1Division of Respiratory Medicine, Department of Medicine, The University of British Columbia, and Vancouver Hospital; 2Department of Surgery and Vancouver Hospital, Vancouver V6H 3Z6; 3Department of Pathology, British Columbia Cancer Agency, Vancouver V5Z 4E6, British Columbia; and 4School of Medicine, University of Miami, Miami, Florida 33136
Submitted 29 August 2002 ; accepted in final form 14 February 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
transforming growth factor-1; idiopathic pulmonary fibrosis
In contrast to interstitial lung diseases, like sarcoidosis or
hypersensitivity pneumonitis, lung biopsies from patients with IPF demonstrate
minimal inflammation, and little evidence exists that lesions of IPF are
preceded by inflammation (37,
38). Furthermore, IPF does not
respond to the current standard therapy of immunosuppressive agents such as
corticosteroids, cyclophosphamide, and azathioprine
(37,
38). Collectively, these
observations provide compelling evidence that inflammatory cells in IPF may
not be important in the pathogenesis of IPF, but the overproduction of
TGF-1 by epithelial cells may be critical to the fibrosis seen in IPF.
The mechanism by which epithelial cells are induced to release TGF-
1 is
not known. In this paper, sliced lung explants from normal rats, which were
relatively free of inflammatory cells, were cultured in the presence of the
retrovirus pMX. The AECs of the explants were successfully transfected with
the cDNA of TGF-
1, carrying a site-directed mutation where cysteines at
positions 223 and 225 were substituted with serines. This mutation results in
the secretion of biologically active TGF-
1
(8). This retrovirus was
designated as pMX-L-s223,225-TGF-
1, whereas the empty vector with no
TGF-
1 cDNA was designated as pMX. In lung explants cultured with pMX,
normal lung architecture was observed. However, 7 days after treatment with
pMX-L-s223,225-TGF-
1, there were increased quantities of active
TGF-
1inthe serum-free conditioned media (CM) overlying the explants. In
addition, 14 days after transfection, the lung explants histologically had
evidence of fibrosis, fibroblast buds, and enlarged air spaces resembling
remodeled lung, as seen in IPF. The same lung explants had increased synthesis
of collagens I, III, IV, and V and fibronectin by Western blot analysis.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of retroviral vectors. The plasmid pPK9A (gift from
Dr. Lalage Wakefield, Laboratory of Chemoprevention, National Institutes of
Health, Bethesda, MD) contained the entire length of the cDNA of LTGF-1
where cysteines in positions 223 and 225 were substituted with serines,
resulting in TGF-
1 protein in its biologically active form upon
secretion. This cDNA was designated as L-s223,225-TGF-
1. The 1.2-kb
TGF-
1 was isolated by BglII digestion and purified with a DNA
gel extraction kit (Qiagen, Mississauga, ON, Canada). The
L-s223,225-TGF-
1 was subcloned into the retrovector pMX. The retrovirus
containing the TGF-
1 gene was designated as pMX-L-s-223,225-TGF-
1,
whereas the retrovirus, pMX with no TGF-
1 cDNA, was designated as pMX.
The retroviruses were produced using the packaging cell line Plat E
(26), and viral titration was
estimated based on the number of infected NIH/3T3 cells using the method as
described by Kitamura
(26).
Animal procedures. Female Sprague-Dawley rats free of respiratory
disease and weighing 200-250 g were obtained from The University of British
Columbia Vivarium. All procedures on the rats were approved by the Canadian
Council of Animal Care. Rats were anesthetized with 0.4 ml of ketamine
(Biomedia-MTC, Cambridge, ON, Canada) and 0.2 ml of rompun (Bayer, Etobicoke,
ON, Canada), which were administered intraperitoneally. With blunt dissection,
the trachea was exposed, and an 18-gauge catheter was inserted. Thoracic and
abdominal cavities were exposed; the inferior vena cava and abdominal aorta
were severed. To remove peripheral blood leukocytes in the pulmonary
circulation, 10 ml of normal saline were injected into the right ventricle
until the lungs turned white. The trachea, lungs, and heart were removed. To
remove alveolar inflammatory cells, the lungs were lavaged with 50-60 ml of
warm normal saline through the trachea. The lungs were then infused with 5 ml
of 40°C 0.4% agarose DMEM [2x solution of serum-free DMEM and 0.8%
agarose solution (GIBCO) at 1:1 concentration at 40°C, supplemented with
hydrocortisone (0.2 µg/ml), retinol acetate (0.2 µg/ml), and 2%
insulin-transferrin-selenium X]. The trachea was closed by tying a thread, and
the lungs were placed into six-well plates. The plates were left on ice
overnight to further solidify the lungs. The lungs were separated from the
heart and gently sliced manually from each lobe with a sterilized scalpel.
Each pair of lungs yielded 40 slices that were 1-2 mm in thickness.
Culture of lung slices. Sterile gelform was made by adding 1.5 ml
of warm 0.4% agarose DMEM into six-well plates. After agarose DMEM was
solidified, a sterilized scalpel was used to remove gelform from the well. Six
lung slices were placed on the top of each gelform, and 1.5 ml of serum-free
DMEM supplemented with hydrocortisone (0.1 µg/ml), retinol acetate (0.1
µg/ml), and 1% insulin-transferrin-selenium X were added on the bottom of
the gelform. Six-well culture plates were incubated at 37°C, 5%
CO2. Medium was changed twice a week, and lung slices were turned
every other day and were collected on days 7 and 14 after
culture. Treatment of the lung slices consisted of media, KGF (25 ng/ml), pMX
[106 50% tissue culture infective dose (TCID50)], pMX
(106 TCID50) plus KGF (25 ng/ml), and
pMX-L-s223,225-TGF-1 (106 TCID50) plus KGF (25
ng/ml) in the absence or presence of anti-TGF-
1 antibody (0.1 µg/ml)
or fetuin (10 µM). The rationale for using fetuin is based on the
observations that for TGF-
1-mediated signal to occur, TGF-
1 must
first bind to the type II TGF-
receptor (T
R-II)
(45). After binding to the
T
R-II, T
R-II recruits and phosphorylates the type I TGF-
receptor before signal transduction by TGF-
(45). The major
cytokine-binding domain in the extracellular component of T
R-II is
within a 19- to 20-amino acid disulfide-looped sequence designated as
TGF-
receptor homology domain 1 (TRH1). Fetuin is a glycoprotein
synthesized by hepatocytes and found in serum
(10). The TRH1 has significant
homology with fetuin, and, therefore, fetuin can bind TGF-
1 and prevent
TGF-
1 from associating with T
R-II. The rationale for using KGF was
based on the following observations. Normally, AECs have a very low
proliferative index and are quiescent
(44,
47). Retroviruses only
transfect proliferating cells
(44). KGF, also referred to as
fibroblast growth factor-7 (FGF-7), is a heparin-binding growth factor
(44,
47). The critical
characteristic of KGF is that the biological activity of KGF is restricted to
epithelial cells because the KGF receptor is expressed only by epithelial
cells (44,
47). In vitro KGF has been
found to be a potent mitogen for type II AECs, and the effects are maximal 2
days after exposure. Selection of AECs as target cells for retroviral gene
transfection was achieved by using KGF to induce AEC proliferation.
For histology, lung slices were fixed in 4% paraformaldehyde for 24 h and
then 70% ethanol before paraffin embedding, staining with hematoxylin and
eosin (H&E) for histology, and Masson's trichrome for distribution of
collagen and elastin (14). All
slides were blinded, and two independent examinations were done and then
collated. For the extent of lung involvement, the lung sections were examined
under low power, revealing the entire lung section that contained normal
looking, as well as fibrotic, lesions. The proportion of lung section stained
green with Masson's trichrome was recorded as a percentage of the overall lung
section. For grading the extent of staining with Masson's trichrome, 0
designated no staining, grade 1 designated detectable color (see
Fig. 2C for an example
of grade 1), and grade 2 designated more green staining and
was between staining grades 1 and 3 (see
Fig. 2C, changes seen
in the right-hand corner, for an example of grade 2 staining).
Grade 3 represented extensive staining with Masson's trichrome (see
Fig. 2, D-G, for an
example of grade 3 staining). The overall score was calculated by
multiplying the percentage of lung involved with the grade.
Immunohistochemistry using anti--smooth muscle actin antibody was done
to identify interstitial fibroblasts or myofibroblasts
(33). Some lung slices from
each condition were used for in situ hybridization, whereas other slices were
collected for protein extraction and Western analysis. CM overlying the
explants were collected in the presence of protease inhibitors, leupeptin (0.5
µg/ml; Amersham, Buckinghamshire, UK), and aprotinin and pepstatin A (1
µg/ml each; both from Sigma, Oakville, ON, Canada), and frozen at -80°C
until ready for TGF-
1 quantitation by ELISA.
|
In situ hybridization to localize L-s223,225-TGF-1.
Tissue sections from paraffin-embedded lung explants were de-waxed in xylene,
rehydrated in a series of graded ethanol concentrations, and treated with
proteinase K solution (20 µg/ml; Boehringer Mannheim, Mannheim, Germany) in
50 mM Tris · HCl, pH 7.4, 10 mM EDTA, and 10 mM NaCl. After recovery of
the puromycin resistance cDNA, the 1.756-kb insert was labeled with
digoxigenin. After preparation of the probe cocktail, 30 µl were
distributed over each section and covered with a coverslip. The negative
control followed the same steps as above, but the labeled probe was replaced
with unlabeled probe. After denaturation of the DNA by heating the slides at
95°C, DNA-DNA hybridization was performed by incubation of slides in a
hybridization oven at 42°C overnight. The anti-digoxigenin-Ap antibody
(Fab fragment from sheep) at 1:500 was applied to the section, which was then
cultured in a humid chamber. Thirty microliters of color solution (4-nitro
blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate) were applied
to the slides, which were left in the dark overnight and stained with 0.5%
neutral red (1% acetic acid).
Quantitation of retrovirus-mediated gene transfection in lung
explants. To determine the approximate transfection efficiency of the
L-s223,225-TGF-1 construct, retrovirus-mediated enhanced green
fluorescent protein (EGFP) gene transfection was performed on lung explants.
Plat E cells (500,000) were cultured with DMEM containing 10% FCS in a 60-mm
dish. Fifty to eighty percent subconfluent Plat E cells were transfected with
pMX-EGFP by Lipofectin (GIBCO). The pMX-EGFP retroviral culture supernatants
were collected after 48 h of culture. The estimated titers of retroviruses
were 1-10 x 106 colony-forming units/ml. Lung slices were
cultured on top of agarose gel in the absence or presence of 1 x
106 TCID50 pMX-EGFP virus. KGF was used to direct the
transfection of pMX-EGFP into AECs. Medium was changed twice a week, and the
lung slices were harvested on day 14. The lung tissue sections placed
on slides were examined by fluorescence microscopy. Five areas per section
were randomly selected for the calculation of the percentage of EGFP
expression cells in the lung explants.
Transfection of pMX retroviruses into alveolar inflammatory cells.
Despite our efforts to remove inflammatory cells from the lungs, it is
recognized that all inflammatory cells from the lungs cannot be totally
eliminated in our model. It is, therefore, possible that inflammatory cells
remaining in the explants could be transfected by the
pMX-L-s223,225-TGF-1 gene in the conditions used. To determine whether
alveolar cells could be transfected with the pMX-L-s223,225-TGF-
1 gene,
the cells in the BAL obtained earlier were cultured with KGF (25 ng/ml) and
pMX-L-s223,225-TGF-
1 (106 TCID50) in
puromycin-selective media. The presence of pMX-L-s223,225-TGF-
1/KGF are
conditions hypothesized to be ideal for selective transfection of AECs. The
plates were examined daily for 14 days for viable cells, and CM were collected
5, 7, and 14 days after culture for detection of TGF-
1 by ELISA. A
positive control for successful transfection with pMX-L-s223,225-TGF-
1
was done by using L2 cells. L2 cells are a simian virus 40 transformed cell
line of normal rat AECs (18)
that were cultured in identical conditions as were the alveolar inflammatory
cells.
Detection of TGF-1 in CM. TGF-
1 protein
quantitation was done using a commercially available ELISA kit that only
detects biologically active TGF-
1
(7). Neutral CM expected to
contain biologically active TGF-
1 were used
(19,
24,
48). However, an aliquot of
the same sample was acidified to remove the LAP-1 from any LTGF-
1
present. The sample was neutralized, and the TGF-
1 content of the
previously acidified CM was quantitated in the same ELISA assay to determine
the total TGF-
1 present in each sample.
Western analysis to detect and quantitate connective tissue proteins, phosphorylated Smad2, and connective tissue growth factor. The lung explants were snap-frozen on dry ice with ethanol and stored at -80°C until protein extraction. Lung explant protein extraction was performed as described previously (48). Briefly, the frozen lungs were pulverized in a chilled mortar and placed in tissue lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (Sigma). The samples were further homogenized in the presence of 0.5% Triton X-100 and then centrifuged at 15,800 g for 10 min at 4°C. The supernatants were collected, and protein levels were determined using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). The protein samples (25 µl) were electrophoresed on 10% SDS-PAGE in a Mini-Protean II Electrophoresis Cell (Bio-Rad). Protein molecular weight markers (Amersham) were run parallel to each blot as an indicator of the molecular weight. Equal loading of protein was evaluated using silver staining (not shown). It is of note that an additional method to silver staining was done to validate equal loading of protein on SDS-PAGE gels using Ponceau S staining solution (Sigma) or Coomassie brilliant blue (Sigma) staining. The separated proteins were transferred at 50 V overnight onto nitrocellulose membrane (GIBCO BRL) in a Mini Trans-Blot chamber with transfer buffer (25 mM Tris · HCl, 192 mM glycine, and 20% methanol). The nitrocellulose membrane was blocked for 1 h using 5% instant skim milk in Tris-buffered saline (TBS). For detection of procollagen I and III (Rockland, Gilbertsville, PA), a 1:3,000 dilution of antibody was used; for collagen IV (Rockland), a dilution of 1:2,500 antibody was used; for collagen V (Cedarlane, Hornby, ON, Canada), a dilution of 1:3,000 antibody was used. A 1:1,000 dilution was used for fibronectin, 1:750 for phosphorylated Smad2 (Upstate Biotechnology, Lake Placid, NY), and 1:500 dilution of connective tissue growth factor (CTGF). After being washed, the nitrocellulose membrane was incubated with horseradish peroxidase linked with the secondary antibody (anti-rabbit or anti-goat immunoglobulin G; Bio-Rad), as recommended by the manufacturer. Finally, the washed blots were exposed to an enhanced chemiluminescence (ECL) detection system (Amersham) and recorded on an autoradiograph (Kodak X-Omat film). Before being reprobed, the nitrocellulose membrane was incubated at 50°C for 30 min with a stripping buffer (100 mM 2-mercap-toethanol, 2% SDS, and 62.5 mM Tris · HCl, pH 6.7). The blots were rinsed twice with TBS. To ensure the removal of antibodies, membranes were incubated with the ECL detection reagents and exposed to film (Kodak). No band was detected, confirming that all antibodies were stripped off the membrane. The same nitrocellulose membrane was blocked using 5% instant skim milk in TBS for detection of the other collagens and fibronectin. Relative absorbance was determined using the Quantity I imaging system (Bio-Rad).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Release of TGF-1 by cells of explants. We next
determined whether transfected cells of the explants release biologically
active TGF-
1. Seven days after explantation and treatment, serum-free CM
overlying the explants were examined for the presence of active TGF-
1
using an ELISA. The CM obtained from lung explants cultured with
L-s223,225-TGF-
1/KGF contained increased quantities of TGF-
1. In
the presence of antibody to TGF-
1, all the TGF-
1 activity in the
CM was neutralized, confirming the specificity of the ELISA. Fetuin binds to
TGF-
1 and prevents the association of TGF-
1 with T
R-II and
does not allow TGF-
to interact with T
R-II. For this reason, in
the presence of fetuin, the quantity of TGF-
1 in the CM was increased
(Table 2). No TGF-
1 was
detected in the CM of explants of control conditions consisting of explants
receiving no treatment, the retrovirus without the TGF-
1 gene, pMX, KGF,
or pMX plus KGF. These findings confirm that cells in the explants treated
with pMX-L-s223,225-TGF-
1/KGF release biologically active TGF-
1.
Total TGF-
1 generated by the explants did not vary significantly among
the various treatments. Fourteen days after explantation and treatment, the CM
from pMX-L-s223,225-TGF-
1/KGF in the absence or presence of fetuin
contained small quantities of active TGF-
1. Because all other evidence
(see Figs. 1,
2,
3,
4,
5) indicated a biological
response to TGF-
1, the decreased detection of TGF-
1, 14 days after
transfection, does not minimize the significance of our findings.
|
|
|
|
Alveolar inflammatory cells are not transfected by
L-s223,225-TGF-1. In the current model of pulmonary
fibrosis, alveolar inflammatory cells were removed by extensive BAL. The
pulmonary vasculature was flushed by forcefully injecting normal saline into
the right ventricle while the inferior vena cava and aorta were severed. This
method is likely to remove not all but a large number of inflammatory cells
from the alveoli and vasculature of the lungs. To determine whether
inflammatory cells remaining in the lungs could be transfected with
pMX-L-s223,225-TGF-
1, cells obtained by BAL were used. These
inflammatory cells were cultured with pMXL-s223,225-TGF-
1/KGF,
conditions that have been demonstrated to be optimum for transfection of the
TGF-
1 gene into AECs of lung explants
(Fig. 1; Tables
1 and
2). Because we had previously
demonstrated that a cell line of AECs called L2 could be transfected by
pMX-L-s223,225-TGF-
1
(46), L2 cells were used as a
positive control. It is of note that the pMX vectors contain the gene for
puromycin resistance, and only cells successfully transfected with the vector
will survive in media containing puromycin. Inflammatory cells did not survive
in selected media containing puromycin (1 µg/ml; not shown), and there was
no evidence of biologically active TGF-
1 in the CM
(Table 3). However, L2 cells
treated with pMX-L-s223,225-TGF-
1/KGF, previously demonstrated to
secrete active TGF-
1 in these conditions
(46), remained viable in
puromycin-selective media (not shown) and released active TGF-
1
(Table 3). These findings, in
addition to the absence of puromycin labeling in inflammatory cells
(Fig. 1), confirm the selective
nature of transfection of the L-s223,225-TGF-
1 gene into AECs without
transfecting any inflammatory cells that remain in the explants.
|
Transfection of AECs with L-s223,225-TGF-1 leads to
pulmonary fibrosis and remodeling. Because lung sections stained with
Masson's trichrome demonstrated the histology as well as distribution of
collagens, the histology presented is that observed with Masson's trichrome.
H&E-stained sections are not shown. All slides were blinded, and two
independent examinations were done and collected. All fields of all lung
sections were examined. Lung explants cultured in media alone
(Fig. 2A) or pMX
(Fig. 2B) retained a
normal alveolar architecture. Lung explants cultured with KGF demonstrated a
previously described characteristic of micropapillary epithelial cell
hyperplasia or "knobby proliferation"
(Fig. 2C)
(44,
47). The knobby appearance has
been correlated with AEC proliferation
(Fig. 2C)
(44,
47). Unlike the normal lung in
which type II AECs are 4% of the alveolar surface
(44,
47), in these areas, all
alveoli appeared to be hyperplastic type II AECs and resembled a cuboidal
epithelial monolayer. The distribution of these changes was patchy, being
present primarily in the periphery of the lung tissue. Lung explants cultured
with media, pMX, or KGF did not demonstrate collagen. Lung explants cultured
with L-s223,225-TGF-
1 in the presence of KGF, designated as
pMX-L-s223,225-TGF-
1/KGF for brevity, demonstrated a patchy distribution
of AEC hyperplasia and interstitial thickening on H&E (not shown) and
significant distribution of collagen by Masson's trichrome
(Fig. 2, D-G). The
histological changes were observed throughout the explants treated with
pMX-L-s223,225-TGF-
1/KGF. However, the severity of changes was observed
primarily on the surface, as deeper cuts into the paraffin-embedded lung
section led to a loss of histological changes, resembling remodeled lung.
These features suggest that the surface of the explants was most accessible to
the retroviral vector and thus transfection. Masson's trichrome demonstrated
extensive collagen deposition in areas of thickened interstitium
(Fig. 2, D-G). In some
sections, fibroblasts were observed (Fig.
2, D-G). Fibroblast buds were also found
(Fig. 2G), which were
lined with hyperplastic type II AECs (small arrow), and extensive fibrosis was
observed subepithelially and within the bud
(Fig. 2G, large
arrow). In another section, evidence of enlarged air spaces reminiscent of
honeycomb cysts was also seen (Fig. 2,
E and F, arrowheads). The relative presence of
collagen, as assessed by the extent of green coloration, was most evident in
explants cultured with KGF and pMX-L-s223,225-TGF-
1 14 days after
transfection. The presence of anti-TGF-
1 antibody seemed to have little
effect on the expression of collagen (Table
4). The total absence of detectable collagen in explants treated
with KGF plus pMX relative to the other control conditions is unclear. It is
of interest that in regions where Masson's trichrome staining was observed,
the overlying alveolar epithelium was hyperplastic in morphology, suggestive
of proliferation (Fig. 2,
D-G, small arrows). Induction of proliferation of AECs
would make these AECs susceptible to pMX-mediated transfection. In the normal
lung, interstitial fibroblasts are not visible. TGF-
induces fibroblast
proliferation, fibroblast recruitment, and differentiation to myofibroblasts,
as well as connective tissue synthesis
(17,
30). In lung sections
receiving no treatment or cultured with pMX
(Fig. 3A), KGF, or pMX
plus KGF (not shown), there was no evidence of immunohistostaining with
-smooth muscle actin. In explants cultured with
pMX-L-s223,225-TGF-
1/KGF, there were areas of substantial connective
tissue with elongated cells that were likely to be fibroblasts. In these
areas,
-smooth muscle actin stained a number of cells within the
thickened interstitium (Fig.
3B, arrows), indicating that the effects of TGF-
1
on interstitial cells can lead to the development of myofibroblast, a cell
phenotype that has more contractile properties than fibroblasts
(14,
17,
33,
36). These changes were not
observed in lung explants receiving no treatment or in explants treated with
KGF, pMX, or KGF plus pMX. Lung sections obtained from explants cultured with
pMX-L-s223,225-TGF-
1/KGF and anti-TGF-
1 antibody or fetuin had
less fibrosis and the minimal number of cells that immunostained with
-smooth muscle actin (not shown). It is of note that examination of
explants on days 7 and 14 showed no evidence of tissue
necrosis by light microscopy. Staining of explants with trypan blue also
demonstrated no evidence of cell necrosis (data not shown).
|
Expression of connective tissue proteins in lung explants. In
fibrotic lesions such as those seen in pulmonary fibrosis, there is increased
synthesis of collagens I, III, and fibronectin, whereas collagen IV expression
is associated with basement membrane synthesis, and collagen V expression is
seen as part of provisional matrix
(11). To verify and quantitate
connective tissue synthesis, proteins from explants were immunoblotted for
collagens I, III, IV, V, and fibronectin. Collagen I and fibronectin
expression was observed in all experimental conditions, but the expression of
these proteins was elevated in lung explants cultured with pMX-L-s233,
225-TGF-1/KGF (Fig. 4).
In the presence of neutralizing antibody to TGF-
1 or fetuin, there was a
decrease in expression of connective tissue proteins
(Fig. 4, lanes 6 and
7, respectively).
Cells of lung explants respond to TGF-1 by expressing
phosphorylated Smad2 and CTGF. An index of target cell response to
TGF-
is the phosphorylation of Smad proteins that mediate intracellular
signals of the TGF-
1 superfamily
(35), more specifically, the
phosphorylation of the COOH-terminal SSXS motif of Smad2 and Smad3
(35). The phosphorylated Smad2
(p-Smad2) antibody is highly specific and sensitive in detecting
TGF-
1-dependent signaling
(35). To confirm that the
TGF-
1 released in lung explants regulates signal transduction, proteins
from the explants were immunoblotted with anti-phosphorylated Smad2 antibody.
Explants receiving no treatment had detectable constitutive expression of
p-Smad2 (Fig. 5, lane
1). The presence of pMX increased the expression of p-Smad2, suggesting
that some nonspecific induction of a TGF-
1-mediated signal occurred in
the presence of pMX. However, there was a more marked increase in p-Smad2 when
pMX-L-s233,225-TGF-
1/KGF was in cultures of the lung explants. The
presence of neutralizing antibody to TGF-
1 did not significantly
decrease the expression of p-Smad2, whereas the presence of fetuin resulted in
some decrease of p-Smad2 expression.
The induction of connective tissue synthesis may occur in response to a
number of cytokines, such as IL-1, PDGF, IGF-1, and FGF-2
(2,
32,
34,
43). Many of these cytokines
are induced by TGF-
(28). Induction of connective
tissue synthesis by TGF-
1, but not other fibrogenic cytokines, is
mediated by CTGF (16,
30). CTGF is a 33- to 38-kDa
cysteine-rich protein that also regulates fibroblast proliferation and
chemotaxis (16,
30). To confirm that
connective tissue synthesis observed in lung explants was mediated by
TGF-
1, the proteins were immunoblotted with anti-CTGF antibodies.
Constitutive CTGF expression was observed in control samples
(Fig. 5, lanes 1-4),
but explants cultured with pMX-L-s233,225-TGF-
1/KGF had a significant
increase in CTGF (Fig. 5,
lane 5). In the presence of neutralizing antibodies to TGF-
1 or
fetuin, CTGF expression was decreased (Fig.
5, lanes 6 and 7, respectively). These findings
confirm that when the TGF-
1 is released by the AECs of the explant, and
it interacts with its T
R-II, then there is induction of connective
tissue synthesis by the cells in the explant. Lastly, the presence of insulin
and KGF in the defined media may be considered components of inflammation.
However, the presence of insulin and KGF without transfection with the
retrovirus pMX-L-s223,225-TGF-
1 had no effect on the structural
integrity of the tissue by light microscopy, increase in the release of
TGF-
1 (Table 2), or
connective tissue synthesis (Fig.
4). In the absence of pMX-L-s223,225-TGF-
1, there was also
no evidence of increase in responsiveness to TGF-
1 by induction of
p-Smad2 and CTGF (Fig. 5).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mechanism by which AECs overproduce TGF-1 in IPF is unclear.
Early lesions of IPF have changes in the alveolar epithelium, suggestive of
injury (12,
38). In addition, the presence
of fibroblast foci is also speculated to be important as the initial lesion in
the pathogenesis of IPF and is a criterion for the diagnosis of usual
interstitial pneumonitis of IPF
(12,
38). Fibroblast foci are small
aggregates of actively proliferating myofibroblasts and fibroblasts and are
hypothesized to be present at sites of AEC injury where there may be
inadequate reepithelialization
(15,
37). The lack of epithelial
cells, in turn, leads to induction of the underlying fibroblast and
myofibroblast proliferation and connective tissue synthesis
(15,
37). Fibroblast foci were not
observed in our lung sections, and this may be because, in this model, AECs
were not injured by pMX or KGF. Although no fibroblast foci were observed,
there were cells in the interstitium that resembled fibroblasts and
protrusions of misshapen lung tissue that resembled lesions, referred to as
fibroblast buds (22).
Furthermore, the presence of KGF has been previously demonstrated to protect
AECs from various injuries
(42,
44,
47). The lack of injury in
this model does not diminish the relevance of the observation that in the
event of AEC release of active TGF-
1, rapid pulmonary remodeling will
follow. It should be noted that even though fibroblasts are the main source of
connective tissue, epithelial cells can also synthesize a variety of
connective tissue proteins
(13). Because TGF-
1 has
been demonstrated to induce epithelial cells to synthesize collagens
(13), it is then possible that
the enhanced connective tissue synthesis observed in the explants may be due
to the effects of TGF-
1 on the AECs as well as underlying
fibroblasts.
The findings from the current model do not support inflammatory cells as
being important in the pathogenesis of pulmonary fibrosis. This is because
inflammatory cells in the pulmonary vessels and alveoli were removed from the
lung before slicing the lung for ex vivo culture. Maintaining viable lung
tissue in culture also overcame the contribution of circulating leukocytes
that could be recruited to the interstitium and alveoli. Furthermore, in situ
hybridization demonstrated successful transfection of
pMX-L-s223,225-TGF-1 into AECs and not inflammatory cells. Lastly, the
lack of viability of rat alveolar inflammatory cells in puromycin-selective
media demonstrates that even if alveolar inflammatory cells, such as
macrophages, were present in the explants, they would not be transfected by
the TGF-
1 gene and contribute to the generation of TGF-
1 in the
explants. Although it is not likely that the inflammatory cells remaining in
the lung were transfected by pMX-L-s225,225-TGF-
1, these cells may
respond to the AEC-derived TGF-
1 by generating a variety of cytokines,
some of which may be fibrogenic in nature. The effects of fibrogenic cytokines
from residual pulmonary inflammatory cells cannot be excluded in the current
model. Despite these considerations, there is a selective nature of AECs as a
source of biologically active TGF-
1 in this model, and it confirms the
role of AECs as being important in the pathogenesis of pulmonary fibrosis and
remodeling.
Observations of others also support the importance of AECs in the
pathogenesis of pulmonary fibrosis. For instance, Munger et al.
(31) demonstrated that the
expression of the integrin v
6 on AECs after lung injury could
result in activation of LTGF-
1 and consequent pulmonary fibrosis.
Adamson et al. (1) observed
that under hyperoxic conditions, there was fi-brosis in areas of AEC necrosis
in lung explants that were free of peripheral blood. There is additional
support for the importance of epithelial cells in the pathogenesis of
pulmonary fibrosis in IPF. In transgenic mice, when human TGF-
was
overexpressed by pulmonary epithelial cells, there was pulmonary fibrosis
without evidence of changes in the number of inflammatory cells in the lungs
(29,
41). It is of interest that in
another rat model of pulmonary fibrosis described by Sime et al.
(40), when the adenovirus with
L-s233,225-TGF-
1 was administered intratracheally, there were increased
quantities of TGF-
1 in fluid retrieved by BAL. In addition, there was
extensive pulmonary fi-brosis
(40). Because adenoviruses can
transfect many types of cells, it is possible that inflammatory as well as
structural cells may have been induced to express TGF-
1 in this study
(40). The findings of Sime et
al. are valuable in confirming that increased quantities of TGF-
1 in the
lung can lead to fibrosis (40)
but do not identify the cellular source of TGF-
1 release. Unlike the
findings of Sime et al. (40),
the findings from the current study demonstrate the role of TGF-
1
release from AECs as being important in the pathogenesis of pulmonary
fibrosis.
To confirm that the increase in connective tissue synthesis was mediated by
TGF-1 generated by the cells of the explant, a number of means were
undertaken. In the presence of neutralizing antibody to TGF-
1 or fetuin,
which is a competitive ligand for the T
R-II
(10), there was a decrease in
connective tissue synthesis. The TGF-
1 antibody or fetuin was not able
to block connective tissue synthesis completely. This could be because the
antibody or fetuin could not adequately reach all areas of the explants that
may have been transfected with pMX-L-s223,225-TGF-
1 or were responding
to TGF-
1. Alternatively, a significant amount of TGF-
1 could
already have been associated with the receptor by the time that the
TGF-
1 antibody or fetuin was added. Further confirmation that the
connective tissue responses were due to TGF-
1 was the expression of
p-Smad2 and CTGF. Induction of p-Smad2, an intracellular enzyme, was increased
in conditions where there was an induction of TGF-
1. Under conditions
meant to neutralize TGF-
1 using TGF-
1 antibody or fetuin, which
would interfere with TGF-
1 binding to its receptor, p-Smad2 was
decreased only in the presence of fetuin but not anti-TGF-
1 antibody. It
is possible that the overproduction of TGF-
1 could have resulted in the
release of other TGF-
isoforms, which may then have phosphorylated
Smad2. However, fetuin, which mimics the TRH1, is not specific to inhibiting
the effects of TGF-
1 but may interfere with the binding of other
TGF-
isoforms to the T
R-II and thus phosphorylation of Smad2.
Lastly, CTGF is critical as an intermediary protein for the synthesis of
connective tissue proteins by TGF-
1 but not other fibrogenic cytokines
(16,
30). Fibroblasts and
epithelial cells respond to TGF-
1 by expressing CTGF which, in turn,
results in collagen synthesis
(12,
36). Collagen synthesis
mediated by cytokines other than TGF-
1 does not induce CTGF
(12,
36). Explants cultured with
pMX-L-s223,225-TGF-
1 had marked increases in CTGF, whereas the presence
of TGF-
1 antibody and fetuin suppressed CTGF expression. These findings
confirm that TGF-
1 generated by the cells of the explant results in
connective tissue synthesis. Although the current model results in successful
transfection of AECs with the TGF-
1 gene followed by release of
biologically active TGF-
1 and a connective tissue response, there are
some limitations in this model. These limitations are the patchy nature of
successful transfection, the limited number of days the tissue can be
maintained in culture, and the reduction of TGF-
1 activity in
conditioned media after 7 days. The reduction in TGF-
1 activity may have
been due to a deterioration of the TGF-
1 secreted, internalization of
TGF-
1 by the cells of the explants, attachment of TGF-
1 to the
TGF-
-binding proteins present on the cells of the explant, or adherence
of TGF-
1 to the plastic of the culture dishes
(48). It is possible that
despite a stable transfection, there may have been a decrease in the synthesis
of TGF-
1bythe AECs. Alternatively, it is possible that although there
was no histological evidence of a decrease in viability of AECs, there may
have been AEC dysfunction after 14 days of transfection. This could lead to a
decrease in the release of active TGF-
1.
The clinical implications of these findings are highly significant. The
previously held dogma described the pathogenesis of pulmonary fibrosis in IPF
to start with a pulmonary injury, followed by recruitment and activation of
inflammatory cells that release proinflammatory and fibrotic cytokines such as
TGF-1, PDGF, IGF-I, FGF-2, IL-1, granulocyte/macrophage
colony-stimulating factor, and TNF-
, leading to chronic inflammation
and fibrosis (15,
22-24,
37). On the basis of this
premise, the treatment for IPF and many other progressive inflammatory and
fibrotic diseases has been the use of anti-inflammatory agents such as
corticosteroids, azathioprine, and cyclophosphamide
(15,
22-24,
37). An example where injury
and inflammation precede fibrosis is the most common animal model of pulmonary
fibrosis induced by the anti-neoplastic antibiotic bleomycin
(20,
21,
25,
48,
49). Bleomycin-induced lung
toxicity is characterized by AEC injury followed by recruitment and activation
of inflammatory cells before enhanced connective tissue synthesis
(20,
21,
25,
48,
49). In addition, we have
demonstrated that after bleomycin treatment, the administration of high doses
of corticosteroids prevents the recruitment of inflammatory cells into the
injured lung (25), and there
is a decrease in collagen synthesis
(39). However, the
distribution of TGF-
1 in its biologically active conformation in IPF
lungs is not the same as in bleomycin-induced pulmonary fibrosis (BPF)
(20,
22,
23,
48). We have demonstrated that
after bleomycin administration, the biologically active form of TGF-
1 is
almost exclusively expressed by alveolar macrophages
(20). The secretion of
biologically active TGF-
1 by alveolar macrophages is critical to the
pathogenesis of pulmonary fibrosis after bleomycin administration
(21,
25,
48,
49). Unlike BPF in lungs of
patients with IPF, biologically active TGF-
1 is expressed by AECs and
epithelial cells lining honeycomb cysts and fibroblast buds
(22-24).
In addition, fluid lining alveolar spaces where the expression of TGF-
1
in epithelial cells is highly expressed contained active TGF-
1
(24). These findings suggest
that in IPF lungs, epithelial cells release TGF-
1. We have also
demonstrated that high doses of corticosteroids do not alter the secretion of
TGF-
1 (25). Furthermore,
in all the explant cultures, the media were supplemented with 0.2 µg/ml of
hydrocortisone. The presence of corticosteroids had no effect on the
generation of active TGF-
1 or connective tissue synthesis, further
confirming that in instances of increased TGF-
1 production, the presence
of corticosteroids is not of benefit. It must be recognized that epithelial
cells are structural cells, and their numbers cannot be altered by any
immunosuppressive agents, and if steroids do not decrease secretion of
biologically active TGF-
1, then this may explain the failure of standard
immunosuppressive therapy for IPF and other progressive fibrotic diseases. It
is then possible that progressive fibrotic diseases like IPF
(15,
22-24,
37), cirrhosis of the liver
(3), or glomerulonephritis
(4) are disorders of epithelial
cells and not inflammatory cells. If this is the case, as our study suggests,
then treatment of progressive fibrotic diseases warrants development of
therapeutic modalities that would interfere with the generation of TGF-
1
or the effects of biologically active TGF-
1.
![]() |
DISCLOSURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|