1 Meakins-Christie Laboratories, Royal Victoria Hospital; and 2 Genetics Unit, Shriners Hospital for Crippled Children, McGill University, Montreal, Quebec, Canada H2X 2P2
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ABSTRACT |
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Bleomycin (BM)-induced pulmonary fibrosis
involves excess production of proteoglycans (PGs). Because transforming
growth factor-1 (TGF-
1) promotes
fibrosis, and interferon-
(IFN-
) inhibits it, we hypothesized
that TGF-
1 treatment would upregulate PG production in
fibrotic lung fibroblasts, and IFN-
would abrogate this effect.
Primary lung fibroblast cultures were established from rats 14 days
after intratracheal instillation of saline (control) or BM (1.5 units).
PGs were extracted and subjected to Western blot analysis.
Bleomycin-exposed lung fibroblasts (BLF) exhibited increased production
of versican (VS), heparan sulfate proteoglycan (HSPG), and biglycan
(BG) compared with normal lung fibroblasts (NLF). Compared with NLF,
BLF released significantly increased amounts of TGF-
1.
TGF-
1 (5 ng/ml for 48 h) upregulated PG expression in both BLF and NLF. Incubation of BLF with anti-TGF-
antibody (1, 5, and 10 µg/ml) inhibited PG expression in a dose-dependent manner.
Treatment of BLF with IFN-
(500 U · ml
1 × 48 h) reduced VS, HSPG, and BG
expression. Furthermore, IFN-
inhibited TGF-
1-induced
increases in PG expression by these fibroblasts. Activation of
fibroblasts by TGF-
1 promotes abnormal deposition of PGs
in fibrotic lungs; downregulation of TGF-
1 by IFN-
may have potential therapeutic benefits in this disease.
lung fibrosis; versican; heparan sulfate proteoglycan; biglycan
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INTRODUCTION |
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THE LUNG EXTRACELLULAR MATRIX (ECM) consists of collagen and elastic fibers interspersed with structural glycoproteins and proteoglycans (PGs; see Ref. 8). PGs, a heterogeneous group of macromolecules, consist of a core protein to which glycosaminoglycan chains are covalently attached. PGs influence lung tissue mechanical properties and, through their interactions with various macromolecules, contribute to a variety of biological functions such as water balance, cell adhesion, cell migration, and growth factor binding within the ECM (1, 20, 33). Versican (VS), the large aggregating chondroitin sulfate containing PG, forms macromolecular aggregates with hyaluronic acid in the lung interstitial matrix. In addition, perlecan, a heparan sulfate proteoglycan (HSPG), and several small leucine-rich repeat PGs, i.e., biglycan (BG), decorin, fibromodulin, and lumican, have been identified in the lung tissue (11, 33, 43). Changes in ECM synthesis and degradation play a part not only in physiological processes such as development, growth, and aging but also in wound healing, inflammation, and fibrosis (33, 37).
Bleomycin (BM)-induced pulmonary fibrosis, a well-established animal
model for the study of human pulmonary fibrosis, is an inflammatory
interstitial lung disease characterized by excessive accumulation of
fibroblasts and ECM molecules, including PGs, in the intraluminal and
interstitial compartments of the lung (8, 40, 43).
Evidence from human studies and animal models indicates that
transforming growth factor (TGF)-1 plays a pivotal role
in mediating pathophysiological changes in fibrotic diseases (37). TGF-
1 stimulates fibroblasts to
synthesize large amounts of ECM proteins. TGF-
1 levels
are upregulated in patients with cryptogenic fibrosing alveolitis and
also in BM-induced lung fibrosis (BLF) in rats (23).
Fibroblasts participate in inflammatory responses and wound repair
through their ability to release cytokines, secrete ECM proteins, and
through cell-cell interactions with other inflammatory cells such as
macrophages. At sites of injury and wound repair, fibroblasts have been
shown to migrate from different anatomic sites and transform into a
less proliferative but more contractile and collagen synthetic
phenotype (14, 32). It has been reported that lung
fibroblasts cultured from BM-induced fibrotic rat lungs produced more
collagen than normal lung fibroblasts (NLF) in culture (30). Furthermore, Raghu et al. (31)
demonstrated that TGF-1 stimulated collagen production
and collagen mRNA levels in fibroblasts derived from normal and
fibrotic human lungs.
Although the effect of TGF-1 on collagen expression in
NLF and BLF has been relatively well defined, the pattern of synthesis of PGs by NLF and BLF and the effects of TGF-
1 on PG
production have not been investigated. This is of particular interest
because PGs are one of the first ECM components to be upregulated in
the fibrotic process and may be critical in establishing the
provisional matrix necessary for subsequent cell migration and
proliferation (33). Moreover, an increase in
TGF-
1, both at the messenger and protein levels, occurs
well before any detectable increase in the expression of collagens in
BM-induced pulmonary fibrosis (19). We have recently
reported marked increases in both the large and small PGs in BM-induced
pulmonary fibrosis in intact rats (12, 40). Based on these
observations, we hypothesized that the upregulation of PGs in fibrotic
lungs may be because of an increase in the biosynthetic activity of
resident fibroblasts. Moreover, we postulated a role for
TGF-
1 in this process, as has been shown in other organ
systems (4, 22).
The observation by Giri and colleagues (16, 41) that
anti-TGF- antibodies and TGF-
-soluble receptors were effective in
inhibiting BLF highlights TGF-
1 as a potential candidate
for therapeutic intervention. Subsequent studies found decorin, a small
PG and a natural inhibitor of TGF-
1, to ameliorate
various fibrotic disorders, including BLF (15).
Interferon-
(IFN-
), a major effector cytokine produced by
activated T lymphocytes and natural killer cells, has immunomodulatory,
antiviral, and antiproliferative properties (3). It
modulates the metabolism of connective tissue cells, inhibiting
fibroblast proliferation and the production of collagenous and
noncollagenous ECM proteins (9). Gurujeyalakshmi and Giri
(17) reported that IFN-
downregulates TGF-
1 and collagen gene expression in the BM model of
lung fibrosis. IFN-
has also been shown to inhibit the growth of
fibroblast cultures and collagen synthesis derived from normal and
fibrotic human lungs (27). However, the role of IFN-
in
providing protection against increases in PG production by BM lung
fibroblasts remains poorly defined.
Therefore, we compared PG production by NLF and BLF. We also
investigated the modulating effect of TGF-1 on PG
production and determined whether endogenous TGF-
-induced increases
in PG production would be abrogated by the addition of neutralizing anti-TGF-
antibodies. Finally, the anti-fibrotic effect of IFN-
on TGF-
1-induced increases in PG expression was studied.
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MATERIALS AND METHODS |
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Chemicals.
Reagents were obtained from the following sources: BM (Blenoxane) from
Bristol-Myers Squibb (Princeton, NJ); agarose from Park Scientific
(Northampton, UK); acrylamide, 6-aminohexanoic acid, benzamidine
hydrochloride, chondroitinase avidin-biotin complex (ABC), EDTA,
N-ethylmaleimide (NEM), guanidine hydrochloride (GuHCl), N,N'-methylene-bisacrylamide,
N,N,N',N'-tetra methyethylenediamine, L-glutamine, phenylmethylsulfonyl fluoride (PMSF),
sodium acetate, Trizma base, Tween 20, and monoclonal antibodies
anti-vimentin and anti- smooth muscle actin (
-SMA) from Sigma
(Oakville, ON, Canada); monoclonal antibodies 12C5 and C17 from
Developmental Studies Hybridoma Bank (Iowa City, IA); hyperfilm, Hybond
enhanced chemiluminescence (ECL) nitrocellulose membrane, molecular
weight standards, ECL Western blotting detection reagents, and
streptavidin-biotinylated horseradish peroxidase (HRP) complex from
Amersham Pharmacia Biotech (QC, Canada); collagenase type I, DNase,
DMEM, FBS, fungizone, IFN-
, penicillin-streptomycin, PBS,
recombinant human TGF-
1, and trypsin from GIBCO
(Burlington, ON, Canada); pan-specific TGF-
antibody and
TGF-
1 enzyme-linked immunosorbent assay kit from R&D Systems.
Experimental design. Pulmonary fibrosis was induced in male Sprague-Dawley rats (weight: 275-350 g) by a single intratracheal instillation of 1.5 units BM in 0.3 ml saline. Control rats received an equal volume of saline. Control and BM rats were killed at 14 days by exanguination under phenobarbitol sodium anesthesia according to standard ethical procedures. Using sterile techniques, the lungs were perfused until they were pale with sterile PBS via the right ventricle. The lungs were removed and trimmed of extraneous tissues and bronchial and vascular structures.
Primary rat lung fibroblast culture. Cultures of rat lung fibroblasts were established by enzymatic dissociation of finely minced lung tissues according to the standard protocol of Phan et al. (30) with slight modifications. Tissue fragments (after mincing into 2- to 4-mm pieces) were digested in DMEM containing trypsin (2.5 mg/ml), collagenase (1 mg/ml), and DNase I (2 mg/ml) and incubated with gentle stirring at 37°C in 5% CO2-95% O2 in humidified air for 30 min. Digestion was carried out for three cycles of 30 min each, with removal of medium and free cells after each cycle and the addition of fresh medium for each successive cycle. Medium removed at the completion of each cycle was mixed with an equal volume of DMEM. Pooled liberated cells, collected and separated from undigested tissue and debris, were filtered through sterile gauze and centrifuged to pellet cells. The pelleted cells were washed two times with PBS and DMEM containing 10% FBS and finally suspended in this media. The cells were then incubated in a 5% CO2 incubator at 37°C. After 24-48 h, unattached cells were removed by washing, and fresh medium was added. After another 48 h, cells were confluent and harvested by trypsinization with 0.25% trypsin-EDTA for 3-5 min at 37°C and then counted and split 1:3. Fibroblasts were maintained in DMEM + 10% FBS containing 2 mM L-glutamine, 0.37 g of sodium bicarbonate/100 ml, 200 units penicillin/ml, 200 µg/ml streptomycin sulfate, and 2.5 µg/ml fungizone. Cells were passaged every 3-5 days, and by the fourth passage they were homogeneous monolayers (morphologically consistent with fibroblast-like cells, phase-contrast light microscopy). Experiments were carried out with fibroblasts between the fourth and sixth passages.
Phenotypic characterization of lung fibroblasts.
Immunofluorescent staining was used to characterize the phenotype of
isolated lung fibroblasts. Lung fibroblasts were grown to subconfluence
on glass coverslips and fixed with ice-cold acetone for 20 min at
20°C. The cells were then treated with 70% ethanol for 5 min at
20°C. The cells were washed in sterile PBS and incubated with mouse
monoclonal antibodies for vimentin (a fibroblast marker) and
-SMA
for 1 h at room temperature. The cells were washed again in PBS
and reincubated with fluorescent goat anti-mouse IgG (Molecular Probes,
Eugene, OR) for 45 min at room temperature. After being washed in PBS,
the coverslips were sealed with crystal mount, and the fluorescence
pattern was examined by confocal microscopy. Negative controls were
processed similarly, except incubation with the primary antibody was omitted.
Determination of cell proliferation and cell viability.
Fibroblast proliferation was assayed by direct cell counting.
Fibroblasts were seeded at a density of 1 × 105 cells
in 25-cm2 culture flasks in DMEM + 0.1% FBS, and the
rates of growth were measured with or without TGF-1 at
24, 48, and 72 h. Cells were trypsinized and placed in a
hemocytometer for actual cell counting using a microscope. Experiments
were performed in triplicate with cells obtained from saline and
BM-exposed rat lungs. Cell viability was determined by the trypan blue
exclusion test.
Characterization of PGs synthesized by fibroblasts in culture.
Fibroblasts were grown to confluence and then serum deprived (0.1%
FBS) for 24 h before stimulation with TGF-1 (5 ng/ml), anti-TGF-
antibody (1, 5, or 10 µg/ml), IFN-
(5, 50, or
500 U/ml), or TGF-
1 + IFN-
(5, 50, or 500 U/ml) for
48 h. The amount of TGF-
1 used in the present study
was based on a previous study on collagen production using fibroblasts
derived from fibrotic human lung (31). At the end of the
experimental period, PGs were fractionated into cell layer and medium
compartments. The cell layer was rinsed three times with PBS and
extracted with ice-cold 4 M GuHCl-50 mM sodium acetate (pH 5.8)-1%
Triton X-100 containing the following proteinase inhibitor cocktail:
100 mM 6-aminohexanoic acid-10 mM EDTA-5 mM benzamidine
hydrochloride-10 mM NEM-0.1 mM PMSF at 4°C overnight. The PG extracts
were then centrifuged at 15,000 revolutions/min for 30 min; the
supernatants were dialyzed exhaustively against 50 mM
Tris · HCl (pH 8.0) containing proteinase inhibitors and
distilled water and concentrated; then protein content was estimated.
Cell culture media were dialyzed as described above, and then protein
content was measured (Bio-Rad protein assay).
Composite agarose-PAGE and Western blotting of VS and HSPG. Electrophoretic separation of large PGs was performed in a composite gel (0.6% agarose-1.2% polyacrylamide), as described previously (40). Samples were run in the gel at 60 volts and then separated at 160 volts until the bromphenol blue marker migrated to 3 cm. Separated PGs were electrophoretically transferred and probed with antibodies to VS or large basement membrane HSPGs. After electrophoresis, separated PGs were transferred to nitrocellulose membranes using a Bio-Rad (Mississauga, ON, Canada) blotter apparatus (20 volts overnight at 4°C). After blocking, membranes were probed with monoclonal antibodies 12C5 (1:2,000) or C17 (1:1,000) to detect VS or large basement membrane HSPGs, respectively, in Tris-buffered saline with Tween [TBST; 10 mM Tris · HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20] for 1 h at room temperature. After washing with TBST, membranes were incubated with a biotinylated rabbit antimouse secondary antibody (1:2,500) for 1 h at room temperature, washed again with TBST, and then incubated in streptavidin-biotinylated HRP complex (1:3,000) for 45 min at room temperature. After washing of membranes, antibody binding was visualized through ECL detection.
SDS-PAGE and Western blotting of BG. Specific polyclonal antipeptide IgG that has been previously shown to interact with BG in human cartilage extracts (34) was used to identify the expression of BG protein expressed by lung fibroblasts. PG extracts were digested with chondroitinase ABC (0.1 U/ml at 37°C for 4 h), run in a 10% SDS-PAGE gel, and then analyzed by immunoblotting. After electrophoresis, the separated proteins were electrophoretically transferred to nitrocellulose membranes and blocked for 1 h at 20°C. After blocking, membranes were washed with TBST and then incubated with primary antibodies for 1 h at room temperature to detect BG (1:500 dilution) core protein. After being washed with TBST, membranes were incubated with a 1:1,000 dilution of biotin-labeled swine anti-rabbit secondary antibody for 1 h at 20°C. After further washing with TBST, membranes were incubated in streptavidin-biotinylated HRP complex (1:5,000) for 45 min at room temperature, washed one time again, and then visualized by ECL detection.
Quantification of immunoblots. Densitometric analysis of both the large and small PGs was accomplished with image analyzer software (Fluorchem; Alpha Innotech, San Leandro, CA), which measures the sum of all the pixel values after background correction. The mean values of three experiments are presented.
Measurement of TGF-1 in cell-conditioned medium.
The concentration of TGF-
1 released by NLF and BLF was
determined using a human TGF-
1 enzyme-linked
immunosorbent assay kit (R&D Systems) that detects rat
TGF-
1 protein. Briefly, NLF and BLF were plated and
grown to confluency in complete medium containing 10% FBS. Next, these
cells were washed in PBS and incubated in a serum-free medium
containing 0.2 mg/ml BSA. The medium was changed every 4 h for a
period of 24 h. After 24 h, the conditioned media were
separated from cells and centrifuged. The cell-free conditioned media
were assayed for TGF-
1 after acidification and
neutralization according to the manufacturer's protocol. All assays
were done in duplicate wells. The assay detects only the active form of
TGF-
1, and the results, normalized to an equal number of
cells, were expressed as nanograms per milliliter per 106 cells.
Statistical analysis.
All data are presented as means ± SD of three observations.
Student's unpaired t-test was used to analyze the
statistical significance of the differences between the results of NLF
and BLF and that of cells treated with or without TGF-1.
One-way ANOVA with post hoc Bonferroni correction was used to determine the significance of dose-response studies with neutralizing
anti-TGF-
antibody and IFN-
. Statistical analyses were performed
using GraphPad Prism software (version 3.0).
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RESULTS |
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Characterization of lung fibroblasts.
Immunofluorescent studies on cultured cells revealed that both NLF
(Fig. 1A) and BLF (Fig.
1B) were strongly positive for vimentin (a fibroblast
marker). There was no systematic difference between NLF and BLF in the
staining pattern of vimentin. We also stained the isolated fibroblasts
with -SMA (a smooth muscle marker). The results demonstrated that
NLF (Fig. 1C) expressed a weak signal for
-SMA, whereas
BLF (Fig. 1D) contained more cells positive for
-SMA.
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Growth rate and cell viability.
In a previous study, we observed a maximal increase in lung PGs at 14 days post-BM treatment (40): lung fibroblasts were isolated at this time point in the current study. The growth rate of
BLF did not differ significantly from NLF (data not shown). Because
TGF-1 is a potent regulator of growth, cell
proliferation was also measured in the presence of exogenously added
TGF-
1. The results revealed no change in proliferation
rate, indicating TGF-
1 did not affect growth rates in
either NLF or BLF. We were also interested in determining whether
TGF-
1 or anti-TGF-
antibody had any cytotoxic effects
on these fibroblasts. To this end, we used the trypan blue exclusion
method to assess cell viability. Our results indicated that neither
TGF-
1 nor anti-TGF-
antibody caused detachment of
cells from the culture flask. More than 95% of cells were attached to
the culture flask and excluded trypan blue stain.
BLF express increased amounts of PGs at the protein levels in vitro. Similar results were obtained for both the cell layer and medium compartments with regard to PG expression; therefore, in the present study, we present the data obtained from the cell layer.
Our data indicate that NLF and BLF differ in their ability to produce PGs in vitro. BLF demonstrated a 7.3-fold increase in the in vitro expression of VS (Fig. 2B) in the cell layer compared with NLF (Fig. 2A). Two immunoreactive bands were detected on composite gels for VS, suggesting that the protein has two isoforms, perhaps because of alternative splicing of the VS gene. We observed a 3.6-fold increase of HSPG in the cell layer of BLF (Fig. 3B) compared with NLF (Fig. 3A). Finally, BLF (Fig. 4B) produced larger amounts (3.5-fold increase) of cell-associated BG compared with NLF (Fig. 4A).
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BLF secrete higher amounts of TGF-1 protein.
To investigate the hypothesis that enhanced expression of PGs in BLF
may be the result of increased expression of TGF-
1, we
measured the levels of TGF-
1 released in serum-free
cell-conditioned media of NLF and BLF. Our results demonstrated that
there was a significant increase (3.4-fold increase) in
TGF-
1 protein secreted by BLF (Fig.
5) compared with NLF.
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TGF-1 upregulates PG expression in NLF and BLF in
vitro.
After exposure to TGF-
1 (5 ng/ml), there was a twofold
increase in VS in the cell layer of BLF (Fig. 1B). BLF
exposed to TGF-
1 exhibited a 3.8-fold increase of HSPG
in the cell layer (Fig. 3B) and a 3.2-fold increase in the
cell-associated BG (Fig. 4B).
Neutralizing antibody to TGF- decreases PG expression in
fibroblasts.
There was a dose-dependent decrease in PG expression in response to
neutralizing antibody. Although the lowest concentration (1 µg/ml) of
anti-TGF-
antibody had no inhibitory effect on PG expression,
incubation with 5 and 10 µg/ml significantly modulated PG production.
As shown in Fig. 1B, anti-TGF-
antibody at 5 and 10 µg/ml inhibited 48.9 and 86.7% of VS expression, respectively, in
the cell layer obtained for BLF. Anti-TGF-
antibody treatment resulted in a 57.4 and 80.8% inhibition of HSPG expression in the cell
layer (Fig. 3B) at 5 and 10 µg/ml, respectively, in BLF. Anti-TGF-
antibody treatment led to a 40.9 and 62.6% inhibition of
BG in the cell layer at 5 and 10 µg/ml (Fig. 4B),
respectively, for BLF.
IFN- reduces PG expression in fibroblasts.
To determine whether IFN-
can suppress PG protein expression in BLF,
we used three different concentrations of IFN-
(5, 50, or 500 U/ml).
We found that the two lower concentrations (5 and 50 U/ml) had no
significant effect in reducing PG production, whereas IFN-
at 500 U/ml markedly decreased PG expression. A 64, 63, and 62% inhibition of
VS (Fig. 2B), HSPG (Fig. 3B), and BG (Fig.
4B) expression, respectively, was observed in the cell layer
of BLF treated with 500 U/ml IFN-
. We were also interested in
determining whether IFN-
treatment would block the
TGF-
1-induced increases in PG expression by fibroblasts.
Whereas the two lower concentrations (5 or 50 U/ml) of IFN-
resulted
in no inhibitory effect on PG expression, IFN-
treatment at a dose
of 500 U/ml resulted in a 74, 61, and 76% inhibition of VS (Fig.
2A), HSPG (Fig. 3A), and BG (Fig. 4A),
respectively, in NLF treated with TGF-
1. Similarly,
IFN-
treatment resulted in a 75, 78, and 67% inhibition of VS (Fig.
2B), HSPG (Fig. 3B), and BG (Fig. 4B)
expression, respectively, in BLF treated with TGF-
1.
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DISCUSSION |
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The most significant findings of the present study are
1) BLF produced larger amounts of all classes of PGs than
NLF, 2) BLF secreted increased amounts of
TGF-1 protein than NLF, 3) exposure of
fibroblasts to exogenous TGF-
1 stimulated these cells to
produce more PGs than untreated cells, 4) anti-TGF-
antibody inhibited the levels of PGs expressed by these fibroblasts in
a dose-dependent fashion, and 5) IFN-
attenuated the
TGF-
1-induced increases in PG expression. In the present
study, we have analyzed PG expression at the protein level. PG
expression at the mRNA level would have provided additional
information; however, the presence of mRNA does not necessarily predict
protein production. A previous study on cytokine regulation of PG
expression in lung fibroblasts has reported that changes in mRNA levels
for the various PGs were not consistent with changes in protein
production (38).
We questioned whether BM induced phenotypic changes in lung
fibroblasts. Previous studies have demonstrated the emergence of
myofibroblasts as the predominant cell type involved in the increased
deposition of ECM proteins and enhanced contractility of lung tissue in
this disease process (13, 24, 26, 44). Our findings are in
agreement with these studies, in so far as the BM fibroblasts showed
positive staining for both vimentin and -SMA and hence a phenotype
more consistent with that of the myofibroblast. Whether this specific
cell type produces relatively increased amounts of PGs upon cytokine
stimulation warrants investigation.
The marked upregulation of PGs by BLF, and the significant response to
TGF-1, are relevant to events in the development of pulmonary fibrosis. Increased VS expression may be of importance in
providing the provisional matrix for cell migration and cell proliferation (2, 33). This provisional matrix may be
required for subsequent collagen deposition (2). In this
regard, VS is one of the early response ECM components to be
upregulated during the development of BM-induced pulmonary fibrosis
(2). Given the observation that HSPG binds to growth
factors and regulates cell growth and cell-matrix interactions
(21), its upregulation by BLF could influence many
cellular functions pertinent to fibrosis. Because the core protein of
BG has been shown to bind to collagens and fibronectin, it may modulate
cell adhesion, cell migration, and collagen fiber assembly during
fibrogenic processes (35). Furthermore, in a recent study
from this laboratory, BG expression was correlated with biomechanical
changes in lung tissue behavior (12). Hence these
molecules may also be important in determining the mechanical changes
observed in this disease process.
During the development of pulmonary fibrosis, fibroblasts are exposed to a number of inflammatory mediators, including eicosanoids, immune complexes, serum components, inflammatory growth and differentiation factors, and cytokines, many of which have the potential to influence fibroblast function (8). In addition, resident fibroblasts participate in the inflammatory response with other inflammatory cells such as macrophages, resulting in fibroblast chemotaxis to sites of injury and excessive ECM synthesis and deposition (14). As a result, fibroblasts attain a different metabolic phenotype with increased biosynthetic activity, as evidenced by increased ECM gene expression (32).
In our study, PG production and secretion were significantly increased in BLF compared with NLF. Our results are consistent with the studies of Phan et al. (30), who reported that the BLF secreted increased amounts of collagen. BM has been reported to increase the synthesis of acidic glycosaminoglycans in cultured fibroblasts derived from carrageenin granuloma (29). Our data are also in agreement with findings of increased fibroblast PG production in other types of fibrotic disorders; human granulation tissue fibroblasts have been shown to produce increased amounts of PGs compared with human gingival fibroblasts (18).
Enhanced ECM synthesis and deposition could be attributed to an increase in fibroblast proliferation and/or synthetic capacity. However, there was no significant difference in growth rates, as measured by in vitro cell proliferation, between NLF and BLF. Similar results were reported by Phan et al. (30), who also showed no change in growth rates of NLF and BLF. Our results are also supported by the studies of Wegrowski et al. (42), who reported that fibroblasts derived from patients with primary hypertrophic osteopathy synthesized more PG with no change in cell proliferation. Thus the observed increases in PG production by BLF can be explained by either increased synthesis or decreased degradation. Our findings indicate an enhancement in PG biosynthetic activity by fibroblasts isolated from BM-exposed rat lungs compared with saline-exposed rat lungs. Fibroblasts may be "primed" by BM to increase PG production. This corroborates our previous in vivo findings of marked upregulation of these PGs in the lung tissue of BM-induced fibrosis in rats (12, 40). Enhanced secretion of PGs by fibroblasts may represent a mechanism to explain increased ECM deposition in this model.
TGF-1 is believed to be a critical mediator
involved in the fibrotic response through its ability to regulate ECM
production (37). Normally, TGF-
1 is
expressed in bronchiolar epithelial cells and interstitial fibroblasts.
However, after tissue injury and inflammation, TGF-
1 is
highly expressed in macrophages and mesenchymal endothelial and
mesothelial cells of the lung (23). TGF-
1
is increased in biopsies from fibrotic lungs (23).
Elevations in TGF-
1 mRNA and protein content precede the
increased expression of collagens in BM-induced pulmonary fibrosis
(19). BM has also been shown to modulate the expression of
TGF-
1 in rat lung fibroblasts (6). These
observations indicate that TGF-
1 contributes to the
general lung fibrotic process. We questioned whether this cytokine
would affect production of PGs by fibroblasts in the BM model. Indeed,
in the current study, TGF-
1 increased the expression of
PGs in both NLF and BLF. These observations are consistent with those
demonstrated in previous in vitro studies using other tissue-specific
fibroblasts. Dermal fibroblasts exposed to TGF-
1 increased their PG levels as follows: expression of VS, HSPG, and BG
was upregulated (22). In general, the response to
TGF-
1 stimulation by both NLF and BLF was similar.
Interestingly, it was reported previously that fibrotic human lung
fibroblasts and NLF exhibited a similar level of increase in collagen
production in response to TGF-
1 treatment
(31).
TGF-1-induced PG production was not coupled to enhanced
cell proliferation. The biological activity of TGF-
1
depends on cell type and culture conditions. Hence, in our experimental
conditions, the proliferative effect of TGF-
1 may not
have been evident. Our findings are in accordance with the studies of
Schonherr et al. (36), who reported that
TGF-
1 increased the synthesis of a large VS-like
chondroitin sulfate PG by arterial smooth muscle cells without changing
the smooth muscle growth rate. The present results suggest that the
stimulation of PG production by TGF-
1 can occur
independently of effects on cell proliferation.
The marked upregulation of PGs by BLF may be because of an
increase in TGF-1 expression by these cells in response
to BM-induced lung injury. Accordingly, we compared the amount of
TGF-
1 protein released by NLF and BLF. The results
indicated that BLF secreted higher amounts of TGF-
1
protein than that of NLF, supporting our hypothesis that increased
expression of PGs in BLF is the result of an increase in the expression
of TGF-
1. Increased expression of TGF-
1
mRNA was observed in BM-induced pulmonary fibrosis in rats
(43). Fibroblasts isolated from fibrotic human gingiva have been demonstrated to produce more TGF-
1 in vitro
than normal fibroblasts, which in turn can stimulate PG production
(39). We investigated the putative autocrine role of
TGF-
1 in the overproduction of PGs in this model using
neutralizing antibody to TGF-
isoforms. The present findings
revealed that the elevated production of PGs by BLF was significantly
inhibited by anti-TGF-
antibody in a dose-dependent manner. At the
highest dose of anti-TGF-
antibody, PG production by BLF was similar
to the basal levels produced by NLF, that is, the enhanced PG synthesis
was totally abrogated. Inhibition by neutralizing antibody to TGF-
of PG production to near normal levels has also been demonstrated in rat fibrotic glomeruli (5). In addition, antibody to
TGF-
reduced the levels of fibronectin, laminin, and chondroitin
sulfate PG in injured rat brain (25). The results of these
studies suggest that increased production of TGF-
plays a key
role in the pathogenesis of fibrogenic diseases, including BLF.
Furthermore, the significant reduction in PG production by NLF with
anti-TGF-
antibody treatment suggests that the constitutive
production of PGs in these fibroblasts is also under autocrine control
by TGF-
.
IFN-, a lymphokine produced by activated T lymphocytes, macrophages,
and natural killer cells, has been shown to modulate the metabolism of
connective tissue cells. Numerous studies have demonstrated that
IFN-
has a regulatory role on collagen accumulation by inhibiting
the synthesis of types I and III collagen and abrogating the
stimulatory effect of TGF-
1 (17). IFN-
has been shown to inhibit the growth of fibroblasts and collagen
synthesis from normal and fibrotic human lungs (27).
Likewise, inhibitory effects of IFN-
on aggrecan and decorin core
protein gene expression in cultured human chondrocytes have been
reported (10). In addition, IFN-
was reported to
inhibit experimental renal fibrosis (28). We also examined
whether IFN-
would interfere with the increased PG expression by
fibrotic lung fibroblasts and fibroblasts treated with
TGF-
1. Our results indicate that IFN-
treatment led
to a marked decrease in the production of PGs by BLF. Because of the
importance of TGF-
1 in the regulation of ECM production, the ability of IFN-
to decrease the production of PGs by fibroblasts exposed to TGF-
1 may have beneficial effects in
controlling the excessive PG accumulation in the fibrotic lung. It is
possible that IFN-
could block the abnormal deposition of PGs in the
fibrotic lung by altering TGF-
1 gene expression and/or
TGF-
signaling. Consistent with this hypothesis, it was reported
that, in the BM-induced model of pulmonary fibrosis, exogenous IFN-
downregulates the transcription of the gene for TGF-
1
(17), which in turn could modulate the turnover of ECM
proteins, including PGs. In contrast, Chen et al. (7)
reported that IFN-
(
/
) mice exhibited reduced inflammation and
fibrosis when treated with BM, suggesting that IFN-
may cause
inflammation early in the development of BM fibrosis, but its
continuous presence either endogenously or administered
exogenously may have a net antifibrotic effect. An exciting study
by Ziesche et al. (45) demonstrated the potency of IFN-
for the treatment of lung fibrosis in humans, revealing an
increase in total lung capacity and improved arterial oxygenation that
ran parallel with decreases in mRNA levels of TGF-
and
connective tissue growth factor.
In conclusion, our study has demonstrated that rat lung
fibroblasts in culture are capable of synthesizing various PGs that are
normally expressed in lung tissue. After BM-induced lung injury, these
fibroblasts respond with an increased expression of PG; TGF-1 further activates these cells. Because
anti-TGF-
antibody reduced the levels of PGs expressed by these
fibroblasts to near- normal levels, our observations indicate an
autocrine role for TGF-
1. Inhibition of endogenous
TGF-
activity may therefore be of importance in controlling the
excessive deposition of ECM components in pulmonary fibrosis. We have
also demonstrated that IFN-
suppresses the excessive production of
PGs by fibrotic lung fibroblasts and fibroblasts exposed to
TGF-
1. This observation supports a potential therapeutic
role for IFN-
via modulation of the abnormal deposition of PGs
observed during pulmonary fibrosis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Barbara Tolloczko and Vasanthi Govindaraju for help in immunofluorescence studies. The monoclonal antibodies 12C5, developed by Dr. R. Asher, and C17, developed by Dr. J. R. Sanes, were obtained from the Developmental Studies Hybridoma Bank of the University of Iowa Department of Biological Sciences, Iowa City, IA.
![]() |
FOOTNOTES |
---|
This work was supported by the J.T. Costello Memorial Research Fund and the Canadian Institutes of Health Research. N. Venkatesan was a recipient of a fellowship from the Canadian Lung Association and the Canadian Institutes of Health Research.
Address for reprint requests and other correspondence: M. S. Ludwig, Meakins-Christie Laboratories, McGill Univ., 3626 Ste. Urbain St., Montreal, QC, Canada H2X 2P2 (E-mail: mara.ludwig{at}mcgill.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
June 5, 2002;10.1152/ajplung.00061.2002
Received 15 February 2002; accepted in final form 16 May 2002.
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