Lung Biology Program and Departments of Pathology and Medicine, Tulane University Medical Center, New Orleans, Louisiana 70112
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ABSTRACT |
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Corticosteroids
(CSs) are commonly used for anti-inflammatory therapy in asthma and in
interstitial lung diseases. In attempting to understand the mechanisms
through which CSs control cell proliferation, we have carried out
experiments to test the effects of dexamethasone (Dex) on the growth of
lung fibroblasts. Using mouse 3T3 fibroblasts as well as early-passage
rat lung fibroblasts (RLFs), we show that the quiescent cells in 1%
serum or in serum-free media proliferate significantly in response to
the addition of 107 to
10
9 M Dex. Increases as
high as fourfold in cell numbers were recorded for the RLFs after 48 h
in culture. A polyclonal antibody to the AB isoform of human
platelet-derived growth factor (PDGF) blocked the proliferative
response. As expected, the fibroblasts produced primarily PDGF-A chain,
and the RLFs exhibited few PDGF-
receptors (PDGF-R
), the receptor
type necessary for binding the AA isoform. Accordingly, we determined
that Dex upregulated PDGF-R
mRNA and protein. Therefore, we can
postulate that Dex-induced fibroblast proliferation is mediated, at
least in part, by PDGF-AA, which binds to the PDGF-R
.
platelet-derived growth factor- receptor; platelet-derived
growth factor isoforms; anti-platelet-derived growth
factor
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INTRODUCTION |
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CORTICOSTEROIDS (CSs) are commonly used in therapeutic regimens for inflammation and fibroproliferative processes (25). We have been studying the mechanisms controlling mesenchymal cell growth in the lungs of rats and mice exposed to asbestos fibers (5, 28, 29) and in lung mesenchymal cells maintained in vitro (2, 7). Our goal is to determine those factors that control proliferation of epithelial and mesenchymal cells during the development of interstitial disease of the lung parenchyma and airway walls. In this regard, it has been postulated that platelet-derived growth factor (PDGF), the potent mesenchymal cell mitogen, plays a role in fibrogenic lung disease (48), and we have participated in this growing area of investigation (2, 3, 29).
Although it is clear that CSs have multiple effects on a wide variety of cell types (43), there is little information available on the interactions between CSs and expression of PDGF receptors and their ligands. For example, early studies (32) on serum-free media that support growth of a fibroblast line showed that dexamethasone (Dex) increased the replicative life span of the cells and induced a "putative growth factor" that increased cell density. More recently, investigators (40) using fibroblasts separated from human gingival tissues showed that Dex increased the mitogenic response to PDGF. Other investigations have shown that CSs block fibroblast growth (17). Such studies raise fundamental questions regarding the mechanisms through which anti-inflammatory agents exert control over cell growth (20, 37), a feature central to the development of fibroproliferative disease.
In the experiments reported here, we have treated a mouse embryo
fibroblast cell line (3T3 cells) and early- passage adult rat lung
fibroblasts (RLFs) with Dex in vitro. We report that the quiescent
fibroblasts exposed to 107
or 10
9 M Dex exhibited up
to fourfold increases in cell numbers. A polyclonal antibody to PDGF
blocked the response to Dex treatment, and Dex caused a rapid
upregulation of the mRNA coding for the PDGF-
receptor (PDGF-R
)
as well as an increase in receptor protein. This could explain the
Dex-induced increase in cell number, since we have shown that these
cells primarily produce the A chain of PDGF, which binds only to the
-receptor.
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MATERIALS AND METHODS |
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Cell Culture
3T3 fibroblasts (CCL-92, American Type Culture Collection, Rockville, MD) were cultured in DMEM (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. All supplements were from GIBCO BRL (Gaithersburg, MD). The cultures were maintained at 37°C in a humidified incubator in an atmosphere of 5% CO2 in air.Primary RLF cells were isolated from adult rat lungs (Charles River Laboratories, Raleigh, NC) as previously described (21), maintained in the same medium under the same conditions as the 3T3 fibroblasts, and used in experiments at passages 3-13.
Cell Proliferation Assays
PDGF-BB and PDGF-AA treatments. RLF or 3T3 cells were seeded at a density of 2-3 × 104 cells/well in two 24-well (2 cm2) tissue culture plates and allowed to grow to subconfluence in DMEM with 10% FBS. The cells were rinsed twice in PBS and refed with DMEM with 1% FBS to establish quiescence for 24-48 h. Triplicate wells in the 24-well plates were treated with 1, 5, 10, and 20 ng/ml of either PDGF-BB or PDGF-AA (R&D Systems, Minneapolis, MN). Stock solutions of PDGF were made up as 1 µg/ml in 4 mM HCl in 1% bovine serum albumin and diluted in DMEM with 1% FBS to the final concentrations just before treatments. Control wells contained DMEM with 1% FBS only or DMEM with 10% FBS. The cells were incubated at 37°C and 5% CO2 in a humidified incubator for either 48 or 96 h. At the end of each incubation period, the cells in each well were trypsinized and counted using a hemacytometer.
Dex treatment.
RLF or 3T3 cells were seeded and quiesced as above. Triplicate wells in
the 24-well plates were treated with
105,
10
7,
10
9, or
10
12 M Dex (Sigma) in DMEM
with 1% FBS. The stock solution (1 mM) of Dex was made in 100%
ethanol and diluted to the final concentration in DMEM with 1% FBS.
Control wells contained DMEM with 1% FBS only or DMEM with 10% FBS.
The cells were incubated at 37°C and 5%
CO2 in a humidified incubator for
either 48 or 96 h. The cells were counted as above.
Dex+anti-PDGF antibody
treatments.
3T3 or RLF cells were seeded and quiesced as in
PDGF-BB and PDGF-AA treatments, and
triplicate wells in the 24-well plates were treated with Dex
(107 M) or PDGF (20 ng/ml)
with and without the addition of 50 µg/ml of polyclonal anti-human
PDGF (Collaborative Biomedical Products, Bedford, MA). This polyclonal
antibody recognizes all three PDGF isoforms and blocks their biological
activity with no apparent cytotoxicity at concentrations of 50-60
µg/ml (3, 22). Antibody-negative cultures of Dex had IgG added.
Control wells contained DMEM with 1% FBS and DMEM with 10% FBS.
Incubation and cell counting were as in PDGF-BB and
PDGF-AA treatments.
Analysis of Total Cell RNA
3T3 or RLF cells were seeded at a density of 4-6 × 105 cells/100-mm tissue culture dish and allowed to grow for 24-48 h until the cells reached ~80% confluency. The cells were then rinsed twice in PBS and refed with SFDM for another 24-48 h. A number of plates were treated with 10Ribonucleoside 5'-triphosphate and deoxyribonucleoside 5'-triphosphate
sets were purchased from Pharmacia Biotech (Piscataway, NJ). All enzymes were purchased from Promega (Madison, WI).
[-32P]dCTP and
[
-32P]UTP were from
ICN Pharmaceuticals (Costa Mesa, CA). All other chemicals used in RNA
analysis work were of molecular biology grade and were purchased from
Sigma or Fisher Scientific unless otherwise noted.
RNase protection assay.
RNase protection assays were carried out as commonly described (1).
Briefly, 10 µg of total cell RNA were used to hybridize to an
[-32P]UTP (3,000 Ci/mmol)-labeled antisense RNA probe of either PDGF-A, PDGF-B, or
GAPDH. Probe for PDGF-A was made by in vitro transcription of
linearized plasmid containing the rat PDGF-A
Sma I (nucleotides 80-613) cDNA
fragment in pBluescript using T3 RNA polymerase. PDGF-B probe was made
by in vitro transcription of linearized plasmid containing the rat
PDGF-B 534-nucleotide cDNA fragment in pBluescript using T7 RNA
polymerase. Both PDGF cDNA fragments containing plasmids were kindly
provided by Dr. D. Katayose, National Heart, Lung, and Blood Institute,
Bethesda, MD (19). GAPDH probe was
made by in vitro transcription of linearized plasmid containing the rat
GAPDH fragment in pBluescript using T7 RNA polymerase. The hybridized
fragments were digested with RNase T1 (Calbiochem-Novabiochem, La
Jolla, CA) and separated on a 7 M, 8% urea polyacrylamide gel. The
bands were visualized by autoradiography and/or by exposure to
an imaging plate. Levels of mRNA were quantitated using a Fuji
phosphorimager (Fuji Medical Systems, Stanford, CT).
Northern analysis.
Northern analysis was carried out as commonly described
(1). Thirty micrograms of total cell
RNA were electrophoresed on a 1% agarose-2.2 M formaldehyde gel,
transferred to a polyvinylidene difluoride membrane (Immobilon-N;
Millipore, Bedford, MA), and hybridized with
[-32P]dCTP (3,000 Ci/mmol)-labeled DNA probes of either PDGF-A, PDGF-B, PDGF-R
[from plasmid pBS802E, which contains the PDGF-R
clone in pBluescript, which was kindly provided by Dr. R. R. Reed (The Johns Hopkins Univ. School of Medicine)], or GAPDH.
Western blot analysis.
3T3 or RLF cells were allowed to grow on 100-mm culture dishes, were
quiesced as for RNA analysis work, were treated with either
107 M Dex in SFDM or SFDM
alone, and were incubated at 37°C and 5% CO2 in a humidified incubator for
48 h. Western blot analysis was carried out with the isolated cell
membrane proteins as described (41), with minor modifications. At the
end of the incubation period, cells were rinsed with cold PBS and
scraped to harvest into cold homogenization buffer (25 mM
Tris · HCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 25 µg/ml of
aprotinin, leupeptin, and pepstatin A). The cells were disrupted by
triturating up and down several times using a syringe fitted with a
27-gauge needle. Nuclei and unbroken cells were removed by
low-speed centrifugation. Cytosol (soluble) and membrane (insoluble)
fractionation was obtained by centrifugation at 100,000 g for 1 h at 4°C. To solubilize
the proteins, the pellet was resuspended in homogenization buffer containing 1% Triton X-100. Equal amounts (protein quantitated by
DC-Bio-Rad assay) of protein were analyzed on an 8-16% gradient of Tris-glycine gel (Novel Experimental Technology, San Diego, CA). The
proteins were then transferred to a nitrocellulose membrane [Hybond enhanced chemiluminescence (ECL); Amersham, Arlington Heights, IL] by a semidry method (in a Bio-Rad transblot semidry transfer cell) and immunoblotted with a polyclonal antibody raised against PDGF-R
(rabbit anti-PDGF-R
; Santa Cruz Biotechnology, Santa Cruz, CA). The immunoreactive bands were visualized
with horseradish peroxidase-linked donkey anti-rabbit Ig
(Amersham) as the secondary antibody and the ECL (Amersham) detection
system.
Statistics
All cell proliferation assays and RNA analysis experiments were carried out at least three separate times.Results are expressed as mean values ± SE. One- or two-factor ANOVA was used to compare data. Differences were considered statistically significant at P < 0.05. Percent increases were calculated as a percentage of unstimulated or untreated cells.
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RESULTS |
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Fibroblast Proliferation
PDGF isoforms.
As has been reported previously (2, 3), 3T3 cells and primary RLFs
divide vigorously in culture under the influence of the BB isoform of
PDGF in a concentration-dependent fashion (Fig.
1, A and
B). The cells respond less to the AA
isoform because AA binds only the -receptor dimer (44), whereas
the BB isoform activates all three receptor dimers (44) (i.e., the
-,
-, and
-receptors). All of the experiments
reported here were repeated a minimum of three separate times, with
triplicate cultures for each concentration. Twenty nanograms per
milliliter of the PDGF isoform induced the maximum increase in cell
numbers, as high as fourfold in some experiments (Fig.
1A). Higher concentrations of
PDGF-BB caused no further increase in cell numbers (not shown). RLFs
typically have lower proliferation rates in culture than 3T3 cells (2,
3) and in these experiments exhibited approximately twofold increases
(Fig. 1B). The AA isoform had
little effect on RLFs, as has been reported previously (2).
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Dex.
Varying concentrations of Dex alone induced proliferation of both 3T3
cells and RLFs (Fig. 2). Compared with the
numbers of cells in low-serum medium,
107 and
10
9 M Dex caused 2.5- to
3-fold increases in 3T3 cell numbers (Fig. 2A) and ~3.5- to 4-fold increases
in RLFs (Fig. 2B). A concentration of 10
5 M Dex appeared to
have some toxicity, whereas
10
12 M Dex showed little
consistent effect on cell numbers (Fig. 2). Additional experiments were
carried out with the same concentrations of Dex in serum-free media to
compare the effects of 1% FBS with no serum. In these experiments,
there were clear increases in cell numbers as reported above for the
low-serum experiments (data not shown).
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PDGF antibody.
In attempting to understand how Dex might induce cell growth, the
cultures were treated with 50 µg/ml of anti-human PDGF-AB antibody.
As expected on the basis of earlier studies (2, 3), this polyclonal
antibody blocked essentially all of the PDGF-induced cell proliferation
at 48 and 96 h of culture. In this set of experiments with 3T3 cells
(Fig.
3A), Dex
alone caused an ~75% increase in cell number, and this was ablated
by antibody treatment. An IgG control and several irrelevant antibodies
to growth factors (such as transforming growth factor-) caused no
changes in cell proliferation (data not shown). Again, Dex alone had a
more substantial effect on RLF growth (~2-fold), and the antibody
blocked this response as well (Fig.
3B), keeping the cell numbers at
control levels. In the Dex-treated cells (both 3T3 and RLF), the
specific antibody prevented cell growth at a statistically significant
level (P < 0.05) at 48 h in culture,
whereas the 96-h cultures showed a similar pattern of reduced growth,
but the significance values reached P < 0.06-0.09.
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Gene Expression
To determine whether Dex increased expression of the mRNAs for the PDGF-A- and -B-chain proteins as well as the PDGF-RRNase protection assays. Both 3T3 cells and RLFs expressed the mRNA for the PDGF-A chain, whereas only 3T3 cells expressed detectable mRNA for the B chain (Fig. 4). This is consistent with earlier observations using Northern analysis and protein assays (22). The levels of mRNA for the A chain were quantitated (data not shown), and in more than three separate trials for each of the cell lines, there was no consistent change in PDGF-A message levels at 4, 16, 24, 48, or 96 h after treatment with three or four concentrations of Dex.
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Northern analysis.
Because the antibody to the PDGF protein blocked the increase in cell
number induced by Dex, we propose that this activity is regulated by
the PDGF ligands or their receptors. RLFs produce little PDGF-B (22),
and no increase in the mRNA for the A or B chain was detected (Fig. 4).
Thus the mRNA levels for the A-chain receptor (PDGF-R) were analyzed
in 3T3 cells and RLFs by Northern analysis. The PDGF-R
message was
increased slightly as soon as 45 min after treatment and continued to
increase for 2 days after treatment (Fig.
5,
A-C). There were more than
twofold increases by 22 h, and expression peaked at about fourfold by
48 h posttreatment (Fig. 5C).
Expression returned to near normal levels by 96 h (Fig. 5C). The increase in expression
induced by Dex was four- to fivefold in several separate experiments.
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Western blot analysis.
Inasmuch as gene expression for the PDGF-R was clearly increased
through the 48-h period after treatment with Dex (Fig. 5), we carried
out a Western analysis of the membrane fraction protein of both the 3T3
and RLF cells. The antibody for the PDGF-R
appeared in a single band
at the predicted molecular mass of 170 kDa (Fig. 6). It is known that 3T3 cells exhibit
large numbers of the
-receptor (2), and this is obvious in the
immunoreactive bands. Treatment with
10
7 and
10
9 M Dex showed an
increased amount of PDGF-R
protein by 48 h posttreatment. RLFs
exhibit few
-receptors (2), and the cells in SFDM demonstrate this
by a complete lack of immunoreactivity. As predicted by the mRNA
analysis (Fig. 5), the PDGF-R
protein was clearly increased in the
immunoreactive bands of the Western blot after treatment with Dex (Fig.
6).
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DISCUSSION |
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Reduction of lung inflammation and treatment of airway hyperresponsiveness associated with asthma can be achieved with therapeutic doses of CSs (25). Patients studied in clinical trials and animal models in laboratory settings offer opportunities for understanding the efficacy of treatment as well as the mechanisms through which these potent agents exert their effects. To study the mechanisms through which any therapeutic agent induces a specific effect, it is often necessary to place the target cells of interest in culture, although the disadvantages of removing any cell type from its natural milieu are obvious. Here we have used a commercially available fibroblast cell line (mouse 3T3) and primary early-passage RLFs derived in our laboratory to determine whether a CS (Dex) affects the growth of these mesenchymal cells in vitro. Dex alone induced a consistent, statistically significant increase in fibroblast growth (Fig. 2), and an antibody to PDGF effectively blocked these proliferative events (Fig. 3).
It is difficult to know at this point in time whether these findings are significant in terms of how asthmatic patients or experimental animals respond to treatment with these drugs. Several papers support our hypothesis that the growth effects of Dex reported here are relevant to events shown in vivo. For example, Rutherford et al. (40) showed that Dex combines synergistically with PDGF-BB to enhance the proliferation of gingival fibroblasts in vitro. These investigators then went on to demonstrate that the same combination of Dex and PDGF-BB induced regeneration of injured periodontium in Cynomolgus monkeys (39). They concluded that this repair was due to new tissue growth, including mesenchymal cells and extracellular matrix (39). The in vitro work of Rutherford et al. (40) provided an impetus for the experiments we have reported here, and it is encouraging to note that our findings are in general agreement, although a preliminary experiment we carried out using PDGF+Dex induced only an additive effect on the fibroblasts in vitro. Whether the combined effects of a CS along with PDGF peptide will affect the lung parenchyma or airway walls in a synergistic fashion is a completely open question at this point in time, although it is clear that the bulk of the literature supports the understanding that Dex blocks inflammation (25) and airway wall remodeling (25, 43, 46).
The complex issue of PDGF-induced mesenchymal cell proliferation has
been approached in many ways, from in vivo studies of wound healing
(33) and atherogenesis (14) to in vitro experiments at the cellular (7)
and molecular levels (20). It has been postulated in several settings
(6, 29) that PDGF plays a central role in mediating pulmonary
fibrogenesis. Now we have found here that Dex induces a mitogenic
effect on lung fibroblasts, possibly by upregulating the PDGF-R. We
interpret this finding to mean that the fibroblasts, both the cell line
and primary cells, can then respond to their own PDGF more efficiently.
Support for this notion is found in Figs. 5 and 6, in which we show
that Dex upregulates gene expression for the PDGF-R
along with the
receptor protein and that an antibody to PDGF blocks Dex-induced cell
proliferation (Fig. 3). The polyclonal antibody used in this study and
in previous experiments from our laboratory has proven to be very
useful for ELISA (3, 22), Western blots (22) (Fig. 6), and cell-growth assays (2, 3, 22) (Fig. 3). It recognizes all three PDGF isoforms in
the assays and is nontoxic at the concentrations used routinely
(50-60 µg/ml) (3, 22). Here, it appears that the antibody has
neutralized this fibroblast-derived AA isoform, thus blocking its
binding to the
-receptors, a scenario that has been reported
previously for asbestos-induced fibroblast growth (3, 22) as discussed
below. PDGF-AA is produced as the predominant isoform by lung
fibroblasts (22) (Fig. 4B). The data
in Fig. 6 show that the PDGF-R
protein is increased by Dex, thus
providing added binding sites for the A-chain ligand. Therefore, we
suggest that the AA isoform released into the medium by the fibroblasts binds specifically to the increased numbers of cell surface
-receptors in an autocrine/paracrine manner, further stimulating
cell proliferation (Figs. 2 and 3). Indeed, it is clear that a variety
of fibroblasts respond vigorously to the BB isoform of PDGF, whereas
primary lung fibroblasts exhibit weaker proliferation under the
influence of the AA isoform, since these cells exhibit fewer
-receptors (2) and the PDGF-
-receptor (PDGF-R
) dimer is the
most abundant receptor spanning fibroblast membranes (2). Such cell
proliferation data can be seen in Fig. 1. The findings that little
PDGF-BB is released by fibroblasts in vitro (22) and that PDGF-
appears to be the receptor subtype upregulated both in vitro and in
vivo by exogenous factors (as discussed below) support this
autocrine/paracrine hypothesis. The molecular elements that control
upregulation of the PDGF-R
but not the PDGF-R
have yet to be
defined. It is our view that all of the isoforms are likely to play a
role in mesenchymal cell proliferation (29); however, we have focused here on the PDGF-R
because the AA isoform is the predominant ligand
released by fibroblasts in vitro.
The mRNA coding for the PDGF-R can be activated by several agents,
although the mechanism controlling this action has not been
established. For example, it has been shown that treating RLFs with
asbestos fibers causes a rapid, concentration-dependent upregulation of
the PDGF-R
mRNA (2), and recent results show a similar response at
sites of lung injury in asbestos-exposed rats (23). In vitro, the RLFs
exhibit a concomitant increase in receptor number and a significant
enhancement of the mitogenic response to the PDGF-AA ligand (2, 22).
Subsequent work from Bonner's laboratory [Lindroos and
colleagues (26, 27)] demonstrated that a similar scenario was
mediated by interleukin-1 (IL-1), a multipotent cytokine released by
several cell types. We do not believe IL-1 is responsible for the Dex
effect shown here. This statement is based on the time-related events
reported for IL-1-induced upregulation of PDGF-R
mRNA at 4 h after
treatment (27). Because we show here that Dex activates the
-receptor mRNA as soon as 45 min after treatment (Fig.
5C), it does not seem likely that IL-1 is directly involved. Likewise, Coin et al. (11) showed that
lipopolysaccharide upregulates the PDGF-R
independent of IL-1.
Interestingly enough, earlier studies on the effects of CSs on
fibroblast growth (8, 32) reported enhanced responses to PDGF. Because
neither of these studies used the individual PDGF isoforms, we are left
to speculate that the CS treatment upregulated the PDGF-R
, thus
explaining the previous findings of increased cell growth (8,
32) and supporting our postulate presented here. The
rapid response to Dex that we show here suggests that the steroid is
acting through the classic mechanism of ligand binding and second
messenger signaling through activation of a glucocorticoid response
element on the PDGF-R
gene (43). It is clear that this pathway leads
to the production of I
B and consequent inactivation of nuclear
factor-
B. Thus CSs downregulate the synthesis of inflammatory
cytokines, several of which could play a role in cell proliferation
(42, 43).
There is a large body of literature regarding the use of CSs in control
of cell growth (25, 43, 45). Generally, CSs are negative growth
regulators, blocking cell proliferation and preventing extracellular
matrix production (17, 25, 30, 35, 36, 43). On the other hand, it is
becoming increasingly clear that CSs are capable of stimulating cell
growth, reportedly through stimulation of growth factors (25, 39) and
upregulation of glucocorticoid receptors (9, 16). Specifically, CSs
have been reported to block cell proliferation by downregulating
hepatocyte growth factor (15), epidermal growth factor (31), and tumor necrosis factor (24), all potent regulators of the cell cycle in
multiple cell types. In contrast, CSs are known to upregulate or
potentiate insulin-like growth factor (12) and epidermal growth factor
in cultures of human fibroblasts (13), and there is a report of
hydrocortisone increasing PDGF-R expression in a malignant cell line
(47). The data we have presented here are consistent with those
suggesting a role for CSs in inducing cell growth, but it should be
obvious that CSs are capable of producing multiple effects dependent on
cell type, growth state, and the addition of other interacting agents.
Finally, we should consider how the combination of CSs and PDGF might be playing a role in fibroproliferative lung disease. With the use of airway wall fibrogenesis in chronic asthma as an example (4), it is not difficult to envision a situation where protracted use of steroid therapy could have an effect on mesenchymal cell growth and enhanced airway remodeling. Although it is clear that CSs have a significant ameliorating effect on airway inflammation and hyperresponsiveness (25, 46), the long-term sequelae of steroid use on airway mesenchymal cell populations remain undefined. Our data presented here suggest that this issue should be pursued. Chronic asthmatics treated extensively with CSs exhibit airway wall fibrosis. This does not implicate steroids by association, but it has been shown that CS treatment can increase collagen in the walls of pulmonary vessels (34) and that long-term steroid use increases the risk of developing atherosclerosis (18). It would be interesting to ask whether chronic steroid therapy plays some role in increased thickening of the airway walls. Inasmuch as PDGF is the most potent mesenchymal cell mitogen yet described (3, 38), it will be essential to establish whether this factor is playing a role in cell proliferation of the airway walls and whether treatment with CSs alters the pathogenesis of airway wall fibrosis in chronic asthma through interactions with peptide growth factors and their receptors.
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ACKNOWLEDGEMENTS |
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We thank Odette Marquez for preparation of the manuscript.
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FOOTNOTES |
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This work was supported by an award from Glaxo Wellcome, Research Triangle Park, NC; by National Institute of Environmental Health Sciences Grant ES-60766; and by the Tulane University Center for Bioenvironmental Research.
Address for reprint requests: A. R. Brody, Lung Biology Program and Dept. of Pathology, Tulane Univ. Medical Center, 1430 Tulane Ave., SL-79, New Orleans, LA 70112-2699.
Received 8 July 1997; accepted in final form 6 January 1998.
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