The invasion of fibroblasts into the submucosal region of the airway in patients with asthma is one of several key features of airway remodeling (22
). In a normal response, fibroblasts are thought to be drawn to the subepithelial compartment of the airway after injury or environmental insult, and these cells begin wound healing through deposition of extracellular matrix proteins and interaction with inflammatory cells (23
). In asthma, this wound healing and remodeling response in the airway becomes deranged and uncontrolled, leading to increased numbers of fibroblasts invading the submucosa and contributing to subepithelial fibrosis with diminished lung function over time (24
). The numbers of myofibroblasts localized to the region correlates with basement membrane thickening (25
), and the thickness of the subepithelial layer and the reticular basement membrane has been shown to be associated with severity of asthma (27
In the present study, we show for the first time that airway fibroblasts isolated directly from patients with asthma and stimulated with IL-13 as a chemoattractant invade a simulated basement membrane in significantly greater numbers than those isolated from normal control subjects. We also demonstrate that the IL-13–induced airway fibroblast invasion response in asthma is mediated by MMPs and TGF-β1. Our data show that both MMPs and TGF-β1 were required for the IL-13–induced asthmatic airway fibroblast invasion, and airway fibroblast secretion of both MMP-2 and TGF-β1 was significantly induced by IL-13, suggesting that both MMPs and TGF-β1 are critical mediators of the IL-13 pathway for invasion in asthma. We postulate that in the Matrigel assay, IL-13 acts a chemoattractant for airway fibroblast invasion. As an IL-13 chemical gradient is established within the transwell plate, the higher concentration of IL-13 in the lower well stimulates invasion of airway fibroblasts. The cytokine binds to the IL-13 receptors on the cell surface of the airway fibroblast, initiating secretion of TGF-β1 and MMPs capable of degrading Matrigel. IL-13–induced TGF-β1 expression in airway fibroblasts may result in phenotypic transition of the fibroblasts to activated myofibroblasts, which have been demonstrated in the oncology literature to be proinvasive (11
). Lee and colleagues (2
) demonstrated that targeted transgenic overexpression of IL-13 in the murine lung resulted in significantly increased expression of MMP-9 mRNA, and IL-13–induced activation of TGF-β1 in vivo
was mediated by an MMP-9–dependent mechanism. Thus, our findings reported here are supported by these data in that IL-13 stimulates airway fibroblast invasion and airway remodeling in asthma through a mechanism that requires the action of MMPs and TGF-β1.
Our data also highlight a significant and fundamental difference between subjects with asthma and normal healthy subjects because airway fibroblasts isolated from subjects with asthma invade the Matrigel with greater numbers and respond to IL-13 with significant differences. One possible explanation for the difference in responses to IL-13 between asthmatic and normal control airway fibroblasts may be differential expression levels of the IL-13 receptor subunits on these cells in the two groups of subjects. IL-13 signaling in the airway fibroblast is mediated through a complex of receptor subunits (IL-13Rα1 and IL-4Rα) localized to the cell-surface (5
). Although some evidence has shown that IL-13 may signal through IL-13Rα2 to induce TGF-β1 and fibrosis in the colon, IL-13Rα2 is generally thought to act as a decoy receptor to negatively regulate IL-13-signaling (28
). Deficiency of IL-13Rα2 in the murine lung has been shown to increase IL-13–induced airway inflammation and features of airway remodeling (6
). Recent reports from research on asthma and airway remodeling lend support to the role of IL-13Rα2 acting as a nonsignaling, inhibitory receptor. Townley and coworkers (30
) used IL-13Rα2-IgGFc fusion protein as a blocking pretreatment to inhibit IL-13–stimulated sensitization to methacholine in the murine airway. The authors found that IL-13Rα2 pretreatment was more effective than salmeterol–fluticasone in preventing IL-13–induced bronchial hyperresponsiveness (30
). Also, pulmonary expression of IL-13 was reported to play a role in esophageal tissue remodeling, because mice that overexpressed IL-13 in the lung, and were also genetically deficient for IL-13Rα2, developed significantly more severe esophageal remodeling than IL-13Rα2–sufficient mice (31
). In the present study, we demonstrate that in human asthma, airway fibroblast cell-surface expression of IL-13Rα2 is suppressed compared with normal control subjects. However, the airway fibroblast cell-surface levels of IL-13Rα1 and IL-4Rα were not significantly changed in subjects with asthma relative to normal control subjects. Our data indicating that significantly higher levels of IL-13Rα2 are expressed overall in asthmatic airway tissue compared with normal control subjects is somewhat in contrast with our data using airway fibroblasts in primary cultures ex vivo
. However, the IL-13Rα2 airway tissue data take into account all cell types that stained positive for IL-13Rα2, including mesenchymal cells, and particularly epithelial cells, which showed marked positive staining for IL-13α2. Our ex vivo
cultures of airway fibroblasts have been well-characterized to be primarily of a fibroblast phenotype, with little (<5%) α-smooth muscle actin–positive staining. We recognize that exposure to mediators, such as IL-13, or even the Matrigel itself, may alter the phenotype of fibroblasts; therefore, work is ongoing to characterize further the phenotype of these cells during the invasion process. Taken together, these data suggest that low expression levels of IL-13Rα2 in human asthmatic airway fibroblasts may contribute to airway remodeling through a lack of regulation of IL-13 signaling, thus stimulating profibrotic processes, such as fibroblast proliferation and invasion.
IL-13 and IL-13 receptor subunit expression has been implicated in cellular invasion in cancer and rheumatoid arthritis. Fujisawa and colleagues (32
) used the Matrigel assay to demonstrate that invasion of pancreatic cancer cells increased significantly after stimulation with IL-13 and transfection with IL-13R
The authors also linked the IL-13–induced invasion response in pancreatic cancer to increased levels of MMP-9, -12, and -14 (32
). In lymphoma cells, IL-13 has been shown to induce MMP-10 expression, and invasion of these cells in the Matrigel assay was demonstrated to be dependent on MMP-10 activity (33
). Thus, these observations are consistent with the present study and suggest that IL-13 and expression of IL-13 receptor subunits modulates cellular invasiveness in a variety of cell types during the pathogenesis of cancer and other diseases, such as asthma.
We also show that IL-13–induced asthmatic airway fibroblast invasion is significantly and inversely correlated with methacholine log2
, an index of AHR. Production of IL-13 and infiltration of IL-13–postive cells in the submucosal region of the airway has been associated with AHR (34
). In addition to TH
2 lymphocytes, IL-13 is secreted by many cell types in the asthmatic airway, including macrophages (36
), mast cells (37
), airway smooth muscle cells (38
), and airway epithelial cells (39
). Our histologic examination of endobronchial biopsy airway tissue sections from these patients indicated the presence of inflammatory cells that may be capable of producing IL-13 and stimulating airway fibroblast invasion. Expression of extracellular matrix by infiltrating airway myofibroblasts in the subepithelial region of the airway has been associated with AHR and may contribute to airway wall thickening and airway lumen narrowing leading to altered airway compliance and AHR (40
). Invasion of airway fibroblasts into the submucosal region of the airway is likely to occur early in the repair response because these fibroblasts are drawn to areas of IL-13 secretion in the airway after epithelial damage and inflammation.
The patients included in the present study were subjects with mild asthma whose disease was controlled without the use of corticosteroids. We have demonstrated that IL-13 differentially increases fibroblast proliferation in this mild population (4
). Subjects with severe asthma are a logical extension of this work to determine if their fibroblasts demonstrate more of an invasive phenotype. A previous report from our laboratory demonstrated that procollagen I expression was significantly increased in subjects with severe asthma, compared with subjects with mild asthma or normal control subjects after exposure to platelet-derived growth factor-BB (PDGF-BB) (41
). The effect of PDGF-BB in severe asthma was attributed to significantly increased levels of the PDGF receptor-β at baseline on the severe asthmatic fibroblasts compared with the mild asthmatic and normal control fibroblasts. This study did not explore the effect of IL-13 on procollagen I expression in asthma. Therefore, ongoing studies in our laboratory will determine if IL-13 stimulates increased collagen deposition in the airways of both mild and severe subjects with asthma and if this effect is associated with increased airway fibroblast invasion and AHR.
In conclusion, we have demonstrated that IL-13 significantly stimulates human asthmatic airway fibroblast invasion in a Matrigel assay compared with that observed for normal control subjects. The mechanism of IL-13–induced airway fibroblast invasion in human asthma required both MMPs and TGF-β1, and we have also shown that airway fibroblasts cultured from subjects with asthma expressed reduced levels of IL-13Rα2 at baseline compared with normal control subjects. Goals of future studies include determining the direct effect that IL-13 receptor subunits expression may have on airway fibroblast invasion through knockdown of IL-4Rα, knockdown of specific MMPs, and characterization of the gene expression profile of invading fibroblasts to define further this feature of airway remodeling.