Impaired synthesis of prostaglandin E2 by lung fibroblasts and alveolar epithelial cells from GM-CSFminus /minus mice: implications for fibroproliferation

Ryan P. Charbeneau1, Paul J. Christensen1, Cara J. Chrisman1, Robert Paine III1,2, Galen B. Toews1,2, Marc Peters-Golden1, and Bethany B. Moore1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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


    INTRODUCTION
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INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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ABSTRACT
INTRODUCTION
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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).


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Fig. 1.   A: granulocyte-macrophage colony-stimulating factor knockout (GM-CSF-/-) pulmonary fibroblasts secrete lower levels of PGE2 than do wild-type (GM-CSF+/+) fibroblasts. Pulmonary fibroblasts from the respective genotypes were isolated and cultured for 14 days. At this time point, cells were trypsinized and plated at 5,000 cells/well. Cell-free supernatants were collected after 48 h and analyzed for PGE2 by specific immunoassay (n = 6 wells per mouse, 3 mice per experiment, representative of 3 independent experiments, P = 0.006). B: GM-CSF-/- AECs secrete lower levels of PGE2 than do GM-CSF+/+ AECs. AECs from the respective genotypes were isolated by the method of Corti et al. (9). Cells were then cultured at 50,000 cells/well for 48 h before medium was changed. Cell-free supernatants were collected after 24 h and were analyzed for PGE2 by specific immunoassay (n = 6, representative of 3 independent experiments, P = 0.02).

Next, we examined the ability of AECs from GM-CSF+/+ and GM-CSF-/- mice to produce PGE2. Cell-free supernatants were collected and analyzed for PGE2 by specific immunoassay. The capacity for PGE2 synthesis by GM-CSF-/- AECs was significantly less (P = 0.02) than that by GM-CSF+/+ AECs (Fig. 1B).

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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Fig. 2.   A: GM-CSF-/- pulmonary fibroblast expression of the key prostanoid synthetic enzymes cyclooxygenase (COX)-1 and COX-2 is diminished compared with GM-CSF+/+ fibroblasts. Pulmonary fibroblasts from the respective genotypes were isolated and grown for 14 days in culture before being reseeded at 2 × 106 cells/well in 6-well plates. Cells were then cultured in the presence (LPS) or absence (Con) of 10 ng/ml LPS for 24 h before cellular lysates were made and immunoblot analyses were performed. Data are representative of 2 independent experiments. B: GM-CSF-/- alveolar epithelial cell (AEC) expression of the key prostanoid synthetic enzymes cytoplasmic phospholipase A2 (cPLA2), COX-1, and COX-2 is reduced compared with GM-CSF+/+ AECs. AECs from the respective genotypes were isolated by the method of Corti et al. (9). Cells were plated at 2 × 106/well in 6-well plates and were cultured in the presence or absence of 10 ng/ml LPS for 24 h. Immunoblot analyses were performed on cell lysates. Data are representative of 2 independent experiments.

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).


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Fig. 3.   GM-CSF-/- pulmonary fibroblasts proliferate more than do GM-CSF+/+ fibroblasts. Pulmonary fibroblasts from the respective genotypes were isolated and grown for 14 days in culture. At this time, cells were trypsinized and plated at 5,000 cells/well in a 96-well plate. Cells were cultured for an additional 48 h after which time cell proliferation was measured by incorporation of [3H]thymidine. Data represent 6 samples per group from 3 individual mice and are representative of 3 independent experiments (P = 0.0046).

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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Fig. 4.   Inhibition of GM-CSF+/+ vs. GM-CSF-/- pulmonary fibroblast proliferation by exogenous PGE2. Pulmonary fibroblasts from the respective genotypes were isolated and grown for 14 days in culture. Cells were then plated at 5,000 cells/well in a 96-well plate and were cultured for an additional 48 h in the presence or absence of 0.1 µM PGE2. Cell proliferation was then measured by incorporation of [3H]thymidine. Data represent n = 6 from 3 individual mice, representative of 3 independent experiments; P < 0.05 for both strains.

AECs are another important source of PGE2 that might affect fibroblast proliferation. To examine functional consequences of diminished PGE2 production by GM-CSF-/- AECs, we studied proliferation of GM-CSF+/+ fibroblasts in coculture with AECs from both genotypes. AECs from the respective genotypes were grown in coculture with GM-CSF+/+ pulmonary fibroblasts for 48 h, and fibroblast proliferation was then measured by incorporation of [3H]thymidine. As expected, fibroblast proliferation was inhibited by AECs of both genotypes, but to a lesser degree (P < 0.04) by GM-CSF-/- AECs (Fig. 5). This is consistent with the observation that GM-CSF-/- AECs produce less PGE2 and thus are less able to inhibit fibroblast proliferation.


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Fig. 5.   Inhibition of GM-CSF+/+ pulmonary fibroblast proliferation by GM-CSF+/+ vs. GM-CSF-/- AECs in coculture. AECs were isolated from the respective genotypes and plated at 50,000 cells/well in a 96-well plate and allowed to adhere for 24 h before the culture medium was removed and fresh medium containing fibroblasts was added. AECs and GM-CSF+/+ pulmonary fibroblasts (5,000 cells/well) were cocultured for 24-48 h, after which time cell proliferation was measured by incorporation of [3H]thymidine. Both GM-CSF+/+ and GM-CSF-/- AECs can inhibit fibroblast proliferation (P < 0.0001 for both). However, GM-CSF-/- AECs are significantly less able to inhibit fibroblast proliferation compared with GM-CSF+/+ AECs (P = 0.04).

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).


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Fig. 6.   GM-CSF addition restores PGE2 production and limits proliferation in fibroblasts from GM-CSF-/- mice. A: fibroblasts were purified from GM-CSF-/- mice and cultured at 5,000 cells/well in 96-well plates in the presence of media alone or media supplemented with 10 ng/ml GM-CSF. After 24 h of culture, supernatants were collected and PGE2 was analyzed by specific immunoassay. Exogenous GM-CSF increases PGE2 production in GM-CSF-/- fibroblasts (n = 6 representative of 2 independent experiments, P = 0.01). B: fibroblasts were purified and cultured as in A. After 24 h of incubation with or without GM-CSF, [3H]thymidine was added for 16 h, and proliferation was assessed. The addition of GM-CSF to the GM-CSF-/- fibroblasts significantly reduced proliferation consistent with the increased production of PGE2 noted in A (n = 6 representative of 2 independent experiments, P = 0.0015). C: GM-CSF addition restores PGE2 production in AECs from GM-CSF-/- mice. AECs were purified from wild-type GM-CSF+/+ mice or mice with transgenic expression of GM-CSF under the SpC promoter (SpC-GM mice). Cells were then cultured at 50,000 cells/well for 48 h before medium was changed. Cell-free supernatants were collected after 24 h and were analyzed for PGE2 by specific immunoassay (n = 4, representative of 2 independent experiments). SpC-GM AECs produce ~7 times the amount of PGE2 made by wild-type cells (P < 0.0001).

To examine the effect of GM-CSF addition to AECs, we utilized a genetically altered mouse that expresses the murine GM-CSF gene as a transgene under the control of the surfactant protein C promoter within GM-CSF-/- mice (SpC-GM mice) as previously described (16). These mice specifically overexpress GM-CSF in the alveolar epithelial cells within in the lung but do not express GM-CSF in any other cell within the body. The AECs in these animals produced approximately seven times more PGE2 than did AECs from normal mice (Fig. 6C). Thus the genetic addition of GM-CSF to AECs augments the production of PGE2 by these cells.

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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Fig. 7.   SpC-GM mice are protected from FITC-induced pulmonary fibrosis. GM-CSF+/+, GM-CSF-/-, or SpC-GM mice were injected with saline or FITC on day 0. On day 21, lungs were harvested and collagen content was determined via hydroxyproline assay. Results are presented as the amount of collagen measured in FITC-treated lungs normalized to the amount of collagen present in saline-treated lungs for each genotype. GM-CSF-/- mice develop worse fibrosis compared with GM-CSF+/+ mice (P < 0.01); however, SpC-GM mice are protected from the development of FITC-induced fibrosis compared with wild-type mice (P < 0.01) and GM-CSF-/- mice (P < 0.001) both (n = 6 animals per group representative of 2 experiments). There was no histological evidence of fibrosis in the saline-treated mice of any genotype.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 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.


    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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 284(6):L1103-L1111