Role of diminished epithelial GM-CSF in the pathogenesis of bleomycin-induced pulmonary fibrosis

Paul J. Christensen, Marc B. Bailie, Richard E. Goodman, Aidan D. O'Brien, Galen B. Toews, and Robert Paine III

Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan School of Medicine and Veterans Affairs Medical Center, Ann Arbor, Michigan 48105


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Evidence derived from human and animal studies strongly supports the notion that dysfunctional alveolar epithelial cells (AECs) play a central role in determining the progression of inflammatory injury to pulmonary fibrosis. We formed the hypothesis that impaired production of the regulatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) by injured AECs plays a role in the development of pulmonary fibrosis. To test this hypothesis, we used the well-characterized model of bleomycin-induced pulmonary fibrosis in rats. GM-CSF mRNA is expressed at a constant high level in the lungs of untreated or saline-challenged animals. In contrast, there is a consistent reduction in expression of GM-CSF mRNA in the lung during the first week after bleomycin injury. Bleomycin-treated rats given neutralizing rabbit anti-rat GM-CSF IgG develop increased fibrosis. Type II AECs isolated from rats after bleomycin injury demonstrate diminished expression of GM-CSF mRNA immediately after isolation and in response to stimulation in vitro with endotoxin compared with that in normal type II cells. These data demonstrate a defect in the ability of type II epithelial cells from bleomycin-treated rats to express GM-CSF mRNA and a protective role for GM-CSF in the pathogenesis of bleomycin-induced pulmonary fibrosis.

lung injury; growth factors; animal model; granulocyte-macrophage colony-stimulating factor; alveolar epithelial cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PULMONARY FIBROSIS is the consequence of a diverse collection of insults to the lung. It is likely that a complex set of cell-cell interactions are critically involved in determining whether the tissue response to the insult is healing, leading to restoration of the normal alveolar architecture, or progression to pulmonary fibrosis. Alveolar epithelial cell (AEC) injury is a universal feature of the pathological processes that lead to pulmonary fibrosis (reviewed in Refs. 20, 22). After injury and loss of type I cells, type II cells proliferate and migrate to cover the denuded alveolar basement membrane. Initially, hypertrophic type II cells restore the integrity of the alveolar epithelium. Regeneration of normal AECs is a critical step that determines whether the response to a pulmonary insult will be restoration of normal alveolar architecture or pulmonary fibrosis (1, 16, 34, 35).

There is limited information concerning the specific contributions of AECs to recovery or fibrosis after lung injury. Impaired or delayed type II cell proliferation and/or differentiation results in obliteration of the alveolar space as a consequence of apposition of denuded basement membranes or the accumulation of granulation tissue within the alveolar space, with subsequent proliferation of mesenchymal cells and deposition of connective tissue (29). Thus in the absence of normal regeneration of the alveolar epithelium, fibrosis ensues, and alveolar gas-exchange units are lost.

Other mechanisms through which AECs determine the outcome of a pulmonary insult are likely to involve regulatory interactions with stromal and inflammatory cells in the local environment. AECs express a number of factors that influence inflammatory cell or mesenchymal cell activity, including PGE2 (7), transforming growth factor (TGF)-beta (21), and insulin-like growth factors (15, 23). Of particular interest in the present context is the expression by AECs of granulocyte-macrophage colony-stimulating factor (GM-CSF). This growth factor exerts potent local regulatory effects on immune and inflammatory cells and plays a complex role in the processes of fibrosis and wound healing. When delivered subcutaneously, GM-CSF induces the accumulation of myofibroblasts (27, 32) and thus may promote fibrosis. In contrast, GM-CSF also enhances wound epithelialization (4, 18), which has beneficial effects on wound repair. In bleomycin-induced pulmonary fibrosis in mice, administration of GM-CSF reduced deposition of hydroxyproline while increasing the number of macrophages recovered by bronchoalveolar lavage (26). Thus GM-CSF plays a role in wound healing either through effects on stromal cells or by changing the number or phenotypic state of mononuclear cells.

The lung is a rich source of GM-CSF. Murine GM-CSF was first purified and cloned from lungs of endotoxin-treated mice (6, 14). Although macrophages, interstitial cells, and epithelial cells in cultured human lung explants all stained positively for GM-CSF (24), work from our laboratory (9) has demonstrated that AECs produce significant amounts of GM-CSF.

Based on the diverse activities of GM-CSF and its expression in AECs, we have formed the hypothesis that in circumstances that lead to pulmonary fibrosis, AEC injury results in diminished expression of GM-CSF and that this decreased expression contributes to the development of fibrosis rather than normal recovery. To test this hypothesis, we used a well-characterized model of bleomycin-induced pulmonary fibrosis in the rat (reviewed in Ref. 31). We now demonstrate that GM-CSF mRNA expression is diminished after bleomycin-induced lung injury and that fibrosis is worsened if GM-CSF activity is neutralized. When type II cells were purified from lungs of rats treated with bleomycin, the expression of GM-CSF mRNA in response to in vitro stimulation with endotoxin was significantly reduced compared with expression in type II cells from control animals. Thus impaired AEC GM-CSF expression is likely to play an important role in the progression to pulmonary fibrosis after bleomycin-induced lung injury.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Specific pathogen-free male Fischer 344 rats weighing 125-150 g were obtained from Charles River Laboratories. Rats were kept in isolation cages and received water and rat chow ad libitum. All protocols were approved by the animal care committees at the University of Michigan School of Medicine and the Ann Arbor Veterans Affairs Medical Center.

Experimental protocol. Briefly, rats were anesthetized via intraperitoneal injection of 20 mg of ketamine and xylazine. The rats were placed in a dorsal recumbent position and suspended by the incisors at a 60° angle. A midline incision was made in the neck, and the trachea was exposed by blunt dissection. A 22-gauge angiocatheter was inserted into the trachea under direct visualization, and bleomycin (0.75 U/100 g) was administered in 200 µl of saline. Control rats received an identical volume of saline. After instillation, the catheter was removed and the incision was closed with a wound clip.

Semiquantitative RT-PCR. We based our RT-PCR method on controlling the concentration of total RNA applied in each sample compared with the relative quantities of specific PCR products among samples (13). Within a given experiment, all samples were subjected to the same reagent mixtures and treatments. The RT reactions were run as bulk reactions, primed with oligo(dT). Products were then divided into aliquots for all PCRs, minimizing the variation caused by differences in RT efficiencies and RNA loading. For RT, samples were mixed with an aliquot of a single reaction mixture with a final concentration of 50 mM KCl, 10 mM Tris·HCl (pH 8.3 at 25°C), 5 mM MgCl2, 1 mM each nucleotide (dGTP, dATP, dTTP, and dCTP; Boehringer Mannheim), 2.5 mM oligo(dT)18, 1 U/ml of RNasin (Promega), and 2.5 U/ml of Moloney murine leukemia virus RT (GIBCO BRL) in addition to RNA (0.05-50 ng/ml). Samples were reverse transcribed for 45 min at 42°C. After heat denaturation and cooling, the Moloney murine leukemia virus reaction products were divided into aliquots (5 µl), placed in 96-well v-bottom polycarbonate plates for each PCR, and stored at -20°C.

PCRs were performed with the use of previously described methods (13). The final concentration of reagents was 2 mM MgCl2, 50 mM KCl, 10 mM Tris·HCl (pH 8.3 at 25°C), 0.25 U/ml of Taq DNA polymerase (GIBCO BRL), and 0.2 mM sense and antisense oligonucleotide primers specific for the cDNA species. The primers for rat GM-CSF were 5'-gaggatgtggctgcagaa-3' (sense) and 5'-agtggctggctatcatgg-3' (antisense). The primers for monocyte chemoattractant protein (MCP)-1 were 5'-tctctgtcacgcttctgg-3' (sense) and 5'-gcttgaggtggttgtgga-3' (antisense). Annealing temperatures and the number of cycles of amplification were determined empirically. Amplification was accomplished with an OmniGene thermal cycler (Hybaid). Amplification was stopped below the plateau phase. Samples of each reaction product were electrophoresed, stained with 25 ng/ml of ethidium bromide, and photographed with long-wave ultraviolet illumination. The DNA was transferred from the gels to positively charged nylon membranes for hybridization with 3'-digoxigenin end-labeled gene-specific oligonucleotide probes homologous to regions internal to the amplification primers. For GM-CSF, the probe was 5'-aagaagctctgagcctcct-3'; for MCP-1 the probe was 5'-tctcagccagatgcagtt-3'. Primers and probes were designed with the aid of the Primer 2 program (Scientific and Educational Software, Stateline, PA) from cDNA sequences (rat GM-CSF, GenBank accession no. U00620; rat MCP-1, GenBank accession no. M57441). All oligonucleotides were synthesized at the University of Michigan DNA Core Facility. Signals were detected by chemiluminescence with anti-digoxigenin-coupled alkaline phosphatase and Lumigen PPD (Boehringer Mannheim) with the use of multiple exposures of X-ray film (Fuji Film). The resulting films were analyzed for band intensity with a video imaging system with the program Image 1.55 (National Institutes of Health, Bethesda, MD). For semiquantitative analysis, sample densitometry values were converted to relative concentrations of RNA by extrapolating from the regression curve of the dilution series of positive controls. Thus relative concentrations were estimated by comparison with the products of stimulated rat splenocytes, which were tested at multiple concentrations to produce a standard curve similar to that used in ELISA.

Determination of hydroxyproline content of lung. The hydroxyproline content of rat lung was determined by standard methods (30). The pulmonary vasculature was perfused with PBS until free of blood. Lung tissue was excised, trimmed free of surrounding conducting airways, and homogenized in 2 ml of PBS. A 1-ml aliquot of lung homogenate was hydrolyzed in 6 N HCl (0.5 ml of homogenate and 0.5 ml of 12 N HCl) at 110°C for 12 h; 50-µl aliquots were added to 1 ml of 1.4% chloramine T (Sigma, St. Louis, MO), 10% n-propanol, and 0.5 M sodium acetate, pH 6.0. After 20 min at 22°C, 1 ml of Ehrlich's solution (1 M p-dimethylaminobenzaldehyde in 70% n-propanol and 20% perchloric acid) was added and allowed to incubate at 65°C for 15 min. Absorbance was measured at 550 nm, and the amount of hydroxyproline was determined against a standard curve generated with the use of known concentrations of reagent-grade hydroxyproline (Sigma).

Rabbit anti-rat GM-CSF antibody. To fully understand the biology of GM-CSF in rat systems, it was necessary to develop new reagents. We have cloned, expressed, and purified functional rat GM-CSF. With this recombinant molecule, we have generated specific antibodies that recognize and neutralize rat GM-CSF. Rat GM-CSF was cloned with the use of the PCR (9). The rat sequence is 81% homologous to the murine GM-CSF cDNA and 65% homologous to the human GM-CSF cDNA. The predicted protein for rat GM-CSF, excluding the putative leader sequence, is 67% homologous to murine GM-CSF, with significant dissimilarity between the rat and murine sequences in areas thought to be critical for receptor binding (5, 28).

Recombinant rat (rr) GM-CSF was expressed in a bacterial expression system (pET System, Novagen, Madison, WI). A fusion protein with six consecutive histidines at the amino terminus was expressed, allowing single-step purification by metal chelation chromatography (Novagen). The eluted protein was detected as a single band at 19 kDa by silver stain of an SDS-PAGE gel and was recognized by the goat anti-murine GM-CSF antibody in Western blot analysis. This purified protein was biologically active, stimulating macrophage proliferation (stimulation index = 60) and macrophage anti-cryptococcal activity (data not shown). The rrGM-CSF was then used to immunize rabbits. The hyperimmunized serum recognized rrGM-CSF in a direct ELISA at titers >1:1,000,000 and blocked the induction of macrophage DNA synthesis at titers of 1:100 and above.

Polyclonal IgG was purified from both nonimmune rabbit serum and anti-rrGM-CSF rabbit serum with a protein A agarose column (Pierce, Rockford, IL) according to the manufacturer's instructions.

Isolation and culture of type II AECs. Type II cells from both bleomycin-treated and saline-treated rats were isolated by elastase cell dispersion and IgG panning (10). Briefly, the rats were anesthetized, the trachea was cannulated, and the pulmonary circulation was perfused free of blood with a balanced salt solution at 4°C. After multiple whole lung lavages with EGTA (1 mM) in a balanced salt solution, porcine pancreatic elastase (4.3 U/ml; Worthington) was instilled via the trachea to release type II cells. Contaminating cells bearing Fc receptors were removed from the cell suspension by panning on plates coated with rat IgG (Sigma). Unless otherwise stated, the cells were plated on tissue culture-treated plastic dishes or in Lab-TEK slides at 2 × 105 cells/cm2 in DMEM supplemented with penicillin-streptomycin (GIBCO BRL) and 10% newborn calf serum (Sigma). Cells were cultured at 37°C in 7% CO2.

Immunofluorescence microscopy. Isolated cells were evaluated by immunofluorescence staining with the lectin Bandeiraea simplicifolia-I (BS-I), which recognizes rat macrophages and some basal bronchial epithelial cells (33); anti-vimentin, which recognizes the intermediate filament protein found in mesenchymal cells (fibroblasts and endothelial cells) and cells of hematopoietic origin but not epithelial cells; anti-cytokeratin 8, which recognizes one of the cytokeratins found in AECs (25) but is not present in macrophages, fibroblasts, and endothelial cells; and rabbit anti-surfactant protein (SP)-A, which recognizes SP-A (gift from Dr. J. Shannon, University of Colorado, Denver, CO). Control cell included mouse ascites fluid and rabbit nonimmune IgG. For immunofluorescence microscopy, cytospin preparations of freshly isolated cells or monolayers of cells adherent to Lab-TEK slides were fixed with methanol (precooled to -20°C) for 20 min followed by brief dipping in acetone (-20°C). After hydration, nonspecific binding sites were blocked by incubation with PBS containing 1% BSA and 10% newborn calf serum. Subsequently, the cells were incubated with primary antibody for 30 min at 37°C. After being washed, the cells were incubated with tetramethylrhodamine B isothiocyanate-conjugated goat anti-murine or goat anti-rabbit IgG for 30 min. The cells were then washed extensively. The lectin BS-I was directly conjugated to FITC. The cells were viewed on a Nikon Labphot 2 microscope equipped for epifluorescence. To determine the proportion of nonepithelial cells in preparation, the number of vimentin-positive cells present among cells in 10 random fields was enumerated, counting at least 400 cells total. Complementary results were obtained by counting the number of cytokeratin-positive cells.

Statistical analysis. Data are expressed as means ± SE. Groups were compared with a two-tailed t-test for unpaired samples with Statview 4.5. Comparisions were deemed significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of GM-CSF mRNA in the lungs of bleomycin-treated rats. To explore the role of GM-CSF in the pathogenesis of pulmonary fibrosis, we used bleomycin to induce pulmonary fibrosis in the rat. Control rats received an identical volume of saline alone. Bleomycin consistently induced marked early inflammation (days 1-3) followed by the development of multifocal fibrosis (data not shown).

GM-CSF is expressed in the normal lung by AECs (9, 24). Bleomycin has major effects on these AECs. Therefore, we examined the expression and time course of GM-CSF mRNA in the lungs of mice treated with bleomycin for the purpose of inducing pulmonary fibrosis. Total lung RNA was extracted on days 1, 3, 5, 7, 10, 14, and 28, and GM-CSF mRNA expression was determined by RT-PCR (Fig. 1). GM-CSF mRNA was readily detected in saline-treated control animals, with no change in the level of expression over the 28 days of the experiment. In contrast, in bleomycin-treated animals, there was a consistent but transient reduction in GM-CSF mRNA expression. This reduction was first apparent on day 3 and was maximal on days 5 and 7, with recovery beginning on day 14. Thus lung expression of GM-CSF was impaired for a discrete period during the phase of acute lung injury and early in the development of fibrosis after instillation of bleomycin.


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Fig. 1.   Time course of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA expression after intratracheal saline or bleomycin challenge. Rats were given intratracheal sterile saline or bleomycin on day 0. Animals were killed, and lung RNA was prepared for PCR analysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and GM-CSF at indicated time points as described in METHODS. A representative experiment of 3 separate experiments is shown.

Effect of neutralizing antibodies to rat GM-CSF on the hydroxyproline content of lungs after bleomycin-induced pulmonary injury. Having demonstrated a reduction in GM-CSF mRNA after bleomycin-induced injury, we sought to determine directly the role of GM-CSF in the development of pulmonary fibrosis in this model. Rats were treated with intratracheal bleomycin or saline as described in METHODS. In addition, rats received either anti-rat GM-CSF IgG or control IgG at 10 mg/dose by intraperitoneal injection on days 0, 4, and 8 (Fig. 2A). On day 14, the animals were weighed, and cell counts and differential counts were determined by total lung lavage. Hydroxyproline content of the lung was determined as a measure of relative fibrosis. Fibrosis is typically rather mild 14 days after bleomycin compared with 21-28 days; however, we chose this relatively early time point to avoid questions concerning persistent effects of the neutralizing antibody at times after the restoration of normal GM-CSF mRNA levels in the bleomycin-treated animals. In saline-challenged animals, there were no differences between control IgG or anti-GM-CSF IgG with regard to cell count, differential count, or hydroxyproline content of the lung. Therefore, the results for these control animals are shown as pooled data. There were significant differences in weight between the control and bleomycin-treated rats at the completion of the experiment (Fig. 2B). The rats treated with anti-GM-CSF lost more weight than those that received control IgG, although the difference did not achieve significance. As expected, animals treated with bleomycin had higher levels of hydroxyproline than saline-treated control animals, indicating early fibrosis (Fig. 3). Bleomycin-treated animals that received anti-GM-CSF IgG developed significantly more hydroxyproline accumulation in the lung compared with animals that received control IgG. Thus neutralization of GM-CSF resulted in more severe fibrosis 2 wk after intratracheal bleomycin.


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Fig. 2.   Effect of neutralizing GM-CSF on weight change in rats treated with intratracheal bleomycin (bleo). A: schematic diagram showing the timing of intraperitoneal administration of anti-GM-CSF (alpha -GM-CSF) or control (Ctl) IgG antibodies to bleomycin-treated rats. BAL, bronchoalveolar lavage; HP, hydroxyproline. B: weight change after intratracheal bleomycin with and without neutralization of GM-CSF. Data are means ± SE expressed as change vs. baseline (day 0) weight; n = 6 rats. * P < 0.05 compared with control. The difference in weight between bleomycin+IgG and bleomycin+anti-GM-CSF was not significant.



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Fig. 3.   Lung hydroxyproline content after intratracheal bleomycin with and without neutralization of GM-CSF. Rats were given intratracheal bleomycin or saline (sal) on day 0, and intraperitoneal anti-GM-CSF IgG (alpha -GM-CSF) or control IgG was administered. Animals were killed, and lung hydroxyproline content was determined on day 14. Data are means ± SE; n = 6 rats. * P < 0.05 compared with bleomycin+control IgG.

Effect of neutralizing antibodies to rat GM-CSF on the number and type of inflammatory cells in the alveolar space after bleomycin-induced pulmonary injury. Bleomycin induces an inflammatory response in the lung that is characterized by the increased numbers of macrophages, neutrophils, and eosinophils recovered by bronchoalveolar lavage. To determine whether the changes in hydroxyproline content induced by neutralization of GM-CSF were associated with changes in alveolar inflammation, total lung lavage was performed on bleomycin-treated rats that had received anti-GM-CSF IgG or control IgG. On day 14 after bleomycin, significantly fewer lavagable cells were recovered from the rats treated with anti-GM-CSF IgG compared with those from control IgG-treated animals (Fig. 4). This difference was largely attributable to a significant reduction in the number of macrophages in the anti-GM-CSF group. There was a trend toward increased numbers of neutrophils and eosinophils in the anti-GM-CSF group, but this difference did not achieve significance. Thus neutralization of GM-CSF during the development of fibrosis after intratracheal bleomycin resulted in an alteration in the number of inflammatory cells in the alveolar space due to a decreased number of macrophages.


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Fig. 4.   Number of inflammatory cells recovered from the alveolar space of rats after intratracheal bleomycin with and without neutralization of GM-CSF. Rats were given intratracheal bleomycin or saline on day 0, and intraperitoneal alpha -GM-CSF or control IgG was administered. On day 14, cell counts were determined in BAL fluid. A: total cell number in BAL fluid; n = 6 rats. * P < 0.05 compared with bleomycin+control IgG. B: no. of macrophages (AM), neutrophils (PMN), lymphocytes (lymph), and eosinophils (eos) in BAL fluid; n = 6 rats. NS, not significant. * P < 0.05 compared with bleomycin+control IgG.

Expression of GM-CSF mRNA by type II cells isolated from bleomycin-injured animals. The reduction in expression of GM-CSF mRNA in lung homogenates of animals that develop pulmonary fibrosis after bleomycin exposure might represent an effect of bleomycin on AEC GM-CSF expression. To address this question, experiments were performed in which AECs were isolated from bleomycin-injured and control rats and stimulated to induce GM-CSF. The expression of GM-CSF mRNA was determined by RT-PCR. Type II cells were isolated from rats 7 days after intratracheal instillation of bleomycin or saline. We chose this time because it is the point at which GM-CSF mRNA expression is decreased in whole lung homogenates. Staining immediately after the isolation procedure revealed that >90% of the cells from both control and bleomycin-treated animals were epithelial cells, with small numbers of contaminating macrophages and fibroblasts (Table 1). Phase-contrast microscopy (data not shown) and immunofluorescence staining for SP-A indicated that there were differences between the type II cells from bleomycin-treated animals and those from the saline-treated control animals (Fig. 5). The type II cells from bleomycin-treated animals were larger, with more heterogeneous staining for SP-A than control cells. This indicates that type II cells from the bleomycin-treated animals have been influenced in the experiment and do not represent selection of an unaffected subpopulation.

                              
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Table 1.   AECs isolated from rats 7 days after bleomycin treatment



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Fig. 5.   Immunfluorescence microscopy showing type II cells from bleomycin-treated and control rats stained for surfactant protein A (SP-A) and cytokeratin 8. Type II cells were isolated 7 days after intratracheal saline (A and C) or bleomycin (B and D). Cytospin preparations of freshly isolated cells were stained for SP-A (A and B) and photographed at equal magnification (×40 objective). Cells were fixed and stained for cytokeratin 8 after 2 days in culture (C and D; ×20 objective).

AECs from bleomycin-injured animals formed confluent monolayers on tissue culture-treated plastic dishes within ~48 h. There was extensive cytokeratin staining throughout the monolayer, with small numbers of contaminating cells (Fig. 5). Thus type II cells can be isolated from bleomycin-injured rats with good yield and high purity. Having obtained viable type II cells from rats on day 7 after bleomycin instillation, it was possible to determine whether altered type II cell function accounted for the reduction in lung GM-CSF mRNA. The relative expression of GM-CSF mRNA by type II cells from bleomycin-injured and control rats was determined by Northern blot analysis (Fig. 6). Type II cells isolated from normal rats expressed abundant GM-CSF mRNA immediately after isolation, whereas cells isolated from bleomycin-injured rats demonstrated reduced expression of GM-CSF mRNA compared with these control animals. Thus the diminished level of GM-CSF mRNA found in the lungs of bleomycin-treated rats is likely to be, in part, a reflection of diminished expression by type II cells.


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Fig. 6.   Expression of GM-CSF mRNA by type II cells isolated from bleomycin-treated and control rats. Rats were innoculated with intratracheal bleomycin or saline, and type II cells were isolated 7 days later. Total cellular RNA was extracted on the day of isolation (day 0) or day 2. Relative expression of GM-CSF mRNA was determined by Northern blot analysis. Equal loading was confirmed by equivalence of 18S and 28S ribosomal bands in the ethidium bromide-stained gel.

We next performed experiments to determine whether type II cells isolated from bleomycin-treated rats were induced to express GM-CSF mRNA normally after stimulation with endotoxin. AECs secrete GM-CSF and express GM-CSF mRNA in response to various inflammatory stimuli (Christensen, unpublished observations). The particular signals evaluated here were tumor necrosis factor (TNF) and lipopolysaccharide (LPS). AECs from bleomycin- or saline-treated rats (n = 4/group) were exposed to LPS (100 ng/ml) or TNF (10 ng/ml) for 3 h on day 2 in culture to induce GM-CSF mRNA expression. In preliminary studies, we have found that this duration of exposure provides optimal induction of GM-CSF mRNA. Total cellular RNA was extracted, and relative GM-CSF mRNA expression was determined by semiquantitative RT-PCR. As shown in Fig. 7A, the TNF-induced expression of GM-CSF mRNA was no different in control type II cells than in type II cells isolated from bleomycin-injured rats. In contrast, after stimulation with LPS, there was a significant reduction in GM-CSF mRNA expression in the type II cells isolated from bleomycin-treated rats compared with that in control animals. These data indicate that after bleomycin treatment in vivo, AECs acquire an abnormality in GM-CSF expression in response to some but not all stimuli. These data also support the conclusion that the change in GM-CSF mRNA expression after bleomycin is attributable, at least in part, to changes in expression at the level of the individual AECs.


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Fig. 7.   Relative expression of GM-CSF (A) and monocyte chemoattractant protein (MCP)-1 (B) mRNA by type II cells from bleomycin- and saline-treated rats stimulated in vitro. Type II cells were isolated from saline control and bleomycin-treated rats on day 7. After 2 days in culture, the cells were exposed to tumor necrosis factor (TNF; 10 ng/ml) or lipopolysaccharide (LPS; 100 µg/ml) for 3 h. Total cellular RNA was extracted, and relative expression of GM-CSF and MCP-1 were determined by semiquantitative RT-PCR as described in METHODS. Data are means ± SE of relative concentrations of stimulated spleen RNA; n = 4 rats/group. * P < 0.05 compared with saline control.

To determine whether the change in GM-CSF expression in AECs isolated from the bleomycin-treated animals was a reflection of a more widespread change in ability to express inflammatory factors, we determined the relative expression of MCP-1 mRNA by the epithelial cells after stimulation with LPS and TNF. As shown in Fig. 7B, the induction of MCP-1 mRNA in response to both LPS and TNF was the same in type II cells isolated from bleomycin-treated and control rats. Thus bleomycin did not induce a global effect on type II cell responses to LPS.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study examines the hypothesis that alteration in AEC expression of GM-CSF plays an important role in the development of pulmonary fibrosis after intratracheal administration of bleomycin in the rat. We found a consistent reduction in expression of GM-CSF mRNA in the lungs of rats during the first week after intratracheal administration of bleomycin. When residual GM-CSF activity was neutralized by the administration of anti-GM-CSF antibody to bleomycin-treated rats, the level of fibrosis was increased, demonstrating that GM-CSF can play a protective role in this model of fibrotic lung disease and indicating that the reduction in GM-CSF expression can contribute to the development of pulmonary fibrosis. In addition, type II AECs isolated 1 wk after bleomycin injury had a specific defect in the capacity to produce GM-CSF mRNA. Together, our data indicate that abnormalities in AEC function may play an important role in determining whether the result of lung injury is fibrosis or restoration of normal lung architecture.

Our studies indicate that, when present at appropriate sites and in appropriate concentrations, GM-CSF may promote normal healing and protect against fibrosis after lung injury. Previous studies in extrapulmonary organs have defined a complex role for GM-CSF in fibrosis and wound healing. The subcutaneous infusion of GM-CSF in rats induced accumulations of macrophages and fibroblasts at the infusion site (27, 32). Fibroblasts of the myofibroblast (fibrotic) phenotype were prominent, suggesting that GM-CSF may be a profibrotic agent. In contrast, when GM-CSF was administered intradermally to leprosy patients with skin wounds, there was enhanced wound healing with increased number and layers of keratinocytes (18). Interestingly, these changes were not found when GM-CSF was delivered subcutaneously, suggesting that the site of activity is a critical determinant of the effect of GM-CSF.

Studies in the context of pulmonary fibrosis indicate that GM-CSF may play an important but complex role in healing in the lung. Overexpression of murine GM-CSF by adenovirally mediated gene transfer in the rat resulted in pulmonary fibrosis (36, 37). However, our studies showing that neutralization of GM-CSF in bleomycin-treated rats worsens pulmonary fibrosis indicate that at appropriate concentrations this cytokine favors normal healing. This postulate is supported by the findings of Piguet et al. (26) that administration of GM-CSF reduced deposition of hydroxyproline in response to bleomycin in mice. Thus our data provide further evidence that GM-CSF plays a complex role in wound healing; whether the inflammatory insult results in scarring or healing is likely to be determined by the quantity, location, and kinetics of GM-CSF expression.

The lung is a relatively rich source of GM-CSF. Murine GM-CSF was cloned from lungs of endotoxin-treated mice (6, 14). GM-CSF is an extremely potent growth factor that acts locally. The protein is not detectable in serum, even during systemic inflammation (12). Not surprisingly, we could not measure the level of GM-CSF protein in the lung. The sensitivity of our assay is not sufficient to measure GM-CSF protein in vivo, even in the resting state.

Intratracheal administration of bleomycin in rats leads to acute lung injury followed by abnormal healing with pulmonary fibrosis (22). Bleomycin causes severe damage to type I epithelial cells followed by type II epithelial cell proliferation and differentiation. Subsequently, fibrosis develops in alveolar regions lined with hyperplastic type II cells (19). Several lines of evidence have indicated that AECs are likely to play a pivotal role in determining whether pulmonary fibrosis develops after an acute injury (34). One means by which AECs might exert this influence is through the expression of GM-CSF. Immunohistochemical detection of GM-CSF in cultured human lung reveals staining of several cells including type II AECs (24). In addition, significant GM-CSF bioactivity has been documented (9) in culture supernatants from rat AECs in vitro. We have now examined type II cells isolated 7 days after in vivo exposure to bleomycin and have found diminished expression of GM-CSF in response to stimulation with LPS. These data suggest that in bleomycin-induced lung injury there is a sustained abnormality in alveolar epithelial GM-CSF expression that contributes to the development of fibrosis.

The ability to isolate and culture type II cells from the lungs of bleomycin-treated animals provides an important new opportunity for mechanistic studies of the role of AECs in pulmonary fibrosis. Our in vitro data provide strong evidence that the decrease in GM-CSF expression found in vivo is not merely a consequence of AEC loss but represents a more widespread effect on the hypertrophic type II cells that survive. In support of this conclusion, in an immune complex model of inflammatory lung injury that does not lead to fibrosis in the rat, we found no change in GM-CSF mRNA expression in vivo or in vitro compared with that in saline-treated control animals (data not shown). Similarly, the expression of MCP-1 mRNA by AECs in vitro was not impaired in cells isolated from bleomycin-injured lungs compared with that in cells from control lungs, indicating that the reduced expression of GM-CSF was not a reflection of a widespread inability of the injured cells to express cytokines. Our studies indicate that the diminished expression of GM-CSF mRNA by AECs from bleomycin-exposed rats is not simply a reflection of a global defect in cytokine expression. The explanation for this "specificity" is not yet clear. It is possible that bleomycin-induced injury may influence the expression of intracellular signaling pathways that lead to GM-CSF transcription. Alternatively, the turnover of GM-CSF mRNA is tightly regulated by sequences in the 3'-untranslated region of the GM-CSF gene. An attractive hypothesis might be that bleomycin treatment results in diminished stability of GM-CSF mRNA in hypertrophic type II AECs. Further studies will need to be performed to answer these questions.

In bleomycin-treated animals in which GM-CSF activity was blocked with neutralizing antibody, we found a decreased number of alveolar macrophages compared with those in animals receiving control IgG. Alveolar macrophages are pluripotent cells; they can increase inflammation and injury through the release of mediators such as TNF or reactive oxygen and nitrogen intermediates. However, these cells also may play an important role in normal recovery through degradation of provisional matrix within the alveolar space or through the elaboration of epithelial cell growth factors such as hepatocyte growth factor/scatter factor. Thus GM-CSF may play its beneficial role after an acute pulmonary insult through effects on alveolar macrophages. GM-CSF can influence macrophage number through induction of proliferation (2, 8), inhibition of apoptosis (3), or stimulation of chemotaxis (24a). In a recent study (11), mutant mice carrying a homozygous null allele of the GM-CSF gene developed progressive accumulation of surfactant lipids and proteins in the alveolar space, likely due to abnormalities in alveolar macrophage function. When the GM-CSF gene was reintroduced into the germ line under a pulmonary epithelial cell-specific promoter, the lungs of these mice remained normal, suggesting that the production of GM-CSF by pulmonary epithelial cells alone is sufficient to reverse this abnormality (17). Finally, GM-CSF itself induces proliferation in keratinocytes. It is possible that this growth factor plays an autocrine role in preservation of the alveolar epithelium.

In summary, we have shown that GM-CSF mRNA expression is diminished after the intratracheal administration of bleomycin in rats before the development of pulmonary fibrosis. Neutralization of GM-CSF activity led to increased fibrosis in response to bleomycin. This change was associated with a decreased number of macrophages in the alveolar space. Finally, type II cells isolated from bleomycin-injured rats demonstrated decreased expression of GM-CSF mRNA in response to in vitro stimulation with LPS, indicating that the in vivo exposure to bleomycin led to an alteration in the ability of hyperplastic type II cells to produce this growth factor. These data provide strong evidence that a defect in AEC expression of GM-CSF can play an important role in determining the progression to fibrosis after lung injury.


    ACKNOWLEDGEMENTS

We thank Steven Wilcoxen and Roderick McDonald for excellent technical assistance and Dr. Marc Peters-Golden for review of an early version of this manuscript.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants 1P50-HL-56402, HL-50496, and HL-07749; by a Merit Review Award (to P. J. Christensen); and by Research Enhancement Award Program funds from the Department of Veterans Affairs.

Address for reprint requests and other correspondence: P. J. Christensen, Pulmonary Section (111G), VAMC, 2215 Fuller Rd., Ann Arbor, MI 48105 (E-mail: pchriste{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. §1734 solely to indicate this fact.

Received 3 January 2000; accepted in final form 20 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 279(3):L487-L495