Interleukin-13 induces collagen type-1 expression through matrix metalloproteinase-2 and transforming growth factor-β1 in airway fibroblasts in asthma

Abstract

Airway remodelling is a feature of asthma that contributes to loss of lung function. One of the central components of airway remodelling is subepithelial fibrosis. Interleukin (IL)-13 is a key T-helper 2 cytokine and is believed to be the central mediator of allergic asthma including remodelling, but the mechanism driving the latter has not been elucidated in human asthma. We hypothesised that IL-13 stimulates collagen type-1 production by the airway fibroblast in a matrix metalloproteinase (MMP)- and transforming growth factor (TGF)-β1-dependent manner in human asthma as compared to healthy controls. Fibroblasts were cultured from endobronchial biopsies in 14 subjects with mild asthma and 13 normal controls that underwent bronchoscopy. Airway fibroblasts were treated with various mediators including IL-13 and specific MMP-inhibitors. IL-13 significantly stimulated collagen type-1 production in asthma compared to normal controls. Inhibitors of MMP-2 significantly attenuated collagen production in asthma but had no effect in normal controls. IL-13 significantly increased total and active forms of TGF-β1, and this activation was blocked using an MMP-2 inhibitor. IL-13 activated endogenous MMP-2 in asthma patients as compared to normal controls. In an ex vivo model, IL-13 potentiates airway remodelling through a mechanism involving TGF-β1 and MMP-2. These effects provide insights into the mechanism involved in IL-13-directed airway remodelling in asthma.

FIGURE 1

COL1A2 mRNA expression was significantly induced by interleukin-13 (50 ng·mL⁻¹) in asthma. COL1A2, COL1A1 and COL3A1 mRNA expression was measured by quantitative real-time reverse transcriptase-PCR in airway fibroblasts isolated from asthmatic (n=15) and normal controls (n=15). Data are presented as fold change from untreated fibroblasts. **: p<0.01 between asthma and normal controls; ^#: p<0.01 within asthmatic subjects.

FIGURE 2

Significant attenuation in interleukin (IL)-13-induced COL1A2 mRNA expression was observed at 48 h when a matrix metalloproteinase (MMP) inhibitor (MMPi) (10 μM), a specific MMP2 inhibitor (MMP2i) (40 μM) and a transforming growth factor-β receptor II (TGF-βRII)-neutralising antibody (Ab) (20 μg mL⁻¹) were added to IL-13-treated airway fibroblasts in asthma subjects (n=15) compared to normal subjects (n=15) and within asthma patients alone as measured by real-time reverse transcriptase-PCR. Data are presented as fold change from fibroblasts from each group treated with IL-13 alone. *: p<0.05 between asthma and normal subjects; #: p<0.05 within the asthma group for each condition.

FIGURE 3

Collagen type-1 protein was induced by interleukin (IL)-13 (50 ng·mL⁻¹), depicted as the ratio of IL-13-treated airway fibroblasts to untreated fibroblasts at 48 h in asthma (n= 18) as compared to normal controls (n= 12) and within asthma alone. Matrix metalloproteinase-2 inhibition (MMP2i) significantly attenuated IL-13-induced collagen type-1, depicted as the ratio of collagen type-I production following treatment with IL-13 plus MMP2i fibroblasts as compared to treatment with IL-13 alone, in asthma subjects as compared to normal subjects and within the asthma group. *: p<0.05 between asthma and normal controls; ^#: p<0.05 within asthmatic subjects for each condition.

FIGURE 4

Matrix metalloproteinase (MMP)-2 activity in normal and asthmatic fibroblasts at baseline and after treatment with interleukin (IL)-13. MMP-2 activity is induced after treatment with IL-13 (50 ng·mL⁻¹) for 48 h in asthma (n=6) as compared to normal controls (n=4) and within asthma alone, as measured by an MMP-2 activation assay. *: p<0.05 between asthma and normal controls; ^#: p<0.05 within asthmatic subjects for the IL-13 condition.

FIGURE 5

Interleukin (IL)-13 stimulates active transforming growth factor (TGF)-β1 production by asthmatic airway fibroblasts. a) Total and endogenously active forms of TGF-β1 from airway fibroblast cell culture supernatants were increased from baseline following stimulation with IL-13 (50 ng mL⁻¹) at 48 h in asthma subjects (n=9) as compared to normal subjects (n=7) and within the asthma group, depicted as the ratio of TGF-β1 production in fibroblasts treated with IL-13 as compared to untreated fibroblasts. Matrix metalloproteinase-2 inhibition (MMP2i) blocked TGF-β1 activation in fibroblasts treated with IL-13 in asthma subjects compared to normal subjects and within the asthma group, depicted as the ratio of TGF-β1 activation after treatment with IL-13 plus MMP2i as compared to treatment with IL-13 alone (IL-13 plus MMP2i/IL-13). b) Bioactive TGF-β1 levels, as represented by luciferase activity in airway fibroblast cell culture supernatants, were elevated following exposure to IL-13 (50 ng mL⁻¹) at 48 h in asthma subjects (n=9) as compared to untreated controls. RLU: relative luciferase units. *: p<0.05 between asthma and normal subjects; ^#: p<0.05 within asthmatic subjects for each condition.

FIGURE 6

Expression of α-smooth muscle actin (SMA) and desmin proteins in cell culture lysates from airway fibroblasts was increased in asthma patients following treatment with interleukin (IL)-13. Cell lysates were prepared from airway fibroblasts from either asthmatic or normal subjects after exposure to IL-13 (50 ng mL⁻¹) or serum-free media (untreated control) for 48 h. a) Representative Western blot of myofibroblast markers in airway fibroblasts in asthma or normal subjects. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) indicates loading control. b) Quantification of data shown in a): mean ± SEM of six asthma subjects and four normal controls. Data are expressed as the ratio of the intensity of bands following IL-13 to untreated control. *: p<0.05 between asthma and normal subjects.