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Am J Physiol Lung Cell Mol Physiol 274: L119-L126, 1998;
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Vol. 274, Issue 1, L119-L126, January 1998

Human bronchial epithelial cells modulate collagen gel contraction by fibroblasts

Tadashi Mio1, Xiang-Der Liu2, Yuichi Adachi3, Ilja Striz4, C. Magnus Sköld2, Debra J. Romberger2, John R. Spurzem2, Mary G. Illig2, Ron Ertl2, and Stephen I. Rennard2

1 Pulmonary Medicine, Chest Disease Research Institute, Kyoto University, Kyoto 601; 3 Department of Pediatrics, Toyama Medical and Pharmaceutical University, Toyama 930-01, Japan; 2 Pulmonary and Critical Care Medicine, University of Nebraska Medical Center, Omaha, Nebraska 68198-5300; and 4 Department of Immunology, Institute of Clinical and Experimental Medicine, 140 00 Prague 4, Czech Republic

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Connective tissue contraction is an important aspect of both normal wound healing and fibrosis. This process may contribute to small airway narrowing associated with certain airway diseases. Fibroblast-mediated contraction of a three-dimensional collagen gel has been considered a model of tissue contraction. In this study, the ability of primary cultured human bronchial epithelial cells (HBEC) obtained by bronchial brushings to modulate fibroblast gel contraction was evaluated. Human lung fibroblasts (HFL1) were cast into type I collagen gels. The gels were floated both in dishes containing a monolayer of HBEC or in dishes without HBEC. Contraction assessed by measuring the area of gels was increased at all time points from 24 h up to 96 h of coculture. At 48 h, coculture of HBEC with fibroblasts resulted in significantly more contraction than fibroblasts alone (36.6 ± 1.2 vs. 20.4 ± 1.7%, P < 0.05). Lipopolysaccharide (LPS, 10 µg/ml) stimulation of the HBEC augmented the contraction (44.9 ± 1.0%, P < 0.05 vs. HBEC). In the presence of indomethacin, the augmentation by LPS was increased further (52.2 ± 4.3%, P < 0.05 vs. HBEC with LPS), suggesting that prostaglandins (PGs) are present and may inhibit contraction. Consistent with this, PGE was present in HBEC-conditioned medium. Bronchial epithelial cell conditioned medium had an effect similar to coculturing. SG-150 column chromatography revealed augmentive activity between 20 and 30 kDa and inhibitory activity between 10 and 20 kDa. Measurement by enzyme-linked immunosorbent assay confirmed the presence of the active form of transforming growth factor (TGF)-beta 2. The stimulatory activity of conditioned medium was blocked by adding anti-TGF-beta antibody. These data demonstrate that, through the release of factors including TGF-beta 2 which can augment and PGE which can inhibit, HBEC can modulate fibroblast-mediated collagen gel contraction. In this manner, HBEC may modulate fibroblast activities that determine the architecture of bronchial tissue.

remodeling; transforming growth factor-beta

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

CONTRACTION OF TISSUE is an essential process in wound healing. In tissues such as skin, contraction speeds wound closure, minimizes scar size, and helps maintain mechanical properties of the tissue. The same process, however, may contribute to the abnormal tissue architecture observed in various forms of fibrosis. In this regard, increased numbers of fibroblasts or myofibroblasts have been reported in lung tissue of patients with pulmonary fibrosis (31) and in the bronchi of patients with asthma (4). Because fibroblasts or myofibroblasts are able to generate traction force, these cells could contribute to tissue contraction frequently associated with fibrotic processes in the lung.

When fibroblasts are cultured in three-dimensional collagen gels, fibroblasts contract the gels. This phenomenon has been considered to be an in vitro model of wound contraction and connective tissue morphogenesis (3, 8). Several biological factors are known that can modulate fibroblast-mediated collagen gel contraction. Platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta ), and fetal calf serum (FCS) have been shown to augment the contraction (3, 17, 29), whereas prostaglandin (PG) E2 and glucocorticoids inhibit contraction (6, 8).

In asthma and chronic bronchitis, airway narrowing is thought to be a major cause of fixed airflow obstruction. Airway epithelial cells are capable of modulating functions of fibroblasts, including chemotaxis, proliferation, and production of extracellular matrix (32, 39). The present study was designed to test the hypothesis that human bronchial epithelial cells (HBEC) modulate fibroblast-mediated collagen gel contraction. The present investigation reveals that both augmentive and inhibitory mediators of fibroblast gel contraction are released by HBEC and that their release can be modulated.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Type I collagen (rat tail tendon collagen, RTTC) was extracted by a previously published method (28). Briefly, tendons were excised from rat tails, and the tendon sheath and other connective tissues were carefully removed. After being washed with Dulbecco's modified phosphate-buffered saline (GIBCO, Grand Island, NY) six times for 24 h and 95% ethanol overnight, type I collagen was extracted in 4 mM acetic acid. Protein concentration was determined by weighing a lyophilized aliquot from each lot of collagen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis routinely demonstrated no detectable proteins other than type I collagen.

Lipopolysaccharide (LPS; Escherichia coli 0127:B8) was purchased from Sigma (St. Louis, MO). Anti-TGF-beta antibody (able to neutralize the biological activity of human TGF-beta 1 and -beta 2 according to the manufacturer's information) was purchased from R&D Systems (Minneapolis, MN). Tissue culture supplements and media were purchased from GIBCO Life Technologies (Grand Island, NY) except as described otherwise. FCS was purchased from Biofluids (Rockville, MD). Other reagents were purchased from Sigma.

Cell culture. Human fetal lung fibroblasts (HFL1) were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured on tissue culture dishes (Falcon; Becton-Dickinson, Franklin Lakes, NJ) with Dulbecco's modified Eagle's medium (DMEM; GIBCO) supplemented with 10% FCS, 100 µg/ml penicillin, 250 µg/ml streptomycin, and 2.5 µg/ml Fungizone. The fibroblasts were then detached by 0.25% trypsin in 0.5 mM EDTA and resuspended in DMEM without serum. Fibroblasts were used between the 10th and 20th passages.

HBEC were obtained from healthy donors and in one case from a fresh autopsy by a modification of a previously published method (22). Informed consent was obtained from each subject in agreement with a protocol approved by the Institutional Review Board for the Protection of Human Subjects at the University of Nebraska Medical Center. Bronchial epithelial cells were obtained by bronchoscopic brushing and were cultured under serum-free conditions using LHC-9-RPMI (a 1:1 mixture of medium RPMI 1640 and LHC-9; see Refs. 2, 24). Cells were plated on collagen-coated tissue culture dishes (Vitrogen 100; Collagen, Palo Alto, CA) at 37°C in a humidified atmosphere of 5% CO2. Cells were passaged one to two times a week at a 1:4 ratio. These cultured cells were identified as epithelial cells by positive staining with monoclonal murine anti-human cytokeratin antibody (MAK-6; Triton, Alameda, CA) using the avidin-biotin complex method (ABC kit; Vector, Burlingame, CA). Cells at the 4th to 8th passages were used for experiments.

Collagen gel contraction assay. Preparation of collagen gels was performed using a previously described method (27). Briefly, collagen gels were prepared by mixing RTTC, distilled water, and four times concentrated DMEM and cell suspensions so that the final mixture resulted in 0.6 mg/ml of collagen, 1 × 105 cells/ml, and a physiological ionic strength of 1× DMEM. Then, 1-ml aliquots of the mixture were put into each well of 12-well tissue culture plates (Falcon). Gelation was usually completed within 10 min at 37°C.

After gelation, 1 ml of control medium or samples was put over the gels, and the gels were then released from the surface of the tissue culture plates using a sterile spatula. The gels were then incubated at 37°C under 5% CO2 with continuous rocking (15 cycles/min) on a plate rocker (Bellco Biotechnology, Vineland, NJ) to prevent reattachment of gels to the bottom of the culture dishes. The contraction of collagen gels was recorded by making a photocopy of the culture dishes. The area corresponding to the gel was then quantified using an Optomax V image analyzer (Optomax, Burlington, MA). Data are expressed as a percentage of area corresponding to each gel at the indicated time compared with the area of the gel immediately after release.

Coculture of HBEC with HFL1. HBEC were cultured in 12-well tissue culture plates (Falcon) with LHC-9-RPMI medium until confluency. Fibroblasts embedded in collagen gels were prepared as described above. After gelation and release, the gels were transferred to separate plates with or without confluent HBEC. Fibroblasts and HBEC were cultured in 2 ml of LHC-D-RPMI medium (growth factor-depleted LHC-9-RPMI). The area of gels was assessed as described above.

HBEC-conditioned medium. HBEC were cultured in 100-mm dishes (Falcon) until confluency with LHC-9-RPMI medium. Cells were washed with LHC-D-RPMI medium two times and then were cultured with 5 ml of LHC-D-RPMI/dish with or without 10 µg/ml of LPS. Supernatant media were harvested after 48 h of culture, floating cells and debris were removed by centrifugation, and the conditioned media were stored at -70°C until use.

Partial characterization. To evaluate the biochemical characteristics of the activity contained in HBEC-conditioned medium that modulates fibroblast-mediated collagen gel contraction, various treatments were performed using a previously described method (37). To determine if low-molecular-weight substances with activity were present, conditioned medium was dialyzed against LHC-D-RPMI overnight at 4°C with three exchanges of medium using dialysis membranes, with an approximate molecular weight cutoff of 1,000 or 10,000 (Spectra/Por; Spectrum, Los Angeles, CA). Sensitivity to trypsin was examined by incubating the conditioned medium with 0.1 mg/ml of trypsin (Sigma) for 2 h at 37°C. After incubation, 0.2 mg/ml of soybean trypsin inhibitor (Sigma) was added to the sample solution to inactivate the trypsin. Parallel samples were treated in the same manner except that trypsin inhibitor was added before incubation. Pepsin digestion was performed by incubating the conditioned medium with 0.1 mg/ml pepsin (Sigma) for 2 h at 37°C after adjusting the pH to 2.5 with acetic acid. After digestion, samples were dialyzed against tris(hydroxymethyl)aminomethane-buffered saline, pH 7.5, with two exchanges of medium and LHC-D-RPMI overnight. Parallel samples were treated in the same manner without adding pepsin. Sensitivity to heat was tested by boiling the sample for 10 min at 100°C. Lipid extraction was done by extracting the sample with 2 vol of ethyl acetate two times. The aqueous phase was lyophilized to remove the remaining ethyl acetate and reconstituted with distilled water to the original volume. The treated samples were then immediately tested for fibroblast gel contraction as described above.

Column chromatography. To examine the possibility of multiple factors contributing to fibroblast-mediated collagen gel contraction modulating activity in the HBEC-conditioned medium and also to determine the approximate molecular weights of those factors, molecular sieve column chromatography was performed. The HBEC-conditioned medium was lyophilized and reconstituted with distilled water to one-tenth of the original volume. The sample then was applied to a Sephadex G-150 column (45 × 2.5 cm, Sephadex G-150 super fine; Pharmacia, Uppsala, Sweden). Hanks' balanced salt solution was used as the running buffer solution. The column was calibrated with ribonuclease A (molecular weight 13,700), chymotrypsinogen A (molecular weight 25,000), ovalbumin (molecular weight 43,000), bovine serum albumin (molecular weight 67,000), and blue dextran 2000 (molecular weight ~2,000,000; gel filtration calibration kit; Pharmacia). All fractions after the void volume were evaluated for fibroblast gel contraction activity in duplicate.

Inhibition of augmentory activity by antibody. One hundred microliters of HBEC-conditioned media were incubated with 50 µl of 1 mg/ml anti-TGF-beta antibody. Samples were then centrifuged at 13,000 revolutions/min for 15 min, and supernatants were tested for fibroblast gel contraction activity. To control for nonspecific inhibition of contraction by the antibody, excess TGF-beta (250 pM) was added to cultures with and without antibody.

Release of PGE and TGF-beta by HBEC. To confirm the production of PGE and TGF-beta by HBEC, cells were cultured with and without LPS and indomethacin as described above. Media were harvested and frozen until assay. PGE concentration in bronchial epithelial cell-conditioned medium was measured by 3H radioimmunoassay using a commercially available kit (Advanced Magnetics, Cambridge, MA). As reported by the manufacturer, cross-reactivity is 100% with PGE2, 50% with PGE1, 6% with PGA2, 3% with PGA1, 1.3% with PGF2alpha , and <1% with any other arachidonic acid metabolite. TGF-beta and TGF-beta 2 were determined by an enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN) that detects the active forms of TGF-beta . To measure TGF-beta , samples were assayed both with and without acidification and neutralization to convert the latent forms of TGF-beta to the active forms.

Statistical analysis. Results are expressed as means ± SE of three separate determinations except as described otherwise. Groups of data were evaluated by analysis of variance (ANOVA). Data that appeared statistically significant were compared by Student's t-test. Values of P < 0.05 were considered significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of cocultured HBEC on fibroblast-mediated collagen gel contraction. Fibroblasts in collagen gels contracted the gels 57 ± 1.8% in 96 h. Coculture with HBEC significantly augmented the fibroblast gel contraction (P < 0.01, ANOVA). The percent decrease in area in gels cultured in the presence of an HBEC monolayer was 82 ± 0.8% in 96 h (Fig. 1).


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  		Fig. 1.   Coculture of human bronchial epithelial cells (HBEC) with fibroblasts augments fibroblast-mediated collagen gel contraction. HBEC were cultured in tissue culture plates until confluency. Fibroblasts were cast in collagen gels, and the gels were transferred to dishes with or without HBEC as described in MATERIALS AND METHODS. Gels were cultured up to 96 h with continuous rocking. Photocopies of gels were taken at the indicated times, and the areas of the gels were quantified by image analyzer. Vertical axis: percent decrease of area of gels compared with that of gels immediately after release. Values represent means ± SE of 3 determinations. open circle , Fibroblasts alone; bullet , coculture.

The fibroblast-mediated gel contraction was significantly increased in the presence of 10 µg of LPS when LPS was added to fibroblasts and HBEC in coculture (Fig. 2). In contrast, LPS added to fibroblast-containing collagen gels in the absence of HBEC showed no effect on fibroblast-mediated gel contraction. The cyclooxygenase inhibitor indomethacin (1 µM) added to fibroblast-containing collagen gels alone had no effect on fibroblast-mediated gel contraction. However, when indomethacin was added with LPS to gels cocultured with HBEC, the fibroblast-mediated gel contraction was significantly augmented (P < 0.05).


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  		Fig. 2.   Lipopolysaccharide (LPS) and indomethacin (Indo) augment fibroblast-mediated gel contraction of fibroblasts and HBEC in coculture. Fibroblasts were embedded in collagen gels and transferred to wells with or without HBEC on the bottom. Gels were then cultured with or without LPS (10 µg/ml) and with or without Indo (1 µM). After 48 h of culture, the areas of the gels were quantified. Open bars, fibroblasts alone; hatched bars, coculture of HBEC and fibroblasts. * P < 0.05 and ** P < 0.01.

Effect of PGE2 on fibroblast-mediated gel contraction. PGE2 is one of the major PGs that HBEC produce, and the production of PGE2 can be blocked in the presence of indomethacin (23, 25). The augmentation of LPS-stimulated HBEC on fibroblast-mediated gel contraction by indomethacin suggests the presence of inhibitory PGs. For this reason, the effect of PGE2 on fibroblast gel contraction was evaluated directly. PGE2 attenuated the fibroblast gel contraction in a concentration-dependent manner (Fig. 3; P < 0.01, ANOVA). The attenuation by PGE2 was persistent during the observation period (Fig. 4). The effect of PGE2 was reversible, however, because the gels that had been incubated with PGE2 contracted at a rate similar to control gels after removal of PGE2 by repeated washing with medium. When PGE2 was added after 24 h of culture with medium, PGE2 was still able to attenuate fibroblast gel contraction. Finally, HBEC spontaneously produced PGE, and the production of PGE was significantly increased after exposure to LPS (Fig. 5). Indomethacin nearly completely inhibited the release of PGE both under basal conditions and when HBEC were incubated with LPS.


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  		Fig. 3.   Concentration-dependent attenuation of fibroblast gel contraction by prostaglandin (PG) E2. Fibroblasts embedded in collagen gels were cultured with various concentrations of PGE2 ranging from 10-10 to 10-6 M. Area of gels was measured after 48 h of incubation.


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  		Fig. 4.   Time course of attenuation of fibroblast-mediated gel contraction by PGE2. Fibroblasts embedded in collagen gels were incubated with 10-7 M PGE2 (bullet ) or without PGE2 (open circle ) for 72 h. One series of gels was incubated with PGE2 for 24 h, and then the gels were washed with medium 3 times by a 30-min incubation for each time (square ). Another parallel series was incubated without PGE2 for the first 24 h, and then PGE2 was added (black-square). Photocopies of gels were taken at indicated times. Area of the gels was quantified by image analyzer.


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  		Fig. 5.   Effect of LPS and Indo on PGE production by HBEC. HBEC were maintained in culture and incubated with and without LPS (10 µg/ml) or Indo (1 µM). Supernatant media were collected after 24 h and assayed for PGE by radioimmunoassay.

Partial characterization of augmentory activity on fibroblast gel contraction in HBEC-conditioned medium. Collagen gel contraction was augmented by HBEC-conditioned medium in a concentration-dependent manner (Fig. 6). The conditioned medium prepared in the presence of 10 µg/ml of LPS showed more activity than the conditioned media prepared without stimulation (P < 0.01, ANOVA). LPS might have augmented the contractile activity present in HBEC supernatants by altering the release of mediators from HBEC or by affecting the activity of mediators in HBEC-conditioned medium. To evaluate this latter possibility, HBEC supernatants were harvested, and after harvest, LPS was added. HBEC-conditioned medium augmented contraction, and HBEC-conditioned medium harvested in the presence of LPS further augmented contraction. Addition of LPS, however, to HBEC-conditioned medium did not increase contractile activity (Fig. 7). Thus the conditioned medium prepared with LPS stimulation was used for partial characterization.


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  		Fig. 6.   Effect of HBEC-conditioned medium on fibroblast-mediated gel contraction. HBEC were cultured until confluency in growth medium and then rinsed two times and incubated with LHC-D-RPMI for 48 h with or without 10 µg/ml of LPS. Supernatant media were centrifuged to remove floating cells and debris and were used as HBEC-conditioned medium. Fibroblast-embedded gels were prepared as described in MATERIALS AND METHODS and cultured with various concentrations of conditioned medium. Area of gels was measured after 48 h of culture. open circle , Unstimulated conditioned medium; bullet , LPS-stimulated conditioned medium.


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  		Fig. 7.   Effect of LPS on fibroblast-mediated collagen gel contraction. Fibroblasts were cast into collagen gels, and contraction was performed as described in MATERIALS AND METHODS with various additions. LPS did not affect fibroblast-mediated gel contraction when it was added under control conditions or when it was added with conditioned medium (CM) harvested from HBEC. In contrast, conditioned medium harvested from LPS-exposed cells had further augmented contraction. DMEM, Dulbecco's modified Eagle's medium.

The conditioned medium lost stimulatory activity with heat treatment (Table 1, P < 0.05). Dialyzed conditioned medium showed increased activity on fibroblast gel contraction compared with the untreated conditioned medium (P < 0.05). The lipid inextractable aqueous phase of conditioned medium also showed increased activity. Both trypsin digestion and pepsin digestion slightly increased the activity. The acid treatment used as a control for the pepsin treatment also augmented contraction activity compared with untreated media, although not as much as pepsin treatment. Taken together, the factors mediating fibroblast gel contraction activity produced by HBEC appear to be heterogeneous and may contain both lipid-inextractable and nondialyzable augmentive factor(s) and lipid- soluble and dialyzable inhibitory factor(s).

                              
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  		Table 1.   Partial characterization of HBEC-conditioned medium

Column fractionation. Molecular sieve chromatography by SG-150 also revealed that the activity was heterogenous in size. There was one distinct augmentory peak between 20 and 30 kDa ( peak A in Fig. 8), and one inhibitory activity between 10 and 20 kDa. However, several smaller augmentory and inhibitory activities that were not further characterized were also suggested.


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  		Fig. 8.   Size fractionation of HBEC-conditioned medium. HBEC- conditioned medium was lyophilized and reconstituted in one-tenth volume. Concentrated sample was fractionated on a SG-150 as described in MATERIALS AND METHODS. Each fraction was assayed for fibroblast-mediated gel contraction activity. Area of the gels was measured after 48 h of incubation. Values represent means of 2 determinations. VO, excluded volume; VT, included volume; A, distinct augmented peak between 20 and 30 kDa.

Identification of TGF-beta as a stimulatory activity released by HBEC. To determine if TGF-beta released by the HBEC could have accounted for the activity stimulating fibroblast-mediated contraction of collagen gels, two approaches were taken. First, the amount of TGF-beta present in epithelial cell supernatant media was measured by ELISA. TGF-beta 1 was not detectable in HBEC supernatant media, but TGF-beta 2 was detectable (Fig. 9A). Importantly, ~10% of the TGF-beta 2 was detectable before activation by acidification, suggesting that it had been activated in cell culture. Second, the contractile activity present in epithelial cell-conditioned medium was inhibited by anti-TGF-beta antibody (Fig. 9B). This inhibitory effect could be overcome by excess TGF-beta , indicating that inhibition was not due to a nonspecific effect of the antibody.


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  		Fig. 9.   HBEC production of transforming growth factor (TGF)-beta 2 and its role in fibroblast-mediated collagen gel contraction. TGF-beta 2 was assayed in conditioned medium harvested from HBEC before and after acidification to activate TGF-beta (A). In addition, the ability of epithelial cell supernatants to augment collagen gel contraction was assayed (B). Both the ability of antibody (Ab) to TGF-beta to block the augmented contraction and the ability of excess TGF-beta to overcome the antibody effect were determined.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The current study demonstrates that HBEC can modulate fibroblast-mediated collagen gel contraction by releasing both augmentive and inhibitory factors. The net augmentive activity for fibroblast gel contraction was increased by LPS stimulation of the bronchial epithelial cells. Partial characterization and column fractionation results indicate that the activity is heterogeneous. One of the inhibitory factors appeared to be PG. One of the PGs known to be released by HBEC, PGE2, inhibited the fibroblast gel contraction. Antibody-neutralizing experiments indicated that one augmentory factor is TGF-beta .

The rearrangement of extracellular matrix is a crucial process in both normal tissue repair and abnormal fibrosis (13, 14). Such processes may promote wound healing by reducing the area of wound. However, in fibrotic disease, the contraction and rearrangement of extracellular matrix may result in altered tissue structure and could cause tissue dysfunction (11). Fibroblasts can generate traction force and are known to cause rearrangement of extracellular matrix (11, 18, 38). In the lung, increased numbers of fibroblasts or myofibroblasts accompanied by increased connective tissue are reported not only in interstitial lung diseases (31) but also in chronic bronchitis and asthma (4).

Bronchial epithelial cells have several important functions. They serve as a physical barrier against exogenous insults and are important in producing and clearing airway secretions. Recently, it has become evident that bronchial epithelial cells can both respond to and release a number of inflammatory mediators (32, 39). In this regard, bronchial epithelial cells are capable of modulating fibroblast activity, including fibroblast chemotaxis, proliferation, and matrix production (21, 36). The present study extends these observations to include fibroblast contraction of collagen gels.

In vivo, fibroblasts reside in an extracellular matrix and are distinctly different from cells cultured in vitro in dish culture. In this regard, when fibroblasts are cultured in a three-dimensional native collagen gel, they acquire a bipolar, spindle-shaped form and have prominent stress fibers and resemble myofibroblasts (7, 9, 40). When collagen gels containing fibroblasts are detached from the underlying surface, the fibroblasts contract the gel (3). When collagen gels are detached immediately after gelation has completed, fibroblasts contract up to ~50% in 1 day in medium containing 10% FCS and in several days in serum-free medium. The rate of contraction can vary, however, with different fibroblast strains, collagen concentration, number of fibroblasts present, and the presence of soluble mediators. Different assay systems are used by different investigators, and their comparability is not fully determined. Nevertheless, many biologically active substances are known to modulate fibroblast gel contraction. TGF-beta (29), PDGF (17), cellular fibronectin (1), endothelin (16), and thrombin (30) have been shown to augment fibroblast-mediated collagen gel contraction, whereas cytochalasin (15), glucocorticosteroids (6), anti-beta 1-integrin antibody (34), beta -adrenergic agonists, and PGE2 (27) have been shown to attenuate fibroblast gel contraction.

Although clearly different from the in vivo conditions, the fibroblast-mediated collagen gel contraction has been considered to be a wound-healing model (3, 14). We have used it as a simple method to evaluate the contractility of fibroblasts and the ability of epithelial cells to modulate that contractility. LPS stimulation of HBEC augmented the net release of fibroblast contractile activity. This represented an increase in procontractile activity greater than a concurrent increase in inhibitory factors such as PGE. It seems likely that other stimuli will also be able to affect such release by HBEC as will conditions of culture, e.g., cell density or differentiated state. The ability of HBEC to modulate the release of factors that alter fibroblast activity suggests that they may play a role in the maintenance of airway structure.

In this study, TGF-beta and PGs are potentially identified as mediators by which bronchial epithelial cells might modulate fibroblast gel contraction. These data are consistent with previously published data describing bronchial epithelial cell release of both TGF-beta and PGE2 (5, 21), a result confirmed by the present study. The concentration of PGE released into the conditioned medium by epithelial cells, moreover, was precisely in the region of the concentration-response range for PGE inhibition of fibroblast-mediated collagen gel contraction. This suggests that LPS modulation of PGE release by HBEC is likely to be one of the mechanisms involved in modulating fibroblast contraction.

Previous studies with bovine bronchial epithelial cells have demonstrated that these cells produced TGF-beta . Up to 5% of the TGF-beta , moreover, was released in the active form (33). Although it was not possible to identify the species of TGF-beta released, TGF-beta 2 mRNA was detectable, but TGF-beta 1 mRNA was not. Results from the current study are consistent with these previous results. TGF-beta 2 was detectable in HBEC supernatants, and ~10% was released in the active form. TGF-beta 1 was not detectable with the immunoassays available.

Although it is likely that PGE and TGF-beta contribute to HBEC modulation of fibroblast-mediated collagen gel contraction, it is likely that other factors released by HBEC also contribute. The identities of all of the factors involved and their relative importance in specific situations will be important topics for future studies. It seems likely, moreover, that the relative production of these mediators may vary with cell culture conditions.

The mechanisms by which fibroblast-mediated gel contraction is altered by HBEC cell supernatants remain to be determined. TGF-beta has been suggested to increase the expression of integrins in various kinds of cells (10, 12, 19, 35), and alpha 2beta 1-integrin is known to be required for fibroblast-mediated collagen gel contraction (34). In addition, cellular fibronectin has also been suggested to play a role in fibroblast-mediated collagen gel contraction (1), and TGF-beta also increases the production of fibronectin. Whereas either or both of these could represent mechanisms for TGF-beta -induced augmentation of contraction, the mechanism(s) for effects of PGE is less well defined. Preliminary studies in our laboratories have suggested that PGE has little effect on fibroblast integrin expression. PGE may modulate fibroblast fibronectin production (26), however. By whatever mechanism(s) the effects are mediated, it is clear PGE and TGF-beta have opposite effects on fibroblast-mediated collagen gel contraction.

The possibility remains, moreover, that at least some of the PGs that inhibited fibroblast-mediated gel contractions were produced by the fibroblasts themselves. Some fibroblasts are known to produce PGs (20), and bovine bronchial epithelial cells have been demonstrated to release a factor that can stimulate fibroblast PG production (21). Thus, while the experiments conducted in the present study with indomethacin suggest a role for PGs, fibroblasts as well as epithelial cells may be a source of this mediator.

In conclusion, the current study demonstrates that HBEC can modulate fibroblast-mediated collagen gel contraction. Stimulation of epithelial cells with LPS can increase the contraction stimulation activity. By such regulatory mechanisms, bronchial epithelial cells might play a role in the development of bronchial fibrosis and the airway narrowing that occur in chronic inflammatory airway disease.

    FOOTNOTES

Address for reprint requests: S. I. Rennard, Univ. of Nebraska Medical Center, 600 South 42nd St., Omaha, NE 68198-5300.

Received 2 January 1996; accepted in final form 10 September 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Asaga, H., S. Kikuchi, and K. Yoshizato. Collagen gel contraction by fibroblasts requires cellular fibronectin but not plasma fibronectin. Exp. Cell Res. 193: 167-174, 1991[Medline].

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3.   Bell, E., B. Ivarsson, and C. Merrill. Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc. Natl. Acad. Sci. USA 76: 1274-1278, 1979[Abstract/Free Full Text].

4.   Brewster, C. E. P., P. H. Howarth, R. Djukanovic, J. Wilson, and S. T. Holgate. Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am. J. Respir. Cell Mol. Biol. 3: 507-511, 1990.

5.   Churchill, L., F. H. Chilton, J. H. Resau, R. Bascom, W. C. Hubbard, and D. Proud. Cyclooxygenase metabolism of endogenous arachidonic acid by cultured human tracheal cells. Am. Rev. Respir. Dis. 140: 449-459, 1989[Medline].

6.   Coulomb, B., L. Dubertret, E. Bell, and R. Touraine. The contractility of fibroblasts in a collagen lattice is reduced by corticosteroids. J. Invest. Dermatol. 82: 341-344, 1984[Medline].

7.   Eddy, R. J., J. A. Petro, and J. J. Tomasek. Evidence for the nonmuscle nature of the "myofibroblast" of granulation tissue and hypertrophic scar. An immunofluorescence study. Am. J. Pathol. 130: 252-260, 1988[Abstract].

8.   Ehrlich, H., and D. J. Wyler. Fibroblast contraction of collagen lattices in vitro: inhibition by chronic inflammatory cell mediators. J. Cell. Physiol. 116: 345-351, 1983[Medline].

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AJP Lung Cell Mol Physiol 274(1):L119-L126
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