Background
Asthmatic inflammation is characterised by extensive airway remodelling involving sub-epithelial fibrosis and thickening of the airway smooth muscle (ASM) layer [
1]. 5–10% of asthmatic patients suffer from severe asthma, which is difficult to control despite receiving high-dose inhaled corticosteroids and long-acting β
2-adrenoceptor (β
2-AR) agonists (LABA), leukotriene modifiers or theophylline [
2]. In severe asthmatic airways, there is more prominent sub-epithelial fibrosis and ASM thickening which contribute to airway obstruction [
3].
Fibrocytes are bone marrow-derived progenitor cells that express leukocytes markers, such as CD45, and mesenchymal proteins, including collagen I [
4]. Migration of fibrocytes towards allergen-exposed airways and their subsequent differentiation into α–smooth muscle actin (SMA)-expressing myofibroblasts may contribute to increased ASM mass and sub-epithelial fibrosis [
5]. Fibrocyte accumulation has also been observed in the airways of severe refractory asthmatic patients [
6]. The CC chemokine receptor 7 (CCR7)/CC chemokine ligand 19 axis is important in the migration of fibrocytes from asthmatic patients with chronic airflow obstruction [
7]. We have previously shown that severe asthma patients have more circulating fibrocytes, with higher myofibroblastic differentiation potential and relative corticosteroid insensitivity in vitro [
8].
The use of LABAs in conjunction with inhaled corticosteroids results in further improvement of symptoms and lung function [
9]. β
2-AR activation induces the production of 3′,5′-cyclic adenosine monophosphate (cAMP) by adenylyl cyclase leading to airway smooth muscle relaxation [
10]. β
2-AR agonists also exert other effects on cellular function such as inhibition of proliferation, α–SMA expression and collagen synthesis in pulmonary fibroblasts [
11,
12]. Corticosteroid insensitivity in peripheral blood mononuclear cells (PBMCs) from patients with severe asthma has been associated with reduced glucocorticoid receptor (GR) nuclear translocation and hyper-phosphorylation of GR, effects that are reversed by β
2-AR agonists [
13].
We hypothesised that β2-AR activation has differential effects on the function of fibrocytes from severe asthma patients compared to healthy subjects and non-severe asthma patients. We determined the effect of the β2-AR agonist, salmeterol, in the presence or absence of the corticosteroid dexamethasone on the number, differentiation potential, and CCR7 and β2-AR expression in fibrocytes from patients with non-severe and severe asthma and healthy subjects. The involvement of cAMP in the downstream signalling was investigated using the phosphodiesterase (PDE) 4 inhibitor rolipram and the cAMP analogue 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) on fibrocytes in cultured non-adherent non-T (NANT) cells from severe asthmatic subjects.
Methods
Subjects
Patients with severe and non-severe asthma and healthy subjects were recruited as previously described [
8], according to the American Thoracic Society guidelines for refractory asthma (Table
1) [
14]. The study was approved by the local ethics committee and informed consent was obtained from each participant. Additional details on the method are provided in the Additional file
1.
Table 1
Clinical characteristics of study subjects
Number | 12 | 9 | 13 |
Age, years | 39.8 ± 2.5 | 52.6 ± 4.2 | 54.4 ± 3.8* |
Gender, Female/Male | 6/6 | 7/2 | 11/2 |
Inhaled corticosteroid dose, μg BDP equivalent | 0 | 150.0 ± 105.2 | 2000 ± 258.2**##
|
Atopy (n) | 5 | 4 | 5 |
Receiving oral corticosteroids | 0 | 0 | 4 |
Pre-bronchodilator FEV1 (L) | 3.4 ± 0.2 | 2.2 ± 0.3 | 1.5 ± 0.2*** |
Pre-bronchodilator FEV1 of predicted value (%) | 94.9 ± 2.5 | 82.3 ± 5.5 | 61.0 ± 5.5*** |
FEV1/FVC (%) | 88.2 ± 2.3 | 69.5 ± 2.0 | 64.5 ± 3.1*** |
Isolation of NANT cells
NANT cells were isolated as previously described [
8]. Briefly, PBMCs were isolated from peripheral blood using Ficoll-Paque™ PLUS density gradient centrifugation (GE HealthCare, Uppsala, Sweden). Adherent cells were depleted by adherence, and T cells in the non-adherent fraction of PBMCs were removed using CD3
+ MicroBeads according to the manufacturer’s instructions (Miltenyi Biotec, California, USA). The NANT cells were then incubated in Iscove’s Modified Dulbecco’s Medium (IMDM; Sigma-Aldrich) supplemented with 30% FBS (Sigma-Aldrich) and 1% BSA in a humidified incubator, at 37
∘C with 5% CO
2, for 3 days in the presence or absence of treatments. NANT cells were counted on a haemocytometer after Trypan blue staining.
Analysis of fibrocytes by flow cytometry
Fibrocytes were identified in the NANT cell population, as cells positive for collagen I and CD45 (Col I
+/CD45
+), by flow cytometry [
8]. NANT cells were fixed using 0.5% paraformaldehyde, washed twice with phosphate buffered saline (PBS) and incubated with an allophycocyanin (APC)-conjugated mouse anti-human CD45 antibody (BD Biosciences, #555485, San Jose, California, USA). In some experiments cells were also stained with a phycoerythrin (PE)–conjugated mouse anti-human CCR7 antibody (BD Biosciences, #150503) 0.125 μg for 30 min. Cells were then permeabilised using 1X BD FACS Permeabilising Solution 2 (BD Biosciences, #347692) 120 μl for 15 min and then stained using 0.015 μg of fluorescein isothiocyanate (FITC)-conjugated mouse anti-human collagen I antibody (Millipore Corporation, #FCMAB412F, Temecula, California) for 30 min.. Differentiating fibrocytes were identified as α–SMA-positive (α–SMA
+) cells, by staining permeabilised NANT cells with 0.125 μg of PE-conjugated mouse anti-human α–SMA antibody (R&D Systems, #IC1420P Abingdon, UK). Cells incubated with isotype-matched antibodies were used as negative controls.
To evaluate the expression of β2-AR on the surface, cells were incubated with 1 μg of unconjugated rabbit IgG anti-human β2-AR (Abcam, #ab61778, Cambridge, UK,) and 0.015 μg of anti-human CD45 antibody for 30 min, followed by a PE–conjugated goat IgG anti-rabbit polyclonal secondary antibody (Abcam, #ab97070) 0.3 μg for 30 min, permeablised and then stained with FITC-conjugated anti-human collagen I antibody 0.5 μg for 30 min.
To evaluate the expression of β2-AR in the whole cell, cells were incubated with anti-human CD45 antibody, permeablised, then stained with 1 μg of unconjugated rabbit IgG anti-human β2-AR and 0.5 μg of FITC-conjugated anti-human collagen I antibody for 30 min, followed by 0.5 μ of PE–conjugated goat IgG anti-rabbit polyclonal secondary antibody g for 30 min.
Analysis of NANT cell apoptosis using Annexin V and propidium iodide (PI) staining
NANT cell apoptosis was determined by Annexin V and propidium iodide (PI) staining using the FITC Annexin V/Dead cell apoptosis kit (Invitrogen, Paisley, UK) according to the manufacturer’s instructions. Briefly, following treatment, NANT cells were washed in PBS, incubated with FITC-conjugated annexin V and PI and analysed by flow cytometry. Annexin V−/PI− were considered live cells, Annexin V+/PI− as early apoptotic and annexin V+/PI+ as late apoptotic cells.
Statistical analysis
Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was carried out using the GraphPad Prism v.5 software package (GraphPad Prism Software Inc., California, USA). Intra-group comparisons of more than two conditions were carried out using the Friedman test followed by Dunn’s post-hoc test and pairwise comparisons using a paired t-test. Inter-group comparisons were carried out using the Mann-Whitney test. p < 0.05 was considered as statistically significant.
Discussion
We have demonstrated that β2-AR activation by salmeterol leads to a reduction in the proliferation, myofibroblastic differentiation and CCR7 expression in fibrocytes from healthy subjects and patients with non-severe asthma. In contrast, salmeterol had no effect on fibrocytes from severe asthma patients indicating defective β2-AR signalling. Increasing intracellular cAMP levels, using the analogue 8-Br-cAMP or inhibiting cAMP degradation using the PDE4 inhibitor, rolipram, led to reduction in the number and differentiation of severe asthma fibrocytes indicating that the defect possibly lies up-stream of cAMP production. Indeed, we showed that fibrocytes from severe asthma patients have reduced baseline surface β2-AR expression which was not reduced further by salmeterol. Co-treatment with dexamethasone increased surface β2-AR density and augmented the inhibition of differentiation by salmeterol in fibrocytes of patients with severe asthma.
The airways of patients with severe asthma patients show increased airway remodelling despite receiving high doses of inhaled and oral corticosteroids and LABAs [
3]. Increased numbers of circulating fibrocytes, with increased capacity to differentiate into myofibroblasts and relative corticosteroid insensitivity may contribute to the increased airway remodelling in severe asthma [
8]. In this study, we show that β
2-AR activation by salmeterol can reduce the proliferation, differentiation and CCR7 expression of fibrocytes from healthy subjects and non-severe asthmatic patients. These findings are consistent with studies in fibroblasts showing inhibition of collagen synthesis, expression of α–SMA and proliferation in response to β
2-AR agonists [
11,
12,
15].
We also show that fibrocytes from severe asthma are less sensitive to the inhibitory effects of salmeterol suggesting that LABAs may be unable to reduce fibrocyte numbers and myofibroblastic differentiation in the airways of patients with severe asthma due to defective β
2-AR signalling. Activation of the β
2-AR leads to G
s-mediated activation of adenylyl cyclase which catalyses the conversion of adenosine triphosphate (ATP) to cAMP leading to activation of PKA and EPAC-dependent pathways [
10]. Increasing intracellular cAMP levels using 8-Br-cAMP mimicked the inhibitory effect of β
2-AR activation on the number and differentiation of fibrocytes. These findings are in line with studies showing reduction in proliferation, myofibroblastic differentiation and chemotaxis of fibroblasts by cAMP [
16‐
18]. Significantly, fibrocytes from patients with severe asthma were also sensitive to the inhibitory effects of the cAMP analogue, whilst inhibiting cAMP hydrolysis using rolipram enhanced the inhibitory effect of salmeterol on these cells. These findings indicate that the signalling pathways downstream to cAMP are intact in severe asthma fibrocytes and that the defect most likely lies at the level of the β
2-AR expression or activation.
We have demonstrated that severe asthmatic fibrocytes show reduced surface β
2-AR expression, whilst they have similar whole cell β
2-AR expression levels, compared to fibrocytes from healthy subjects. Moreover, salmeterol failed to reduce the surface β
2-AR expression in fibrocytes from patients with severe asthma, possibly due to the already low β
2-AR surface density. These findings indicate a possible defect in the membrane localisation but not the overall expression of the β
2-AR in severe asthma fibrocytes. As this occurs in the absence of salmeterol in vitro, it is possible that this is due to chronic exposure to systemic LABAs or endogenous circulating catecholamines and inflammatory mediators in vivo [
19,
20]. Prolonged β
2-AR activation triggers its uncoupling from adenylyl cyclase, internalisation of the uncoupled receptors, and increased degradation of internalized receptors, leading to desensitisation [
21]. Under normal conditions β
2-AR are subsequently resensitised by recycling back to the cell membrane to interact with agonists [
22]. Defective β
2-AR resensitisation has been shown to cause reduced baseline surface expression and attenuated agonist-induced activation in ASM cells of subjects with fatal asthma [
23].
The inhibitory effect of salmeterol on severe asthma fibrocyte differentiation was restored by co-incubation with the corticosteroid, dexamethasone. However, salmeterol and dexamethasone did not modulate the total number of fibrocytes from severe asthma patients in culture and this may be a result of the increased capacity of fibrocytes from patients with severe asthma to survive, as we have previously demonstrated [
8]. Resistance to the anti-proliferative effects of corticosteroids, due to reduced expression of CCAAT/enhancer binding protein α (C/EBPα), was also reported in airway smooth muscle cells from patients with asthma [
24]. Corticosteroids have been shown to potentiate β
2-AR agonist effects by increasing β
2-AR density through up-regulation of transcription [
25]. In agreement with these findings, we demonstrated up-regulation of the surface β
2-AR MFI ratio, indicating increased surface receptor density, in response to dexamethasone and salmeterol in severe asthma fibrocytes. Nonetheless, the superior effect of salmeterol and dexamethasone combination may also be a result of potentiation of glucocorticoid receptor (GR) function by salmeterol, as β
2-AR agonists are known to induce GR nuclear translocation and modulate phosphorylation of GR, in PBMCs, lung fibroblasts and airway smooth muscle cells [
13,
26,
27]. A combination of the corticosteroid, budesonide and the β
2-AR agonist, formoterol, has been shown to inhibit transforming growth factor (TGF)–β1-induced collagen I synthesis, whilst either drug alone had no effect [
28]. Moreover, a salmeterol and fluticasone propionate combination inhibits TGF–β1-induced α-SMA expression in lung fibroblasts [
11]. Thus, the combination of β
2-AR agonists and corticosteroids may be more potent in controlling airway remodelling in severe asthma, than either drug alone.
We showed that rolipram, a first generation selective PDE4 inhibitor [
29], enhanced the inhibitory effect of salmeterol on the differentiation of fibrocytes from both healthy subjects and severe asthma patients. Rolipram has been shown to reverse endotoxin-induced airway hyperresponsiveness and bronchoconstriction in vivo [
30], whilst PDE4 knockdown attenuates basic fibroblast factor and interleukin-1β-induced proliferation, and transforming growth factor-β-induced myofibroblastic differentiation of fibroblasts [
31]. Also, combination of the β
2-AR agonist, indacaterol, and the PDE4 inhibitor, roflumilast, inhibits the expression of endothelin-1, connective tissue growth factor and α-SMA, and the secretion of fibronectin from normal human lung fibroblasts, more strongly than with either drug alone [
32]. Significantly, rolipram was also shown to overcome β
2-AR desensitisation in ASM cells [
33].