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Background: Mechanical strain and cytokine stimulation are two important mechanisms leading to airway remodeling in asthma. The effect of mechanical strain on cytokine secretion in airway fibroblasts is not known. The aim of this study was to determine whether bronchial and nasal fibroblasts differentially alter cytokine secretion in response to mechanical strain.
Methods: We measured secretion of the pro-fibrotic cytokine, interleukin-6 (IL-6), and the pro-inflammatory cytokines, IL-8 and monocyte chemotactic protein 1, before and after mechanical strain in bronchial fibroblasts obtained from asthmatic patients [asthmatic bronchial fibroblasts (BAF)] and normal volunteers [normal bronchial fibroblasts (BNF)], and in nasal fibroblasts (NF) obtained from nasal polyps. Cells were grown on flexible membranes and a mechanical strain of 30% amplitude, 1 Hz frequency was applied for 3, 6 and 24 h. Control cells were unstrained. IL-6, IL-8 and monocyte chemotactic protein 1 was measured after 24 h strain using enzyme-linked immunoassay; mRNA was measured by real time polymerase chain reaction. We also measured mRNA for versican, a matrix proteoglycan, known to be upregulated in the asthmatic airway wall.
Results: In unstrained conditions, no differences in cytokine secretion were observed. After 24 h strain, BAF secreted more IL-6 than BNF. Mechanical strain increased IL-8 mRNA in BAF, BNF and NF; and IL-6 and versican mRNA, in BAF, only.
Conclusions: Cytokine responses to mechanical strain varied in different airway fibroblast populations, and depended on the site of origin, and the underlying inflammatory state. Strain resulted in IL-6 upregulation and increased message for extracellular matrix protein in bronchial fibroblasts from asthmatic patients only, and may reflect these patients’ propensity for airway remodeling.
Asthma is a disease characterized by bronchoconstriction, airway inflammation, and airway remodeling. Airway inflammation involves the activation of various types of cells including inflammatory cells, such as eosinophils and T lymphocytes, and structural cells, such as airway epithelial cells, airway smooth muscle cells, and fibroblasts. These cells secrete different types of inflammatory mediators, including pro-inflammatory and pro-fibrotic cytokines and chemokines (1). While the role of pro-inflammatory mediators in contributing to asthma pathogenesis has been relatively well investigated, less attention has been directed at the potential importance of pro-fibrotic mediators, such as interleukin-6 (IL-6), in contributing to airway remodeling (2, 3).
Structural changes in the asthmatic airway wall include increased deposition of various extracellular matrix (ECM) components, such as collagen, elastin, glycoproteins and proteoglycans (PG) (4–7). Fibroblasts are the putative cell responsible for matrix deposition (8). There is information available in the literature documenting the ability of fibroblasts to secrete chemokines and cytokines (9, 10), thereby suggesting the potential for an autocrine mechanism, wherein the release of cytokines by fibroblasts results in increased matrix secretion.
A further stimulus to change in the asthmatic airway wall is thought to be excessive mechanical strain. It has been hypothesized that airway constriction and heterogeneous distribution of ventilation results in increased mechanical strain or stress on the structural cells of the asthmatic airway wall (11). Mechanical strain has been shown to affect the production of ECM components, upregulating type I collagen in pulmonary fibroblasts, and type III and IV collagen in co-cultures of bronchial epithelial cells and lung fibroblasts (12, 13). We have shown in bronchial fibroblasts obtained from asthmatic subjects, that mRNA and protein for the large, aggregating PG, versican, is increased in response to mechanical strain, in comparison to cells obtained from normal volunteers (14, 15). Mechanical strain also results in upregulation of inflammatory cytokines, such as IL-8 and monocyte chemotactic protein 1 (MCP-1), in airway epithelial and airway and vascular smooth muscle cells (16–19). However, there is no information currently available on whether mechanical strain induces cytokine production in bronchial fibroblasts. If excessive mechanical strain of bronchial fibroblasts results in increased secretion of cytokines, especially pro-fibrotic cytokines, then the fibroblast response to mechanical strain could lead to increased inflammation and/or signal to upregulate matrix deposition.
To investigate this question, we studied the effect of excessive mechanical strain on pro-inflammatory and pro-fibrotic cytokine and chemokine secretion in different types of airway fibroblasts. We questioned whether underlying inflammation would be necessary for the response and therefore, compared the response of bronchial fibroblasts from asthmatic patients to that of fibroblasts from normal volunteers. We also questioned whether there are phenotypic differences in cytokine secretion in different airway fibroblast populations, both in terms of basal secretion and in terms of response to mechanical stimulation, and so examined fibroblasts of nasal origin. We measured changes in IL-6, IL-8, and MCP-1, and assessed changes in versican mRNA as a marker of matrix upregulation.
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The present study shows that excessive mechanical strain leads to differences in cytokine and chemokine message and secretion in various types of airway fibroblasts. IL-8 mRNA was increased in response to mechanical strain in all fibroblasts studied. On the other hand, IL-6 message was increased in bronchial fibroblasts from asthmatic patients only. Further, bronchial fibroblasts from asthmatic patients secreted more IL-6 after excessive mechanical strain than did bronchial fibroblasts from normal volunteers. Along with the upregulation of the pro-remodeling cytokine, IL-6, message for the ECM proteoglycan, versican, was increased in response to excessive mechanical strain in these same cells.
Fibroblast populations from different parts of the airway tree have been shown to demonstrate different cytokine profiles. Kotaru et al. (23) reported that fibroblasts isolated from the proximal airways behave differently than fibroblasts isolated from the distal lung, in terms of cytokine and collagen secretion, and response to cytokine stimulation. In the current experiment, no differences in basal cytokine secretion were evident; however, the cytokine response to mechanical strain varied amongst the different fibroblast populations. The ability of lung cells to respond to mechanical stimulation with upregulation of cytokines has been previously demonstrated in bronchial and alveolar epithelial cells (16, 18, 24), but not in bronchial fibroblasts. To our knowledge, no previous data are available on the effects of mechanical strain on nasal fibroblasts.
The mechanism by which fibroblasts upregulate cytokine secretion in response to mechanical strain is not known, but one putative pathway is via stimulation of MAP kinase phosphorylation. A number of studies have now shown that mechanical strain causes activation of MAP kinase signaling in various types of fibroblasts (lung, chick embryo, cardiac) including a study we have published in these same populations of fibroblasts (15, 25, 26). Furthermore, in that study, we showed that bronchial fibroblasts obtained from asthmatic patients had a different profile of MAP kinase phosphorylation in response to mechanical strain, than fibroblasts obtained from normal volunteers. In airway smooth muscle cells, mechanical strain has been shown to cause increases in IL-8 through activation of ERK 1/2 and p38 kinases (19). It seems plausible that these signaling pathways are involved in the response we document in the current experiment. Another interesting question is whether mechanical strain influences fibroblast differentiation. Along these lines, Choe et al. (27) have shown that mechanical strain of lung fibroblasts resulted in myofibroblast differentiation (as assessed by number of α smooth muscle actin positive cells present in a tissue engineered airway wall model containing epithelial cells and human fetal lung fibroblasts). Whether fibroblasts from asthmatic patients would be more sensitive to this effect is not known.
Airway fibroblasts from asthmatic and control subjects responded differently to mechanical strain, in terms of upregulation of cytokine message and secretion. IL-8 message was increased in response to excessive mechanical strain in all three fibroblast populations, BNF, BAF and NF. Vlahakis et al. (16) showed a significant increase in IL-8 in alveolar epithelial cells submitted to a 30% stretch amplitude, while Oudin and Pugin (18) showed an IL-8 response in bronchial epithelial cells submitted to a 20% stretch. In our study, it is interesting to note that the upregulation of IL-8 mRNA was most sustained in the asthmatic cells. Further, BAF showed enhanced IL-8 message even in those cells not submitted to the strain stimulus, simply with time in culture. After 24 h of mechanical strain, IL-6 protein was significantly greater in BAF as compared to BNF. IL-6 message was increased after 3 h of mechanical strain in BAF, only. These differences may reflect differences in the underlying inflammatory milieu and subsequent alterations in phenotype. Cytokines are up-regulated in the inflammatory state characteristic of asthmatic disease. IL-8 is an important member of the chemokine family, mediates the activation and migration of neutrophils and is involved in the initiation and amplification of inflammation (28). IL-6 contributes to the inflammatory process, but may also play an important role as a pro-remodeling cytokine. Kuhn et al. (29) reported that IL-6 transgenic mice showed significant airway remodeling and sub-epithelial collagen accumulation. Other investigators have reported that IL-6 or IL-6 like cytokines have relevance to asthmatic airway remodeling (2, 30). Our observation that mechanical strain up-regulates IL-6 in the BAF only, may reflect the tendency for these cells to contribute to the remodeling characteristic of this disease.
We investigated the effect of mechanical strain on versican message as a marker of extra-cellular matrix. Versican is a large aggregating proteoglycan, which deposition is increased early in the fibrotic process (31). It forms a provisional matrix upon which subsequent collagen and other fibrotic proteins are laid down (32). We have shown in recently published experiments, investigating similar populations of bronchial fibroblasts obtained from asthmatic and control subjects, that versican protein and message is upregulated in response to excessive mechanical strain (14, 15). In the current experiment, we corroborate our previous findings.
The observation that mechanical strain differentially up-regulates pro-remodeling cytokines and extra-cellular matrix proteins in airway fibroblasts from asthmatic patients has particular relevance for asthma. Asthma is characterized by remodeling of the airway wall which contributes to abnormal airway physiology, including airway narrowing and airway hyper-responsiveness. In addition, asthmatic airways are subject to excessive mechanical stimulation as a consequence of heterogeneous airway narrowing and ventilation distribution (11). In the current study, excessive mechanical strain resulted in increased secretion of the pro-remodeling cytokine, IL-6, and the extra-cellular matrix protein, versican. One can postulate an autocrine mechanism whereby mechanical strain causes increased secretion of IL-6 which leads to enhanced matrix deposition. Alternately, the strain itself may result in increased ECM secretion, independent of the cytokine effect. Perhaps these two effects function synergistically.
In conclusion, we show that bronchial fibroblasts from asthmatic patients respond in a unique manner to excessive mechanical stimulation, with increased secretion of pro-inflammatory and pro-remodeling cytokines, co-incident with, or perhaps causal to, the up-regulation of matrix components. This phenotype, specific to the BAF, likely contributes to the airway remodeling characteristic of this disease.