1 Department of Medicine, Vancouver Hospital and Health Sciences Centre, University of British Columbia, Vancouver, British Columbia V6Z 3Z6, Canada; 2 University of Miami School of Medicine, Miami, Florida 33136; and 3 GlaxoSmithKline, Stevenage, Herts SG1 2NY, United Kingdom
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ABSTRACT |
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Fibrosis around the smooth
muscle of asthmatic airway walls leads to irreversible airway
obstruction. Bronchial epithelial cells release granulocyte/macrophage
colony-stimulating factor (GM-CSF) in asthmatics and are in close
proximity to airway smooth muscle cells (ASMC). The findings in this
study demonstrate that GM-CSF induces confluent, prolonged,
serum-deprived cultures of ASMC to increase expression of collagen I
and fibronectin. GM-CSF also induced ASMC to increase the expression of
transforming growth factor (TGF)- receptors type I, II, and III
(T
R-I, T
R-II, T
R-III), but had no detectable effect on the
release of TGF-
1 by the same ASMC. The presence of GM-CSF also
induced the association of TGF-
1 with T
R-III, which enhances
binding of TGF-
1 to T
R-II. The induction of T
Rs was parallel
to the increased induction of phosphorylated Smad2 (pSmad2) and
connective tissue growth factor (CTGF), indicative of TGF-
-mediated
connective tissue synthesis. Dexamethasone decreased GM-CSF-induced
T
R-I, T
R-II, T
R-III, pSmad2, CTGF, collagen I, and
fibronectin. In conclusion, GM-CSF increases the responsiveness of ASMC
to TGF-
1-mediated connective tissue expression by induction of
T
Rs, which is inhibited by corticosteroids.
airway remodeling; corticosteroids; irreversible airway
obstruction; phosphorylated Smad2; connective tissue growth factor; granulocyte/macrophage colony-stimulating factor; transforming growth
factor-; airway smooth muscle cells
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INTRODUCTION |
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A FUNCTIONAL ABNORMALITY in asthma is obstruction to airflow as a consequence of recruitment of inflammatory cells, mucus plugging, and airway wall thickening (17, 30). The airways are thickened by inflammatory cell infiltrates consisting of eosinophils, lymphocytes and mast cells, mucosal edema, and vasodilation (17, 30). Although many of these changes are reversible (30), airway obstruction in asthmatics may also be irreversible due to airway remodeling (30). Remodeling of airways occurs when myofibroblasts just beneath the bronchial epithelium proliferate and synthesize increased amounts of collagens I, III, and V and fibronectin (30). In addition, airway remodeling occurs from increases in collagens, elastins, fibronectin, laminin, hyaluronan, and versican around airway smooth muscle cells (ASMC) (27). ASMC may become hyperplastic and hypertrophied, which also contributes to structural changes, airway narrowing, and obstruction (27). On the basis of observations made on the pathogenesis of fibrosis at other sites, it is speculated that a number of cytokines and growth factors may be important in regulating the recruitment and proliferation of fibroblasts and connective tissue synthesis by fibroblasts, myofibroblasts, and ASMC (24, 33). The source of cytokines that regulate fibroblasts could be the structural cells themselves such as bronchial epithelial cells (BEC) and ASMC (3). Alternatively, the inflammatory cells recruited to the airways or plasma protein and platelets that leak into the airways could be additional sources of fibrogenic cytokines (3, 17).
Two cytokines of particular interest in the context of airway
remodeling are granulocyte/macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF)-. GM-CSF is a
22-25-kDa homodimeric glycoprotein originally described to induce
proliferation and differentiation of bone marrow cells (13,
15). GM-CSF is produced and released by a number of cells
(13, 35), such as fibroblasts, keratinocytes, BEC,
vascular cells, and ASMC (6, 7, 13, 25, 26, 35). With the
use of immunohistochemistry and Northern analysis, the expression
of GM-CSF is increased in the BEC of asthmatics compared with
nonasthmatics (26). Isolated BEC from asthmatic airways
release more GM-CSF than BEC from nonasthmatic controls
(26). It is speculated that, in asthma, the increased presence of GM-CSF in the airways leads to survival of eosinophils, which have been demonstrated to have an important role in regulating a
number of events in asthma (6, 7, 25, 26). Recently, an
additional effect of GM-CSF has been described, demonstrating the
induction of connective tissue synthesis (8, 31, 36). For
example, in a rat model, subcutaneous instillation of GM-CSF led to
fibroblast accumulation and stimulation of
-smooth muscle actin
synthesis in myofibroblasts (31). In another rat model, intratracheal administration of an adenovirus containing the GM-CSF gene led to irreversible pulmonary fibrosis (36). The
mechanism by which GM-CSF induces collagen synthesis is not well
understood but could be due to induction of TGF-
, a regulator of
connective tissue synthesis (4, 24, 33). GM-CSF induced
mRNA of TGF-
1 by vascular smooth muscle cells (29) and
leiomyoma cells (10). GM-CSF also caused myometrial smooth
muscle cells to release TGF-
1 (10).
TGF- is a multifunctional polypeptide (4, 24, 33) that
is present as three isoforms in mammals, but TGF-
1 is the isoform
most commonly associated with disorders characterized by inflammation
and fibrosis (4). Signal transduction of TGF-
1 is
mediated when TGF-
1 associates with TGF-
receptor type II (T
R-II), a 73-kDa serine/threonine receptor that is constitutively phosphorylated (5, 14). On binding TGF-
1, T
R-II
phosphorylates TGF-
receptor type I (T
R-I), a 53-kDa protein,
leading to signal transduction mediated by a number of intracellular
proteins that include the Smads (5, 14, 28). TGF-
receptor type III (T
R-III), or
-glycan, is a 250-350-kDa
proteoglycan that also binds TGF-
1 but has no definite signaling
capability (5, 14). However, the association of TGF-
with T
R-III enhances the interaction of TGF-
with T
R-II and
subsequent signal transduction (5, 14). With the use of
immunohistochemistry, we have previously demonstrated that all isoforms
of TGF-
, T
R-I, and T
R-II, were expressed by ASMC of human and
rat lungs (21, 23). We have also demonstrated that
subconfluent bovine ASMC or mechanically wounded monolayers of ASMC
release biologically active TGF-
1, that in an autocrine fashion,
induces collagen I synthesis (12). Furthermore, the
addition of TGF-
1 to ASMC also led to collagen synthesis in a
dose-dependent fashion (12). Despite the fibrogenic effects of TGF-
1 on ASMC, previous studies using Northern blot analysis and immunohistochemistry have not demonstrated evidence of
increased expression of TGF-
1 in ASMC of patients with asthma compared with nonasthmatic airways (2, 11, 32).
Furthermore, there has been no correlation of expression of TGF-
1
with the severity of asthma and airway remodeling (11).
Unlike our observations, these findings do not demonstrate the release
of biologically active TGF-
1 in a model simulating ASMC injury
(12). The release of active TGF-
1 is important in
autocrine regulation of collagen synthesis by ASMC (12).
In addition, the studies mentioned earlier do not provide any evidence
on the regulation of T
Rs, which might be equally important in
TGF-
1-mediated effects.
BEC, which express and release GM-CSF during episodes of asthma, are in
close proximity to ASMC. It is, then, conceivable that GM-CSF
previously demonstrated to induce TGF-1 (10, 29) might
regulate ASMC to synthesize and release TGF-
1, which, in turn, would
induce collagen synthesis in the ASMC layer of bronchi. In this study,
we demonstrate that GM-CSF induces collagen I and fibronectin
expression by bovine confluent, prolonged, serum-deprived cultures of
ASMC in a dose-dependent fashion. The presence of GM-CSF does not lead
to detectable release of TGF-
1 by ASMC. However, the presence of
GM-CSF in confluent, prolonged, serum-deprived cultures of ASMC
increases the density of TGF-
1 on the ASMC and expression of
T
R-I, T
R-II, and T
R-III, as well as the association of
TGF-
1 with T
R-III. The presence of GM-CSF also induces
TGF-
-mediated signal transduction necessary for connective tissue
synthesis. These findings, for the first time, demonstrate that
regulation of T
Rs on ASMC without evidence of release of TGF-
1
can lead to enhanced connective tissue expression that is mediated by
TGF-
1.
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MATERIALS AND METHODS |
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Materials.
-Modified Eagle's medium (
-MEM), fetal calf serum (FCS), and
other cell culture reagents were purchased from GIBCO-BRL (Burlington, ON, Canada). Anti-T
R-I and anti-T
R-II antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). To block the
interaction of TGF-
1 with T
R-II, anti-T
R-II antibody from R&D
Systems (Minneapolis, MN) was used. Anti-collagen type I and
anti-fibronectin antibodies were from Cedarlane Laboratories (Hornby,
ON, Canada). Anti-phosphorylated Smad2 antibody and anti-T
R-III
antibody were purchased from Upstate Biotechnology (Lake Placid, NY).
Cell culture.
Bovine tracheas were obtained from the local slaughterhouse. An
explanted culture of the smooth muscle tissue was established as
described previously with some modification (12). Briefly, the associated fat and connective tissues were removed in cold Krebs-Ringer solution (in mM: 118 NaCl, 4.8 KCl, 2.5 CaCl2
2 H2O, 1.2 MgSO4, 25 NaCHO3, 1.15 KH2PO4, 5.6 dextrose, and 12.6 HEPES) with
antibiotic-antimycotic reagents containing 100 U/ml of penicillin G,
100 µg/ml of streptomycin, 0.25 µg/ml of amphotericin B, and 0.25 µg/ml of sodium desoxycholate (GIBCO-BRL). The smooth muscle was then
isolated, cut into 1-2-mm cubic size, and placed on culture dishes
with a minimal volume of -MEM supplemented with 10% FCS and
antibiotic-antimycotic reagents. In an incubator at 37°C in a
humidified atmosphere (5% CO2-balanced air), ASMC migrated
from the tissue explants the following day. When cells were approaching confluence in some parts of the dish at 6-10 days after being explanted, the explants were removed, and the ASMC were passaged with
0.05% trypsin/0.53 mM EDTA. For the experiments, ASMC in passages 1-3 were grown to confluence in
-MEM with
10% FCS and antibiotic-antimycotic reagents and then changed to
serum-free
-MEM containing antibiotics and ITS (10 µg/ml of
insulin, 5.5 µg/ml of transferrin, and 5 ng/ml of sodium selenite,
from Sigma) for 10 days before addition of GM-CSF with or without
10
4 M dexamethasone (SABEX, Boucherville, QC, Canada). It
is of note that we have previously demonstrated that when ASMC are
subconfluent in culture, they release biologically active TGF-
1
(12). In these conditions, the release of TGF-
1 results
in the synthesis of collagen I (12). However, when ASMC
were grown to confluence and kept in serum-free conditions for 10 days,
there was no active TGF-
1 present in the conditioned media (CM)
(12). Such conditions also had minimal or no collagen I
synthesis (12) and were considered suitable to evaluate
the effects of GM-CSF on the release of TGF-
1 or connective tissue
synthesis by GM-CSF. Henceforth, when we refer to confluent monolayers
of ASMC, the term refers to prolonged, serum-deprived, confluent ASMC.
Collection of CM.
At various times after cells were incubated, the overlying CM were
removed, stored in sterile, siliconized Eppendorf tubes in the presence
of the protease inhibitors, including 5 µg/ml of leupeptin
(Boehringer Mannheim), 5 µg/ml of aprotinin (Sigma), 5 µg/ml of
pepstatin A (Sigma), and 1 mM PMSF (Sigma), and frozen at 86°C
until ready for use.
TGF-1 receptor assay by flow cytometry.
Twenty-four hours after being treated with GM-CSF or normal
saline, ASMC were removed from the culture dishes using a nonenzymatic cell disassociation solution (Sigma) and then stained with Fluorokine Cytokine Flow Cytometry Reagents (R&D Systems) as instructed by the
manufacturer. Briefly, each sample in both the control and the GM-CSF
group was divided into two aliquots of 1 × 105 cells.
The first aliquot was incubated at 4°C for 1 h with biotinylated recombinant human (rh)TGF-
1, which binds to the cell
predominantly via specific cell surface receptors. The cells were then
incubated with avidin-fluorescein, which attaches to the cell-bound
biotinylated TGF-
1, for 30 min at 4°C in the dark. The other
aliquot, which served as the negative staining control, was stained
exactly as the first aliquot except biotinylated negative control
reagent was used instead of biotinylated rhTGF-
1. The cells were
washed twice with RDF1 buffer to remove unreacted
avidin-fluorescein and resuspended in RDF1 buffer for final flow
cytometric analysis using Coulter Epics XL-MCL Flow Cytometer and Expo
32 software. Each sample was normalized by its individual negative
staining control before obtaining a mean and SE and applying
statistical analysis. The specificity of the reaction was confirmed
using anti-TGF-
1 antibody.
TGF-1 assay by ELISA.
TGF-
1 is secreted in a biologically latent form due to a noncovalent
association of the latency associated peptide-1 (LAP-1) with the
NH2-terminal 25-kDa portion of the protein. In this form, TGF-
1 is called latent TGF-
1 (LTGF-
1). To convert LTGF-
1 to a biologically active TGF-
1, the LAP-1 must be removed, which can be
achieved by acidification of the CM. The current assay detects TGF-
1
only in its active conformation. Each sample was divided into two
aliquots in which one aliquot of CM had a neutral pH containing
biologically active TGF-
1. To detect total TGF-
1 in each sample,
the other aliquot of CM was acidified by 1 N HCl for 10 min,
neutralized with 1.2 N NaOH/0.5 M HEPES, and used in the assay as
described. DuoSet TGF-
1 ELISA kit (R&D Systems) was used to
determine TGF-
1 in neutral CM (representing active TGF-
1) or CM
that were acidified and subsequently neutralized (representing total
TGF-
1) according to the manufacturer's instructions. Briefly,
anti-TGF-
1 capture antibody was coated onto a 96-well microplate
(Costar) overnight at room temperature. The antibody was diluted to a
working concentration of 2 µg/ml, and 100 µl of the preparation
were added to each well. After being washed with wash buffer and
blocked with block buffer, 100 µl of sample or the standard,
rhTGF-
1, were added and incubated for 2 h at room temperature.
The plate was washed before adding 100 µl of the detection antibody
(biotinylated chicken anti-human TGF-
1). The TGF-
1 binding was
colored by streptavidin-horseradish peroxidase, and the optical
density was read using a microplate reader at 450 nm with correction at
540 nm.
Western blot and immune detection.
After collection of the CM, ASMC were washed with phosphate-buffered
saline and then detached by trypsinization. Cell numbers were
determined using a hemocytometer. Whole cell protein was extracted on
ice with triple-detergent lysis buffer (50 mM Tris · HCl, pH
8.0, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 5 mg/ml of sodium
deoxycholate) in the presence of the protease inhibitors (as above). In
addition to these inhibitors, the samples used for detection of
phosphorylated Smad2 (pSmad2) were extracted in the presence of NaF (1 mM) and Na3VO4 (1 mM), potent phosphatase inhibitors. Protein concentration was calculated using the Bradford method with Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA).
Protein extracts were separated by SDS-PAGE [gel concentrations were
8% for collagen I, fibronectin, and TR-III; 10% for T
R-II and
pSmad2; and 12% for T
R-I and connective tissue growth factor (CTGF)] as per Laemmli's method and then transferred onto a
polyvinylidene difluoride (PVDF) membrane (Bio-Rad) using CAPS transfer
buffer (25 mM CAPS, pH 10, 20% methanol) (12).
The equal loading of proteins was confirmed before immunoblotting using
Ponceau S staining solution (Sigma). Briefly, after transfer of the
protein from SDS-PAGE, the PVDF membrane was immersed in Ponceau S
staining solution for 5 min and rinsed, and the protein bands were
visualized. Once equal loading was established, the membrane was
prepared for immunoblotting. After blocking with 5% milk in
Tris-buffered saline containing 0.05% Tween 20 overnight at 4°C, the
membrane was incubated with primary antibody at various dilutions. The dilutions were: for collagen I, 1:3,000; fibronectin, 1:3,000; T
R-I,
1:500; T
R-II, 1:500; T
R-III, 1:600; CTGF, 1:500; and pSmad2,
1:500 for 1 h at room temperature or overnight at 4°C. This was
followed by incubating the blots with a secondary antibody (Santa Cruz)
at a dilution of 1:8,000 for 1 h at room temperature. The proteins
on the membrane were then immunodetected by the ECL system (Amersham,
Arlington Heights, IL) according to the manufacturer's instructions.
Relative absorbance of the resultant bands was determined using the
Quantity One imaging system (Bio-Rad) normalized with data of untreated
control and expressed as fold of control.
Immunoprecipitation of TGF-1-associated proteins.
One hundred micrograms of total cell lysate prepared as described above
were precleared for 1 h at 4°C with 0.25 µg of normal rabbit
IgG and 20 µl of Protein A/G plus-Agarose (Santa Cruz). The
precleared extract was incubated with 1 µg of rabbit anti-human TGF-
1 polyclonal IgG (Santa Cruz) at 4°C for 1 h, then 20 µl of Protein A/G plus-Agarose was added and incubated at 4°C on a
rocker platform overnight. The pellet was washed with triple-detergent lysis buffer four times and then resuspended in 40 µl of nonreducing sample buffer. After being boiled for 3 min, 20 µl of the sample were
loaded on 8% polyacrylamide gel, and electrophoresis and immunoblotting were done as described above. Beads, both unused and
precleared, were mixed with the sample buffer and served as negative control.
Statistical analysis. The results were expressed as means ± SE. All P values (two-tailed) were based on Wilcoxon's signed rank test. Results were considered statistically significant when P < 0.05.
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RESULTS |
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GM-CSF regulation of connective tissue expression and release of
TGF-1 by ASMC.
We previously demonstrated that addition of TGF-
1 to monolayers of
ASMC induced collagen I synthesis in a dose-dependent fashion
(12). Similar to TGF-
1, the presence of GM-CSF also induced collagen I synthesis in a dose-dependent manner (Fig. 1,
A and B). In
addition, TGF-
1 increased the expression of fibronectin by
a greater magnitude than GM-CSF at an equivalent amount of 0.05 and 0.1 ng/ml. We next determined whether the increase in connective tissue
protein by ASMC treated with GM-CSF was associated with an increased
release of TGF-
1 by the ASMC. The presence of GM-CSF at a number of
concentrations and for a number of lengths of incubation time had no
effect on the secretion of TGF-
1 in the active or latent form by
confluent ASMC in culture (Fig. 2, A-C).
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GM-CSF regulation of TRs, pSmad2, and CTGF.
To study the changes in TGF-
signal transduction, we first
determined the expression of T
Rs using flow cytometry and Western analysis. Flow cytometry revealed that the presence of GM-CSF in
cultures of ASMC significantly increased the intensity of fluorescence from labeled TGF-
1, reflecting an increased density of TGF-
1 receptor on the ASMC compared with untreated ASMC (Fig. 3A,
P < 0.05). In addition,
the presence of GM-CSF increased the expression of T
R-I, T
R-II,
and T
R-III compared with untreated control ASMC as detected by
Western analysis (Fig. 3B). Next, we determined whether
there was an increase in T
R-III associated with TGF-
1 by
immunoprecipitating ASMC proteins using TGF-
1 antibody followed by
Western analysis using anti-T
R-III or anti-TGF-
1 antibody. ASMC
protein extracts immunoprecipitated with TGF-
1 all expressed the
same quantity of TGF-
1 (Fig. 3C, bottom
blots). ASMC cultured alone had a minor quantity of T
R-III when
immunoblotted with anti-T
R-III antibody (Fig. 3C,
top blots). However, in the presence of GM-CSF, the quantity
of T
R-III was markedly increased (Fig. 3C, top
blots), indicating that in the presence of GM-CSF, there is an increase
in the number of T
R-III associated with TGF-
1. We next determined
whether GM-CSF also increased TGF-
-mediated downstream signal
transduction. An index of target cell response to TGF-
is the
phosphorylation of the COOH-terminal SSXS motif of Smad2 and -3 that
mediates intracellular signals of the TGF-
superfamily
(28). ASMC cultured with GM-CSF had induction of pSmad2
compared with untreated controls (Fig. 3D). Induction of connective tissue proteins by TGF-
is mediated by CTGF, a
33-38-kDa cysteine-rich protein (16). To confirm that
connective tissue synthesis was mediated by TGF-
1, the protein from
ASMC was immunoblotted with anti-CTGF antibodies. ASMC cultured with
GM-CSF had an induction of CTGF (Fig. 3D). To further
confirm that the GM-CSF-induced above effects are via T
Rs,
GM-CSF-treated ASMC were cultured with or without T
R-II antibody,
which interrupts the TGF-
1/T
R-II interaction. The increased
pSmad2 by GM-CSF was inhibited in the presence of 20 µg/ml of
T
R-II antibody (Fig. 3E). It is of note that in the
presence of GM-CSF, the magnitude of increases in the expression of
T
Rs, collagen I, fibronectin, pSmad2, and CTGF is modest. However,
the results are highly reproducible. Furthermore, there is a
statistically significant difference in the expression of all these
proteins when ASMC treated with GM-CSF are compared with ASMC receiving
no treatment.
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Effects of dexamethasone on GM-CSF-induced expression of TRs,
pSmad2, CTGF, and connective tissue proteins.
Corticosteroids are used as standard therapy in the treatment of asthma
and have been demonstrated to inhibit collagen I synthesis (18,
31, 36). There were no significant differences in the expression
of collagen I, fibronectin, pSmad2, CTGF, T
R-I, T
R-II, and
T
R-III between the extracts from ASMC treated with dexamethasone (10-4 M) and with normal saline (Table
1). However, the presence of
dexamethasone inhibited collagen I and fibronectin expression induced
by GM-CSF (Fig. 4, A and
B). Because the induction of connective tissue synthesis by GM-CSF was demonstrated to be on the
basis of increase in T
Rs, it was next determined whether the
presence of dexamethasone altered T
Rs. The presence of dexamethasone inhibited the increased expression of T
R-I, T
R-II, and
T
R-III (Fig. 3B) as well as pSmad2 and CTGF
(Fig. 3D). These findings demonstrate that dexamethasone
inhibits the expression of GM-CSF-mediated induction of T
Rs and
TGF-
1-mediated signal transduction, important for collagen
synthesis. The presence of dexamethasone did not have an effect on
GM-CSF-mediated increase of association of TGF-
1 with T
R-III
(Fig. 3C, top band). The use of corticosteroids
in asthmatics can vary from being used chronically or at various time
intervals after the onset of an exacerbation of asthma
(9). To simulate these possibilities, in some instances,
ASMC were pretreated with dexamethasone by 1.5 h before the
addition of GM-CSF. In other instances, dexamethasone was added
concomitantly or at a number of intervals after the addition of GM-CSF
(Fig. 4, A and B). The GM-CSF-induced increases
in collagen I and fibronectin were inhibited by dexamethasone added at
all time intervals (Fig. 4, A and B).
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DISCUSSION |
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Although a vast spectrum of cytokines is induced and released in
asthma, some cytokines have been reported to be fibrogenic, such as
TGF- and GM-CSF (3, 8, 17, 25, 27, 30, 31, 36).
However, this is the first observation to demonstrate that GM-CSF
increased induction of TGF-
receptors T
R-I, T
R-II, and
T
R-III, as well as downstream signal transduction, as evidenced by
increases in pSmad2 and CTGF. The connective tissue effects of GM-CSF
mediated by TGF-
1 occurred without a detectable release of
biologically active TGF-
1. These findings are highly significant to
our understanding of the biology of TGF-
1. Because TGF-
1 is
secreted as a biologically latent protein, the most important mechanism
in the regulation of the effects of TGF-
1 is the conversion of
latent TGF-
to its biologically active form (19, 37). However, the current findings demonstrate that an increase in the
receptors to TGF-
can be equally important in regulating the effects
of TGF-
1 in the pathogenesis of fibrosis (22, 23). In
the context of airway remodeling, previous observations did not report
differences in expression of TGF-
1 in airways of asthmatics and
normal controls (2, 11, 32). However, the findings of
others do not take into account that biologically active TGF-
1 may
be released during ASMC injury (12) or that there may be induction of T
Rs by cytokines present in asthmatic airways
(11, 32). A cytokine reported to be released by BEC during
an episode of asthma is GM-CSF (26). On the basis of
current findings, the release of GM-CSF by bronchial epithelium, which
is in direct contact with ASMC, could then induce expression of
T
R-I, T
R-II, and T
R-III. Of equal importance is the
observation that GM-CSF increased the association of TGF-
1 with
T
R-III. When TGF-
1 is associated with T
R-III, the T
R-III
presents TGF-
1 to the signal-transducing complex composed of T
R-I
and T
R-II (5). It has been demonstrated that binding of
TGF-
to T
R-III enhances Smad2 phosphorylation, an indication of
TGF-
-mediated signaling (14). Collectively, the
findings demonstrate that enhanced association of TGF-
1 to ASMC
leads to induction of connective tissue synthesis. This then suggests
that in patients with asthma, the presence of GM-CSF can induce
fibrotic effects via a TGF-
1 pathway by increasing the expression of
T
Rs, despite the lack of apparent increase in TGF-
1 expression
(2). Alternatively, it is possible in the current model
that GM-CSF induced the release of active TGF-
1, but the quantity
was so low that it was not detectable by the assay used. However, since
there is an increase in T
R-I, T
R-II, and T
R-III on ASMC, these
cells are likely to be more responsive to the effects of TGF-
1 in
the CM even if the increase in the quantity is not detectable.
The use of corticosteroids in many asthmatics has been demonstrated to
relieve acute airway obstruction, and, when used chronically, corticosteroids inhibit irreversible airway obstruction attributed to
increased connective tissue deposition (30, 32). The
findings in the current study demonstrate that when dexamethasone is
present concomitantly with GM-CSF, there is a reduction of TR-I,
T
R-II, T
R-III, collagen I, and fibronectin expression.
Dexamethasone did not affect the increase in the association of
TGF-
1 with T
R-III induced by GM-CSF. However, in the same culture
conditions, pSmad2 and CTGF, both indicators of TGF-
-mediated
effects, were decreased. The later findings strongly suggest that the
effect of corticosteroids on connective tissue synthesis is the result of inhibiting GM-CSF regulation of T
R-I and T
R-II, despite a lack
of effect on the association of TGF-
1 with T
R-III.
The current findings demonstrate a unique interaction between two
totally different cytokines, GM-CSF and TGF-1, commonly found at
sites of injury and fibrosis (8, 13, 19, 20, 21, 23, 25, 29, 31,
33, 36, 37). For example, in models of pulmonary fibrosis
induced by the antineoplastic antibiotic bleomycin, there is increase
of both GM-CSF (1, 36) and TGF-
1 (20, 37).
Furthermore, after intratracheal administration of an adenovirus
carrying the gene for GM-CSF to rats, there was an increase in TGF-
1
in the bronchoalveolar lavage fluid (12). In those
instances where both GM-CSF and TGF-
1 are increased, the induction
of T
Rs by GM-CSF could lead to a synergistic effect of GM-CSF and
TGF-
1 on cells for connective tissue synthesis.
In conclusion, we have demonstrated that GM-CSF, a prevalent cytokine
released by BEC in asthma, stimulates confluent, prolonged, serum-deprived culture of ASMC to synthesize connective tissue proteins
by induction of TRs type I, II, and III and association of TGF-
1
with T
R-III. This effect of GM-CSF on ASMC is reversible by
corticosteroids, confirming the importance of using corticosteroids in
asthma to prevent irreversible airway obstruction.
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ACKNOWLEDGEMENTS |
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We thank Valerie Romanchuk for preparation of the manuscript.
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FOOTNOTES |
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The work presented in this paper was supported by a grant from the Canadian Institute of Health Research-Pharmaceutical Medical Association of Canada, GlaxoSmithKline.
Address for reprint requests and other correspondence: N. Khalil, Division of Respiratory Medicine, Dept. of Medicine, Univ. of British Columbia, Jack Bell Research Centre, Rm. 350, 2660 Oak St., Vancouver, BC V6H 3Z6, Canada (E-mail: nkhalil{at}interchange.ubc.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.
First published December 6, 2002;10.1152/ajplung.00091.2002
Received 26 March 2002; accepted in final form 26 November 2002.
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