American Journal of Respiratory Cell and Molecular Biology

Elastin synthesis and degradation in the airway and lung parenchyma contribute to airway mechanics, including airway patency and elastic recoil. IL-13 mediates many features of asthma pathobiology, including airway remodeling, but the effects of IL-13 on elastin architecture in the airway wall are not known. We hypothesized that IL-13 modulates elastin expression in airway fibroblasts from subjects with allergic asthma. Twenty-five subjects with mild asthma (FEV1, 89 ± 3% predicted) and 30 normal control subjects (FEV1, 102 ± 2% predicted) underwent bronchoscopy with endobronchial biopsy. Elastic fibers were visualized in airway biopsy specimens using Weigert’s resorcin-fuchsin elastic stain. Airway fibroblasts were exposed to IL-13; a pan-matrix metalloproteinase (MMP) inhibitor (GM6001); specific inhibitors to MMP-1, -2, -3, and -8; and combinations of IL-13 with MMP inhibitors in separate conditions in serum-free media for 48 hours. Elastin (ELN) expression as well as MMP secretion and activity were quantified. Results of this study show that elastic fiber staining of airway biopsy tissue was significantly associated with methacholine PC20 (i.e., the provocative concentration of methacholine resulting in a 20% fall in FEV1 levels) in patients with asthma. IL-13 significantly suppressed ELN expression in asthmatic airway fibroblasts as compared with normal control fibroblasts. The effect of IL-13 on ELN expression was significantly correlated with postbronchodilator FEV1/FVC in patients with asthma. MMP inhibition significantly stimulated ELN expression in patients with asthma as compared with normal control subjects. Specific inhibition of MMP-1 and MMP-2, but not MMP-3 or MMP-8, reversed the IL-13–induced suppression of ELN expression. In asthma, MMP-1 and MMP-2 mediate IL-13–induced suppression of ELN expression in airway fibroblasts.

Our results suggest that IL-13 contributes to airway obstruction and loss of elastin in asthma through a process requiring matrix metalloproteinase (MMP)-1 and MMP-2 to suppress elastin mRNA expression. These data suggest a novel role for MMPs in elastin gene regulation.

Airway remodeling is a heterogeneous set of tissue responses to chronic inflammation in asthma, resulting in airway narrowing and irreversible airflow obstruction (1). Over time, the cumulative burden of structural changes can result in accelerated lung function decline, beginning as early as childhood. Evidence has linked structural airway changes to extracellular matrix deposition, including subepithelial fibrosis. In contrast, additional studies demonstrate that matrix loss, specifically the loss of elastin, also occurs within the spectrum of remodeling, manifested as exaggerated airways collapsibility (2, 3). Elastin loss can also account for the reduction in lung elastic recoil recently observed in asthma (3, 4).

Elastin is fundamentally important to lung architecture and is directly responsible for airway distensibility (for review see Refs. 5, 6). A decrease in lung elastin can affect airway mechanics by decreasing airway stiffness and increasing airway narrowing. Furthermore, as the tethering effect of elastic fibers is lost, airway smooth muscle may become predisposed to exaggerated shortening (7). Elastic fibers are required to maintain airway patency during respiration; both superficial and deeper elastin network layers can be disrupted in subjects with asthma (2, 3, 8). We have observed the loss of airway elastic recoil in the airways of subjects with asthma (9). Similarly, biopsies of asthmatic airways demonstrate elastin matrix loss (3). Loss of elastin may be due to reduced expression, enhanced destruction, or both. We have demonstrated that loss of elastin in asthma was associated with increased matrix metalloproteinase (MMP)-9 expression (3). We hypothesize that loss of elastin in tissue may be responsible for the airways collapsibility and loss of elastic recoil observed in asthma (3, 4) and that the action of MMPs may modulate this process.

The airway fibroblast is a key cell type that links inflammation to structural airway changes in the lung. These cells, along with airway smooth muscle cells, produce elastin, alter lung architecture after injury, stimulate inflammation, and respond to cytokines relevant to asthma such as IL-4, IL-13, and transforming growth factor-β1 (1013). Specific fibroblast responses include mitogen-stimulated proliferation, invasion, and collagen production (1214). IL-13, a critical effector cytokine in asthma, mediates its effects through several cells, including airway fibroblasts, and plays central roles in airway obstruction, airways hyperresponsiveness (AHR), mucus metaplasia, inflammatory cell infiltration, and fibrosis (15, 16). Transgenic murine models of IL-13 overexpression exhibit extensive airway remodeling (17).

In human asthma, airway fibroblasts link inflammation to structural airway changes and are crucial to elastin matrix regulation. We have made the novel observation that IL-13 suppresses elastin gene expression in airway fibroblasts isolated directly from human subjects, and this effect is significant in subjects with asthma as compared with healthy control subjects. The mechanisms by which IL-13 regulates elastin gene expression in human asthma are unknown, but in this study we hypothesize that MMPs mediate IL-13–induced suppression of matrix expression in airway fibroblasts isolated directly from subjects with asthma.

Subject Recruitment

Fifty-five subjects were recruited from the Denver, CO, and Durham, NC, communities. The subjects with asthma fulfilled criteria for asthma (18) exhibiting a provocative concentration of methacholine resulting in a 20% fall in FEV1 (PC20) of <8 mg/ml and reversibility, as demonstrated by at least a 12% and 200-ml increase in FEV1 or FVC with inhaled albuterol. All subjects provided consent. This protocol was approved by the Duke University and National Jewish Health Institutional Review Board.


Subjects underwent bronchoscopy with endobronchial biopsy as previously described (3, 14).

Airway Tissue Staining

Biopsy tissue was fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with Weigert’s resorcin-fuchsin stain to visualize elastic fibers as previously described (19). Images of stained airway tissue sections were imported into ImageJ software (National Institutes of Health, Bethesda, MD). Relative quantities of elastic fiber staining were determined by calculating the percentage of staining within the total tissue area.

Fibroblast Culture

Endobronchial tissue was placed on collagen-coated plates, and airway fibroblasts were cultured as previously described with modifications (14). Because numerous passages can induce cellular senescence, only cells from the first three passages were used for experimentation (20). Normal and asthmatic airway fibroblasts were cultured and passaged under identical conditions.

Mediator Exposure

Airway fibroblasts were plated at 40,000 cells/well onto non–collagen coated 12-well plates as previously described (14) and incubated until 7 days past confluence. At baseline, these cells express α-smooth muscle actin and secrete collagen type 1, indicating a myofibroblast phenotype (13). After 24 hours in serum-free media, mediators were added, and cells were incubated for 48 hours. Conditions included 50 ng/ml IL-13 (ProSpec, Rehovot, Israel) and 10 μM MMP inhibitors (GM6001 [a broad-spectrum inhibitor of MMPs], MMP Inhibitor I, MMP-2 Inhibitor-1, and MMP-3 Inhibitor-1; Calbiochem, San Diego, CA). Combinations of IL-13 plus GM6001 and IL-13 plus inhibitors of MMP-1, -2, -3, and -8 were also studied. Chosen mediators were based on our previous work and other literature that suggested significant effects upon elastin synthesis or destruction (3, 5).

Quantitative Real-Time PCR

Quantitative real-time PCR was performed to demonstrate relative elastin (ELN) mRNA levels by normalization to glyceraldehyde-3-phosphate dehydrogenase mRNA levels in exposed cells relative to unexposed cells using the ΔΔCT method. Data are shown as mean percentage of fold change from the unexposed control.

Western Blot Analyses

Cell lysates from cultured airway fibroblasts were collected at 48 hours after IL-13 (50 ng/ml) or serum-free media (untreated control) treatments. Samples were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-human elastin and β-actin antibodies (Santa Cruz Biotechnology, Dallas, TX). The blots were developed by enhanced chemiluminescence, documented with the Kodak Image Station 4000 MM PRO, and quantified using Carestream Molecular Image software (version 5.0).

MMP-1 and Tissue Inhibitor of Metalloproteinase-1 and -2 ELISAs and MMP-1 Activity Assay

Supernatants from cultured airway fibroblasts were collected at 48 hours after treatment with IL-13 (50 ng/ml). Measurement of MMP-1 and tissue inhibitor of metalloproteinase (TIMP)-1 and -2 protein levels was performed using ELISA (R&D Systems, Minneapolis, MN). Endogenous MMP-1 activity levels were measured using a fluorimetric enzyme activity assay (R&D Systems). Active MMP-1 concentrations based on mean absorbance at 405 nm after 17-hour incubation were reported.

Statistical Analyses

Analyses were performed using JMP (SAS, Cary, NC) and Prism (GraphPad, La Jolla, CA) statistical software. Mean values from each condition were used for analyses. Baseline values were compared between groups, and differences are reported before using fold-change from baseline as the comparison variable. The fold change from baseline after exposure to mediators was used as the most relevant comparison to take baseline differences into account. Mediator-induced ELN expression relative to the unexposed control was compared within groups using a two-way ANOVA. Paired comparisons were evaluated using a two-tailed paired t test. If the overall P value for the comparisons was less than 0.05, individual contrasts determined significance within and between groups. Correlations were performed for PC20 and post-bronchodilator ratio of FEV1/FVC data for each subject to determine the Pearson’s correlation coefficient. For most analyses, data are expressed as means ± SEM because they were normally distributed; significance is denoted by P < 0.05. Data that were not normally distributed were expressed as medians (interquartile range [IQR]) and compared using a two-tailed Wilcoxon rank sum test; significance is denoted by P < 0.05.


Subject characteristics are shown in Table 1. The subjects with asthma were considered of mild severity because their mean percentage predicted FEV1 was 89 ± 3%. The subjects with asthma exhibited AHR with a mean methacholine PC20 of 1.4 ± 0.4 mg/ml. The normal subjects exhibited normal airways responsiveness, with a methacholine PC20 of >16 mg/ml (P < 0.01). The subjects with asthma exhibited significantly elevated fraction of exhaled nitric oxide (FeNO) as compared with the normal subjects (59.2 ± 19.5 ppb for asthma and 11.8 ± 1.3 ppb for normal; P < 0.01), suggesting significantly increased Th2-mediated airway inflammation in the subjects with asthma. The subjects with asthma used albuterol as needed and no controller medications. The normal subjects took no chronic medications.

Table 1. Subject Characteristics

 Subjects with Asthma (n = 25)Control Subjects (n = 30)P Value
Sex (female/male)14/1121/90.37
Age, yr29 ± 228 ± 20.70
FEV1, liters3.3 ± 0.13.5 ± 0.10.38
FEV1, % pred.89 ± 3102 ± 2<0.01
FVC, liters4.4 ± 0.24.3 ± 0.20.62
FVC, % pred.101 ± 3105 ± 20.28
FEV1/FVC82 ± 185 ± 10.06
Post-bronchodilator FEV1/FVC74 ± 285 ± 1<0.01
PC20, mg/ml1.4 ± 0.44>16<0.01
FeNO, ppb59.2 ± 19.511.8 ± 1.3<0.01

Definition of abbreviations: ACQ, Asthma Control Questionnaire; FeNO, fraction of exhaled nitric oxide; PC20, provocative concentration of methacholine resulting in a 20% fall in FEV1; pred., predicted; SABA, short-acting β agonist.

*A, Asian; AA, African American; W, white.

Elastic Fiber Staining Is Associated with AHR in Asthma

Sections of airway biopsy tissue from asthma and healthy control subjects were stained with Weigert’s elastic fiber stain. Mean percentage area of elastic fiber staining was significantly and positively correlated with methacholine PC20 in asthma (r = 0.66; P < 0.01) (Figure 1A). Elastic fiber staining in airway biopsy tissue was reduced in patients with asthma who had low methacholine PC20 values (Figures 1B and 1C) as compared with patients with asthma who had relatively high methacholine PC20 values (Figures 1D and 1E) and healthy subjects (Figures 1F and 1G).

Effect of IL-13 on ELN mRNA and Elastin Protein Expression in Airway Fibroblasts

Baseline ELN mRNA expression in airway fibroblasts in asthma was not significantly different as compared with normal control airway fibroblasts (P > 0.05) (see Figure E1 in the online supplement). IL-13 significantly suppressed ELN mRNA expression in asthmatic airway fibroblasts as compared with unexposed control fibroblasts (mean percentage fold change of ELN expression, −44.0 ± 7%; P < 0.01), and the magnitude of suppression in asthmatic airway fibroblasts was significantly greater than that observed in normal control airway fibroblasts (mean percentage fold change in ELN expression, −44.0 ± 7% for subjects with asthma and −16.1 ± 10% for normal control subjects; P < 0.05). In normal control subjects, IL-13 did not significantly alter ELN mRNA levels relative to untreated control (P > 0.05) (Figure 2A).

Elastin protein expression in airway fibroblasts, as detected by Western blot (Figure 2B), was not significantly changed with IL-13 relative to unexposed control in cells from subjects with asthma or from normal subjects (P > 0.05), although densitometry measurements indicated a slight reduction of elastin expression in immunoblots of both subjects with asthma and normal subjects (mean band intensity: 525,628.5 ± 201,243 for control subjects and 478,493.2 ± 179,217 for IL-13 in subjects with asthma; 723,506.5 ± 465,263 for control subjects and 640,562 ± 355,344 for IL-13 in normal subjects [P > 0.05]) (Figure 2C).

IL-13–Stimulated ELN Expression Correlates with Post-Bronchodilator FEV1/FVC in Asthma

IL-13–induced ELN gene expression in airway fibroblasts was significantly and positively associated with post-bronchodilator FEV1/FVC (r = 0.58; P < 0.01) (Figure 3A). No significant association between post-bronchodilator FEV1/FVC and airway fibroblast IL-13–induced ELN gene expression was observed in normal subjects (r = 0.10; P > 0.05) (Figure 3B).

Inhibition of MMPs Reverses the Suppressive Effect of IL-13 on ELN Expression

As described above, baseline ELN mRNA expression in airway fibroblasts in asthma did not differ significantly from that of normal control airway fibroblasts (Figure E1). Similarly, the addition of a pan-MMP inhibitor (GM6001) alone did not significantly stimulate ELN mRNA expression in airway fibroblasts from subjects with asthma as compared with airway fibroblasts from normal control subjects (median percentage fold change in ELN expression, 0.0% [IQR, −44.5 to 79.8] for subjects with asthma and −41.0% [IQR, −54.9 to −4.07] for normal subjects; P > 0.05) (Figure 4A). Within asthma, treatment with the pan-MMP inhibitor significantly stimulated ELN gene expression in airway fibroblasts as compared with IL-13 treatment alone (mean percentage fold change in ELN expression, −34.4 ± 7% for IL-13 and 1.0 ± 15% for pan-MMP inhibitor; P < 0.05) (Figure 4B). No significant difference was observed between IL-13 and pan-MMP inhibitor treatment in airway fibroblasts from normal control subjects (P > 0.05) (Figure 4C).

The effect of the pan-MMP inhibitor was also tested in the presence of IL-13. The combination of pan-MMP inhibitor and IL-13 resulted in airway fibroblast ELN mRNA levels that were not significantly different from untreated control subjects for both asthma and normal control groups (mean percentage fold change in ELN expression, −6.7 ± 13% for subjects with asthma [P > 0.05] and 0.2 ± 21% for normal subjects [P > 0.05]) (Figures 4B and 4C). However, in asthma, a significant increase in ELN gene expression after treatment with the combined pan-MMP inhibitor and IL-13 was observed as compared with IL-13 alone (mean percentage fold change in ELN expression, −34.4 ± 7% for IL-13 alone; −6.7 ± 13% for combined MMP inhibitor and IL-13 [P < 0.05]) (Figure 4B). Thus, the combination of pan-MMP inhibitor with IL-13 reversed the suppressive effect of IL-13 alone.

MMP-1 and MMP-2 Mediate IL-13–Induced Suppression of ELN mRNA Expression

Because the pan-MMP inhibitor is a broad-spectrum inhibitor of several enzymes in the MMP family, these data prompted us to investigate the effects of inhibitors of specific MMPs on elastin gene expression in airway fibroblasts. We used MMP inhibitor I, which specifically inhibits MMP-1 and MMP-8 at low concentrations (half maximal inhibitory concentration [IC50] = 1 μM) and MMP-3 and MMP-9 at very high concentrations (IC50 > 30 μM). MMP-2 inhibitor-1 specifically inhibits MMP-2. MMP-3 inhibitor-1 specifically inhibits MMP-3. These specific inhibitors were tested alone and in combination with IL-13 (50 ng/ml) on airway fibroblasts from a group of patients. To discern whether effects of the MMP inhibitor I at the 10 μM concentration were due to inhibition of either MMP-1 or MMP-8, we also tested a specific inhibitor of MMP-8 (MMP-8 inhibitor I). No significant effects were observed after incubation of airway fibroblasts with the specific MMP-8 inhibitor alone or combined with IL-13 (Figure E2). Therefore, we conclude that any effects of the MMP inhibitor I in our experiments can be attributed to specific inhibition of MMP-1.

Inhibition of MMP-1 or MMP-2, but neither MMP-3 nor MMP-8, stimulated significantly increased ELN expression in airway fibroblasts in subjects with asthma as compared with normal subjects. Alone, inhibition of MMP-1 resulted in no significant change in ELN expression in subjects with asthma as compared with normal subjects (P > 0.05) (Figure E3). However, when combined with IL-13, the MMP-1 inhibitor resulted in a significant increase in ELN expression in subjects with asthma as compared with normal control subjects (median percentage fold change in ELN expression, −0.44% [IQR, −45.9 to 116.9] for subjects with asthma and −49.07% [IQR, −69.7 to −29.4] for normal subjects; P < 0.05) (Figure 5A).

Similarly, the MMP-2 inhibitor alone significantly increased ELN expression in subjects with asthma as compared with normal subjects (mean percentage fold change in ELN expression, −3.1 ± 13% for subjects with asthma and −46.5 ± 11% for normal subjects; P < 0.05 for inhibitor alone) (Figure 5B). MMP-2 inhibitor combined with IL-13 also resulted in significantly increased ELN expression in subjects with asthma (median percentage fold change in ELN expression, −23.2% [IQR, −46.3 to 48.9] for subjects with asthma and −73.8% [IQR, −90 to −22.9] for normal subjects; P < 0.05) (Figure 5C).

Within asthma, the combination of IL-13 and the inhibitors of either MMP-1 or MMP-2 reversed the effect of IL-13 in that ELN mRNA levels significantly increased relative to the effect of IL-13 alone (P < 0.05) (mean percentage fold change in ELN expression, −31.7 ± 9% for IL-13 alone, 10.9 ± 24% for MMP-1 inhibitor alone, 39.4 ± 30% for combined MMP-1 inhibitor and IL-13, −36.4 ± 9% for IL-13 alone, 4.0 ± 16% for MMP-2 inhibitor alone, and 16.3 ± 25% for combined MMP-2 inhibitor and IL-13) (Figures 6A and 6B). No significant effect of MMP-1 or MMP-2 inhibitors as compared with IL-13 alone was observed in airway fibroblasts from normal patients (Figure E4). No significant effect on ELN mRNA levels in airway fibroblasts in subjects with asthma as compared with normal control subjects or within subjects with asthma was appreciated with specific inhibition of MMP-3 alone or combined with IL-13 (P > 0.05) (Figure E5).

IL-13 Stimulates Increased MMP-1 Secretion and Activity in Airway Fibroblasts in Asthma

We have previously shown that IL-13 stimulates significantly increased MMP-2 production (12) and activity (13) in asthmatic airway fibroblasts as compared with normal airway fibroblasts and as compared with untreated control cells. Because inhibition of MMP-1 alters IL-13–induced ELN expression in asthmatic airway fibroblasts (Figure 6A), we measured production and activity of MMP-1 in airway fibroblasts from subjects with asthma and normal subjects after exposure to IL-13. IL-13 significantly stimulated total MMP-1 secretion from airway fibroblasts from subjects with asthma as compared with cells from normal control subjects (mean MMP-1 production, 73.89 ± 21.8 ng/ml with IL-13 in airway fibroblasts from subjects with asthma compared with 19.85 ± 4.1 ng/ml with IL-13 in airway fibroblasts from normal control subjects; P < 0.05) (Figure 7A). Similarly, the effect of IL-13 on MMP-1 activity was significantly increased in airway fibroblasts from subjects with asthma as compared with cells derived from normal healthy control subjects (P < 0.05) and as compared with fibroblasts from untreated control subjects with asthma (P < 0.05) (mean active MMP-1, 1.27 ± 0.6 ng/ml for untreated control cells from subjects with asthma; 3.26 ± 1.1 ng/ml for IL-13–treated cells from subjects with asthma compared with 2.46 ± 0.9 ng/ml for normal airway fibroblasts from untreated control subjects; 1.48 ± 0.6 ng/ml for IL-13–treated airway fibroblasts from normal subjects) (Figure 7C). The effect of IL-13 to induce increased MMP-1 secretion or activity was elevated in subjects with asthma as compared with normal control subjects; however, this effect was not significant in the small numbers of patients that we analyzed for this study (Figures 7B and 7D). IL-13 exerted no significant effect on the production of TIMP-1 or -2, endogenous inhibitors of MMP-1 and MMP-2, respectively, by airway fibroblasts (P > 0.05, comparing IL-13 with fibroblasts for untreated control subjects with asthma or comparing subjects with asthma with normal subjects for TIMP-1 or TIMP-2) (Figures E6A and E6B).

Airway remodeling can lead to permanent biomechanical and pathologic alterations of airways. Despite extended courses of steroids, some patients manifest persistent airway obstruction. Previously, we have shown that elastin fiber degradation in the airways was associated with loss of elastic recoil in asthma (3). We demonstrate in this study that elastin loss occurs in the airways of patients with allergic asthma along with airways obstruction and AHR. These data suggest that elastin synthesis is impaired and/or elastin degradation is augmented with increased AHR and remodeling in asthma.

The mechanisms directing elastin loss in asthma are unclear. In this study, we show that IL-13, a key cytokine that directs airway remodeling in allergic asthma (15, 16), suppresses elastin mRNA expression in airway fibroblasts isolated from subjects with mild allergic asthma. Our data suggest that IL-13 is a critical modulator of elastin mRNA, and this process occurs through effects of MMPs on elastin mRNA levels. The MMPs have emerged as key mediators of extracellular matrix remodeling in asthma and are an important link between IL-13 and airway elastin, but most reported effects are manifested in protein degradation, not mRNA expression (21). Our study shows that MMPs may also directly or indirectly modulate mRNA expression. Although we have not fully characterized the cellular signaling that accounts for IL-13–directed ELN mRNA suppression via MMP activity, one possible mechanism may be through the heparin-binding epidermal growth factor (HB-EGF)/EGF receptor (EGFR) pathway. It is plausible that MMP-1 and MMP-2, induced by IL-13 in airway fibroblasts, mediate proteolytic cleavage of membrane-tethered HB-EGF at the cell surface (2224). The shed HB-EGF binds to EGFR and, through autocrine signaling, suppresses ELN mRNA expression, as previously demonstrated by DiCamillo and colleagues (25) and Liu and colleagues (26) in lung fibroblasts (25, 26). Levels of HB-EGF are increased in bronchoalveolar lavage fluid after allergen stimulation (27). Also, IL-13 stimulates proteolytic release of HB-EGF in airway epithelial cells (28). Thus, we postulate that in patients with allergic asthma, elevated responsiveness to IL-13 by airway fibroblasts stimulates MMP1 and MMP2 secretion, which acts indirectly to initiate negative regulation of ELN transcription through shedding of HB-EGF (or other mediators) rather than acting directly to degrade positive regulators of elastin gene expression. Future experiments will determine the role of HB-EGF and EGFR in IL-13–directed ELN suppression in airway fibroblasts in asthma.

Murine models of tobacco-induced emphysema show that IL-13 overexpression induces MMP-2, MMP-9, MMP-12, MMP-13, and MMP-14 (21). In this model, the effects of IL-13–related emphysema were attenuated by MMP blockade. Although matrix destruction by MMPs is a potential cause of elastin loss in patients with asthma, we present evidence for the ability of MMPs to mediate suppression of elastin mRNA levels in the airway fibroblast. Thus, IL-13, through its influence on MMP-1 and -2, is linked not only to elastin protein integrity but also to its production. Furthermore, we have previously shown that IL-13 stimulates increased COL1A2 expression and cellular invasion by airway fibroblasts in asthma through a mechanism requiring MMP-2 activity (12, 13). Therefore, MMP-2 has emerged as an important signaling mediator of several different features of IL-13–induced airway remodeling in asthma, including subepithelial fibrosis and loss of elastin in the submucosa.

We propose that loss of elastin in the asthmatic airway is a facet of remodeling that can lead to significant physiologic changes, including airflow obstruction, increased airways collapsibility, and loss of elastic recoil. Indeed, IL-13–induced suppression of elastin gene expression in airway fibroblasts was significantly correlated with post-bronchodilator FEV1/FVC, an established measure of airways obstruction and possibly remodeling (29, 30). Our data suggest that IL-13–induced suppression of elastin gene expression in airway fibroblasts could be associated with airways obstruction in asthma.

These data are in agreement with another study that demonstrated significant associations between elastic fiber assembly patterns and post-bronchodilator FEV1% predicted (31). Our data suggest that increased elastin gene expression is associated with increased post-bronchodilator FEV1/FVC, or less airway obstruction, and that elastic fiber staining in airway biopsy tissue is directly correlated with methacholine PC20, or inversely correlated with AHR in asthma. We hypothesize that airways collapsibility associated with reduced airway elastin increases susceptibility to not only methacholine but also to other mediators known to cause bronchoconstriction, such as allergens and airway irritants. Furthermore, we have previously shown that IL-13–induced airway fibroblast invasion in asthma was inversely associated with methacholine PC20 (12). Taken together, these data suggest that airway fibroblasts isolated from subjects with mild asthma exhibiting the most severe AHR are more susceptible to IL-13–directed signaling than those with less severe AHR, associating AHR with airway remodeling as manifested by airway wall thickening and stiffening due to increased subepithelial fibrosis and reduced elastin gene expression. Airway collapsibility and loss of elastic recoil may, in part, represent the spectrum of changes caused by IL-13 in asthma; the physiologic manifestation of increased airway collapsibility due to loss of elastin versus increased airway wall stiffness due to increased collagen production, which can each modulate FEV1/FVC and AHR, will be determined by which process is dominant. This latter issue may be the most important in understanding the phenotypic change seen in asthmatic airway fibroblasts.

A previous study has shown an inverse relationship between methacholine PC20 and elastic fiber staining in the large airways in mild asthma (32). Although our study was similar to this previous study in the methods of investigating extracellular matrix in airway biopsy tissue in a small group of patients with mild asthma on no controller medications, we observed an opposite association between elastic fiber staining and methacholine PC20. Because asthma is a heterogeneous disease characterized by several different clinical, inflammatory, and molecular phenotypes (33, 34), many different factors, such as T helper 2–driven inflammation or obesity, may affect the association of elastin fiber staining and AHR. Larger cohorts of patients are required to discern the exact relationship between elastic fiber expression and AHR in specific phenotypes of patients with asthma.

In summary, we have demonstrated that a reduction of elastic fiber staining is associated with increasing AHR in a cohort of subjects with mild allergic asthma. One possible mechanism for this observed elastin loss in asthma might be directed through IL-13–induced suppression of ELN mRNA expression in airway fibroblasts. Inhibition of MMP-1 and MMP-2 significantly reverses the IL-13–induced elastin suppression, pointing to an important role for MMPs beyond protein destruction, at the level of elastin gene regulation. Finally, IL-13–induced suppression of ELN mRNA expression was correlated with increased airways obstruction, suggesting that loss of elastin contributes to IL-13–induced airway wall stiffening and remodeling in asthma and may partially account for the observed process of airways collapsibility in asthma.

The authors thank Denise Beaver, R.R.T., R.C.P., of Duke University Medical Center for excellence in recruiting patients and coordinating this study, and Joseph Thomas, M.S., M.B.A., of Duke University Medical Center and Juno Pak, B.S., of National Jewish Health for technical assistance with fibroblast cultures and other experiments during the preparation of this manuscript.

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Correspondence and requests for reprints should be addressed to Jennifer L. Ingram, Ph.D., Duke University Medical Center MSRB1, Research Drive, Durham, NC 27710. E-mail:

*Presently with AstraZeneca Pharmaceuticals, Wilmington, DE.

Presently with Department of Medicine, University of Arizona, Tucson, AZ.

§Presently with Department of Pediatrics, University of Utah, Salt Lake City, UT.

This work was supported by National Institutes of Health/National Heart, Lung and Blood Institute grants 2R01 HL064619–071A and HL-05–009.

Author Contributions: Conception and design of experiments: J.L.I., D.S., M.E.S., and M.K. Acquisition, analysis, and interpretation of data: J.L.I., D.S., T.D.C., D.F., K.H., R.W.S., M.G., A.M., R.F., N.L.L., L.Q., M.E.S., and M.K. Drafting the manuscript for important intellectual content: J.L.I., D.S., R.F., M.E.S., and M.K.

This article has an online supplement, which is accessible from this issue's table of contents at

Originally Published in Press as DOI: 10.1165/rcmb.2014-0290OC June 13, 2015

Author disclosures are available with the text of this article at


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