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Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The functional activity of a macrophage is likely determined by its maturational path and the local cytokine milieu. GM-CSF is a 23-kDa glycoprotein member of the hemopoietic cytokine family, which regulates the proliferation and differentiation of cells in the granulocyte-macrophage lineage (15). GM-CSF also has potent effects on the function of mature hemopoietic cells. The lung is a rich source of GM-CSF, but the functional aspects of GM-CSF in lung tissue injury and repair/fibrosis remain uncertain.
GM-CSF likely plays a complex role in the processes of fibrosis and tissue repair at epithelial surfaces. Intradermal administration of GM-CSF to leprosy patients with skin wounds leads to enhanced wound healing with increased numbers and layers of keratinocytes (16). GM-CSF stimulates keratinocyte proliferation at nanogram per milliliter concentrations (17, 18). Transgenic overexpression of GM-CSF in the lung under the control of the surfactant protein C promoter results in enhanced lung growth and alveolar type II cell hyperplasia (19). In bleomycin-induced pulmonary fibrosis in mice, administration of GM-CSF-neutralizing antisera increases the numbers of macrophages recoverable by bronchoalveolar lavage (BAL)3 and increases the deposition of hydroxyproline (20). Conversely, when overexpressed in the lung using a recombinant replication-defective adenoviral vector, GM-CSF is fibrogenic, leading to accumulation of macrophages and eosinophils in the lung, lung tissue injury, and varying degrees of fibrosis (21).
Our understanding of the role that GM-CSF plays in the complex interplay of the processes of repair and fibrosis has been limited by the inability to eliminate GM-CSF from the system completely and irreversibly, especially over the duration required to assess the fibrotic response. The development of transgenic mice with a targeted deletion of the GM-CSF gene allows the development of an animal model to test the importance of GM-CSF in lung injuries that progress to fibrosis. Intratracheal instillation of the chemotherapeutic agent bleomycin in C57BL/6 mice is a widely accepted animal model for studying the lung fibrotic response. In response to this insult, numerous cytokines, chemokines, and growth factors are produced that mediate the fibrotic/repair processes. In this study we investigate the importance of GM-CSF for the development of bleomycin-induced fibrosis. Comparing GM-CSF-/- mice with GM-CSF+/+ (wild-type) controls, we evaluate the importance of GM-CSF for collagen deposition, inflammatory cell recruitment, and leukotriene and PG production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A breeding pair of GM-CSF-/- mice, generated by G. Dranoff and bred extensively onto the C57BL/6 background, were obtained from J. A. Whitsett (Cincinnati, OH) and have been previously described (22). The mice were bred in the University Laboratory Animal Medicine facilities under specific pathogen-free conditions at the University of Michigan. Control C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). As GM-CSF-/- mice may be larger than age-matched controls, animals were matched for weight (20–25 g) in all experiments. The University of Michigan Committee on the Use and Care of Animals approved these experiments.
Bleomycin injection
Control and GM-CSF-/- mice were anesthetized with sodium pentobarbital. A single incision was made at the neck, and the salivary glands were parted by blunt dissection. The muscle covering the trachea was snipped to expose the tracheal rings. A single 30-µl injection containing 0.025 U of bleomycin (Sigma, St. Louis, MO) diluted in normal saline was injected using a Tridak stepper (Brookfield, CT) and a 30-gauge needle.
Hydroxyproline assays
Hydroxyproline is a modified amino acid found at a uniquely high percentage in collagen. Therefore, we determined the hydroxyproline content of the lungs as a quantitative measure of collagen deposition. Mice were euthanized by CO2 asphyxiation and perfused via the heart with 5 ml of normal saline. Individual lung lobes were removed, taking care to avoid the large conducting airways. The isolated lobes were homogenized in 1 ml of PBS, and hydrolyzed by the addition of 1 ml of 12 N HCl. Samples were then baked at 110°C for 12 h. Aliquots (5-µl) were then assayed by adding chloramine-T solution for 20 min followed by development with Erlich’s reagent at 65°C for 15 min as previously described (23). Absorbance was measured at 550 nm, and the amount of hydroxyproline was determined against a standard curve generated using known concentrations of hydroxyproline standard (Sigma).
Histology
Animals were euthanized and perfused via the left ventricle with 3 ml normal saline. Lungs were inflated with 1 ml 10% neutral buffered formalin, removed, and fixed overnight in formalin before being dehydrated in 70% ethanol. Lungs were then processed using standard procedures and embedded in paraffin. Sections (3–5 µm) were cut, mounted on slides, and stained with Masson’s Trichrome Blue for collagen deposition.
Collagenase digestions of whole lung
To analyze lung leukocytes, lungs were excised, minced, and enzymatically digested for 30 min using 15 ml/lung of digestion buffer (RPMI, 5% FCS, antibiotics, 1 mg/ml collagenase) (Boehringer Mannheim, Chicago, IL) and 30 µg/ml DNase (Sigma). The cell suspension and undigested fragments were further dispersed by repeated passage through the bore of a 10-ml syringe without a needle. The total cell suspension was pelleted, and any contaminating erythrocytes were eliminated by lysis in ice-cold NH4Cl buffer (0.829% NH4Cl, 0.1% KHCO3, and 0.0372% Na2 EDTA, pH 7.4). The pellet was resuspended in 5 ml of complete medium (RPMI, 10% FCS, 1% penicillin/streptomycin) and dispersed by 20 passages through a 5-ml syringe. Then the dispersed cells were filtered through a Nytex filter (Tetko, Kansas City, MO) to remove clumps. The total volume was brought up to 10 ml with complete media. An equal volume of 40% Percoll was added and the cells were centrifuged at 3000 rpm for 30 min. The cell pellets were resuspended in complete media and counted on a hemocytometer in the presence of trypan blue. Cells were >90% viable by trypan blue exclusion. Cytospins of recovered cells were prepared for differential staining as described below.
BAL
Mice were euthanized via CO2 asphyxiation, the trachea was cannulated with polyethylene tubing (PE50, Intramedic; Clay Adams, Parsippany, NJ) attached to a 25-gauge needle on a tuberculin syringe, and the lungs were lavaged twice with 0.75 ml PBS/5 mM EDTA (Sigma) for a total lavage volume of 1.5 ml. In >95% of the mice, the recovery volume was 1.3–1.4 ml. The BAL fluid was spun at 1500 rpm, and the supernatant was removed. The pelleted cells were collected, and erythrocytes were lysed as described above in ice-cold NH4Cl buffer for 3 min before being washed and pelleted in complete media. Total BAL cells were enumerated by counting on a hemocytometer in the presence of trypan blue. Cytospins were prepared from resuspended BAL cells.
Differential staining
Cytospins of either collagenase digestions or BAL were made by centrifuging 50,000–100,000 cells onto microscope slides using a Shandon Cytospin 3 (Shandon, Astmoore, U. K.). The slides were allowed to air dry and were then stained using a modified Wright-Giemsa (WG) stain. For WG staining, the slides were fixed/prestained for 2 min with a one-step methanol-based WG stain (Harleco; EM Diagnostics, Gibbstown, NJ) followed by steps 2 and 3 of the Diff-Quick whole blood stain (Diff-Quick; Baxter Scientific, Miami, FL). This modification of the Diff-Quick stain procedure improves the resolution of eosinophils from neutrophils in the mouse. A total of 300 cells were counted from randomly chosen high power microscope fields for each sample. The differential percentage was multiplied by the total leukocyte number to derive the number of monocyte/macrophages, neutrophils, and eosinophils per sample.
FACS analysis
Lung cells (1 x 106) pooled from the
collagenase digestions of three separate animals were incubated for 15
min on ice in Fc block (PharMingen, San Diego, CA) before washing and
centrifugation. Then cells were stained in a 100-µl total volume with
1-µg combinations of the following Abs: CD45 (YW62.3; Caltag
Laboratories, Burlingame, CA), CD4 (RM4-4; PharMingen), CD8 (53-6.7;
PharMingen), CD19 (1D3; PharMingen), TCR ß (H57-597; PharMingen), TCR
(GL3; PharMingen), or DX5 (Phar-Mingen). Stained samples were
stored in the dark at 4°C until analysis on a flow cytometer
(FACScan; Becton Dickinson, Mountain View, CA). All samples were
stained with CD45 to identify a leukocyte-specific gate. The absolute
number of a type of leukocyte in the lungs was determined as the
percentage of that cell type times the total number of cells in the
lungs.
Determination of PGE2 synthesis by isolated alveolar macrophages
Following anesthesia and euthanasia by exsanguination, the tracheobronchial tree was exposed and alveolar macrophages were harvested by BAL using two 0.75-ml lavages in PBS containing 5 mM EDTA. Isolated alveolar cells were resuspended in serum-free DMEM at 0.5 x 106/ml and plated at 0.2 ml/well in 96-well plates. Nonadherent cells were removed by washing twice with DMEM, and adherent cells were cultured in DMEM containing 10% FCS overnight, with or without exogenous murine recombinant GM-CSF (17 ng/ml; R&D Systems, Minneapolis, MN). Following overnight culture, the alveolar macrophages were washed three times in DMEM and subsequently stimulated with Ca2+ ionophore A23187 (1 µM) for 30 min to trigger arachidonate metabolism. Cell-free supernatants were analyzed by enzyme immunoassay (EIA) for the predominant cyclooxygenase (COX) product PGE2 (Cayman Chemicals, Ann Arbor, MI).
PGE2 and leukotriene C4 (LTC4) analysis in lung homogenates
Mice were euthanized and the lungs were harvested as described above. Lung tissue was homogenized (Tissue Tearor, model 985-370; Biospec Products, Bartlesville, OK) at level 5 in a 1-ml volume of ice-cold DMEM. Following homogenization, tissue fragments and intact cells were further fragmented by sonication (Sonifier model 250; Branson Ultrasonics, Danbury, CT) on ice for 30 s at power level 1. Lipids were then methanol extracted from the homogenate using Sep-Pak cartridges (Waters). The samples were dried down under a steady stream of nitrogen to evaporate the solvent, then resuspended in 250 µl of DMEM. PGE2 and LTC4 levels were measured by EIA (Cayman Chemicals).
In vivo indomethacin studies
C57BL/6 mice were anesthetized and injected with 0.025 U bleomycin intratracheally (i.t.) as described. Mice were divided into two groups with one group receiving daily i.p. injections of the PG synthesis inhibitor, indomethacin, at a dose of 1.2 mg/kg as previously described (24). Indomethacin injections were started at day 10 following bleomycin injection to specifically inhibit PG synthesis during the postinflammatory, fibroproliferative phase of the bleomycin response.
Statistics
Statistical significance was analyzed using the InStat 2.01 program (GraphPad Software, San Diego, CA) on a Power Macintosh G3. Student t tests were run to determine p values. Values of p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Weight-matched GM-CSF-/- mice and C57BL/6
wild-type mice were injected with bleomycin or saline as a control.
Animals were euthanized at day 21 postinoculation, and lungs were
analyzed histologically for the extent of fibrosis (Fig. 1
). Lung tissues were stained with
Masson’s Trichrome Blue to assess mature collagen deposition. No
fibrotic changes were noted in saline-treated wild-type (A)
or GM-CSF-/- mice (B). Following
bleomycin injection, GM-CSF-/- animals
(D) demonstrated more severe and more extensive fibrosis
than their wild-type counterparts (C). Thus, histological
analysis of wild-type and GM-CSF-/- mice
documented the presence of more severe fibrosis in
GM-CSF-/- mice.
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GM-CSF-/- mice treated with bleomycin contained
significantly more lung hydroxyproline than bleomycin-treated wild-type
mice (*, p = 0.02). There were no significant
differences in hydroxyproline content in wild-type or
GM-CSF-/- mice injected with saline (Fig. 2).
Thus, fibrosis in response to bleomycin was significantly more severe
in the GM-CSF-/- mice than in wild-type
mice.
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To characterize the inflammatory response to bleomycin, we
performed a kinetic analysis in wild-type mice to determine when the
peak inflammatory response occurred. Wild-type mice were injected with
either saline or bleomycin on day 0. Animals were then euthanized at
days 1, 3, 7, 14, and 21. BAL fluid was collected at each time point,
and the number of total leukocytes was determined. The peak
inflammatory response to bleomycin in wild-type mice occurred at day 7
postinjection (Fig. 3A).
Results were similar in the GM-CSF-/- mice,
where bleomycin injection resulted in a peak inflammatory response at
day 7 as well (Fig. 3B). The total number of BAL cells in
GM-CSF-/- mice was greater at day 7 than that
in wild-type mice (5.94 ± 1.5 x 106
vs 1.05 ± 0.01 x 106). Baseline
numbers of BAL cells were also higher in the
GM-CSF-/- mice (1.02 ± .36 x
106 vs 0.078 ± 0.02 x
106 for wild type). Because day 7 was the peak
response for both groups of mice, this time point was chosen for
further study of the inflammatory response to bleomycin.
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Histological analysis revealed that the inflammatory response
involved both the alveolar and interstitial spaces of the lung.
Therefore, we performed collagenase digestions on excised whole lung
lobes to collect both alveolar and interstitial cells from
bleomycin-treated and untreated mice. At baseline, lungs from wild-type
mice contained 10.1 ± 1.32 x 106 total leukocytes,
whereas GM-CSF-/- animals had 17.7 ± 1.8
x 106 leukocytes (Fig. 4).
Therefore, consistent with results of BAL,
GM-CSF-/- mice had more leukocytes resident in
their lungs at baseline than did wild-type mice (*, p
= 0.036). When treated with bleomycin, lungs from wild-type mice
contained 21.27 ± 1.8 x 106 leukocytes on day 7,
whereas lungs from GM-CSF-/- mice
contained 27.53 ± 1.36 x 106 leukocytes
(Fig. 4) (**, p = 0.019). The total number of newly
recruited leukocytes was determined by subtracting the number of
resident cells (untreated mice) from the total number of lung
leukocytes in bleomycin-treated mice. On day 7 following
bleomycin, wild-type mice had recruited 11.17 x 106
leukocytes per mouse whereas GM-CSF-/- animals
had recruited 9.83 x 106 leukocytes. Thus, although
lungs from GM-CSF-/- mice contained larger
numbers of resident leukocytes and larger numbers of total leukocytes
following bleomycin-induced injury than wild-type mice, they did not
recruit larger numbers of leukocytes to their lungs than wild-type mice
in response to the injury.
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The recovered cells were analyzed both by differential counting
and flow cytometry. Table I gives values
for inflammatory cell numbers in the lung at baseline compared with
bleomycin treatment at day 7. Table II
compares total lymphocyte populations in the same groups of mice. Cells
of monocytic origin accounted for the largest numbers of cells in the
lung digests. By differential analysis, we consistently observed more
monocytic cells at baseline in GM-CSF-/-
animals than in wild-type controls (#, p = 0.04).
Following bleomycin treatment, the absolute numbers of monocytes and
macrophages further increases. Similarly, neutrophils are increased in
GM-CSF-/- animals compared with wild-type
animals at baseline and following bleomycin. Eosinophils are absent in
GM-CSF-/- animals. Interestingly, although
absolute numbers of lymphocytes are increased in wild-type mice
following bleomycin, they decrease or are unchanged in
GM-CSF-/- animals following bleomycin. Given
that there are few significant differences in the magnitude of the
inflammatory response in the wild-type and
GM-CSF-/- mice, we hypothesized that the
differences in the fibrotic responses between wild-type and
GM-CSF-/- mice might be due to altered
production of pro- or anti-inflammatory mediators.
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One possibility to explain the increased fibrotic response in
GM-CSF-/- mice was elevated leukotriene levels.
Leukotrienes have a variety of possible proinflammatory/profibrotic
actions. To investigate this possibility, lung homogenates from
wild-type or GM-CSF-/- mice were analyzed for
the predominant murine lung leukotriene product,
LTC4, at day 7 following bleomycin treatment.
GM-CSF-/- mice had significantly reduced levels
of LTC4 (Fig. 5, p = 0.01) as compared with wild-type animals,
demonstrating that in these mice enhanced leukotriene synthesis is not
responsible for the exaggerated fibrosis; indeed, it occurs despite a
reduction in LTC4.
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Another possible mechanism for the more aggressive fibrotic
response in GM-CSF-/- mice than in the
wild-type mice was defective production of an antifibrotic mediator in
the GM-CSF-/- mice. PGE2
potently down-regulates fibroblast proliferation and collagen synthesis
(11, 12, 13, 14). To assess whether a defect in
PGE2 production existed in the
GM-CSF-/- mice, wild-type and
GM-CSF-/- mice were injected with saline or
bleomycin, and whole lung homogenates were assayed for
PGE2 levels at days 7 and 21. The lungs of
GM-CSF-/- animals contained lower levels of
PGE2 than wild-type animals at both time points
for both the saline- and bleomycin-treated mice (Fig. 6). Thus, increased fibrosis in
GM-CSF-/- mice treated with bleomycin was
associated with relatively impaired PGE2
production at both days 7 and 21.
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Because monocyte/macrophages accounted for the largest number of
inflammatory cells in the lung, and pulmonary macrophages have a high
capacity for PGE2 synthesis, we assessed whether
a defect in PGE2 production existed in pulmonary
macrophages in GM-CSF-/- mice. To assess
maximal capacity for PGE2 synthesis, alveolar
macrophages isolated from BAL of wild-type and
GM-CSF-/- mice were stimulated with calcium
ionophore A23187 (1 µM) for 30 min, and PGE2
was measured in the supernatant by EIA. PGE2
production by unstimulated cells was below the level of assay detection
in both wild-type and GM-CSF-/- mice (data not
shown). Stimulated alveolar macrophages from bleomycin-untreated
GM-CSF-/- mice produced significantly less
PGE2 than did cells from wild-type animals (Fig. 7, left). Bleomycin treatment
was associated with a significant reduction in alveolar macrophage
capacity for PGE2 synthesis in wild-type animals
(Fig. 7, right). Cells from
GM-CSF-/- animals treated with bleomycin were
even more profoundly impaired in PGE2
production.
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, right). GM-CSF
administration restored the levels of PGE2 seen
in alveolar macrophages from GM-CSF-/- animals
back to the level seen in cells from wild-type animals. Furthermore,
GM-CSF treatment superinduced the alveolar macrophages from wild-type
animals to produce more PGE2 upon stimulation
(Fig. 8, left).
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To determine whether the increased fibrosis seen in the
GM-CSF-/- mice could be causally linked to the
diminished PGE2 levels observed, experiments were
performed in which PG synthesis was pharmacologically blocked by the in
vivo administration of the COX inhibitor, indomethacin. Wild-type mice
were treated with bleomycin on day 0, and then given daily i.p.
administrations of indomethacin starting at day 10 postbleomycin. Fig. 9 demonstrates that indomethacin
treatment started at day 10 significantly increases bleomycin-induced
pulmonary fibrosis (*, p = 0.02) at day 21. The level
of fibrosis seen in the day 10 indomethacin-treated mice was comparable
to that measured in the GM-CSF-/- mice treated
with bleomycin (see Fig. 2
). Thus, pharmacological inhibition of
PGE2 synthesis worsens bleomycin-induced
pulmonary fibrosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several aspects of the animal model used in this study are worthy of
mention. First, the GM-CSF-/- mice lack GM-CSF
at the genomic, mRNA, and protein levels. Thus, the lack of GM-CSF is
specific, absolute, and irreversible. The use of animals that are
genetically deficient in GM-CSF offers a definitive system for studying
the role of GM-CSF in the evolution of pulmonary fibrosis. This
approach avoids the use of neutralizing Abs, thereby eliminating the
concerns about neutralization efficiency and complement-mediated tissue
injury. Second, young animals were used in all studies. As
GM-CSF-/- mice age, they develop progressive
accumulation of surfactant lipids and proteins in the alveolar space
(27). However, minimal or no evidence of pulmonary
alveolar proteinosis was noted in the young animals used in our studies
(Fig. 1, A and B). Most importantly, no fibrotic
abnormalities are found at baseline in these mice. No histological
findings suggestive of fibrosis are noted, and normalized
hydroxyproline content in untreated wild-type and
GM-CSF-/- mice do not differ (Fig. 2). Third,
bleomycin-induced pulmonary fibrosis is a clinically relevant model of
human disease (28).
Our findings documenting a beneficial role for GM-CSF in repair following lung injury are at variance with previously reported studies that used rats, in which GM-CSF was overexpressed in their lungs. High, transient overproduction of murine GM-CSF in rat lungs led to the accumulation of eosinophils and macrophages, was associated with tissue injury, and was followed by histological evidence of fibrosis (21). Adenoviral delivery of murine GM-CSF to rat lungs resulted in an increase in granuloma formation, fibroblast accumulation, and expression of the fibrogenic cytokine TGF-ß1 (29). Several features must be considered when evaluating these previously published studies. Concentrations of GM-CSF induced in this model were supraphysiological, and high levels of GM-CSF were expressed transiently during the early post injury phase. Furthermore, the injury associated with an adenoviral vector alone complicated interpretation. Finally, the murine GM-CSF gene was inserted in rat lungs. Thus, GM-CSF likely plays a complex role in the processes of fibrosis and repair following injury. Whether the process results in scarring or healing is likely determined by the location, timing, and amount of GM-CSF produced.
The profibrotic phenotype observed in GM-CSF-/- mice can be explained by the absence of an anti-inflammatory/antifibrotic agent and/or the presence of a proinflammatory/profibrotic agent. We have excluded the possibility that GM-CSF-/- mice produce increased levels of the proinflammatory/profibrotic eicosanoid, LTC4. However, we cannot exclude the possibility that alternative proinflammatory or profibrotic mediators might be overexpressed in the GM-CSF-/- animals. This will be of interest for future studies. One might have predicted, given the decreased leukotriene levels in the GM-CSF-/- mice, that arachidonic acid metabolism might be shunted to favor COX products. Instead, we found that GM-CSF-/- animals also have impaired synthesis of PGE2 in their lungs compared with wild-type animals at both early and late time points in the course of this disease model. Earlier work from our laboratories has demonstrated that GM-CSF augments the release of arachidonic acid in alveolar macrophages via increased phospholipase A2 (PLA2) activity (30). Therefore, it seems likely that diminished availability of arachidonic acid may be a plausible mechanism to explain the in vivo diminution of both LTC4 and PGE2 in GM-CSF-/- mice.
Although PGE2 is produced by immune and inflammatory cells, fibroblasts, epithelial cells, and endothelial cells, we have directed our studies toward monocytes/macrophages because of their prominence in the pulmonary response to bleomycin and because GM-CSF is known to have profound effects on mononuclear cell function. Alveolar macrophages purified from GM-CSF-/- animals exhibit an impaired ability to synthesize the antifibrogenic lipid, PGE2. The defect was correctable with exogenous (recombinant) GM-CSF, demonstrating that GM-CSF deficiency was indeed responsible for the PGE2 synthesis defect. Furthermore, we show that a fibrosing insult itself (bleomycin) blunts the ability of alveolar macrophages purified from either GM-CSF-/- or control mice to synthesize PGE2. These data suggest that diminished alveolar macrophage PGE2 production may be a generalized hallmark of fibrotic disease, and that this defect is exaggerated in GM-CSF-/- animals. Patients with the progressive fibrotic disorder idiopathic pulmonary fibrosis (IPF) have sustained reduction of PGE2 levels (10, 31). Whether the PGE2 synthesis defect identified in macrophages from GM-CSF-/- mice extends to other lung cells, including epithelial cells and fibroblasts themselves, is the subject of future studies.
The defect in PGE2 production contributes to the exuberant proliferation of fibroblasts associated with fibrotic lung disease. PGE2 is a known inhibitor of both fibroblast proliferation (11, 12) and fibroblast collagen synthesis (13, 14). In addition, PGE2 has been documented to promote the degradation of collagen, the major extracellular matrix component of fibrotic tissue (32). Therefore, decreased production of PGE2 could lead to unchecked fibroblast proliferation and deposition of extracellular matrix. Interestingly, fibroblasts purified from the lungs of patients with IPF have decreased PGE2 production and decreased expression of COX-2, the inducible isoform of COX, as compared with fibroblasts isolated from normal human lung (10). Taken together, these data from GM-CSF-/- mice and humans with IPF raise the possibility that diminished PGE2 production is a central mechanism of pulmonary fibrotic diseases in general. Our indomethacin experiments demonstrate that pharmacologic blockade of PGs can increase bleomycin-induced pulmonary fibrosis. Further support for the notion that PGE2 exerts important down-regulatory actions on the evolution of pulmonary fibrosis comes from recent preliminary work showing that COX-2-/- mice have an enhanced fibrotic response to bleomycin (33).
GM-CSF has profound effects on the proliferation, differentiation, and survival of hemopoietic cells in the granulocyte-macrophage lineage, especially macrophages, neutrophils, and eosinophils (15). Eosinophils are common participants in pulmonary fibrosis and portend a worse outcome in human IPF (34). They have been strongly implicated in the pathogenesis of fibrosis in the bleomycin model (25, 26). However, eosinophils are totally absent in the inflammatory response to bleomycin in GM-CSF-/- mice at day 7. Thus, our data demonstrate that eosinophils are not required for this fibrotic host response to lung injury; in the absence of GM-CSF (and the absence of eosinophils) the fibrosis is worse than in wild-type mice.
The role of alveolar macrophages in the pathogenesis of pulmonary fibrosis is quite complex. In some settings, these inflammatory cells may contribute to the initial lung injury that eventually leads to fibrosis. Alternatively, by the removal of provisional matrix from the alveolar space, or by secreting factors that promote repair (such as hepatocyte growth factor) or limit collagen production (such as PGE2), alveolar macrophages may promote normal repair rather than fibrosis. Interestingly, despite the absence of a factor that is mitogenic (15) and chemotactic (35) for alveolar macrophages, GM-CSF-/- mice have more cells in their lungs at baseline than wild-type animals. Furthermore, baseline levels of neutrophils, B, NK, and T cells are slightly elevated in GM-CSF-/- mice. One possibility to explain this phenomenon is that the GM-CSF-/- mice may have cells of a more immature phenotype as compared with wild-type mice, and that this results in the loss of a feedback inhibition loop that would limit cellular recruitment to the lung. Despite this, wild-type and GM-CSF-/- mice recruit similar numbers of cells into the lung during the first week following bleomycin injury. As a result, GM-CSF-/- mice have 23% more cells in their lungs at day 7 following bleomycin than wild-type mice. We have demonstrated that alveolar macrophages from GM-CSF-/- mice produce significantly less PGE2 than macrophages from wild-type mice. Whether other abnormalities in either proinflammatory or antifibrotic activities of the pulmonary macrophages from GM-CSF-/- mice contribute to the severity of pulmonary fibrosis in these mice awaits further study.
Bleomycin-induced fibrosis is significantly worse in GM-CSF-/- mice than in their wild-type counterparts. These studies indicate that GM-CSF is an important regulator of the number and phenotypic state of local mononuclear cells. Alveolar macrophage synthesis of the antifibrotic prostanoid PGE2 is crucially dependent on the presence of GM-CSF. This loss-of-function experiment documents an important role for GM-CSF in processes that minimize the development of fibrosis or alternatively preserve normal alveolar architecture following lung injury.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bethany B. Moore, University of Michigan, 6301 MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0642.
3 Abbreviations used in this paper: BAL, bronchoalveolar lavage; LTC4, leukotriene C4; i.t., intratracheal(ly); WG, Wright-Giemsa; EIA, enzyme immunoassay; COX, cyclooxygenase; IPF, idiopathic pulmonary fibrosis.
Received for publication December 20, 1999. Accepted for publication July 10, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-mediated lung cytokine networking and eosinophil recruitment in pulmonary fibrosis. J. Immunol. 158:954.[Abstract]
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R. J. Soberman and P. Christmas Revisiting prostacyclin: new directions in pulmonary fibrosis and inflammation Am J Physiol Lung Cell Mol Physiol, August 1, 2006; 291(2): L142 - L143. [Full Text] [PDF]
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G. Y. Park and J. W. Christman Involvement of cyclooxygenase-2 and prostaglandins in the molecular pathogenesis of inflammatory lung diseases Am J Physiol Lung Cell Mol Physiol, May 1, 2006; 290(5): L797 - L805. [Abstract] [Full Text] [PDF]
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V. Y. Lee, C. Schroedl, J. K. Brunelle, L. J. Buccellato, O. I. Akinci, H. Kaneto, C. Snyder, J. Eisenbart, G. R. S. Budinger, and N. S. Chandel Bleomycin induces alveolar epithelial cell death through JNK-dependent activation of the mitochondrial death pathway Am J Physiol Lung Cell Mol Physiol, October 1, 2005; 289(4): L521 - L528. [Abstract] [Full Text] [PDF]
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A. Nakatani-Okuda, H. Ueda, S.-i. Kashiwamura, A. Sekiyama, A. Kubota, Y. Fujita, S. Adachi, Y. Tsuji, T. Tanizawa, and H. Okamura Protection against bleomycin-induced lung injury by IL-18 in mice Am J Physiol Lung Cell Mol Physiol, August 1, 2005; 289(2): L280 - L287. [Abstract] [Full Text] [PDF]
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