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 |
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)-
2. The stimulatory
activity of conditioned medium was blocked by adding anti-TGF-
antibody. These data demonstrate that, through the release of factors
including TGF-
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-
 |
INTRODUCTION |
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-
(TGF-
), 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 |
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-
antibody (able to neutralize the biological activity of
human TGF-
1 and
-
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-
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-
(250 pM) was added to cultures with and without
antibody.
Release of PGE and TGF-
by
HBEC. To confirm the production of PGE and TGF-
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
PGF2
, and <1% with any other
arachidonic acid metabolite. TGF-
and
TGF-
2 were determined by an
enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN)
that detects the active forms of TGF-
. To measure TGF-
, samples
were assayed both with and without acidification and neutralization to
convert the latent forms of TGF-
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 |
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. , Fibroblasts alone;
, coculture.
|
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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.
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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 ( ) or without
PGE2 ( ) 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 ( ). Another parallel series was incubated
without PGE2 for the first 24 h,
and then PGE2 was added ( ).
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.
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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. , Unstimulated conditioned medium; ,
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.
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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).
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.
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Identification of TGF-
as a
stimulatory activity released by HBEC. To determine if
TGF-
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-
present in
epithelial cell supernatant media was measured by ELISA.
TGF-
1 was not detectable in
HBEC supernatant media, but
TGF-
2 was detectable (Fig.
9A).
Importantly, ~10% of the TGF-
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-
antibody (Fig.
9B). This inhibitory effect could be
overcome by excess TGF-
, 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)- 2 and its role in
fibroblast-mediated collagen gel contraction.
TGF- 2 was assayed in
conditioned medium harvested from HBEC before and after acidification
to activate TGF- (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- to block the augmented contraction and the ability of
excess TGF- to overcome the antibody effect were determined.
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 |
DISCUSSION |
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-
.
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-
(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-
1-integrin antibody (34),
-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-
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-
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-
. Up to 5% of the TGF-
, moreover, was released in the active form (33). Although it
was not possible to identify the species of TGF-
released, TGF-
2 mRNA was detectable, but
TGF-
1 mRNA was not. Results
from the current study are consistent with these previous results. TGF-
2 was detectable in HBEC
supernatants, and ~10% was released in the active form.
TGF-
1 was not detectable with
the immunoassays available.
Although it is likely that PGE and TGF-
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-
has been
suggested to increase the expression of integrins in various kinds of
cells (10, 12, 19, 35), and
2
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-
also increases the production of
fibronectin. Whereas either or both of these could represent mechanisms
for TGF-
-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-
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.
 |
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