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Keywords:

  • asthma;
  • inflammation;
  • inhaled corticosteroids;
  • lung fibroblast;
  • remodelling;
  • β2-agonists

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Background:  Bronchial asthma is characterized by lower airway inflammation and remodelling. Anti-inflammatory treatment with inhaled corticosteroids (ICS) provides the mainstay of asthma therapy together with bronchodilation induced by short- and long-acting inhaled β2-agonists. Lower airway fibroblasts may play a critical role in airway inflammation and remodelling, suggesting that they might represent an important target for the major anti-asthmatic drugs. The aim of our study was to investigate the effects of beclomethasone dipropionate (BDP), salbutamol and formoterol either alone or in combination on in vitro cultures of human bronchial fibroblasts.

Methods:  Fibroblasts were cultured in the presence of pro-inflammatory and proliferative stimuli, BDP, salbutamol and formoterol. The effects of drugs on cell proliferation were ascertained by 3H-thymidine incorporation. CD90 and CD44 expression were detected by flow cytometry and fibronectin secretion using the enzyme-linked immunosorbent assay technique.

Results:  This study showed that BDP alone has significant anti-proliferative effects on lung fibroblasts treated with basic fibroblast growth factor and the combination of BDP with formoterol or salbutamol strengthen these effects. Short-acting β2-agonist (SABA) or long-acting β2-agonist (LABA) by themselves did not show any significant effect on the different cultures. CD44 and CD90 expression and fibronectin production were modulated by pro-inflammatory and proliferative stimuli; the addition of the drugs brought them back near to the basal level.

Conclusions:  From this in vitro study, we can conclude that BDP, when combined with salbutamol or formoterol, exhibits enhanced anti-remodelling activity in bronchial fibroblasts, providing new insights on the additive effects of ICS and SABAs and LABAs for asthma therapy.

Abbreviations:
BDP

beclomethasone dipropionate

ECM

extracellular matrix

FBS

foetal bovine serum

FGFb

basic fibroblast growth factor

ICS

inhaled corticosteroid

IL-4

interleukin-4

LABA

long-acting β2-agonist

SABA

short-acting β2-agonist

TGF-β

transforming growth factor-β

Bronchial asthma is a chronic inflammatory disorder of the airways in which many cells and elements play a crucial role. The chronic inflammation is associated with increased airway responsiveness leading to recurrent episodes of wheezing, breathlessness, chest tightness and coughing, particularly at night or early in the morning. These episodes are usually associated with widespread but variable airflow obstruction that is often reversible either spontaneously or after treatment (1). Asthma represents a profound public health problem worldwide.

From the functional point of view, nonsmoking asthmatic patients may have a significantly greater decline in lung function compared with nonasthmatic subjects and may develop chronic irreversible (fixed) airflow limitation (2, 3). This has been related to the physiological consequences of chronic airway inflammation causing airway remodelling, a collective term to include airways epithelial basement membrane thickening, airway wall oedema, airway smooth muscle hyperplasia and hypertrophy (4).

Anti-inflammatory treatment with inhaled corticosteroids (ICS) provides the mainstay of asthma therapy together with bronchodilation induced by inhaled Short-acting β2-agonists (SABAs) and long-acting β2-agonists (LABAs). Several clinical studies have shown that adding LABAs to the treatment of symptomatic patients despite regular treatment with ICS produces beneficial effects on symptoms, lung function and asthma exacerbations (1, 5, 6). On the other hand, the SABA plus ICS combination has been recently clinically reevaluated (7).

Both β2-agonists may inhibit airway inflammation, probably increasing intracellular cAMP levels and inhibiting activation of eosinophils and other inflammatory cells trafficking as described in previous studies (8–11). The main target of glucocorticoid action is inhibition of inflammatory cell activation, demonstrated for T-cell proliferation, blood mononuclear cell cytokine production and eosinophil chemokine production and survival (12–16). Structural cells such as fibroblasts are involved in the maintenance of bronchial wall integrity, and may also participate to the airway inflammatory response representing an important target for anti-asthmatic drugs (17, 18).

Lower airway fibroblasts may play a critical role in inflammation of the airways, as they are involved in continuous cycles of proliferation, activation and cross-talk with inflammatory cells. Fibroblasts possess a very high basal level of surface expression of CD90, a member of the group of adhesion molecules in the immunoglobulin super-family that is further induced during the early phase of wound healing, suggesting an activation of its expression by inflammatory mediators. Moreover, CD90 is involved in the attachment of fibroblasts to extracellular matrix (ECM) components, such as fibronectin and collagen I (19).

Another cell surface protein involved in fibroblast proliferation and migration during airway remodelling is CD44, a receptor for matrix proteins supporting cell motility, including fibronectin and glycosaminoglycan hyaluronan (20, 21).

Besides being a major component of the ECM and representing a remarkable adhesive substrate for epithelial cells and fibroblasts, fibronectin and its soluble fragments are very active in promoting the directional migration of these cells in the lung. Not only can fibronectin affect proliferation directly, but it may also function as a reservoir or carrier for growth factors released during the matrix degradation process (22).

In a previous study published by our group in 2001, we had shown that fluticasone propionate displays novel anti-inflammatory effects on human lung fibroblasts during their myofibroblastic differentiation (23).

Profita et al. (24) investigated the effects of beclomethasone dipropionate (BDP) alone or combined with salbutamol or formoterol on sputum cells isolated from mild to moderate asthmatics, showing a synergistic anti-inflammatory effect. From their study, it was observed that SABA (salbutamol) may produce the same beneficial effects in vitro as a LABA when added to BDP: this concept, in their opinion, might support the use of salbutamol combined with BDP instead of using salbutamol alone in the symptomatic relief of mild asthma.

Based on these results, the focus of our study was to evaluate in vitro how BDP, salbutamol and formoterol alone or in combination affect fibroblast proliferation, ECM component secretion and cell surface receptor expression.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Cell proliferation

Primary fibroblast cultures (LFIB) named L1FIB and L2FIB were obtained from the healthy part of human bronchi derived from lobectomies of patients who underwent surgery. Fibroblasts obtained were part of our cellular bank and already used in previous studies.

Bronchial tissue was cut into small fragments and incubated in RPMI-1640 (Euroclone, Milan, Italy) medium containing a mixture of 10 UI/ml DNAse, 500 UI/ml collagenase type IV and 30 UI/ml hyaluronidase (all enzymes purchased from Sigma-Aldrich, Milan, Italy) on a magnetic stirrer for 2 h at 37°C. The cells were then cultured in RPMI supplemented with 10% foetal bovine serum (FBS), glutamine and antibiotics (Euroclone, Milan, Italy) for at least 24 h.

In addition, two different human foetal lung fibroblast cell lines, HFL1 and ICIG7, already used in our laboratory in previous studies, were tested (25, 26). Fibroblast cultures were characterized by flow cytometry using the specific antibody CD90 (Instrumentation Laboratory, Milan, Italy). All cultures used in the study were >95% CD90+.

Fibroblasts were plated in 96-well microtitre plates at a density of approximately 5 × 103 cells/well in 0.2 ml of RPMI plus 10% FBS and allowed to attach. After 24 h of incubation, the medium was replaced by 0.2 ml FBS-free RPMI. The day after the medium was replaced with RPMI with 2% FBS and the following substances were added and lasted 48 h: BDP (1.39 × 10−5 M), salbutamol (1.75 × 10−5 M) or formoterol (5.59 × 10−7 M), alone or combined, in the presence or absence of the proliferative stimulus basic fibroblast growth factor (FGFb) (10 ng/ml), or pro-inflammatory stimuli, such as transforming growth factor-β (TGF-β) (10 ng/ml) or interleukin-4 (IL-4) (10 ng/ml). [3H]-thymidine was added in the last 18 h. The medium was then removed, the cells harvested and thymidine incorporation was measured by a beta-counter and expressed as count per minute to measure fibroblast proliferation. All experiments were performed at least in triplicate.

Phenotypic analysis of cells

Flow cytometry was used to calculate the number of surface molecules on fibroblast cultures stimulated as described above. The following fluorescein isothiocyanate-labelled monoclonal antibody (mAb) was used: CD44 (Al-Immunotools, Friesoythe, Germany) a surface molecule which mediates leukocitary adhesion. In addition CD90 (Thy-1), a phycoerythrin-labelled mAb (Immunotech, Marseille, France), was tested. Briefly, 0.05 ml medium containing 1 × 105 cells was incubated with 10 ng/ml mAbs for 30 min at 4°C, followed by extensive washing and analysis on a Coulter EPICS XL flow cytometer (Beckman Coulter S.p.A., Milan, Italy). Analyses were carried out on cells gated to exclude nonviable cells.

ELISA analysis

Fibroblast culture supernatants were used to investigate the production of fibronectin by enzyme-linked immunosorbent assay (ELISA) technique. Enzyme-linked immunosorbent assay was performed using the QuantiMatrixTM human fibronectin ELISA kit (Chemicon International, Temecula, CA, USA) according to the manufacturer’s protocols.

Statistical analysis

Statistical analysis was performed by using a Mann–Whitney U-test and results were expressed as median ± SEM. Differences were considered significant at P < 0.05 (*) and highly significant at P < 0.01 (**).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Cell proliferation

Preliminary cell proliferation tests in our laboratory have shown that the most efficient drug concentrations were 1.39 × 10−5 M for BDP and 1.75 × 10−5 M and 5.59 × 10−7 M respectively for salbutamol (S) and formoterol (F). These concentrations were therefore used in all the experiments performed. Formoterol or salbutamol by themselves did not show any significant effect in the different cultures (data not shown).

Basic fibroblast growth factor increased cell proliferation both on LFIB and on the two cell lines (P < 0.01), and BDP alone or BDP combined with salbutamol or formoterol significantly down-regulated this effect (P < 0.01) (Fig. 1A–C). The treatment of both cell lines and LFIB with FGFb + BDP + S/F compared with FGFb + BDP showed a significantly greater down-regulative effect (P < 0.05).

image

Figure 1.  Effects on proliferation in different conditions; (A) LFIB; (B) ICIG7 and (C) HFL1; CTRL, not treated; FGFb, basic fibroblast growth factor; BDP, beclomethasone dipropionate; S, salbutamol; F, formoterol; *P < 0.05; **P < 0.01.

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A significant difference between BDP + S and BDP + F has been observed only on HFL1 cell line. Culture stimulation by IL-4 and TGF-β did not affect proliferation (P = 0.57 and P = 0.081 respectively, data not shown).

CD90

Primary fibroblast cultures cells, treated with FGFb, significantly reduced CD90 expression (P < 0.01) (Fig. 2), while it was significantly increased both with BDP alone (P < 0.01) or BDP combined with salbutamol or formoterol (P < 0.01). The treatment of cells with FGFb + BDP + S/F compared with FGFb + BDP showed a significantly greater up-regulative effect (P < 0.05).

image

Figure 2.  CD90 expression on LFIB cells in different conditions: CTRL, not treated; FGFb, basic fibroblast growth factor; BDP, beclomethasone dipropionate; S, salbutamol; F, formoterol. Data are expressed as mean fluorescence channel (MFC) compared with the mean fluorescence channel of isotype control antibody (MFC ctrl); *P < 0.05; **P < 0.01.

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Formoterol or salbutamol alone did not show any significant effect on CD90 expression after stimulation with FGFb. Interleukin-4 reduced CD90 expression on all cell lines (P < 0.01), and the drugs, either alone or in combination, were not able to modify this effect (data not shown). Cell culture treatment with TGF-β did not modify CD90 expression (data not shown).

CD44

CD44 expression was significantly increased by FGFb only on ICIG7 (P < 0.01) (Fig. 3A) while this effect was decreased when cells were treated with BDP alone (P < 0.01) or BDP combined with salbutamol or formoterol (P < 0.01). The treatment of cells with FGFb + BDP + S/F compared with FGFb + BDP showed a significantly greater down-regulative effect (P < 0.05).

image

Figure 3.  CD44 expression in different conditions: (A) ICIG7 treated with FGFb; (B) ICIG7 treated with IL-4 and (C) LFIB treated with TGF-β. CTRL, not treated; FGFb, basic fibroblast growth factor; TGF-β, transforming growth factor-β; IL-4, interleukin-4; BDP, beclomethasone dipropionate; S, salbutamol; F, formoterol. Data are expressed as mean fluorescence channel (MFC) compared with the mean fluorescence channel of isotype control antibody (MFC ctrl); *P < 0.05; **P < 0.01.

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Interleukin-4 down-regulated CD44 expression only on ICIG7 cells (P < 0.01). A significant increase was observed when cells were treated with BDP alone or BDP combined with either salbutamol or formoterol (P < 0.01). The treatment of cells with IL-4 + BDP + S/F compared with IL-4 + BDP showed a significantly greater up-regulative effect (P < 0.01) (Fig. 3B).

Transforming growth factor-β down-regulated CD44 on LFIB (P < 0.01); BDP alone or BDP combined with salbutamol was the only set of two conditions effective in up-regulating CD44 (P < 0.05). The treatment of cells with TGF-β + BDP + S compared with TGF-β + BDP showed a significantly greater up-regulative effect (P < 0.05) (Fig. 3C). Formoterol or salbutamol by themselves did not show any significant effect on CD44 expression after stimulation with FGFb, IL-4 or TGF-β (data not shown).

Fibronectin

Basic fibroblast growth factor increased fibronectin secretion on LFIB in untreated cells (P < 0.05). The addition of BDP + S or BDP + F to cells treated with FGFb significantly reduced its production (P < 0.05) (Table 1). Fibronectin was augmented by treatment with TGF-β only on LFIB (P < 0.05) (Table 1). A remarkable reduction in the secretion of fibronectin was observed when BDP was combined with salbutamol or formoterol (P < 0.05). By contrast, BDP alone showed only a nonsignificant slight reduction in fibronectin production.

Table 1.   Production of fibronectin by human lung fibroblasts
 P+BDPP+BDP + SP+BDP + FP
  1. Production of fibronectin by primary human lung fibroblasts (LFIB) under different experimental conditions: CTRL, not treated; FGFb, basic fibroblast growth factor; IL-4, interleukin-4; TGF-β, transforming growth factor- β; BDP, beclomethasone dipropionate; S, salbutamol; F, formoterol.

  2. Fibronectin amount is expressed in nanograms per milliliter; NS, not significant. *P < 0.05.

CTRL810.6 ± 69.3       
FGFb1509.9 ± 205*1213.9 ± 87NS616.7 ± 49.9*818.1 ± 49.9*
TGF-β1848 ± 140.2*1286 ± 67.2NS637.1 ± 19.4*876.6 ± 64.2*
IL-4538.8 ± 2.6NS698.6 ± 67.6NS794.3 ± 127.1NS883.3 ± 3.4NS

No relevant results were observed after treatment with IL-4. Fibronectin expression after stimulation with the different stimuli was not affected by the treatment with formoterol or salbutamol alone (data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

As a first general conclusion, the present study has confirmed the anti-proliferative effect of ICS on human lung fibroblasts. The study has also demonstrated that the combination of ICS and LABA or SABA potentiates these effects, whereas LABA and SABA by themselves are not effective on this cell type.

The role of fibroblasts in airway remodelling has grown in the last few years and they are now considered the major cause of basal membrane thickening in asthma. We previously demonstrated that fluticasone propionate inhibits differentiation from fibroblast to myofibroblast in vitro and this effect is further enhanced by the addition of salmeterol (27). This view is also supported by a study published in 2002 by Spoelstra et al. on the anti-inflammatory effects of formoterol and budesonide on human lung fibroblasts demonstrating a similar effect, although using a different model (28).

In a study conducted in vivo in 1998 by Hoshino et al., it was observed that BDP alone prevented remodelling of the airways by reducing lamina reticular thickness by modulation of insulin-like growth factor expression with consequent inhibition of the airway infiltration by inflammatory cells (29).

The beneficial effects of different LABAs in combination with ICS have been recently studied, suggesting an additive effect on fibroblasts (17, 30, 31), although this evidence was lacking for BDP when combined with LABA or SABA.

The present in vitro study demonstrated that β2-agonists, when given alone, do not exert any significant anti-proliferative effect on bronchial fibroblasts; however, an additive effect on ICS was identified. This is of interest as the ICS + β2-agonists combination is confirmed to have superior anti-inflammatory activity when compared with ICS alone. Interestingly, for the first time, we demonstrated that LABA and SABA when combined with the same ICS show similar additive effects on inhibiting the proliferation of lung fibroblasts. Only HFL1 cell proliferation showed a significant difference between treatment with BDP + S or BDP + F.

We investigated whether CD90 is, in some way, modulated by ICS and β2-agonists. Our results show that FGFb, which exerts a pro-fibrogenic stimulus, down-regulates CD90 expression, as already reported by Hagood et al. (32) and the drugs up-regulate its expression.

In our experiments, we observed that a pro-fibrotic stimulus such as FGFb up-regulated CD44 expression whereas the pro-inflammatory stimuli used (TGF-β and IL-4) caused its down-regulation. In both cases, the drugs brought CD44 expression towards the CTRL levels.

CD44 modulation is therefore a valuable model as its behaviour changes depending on the kind of stimulation present. It was therefore possible to explore the additive effects of β2-agonists on ICS with respect to both enhancing and decreasing CD44 expression.

The cells were stimulated with TGF-β, an important pro-inflammatory cytokine, whose levels are increased in asthmatic airways (33), leading to the deposition of fibronectin in the ECM (34). In our study, both ICS and β2-agonists reduced TGF-β-induced fibronectin production, demonstrating their ability to reduce ECM deposition.

In conclusion, in our study, salbutamol (SABA) and formoterol (LABA) showed similar additive anti-proliferative and anti-remodelling activities when added to BDP. The heterogeneity in cell lines used and the different results obtained permit us only to hypothesize what might be the real effects of these drugs in vivo, therefore our inferences need to be confirmed in a controlled clinical trial to fully evaluate the potential anti-remodelling effects of the ICS + β2-agonists combinations.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

This work has been partially supported by ARMIA, GA2LEN and Chiesi Farmaceutici S.p.A.

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
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