Laboratories of 1 Pulmonary Pathobiology, 2 Experimental Pathology, and 3 Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
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ABSTRACT |
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Platelet-derived growth factor (PDGF) is a potent
mitogen for mesenchymal cells. Induction of the PDGF receptor-
(PDGF-R
) in vitro enhances PDGF-induced mitogenesis and chemotaxis.
Thus we investigated whether the PDGF-R
is induced in vivo during pulmonary fibrogenesis using a vanadium pentoxide
(V2O5)
model of lung injury. PDGF-R
mRNA expression was induced 24 h
postinstillation. PDGF-R
mRNA was constitutively expressed and did
not increase. Western blotting showed upregulation of PDGF-R
protein
by 48 h, and immunohistochemical analysis localized PDGF-R
primarily in mesenchymal cells residing within fibrotic lesions. Upregulation of
PDGF-R
in vivo preceded mesenchymal cell hyperplasia (3-7 days)
and collagen deposition by day 15.
Supernatants from alveolar macrophages treated with
V2O5
in vitro released upregulatory activity for PDGF-R
on cultured lung
myofibroblasts, and this activity was blocked by the
interleukin-1-receptor antagonist. These data suggest that
interleukin-1
-mediated induction of PDGF-R
in vivo is important
to lung myofibroblast hyperplasia during fibrogenesis.
lung fibrosis; vanadium pentoxide; platelet-derived growth
factor-receptor system; interleukin-1; transforming growth factor-1
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INTRODUCTION |
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THE PROLIFERATION of fibroblasts in the lung is a key
component of lung fibrosis. The disease may be initiated by exposure to
a variety of factors including chemotherapeutic drugs such as bleomycin
(27, 30), man-made fibers such as asbestos (4, 5), and metals such as
vanadium and cadmium (10, 25). Myofibroblast hyperplasia is an early
event in the fibrotic process and generally precedes extracellular
matrix deposition (21). The factors that initiate and perpetuate the
myofibroblast growth response during fibrosis have not been fully
clarified. However, several growth-promoting cytokines are increased
within bronchoalveolar lavage fluid during the early stages of
pulmonary fibrogenesis, and these agents include platelet-derived
growth factor (PDGF), interleukin-1 (IL-1
), tumor necrosis
factor-
, transforming growth factor-
(TGF-
), and insulin-like
growth factor-1 (for a review see Ref. 16).
PDGF is a major mitogen and chemoattractant for cells of mesenchymal
origin, including fibroblasts, myofibroblasts, and smooth muscle cells
(reviewed in Ref. 13). Several studies have emphasized the importance
of PDGF in fibroproliferative diseases such as idiopathic pulmonary
fibrosis (1), obliterative bronchiolitis (14), and atherosclerosis (26)
in humans. Mesenchymal cells produce PDGF-AA, whereas macrophages
secrete primarily PDGF-AB and PDGF-BB (6). PDGF isoforms stimulate cell
replication and chemotaxis by interacting with cell-surface receptor
subtypes termed PDGF receptor- (PDGF-R
) and PDGF-R
(28). PDGF
binding initiates PDGF-receptor dimerization that results in
autophosphorylation of tyrosine residues within the intracellular
domain of the receptor (13). PDGF-AA binds to PDGF-R
and does not
bind to PDGF-R
(28). The expression of PDGF-R
on adult
fibroblasts is relatively low compared with the normally abundant
PDGF-R
, yet PDGF-R
is highly expressed during development (23). A
recent study (7) with PDGF-A-deficient mice demonstrated that
PDGF-AA and its receptor (PDGF-R
) are essential for the development
of myofibroblasts in the lung, and surviving offspring develop a fatal
emphysema due to reduced elastin deposition. PDGF-R
is necessary for
maximal mitogenic and chemotactic responses to PDGF (8, 20, 24, 29),
and this is likely due to unique signaling events mediated via the
heterodimeric
-receptor complex compared with the
-receptor complex (29).
Upregulation of the PDGF-receptor system has been proposed as a
mechanism of mesenchymal cell hyperplasia in fibroproliferative diseases other than lung fibrosis. Animal models of disease associated with induction of PDGF-R include liver fibrosis (31) and
atherosclerosis (26). In contrast, induction of PDGF-R
has been
reported in vascular hypertension in rats (17). In vitro, we have
reported that PDGF-R
on rat lung myofibroblasts is upregulated by
IL-1
(20), basic fibroblast growth factor (2), lipopolysaccharide (8), and asbestos fibers (5). Induction of PDGF-R
by asbestos fibers
(5) could be due to surface-bound endotoxin (8). TGF-
1
downregulates PDGF-R
and suppresses PDGF-mediated responses (3).
Alveolar macrophages stimulated with fibrogenic particles in vitro
release an upregulatory factor(s) for PDGF-R
on lung myofibroblasts,
and this activity is due mainly to IL-1
(19). Induction of the
PDGF-R
subtype in vitro enhances PDGF-mediated mitogenesis and
chemotaxis of lung myofibroblasts (8, 20, 24).
The induction of PDGF-R has not been studied in vivo during the
progression of pulmonary fibrogenesis. We investigated possible alterations of the PDGF-receptor system in a rat model of pulmonary fibrosis induced by vanadium pentoxide
(V2O5).
Occupational exposure to
V2O5
is common in petrochemical industries (18), and vanadium-containing emission dusts released from oil-fired, electricity-generating plants
contribute to environmental exposure in humans (9). We previously
reported that a V2O5-containing emission
dust caused PDGF-R
upregulation 24 h postinstillation in rats
(19).
V2O5 elicited a fibrogenic response as measured by trichrome staining within
15 days after instillation. Upregulation of PDGF-R
in vivo peaked
during the early proliferative phase (24-48 h) after lung injury
but returned to control levels before collagen deposition within
fibrotic lesions. Macrophages treated in culture with
V2O5 released a factor(s) that upregulated PDGF-R
on myofibroblasts in
vitro, and the majority of this upregulatory activity was blocked by
the IL-1-receptor antagonist protein (IRAP). We postulate that induction of PDGF-R
through an IL-1
-dependent mechanism could be
important to mesenchymal cell hyperplasia in vivo during lung fibrogenesis.
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METHODS |
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Reagents.
V2O5
was purchased from Aldrich Chemical (Milwaukee, WI). Human recombinant
PDGF-AA, rabbit anti-murine PDGF-R, and rabbit anti-human PDGF-R
were purchased from Upstate Biotechnology (Lake Placid, NY).
125I-PDGF-AA was purchased from
Biomedical Technologies (Stoughton, MA). Human recombinant IL-1
and
human recombinant IRAP were purchased from R&D Systems (Minneapolis,
MN). Fetal bovine serum (FBS; heat inactivated) and bovine serum
albumin (BSA; Cohn fraction V) were purchased from Sigma Chemical (St.
Louis, MO). The human PDGF-R
cDNA probe was kindly provided by Dr.
Carl-Henrik Heldin (Ludwig Institute for Cancer Research,
Uppsala, Sweden), and the rat PDGF-R
cDNA probe was the kind gift of
Dr. Yutaka Kitami (Ehime University, Ehime, Japan).
Intratracheal instillation.
Sprague-Dawley rats (Charles River) weighing ~200 g were instilled
intratracheally with 200 µl of sterile saline or 2 mg/kg (0.4 mg/rat)
of
V2O5
suspended in saline. This protocol was similar to a previously
published intratracheal instillation procedure (10).
V2O5
suspensions were vortexed thoroughly, then bath sonicated for 30 min at
25°C before instillation. At 24, 48, and 72 h and 6 and 15 days
postinstillation, the animals (three saline instilled and five
V2O5
instilled per time point) were overdosed with an intraperitoneal
injection of Nembutal, and the lungs were removed en bloc. The left
lung was instilled with buffered Formalin in phosphate-buffered saline
(PBS), pH 7.2; the trachea was tied off; and the lungs were immersed in Formalin overnight. After fixation, the lung tissues were embedded in
paraffin. Four-micrometer-thick sections were mounted and stained with
hematoxylin and eosin, Masson's trichrome for collagen, and Verhoeff's stain for elastin and PDGF-R (see
Immunohistochemistry). The right
lobes of the lung were homogenized with a motor-driven tissue grinder
in 10 ml of TRI reagent (Molecular Research Center, Cincinnati, OH). Total RNA was isolated according to the
manufacturer's specifications, except that the aqueous phase from the
first chloroform separation was extracted an additional time with TRI
reagent to inhibit endogenous ribonuclease activity. The protein
fraction from the TRI reagent procedure was separated according to the manufacturer's specifications, frozen at
80°C, and
processed for Western blot analysis of PDGF receptors as described in
Western blot analysis. In the present
study, we did not use a nonfibrogenic particle of similar particle
diameter to
V2O5.
However, we previously reported that rats exposed to iron particles
(>93% iron and a mean diameter of 3 µm) developed no acute
inflammation or fibrosis compared with fibrogenic chrysotile asbestos
fibers (4).
Isolation of pulmonary myofibroblasts.
Early-passage rat lung myofibroblasts from male Sprague-Dawley rats
were isolated and characterized as described previously (8). Cell
isolates at passage 1 or
2 were plated onto
3-aminopropyltriethoxysilane-coated glass chamber slides and grown to
confluence, then fixed briefly in ice-cold acetone. Fixed cells were
then subjected to overnight incubation with a murine monoclonal
antibody to the antigen of interest, followed by a biotinylated horse
anti-mouse antibody, avidin-biotin immunoperoxidase, and
3,3'-diaminobenzidine chromogen (all from Vector Laboratories,
Burlingame, CA). An irrelevant monoclonal antibody
(anti-5-bromo-2'-deoxyuridine; Becton-Dickinson, San Jose, CA) at
an equivalent immunoglobulin G concentration served as a control for
nonspecific immunoreactivity. Concentrations of primary antibodies were
set by titration on appropriate rat-derived positive control cells.
Cells stained positively for vimentin and -smooth muscle actin and
negatively for factor VIII, desmin, and rat leukocyte common antigen
(OX 1). In addition, examination of glutaraldehyde-fixed cell pellets
by transmission electron microscopy showed ultrastructural features
consistent with a myofibroblast phenotype (abundant intermediate
filaments and rough endoplasmic reticulum and lack of Weibel-Palade
bodies characteristic of endothelial cells). Cells were grown to
confluence in 10% FBS-Dulbecco's modified Eagle's medium (DMEM)
before being seeded for the assays described in
Western blot analysis and
125I-PDGF-AA binding
assay.
Isolation of rat alveolar macrophages.
Alveolar macrophages from male Sprague-Dawley rats were obtained by
bronchoalveolar lavage as previously described (6). Macrophages
(30-35 × 106) that were
>95% viable (as determined by trypan blue exclusion) were suspended
in serum-free DMEM (0.25% BSA) and cultured in 175-cm2 flasks coated with
poly(2-hydroxyethyl methacrylate) (Sigma Chemical) that prevents
macrophage attachment to the tissue culture surface and activation via
adherence. After 1 h of equilibration in culture at 37°C in 5%
CO2, the macrophages were treated
with 0.01, 0.1, or 1 µg/cm2
(0.3-30 µM) of
V2O5.
After 24 h, the culture medium was centrifuged at 1,500 revolutions/min
to pellet the macrophages, and the supernatant was filtered (0.45 µm)
and stored at 20°C. These concentrations of
V2O5
did not significantly affect cell viability as determined by trypan
blue exclusion.
Northern blot analysis. Total lung RNA
isolated with TRI reagent (see Intratracheal
instillation) was electrophoresed (20 µg/lane) in
1% agarose-2 M formaldehyde gels and capillary transferred onto
Immobilon S membranes (Millipore, Bedford, MA). RNA from the in vitro
experiments was hybridized to a
32P-labeled cDNA probe for the
PDGF-R or PDGF-R
subunit.
[
-32P]dCTP
(Amersham, Arlington Heights, IL) was used to label the cDNA with a
Prime-It II random-primer labeling kit (Stratagene, La Jolla, CA). The
autoradiographic signal was visualized with a phosphorimaging system.
Western blot analysis. Total lung
protein collected after in vivo
V2O5
instillation (see Intratracheal
instillation) was separated from RNA and DNA
according to the manufacturer's directions and mixed 1:4 with lysis
buffer A [20 mM
tris(hydroxymethyl)aminomethane (Tris), pH 7.5, 5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, and 20 µg/ml of pepstatin, leupeptin,
and aprotinin]. The mixture was centrifuged at 100,000 g for 30 min (Beckman TLA.3
fixed-angle rotor), and the pellet was resuspended in 0.25 ml of lysis
buffer B (lysis
buffer A supplemented with 1% Triton
X-100) and probe sonicated for 30 s. After a second centrifugation at
100,000 g for 30 min, the supernatant
(membrane fraction) was removed and stored at 80°C. In
another set of experiments in which the cell lysates were harvested in
vitro, rat lung myofibroblasts were grown to confluence in
75-cm2 flasks and rendered
quiescent for 24 h in serum-free defined medium (SFDM). The SFDM
consisted of Ham's F-12 medium with
CaCl2 and 0.25% BSA and was
supplemented with an insulin-transferrin-selenium mixture (Boehringer
Mannheim). Myofibroblasts were treated with 0.25×
macrophage-conditioned medium (MØCM; see
Isolation of rat alveolar
macrophages) collected from macrophages treated with V2O5.
Some cultures received
V2O5
alone (0.01-1 µg/cm2) to
test the direct effect of this metal on myofibroblast PDGF-R
expression. IL-1
(2 ng/ml) was used as a positive control for PDGF-R
upregulation (20). Parallel flasks of all agents were set up
to test the effect of an IRAP on PDGF-receptor expression. After
incubation for 24 h at 37°C, the cells were washed with PBS, and
250 ml of lysis buffer B were added to
cover the surface of the attached cells for 20 min. Lysates were stored
at
80°C. Twenty microliters of each sample were mixed with
sample buffer [0.5 M Tris · HCl, pH 6.8, 10%
sodium dodecyl sulfate (SDS), 0.1% bromphenol blue, and 20%
glycerol], boiled for 5 min, and sonicated for 30 s before
electrophoresis in a 2-15% Tris-glycine SDS-polyacrylamide gel
(Integrated Separation Systems, Hyde Park, MA) for 2 h at 130 V and 30 mA. The proteins were transferred to
nitrocellulose membrane (Hybond, Amersham, Arlington Heights, IL). The
membrane was blocked with 3% milk-PBS for 1 h before overnight
incubation at 4°C with a 1:500 dilution of rabbit anti-mouse
PDGF-R
or PDGF-R
antibody (UBI, Lake Placid, NY). After being
washed three times with PBS-Tween, a 1:2,000 dilution of secondary
horseradish peroxidase-conjugated swine anti-rabbit antibody (Dako,
Carpinteria, CA) was added for 1.5 h. An enhanced chemiluminescence
luminol kit (Amersham, Arlington Heights, IL) was used for detection of
bound secondary antibody.
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Immunohistochemistry. Lung tissue was
fixed overnight in 10% neutral buffered Formalin. Testes from
6-day-old rats were used as a positive control tissue for PDGF-R
expression because the connective tissue surrounding the seminiferous
tubules stains intensely for this receptor (11). Immunohistochemistry
was performed with the avidin-biotin-peroxidase method. Tissue sections
(6 µm) were dehydrated through a series of graded alcohol solutions
to 1× automation buffer (AB) consisting of 5% NaCl and 2% HCl
(Biomeda, Foster City, CA). Endogenous peroxidase was blocked in 3%
(vol/vol) H2O2
for 15 min. After a 1× AB wash, the slides were immersed in 10 mM
citrate buffer (pH 6.0) and heated in a microwave oven (750 W) for 5 min. This procedure was repeated two times at 1-min intervals to add
fresh citrate buffer. After cooling for 15 min, the slides were rinsed
in distilled H2O and incubated in
1× AB for 5 min. Sections were blocked with normal goat serum
(Vector Laboratories) for 20 min at room temperature, then incubated
for 1 h at room temperature with a rabbit anti-murine PDGF-R
antibody (UBI) diluted 1:100 in 1% BSA. Sections were washed two times with AB and then incubated for 30 min with a 1:400
dilution of biotinylated secondary goat anti-rabbit immunoglobulin G
(Vector Laboratories). The slides were washed again and incubated with the Elite Avidin-Biotin Complex (Vector Laboratories) for 30 min. Visualization of the antibody complex was done with a diaminobenzidine tablet (10 mg; Sigma Chemical) dissolved in 20 ml of 1× AB
containing 12 µl of 30%
H2O2
for 6 min in the dark. The slides were then rinsed in running tap
water, counterstained with Harris hematoxylin (Harelco, Gibbstown, NJ),
dehydrated through a series of graded alcohols to xylene, and
coverslipped with Permount (Fisher Scientific, Fair Lawn, NJ). Some
paraffin-embedded sections were stained for elastin (Verhoeff's stain)
or desmin as a smooth muscle cell phenotypic marker or vimentin (Clone
LN6, Accurate Antibodies, Westbury, NY) as a marker of fibroblast
phenotype. Mature collagen was detected by Masson's trichrome stain.
125I-PDGF-AA binding assay.
Rat lung myofibroblasts were seeded in 24-well plates and grown to
confluence in 10% FBS-DMEM, then rendered quiescent in SFDM for 24 h.
The cells were then treated with fresh SFDM supplemented with
0.25× MØCM (from control or
V2O5-stimulated
macrophages), V2O5
alone (0.01-1 µg/cm2), or
IL-1 (2 ng/ml) in the absence or presence of 2 µg/ml of IRAP.
After 24 h, the cultures were chilled to 4°C, rinsed in ice-cold
binding buffer (Ham's F-12 with
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, CaCl2, and 0.25% BSA), and
incubated with 2 ng/ml of
125I-PDGF-AA (specific activity
125 µCi/µg) in the absence or presence of 400 ng/ml of PDGF-AA
(nonspecific binding) for 3 h at 4°C on an oscillating platform.
Cells were then rinsed three times in ice-cold binding buffer and
solubilized (1% Triton X, 0.1% BSA, and 0.1 N NaOH; 1 ml/well), and
cell-associated radioactivity was measured in a gamma counter.
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RESULTS |
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PDGF-R mRNA expression is upregulated in vivo after
instillation with
V2O5.
Total lung mRNA was analyzed for PDGF-receptor genes at several time
points (24, 48, and 72 h and 6 and 15 days) after the intratracheal
instillation of
V2O5.
PDGF-R
expression in the whole lung was low and barely detectable by
Northern blot analysis. PDGF-R
mRNA expression was markedly
increased 24 h after
V2O5 instillation (8- to 10-fold above the saline control lung), but this
transient induction returned to control levels of expression by 72 h
postinstillation (Fig. 1). No significant change in
PDGF-R
mRNA expression was observed in rats that were instilled with saline alone. In contrast to PDGF-R
expression, PDGF-R
was highly expressed constitutively, and instillation of
V2O5
did not change the expression of this receptor subtype (Fig.
2).
In vivo expression of PDGF-R protein is increased
after V2O5
instillation and is localized in early fibrotic lesions.
Western blotting of total lung protein homogenates was performed with
antibodies specific to either PDGF-R
or PDGF-R
. Total lung
PDGF-R
protein was strongly upregulated 24 h postinstillation of
V2O5
and was maximally expressed at 48 h (Fig.
3). PDGF-R
protein levels were
constitutively high, and the levels did not significantly change after
V2O5
instillation. Immunohistochemical analysis of the PDGF-R
was
performed on paraffin-embedded sections of
V2O5-instilled
rat lung with the same rabbit anti-murine PDGF-R
antibody that was
employed in the Western blotting procedure. In agreement with the
Western blot analysis, PDGF-R
protein was detected 24 and 48 h
postinstillation of
V2O5.
PDGF-R
was visualized (brown staining) in inflammatory foci that
were characterized by inflammatory cell infiltration (primarily
macrophages, neutrophils, and lymphocytes) and thickened foci composed
of epithelial and mesenchymal cells (Fig.
4). Weak PDGF-R
staining was observed 6 days postinstillation of
V2O5
in proliferative lesions characterized by myofibroblast hyperplasia. As
a negative control, the staining procedure was also performed with
normal rabbit serum. Testes from 6-day-old rats were used in the
immunohistochemical procedure as a positive control because PDGF-R
expression has been shown to be high during development of the
mesenchyme surrounding the semiferous tubules of the testes (11). In
agreement with this earlier study, we observed strong PDGF-R
staining in the connective tissue surrounding the seminiferous tubules
of the testes but no staining within Sertoli cells (Fig. 4).
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V2O5
upregulates PDGF-R on cultured rat lung myofibroblasts
through a macrophage-dependent pathway involving IL-1
.
To address the mechanism whereby
V2O5
causes upregulation of PDGF-R
in vivo, we investigated the
possibility that 1)
V2O5 might directly induce PDGF-R
on cultured lung myofibroblasts or
2)
V2O5
induction of PDGF-R
on myofibroblasts could require the presence of
alveolar macrophage-derived inflammatory mediators. Therefore,
confluent cultures of rat lung myofibroblasts were treated in vitro
directly with
V2O5
or with MØCM from macrophages that had been stimulated with
V2O5
for 24 h. As a positive control for PDGF-R
upregulation, some
myofibroblasts were stimulated with IL-1
(2 ng/ml). Parallel
cultures of myofibroblasts were coincubated with IRAP.
V2O5
alone did not significantly increase PDGF-R
expression as determined
by Western blot analysis (Fig. 6). However,
V2O5
stimulated macrophages to release a factor(s) that strongly upregulated
PDGF-R
on myofibroblasts, and the majority of this activity
(>75%) was blocked by IRAP (Fig. 6). IRAP alone did not cause any
change in PDGF-R
expression (data not shown). PDGF-R
expression
was not changed by any of these treatments. Similar experiments were
performed with an 125I-PDGF-AA
binding assay to more accurately measure upregulation of the
cell-surface PDGF-AA binding site (i.e., PDGF-R
; Fig. 7). These data confirmed the Western
blotting experiments in Fig. 6 and showed that
V2O5
stimulated alveolar macrophages to release a factor(s) that strongly
upregulated the number of
125I-PDGF-AA binding sites on
cultured myofibroblasts (seven- to eightfold increase in upregulatory
activity between control MØCM and MØCM from
V2O5
treatment). IRAP blocked this upregulatory activity by >70%.
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DISCUSSION |
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The hyperplastic growth of myofibroblasts is a key event in the
progression of pulmonary fibrosis. In this study, we have shown that
PDGF-R, but not PDGF-R
, is induced during the
inflammatory and early proliferative stages of fibrogenesis in rats
instilled with
V2O5.
Because the expression of PDGF-R
is required for maximal PDGF-stimulated chemotaxis and mitogenesis of myofibroblasts in vitro
(8, 20, 24), our findings suggest that induction of PDGF-R
in vivo
is a mechanism that contributes to enhanced myofibroblast proliferation
in the fibrotic lung. Furthermore, we showed that
V2O5
stimulated cultured alveolar macrophages to release a factor(s) that
upregulated the PDGF-R
on lung myofibroblasts in vitro and that the
majority of this activity was due to IL-1
. PDGF-R
induction
appears to be a novel mechanism of lung fibroblast hyperplasia during
pulmonary fibrogenesis.
Previous studies from our laboratory (19, 20) have shown that IL-1
is a potent inducer of PDGF-R
on myofibroblasts in vitro. Other
investigators have demonstrated with rat models that IL-1
gene
expression in vivo occurs within 4 h after the instillation of vanadium
sulfate (9) and that increased protein levels of IL-1 in lavage fluid
are maximal within 24 h after the inhalation of asbestos fibers (12) or
the instillation of bleomycin (15). In humans, alveolar macrophages
from patients with idiopathic pulmonary fibrosis or individuals exposed
to asbestos release enhanced levels of IL-1
(33). Induction of
PDGF-R
gene expression by IL-1
occurs 4 h after treatment of lung
myofibroblasts in vitro, and cell-surface PDGF-R
appears within 12 h
(20). Thus the temporal expression of IL-1
in vivo during fibrosis
induced by fibrogenic agents, including vanadium, suggests that this
proinflammatory cytokine is a likely candidate for turning on PDGF-R
after lung injury. In the present study, we showed that
V2O5
stimulates macrophages to release a factor(s) that upregulates
PDGF-R
on lung myofibroblasts in culture and that this activity is
due mainly to IL-1
.
V2O5 did not directly stimulate PDGF-R
, and, therefore, IL-1
may be
the major inducer of PDGF-R
in vivo after lung injury.
We did not measure the induction of PDGF in the
V2O5
model of fibrosis. However, the temporal expression of PDGF in rats has been investigated after the instillation of bleomycin (30). In these
studies, the induction of PDGF occurs within 2-3 days after lung
injury. This temporal induction of PDGF coincides with the upregulation
of PDGF-R in the present study. Thus it appears that PDGF and the
inducible PDGF-R
are coregulated during the progression of fibrosis.
It is important to keep in mind that upregulation of PDGF-R
has a
significance beyond simply increasing the overall number of PDGF
receptors at the cell surface of mesenchymal cells. Indeed, even a
relatively small increase in cell-surface PDGF-R
relative to
PDGF-R
(1:10 ratio) renders cells severalfold more responsive to
PDGF (19, 20). Indeed, Seifert et al. (29) have shown that maximal
mitogenic responses to PDGF isoforms require the presence of PDGF-R
in combination with PDGF-R
and that the PDGF heterodimeric
-receptor complex is a more potent inducer of Swiss 3T3
fibroblast proliferation compared with the PDGF
-receptor complex. Another study from our laboratory (24) emphasized that maximal
chemotaxis of lung myofibroblasts requires the presence of PDGF-R
.
During fibrosis, it is now apparent that two changes in the PDGF system
occur: 1) induction of PDGF-A and -B
chain genes (30) and 2) a
coordinated upregulation of the normally suppressed PDGF-R
gene
(reported in the present study).
PDGF-R expression was turned off before the appearance of mature
collagen within fibrotic lesions as determined by trichrome staining.
This may be due to the expression of TGF-
1, which stimulates collagen expression and also serves to downregulate PDGF-R
on lung
fibroblasts (3). We did not measure TGF-
1 levels in the present
study, but another study (27) has shown that TGF-
1 expression occurs
within 5-7 days during fibrogenesis caused by the instillation of
bleomycin. Previous work from our laboratory (3) showed that TGF-
1
acts at the transcriptional level to suppress PDGF-R
gene expression
and that TGF-
1 inhibits lung fibroblast proliferation, at least in
part, by suppression of PDGF-R
. Thus TGF-
1 has multifunctional
roles in the fibroproliferative process: inhibition of fibroblast
proliferation in concert with activation of genes encoding
extracellular matrix proteins such as collagen. Other investigators
(32) have shown that development of interstitial lung disease in rats
requires expression of both PDGF and TGF-
1. This could be explained
by the fact that PDGF is important to the early proliferative phase of
fibrosis, whereas TGF-
1 drives the matrix deposition phase of the
disease process.
The data presented in the present study, taken together with earlier in
vitro observations of PDGF-R upregulation (8, 20), suggest that
induction of this receptor is important to mesenchymal cell hyperplasia
in vivo during lung fibrogenesis. Induction of PDGF-R
in vitro has
been demonstrated on cultured human lung fibroblasts in response to
thrombin (22), human bronchial smooth muscle cells stimulated with
basic fibroblast growth factor (2), and rat lung myofibroblasts treated
with IL-1
(20). However, induction of PDGF-R
rather than
PDGF-R
appears to be important to some other proliferative diseases.
During liver fibrosis, adipocytes differentiate into myofibroblasts,
and this is accompanied by the appearance of PDGF-R
(31). During
atherosclerosis, PDGF-R
is upregulated with no apparent change in
PDGF-R
(26). Thus, although induction of PDGF-R
appears to be
important to the progression of lung fibrosis, it may not occur in all
fibroproliferative disease states.
Elastin staining indicated that alveolar myofibroblasts (contractile
interstitial cells) were present within fibrotic lesions during
V2O5-induced
fibrogenesis. Alveolar myofibroblasts constitute the major source of
elastin fibers in the lung parenchyma (7), and fibrotic lesions stained
positively for both elastin and collagen (Fig. 5). Furthermore, our
early-passage isolates of rat lung mesenchymal cells that possessed
inducible PDGF-R stained positively for
-smooth muscle actin and
vimentin and contained abundant intermediate filaments as determined by
ultrastructural analysis (8). Thus these cells are most likely a
myofibroblast phenotype and represent the target cell that we observed
in vivo within fibrotic lesions.
In summary, we have shown that induction of PDGF-R occurs in vivo
during the early events associated with myofibroblast proliferation after vanadium-induced lung injury. This is the first report of PDGF-R
induction in vivo during the multistep progression of fibrogenesis. Expression of PDGF-R
remained unchanged during fibrogenesis. Induction of PDGF-R
is due, in part, to a
macrophage-dependent pathway involving IL-1
. Because PDGF-R
expression is necessary for maximal PDGF-stimulated mitogenic and
chemotactic responses in vitro, the present study suggests that
upregulation of PDGF-R
in vivo is a novel mechanism of myofibroblast
hyperplasia that contributes to the development of fibrotic lesions
after lung injury.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Robert Maronpot and Julie Foley for
providing technical support in platelet-derived growth
factor-receptor- immunohistochemistry and lung pathology. We thank
Dr. Darryl Zeldin and Dr. Robert Maronpot for comments during the
preparation of the manuscript. The technical assistance of Herman Price
in performing intratracheal instillations is greatly appreciated.
![]() |
FOOTNOTES |
---|
Address for reprint requests: J. C. Bonner, National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709.
Received 4 June 1997; accepted in final form 16 September 1997.
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