Lung myofibroblasts as targets of salmeterol and fluticasone propionate: inhibition of {alpha}-SMA and NF-{kappa}B

Soria Baouz1, Julien Giron-Michel1, Bruno Azzarone1,2, Massimo Giuliani2, Francesca Cagnoni3, Susanna Olsson3, Renato Testi4, Giulio Gabbiani5 and G. Walter Canonica3

1 Institut National de la Santé et de la Recherche Médicale 506 and 2 Institut National de la Santé et de la Recherche Médicale Unité 542, Bat. Lavoisier, Hospital Paul Brousse, Villejuif, France
3 Department of Allergy and Respiratory Diseases, Department of Internal Medicine, University of Genoa, Genoa, Italy
4 Medical Department, Glaxo SmithKline, Verona, Italy
5 Department of Pathology, Faculty of Medicine, 1211 Geneva 4, Switzerland

Correspondence to: B. Azzarone; E-mail: bazzarone{at}hotmail.com


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Lung myofibroblasts play a major role in the pathophysiology of asthma, contributing not only to tissue remodelling but also to airway inflammation. Nevertheless, only recently, attention has been focused on these cells as potential targets for anti-allergic drugs. Herein, we analysed the pharmacological response of lung myofibroblasts to ß2-agonists associated or not to inhaled corticosteroids, investigating their effects on (i) the constitutive and transforming growth factor-ß (TGF-ß)-induced expression of {alpha}-smooth muscle actin ({alpha}-SMA), the main functional marker of myofibroblastic differentiation and contractility; (ii) isometric contraction and (iii) tumour necrosis factor-{alpha} (TNF-{alpha})-induced nuclear translocation of the pro-inflammatory transcription factor nuclear factor-{kappa}B (NF-{kappa}B). The ß2-agonist salmeterol (SMl) has on human lung myofibroblasts new direct anti-contractile/anti-inflammatory effects that are amplified by the combined use of low concentrations of the glucocorticoid fluticasone propionate (FP). First, SMl and/or FP (10–12 M) inhibits the constitutive and TGF-ß-induced expression of {alpha}-SMA. Second, the two drugs block the TNF-{alpha}-induced nuclear translocation of the pro-inflammatory transcription factor NF-{kappa}B. Finally, SMl decreases TNF- {alpha}-induced production of the inflammatory cytokine IL-6. The complementary anti-inflammatory/ anti-contractile effects displayed by SMl and FP on lung myofibroblasts in vitro may be related to the improvement in lung function and symptom control obtained in vivo by the early use of low doses of glucocorticoids in combination with long-acting ß2-agonists.

Keywords: ß2-agonists, airway inflammation, airway remodelling, asthma


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Asthma is characterized by inflammation, airway remodelling, variable airway obstruction and hyper-responsiveness. In the past 15 years, many studies have established the role of inflammatory cells, such as eosinophils, in the pathogenesis of asthma, whereas the potential role of resident cells has only been acknowledged recently (1, 2). It is well recognized that airway remodelling in asthma is the main consequence of altered fibroblast behaviour (3), and until recently, these remodelling changes have been considered to be secondary phenomena, developing late in the disease process as a consequence of persistent inflammation. An alternative view of asthma pathogenesis has been recently proposed by emphasizing the importance of the airway microenvironment (the epithelial mesenchymal trophic unit) in the origins of the disease (4). Indeed, airway myofibroblasts and other resident cells are able to produce many different chemokines and cytokines upon stimulation. These products can, in turn, activate or attract inflammatory cells in the lungs, favouring the establishment of chronic disease (57). Moreover, human lung fibroblasts may be directly activated by Th2 cytokines such as IL-4 and IL-13, which induce signal pathways leading to the production of several pro-inflammatory molecules (89).

The main goals of asthma treatment are the inhibition of ongoing inflammation and bronchodilation. The prototype anti-asthmatic agent is glucocorticoids. These are typically inhaled when asthma is stable. Bronchodilatory drugs such as ß2-agonists are administered locally in the lungs by inhalation and relax smooth muscle. Long-acting ß2-agonists also appear to have anti-inflammatory effects (1012).

Longtime, lung fibroblasts have not been considered to be potential targets for anti-allergic drugs, although extensive studies have sought to identify the direct inhibitory actions of corticosteroids and ß2-agonists on several inflammatory and structural cells implicated in pulmonary and airway disease (13). Only recently, different groups have approached this problem investigating the response of lung fibroblasts to anti-allergic drugs showing the modulation of important pro-inflammatory molecules (1417). In this respect, we have recently shown that in lung myofibroblasts fluticasone propionate (FP) inhibits JAK/STAT signalling induced by Th2 cytokines and {alpha}-smooth muscle actin ({alpha}-SMA) synthesis, but the efficiency is inversely related to the degree of myofibroblastic differentiation (18). Moreover, FP efficiently decreases basal and tumour necrosis factor-{alpha} (TNF-{alpha})-induced nuclear translocation of the p65 subunit of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) in lung myofibroblasts independently of their degree of differentiation (18).

On the basis of these data, we extended our analysis and we investigated whether the long-acting ß2-agonist salmeterol (SMl) is also effective on human lung fibroblasts/myofibroblasts. Indeed, these cells display specific high-affinity receptors for these compounds (19) that may control collagen synthesis (20), showing that ß2-agonists can specifically act on human lung myofibroblasts (1417).

We analysed the influence of long-acting ß2-agonists, inhaled glucocorticoids or both on lung myofibroblast behaviour, to determine whether their interaction affects the ongoing inflammatory and remodelling processes of the airways, which could also explain their beneficial effects in asthmatic patients.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells
Twenty primary cultures from human airways of normal donors (embryonic, foetal and adult) or from patients with Th2 lesions were established in primary culture by enzymatic digestion of the tissues. Phenotypic analysis of the fibroblastic cultures identified three types of cells: (i) cultures expressing <5% of {alpha}-SMA-positive cells (essentially from normal donors), (ii) cultures expressing between 25 and 40% {alpha}-SMA-positive cells (from pathological samples) and (iii) fully differentiated cultures expressing 100% {alpha}-SMA-positive cells (from pathological samples). All these myofibroblastic primary cultures expressed the fibroblastic marker ASO2. For this study, we have chosen fibroblastic lung primary cultures that represent the aforementioned different groups of myofibroblastic differentiation.

The origin and characteristics of cultured foetal lung fibroblasts (ICIG7) representing the very early step of myofibroblastic differentiation (5% {alpha}-SMA-positive cells) have been previously described (11). When these cells are transferred to an adhesion surface displaying an increased mechanical tension, they undergo an irreversible myofibroblastic differentiation, confirming therefore previous data showing that this treatment induces both in vitro and in vivo the generation of myofibroblasts (21, 22). This myofibroblastic subset (MyoICIG7 cells) is characterized by a shortened in vitro lifespan [25–30 population doubling level (p.d.l.)] when compared with the parental ICIG7 cells (50–55 p.d.l.).

Bronco 5 primary cultures were obtained from the ‘normal’ lung tissue neighbouring a bronchial carcinoma of a 63-year-old patient. These cells can be classified as mildly differentiated myofibroblasts (30% of {alpha}-SMA-positive cells).

The primary adult lung myofibroblasts FPA were derived from an in vivo massive stromal reaction to a melanoma lung metastasis (of a 54-year-old patient) displaying the characteristic of a typical Th2 microenvironment. Indeed, after the enzymatic digestion of the bioptic sample, two types of cells emerged in culture: (i) cells growing in suspension and identified as activated CD4 lymphocytes expressing the Th2 phenotype (IL-4+, IL-5+ and IL-2) and (ii) adherent bipolar myofibroblastic cells (100% {alpha}-SMA-positive cells) termed FPA.

All cells were cultured in DME (EuroBio SA, Les Ulis, France) supplemented with 10% FCS (Biological Industries, Kibbutz Beit Haemek, Israel), 2 mM glutamine and 1% antibiotics (GIBCO BRL, Cergy-Pontoise, France) in a 37°C, 5% CO2 incubator.

Cytokines and antibodies
Human recombinant (r)-transforming growth factor-ß (TGF-ß) and r-TNF-{alpha} were purchased from R&D Systems (Abingdon, UK). The anti-p65 NF-{kappa}B subunit was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-human {alpha}-SMA 1A4 mAb was from DAKO (Glostrup, Denmark). Alexa Fluor488 GAR and Alexa Fluor594 GAM were purchased from Molecular Probes (Leiden, The Netherlands). SMl and FP were kindly provided by Medical Department, Glaxo SmithKline, Verona, Italy. The drugs were diluted to a concentration of 10–2 M in dimethylsulfoxide and stored at –20°C. The ß2-adrenergic receptor 2-AR) blocker propranolol hydrochloride was purchased from Calbiochem (France). The drug was diluted at a concentration of 10–2 M in MeOH and stored at –20°C.

Confocal microscopy on {alpha}-SMA and p65 NF-{kappa}B expression
To determine the pattern of {alpha}-SMA, 24-h myofibroblastic cultures were incubated with 10–8 M SMl and/or 10–8–10–12 M FP with or without TGF-ß (5 ng ml–1 for 72 h at 37°C). To detect p65 NF-{kappa}B nuclear translocation, 48-h lung myofibroblast cultures were pre-incubated in serum-free medium containing 10–8 M SMl and/or 10–8–10–12 M FP for 1 h at 37°C, and then stimulated for 15 min with TNF-{alpha} (10 ng ml–1). Cultures without the drugs and/or TNF-{alpha} were set up as controls. Subsequently, cells from both experiments were washed, permeabilized and processed for laser scanning confocal microscopy as previously described (18).

In some experiments, we analysed whether the effects of the ß2-agonists were dependent upon binding of the drug to the ß2-AR, by pre-incubating the cells, 30 min prior to stimulation with SMl, with 10–8 M of the ß2-antagonist, propranolol, as previously reported (17).

The concentrations of the drugs employed in these above-mentioned assays are in the physiological range (17) that probably occurs in airway lining fluid during inhalation therapy (23). A total of 10–8 M SMl and/or 10–8–10–12 M FP did not affect cellular proliferation and survival, while use of unphysiologically (17) higher concentrations (10–6–10–7 M) induced apoptosis (DIOC6 assay) and reduced proliferation (CFDA assay).

Statistical analysis and quantitative confocal microscopy
A computerized densitometric analysis based on the evaluation of the optic density of the red ({alpha}-SMA) and yellow (NF-{kappa}B) staining was performed on different samples. The Student's t-test was used for statistical analysis. P-values of ≤0.05 were considered to represent significance.

Reverse transcription–PCR analysis of {alpha}-SMA
The following primers were used to detect {alpha}-SMA: 5'-GTC CAC CGC AAA TGC TTC TAA-3' (upstream) and 5'-AAA ACA CAT TAA CGA GTC AG-3' (downstream). The amplification product was a 141-bp fragment (annealing temperature: 58°C; 30 cycles). The following primers were used to amplify glyceraldehyde-3-phosphate dehydrogenase: 5'-GGT GAA GGT CGG AGT CAA CGG A-3' (upstream) and 5'-GAG GGA TCT CGC TCC TGG AAG A-3' (downstream). These primers amplified a 240-bp fragment (annealing temperature: 60°C; 20 cycles). Total RNA isolation and semi-quantitative reverse transcription (RT)–PCR for the {alpha}-SMA PCR products were performed as previously described (18).

Nuclear protein extraction and EMSA
MyoICIG7 myofibroblasts were pre-incubated or not in serum-free medium with 10–8 M FP or with 10–8 M SMl for 1 h at 37°C and then stimulated or not for 15 min with TNF-{alpha} (10 ng ml–1). Nuclear extraction of MyoICIG7 cells and EMSA analysis of NF-{kappa}B DNA-binding activity of these extracts were performed as reported previously (24, 25).

IL-6 production
IL-6 was quantified in cell-free culture supernatants from 72-h samples treated or not with SMl and/or fluticasone, using standard ELISA kits (R&D Systems) according to the manufacturer's instructions. The lower limit of detection was 1–8 pg ml–1.

Deformable silicone substrates and single-cell force measurement
Deformable silicone substrates were essentially prepared as previously described (26). A total of 50 µl of silicone (polydimethyl siloxane; 30 000 cSt; Dow Corning, Midland, MI, USA) was deposited onto a 35-mm round glass coverslip, which was placed into a six-well plate and centrifuged at 1000 r.p.m. for 2 min using a swinging rotor. The silicone surface was then cross-linked by passing it through a Bunsen flame. A rubber ring was sealed with a polyvinylsiloxane dental resin (President MicroSystems; Coltène, Altstätten, Switzerland) on the coverslip, resulting in a small chamber containing at the bottom the cross-linked silicone. Silicone substrates were equilibrated with 0.1% gelatin in Tris–HCl buffer, pH 8.4, sterilized by UV light exposure and left overnight in the incubator at 37°C. The flaming time of 1 s was used; this restricted wrinkle formation to fibroblasts with high contractile force (27).

To visualize the contractile activity of single cells, lung myofibroblasts were cultured for 4 days in the growth medium on these deformable silicone substrates, which were produced in order to obtain an optimal resistance, as previously described. Image sequences were obtained with an inverted microscope (Axiovert 135 Zeiss) equipped with a x63 objective B/W camera (BC-2; AVT Horn), frame grabber (Meteor PCI; Matrox Electronic Systems Ltd) and KS400 software (Zeiss). SMl and fluticasone were added after 60 min control recording in serum-free MEM.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Effects of SMl and FP on {alpha}-SMA expression in human lung myofibroblasts
RT–PCR analysis.
In these experiments (Fig. 1) we used primary cultures of mildly differentiated lung myofibroblasts expressing ~20–30% {alpha}-SMA-positive cells (Bronco 5) and fully differentiated ICIG7 myofibroblastic cultures (MyoICIG7). The specific {alpha}-SMA transcript (141 bp) was present in both myofibroblastic cell cultures. Densitometric analysis (representing the mean of three different experiments) showed that TGF-ß caused a 2-fold increase in the level of {alpha}-SMA mRNA in Bronco 5 and MyoICIG7. In both myofibroblastic cultures, SMl inhibited constitutive and TGF-ß-induced {alpha}-SMA gene transcription.



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Fig. 1. Detection of {alpha}-SMA in human bronchial myofibroblasts at different stages of differentiation by RT–PCR (upper panels: {alpha}-SMA transcripts 141 bp; lower panels: housekeeping gene glyceraldehyde-3-phosphate dehydrogenase 120 bp). These figures are representative of three different experiments. Densitometric analysis from three different experiments.

 
Confocal microscopy.
As {alpha}-SMA is a stable protein with a half-life of ~72 h (28) and as the two drugs are able to inhibit {alpha}-SMA transcription within this time, we investigated their ability to act on protein synthesis.

Densitometric analysis of confocal images (from three different samples) showed that in basal culture conditions 33.7% (±3.1%) of Bronco 5 cells expressed detectable amounts of {alpha}-SMA (Fig. 2A). TGF-ß strongly increased both the percentage of positive cells (96.7 ± 1.5%) and the intensity of the specific anti-{alpha}-SMA staining per cell. SMl 10–8 M (39.3 ± 2.9%) and FP 10–12 M (29.3 ± 3.2%) only partially inhibited TGF-ß-induced {alpha}-SMA expression, whereas the combined use of the two drugs almost totally abolished {alpha}-SMA expression (2.3 ± 0.6%). Similar results were obtained with MyoICIG7 cells. Indeed, 74 ± 3.5% of MyoICIG7 cells were {alpha}-SMA positive in basal culture conditions compared with 95.3 ± 2.5% in the presence of TGF-ß. SMl 10–8 M (10.7 ± 2.1%) and FP 10–12 M (20 ± 1%) efficiently decreased TGF-ß-induced {alpha}-SMA expression, whereas their combined use totally inhibited it (1 ± 1.7%). In all cases, the P-value was ≤0.05 (Student's t-test), indicating a statistical significance. We have previously reported that 10–8 M FP inhibits {alpha}-SMA expression (13) and here we show that the drug is still fully active at 10–12 M and able to show additive effects with SMl. Moreover, the complementary effect on the TGF-ß-induced expression of {alpha}-SMA was also observed in aged myofibroblastic cultures (data not shown) which become resistant to FP alone (18). Interestingly, 10–8 M SMl and/or 10–8–10–12 M FP counteracted the growth inhibition (25–30%) induced by TGF-ß (data not shown).



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Fig. 2. (A) Detection of {alpha}-SMA (red staining) in human bronchial myofibroblasts at different stages of differentiation by confocal microscopy (Bronco 5 and MyoICIG7). Densitometic analysis of confocal pictures on three different samples. Comparison by Student's t-test of basal with TGF-ß groups, TGF-ß with FP + TGF-ß, SMI + TGF-ß, FP/SMI + TGF-ß groups and FP + TGF-ß or SMI + TGF-ß with FP/SMI + TGF-ß groups, gave a P-value of <0.05, indicating statistical significance. (B) Measurement of isometric contraction on deformable silicone substrates on bronchial myofibroblasts at different p.d.l. Young (p.d.l. 8) and aged (p.d.l. 25) Bronco 5 cells produce wrinkles on deformable silicone substrates during 60 min recording. Treatment with FP does not modify the number and the intensity of the wrinkles, whereas wrinkles completely disappear after 30 min treatment with SMl in young cultures and are still consistently decreased in aged cultures. This picture is representative of three different experiments.

 
Modulation of isometric contraction on silicon gel by SMl and FP.
Subsequently, we investigated whether bronchial myofibroblasts were sensitive to the main pharmacological property of ß2-agonists: the bronchodilatory effect which is obtained by blocking the contractile activity and has been reported only in smooth muscle cells (SMCs).

As illustrated in Fig. 2(B), Bronco 5 developed within 24 h, an efficient isometric contractile activity on silicon gel as shown by the formation of several white wrinkles, that appear to be stable on time both in young [population doubling level (p.d.l.) 8] and aged cultures (p.d.l. 25). Incubation with FP did not affect the contractile activity, whereas addition of SMl 10–8 M induced, within 30 min, the total disappearance of the wrinkles in young cultures and a significant decrease in aged ones.

Effects of SMl and FP on NF-{kappa}B expression in human lung myofibroblasts
ß2-Agonists can induce the nuclear translocation and functional activation of the glucocorticoid receptor in lung fibroblasts, even in the total absence of corticosteroids (17). We investigated whether SMl was able to mimic one of the main anti-inflammatory effects of glucocorticoids: the inhibition of the transcription factor NF-{kappa}B (13).

Confocal microscopy.
We used confocal microscopy to study the constitutive and the TNF-{alpha}-induced activation of NF-{kappa}B in mildly differentiated (Bronco 5) and terminally differentiated human lung myofibroblasts (MyoICIG7).

Figure 3 shows different overlay pictures; the p65 subunit of the NF-{kappa}B complex is shown in green and nuclei are shown in red (propidium iodide). The green staining represents the presence of the native p65 subunit in the cytoplasm, whereas the yellow staining shows activated NF-{kappa}B in the nucleus. Densitometric analysis (from three different samples) showed that in basal culture conditions the p65 subunit of NF-{kappa}B displayed a nuclear localization in ~10.2 ± 3% of Bronco 5 cells and 8.8 ± 4% of MyoICIG7 cells. Treatment with TNF-{alpha} strongly increased the percentage of cells expressing an activated NF-{kappa}B: 51 ± 13% in Bronco 5 cells and 44 ± 7.6% in MyoICIG7 cells. When used separately, the two drugs partially counteract this induction. SMl appeared to be less efficient as it decreased the percentage of yellow nuclei to 30 ± 7.5% (Bronco 5) and 32 ± 2.3% (MyoICIG7), whereas FP decreased these percentages to 16.2 ± 2% (Bronco 5) and 17.6 ± 2.7% (MyoICIG7).



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Fig. 3. Confocal microscopy of the nuclear translocation of the p65/NF-{kappa}B in lung myofibroblasts at different stages of differentiation. Propidium iodide stains nuclei red. Cytoplasmic p65/NF-{kappa}B is stained in green and nuclear NF-{kappa}B in yellow. Densitometric analysis of p65/NF-{kappa}B nuclear translocation performed on three different samples. Comparison by Student's t-test of basal with TNF groups, TNF with FP + TNF, SM + TNF, FP/SMI + TNF groups and FP + TNF or SMI + TNF with FP/SMI + TNF, gave a P-value of <0.05, indicating statistical significance.

 
Interestingly, combined treatment further inhibited both constitutive and TNF-{alpha}-induced nuclear localization of the p65 subunit of NF-{kappa}B, decreasing the percentage of yellow nuclei to 1.4 ± 2.4% (Bronco 5) and 7 ± 1.8% (MyoICIG7). In all cases, the P-value was ≤0.05 (Student's t-test).

Gel shift assay.
To confirm the inhibitory effect of SMl and FP on NF-{kappa}B activation, we performed gel retardation assays using nuclear proteins extracted from lung myofibroblasts (Fig. 4A). An oligonucleotide encoding the {kappa}B sequence was used as a probe (Fig. 4). As a control, we used an extract from a T cell line that had been activated for 3 h with phorbol myristate acetate plus ionomycin. Myofibroblastic cells displayed constitutive {kappa}B-binding activity, which co-migrated with the T cell NF-{kappa}B dimer previously identified as p65/p50 (18). Quantitative PhosphorImager analysis (from two different samples) showed that TNF-{alpha} treatment increased the binding of p65/p50 (+64 ± 4.3%), whereas treatment with SMl (–66 ± 1.2%) and FP (–43 ± 3.3%) decreased the percentage of binding.



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Fig. 4. (A) EMSA analysis of NF-{kappa}B nuclear complexes in lung myofibroblasts. A nuclear extract from the human T cells pre-activated with phorbol myristate acetate and ionomycin was used as an internal control. The arrow indicates the p65/p50 dimer. Densitometric analysis from two different experiments. Comparison by Student's t-test of basal with TNF group and TNF with SMI + TNF or FP + TNF groups gave a P-value of <0.05, indicating statistical significance. (B) ELISA analysis of IL-6 (pg ml-1) production after 72 h in culture. Comparison by Student's t-test from three different experiments of basal with TNF group and TNF with SMI + TNF or FP/SMI + TNF groups gave a P-value of <0.05, indicating statistical significance. (C) Confocal microscopy of the nuclear translocation of the p65/NF-{kappa}B in lung FPA myofibroblasts. Propidium iodide stains nuclei red. Cytoplasmic p65/NF-{kappa}B is stained in green and nuclear NF-{kappa}B in yellow. Densitometric analysis of p65/NF-{kappa}B nuclear translocation performed on three different samples. Comparison by Student's t-test of basal with TNF groups, TNF with SMI + TNF and SMI + TNF with propranolol hydrochloride (Prop)/SMI + TNF, gave a P-value of <0.05, indicating statistical significance.

 
The inhibitory effects of SMl on NF-{kappa}B activation were confirmed by investigating the expression of IL-6, an NF-{kappa}B-activated gene (13, 29). ELISA assay (Fig. 4B) showed that MyoICIG7 cells secrete detectable amounts of IL-6 (350 pg ml–1). Treatment with TNF-{alpha} for 72 h strongly increased IL-6 production (12-fold). SMl did not modify constitutive IL-6 secretion, whereas severely affected TNF-{alpha}-induced IL-6 production (–90%) which is under the control of NF-{kappa}B (25). Combined SMl and FP treatment had no additional inhibitory effect on IL-6 constitutive or TNF-{alpha}-induced production.

Finally, we analysed whether the effects of SMl were dependent upon binding of the drug to the ß2-AR by pre-incubating the cells with the ß2-AR antagonist, propranolol. In Fig. 4(C), densitometric analysis of confocal pictures (from three different samples) shows that FPA cells (5 ± 1.3%) constitutively display nuclear localization of the p65 NF-{kappa}B subunit (nuclear yellow punctate staining). Treatment with TNF-{alpha} induces a massive increase in the intensity of nuclear localization of the p65 and in the percentage (97 ± 2.8%) of positive cells. The effects of TNF-{alpha} were not modified by pre-incubation with propranolol (96 ± 3.7%) whereas SMl treatment inhibited TNF-{alpha}-induced nuclear localization of p65 (6 ± 2.4%). When propranolol (108 M) was administered to the cells 30 min prior to stimulation with SMl and TGF-ß, it had a powerful inhibitory effect (89 ± 1.7%) on the SMl-induced decrease of p65 nuclear localization. These data demonstrate that SMl-induced inhibition of NF-{kappa}B activation is mediated by a functional interaction with its respective receptor, the ß2-AR.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we show that the ß2-agonist SMl has anti-contractile and potential anti-inflammatory effects on human lung myofibroblasts. It inhibits, at the transcriptional and translational levels, the expression of {alpha}-SMA, the most important marker of differentiation of fibroblasts into contractile myofibroblasts. Moreover, SMl decreases both the constitutive and TNF-{alpha}-induced nuclear localization of the pro-inflammatory transcription factor NF-{kappa}B as well as the TNF-{alpha}-induced secretion of IL-6, which is mediated by NF-{kappa}B (29). The SMl-induced inhibition of NF-{kappa}B activation is mediated by a functional interaction with its specific receptor the ß2-AR as shown by the antagonistic effects observed pre-incubating lung myofibroblasts with the ß2-AR blocker propranolol. The effects on {alpha}-SMA and NF-{kappa}B are amplified by the combined use of very low concentrations of FP (10–12 M). Although the beneficial effects of long-acting ß2-agonists in asthma have long been attributed to clinical parameters mainly concerning bronchodilation, there is now increasing in vitro evidence that they may also exert some anti-inflammatory effects (12, 30, 31). Our results strengthen this concept, extending the panel of anti-inflammatory effects displayed by ß2-agonists (12, 1417) and the additive effects between long-acting ß2-agonists and inhaled corticosteroids. Indeed, the inhibition of NF-{kappa}B, which controls the activation of many inflammatory molecules (13), is a new observation that may be related to the capacity of ß2-agonists to induce the functional nuclear translocation of the glucocorticoid receptor in lung fibroblasts (32). Interestingly similar results have been reported in lung biopsies of allergic patients, where formeterol inhibits the activation of NF-{kappa}B in epithelial cells (29).

Moreover, this ‘glucocorticoid-like’ inhibition of NF-{kappa}B may explain some of the anti-inflammatory effects recently attributed to ß2-agonists (12, 30, 33). In adult diffuse proliferative IgA nephropathy, early treatment with corticosteroids leads to the disappearance of {alpha}-SMA-positive mesangial cells and the regression of renal injury (34). Thus, our finding that SMl inhibits {alpha}-SMA expression suggests that it could have a role in delaying the appearance of myofibroblasts, the onset of remodelling and consequently the sub-epithelial fibrosis. Furthermore, the main pharmacological property of ß2-agonists: the bronchodilatatory effect which is obtained by blocking the contractile activity of SMCs (35), is also observed in bronchial myofibroblasts. Indeed, our data demonstrate that SMl rapidly and totally inhibits isometric contraction of these cells. Thus, it is likely that in vivo the relief from bronchoconstriction is probably obtained by the combined action of ß2-agonists both on SMC (36) and on myofibroblasts. Interestingly, this effect in vitro is also observed in very aged cells suggesting that in vivo relaxation could be obtained also in long-term established fibrotic lesions.

It has recently been proposed that the anti-inflammatory effect of glucocorticoids is mainly achieved by inhibiting the functions of both resident lung cells and infiltrated inflammatory cells (29).

Myofibroblasts are resident lung cells that can contribute to the pathogenicity of asthma not only by favouring lung tissue remodelling through the synthesis of extracellular matrix proteins but also by enhancing bronchial constriction through the expression of contractile proteins. Moreover, resident lung cells may play a major role in the establishment of chronic inflammation, directly through the production of inflammatory molecules (8, 9, 37), but also contributing to transmigration and activation of bone marrow-derived inflammatory cells (38), highlighting the importance of these cells as potential targets for combined anti-allergic therapy (1418). In conclusion, these data and previous ones suggest that combined early treatment with glucocorticoids and a long-acting ß2-agonist improves asthma symptoms not only by inhibiting classical inflammatory signals (1012) but also by acting on airway myofibroblasts, perhaps delaying the onset of sub-epithelial fibrosis and the secretion of inflammatory factors and contributing to bronchodilation.


    Acknowledgements
 
This work was partly supported by grants from Glaxo SmithKline SpA, Italy (FLIC25 study), Association Nouvelles Recherches Biomedicales (Villejuif, France), Associazione Ricerca Malattie Immunologiche e Allergiche, Italian Ministry of University and Scientific and Technologic Research (Italy), Swiss Science Foundation N°31/61336.00 and by Foundation Novartis. S.B. is a recipient of a fellowship from Associazione Ricerca Malattie Immunologiche e Allergiche. S.O. was supported by the Swedish Society for Medical Research.


    Abbreviations
 
ß2-AR   ß2-adrenergic receptor
FP   fluticasone propionate
NF-{kappa}B   nuclear factor-{kappa}B
p.d.l.   population doubling level
r   recombinant
RT   reverse transcription
{alpha}-SMA   {alpha}-smooth muscle actin
SMC   smooth muscle cell
SMl   salmeterol
TGF   transforming growth factor
TNF-{alpha}   tumour necrosis factor-{alpha}

    Notes
 
Transmitting editor: L. Moretta

Received 10 November 2004, accepted 23 August 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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