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 β2-adrenoceptor (β2-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]. β2-AR activation induces the production of 3′,5′-cyclic adenosine monophosphate (cAMP) by adenylyl cyclase leading to airway smooth muscle relaxation [10]. β2-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 β2-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
Full size table

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% CO2, 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.

Results

Effect of salmeterol on fibrocyte number, differentiation and CCR7 expression

At the time of isolation from blood (day 0) the total number of NANT cells were not significantly different between groups, however there were higher percentages of fibrocytes in severe asthma (51 ± 11 × 10 3; 15% ± 2.2) compared to non-severe asthma (43 ± 5 × 10 3; 6.7% ± 1 and healthy subjects (15 ± 4.2 × 10 3; 4.1% ± 0.9). For experiments, 1 × 106 NANT cells per well were seeded at day 0. After three days in culture, there was a concomitant increase in the percentage of fibrocytes within each group (Additional file 2: Figure S3A), suggesting proliferation of fibrocytes in culture. In contrast to day 0, at day 3 there were no significant differences in the percentage of fibrocytes across the groups. However, there was an higher percentage of differentiated (α–SMA+) fibrocytes in the severe asthma group compared to non-severe asthma patients and healthy subjects (Additional file 2: Figure S3B) confirming our previous observations [8]. As the vast majority of α–SMA+ cells also expressed the fibrocyte markers Col I and CD45, we chose to identify differentiating fibrocytes by single α–SMA staining (Additional file 3: Figure S1).

Incubation with salmeterol (10−9 - 10−7 M) for 3 days led to a decrease in the number of fibrocytes (Fig. 1a ) and differentiating fibrocytes (Fig. 1b ) from healthy subjects and non-severe asthma patients. Salmeterol did not modulate the percentage of apoptotic NANT cells (Additional file 4: Figure S2). As we have shown that fibrocytes proliferate in culture the reduction in fibrocyte number by salmeterol is possibly a result of inhibition of proliferation. In contrast, salmeterol did not modulate the number of fibrocytes (Fig. 1a) and differentiating fibrocytes (Fig. 1b) from patients with severe asthma. We also show that there is an increase in the absolute number of fibrocytes in culture at day 3 in all three groups suggesting proliferation of fibrocytes in culture. Pre-treatment with salmeterol inhibited this proliferation in fibrocytes from healthy subjects and non-severe asthmatics but the anti-proliferative effect of salmeterol was not observed in severe asthma. The effect of salmeterol on the absolute numbers of fibrocytes and differentiating fibrocytes is shown in Additional file 2: Figure S3. Salmeterol also led to a decrease in the percentage of CCR7-positive fibrocytes (Fig. 1c) and CCR7 MFI ratio (Fig. 1d) in NANT cells from patients with non-severe asthma but had no significant effect on those from healthy subjects and patients with severe asthma.

Fig. 1
figure 1

Effect of salmeterol on the number, differentiation and CCR7 expression of fibrocytes. NANT cells from healthy subjects (n = 8–9) and patients with non-severe (n = 7) or severe asthma (n = 7–9) were treated with salmeterol (10−9- 10−7 M) for 3 days. Fibrocyte (Col I+/CD45+ cells; a) and differentiating fibrocyte (α-SMA+ cells; b) number, and percentage (c) and median fluorescence intensity (MFI; d) of CCR7+ fibrocytes (Col I+/CD45+/CCR7+ cells) were determined. Data points represent mean ± SEM. * p < 0.05 and ** p < 0.01 versus vehicle-treated cells for each group. # p < 0.05 healthy versus severe asthma and & p < 0.05 non-severe asthma versus severe asthma

Full size image

The suppressive effect of salmeterol on the number of fibrocytes and differentiating fibrocytes from healthy subjects was prevented in the presence of the β2-AR-specific antagonist ICI-118,551 (10−5 M; Additional file 5: Figure S4), confirming that the effects of salmeterol are mediated by β2-AR activation. The above findings suggest altered β2-AR signalling in fibrocytes from severe asthma.

Effect of cAMP modulation on fibrocyte number and differentiation

Increasing intracellular cAMP levels using the analogue 8-Br-cAMP (10−4 – 10−3 M) led to a concentration-dependent decrease in the number of fibrocytes (Fig. 2a) and differentiating fibrocytes (Fig. 2b) from both healthy subjects and severe asthma patients. Also, the PDE4 inhibitor rolipram (10−5 M), which inhibits PDE4-dependent cAMP hydrolysis, alone and in combination with salmeterol reduced fibrocyte number and differentiation (Fig. 2c-d). The combination of salmeterol and rolipram had a greater inhibitory effect on the number of fibrocytes and differentiated fibrocytes than either drug alone, although these differences did not reach statistical significance. Together these data suggest that the defect in β2-AR signalling in severe asthma fibrocytes possibly lies upstream of cAMP production.

Fig. 2
figure 2

Effect of cAMP modulation on fibrocyte number and differentiation. NANT cells from healthy subjects (n = 4) and severe asthma patients (n = 4) were treated with 8-Br-cAMP (10−4- 10−3 M for 3 days (a-b). Alternatively, NANT cells from severe asthma patients were treated with rolipram (10−5 M) in the presence or absence of salmeterol (10−9 M) for 3 days (c-d). The number of fibrocytes (Col I+/CD45+ cells; a and c) and differentiating fibrocytes (α-SMA+ cells; b and d) were determined. Data points represent mean ± SEM. * p < 0.05 versus vehicle-treated cells for each group

Full size image

β2-AR expression in fibrocytes from healthy subjects and severe asthma patients

The baseline, surface and whole cell, expression of β2-AR was determined in fibrocytes, in the NANT cells of healthy subjects and severe asthma patients at day 0 by staining with an anti-β2-AR antibody before or after permeabilisation, respectively (Fig. 3a-d). Both the percentage of β2-AR-positive fibrocytes (Fig. 3e) and the β2-AR MFI ratio (Fig. 3f) were lower on the surface of circulating fibrocytes of patients with severe asthma compared to healthy subjects, although whole cell β2-AR expression was not significantly different between the two groups (Fig. 3g and h).

Fig. 3
figure 3

Baseline β2-AR expression in fibrocytes from healthy subjects and patients with severe asthma. β2-AR expression in fibrocytes from untreated NANT cells of healthy subjects (n = 11) and severe asthma patients (n = 6) was determined by staining with antibodies for collagen I (Col I), CD45 and β2-AR (c-d), or their respective isotype controls (a-b) at day 0. Representative flow cytometry scatter plots from one experiment are shown. Surface (e-f) and whole cell (g-h) β2-AR expression was determined as percentage of β2-AR+ fibrocytes (Col I+/CD45+/β2-AR+ cells; e, g) and median fluorescence intensity (MFI) ratios (f, h). Horizontal lines represent the median of each group. * p < 0.05, ** p < 0.01

Full size image

Incubation of NANT cells from healthy subjects with salmeterol (10−8 M) for 3 days led to a reduction in the percentage of fibrocytes positive for surface β2-AR (Fig. 4a) and the surface β2-AR MFI ratio (Fig. 4b). Salmeterol had no effect on the percentage of fibrocytes positive for whole cell β2-AR (Fig. 4c) but increased the whole cell MFI ratio (Fig. 4d), in NANT cells from healthy subjects. In contrast, salmeterol had no effect on the percentage of β2-AR-positive fibrocytes (ColI+/CD45+/ β2-AR+) or the MFI ratio, on the surface (Fig. 4e and f) or in the whole cell (Fig. 4g and h), in NANT cells from patients with severe asthma. These data suggest loss of baseline surface β2-AR expression and attenuated agonist-induced β2-AR down-regulation in fibrocytes from severe asthma.

Fig. 4
figure 4

Effect of salmeterol on β2-AR expression in fibrocytes from healthy subjects and patients with severe asthma. NANT cells from healthy subjects (a-d; n = 4) and severe asthma patients (e-h; n = 4) were treated with salmeterol (10−8 M) for 3 days. Surface (a-b and e-f) and whole cell (c-d and g-h) β2-AR expression was determined as percentage of β2-AR+ fibrocytes (Col I+/CD45+/β2-AR+ cells; a, c, e, g) and median fluorescence intensity (MFI) ratios (b, d, f, h). * p < 0.05 and ** p < 0.01

Full size image

Effect of dexamethasone on salmeterol-induced effects on fibrocyte function

To investigate whether the addition of a corticosteroid modulates the effects of β2-AR activation on fibrocytes of patients with severe asthma, NANT cells were incubated with a submaximal dose of salmeterol (10−9 M) in combination with dexamethasone (10−7 M). For all groups there was an increase in the total the number fibrocytes at day 3 as described above. Although dexamethasone or salmeterol alone had no effect on fibrocyte number and differentiation, the number of differentiating fibrocytes (Fig. 5b), but not total fibrocytes (Fig. 5a), was reduced by the combination of salmeterol and dexamethasone. Salmeterol and dexamethasone, individually or in combination, had no effect on fibrocytes positive for surface β2-AR (Fig. 5c). However, dexamethasone alone led to a non-statistically-significant increase in the β2-AR MFI ratio whilst the combination of salmeterol and dexamethasone significantly augmented the β2-AR MFI ratio (Fig. 5d). Thus, dexamethasone may potentiate the effects of salmeterol on severe asthma fibrocytes by increasing surface β2-AR density.

Fig. 5
figure 5

Effect of salmeterol and dexamethasone combination on the number, differentiation and β2-AR expression of fibrocytes from severe asthma patients. NANT cells from patients with severe asthma (n = 4–7) were treated with salmeterol (10−9 M) in the presence or absence of dexamethasone (Dex; 10−7 M) for 3 days. The number of fibrocytes (Col I+/CD45+ cells; a) and differentiating fibrocytes (α-SMA+ cells; b), as well as the percentage (Col I+/CD45+/β2-AR+; c) and median fluorescence intensity (MFI) ratio (d) of β2-AR–positive fibrocytes were determined. Bars represent mean ± SEM. * p < 0.05

Full size image

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 β2-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 β2-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 β2-AR signalling. Activation of the β2-AR leads to Gs-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 β2-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 [16,17,18]. 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 β2-AR expression or activation.

We have demonstrated that severe asthmatic fibrocytes show reduced surface β2-AR expression, whilst they have similar whole cell β2-AR expression levels, compared to fibrocytes from healthy subjects. Moreover, salmeterol failed to reduce the surface β2-AR expression in fibrocytes from patients with severe asthma, possibly due to the already low β2-AR surface density. These findings indicate a possible defect in the membrane localisation but not the overall expression of the β2-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 β2-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 β2-AR are subsequently resensitised by recycling back to the cell membrane to interact with agonists [22]. Defective β2-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 β2-AR agonist effects by increasing β2-AR density through up-regulation of transcription [25]. In agreement with these findings, we demonstrated up-regulation of the surface β2-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 β2-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 β2-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 β2-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 β2-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 β2-AR desensitisation in ASM cells [33].

Conclusions

In this study we have demonstrated that fibrocytes from patients with severe asthma show reduced surface β2-AR expression and consequently insensitivity to the inhibitory effects of β2-AR agonists. These findings, in conjunction with our previous findings showing corticosteroid insensitivity in severe asthma fibrocytes, highlight a possible mechanism underlining the accumulation of fibrocytes in the circulation and lung tissue of severe asthma patients [6, 8]. Moreover, we show that combination of β2-AR agonists with corticosteroids are more effective in suppressing fibrocyte number and differentiation than each drug alone highlighting the importance of combination therapy in the treatment of remodelling in severe asthma.