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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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)- (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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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.
|
|
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.
|
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.
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adamson, I,
Young L,
and
Bowden D.
Relationship of alveolar epithelial injury and repair to the induction of pulmonary fibrosis.
Am J Pathol
130:
377-383,
1988[Abstract].
2.
Akagawa, KS,
Kamoshita K,
and
Tokunaga T.
Effects of granulocyte-macrophage colony-stimulating factor and colony-stimulating factor-1 on the proliferation and differentiation of murine alveolar macrophages.
J Immunol
141:
3383-3390,
1988
3.
Bratton, DL,
Hamid Q,
Boguniewicz M,
Doherty DE,
Kailey JM,
and
Leung DY.
Granulocyte macrophage colony-stimulating factor contributes to enhanced monocyte survival in chronic atopic dermatitis.
J Clin Invest
95:
211-218,
1995[ISI][Medline].
4.
Braunstein, S,
Kaplan G,
Gottlieb AB,
Schwartz M,
Walsh G,
Abalos RM,
Fajardo TT,
Guido LS,
and
Krueger JG.
GM-CSF activates regenerative epidermal growth and stimulates keratinocyte proliferation in human skin in vivo.
J Invest Dermatol
103:
601-604,
1994[Abstract].
5.
Brown, CB,
Hart CE,
Curtis DM,
Bailey MC,
and
Kaushansky K.
Two neutralizing monoclonal antibodies against human granulocyte-macrophage colony-stimulating factor recognize the receptor binding domain of the molecule.
J Immunol
144:
2184-2189,
1990
6.
Burgess, AW,
Camakaris J,
and
Metcalf D.
Purification and properties of colony-stimulating factor from mouse lung-conditioned medium.
J Biol Chem
252:
1998-2003,
1977[Abstract].
7.
Chauncey, JB,
Peters-Golden M,
and
Simon RH.
Arachidonic acid metabolism by rat alveolar epithelial cells.
Lab Invest
58:
133-140,
1988[ISI][Medline].
8.
Chen, G,
Curtis J,
Mody C,
Christensen P,
Armstrong L,
and
Toews G.
Effect of granulocyte-macrophage colony-stimulating factor on rat alveolar macrophage anticryptococcal activity in vitro.
J Immunol
152:
724-734,
1994
9.
Christensen, P,
Armstrong L,
Fak J,
Chen G,
McDonald R,
Toews G,
and
Paine R.
Regulation of rat pulmonary dendritic cell immunostimulatory activity by alveolar epithelial cell-derived GM-CSF.
Am J Respir Cell Mol Biol
13:
426-433,
1995[Abstract].
10.
Dobbs, LG,
Gonzalez R,
and
Williams MC.
An improved method for isolating type II cells in high yield and purity.
Am Rev Respir Dis
134:
141-145,
1986[ISI][Medline].
11.
Dranoff, G,
Crawford AD,
Sadelain M,
Ream B,
Rashid A,
Bronson RT,
Dickersin GR,
Bachurski CJ,
Mark EL,
Whitsett JA,
and
Mulligan RC.
Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis.
Science
264:
713-716,
1994[ISI][Medline].
12.
Gasson, JC.
Molecular physiology of granulocyte-macrophage colony-stimulating factor.
Blood
77:
1131-1145,
1991[ISI][Medline].
13.
Goodman, R,
Nestle F,
Naidu Y,
Green J,
Thompson C,
Nickoloff B,
and
Turka L.
Keratinocyte-derived T cell costimulation induces preferential production of IL-2 and IL-4 but not interferon-.
J Immunol
152:
5189-5198,
1994
14.
Gough, NM,
Gough J,
Metcalf D,
Kelso A,
Grail D,
Nicola NA,
Burgess AW,
and
Dunn AR.
Molecular cloning of cDNA encoding a murine haematopoietic growth regulator, granulocyte-macrophage colony stimulating factor.
Nature
309:
763-767,
1984[ISI][Medline].
15.
Griffin, M,
Bhandari R,
Hamilton G,
Chan YC,
and
Powell JT.
Alveolar type II cell-fibroblast interactions, synthesis and secretion of surfactant and type I collagen.
J Cell Sci
105:
423-432,
1993
16.
Haschek, WM,
and
Witschi H.
Pulmonary fibrosisa possible mechanism.
Toxicol Appl Pharmacol
51:
475-487,
1979[ISI][Medline].
17.
Huffman, JA,
Hull WM,
Dranoff G,
Mulligan RC,
and
Whitsett JA.
Pulmonary epithelial cell expression of GM-CSF corrects the alveolar proteinosis in GM-CSF-deficient mice.
J Clin Invest
97:
649-655,
1996
18.
Kaplan, G,
Walsh G,
Guido L,
Meyn P,
Burkhardt R,
Abalos R,
Barker J,
Frindt P,
Fajardo T,
Celona R,
and
Cohn Z.
Novel responses of human skin to intradermal recombinant granulocyte/macrophage-colony-stimulating factor: Langerhans cell recruitment, keratinocyte growth, and enhanced wound healing.
J Exp Med
175:
1717-1728,
1992[Abstract].
19.
Kawamoto, M,
and
Fukuda Y.
Cell proliferation during the process of bleomycin-induced pulmonary fibrosis in rats.
Acta Pathol Jpn
40:
227-238,
1990[Medline].
20.
Kawanami, O,
Ferrans V,
and
Crystal R.
Structure of alveolar epithelial cells in patients with fibrotic lung disorders.
Lab Invest
46:
39-53,
1982[ISI][Medline].
21.
Khalil, N,
O'Connor R,
Flanders K,
Shing W,
and
Whitman C.
Regulation of type II alveolar epithelial cell proliferation by TGF- during bleomycin-induced lung injury in rats.
Am J Physiol Lung Cell Mol Physiol
267:
L498-L507,
1994
22.
Kuhn, C.
Pathology.
In: Pulmonary Fibrosis, edited by Phan S,
and Thrall R.. New York: Dekker, 1995, p. 59-83.
23.
Mouhieddine, OB,
Cazals V,
Maitre B,
Le BY,
Chadelat K,
and
Clement A.
Insulin-like growth factor-II (IGF-II), type 2 IGF receptor, and IGF- binding protein-2 gene expression in rat lung alveolar epithelial cells: relation to proliferation.
Endocrinology
135:
83-91,
1994[Abstract].
24.
Nakata, K,
Kiyoko S,
Fukayama M,
Hayashi Y,
Kadokura M,
and
Tokunaga T.
Granulocyte-macrophage colony-stimulating factor promotes the proliferation of human alveolar macrophages in vitro.
J Immunol
147:
1266-1272,
1991
24a.
O'Brien, AD,
Standiford TJ,
Christensen PJ,
Wilcoxen SE,
and
Paine R.
Chemotaxis of alveolar macrophages in response to signals derived from alveolar epithelial cells.
J Lab Clin Med
131:
417-424,
1998[ISI][Medline].
25.
Paine, R,
Ben-Ze'ev AB,
Farmer SR,
and
Brody JS.
The pattern of cytokeratin synthesis is a marker of type 2 cell differentiation in adult and maturing fetal lung alveolar cells.
Dev Biol
129:
505-515,
1988[ISI][Medline].
26.
Piguet, P,
Grau G,
and
de Kossordo S.
Role of granulocyte-macrophage colony-stimulating factor in pulmonary fibrosis in mice by bleomycin.
Exp Lung Res
19:
579-587,
1993[ISI][Medline].
27.
Rubbia-Brandt, L,
Sappino A,
and
Gabbiani G.
Locally applied GM-CSF induces the accumulation of -smooth muscle actin containing myofibroblasts.
Virchows Arch
60:
73-82,
1991.
28.
Shanafelt, AB,
Johnson KE,
and
Kastelein RA.
Identification of critical amino acid residues in human and mouse granulocyte-macrophage colony stimulating factor and their involvement in species specificity.
J Biol Chem
266:
13804-13810,
1991
29.
Simon, R,
and
Paine R.
Participation of pulmonary alveolar epithelial cells in lung inflammation.
J Lab Clin Med
126:
108-118,
1995[ISI][Medline].
30.
Thrall, R,
McCormack J,
Jack R,
McReynolds R,
and
Ward P.
Bleomycin-induced pulmonary fibrosis in the rat: inhibition by indomethacin.
Am J Pathol
95:
117-128,
1979[Abstract].
31.
Thrall, R,
and
Scalise P.
Bleomycin.
In: Pulmonary Fibrosis, edited by Phan S,
and Thrall R.. New York: Dekker, 1995, p. 231-292.
32.
Vyalov, S,
Desmouliere A,
and
Gabbiani G.
GM-CSF-induced granulation tissue formation: relationships between macrophage and myofibroblast accumulation.
Virchows Arch
63:
231-239,
1993.
33.
Warner, R,
Paine R,
Christensen P,
Marletta M,
Richards M,
Wilcoxen S,
and
Ward P.
Lung sources and cytokine requirements for in vivo expression of inducible nitric oxide synthase.
Am J Respir Cell Mol Biol
12:
649-661,
1995[Abstract].
34.
Witschi, H.
Role of the epithelium in lung repair.
Chest
99:
22S-25S,
1991[Medline].
35.
Witschi, HR,
Haschek WM,
Klein SA,
and
Hakkinen PJ.
Potentiation of diffuse lung damage by oxygen: determining variables.
Am Rev Respir Dis
123:
98-103,
1981[ISI][Medline].
36.
Xing, Z,
Ohkawara Y,
Jordana M,
Graham FL,
and
Gauldie J.
Transfer of granulocyte-macrophage colony-stimulating factor gene to rat lung induces eosinophilia, monocytosis, and fibrotic reactions.
J Clin Invest
97:
1102-1110,
1996
37.
Xing, Z,
Tremblay GM,
Sime PJ,
and
Gauldie J.
Overexpression of granulocyte-macrophage colony-stimulating factor induces pulmonary granulation tissue formation and fibrosis by induction of transforming growth factor-1 and myofibroblast accumulation.
Am J Pathol
150:
59-66,
1997[Abstract].