1 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109; and 2 Pulmonary Section of the Department of Veterans Affairs Medical Center, Ann Arbor, Michigan 48108
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ABSTRACT |
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Prostaglandin
E2 (PGE2) is a potent suppressor of fibroblast
activity. We previously reported that bleomycin-induced pulmonary fibrosis was exaggerated in granulocyte-macrophage
colony-stimulating factor knockout (GM-CSF/
) mice
compared with wild-type (GM-CSF+/+) mice and that increased
fibrosis was associated with decreased PGE2 levels in lung
homogenates and alveolar macrophage cultures. Pulmonary fibroblasts and
alveolar epithelial cells (AECs) represent additional cellular sources
of PGE2 within the lung. Therefore, we examined fibroblasts
and AECs from GM-CSF
/
mice, and we found that they
elaborated significantly less PGE2 than did cells from
GM-CSF+/+ mice. This defect was associated with reduced
expression of cyclooxygenase-1 and -2 (COX-1 and COX-2), key enzymes in
the biosynthesis of PGE2. Additionally, proliferation of
GM-CSF
/
fibroblasts was greater than that of
GM-CSF+/+ fibroblasts, and GM-CSF
/
AECs
were impaired in their ability to inhibit fibroblast proliferation in
coculture. The addition of GM-CSF to fibroblasts from
GM-CSF
/
mice increased PGE2 production and
decreased proliferation. Similarly, AECs isolated from
GM-CSF
/
mice with transgenic expression of GM-CSF under
the surfactant protein C promoter (SpC-GM mice) produced more
PGE2 than did AEC from control mice. Finally, SpC-GM mice
were protected from fluorescein isothiocyanate-induced pulmonary
fibrosis. In conclusion, these data demonstrate that GM-CSF regulates
PGE2 production in pulmonary fibroblasts and AECs and thus
plays an important role in limiting fibroproliferation.
granulocyte-macrophage colony-stimulating factor; idiopathic pulmonary fibrosis; cyclooxygenase-1; cyclooxygenase-2
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INTRODUCTION |
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IDIOPATHIC PULMONARY FIBROSIS (IPF) is a disease associated with high morbidity and mortality for which standard treatment, namely glucocorticoids and immunosuppressive therapy, is largely ineffective (1). This is consistent with the emerging view that a dysfunctional fibroproliferative response to some inciting form of lung injury is more important in the pathogenesis of IPF than is inflammation (31). The lung injury that is incurred in IPF results in significant damage to alveolar epithelial cells (AECs), disruption of basement membranes, fibroproliferation, extracellular matrix deposition, and the genesis of fibroblastic foci comprising phenotypically altered fibroblasts (1, 31). Consequently, the normal cell-cell interactions that are critical for mitigating fibrogenesis are disrupted.
Though best known for its ability to regulate immune and inflammatory cells, granulocyte-macrophage colony-stimulating factor (GM-CSF) also plays a key role in modulating wound repair and fibrosis. Experimental studies of wound healing demonstrate that GM-CSF facilitates wound contraction, recruits inflammatory cells, induces keratinocyte proliferation, and promotes reepithelialization (14). Furthermore, clinical studies indicate that recombinant human GM-CSF can promote the healing of refractory wounds of various sorts (12, 22, 28).
In initial studies examining the role of GM-CSF in bleomycin-induced
pulmonary fibrosis in mice, the intraperitoneal injection of GM-CSF
resulted in reduced collagen deposition in whole lung homogenates and
decreased fibrosing alveolitis histologically, whereas administration
of anti-GM-CSF antibody had the opposite result (29). We
have confirmed that anti-GM-CSF antibody worsens pulmonary fibrosis
(7); moreover, we have reported that GM-CSF expression is
diminished in whole lung and in AECs isolated from bleomycin-treated
rats (7). We subsequently reported that bleomycin-induced pulmonary fibrosis was exacerbated in mice with a targeted deletion of
the GM-CSF gene (GM-CSF/
mice), as determined by both
quantitative and histologic analysis (23). Interestingly,
levels of the antifibrotic eicosanoid prostaglandin E2
(PGE2) were significantly diminished in whole lung
homogenates and alveolar macrophage cultures from
GM-CSF
/
mice treated with bleomycin relative to those
of wild-type (GM-CSF+/+) mice (23). Addition
of exogenous GM-CSF to alveolar macrophage cultures from both
GM-CSF
/
and GM-CSF+/+ mice resulted in
significant increases in PGE2 production (23).
As IPF is increasingly being perceived as an
"epithelial-fibroblastic" disease (31), we wished to
further investigate the exaggerated susceptibility of
GM-CSF/
mice to bleomycin-induced pulmonary fibrosis by
specifically examining the function of their fibroblasts and AECs.
Because our previous work (23) suggested that regulation
of macrophage PGE2 synthesis represents one potential
mechanism by which GM-CSF modulates fibrosis, we hypothesized that a
similar defect in PGE2 synthesis exists in fibroblasts and
AECs from GM-CSF
/
mice and that key prostanoid
synthetic enzymes may be deficient. In this report we provide further
compelling evidence that GM-CSF plays an important role in regulating
fibrogenesis by its ability to affect the production of the potent
antifibrotic eicosanoid PGE2.
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MATERIALS AND METHODS |
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Mice.
Breeding pairs of GM-CSF/
and GM-CSF
/
mice with transgenic expression of GM-CSF under the SpC promoter
(SpC-GM mice) backcrossed eight generations onto the C57BL/6 background
were obtained from G. Dranoff and J. A. Whitsett (Cincinnati, OH)
and have been previously described (10, 15, 16). The mice
were bred in the University of Michigan Laboratory Animal Medicine
facilities under specific pathogen-free conditions. Control
C57BL/6 mice were obtained from Jackson Laboratories (Bar
Harbor, ME). Mice were used at 4-6 wk of age. All experiments were
approved by the University of Michigan Committee on the Use and Care of Animals.
Fibroblast purification. Murine lungs were perfused with 5 ml of normal saline and removed under aseptic conditions. Lungs were minced with scissors in DMEM complete media containing 10% fetal calf serum. Lungs from a single animal were placed in 10 ml of media in 100-cm2 tissue culture plates. Fibroblasts were allowed to grow out of the minced tissue, and when cells reached 70% confluence they were passaged using trypsin digestion. Fibroblasts were grown for 10-14 days (2-3 passages) before being used and were always used before passage 6.
AEC purification. Type II AECs were isolated by the method developed by Corti et al. (9). After anesthesia and heparinization, the mouse was exsanguinated, and the pulmonary vasculature was perfused via the right ventricle with 0.9% NaCl until the effluent was free of blood. The trachea was cannulated with 20-gauge tubing, and the lungs were filled with Dispase (1-2 ml, Worthington). Subsequently, 0.45 ml of low-melting-point agarose was infused via the trachea, and the lungs were placed in iced PBS for 2 min to harden the agarose. The lungs were then placed in 2 ml of Dispase and incubated for 45 min at 24°C. The lung tissue was subsequently teased from the airways and minced in DMEM with 0.01% DNase. The lung mince was gently swirled for 10 min and passed successively through 100-, 40-, and 25-µm nylon mesh filters. The cell suspension was collected by centrifugation and incubated with biotinylated antibodies (anti-CD32 and anti-CD45) recognizing bone marrow-derived cells. The cell suspension was incubated with streptavidin-coated magnetic particles and was then placed in a magnetic tube separator for removal of the bone marrow-derived cells. Mesenchymal cells were removed by overnight adherence in a petri dish. The nonadherent cells after this initial plating were plated at a density of 50,000 cells/well on 96-well plates coated with fibronectin. Cells were maintained in DMEM with penicillin-streptomycin and 10% fetal calf serum at 37°C in 5% CO2. The final adherent population included only 4% nonepithelial cells at day 2 in culture as determined by intermediate filament staining.
Enzyme immunoassay. Cell-free pulmonary fibroblast and AEC supernatants were collected following overnight culture and were analyzed by enzyme immunoassay for the predominant cyclooxygenase (COX) product PGE2, using a commercially available kit from Cayman Chemicals (Ann Arbor, MI).
Immunoblot analysis. Fibroblasts or AECs, after having been stimulated for 24 h with 10 ng/ml LPS, were detached by scraping into ice-cold lysis buffer (50 mM Tris · HCl, 25 mM KCl, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM leupeptin, pH 7.4), and lysates were prepared by sonication. Approximately 15 µg of total cellular protein (determined by a modified Coomassie blue dye binding assay; Pierce Chemicals, Rockford, IL) were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. Rainbow molecular weight markers as well as standards for cytosolic phospholipase A2 (cPLA2), COX-1, and COX-2 were run in parallel. After transfer to nitrocellulose membranes, membranes were incubated overnight with anti-cPLA2 (1:5,000 dilution; Genetics Institute, Cambridge, MA), anti-COX-1 (1:5,000 dilution; kind gift from Dr. W. Smith, Michigan State University) (32), or anti-COX-2 antisera (1:10,000 dilution; Cayman Chemical), followed by peroxidase-conjugated goat anti-rabbit IgG (1:5,000). Proteins of interest were detected by the enhanced chemiluminescence method (Amersham Pharmacia Biotech, Piscataway, NJ).
Fibroblast proliferation assays.
Fibroblasts purified from lungs of GM-CSF+/+ and
GM-CSF/
mice were cultured at 5,000 cells/well in
96-well flat-bottomed tissue culture plates in complete media (DMEM,
10% fetal calf serum, and 1% penicillin-streptomycin). Cells were
allowed to grow for 48 h in the presence or absence of
PGE2, after which time 10 µCi of
[3H]thymidine was added to each well for a final 16 h. Cells were harvested onto glass fiber filters using a cell
harvester, and incorporated radioactivity was determined by
beta-scintillation counting.
AEC-fibroblast proliferation assays. For fibroblast-AEC cocultures, AECs were purified as described and plated onto fibronectin-coated plates (50,000 cells/well) on day 2 postisolation. AECs were allowed to adhere for 24 h before being washed three times with 1× PBS. Fresh medium (DMEM, 10% fetal calf-serum, and 1% penicillin-streptomycin) containing fibroblasts was added (5,000 cells/well), and fibroblasts were allowed to grow in the presence or absence of AECs for 24-48 h. [3H]thymidine was added (10 µCi/well, Amersham) during the final 16 h of culture, and incorporated radioactivity was determined with a beta-scintillation counter. As purified AECs grow poorly in culture and incorporate only low levels of [3H]thymidine, proliferation counts reflect primarily fibroblast numbers (21, 37). Control cultures of AECs alone consistently incorporated <5% of the total counts measured in coculture.
Fluorescein isothiocyanate-induced pulmonary fibrosis. Pulmonary fibrosis was induced experimentally by the intratracheal injection of fluorescein isothiocyanate (FITC; Sigma, St. Louis, MO) (8, 24). Mice were anesthetized with pentobarbital sodium. The trachea was exposed and entered with a needle under direct visualization. FITC was dissolved in saline (21 mg of FITC in 10 ml of saline), vortexed extensively, and sonicated for 30 s before the slurry was transferred to multiuse vials. The vials were vortexed extensively before 50-µl aliquots were removed for intratracheal injection via a 26-gauge needle. Mice of each genotype were injected with either FITC or saline. On day 21 postinjection, mice were euthanized by CO2 asphyxiation and perfused via the heart with 5 ml of normal saline. Individual lung lobes were removed, and all five lobes from a single mouse were homogenized in 1 ml of saline and hydrolyzed by the addition of 1 ml of 12 N HCl. Samples were then baked at 100°C for 12 h. We then assayed aliquots (5 µl) by adding chloramine-T solution for 20 min followed by development with Erlich's reagent at 65°C for 15 min as previously described (33). Absorbance was measured at 550 nm, and the amount of hydroxyproline was determined against a standard curve generated using a known concentration of hydroxyproline standard. Results are presented as the values obtained in FITC-treated mice normalized to the values obtained in saline-treated mice.
Data analysis. Statistical significance was analyzed with the InStat 2.01 program (GraphPad Software) on a Power Macintosh G3. We used Student's t-tests to determine P values when comparing two groups. When comparing three or more groups, we performed ANOVA with a post hoc Bonferroni test to determine which groups showed significant differences. P < 0.05 was considered significant.
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RESULTS |
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Impaired production of PGE2 by
GM-CSF/
fibroblasts and AECs.
We first examined the ability of cultured fibroblasts from
GM-CSF+/+ and GM-CSF
/
mice to elaborate
PGE2. Cell-free supernatants were collected from
fibroblasts after 48 h and analyzed for PGE2 by
specific immunoassay. Basal levels of PGE2 produced by
GM-CSF
/
fibroblasts were approximately threefold lower
than those from GM-CSF+/+ fibroblasts (Fig. 1A,
P = 0.006).
|
Reduced expression of the key prostanoid synthetic enzymes
cPLA2, COX-1, and COX-2.
Given the impaired production of PGE2 by
GM-CSF/
pulmonary fibroblasts and AECs, we sought to
determine whether there was diminished expression of one or more of the
key prostanoid synthetic enzymes. In this analysis, we considered
cPLA2, the key enzyme responsible for hydrolysis of
arachidonic acid from membrane phospholipids, and both isoforms of
cyclooxygenase (COX-1 and COX-2), which initiate prostaglandin
synthesis from arachidonate. Immunoblot analysis of
GM-CSF+/+ pulmonary fibroblast lysates revealed (Fig.
2A) low-level expression of
all three enzymes. Stimulation with LPS resulted in a modest increase
in expression of cPLA2 and a dramatic increase in COX-2. LPS did not affect COX-1 levels. Analysis of GM-CSF
/
fibroblasts revealed relatively reduced basal levels of COX-1 and COX-2
and slightly higher levels of cPLA2. LPS augmented
expression of cPLA2 and COX-2, but to a far lesser extent
than in GM-CSF+/+ fibroblasts. COX-1 levels were again not
affected by LPS in the GM-CSF
/
fibroblasts. Immunoblot
analysis of GM-CSF+/+ AEC lysates (Fig. 2B)
revealed expression of cPLA2 and COX-1, but low basal
levels of COX-2. Stimulation with LPS resulted in a significant
increase in COX-2 levels but did not increase cPLA2 or
COX-1 expression. Analysis of GM-CSF
/
AEC lysates
revealed relatively reduced basal levels of cPLA2 and
COX-1. LPS stimulation increased cPLA2 and COX-2 levels
only minimally and had no effect on COX-1 levels. Thus GM-CSF
deficiency results in diminished basal and/or induced expression of key
prostanoid synthetic enzymes in both cell types.
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Fibroblasts from
GM-CSF/
mice exhibit increased proliferation.
Because pulmonary fibroblasts from GM-CSF
/
mice
exhibited a reduced capacity for synthesis of PGE2, which
can act in an autocrine fashion to downregulate fibroproliferation, we
hypothesized that GM-CSF
/
fibroblasts would proliferate
to a greater extent than GM-CSF+/+ fibroblasts.
Accordingly, we isolated pulmonary fibroblasts from mice of both
genotypes and examined their proliferation in response to 10% serum.
As estimated by incorporation of [3H]thymidine,
proliferation of GM-CSF
/
fibroblasts was greater
(P = 0.0046) than that of GM-CSF+/+
fibroblasts (Fig. 3).
|
Effect of exogenous PGE2 on
GM-CSF+/+
vs.
GM-CSF/
pulmonary fibroblast proliferation.
The increased proliferation observed (Fig. 3) in
GM-CSF
/
fibroblasts could, in principle, be related to
a diminished responsiveness to the suppressive effects of endogenous
PGE2. Fibroblasts were isolated from
GM-CSF
/
and GM-CSF+/+ mice. Cells were then
cultured for 48 h in the presence of 0.1 µM PGE2,
after which time proliferation was estimated by incorporation of
[3H]thymidine. The dose of 0.1 µM PGE2 was
chosen because this is a physiological concentration that represents an
optimal concentration for PGE2 receptor (EP) binding and
signaling. Indeed, dose response experiments (not shown) demonstrated
that this was the optimal dose for fibroblast inhibition assays in
wild-type cells. Fibroblasts of both genotypes were inhibited by
PGE2 (Fig. 4). There was no significant difference in the degree to which GM-CSF
/
fibroblasts were inhibited compared with GM-CSF+/+
fibroblasts. This lack of differential inhibition of fibroblast proliferation by exogenous PGE2 is consistent with our
finding that cells from the two genotypes had similar levels of mRNA
expression for PGE2 receptors EP1-4 (data not shown).
Thus enhanced proliferation of GM-CSF
/
fibroblasts is
not due to defects in PGE2 responsiveness but, rather,
reflects reduced production of PGE2 by these cells.
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Effect of GM-CSF add-back to fibroblasts and AECs.
We next wanted to determine whether the addition of GM-CSF to cultures
of GM-CSF/
fibroblasts and GM-CSF
/
AECs
could increase the production of PGE2 in these cells. To test the effects of exogenous GM-CSF administration on fibroblasts, we
added exogenous GM-CSF to GM-CSF
/
fibroblast cultures
in vitro. The results are shown in Fig.
6A and demonstrate that
exogenous GM-CSF administration increases the production of
PGE2 in these fibroblast cultures. This results in
decreased proliferative capacity in fibroblast cultures as well (Fig.
6B).
|
Transgenic overexpression of GM-CSF exclusively in AECs protects
from experimental pulmonary fibrosis.
We have previously shown that GM-CSF/
mice develop a
more exuberant fibrotic response to bleomycin than do wild-type
controls. Fibrosis can be induced experimentally by many agents, and we have previously documented that the particulate antigen FITC induces acute lung injury followed by pulmonary fibrosis (8, 24). We now report that GM-CSF
/
mice are more susceptible to
FITC-induced fibrotic responses (Fig. 7)
compared with wild-type mice. Interestingly, however, SpC-GM mice are
protected from FITC-induced pulmonary fibrosis compared with wild-type
animals (Fig. 7). This protection is likely related, at least in part,
to the increased production of PGE2 in these mice, as
levels of PGE2 in lung homogenates from SpC-GM mice
(20.6 ± 6.1 ng/ml) were approximately four times higher than levels in wild-type mice (5.1 ± 1.6 ng/ml) when measured during the fibroproliferative period (day 10, P = 0.06). Thus overproduction of PGE2 in vivo in the SpC-GM
mice can be attributed to its greater elaboration by AECs (Fig.
6C).
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DISCUSSION |
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In this study, we found that pulmonary fibroblasts and alveolar
epithelial cells isolated from mice with a targeted deletion of the
GM-CSF gene elaborate less of the antifibrotic prostanoid PGE2. This deficiency was associated with decreased basal
and LPS-stimulated levels of COX-1, COX-2 and, in the case of
GM-CSF/
AECs, cPLA2 key enzymes in the
biosynthesis of PGE2. GM-CSF
/
pulmonary
fibroblasts exhibited greater proliferation than did GM-CSF+/+ fibroblasts. GM-CSF
/
AECs were
less able to inhibit proliferation of pulmonary fibroblasts in
coculture than were GM-CSF+/+ AECs. Furthermore, we
demonstrated that the addition of GM-CSF back to AECs and fibroblasts
from GM-CSF
/
mice can restore PGE2
production and decrease fibroproliferation. Finally, we demonstrate
that overexpression of GM-CSF exclusively in AECs, resulting in
pulmonary overproduction of PGE2 in vivo, is sufficient to
protect mice from experimental pulmonary fibrosis.
There is abundant evidence in the literature that GM-CSF is important in wound repair. Experimental studies of wound healing have found that GM-CSF facilitates wound contraction, recruits inflammatory cells, induces keratinocyte proliferation, and promotes reepithelialization (14). GM-CSF has been used clinically to facilitate the healing of incisional wounds in animal models (6), as well as postsurgical and radiation-induced wounds (12). Additionally, randomized placebo-controlled trials have confirmed the utility of GM-CSF in chronic leg ulcers (22). As fibrotic lung diseases may be viewed as a manifestation of dysfunctional wound repair, a pathogenic role for GM-CSF is not surprising.
Nearly 10 years ago, Piquet et al. (29) found that
bleomycin-induced pulmonary fibrosis in mice was associated with
initially increased levels of GM-CSF mRNA followed by diminished levels at a later time point. They demonstrated that administration of GM-CSF
at days 7-15 protected mice from
bleomycin-induced pulmonary fibrosis, whereas anti-GM-CSF antibody
exacerbated collagen deposition. Recently our laboratory provided
additional data (7) supporting the important role of
GM-CSF in pulmonary fibrosis. Because AECs are a prominent source of
GM-CSF in the lung, we hypothesized that bleomycin-induced lung injury,
which damages AECs, would result in diminished levels of GM-CSF. We
found that GM-CSF levels were indeed reduced in rat lung homogenates
postbleomycin administration, with levels beginning to recover on
day 14. Type II AECs isolated from bleomycin-injured animals
also exhibited diminished capacity for GM-CSF synthesis, both basally
and on stimulation with LPS. Administration of anti-GM-CSF antibody to
rats at days 0, 4, and 8 postbleomycin
resulted in increased lung hydroxyproline levels, findings consistent
with those of Piquet et al. (29). In addition, we have
demonstrated that GM-CSF/
mice develop more severe
pulmonary fibrosis following bleomycin injury (23). These
data collectively suggest that GM-CSF is beneficial in the setting of
pulmonary fibrosis.
However, some investigators have raised the possibility that GM-CSF
might, in fact, contribute to pulmonary fibrosis. This suggestion is
based on experiments in which very high level murine GM-CSF expression
was induced in rat lungs by adenovirally mediated gene transfer
(35, 36). These investigators found that adenoviral transfer of GM-CSF to the lungs of rats led to the accumulation of
eosinophils and macrophages, tissue injury, and histological evidence
of fibrosis (35). They also found that adenoviral delivery of murine GM-CSF to rat lungs resulted in an increase in granuloma formation, fibroblast accumulation, and expression of the fibrogenic cytokine transforming growth factor-1 (36). In contrast
to these findings, several pieces of information suggest that GM-CSF itself is not directly profibrotic in the lung. First, transgenic overexpression of GM-CSF in the lung under control of the surfactant protein C promoter for the life of an animal does not result in pulmonary fibrosis (15, 16). Likewise, adenovirally
mediated transfer of murine GM-CSF into mice (38) or
aerosol treatment with GM-CSF in mice for extended periods
(30) does not induce pulmonary fibrosis. There are several
potential explanations for these apparently contradictory findings that
are related to species, timing, and dose. In the studies by
Xing et al. (35, 36), adenovirally transferred murine
GM-CSF was expressed at very high levels in the lungs of rats
transiently and at early time points. It is therefore possible that the
species divergence between cytokine and receptor or a host response to
the murine protein may have had some effect in these experiments.
Furthermore, the extremely high levels of GM-CSF used in these studies
suggest that the beneficial effects of GM-CSF may be realized only at
lower concentrations. Our previous studies have examined lung injury
induced by the fibrogenic agent bleomycin. It is possible that the
injury induced by adenoviral administration results in a different
pathological set of events. Finally, the timing of the GM-CSF
administration may be critical. The adenoviral gene transfer
experiments delivered GM-CSF simultaneously with the adenoviral
administration and induced only transient expression. This could be
considered an early time point postinjury, whereas previous experiments
have shown that exogenous GM-CSF was effective when given during the
fibroproliferative phase of bleomycin injury (days
7-15), a relatively later time point. Finally, our
experiments using SpC-GM mice demonstrate clearly that these mice are
protected from FITC-induced pulmonary fibrosis and that this protection
correlates with increased production of PGE2. Thus we
believe that, together, these data support the hypothesis that
exogenous GM-CSF may be beneficial in the treatment of pulmonary
fibrosis if given in appropriate doses and at appropriate time points postinjury.
The antifibrotic properties of PGE2 have been well
documented. PGE2 is known to inhibit fibroblast
proliferation in response to numerous mitogens (3, 11), as
well as to inhibit collagen synthesis (13, 19) and promote
its degradation (2). Bleomycin-induced pulmonary fibrosis
is exacerbated in COX-2/
mice (18) and in
normal mice treated with the COX inhibitor indomethacin
(23). Fibroblasts isolated from the lungs of
bleomycin-treated fibrotic rats have been shown to exhibit reduced
capacity for PGE2 synthesis (25). Furthermore,
reduced levels of PGE2 have been found in bronchoalveolar
lavage fluid (4), as well as conditioned medium obtained
from alveolar macrophages (26) and lung fibroblasts
isolated from patients with IPF (34).
A link between GM-CSF and PGE2 has been previously
established in vitro in macrophages, where GM-CSF was shown to increase the synthesis of a range of eicosanoids, including PGE2
(5). Utilizing GM-CSF/
mice, we performed
additional experiments to elucidate this relationship in vivo in the
context of fibrotic lung disease (23). We found that
GM-CSF
/
mice manifested an increased susceptibility to
bleomycin-induced pulmonary fibrosis as measured by both hydroxyproline
deposition and histological analysis. Furthermore, alveolar macrophages
isolated from GM-CSF
/
mice exhibited a diminished
capacity for the synthesis of PGE2, which was ameliorated
by the administration of exogenous GM-CSF. To our knowledge, this was
the first data linking GM-CSF and PGE2 within the context
of pulmonary fibrosis.
As pulmonary fibroblasts and AECs produce significant quantities of
PGE2 and are central effector cells in fibrotic lung
responses, the present study addressed PGE2 synthesis in
these cell types from GM-CSF/
mice. As previously
observed for lung macrophages, we found that GM-CSF
/
pulmonary fibroblasts elaborate less PGE2 than do
GM-CSF+/+ fibroblasts. This was associated with a relative
basal deficiency of COX-1 in the GM-CSF
/
fibroblasts.
Increased levels of COX-1 were not induced with LPS in either genotype,
consistent with the constitutive nature of COX-1. The inducible
isoform, COX-2, was upregulated by LPS in both genotypes, but to a
lesser degree in the GM-CSF
/
fibroblasts. Cytosolic
PLA2 levels were not deficient in GM-CSF
/
fibroblasts. This was of interest given that previous work from our
laboratory (5) demonstrated increased expression of
cPLA2 by alveolar macrophages treated with exogenous
GM-CSF. However, this effect was not observed in peritoneal macrophages
or peripheral blood monocytes, suggesting that regulation of
cPLA2 by GM-CSF is likely a cell-specific phenomenon.
Similarly, AECs from GM-CSF
/
mice demonstrated reduced
basal or LPS-induced expression of all three key prostanoid synthetic enzymes.
The effects of PGE2 are mediated by a family of G
protein-coupled receptors, EP1-4. As there is precedent for EP
receptor expression being regulated by PGE2 levels
(17), it is possible that increased proliferation of
GM-CSF/
fibroblasts reflected altered EP expression
and, therefore, diminished responsiveness to this suppressive
prostanoid. Our data do not support this hypothesis. Pulmonary
fibroblasts from both genotypes were inhibited to the same degree by
exogenous PGE2, and their EP receptors were expressed similarly.
Although cultured AECs are known to produce PGE2 (7,
10, 19, 25), Pan et al. (27) recently reported that
rat AECs inhibit human pulmonary fibroblast proliferation in coculture primarily by stimulating fibroblast PGE2 synthesis. We have
recently verified the importance of AEC-derived PGE2 with
respect to pulmonary fibroblast inhibition through the use of
COX-deficient mice (20). Our present studies utilizing
GM-CSF/
AECs in coculture with GM-CSF+/+
fibroblasts (Fig. 5) and the in vivo protection from FITC-induced fibrosis seen in SpC-GM mice further emphasize the importance of AECs
as a source of the antifibrotic prostanoid PGE2.
In sum, the previous and current studies suggest that GM-CSF production is crucial in limiting fibrotic responses. Furthermore, the kinetic results that have been reported support the hypothesis that GM-CSF expression drives the production of PGE2 in vivo. Neutralization of GM-CSF from days 0 to 8 postbleomycin is detrimental (29). The administration of GM-CSF from days 7 to 15 is beneficial (29), thus GM-CSF likely needs to be present at least by day 7 postbleomycin to be effective. This time point correlates well with the onset of fibroproliferation following bleomycin. We have demonstrated that the neutralization of PGE2 starting at day 10 postbleomycin during the fibroproliferative phase is also detrimental (23). Therefore, we suggest that GM-CSF expression by day 7 likely drives PGE2 production during the fibroproliferative phase (days 7-15), and thus this is the time point when the antifibrotic effects of PGE2 would be most beneficial.
In conclusion, pulmonary fibroblasts and AECs from
GM-CSF/
mice exhibit a reduced capacity for synthesis
of the antifibrotic prostanoid PGE2 with functional
consequences for fibrogenesis. These results extend our understanding
of the cells and mechanisms that are responsible for the antifibrotic
effects of GM-CSF in models of pulmonary fibrosis. Further elucidation
of the interplay between cytokines and eicosanoids may have important
implications for treatment of pulmonary fibrosis.
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ACKNOWLEDGEMENTS |
---|
The authors thank Carol Wilke and Teresa Marshall for expert
technical assistance, Jeff Whitsett for the gift of both the GM-CSF/
and SpC-GM mice, and William Smith for the gift
of the anti-COX-1 antibody.
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FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute Specialized Center of Research Grant in the pathobiology of fibrotic lung disease (P50HL-56402), National Institutes of Health Grants CA-79046 (B. B. Moore) and HL-51082 (G. B. Toews), as well as by Merit Review Awards from the Medical Research Service (G. B. Toews, P. J. Christensen, and R. Paine III) and Research Enhancement Award Program funds from the Department of Veterans Affairs.
Address for reprint requests and other correspondence: B. B. Moore, 6301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0642 (E-mail: Bmoore{at}umich.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 21, 2003;10.1152/ajplung.00350.2002
Received 21 October 2002; accepted in final form 13 February 2003.
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