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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 142-149, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0121OC

Mechanical Stress Triggers Selective Release of Fibrotic Mediators from Bronchial Epithelium

Daniel J. Tschumperlin, Jonathan D. Shively, Tadashi Kikuchi and Jeffrey M. Drazen

Pulmonary and Critical Care Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School; and Physiology Program, Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts

Address correspondence to: Jeffrey M. Drazen, Pulmonary and Critical Care Division, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115. E-mail: jdrazen{at}nejm.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transforming growth factor-ß (TGF-ß) and endothelin (ET) are found in elevated amounts in the airways of individuals with asthma. The cellular source of these peptides and their role in mediating the airway fibrosis of chronic asthma are unknown. In response to mechanical stresses similar to those occurring in vivo during airway constriction, bronchial epithelial cells increase the steady-state level of mRNA for both ET-1 and ET-2, followed by increased release of ET protein. Mechanical stress also enhances release of TGF-ß2 from a preformed cell-associated pool. TGF-ß2 and ET act individually and, more importantly, synergistically to promote fibrotic protein synthesis in reporter fibroblasts. To confirm the role of these intermediates in stress-induced fibrosis, conditioned medium from mechanically stressed bronchial epithelial cells was shown to elicit fibrotic protein synthesis in reporter fibroblasts; this effect was significantly inhibited by combined treatment with ET receptor antagonists and a neutralizing antibody to TGF-ß2. These data are consistent with a primary pathogenic role for mechanical stress–induced release of both TGF-ß2 and ET in the subepithelial fibrosis that characterizes chronic asthma.

Abbreviations: bronchial epithelial basal media supplemented with growth factors, BEGM • Dulbecco's modified Eagle's medium, DMEM • enzyme-linked immunosorbent assay, ELISA • endothelin, ET • normal human bronchial epithelial, NHBE • phosphate-buffered saline, PBS • platelet-derived growth factor, PDGF • reverse transcriptase–polymerase chain reaction, RT-PCR • trichloroacetic acid, TCA • transforming growth factor, TGF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Inflammatory cells, through the products of their cellular activation, are usually implicated as the key link between airway inflammation and the subepithelial fibrosis that characterizes chronic asthma (1–3). Our previous results have questioned the role of such cells by showing that mechanical stress applied to human bronchial epithelial cells, similar in magnitude to that which prevails in an airway during asthmatic bronchoconstriction but absent from normal airways, is a sufficient stimulus alone to generate a response that could replicate the subepithelial fibrosis of chronic asthma (4). Although we established this phenomenology, the mechanistic links between the deforming stimulus and the fibrotic process have not been elucidated. In this report we show that mechanical deformation increases steady-state levels of mRNA for endothelins 1 and 2 (ET-1, ET-2), enhances microenvironmental availability of ET protein, and triggers enhanced release of latent and active transforming growth factor-ß2 (TGF-ß2) protein, without an increase in TGF-ß2 mRNA. These two differentially regulated mediators act synergistically to promote a profibrotic microenvironment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Normal human bronchial epithelial (NHBE) cells were obtained from Clonetics-BioWhittaker (Walkersville, MD), and human lung fibroblasts (CCL-186) were obtained from the American Type Culture Collection (Rockville, MD). Plasmin, ET-1, ET-2, and ET receptor antagonists (BQ 788 and BQ 485) were purchased from Calbiochem (San Diego, CA). The enzyme-linked immunosorbent assay (ELISA) kit for measuring endothelin was from Peninsula Laboratories (San Carlos, CA). Recombinant TGF-ß2, neutralizing antibody for TGF-ß2, and ELISA kits for TGF-ß2, TGF-ß1, platelet-derived growth factor (PDGF) AB, and PDGF BB were from R&D Systems (Minneapolis, MN). Rabbit polyclonal antibody (sc-90) for immunblot detection of TGF-ß2 was from Santa Cruz Biotechnology (Santa Cruz, CA). [3H]proline was from New England Nuclear (PerkinElmer Life Sciences, N. Billerica, MA).

Cell Culture
NHBE cells were expanded on tissue culture–treated plastic in bronchial epithelial basal media supplemented with growth factors (BEGM; Clonetics-BioWhittaker). Cells at passage 4 were then transferred to six-well microporous polyester inserts (0.4 µm pore size, Transwell-Clear; Corning Costar, Corning, NY) and fed with a 1:1 mixture of BEGM and Dulbecco's modified Eagle's medium (DMEM) as previously described (5). After reaching confluence, cells were cultured at an air–liquid interface for 10–14 d to allow mucociliary differentiation (4). Sixteen hours before the initiation of mechanical stress the cells were fed with a serum-free minimal medium containing a 1:1 mixture of bronchial epithelial basal media and DMEM supplemented with insulin (5.7 µg/ml), transferrin (5 µg/ml), penicillin (100 units/ml), and streptomycin (100 µg/ml).

Normal human lung fibroblasts were expanded on tissue culture–treated plastic, then seeded at 5,000 cells/well on tissue culture–treated six-well plates and grown to confluence in {alpha}-modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin (4).

Application of Mechanical Stress
To expose cells to mechanical stress, silicon plugs with an access port for pressure application were press fit into the top of each transwell (Figure 1) , creating a sealed pressure chamber over the apical surface of the NHBE cells (4). Each plug was connected to a 5% CO2 (balance room air) pressure cylinder via a humidified chamber maintained at 37°C. The pressure in the apical chamber was increased by 30 cm H2O for the indicated duration, while the basal surface and medium remained at atmospheric pressure. The resulting apical-to-basal transcellular pressure produced a continuous compressive stress comparable to that present in the airway epithelium during bronchoconstriction (6, 7), orders of magnitude higher than the stress experienced by the airway epithelium during breathing.



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  		Figure 1. Device schematic and experimental timeline. (A) NHBE cells were grown at air–liquid interface on microporous substrates to express a mucociliary phenotype. An apical-to-basal transcellular pressure gradient was applied to compress the cells. (B) NHBE cells were preconditioned in minimal medium for 16 h, at which point mechanical stress was initiated and continuously applied for 8 h. Samples of media were collected at 0, 4, 8, and 24 h after the onset of mechanical stress, and from time-matched controls, to evaluate the release of soluble factors stimulated by mechanical stress. At identical times cell lysates were collected and the total cellular RNA was extracted for RT-PCR. Conditioned medium collected at 24 h was also used to compare the profibrotic capacity of the medium from stressed and control NHBE cells. Cell lysates for Western analysis were obtained after 8 and 24 h from stressed and control cells. (C) NHBE cells were preconditioned in minimal medium as above, then exposed to mechanical stress for varying durations. All samples were then collected 24 h after the onset of stress.

 
ELISA
Medium from the basal compartment of control wells was collected at the onset of the experiment (after incubation in serum-free medium for 16 h). Further samples were collected after mechanical stress of 4 and 8 h duration, and 16 h after the cessation of 8 h stress (24 h time point, Figure 1). Parallel time controls not exposed to mechanical stress were collected for comparison. Medium samples were assayed for the presence of ET, TGF-ß1, TGF-ß2, PDGF AB, and PDGF BB according to the manufacturer's instructions. The TGF-ß ELISAs were reactive only with the active form of the respective peptide. Therefore, total TGF-ß1 and TGF-ß2 were assessed after acid activation of medium samples, whereas the spontaneously active fractions of these peptides were measured by omitting the acid activation step. The ET ELISA was equally sensitive to ET-1, ET-2, and the ET-1 precursor bigET-1, but was not reactive with ET-3. To test the independence of ET and TGF-ß regulation, mechanical stress was applied in the presence of either a neutralizing antibody specific to TGF-ß2 (1 µg/ml) or antagonists that block signaling through ETA and ETB receptors (BQ 485 and BQ 788, 1 µm each).

Reverse Transcription–Polymerase Chain Reaction
Total RNA was purified from NHBE cell lysates after exposure to mechanical stress for the times noted above (RNeasy; Qiagen, Valencia, CA). Equal amounts of RNA were reverse-transcribed to cDNA using M-MLV reverse transcriptase (RT; Promega, Madison, WI). Polymerase chain reaction (PCR) was performed using Taq polymerase (Promega) and gene-specific primers (GeneLink, Hawthorne, NY) designed from published sequences (Table 1). Amplified PCR products were visualized after electrophoresis in 1.2% agarose gels containing ethidium bromide. Amplification of the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) was used to demonstrate that equal amounts of RNA were reverse transcribed and added to each RT-PCR reaction. Cycle numbers for each primer set (Table 1) were individually selected based on preliminary experiments to ensure evaluation of PCR products in the linear amplification range and avoid saturation.


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  		TABLE 1 PCR Primers

 
Western Blotting
NHBE cell lysates were collected under control conditions and after mechanical stress to analyze the distribution of TGF-ß2 by Western blotting. The basal medium was aspirated and the apical surface was washed two times with ice-cold phosphate-buffered saline (PBS). Two separate methods were then used to ascertain the location of TGF-ß2 within cultured human bronchial epithelial cells. In the first technique, adapted from Dallas and colleagues (8), cells were lysed in modified radio immunoprecipitation buffer containing 150 mM NaCl, 50 mM tris-HCl, 0.5% sodium deoxycholate, and 1% NP-40. Cell lysates were frozen immediately at -70°C for later analysis. Total protein in each cell lysate was quantified with the Bio-Rad protein assay (Bio-Rad, Hercules, CA) and equal amounts of protein were aliquoted for further study. After centrifugation for 10 min at 20,000 x g, the deoxycholate-soluble supernatant (cell associated proteins) and insoluble pellet (predominantly extracellular matrix) were collected for separate analysis. The pellet was washed twice with PBS, then 100 µl of plasmin (0.1 U/ml) in 50:50 BEGM:DMEM was added to both the pellet and supernatant. Plasmin digestion was allowed to proceed for 1 h at 37°C. For the pellet samples, plasmin-released proteins were collected in the soluble portion after centrifugation. These proteins were concentrated by precipitation in 80% ethanol followed by centrifugation for 5 min at 20,000 x g.

In the second method, adapted from Taipale and colleagues (9), extracellular matrices were isolated by washing the cells three times with 0.5% sodium deoxycholate in 10 mM Tris-HCl buffer, pH 8.0 at 4°C for 10 min. The first wash solution was collected and frozen for further analysis of the cell-associated fraction. After an additional wash in PBS, the substrate-attached matrix was allowed to dry overnight. This extracellular matrix was collected by scraping into nonreducing sample buffer (see below).

For both methods, samples were mixed 1:1 with 2x nonreducing sample buffer (100 mM tris-HCl, 5% SDS, 0.1% bromophenol blue, 20% glycerol), boiled for 5 min, and separated by SDS-PAGE on 10% gels (BioRad). Proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA), blocked with 5% milk, and incubated overnight at 4°C with the primary antibody (1:200 dilution), an affinity-purified rabbit polyclonal raised against the carboxy terminus of human TGF-ß2. Bands were visualized with horseradish peroxidase–conjugated anti-rabbit IgG (1:2,000) and enhanced chemiluminescence. Blots were quantified, where applicable, by standard densitometry.

[3H]Proline Incorporation Assay
Synthesis of fibrous protein was estimated from the incorporation of [3H]proline into trichloroacetic acid (TCA)-precipitated proteins in the media as previously detailed (4). Confluent human lung fibroblasts were incubated for 24 h in the presence of 3 µCi/ml [3H]proline in low-serum medium (0.5%) containing growth factors at defined concentrations, or medium conditioned by exposure to NHBE cells. Bronchial epithelium conditioned medium was collected 24 h after the onset of 8 h duration stress, and from parallel time-matched controls. After centrifugation (250 x g, 5 min) the resulting supernatant was incubated for 1 h at 37°C alone or with either a neutralizing antibody specific to TGF-ß2 (1 µg/ml), endothelin receptor antagonists BQ 788 and BQ 485 (1 µm each), or all three compounds together. The medium was then added undiluted to fibroblasts. In preliminary experiments, the TGF-ß2–neutralizing antibody and ET receptor antagonists were found to have no effect on baseline [3H]proline incorporation by fibroblasts at the concentrations employed.

For both growth factor and conditioned media stimulation, 1.0 ml of medium from each fibroblast well was precipitated overnight in 15% TCA at 4°C. The resulting pellet was collected by centrifugation (20,000 x g at 4°C), washed three times in 10% TCA, resuspended in 0.5 M NaOH overnight, and neutralized with 0.5 M HCl. Total [3H]proline incorporation was then assayed by scintillation spectroscopy.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mechanical Stress Triggers Release of ET and TGF-ß2 Protein
NHBE cells grown in air–liquid interface cultures released measurable amounts of ET, TGF-ß2, and PDGF AB into the basal medium (Figure 2A) . TGF-ß1 and PDGF BB were undetectable by ELISA (TGF-ß1 and PDGF BB sensitivity 7 and 15 pg/ml, respectively). Compared with time-matched controls, mechanical stress caused a significant increase in soluble ET at 4 h that was amplified at 8 and 24 h. Mechanical stress also induced an increase in soluble levels of total TGF-ß2 that reached significance at 8 h and was further amplified at 24 h. Constitutive release of PDGF AB into the media by NHBE cells was not affected by mechanical stress at any time point tested. The release of both TGF-ß2 and ET was highly dependent on the duration of the stress applied (Figure 2B). The most dramatic effects occurred at the earliest time points, suggesting that even relatively short durations of mechanical stress commit NHBE cells to increasing the microenvironmental availability of fibrotic mediators.



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  		Figure 2. Mechanical stress triggers selective release of fibrotic mediators. (A) Basal medium collected from NHBE cells exposed to continuous mechanical stress (triangles) were compared with time-matched controls (squares) by ELISA. Stress increased soluble levels of ET and TGF-ß2, but did not alter release of PDGF AB. TGF-ß1 and PDGF BB were also assayed by ELISA, but were not detectable (not shown). For the TGF-ß ELISAs, media samples were activated by acid treatment to measure both latent and spontaneously active TGF-ß. Data are presented as mean ± SD and are the result of 3–4 separate experiments. *P < 0.05, **P < 0.01. (B) NHBE cells were exposed to continuous mechanical stress for the labeled durations. Samples were collected 24 h after the onset of stress and assayed for ET and total TGF-ß2. For both peptides, the amount released was significantly dependent on the duration of pressure application, as determined by ANOVA (P < 0.001). In both cases, significant differences from control (0 duration) were obtained for all durations >= 1 h (**P < 0.05, *P < 0.01). Data are mean ± SD, n = 3.

 
Mechanical Stress Regulates Steady–State Level of ET Message
To examine expression of the genes encoding TGF-ß and ET, we collected RNA from NHBE cells at the onset of mechanical stress, after 4 and 8 h of continuous stress, and 16 h after the completion of 8 h of applied stress. The RNA was reverse-transcribed and the relative amount of message for each factor was assessed after amplification by PCR. Expression of both ET-1 and ET-2 was dramatically elevated after 4 and 8 h of mechanical stress (Figure 3A) . ET-1 message remained elevated 16 h after the cessation of pressure, whereas ET-2 returned to near control levels. NHBE cells did not express measurable amounts of ET-3 message. We confirmed the functionality of the ET-3 primers in A549 cells (data not shown).



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  		Figure 3. Mechanical stress regulates steady-state ET-1 and ET-2 message levels and recruits latent TGF-ß2 from cell-associated stores. (A) Total RNA was extracted from NHBE cells after application of stress for the labeled durations, or from unstimulated cells at the onset of pressure application. As shown by RT-PCR, mechanical stress produced a dramatic increase in steady-state levels of mRNA for ET-1 and ET-2 while eliciting little effect on TGF-ß1 and TGF-ß2. GAPDH is shown to demonstrate equal amplification and loading of samples. Results are representative of three independent experiments. (B) NHBE cells were lysed in deoxycholate buffer, separated into extracellular matrix-associated (pellet) and cell-associated (soluble) components by centrifugation, and probed for TGF-ß2 by Western blotting. The major immunoreactive band in the deoxycholate-soluble fraction was slightly larger than the expected size of ~ 25 kD for TGF-ß2. No immunoreactive band was found in the pellet fraction at this size. (C) After 8 h of mechanical stress (+), the amount of TGF-ß2 in the soluble cell-associated fraction was significantly reduced compared with unstimulated controls (-), but returned to control levels 16 h later (24 h time point). Data are presented as mean ± SD, n = 3, *P < 0.05. (D) Media assayed for TGF-ß2 without acid activation (filled bars) resulted in levels ~ 15% of that measured in activated (open bars) samples (control 13.4 ± 1.9%; mechanical stress 15.9 ± 1.3%, P > 0.05). Mechanical stress produced a statistically significant increase in spontaneously active TGF-ß2 from 64 ± 10 to 181 ± 26 pg/ml (P < 0.01). Data are mean ± SD, n = 4.

 
Steady-state levels of the mRNA encoding both TGF-ß1 and TGF-ß2 remained unchanged throughout the time course of mechanical stress (Figure 3A). The detection of mRNA encoding TGF-ß1 contrasts with the absence of TGF-ß1 peptide in NHBE conditioned medium and cell lysates (not shown).

Mechanical Stress Mobilizes Predominantly Latent TGF-ß2 from Cell-Associated Stores
We collected cell lysates and examined the deoxycholate-insoluble pellet (primarily extracellular matrix) and soluble supernatant for the presence of TGF-ß2 peptide, following the technique of Dallas and colleagues (8). Expression was found exclusively in the deoxycholate-soluble fraction (Figure 3B), suggesting association of TGF-ß2 with the bronchial epithelial cells rather than the extracellular matrix. These results were independent of plasmin digestion, which is known to release TGF-ß1 from the extracellular matrix pellet (8, 9). For further confirmation, matrix- and cell-associated fractions were isolated using a second technique adapted from Taipale and colleagues (9), which also demonstrated no TGF-ß2 immunoreactive peptide in isolated extracellular matrices compared with a strong band in cell-associated fraction (data not shown). These results strongly indicate that NHBE cells maintain a large pool of preformed, cell-associated TGF-ß2. After 8 h of mechanical stress the amount of TGF-ß2 in this cell-associated pool was significantly reduced compared with that found in controls (Figure 3C). By the 24 h time point, the amount of TGF-ß2 in the cell-associated fraction was restored to control levels.

To assess whether the TGF-ß2 released by mechanical stress was latent or active, we assayed media by ELISA both with and without acid activation. Stress significantly increased the amount of spontaneously active TGF-ß2 in the medium (Figure 3D). However, in both control and stressed conditions, the amount of TGF-ß2 measured without activation was relatively constant at ~ 15% of the total measured in activated samples.

ET and TGF-ß2 Are Released Independently of Each Other in Response to Mechanical Stress
As shown in Figure 4 , stress-induced release of TGF-ß2 was unaffected by the presence of ET receptor antagonists. To further confirm the independence of TGF-ß2 release from ET stimulation, we applied ET-2 to NHBE cells for 24 h at a concentration similar to that released into NHBE media during mechanical stress (250 pg/ml) and at a 100-fold excess (25,000 pg/ml). Neither concentration affected the release of TGF-ß2. The converse was also true, as application of mechanical stress in the presence of a neutralizing antibody to TGF-ß2 had no effect on ET release. The receptor antagonists and neutralizing antibody were used at concentrations ~ 1,000-fold greater than the amount of relevant peptide released by NHBE cells in response to stress (Figure 2). At identical concentrations these inhibitors effectively blocked stimulation of fibrotic protein synthesis by fibroblasts in response to the relevant peptide (results not shown).



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  		Figure 4. Release of ET and TGF-ß2 are independent responses to mechanical stress. NHBE cells were exposed to mechanical stress (MS) for 24 h in the presence of ET receptor antagonists BQ 788 and BQ 485 (ETRA, 1 µm each), or 8 h in the presence of a neutralizing antibody specific to TGF-ß2 (antiTGF, 1 µg/ml). Inhibition of the ET signaling pathway had no effect on the stress-induced TGF-ß2 release, and vice versa. Results were confirmed by stimulating NHBE cells for 24 h with a concentration of ET-2 similar to that found in the medium (250 pg/ml) and with a 100-fold higher concentration (25,000 pg/ml).

 
ET-2 and TGF-ß2 Enhance Fibrotic Protein Synthesis Both Individually and Synergistically
Both ET-1 and ET-2 stimulated fibroblasts to incorporate [3H]proline into TCA-precipitable proteins in a dose-dependent fashion, with a peak response at 25,000 pg/ml (Figure 5A) . There was no significant difference in response of fibroblasts to ET-1 and ET-2 except at the lowest concentration, and although the two were different from each other at this concentration, neither differed significantly from control. TGF-ß2 also elicited a dose-dependent increase in [3H]proline incorporation, and did so at lower concentrations and with a higher peak incorporation, suggesting that TGF-ß2 is a more potent stimulator of fibrotic protein synthesis than ET (Figure 5B).



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  		Figure 5. ET-2 and TGF-ß2 stimulate fibrotic protein synthesis alone and synergistically. Human lung fibroblasts were incubated in low serum (0.5%) medium containing the indicated concentrations of ET-1, ET-2, or TGF-ß2 in the presence of 3 µCi/ml [3H]proline. Incorporation of [3H]proline into TCA-precipitable proteins was used to assess fibrotic protein synthesis. (A) ET-1 and ET-2 were equally effective profibrotic agents. (B) TGF-ß2 exhibited higher peak incorporation and was effective at lower concentrations than either ET-1 or ET-2. (C) A concentration of ET-2 similar to that released by NHBE cells in response to mechanical stress (250 pg/ml) was relatively ineffective at stimulating fibrotic protein synthesis, but interacted synergistically with TGF-ß2 at concentrations of 100 and 1,000 pg/ml. Data are presented as mean ± SEM, with n = 4–8. (D) NHBE conditioned media was collected 24 h after the onset of a mechanical stress of 8 h duration (MS), and from parallel time-matched controls. Media samples were incubated for 1 h at 37°C alone or with either a neutralizing antibody specific to TGF-ß2 at final concentration of 1 µg/ml (antiTGF), ET receptor antagonists BQ 788 and BQ 485 at final concentrations of 1 µm each (ETRA), or all three compounds together (Both). Medium from stressed NHBE cells stimulated significantly more fibrous protein synthesis than did medium from control cells (*P < 0.05). The profibrotic effect was partially blocked by treatment with either anti–TGF-ß2 or ET receptor antagonists, and combined treatment directed at both pathways significantly attenuated the fibrotic stimulus by ~ 60% (*P < 0.05). Data are presented as mean ± SEM, with n = 4.

 
Although the concentration of TGF-ß2 released into the media by compressed NHBE cells (Figure 2A) was near the plateau phase of the dose–response curve (Figure 5B), the concentration of ET released in response to mechanical stress (Figure 2A) elicited little effect on fibroblast [3H]proline incorporation (Figure 5A). However, when this low dose of ET-2 was combined with submaximal and maximal effective doses of TGF-ß2, there was an enhanced fibrotic response (Figure 5C).

The Profibrotic Effect of NHBE Conditioned Medium is Partially Dependent on TGF-ß2 and ET
Conditioned medium from mechanically stressed NHBE cells stimulated a significant increase in fibrotic protein synthesis in human lung fibroblasts compared with medium collected from time-matched control NHBE cells (Figure 5D). Treatment of mechanical stress–conditioned medium for 1 h with either a neutralizing antibody specific to TGF-ß2 or receptor antagonists that block signaling through ETA and ETB receptors resulted in a partial reduction of the profibrotic stimulus that did not reach statistical significance. Combined treatment further reduced the profibrotic effect, eliminating ~ 60% of the difference between conditioned medium from stressed cells compared with that collected from control cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
NHBE cells respond to mechanical stress by releasing increased amounts of TGF-ß2 and ET, and these peptides interact synergistically to drive fibrotic protein synthesis. Stress applied to NHBE cells for as little as 1 h led to a significant increase in the accumulation of TGF-ß2 and ET at 24 h, demonstrating that mechanical stress evokes rapid and long-lasting effects on NHBE cell phenotype and growth factor production. Although these peptides act synergistically once released, regulation of their synthesis and release are at distinct levels, and production of each peptide is independent of the other. Thus the stimulation by mechanical stress is multidimensional, likely working through a specific array of transduction systems; the specificity of the response is demonstrated by our observation that release of PDGF AB is unaffected by mechanical stress. TGF-ß and ET are known to be present at elevated concentrations in the asthmatic airway (10–16), and although multiple sources and mechanisms may contribute to this pool (17–19), our data clearly demonstrate that signals initiated by mechanical stress are capable of producing targeted, locally high concentrations of these profibrotic factors directly at the focal point of subepithelial fibrosis.

TGF-ß is a potent mediator of fibrosis (20) and has been implicated in multiple inflammation and injury-induced pulmonary fibrosis models (17, 21–25). TGF-ß1 is typically synthesized as a latent peptide complex that is stored in the extracellular matrix (8, 9, 26, 27). The airway wall exhibits prominent immunostaining for TGF-ß2 and TGF-ß3 (relative to TGF-ß1), with staining concentrated in the interstitial spaces of the bronchial epithelial layer of human (23, 28) and mouse airways (29). Our data demonstrate that NHBE cells differentiated at an air–liquid interface to a mucociliary phenotype produce TGF-ß2 but not TGF-ß1, and possess large stores of TGF-ß2 that are associated with the cell surface and/or cytoplasm. Our finding that NHBE cells preferentially store TGF-ß2 in association with the cell layer rather than the extracellular matrix is unique, and suggests that mechanisms distinct from those described for TGF-ß1 storage are employed by NHBE cells to store latent TGF-ß2.

The qualitative observation of a relatively constant level of steady-state TGF-ß2 message observed during application of mechanical stress argues in favor of regulation by a post-transcriptional mechanism in our system. The finding that mechanical stress reduces the amount of TGF-ß2 in cell lysates and subsequently increases soluble levels of TGF-ß2 is consistent with the mobilization of preformed TGF-ß2 from cell-associated stores by mechanical stress. The TGF-ß2 released is predominantly latent, and the fraction in a latent form remains constant as the total soluble TGF-ß2 available in the media increases. These findings contrast with what is known about TGF-ß1, which is regulated by mechanical stimuli at the transcriptional level (30–35).

ET-1 is regulated by mechanical stimuli in a variety of tissues (34, 36–39) and contributes to tissue remodeling and fibrosis induced by mechanical forces (38, 40). Control of ET-1 is thought to occur predominantly at the transcriptional level, under the influence of protein kinase C signaling and an activator protein-1 binding site in the 5'-flanking promoter sequence (41–45). Our data indicate that mechanical stress applied to NHBE cells dramatically increases the abundance of mRNA encoding both ET-1 and ET-2. To our knowledge, this is the first demonstration that ET-2 is a mechanoresponsive gene. The mechanisms regulating ET-2 transcription are not known (46, 47), but preliminary indications are that the transcriptional control mechanisms for ET-1 and ET-2 are distinct (46–49).

Mechanically induced changes in the soluble levels of ET protein preceded the release of TGF-ß2, consistent with a possible autocrine role for ET. In addition, there is evidence that ET can modulate TGF-ß synthesis (50), and conversely, numerous studies have demonstrated that TGF-ß can influence the synthesis and release of ET (51–53). Our results strongly indicate that the stress-induced release of TGF-ß2 is independent of ET release, and vice versa (Figure 4), although we cannot rule out a common upstream mediator that regulates both pathways.

We found that TGF-ß2 has a profibrotic capacity similar to that previously reported for TGF-ß1 and ß3 (54, 55), and provides a more potent stimulus for fibrotic protein synthesis than either ET-1 or ET-2. Although the soluble level of ET elicited by mechanical stress was not sufficient alone to increase fibrotic protein synthesis significantly, it provided a synergistic stimulus when combined with TGF-ß2, suggesting that these mediators act in concert to account for the fibrotic response observed in asthmatic airways. The profibrotic effect of mechanical stress was substantially, though not totally, neutralized by the combination of a TGF-ß2–neutralizing antibody and ET receptor antagonists, providing strong support for the hypothesis that these agents act synergistically to activate the fibrotic responses to mechanical stress (4). The incomplete inhibition indicates that other factors, such as HB-EGF (5), also likely contribute to the fibrotic response to mechanical stress. In conclusion, these findings offer compelling evidence that the bronchial epithelium contributes to the fibrotic environment of the airway wall, and that the mechanical forces that accompany bronchoconstriction, acting through the bronchial epithelium, are key mediators of the subepithelial fibrosis that characterizes the asthmatic airway wall.


   Acknowledgments
 
This work was supported by NIH HL-33009. D.J.T. is a Parker B. Francis Fellow in Pulmonary Research. The authors wish to thank Omer Kalayci for critical reading of the manuscript, and John Munger for helpful suggestions.

Received in original form July 17, 2002

Received in final form August 30, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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