha}, there was a near-complete reversal of the repression of ICAM-1 by PGE2 or forskolin. Similarly, pretreatment of ASM cells with short- and long-acting β2-adrenoceptor agonists also resulted in significant 60–70% repression of IL-1β-induced ICAM-1 in cells infected with Ad5.CMV.null, and this was again similar to the effects in naïve cells. However, after infection with Ad5.CMV.PKI{alpha}, this repression of ICAM-1 expression was prevented, such that there was no significant difference from IL-1β-treated cells (Fig. 5A).


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  		Table 3. Effect of Ad5.CMV.PKI{alpha} and Ad5.CMV.null on GM-CSF and IL-8 release from ASM cells

 

Figure 5
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  		Fig. 5. Effect of Ad5.CMV.PKI{alpha} on repression of IL-1β-induced ICAM-1, GM-CSF, and IL-8 expression by cAMP-elevating agents in human ASM cells. A: ASM cells were infected with Ad5.CMV.PKI{alpha} and Ad5.CMV.null at 300 MOI for 24 h. After incubation for 24 h in serum-free medium, cells were stimulated with PGE2 (1 µM) and forskolin (10 µM; left) or salbutamol (10 µM), salmeterol (0.1 µM), and formoterol (10 nM; right) for 30 min before stimulation with IL-1β (1 ng/ml). Top: after 18 h, proteins were harvested for Western blot analysis of ICAM-1 and GAPDH expression. Representative blots are shown. Data from densitometric analysis of Western blots are means ± SE (n = 6). Middle and bottom: supernatants were assayed for GM-CSF and IL-8 release by ELISA. Values are means ± SE [n = 5- 7 (left) and n = 4 (right) for GM-CSF and n = 8 for IL-8]. B: ASM cells were infected, or not, with 300 MOI of Ad5.CMV.PKI{alpha} or Ad5.CMV.null. After 24 h, cells were transferred to serum-free medium for a further 24 h. Cells were then treated with IL-1β (1 ng/ml) in the absence or presence of PGE2 (1 µM) or salmeterol (0.1 µM). After 18 h, cells were analyzed for MTT assay. OD, optical density. Values are means ± SE (n = 4). Significance within a treatment group (naïve, null, or PKI{alpha}) was determined by 1-way ANOVA with Dunnett's post test. Paired t-test was used to test significance between pairs of null- and PKI{alpha}-infected samples. *P < 0.05. **P < 0.01.

 
Examination of cells infected with Ad5.CMV.null for GM-CSF release yielded data similar to the findings reported for naïve cells in Fig. 1. Thus GM-CSF release was suppressed by >85% with PGE2 or forskolin (Fig. 5A). However, infection with Ad5.CMV.PKI{alpha} abolished this repression, such that the release of GM-CSF was not significantly different from IL-1β-treated cells. Similarly, pretreatment of Ad5.CMV.null-infected ASM cells with salbutamol, salmeterol, or formoterol significantly repressed IL-1β-induced GM-CSF by 60–75% in a manner that was consistent with naïve cells (cf. Fig. 5A with Fig. 1). After infection with Ad5.CMV.PKI{alpha}, the ability of short- and long-acting β2-adrenoceptor agonists to suppress GM-CSF release was lost, and no significant change from IL-1β-treated cells was observed (Fig. 5A).

As described in Fig. 3, IL-1β-induced release of IL-8 was repressed by PGE2 and forskolin, but not by β2-adrenoceptor agonists. Therefore, the role of PKA in the repression of this chemokine by PGE2 and forskolin was examined. After infection of cells with Ad5.CMV.null, PGE2 and forskolin significantly repressed IL-1β-induced IL-8 release in a manner that was consistent with their effect on naïve cells (Figs. 3 and 5A). By contrast, infection of cells with Ad5.CMV.PKI{alpha} abolished the suppression of IL-1β-induced IL-8 release by PGE2 and forskolin, such that there was no significant difference from IL-1β-treated cells (Fig. 5A).

The data presented in Fig. 4D and the studies of Misior et al. (30) indicate that PKI{alpha} overexpression leads to increases in ASM cell number. Normalization of all data from Western blot analysis of ICAM-1 to GAPDH will take into account any differences in the overall cell density (Fig. 5A). However, since cytokine production will be affected by the total cell number, we used MTT to assess the effect of virus and PKI{alpha} (Fig. 5B). As described in Fig. 4D, there were no differences in MTT between naïve cells and cells infected with Ad5.CMV.null. However, cells infected with Ad5.CMV.PKI{alpha} revealed modest, but significant, increases in MTT compared with the equivalent Ad5.CMV.null-infected cells (Fig. 5B). This again confirms a role for PKA in the suppression of ASM growth. However, in the analyses presented in Fig. 5A, the ability of PKI{alpha} to reverse the inhibition of GM-CSF release by PGE2, forskolin, or β2-adrenoceptor agonists is compared with cells that were also treated with PKI{alpha} virus (i.e., 100%). Since all cells reveal the same increase in MTT activity (Fig. 5B), the analyses presented in Fig. 5A remain valid.

Effect of β2-adrenoceptor agonists and forskolin on NF-{kappa}B-dependent transcription. Since GM-CSF, IL-8, and ICAM-1 are highly NF-{kappa}B-dependent genes in human ASM cells (7), the effect of β2-adrenoceptor agonists and forskolin was examined on NF-{kappa}B-dependent transcription as a possible mechanism of repressive action (43). ASM cells were infected with Ad5.NF-{kappa}B-luc to allow for the examination of NF-{kappa}B-dependent activity. After stimulation with IL-1β, reporter activity was strongly induced by ≥716 ± 147 fold (Table 4). In the presence of β2-adrenoceptor agonists or forskolin, there was no significant effect on IL-1β-induced NF-{kappa}B-dependent transcription (Table 4), suggesting that generic repression of NF-{kappa}B-dependent transcription does not account for the cAMP-mediated repression of these genes.


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  		Table 4. Effect of β2-adrenoceptor agonists and forskolin on NF-{kappa}B-dependent transcription in human ASM cells

 

   DISCUSSION
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Because of their ability to manufacture and elaborate a variety of inflammatory mediators, ASM cells are an important target for anti-inflammatory therapeutic interventions (22). In this context, although inhaled glucocorticoids are certainly capable of repressing numerous inflammatory outputs (31), the effect on synthetic functions of β2-adrenoceptor agonists is considerably less clear. Thus TNF{alpha} induces the expression of the adhesion molecules ICAM-1 and VCAM-1 on ASM cells, and this is reduced by cAMP-elevating agents, including isoprenaline (32). Similar effects were also reported for TNF{alpha}-induced RANTES release, whereas IL-6 output was increased by β2-adrenoceptor agonist (1, 2). In the context of IL-1β-stimulated ASM cells, β2-adrenoceptor agonists again appear to reduce the expression of RANTES, eotaxin, GM-CSF, and, to a lesser extent, IL-8 (18). In the present study, we confirm that IL-1β is a robust activator of GM-CSF, IL-6, IL-8, and ICAM-1 expression in human ASM cells, and this supports the proinflammatory potential of ASM cells. Furthermore, we show that clinically important β2-adrenoceptor agonists, including the short-acting agonist salbutamol and the long-acting β2-adrenoceptor agonists salmeterol and formoterol, repress the expression of GM-CSF and ICAM-1 in IL-1β-stimulated ASM cells. However, there was no effect of β2-adrenoceptor agonists on IL-6 or IL-8 expression, and this clearly indicates that these genes show differential regulation. These differences in sensitivity are highlighted further by the finding that whereas GM-CSF and ICAM-1 expression was profoundly inhibited by forskolin and PGE2, which acts via the Gs-coupled EP2 and EP4 receptors in these cells (8, 10), there was considerably less effect on IL-8, whereas IL-6 was completely unaffected. Thus, in contrast to previous reports, particularly with respect to IL-6 in airway epithelial cells or TNF{alpha}-treated ASM cells (2, 12, 37), we did not detect adverse, proinflammatory, effects of β2-adrenoceptor agonists or other cAMP-elevating agents on the effector genes studied here.

In terms of the pathway or pathways that elicit these repressive effects on GM-CSF and ICAM-1, we have shown that repression is not only observed with β2-adrenoceptor agonists, but also with other compounds, including PGE2, forskolin, rolipram, and the cell-permeant cAMP analog 8-Br-cAMP. Taken together, these data implicate the classical cAMP cascade. However, alternative coupling of the β2-adrenoceptor may lead to Src and MAPK activation, and cAMP can also activate multiple effector molecules, including exchange factor activated by cAMP, and PKG, as well as PKA (15). Indeed, β2-adrenoceptor agonists have been shown to induce actin depolymerization in human ASM, in part via a PKA-independent pathway that involves Src kinase (19). Therefore, we sought to test the role of PKA in the repression of GM-CSF, IL-8, and ICAM-1 in response to β2-adrenoceptor agonists or other cAMP-elevating agents. However, since commonly used pharmacological inhibitors of PKA, such as H-89, are known to be unreliable (11, 36), we have utilized adenoviral-mediated overexpression of PKI{alpha} as a highly selective means of inhibiting PKA (29). Thus inhibition of GM-CSF release and ICAM-1 expression by the β2-adrenoceptor agonists PGE2 and forskolin was almost totally reversed by adenoviral overexpression of PKI{alpha}. Consequently, other than the classical cAMP-PKA pathway, our data do not reveal major, or indeed any, roles for alternative pathways by which β2-adrenoceptor agonists are reported to act (15). In this context, the lesser, often nonsignificant, effects of rolipram may be attributed to low basal levels of cAMP synthesis in these experiments; also, higher levels of adenylyl cyclase activation would allow the effect of phosphodiesterase-4 inhibition to become more clearly manifest. Certainly, the finding that rolipram is the weakest activator of CRE-dependent transcription supports the idea that this compound has relatively low efficacy as an activator of the cAMP-PKA cascade in human ASM cells. In the context of IL-8, our data highlight considerable differential effects between classes of agent (β2-adrenoceptor agonist, forskolin, and PGE2) that are known to generate cAMP. Thus PGE2, forskolin, and 8-Br-cAMP produced ~50% repression of IL-8 release, whereas the three β2-adrenoceptor agonists were without effect. Although the overall repression was greater than for IL-8, similar differences in efficacy between the various agonists were also observed for GM-CSF and ICAM-1. Since the repression elicited by PGE2 and forskolin was prevented by PKI{alpha}, it is possible that differential activation of the cAMP-PKA cascade by the various agonists may yield differential functional effects. However, analysis of CRE-dependent transcription indicates similar levels of activation by each class of compounds, suggesting that simple variation of the core cAMP-PKA cascade is insufficient to explain the functional differences observed at the level of inflammatory gene expression. Although reasons for this effect are not clear, it is possible that differential compartmentalization of the cAMP cascade may provide an explanation (41, 42). Furthermore, the modulation of inflammatory gene expression, for example, by β2-adrenoceptor agonists and cAMP, operates at multiple transcriptional, posttranscriptional, and also posttranslational levels. Thus it is highly likely that the different cAMP-elevating agents may achieve differential levels of repression depending on the extent to which these three levels of gene control are modulated. Importantly, the mechanisms by which cAMP achieves repression, or occasionally induction, of inflammatory gene expression are, for the most, part poorly elucidated, and this precludes rational targeted molecular analyses to examine these differential effects. Furthermore, these events may depend on the nature of the inflammatory stimulus, as well as the exact cell culture details that are used. Thus Ammit et al. (1) showed that cAMP further elevates IL-6 release when TNF{alpha} is used as a stimulus, whereas we find no effect when IL-1β is substituted. Conversely, Hallsworth et al. (18), despite showing inhibition by isoprenaline, salbutamol, and 8-Br-cAMP, report no effect of forskolin on the IL-1β-induced release of IL-8. In the present study, we observed a partial (~50%) inhibition of IL-8 release by forskolin, which is consistent with the effect of the other cAMP-elevating agents/cAMP analog and the blockade by PKI{alpha} overexpression.

One specific mechanism that has been advanced to explain the inhibition by β2-adrenoceptor agonists and other cAMP-elevating agents on inflammatory gene expression is via repressive effects operating at the level of the transcription factor NF-{kappa}B (43). In the present study, we show that two NF-{kappa}B-dependent genes, IL-6 and IL-8, are not affected by β2-adrenoceptor agonists, whereas two other NF-{kappa}B-dependent genes, GM-CSF and ICAM-1, are strongly repressed (7). Thus it seems difficult to confine the observed repression solely to effects mediated via NF-{kappa}B. To further investigate this matter, we took advantage of a highly NF-{kappa}B-dependent luciferase reporter that we had previously characterized in human ASM cells (7). Analysis of this reporter revealed profound activation by IL-1β, but no further effect of salbutamol, salmeterol, formoterol, or forskolin. We therefore conclude that the repressive effects of these compounds are unlikely to involve a generic effect leveled at the activation of NF-{kappa}B. However, the possibility that promoter-specific inhibition, which could still involve NF-{kappa}B, is not excluded by these data, and this could only be addressed by detailed molecular analyses of each promoter.

In conclusion, our data support the existence of anti-inflammatory effects of β2-adrenoceptor agonists and other cAMP-elevating agents acting on human ASM cells. Thus, in the inflammatory milieu in the lung, prostaglandin, particularly PGE2, formation is predicted to downregulate inflammatory gene expression. However, in asthma, the administration of β2-adrenoceptor agonists leads to bronchodilatation, and this additional elevation of cAMP will further promote the repression of inflammatory gene expression. Although clinical observations clearly show that β2-adrenoceptors agonists are not anti-inflammatory (21, 38), the present observations may be important, since this effect could offset the enhanced expression of inflammatory genes that have been reported for airway epithelial cells (13, 27). Importantly, our data highlight the fact that repression of inflammatory gene expression via the cAMP-PKA cascade may operate in a very gene-specific and agonist-dependent manner; thus considerable further work is necessary before such effects could be harnessed therapeutically. Consistent with the variable effects observed on different NF-{kappa}B-dependent genes, our data do not support a generic role of the cAMP-PKA pathway in inhibiting the activation of NF-{kappa}B. Consequently, further investigations are required to tease out the molecular details that underlie the differential effects of β2-adrenoceptor agonists and cAMP elevation on inflammatory gene expression.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
DISCLOSURES
 REFERENCES
 
M. A. Giembycz and R. Newton are Alberta Heritage Foundation for Medical Research Senior Scholar and Scholar, respectively. R. Newton is a Canadian Institutes of Health Research (CIHR) New Investigator. M. Kaur is the recipient of a National Heart and Lung Institute Foundation Studentship. Work in the laboratories of M. A. Giembycz and R. Newton is supported by operating grants from CIHR.


   DISCLOSURES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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 DISCLOSURES
REFERENCES
 
Work in the laboratories of M. A. Giembycz and R. Newton is also supported by AstraZeneca, GlaxoSmithKline, and Nycomed.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Newton, Dept. of Cell Biology & Anatomy, Faculty of Medicine, Univ. of Calgary, 3330 Hospital Dr. NW, Calgary, AB, Canada T2N 4N1 (e-mail: rnewton{at}ucalgary.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

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