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
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Abstract |
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Keywords: ß2-agonists, airway inflammation, airway remodelling, asthma
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Introduction |
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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 -smooth muscle actin (
-SMA) synthesis, but the efficiency is inversely related to the degree of myofibroblastic differentiation (18). Moreover, FP efficiently decreases basal and tumour necrosis factor-
(TNF-
)-induced nuclear translocation of the p65 subunit of the transcription factor nuclear factor-
B (NF-
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.
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Methods |
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The origin and characteristics of cultured foetal lung fibroblasts (ICIG7) representing the very early step of myofibroblastic differentiation (5% -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 [2530 population doubling level (p.d.l.)] when compared with the parental ICIG7 cells (5055 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 -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% -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- were purchased from R&D Systems (Abingdon, UK). The anti-p65 NF-
B subunit was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-human
-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 102 M in dimethylsulfoxide and stored at 20°C. The ß2-adrenergic receptor (ß2-AR) blocker propranolol hydrochloride was purchased from Calbiochem (France). The drug was diluted at a concentration of 102 M in MeOH and stored at 20°C.
Confocal microscopy on -SMA and p65 NF-
B expression
To determine the pattern of -SMA, 24-h myofibroblastic cultures were incubated with 108 M SMl and/or 1081012 M FP with or without TGF-ß (5 ng ml1 for 72 h at 37°C). To detect p65 NF-
B nuclear translocation, 48-h lung myofibroblast cultures were pre-incubated in serum-free medium containing 108 M SMl and/or 1081012 M FP for 1 h at 37°C, and then stimulated for 15 min with TNF-
(10 ng ml1). Cultures without the drugs and/or TNF-
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 108 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 108 M SMl and/or 1081012 M FP did not affect cellular proliferation and survival, while use of unphysiologically (17) higher concentrations (106107 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 (-SMA) and yellow (NF-
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 transcriptionPCR analysis of -SMA
The following primers were used to detect -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
-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 108 M FP or with 108 M SMl for 1 h at 37°C and then stimulated or not for 15 min with TNF- (10 ng ml1). Nuclear extraction of MyoICIG7 cells and EMSA analysis of NF-
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 18 pg ml1.
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 TrisHCl 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.
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Results |
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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 -SMA (Fig. 2A). TGF-ß strongly increased both the percentage of positive cells (96.7 ± 1.5%) and the intensity of the specific anti-
-SMA staining per cell. SMl 108 M (39.3 ± 2.9%) and FP 1012 M (29.3 ± 3.2%) only partially inhibited TGF-ß-induced
-SMA expression, whereas the combined use of the two drugs almost totally abolished
-SMA expression (2.3 ± 0.6%). Similar results were obtained with MyoICIG7 cells. Indeed, 74 ± 3.5% of MyoICIG7 cells were
-SMA positive in basal culture conditions compared with 95.3 ± 2.5% in the presence of TGF-ß. SMl 108 M (10.7 ± 2.1%) and FP 1012 M (20 ± 1%) efficiently decreased TGF-ß-induced
-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 108 M FP inhibits
-SMA expression (13) and here we show that the drug is still fully active at 1012 M and able to show additive effects with SMl. Moreover, the complementary effect on the TGF-ß-induced expression of
-SMA was also observed in aged myofibroblastic cultures (data not shown) which become resistant to FP alone (18). Interestingly, 108 M SMl and/or 1081012 M FP counteracted the growth inhibition (2530%) induced by TGF-ß (data not shown).
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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 108 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-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-B (13).
Confocal microscopy.
We used confocal microscopy to study the constitutive and the TNF--induced activation of NF-
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-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-
B in the nucleus. Densitometric analysis (from three different samples) showed that in basal culture conditions the p65 subunit of NF-
B displayed a nuclear localization in
10.2 ± 3% of Bronco 5 cells and 8.8 ± 4% of MyoICIG7 cells. Treatment with TNF-
strongly increased the percentage of cells expressing an activated NF-
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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Gel shift assay.
To confirm the inhibitory effect of SMl and FP on NF-B activation, we performed gel retardation assays using nuclear proteins extracted from lung myofibroblasts (Fig. 4A). An oligonucleotide encoding the
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
B-binding activity, which co-migrated with the T cell NF-
B dimer previously identified as p65/p50 (18). Quantitative PhosphorImager analysis (from two different samples) showed that TNF-
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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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-B subunit (nuclear yellow punctate staining). Treatment with TNF-
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-
were not modified by pre-incubation with propranolol (96 ± 3.7%) whereas SMl treatment inhibited TNF-
-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-
B activation is mediated by a functional interaction with its respective receptor, the ß2-AR.
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Discussion |
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Moreover, this glucocorticoid-like inhibition of NF-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
-SMA-positive mesangial cells and the regression of renal injury (34). Thus, our finding that SMl inhibits
-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.
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Acknowledgements |
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Abbreviations |
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ß2-AR | ß2-adrenergic receptor |
FP | fluticasone propionate |
NF-![]() | nuclear factor-![]() |
p.d.l. | population doubling level |
r | recombinant |
RT | reverse transcription |
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SMC | smooth muscle cell |
SMl | salmeterol |
TGF | transforming growth factor |
TNF-![]() | tumour necrosis factor-![]() |
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Notes |
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Received 10 November 2004, accepted 23 August 2005.
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References |
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