1 Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039; and 2 Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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The human
surfactant protein (SP)-C gene promoter was used to direct expression
of mouse granulocyte macrophage colony-stimulating factor (GM-CSF;
SP-C-GM mice) in lung epithelial cells in GM-CSF-replete (GM+/+) or
GM-CSF null mutant (GM/
) mice. Lung weight and volume were significantly increased in SP-C-GM mice compared with GM+/+ or
GM
/
control mice. Immunohistochemical staining
demonstrated marked type II cell hyperplasia, and immunofluorescent
labeling for proliferating cell nuclear antigen was increased in type
II cells of SP-C-GM mice. Abundance of type II cells per mouse lung was
increased three- to fourfold in SP-C-GM mice compared with GM+/+ and
GM
/
mice. GM-CSF increased bromodeoxyuridine labeling of
isolated type II cells in vitro. Type II cells, alveolar macrophages, and endothelial and bronchiolar epithelial cells were stained by
antibodies to the GM-CSF receptor
-subunit in both GM+/+ mice and
GM-CSF gene-targeted mice that are also homozygous for the SP-C-GM
transgene. High levels of GM-CSF expression in type II cells of
transgenic mice increased lung size and caused type II cell
hyperplasia, demonstrating an unexpected role for the molecule in the
regulation of type II cell proliferation and differentiation.
proliferation; alveolar macrophage; morphometric analysis; lung injury and repair; lung growth factor
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INTRODUCTION |
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THE ALVEOLAR SURFACE of the mammalian lung is lined by type II and type I epithelial cells, providing an extensive surface area required for gas exchange. Type II cells synthesize and catabolize pulmonary surfactant and serve as progenitor cells for repair after alveolar injury (15). Type I epithelial cells are terminally differentiated, squamous cells that cover ~90% of alveolar surfaces. The basal surfaces of type I cells are closely apposed to an encapsulating capillary network surrounding each alveolus, enhancing efficient O2-CO2 exchange between alveolar gases and blood. After alveolar injury, type II cells proliferate and differentiate into type I cells to repopulate the denuded basal lamina. The mechanisms involved in alveolar repair and remodeling in vivo are not known but are likely mediated by various growth factors and their receptors that direct proliferation or differentiation of type II epithelial cells (22, 23, 30, 34).
Type II cells synthesize, secrete, and recycle the components of
pulmonary surfactant (32). Pulmonary surfactant is a complex mixture of
phospholipids and proteins enriched in dipalmitoylphosphatidylcholine and associated surfactant proteins (SP)-A, -B, -C, and -D and is
critical for reducing surface tension at the air-liquid interface in
the alveolus. Recent gene-targeting experiments demonstrated the vital
role of granulocyte macrophage colony-stimulating factor (GM-CSF) in
pulmonary surfactant metabolism. Mice deficient in GM-CSF
(GM/
) or the GM-CSF receptor
-subunit exhibited
disrupted surfactant metabolism characterized by accumulation of
surfactant phospholipids and proteins similar to that seen in the human
disease pulmonary alveolar proteinosis (10, 20, 27, 28). Surfactant lipid and protein concentrations were corrected when GM-CSF was expressed under control of the human SP-C gene promoter in lung epithelial cells of GM
/
mice, demonstrating that local
expression of GM-CSF was sufficient to correct the proteinosis
phenotype (2).
In the present study, morphometric and immunohistochemical analyses were used to determine the effects of GM-CSF on lung growth and cellularity in transgenic mice expressing GM-CSF in lung epithelial cells. High levels of GM-CSF expression in the lung increased lung size and caused type II epithelial cell hyperplasia.
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MATERIALS AND METHODS |
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Construction of the SP-C-GM-CSF chimeric gene and
generation of transgenic mice. A chimeric gene was
constructed and used to generate SP-C-GM-CSF transgenic mice, as
described previously (12). Briefly, the coding region of mouse GM-CSF
cDNA was isolated. The ends were modified with addition of
EcoR I linkers and were inserted into
the EcoR I site of p3.7-tpA to
generate p3.7GM-tpA. The GM-CSF cDNA was aligned in the 5' to
3' orientation with the human SP-C (3.7SP-C) promoter, creating
an SP-C-GM-CSF (SP-C-GM) chimeric transgene construct. The 3.7SP-C
promoter has been used previously in transgenic mouse models to achieve
high levels of transgene expression restricted to pulmonary epithelial
cells (11). The SP-C-GM sequences were isolated for pronuclear
injection of GM-CSF-replete (GM+/+) FVB/N ova fertilized with GM-CSF
null mutant (GM/
) C57Bl/6/129S sperm, generating founder
mice with the GM+/
,SP-C-GM+/
genotype. To determine
genotypes, tail DNA was digested with
BamH I and was Southern blotted, using
GM-CSF cDNA to probe for GM+ endogenous, GM-null mutant, and SP-C-GM alleles, also described previously (12). Animals from two separate founder lines of SP-C-GM mice were analyzed in this study; histological analysis was also performed on four additional founders that either failed to breed or that did not transmit the SP-C-GM transgene to their
progeny. GM+/
,SP-C-GM+/
founder mice were backcrossed to
GM
/
mice producing GM
/
,SP-C-GM+/
and
GM+/
,SP-C-GM+/
progeny, which were then bred to obtain
homozygote mice of either the GM+/+,SP-C-GM+/+ or
GM
/
,SP-C-GM+/+ genotype. Lines of GM+/+ or
GM
/
control mice were derived by backcrossing
SP-C-GM
/
littermates. Mice used in this study were of
generation F8 or greater.
Bronchoalveolar lavage and measurement of proteins by
enzyme-linked immunosorbent assay. Mice were
anesthetized by intraperitoneal injection of pentobarbital sodium and
were killed by aortic exsanguination. Bronchoalveolar lavage (BAL) was
performed using three 1-ml aliquots of phosphate-buffered saline (PBS),
which were pooled, measured for recovery volume, and stored at
20°C until assay. GM-CSF was measured using the Endogen
GM-CSF Minikit enzyme-linked immunosorbent assay (ELISA; Endogen,
Cambridge, MA). Horseradish peroxidase (HRP)-streptavidin (Zymed
Laboratories, South San Francisco, CA) was diluted to 1:8,000.
Substrate was the Dako TMB One-Step Substrate System (Dako,
Carpinteria, CA). Plates were read at 450 nm using a Dynatech plate
reader (Dynatech Laboratories, Chantilly, VA). The lower limit of
detection with this system is <5 pg/ml; thus any samples falling
below this were reported as not detectable. All samples >500 pg/ml
were diluted and repeated.
Endotoxin treatment of mice. Lung injury was induced by in vivo administration of intratracheal lipopolysaccharide (LPS) to GM+/+ FVB/N mice. LPS from Salmonella typhimurium (Sigma Chemical, St. Louis, MO) was diluted to a working concentration of 100 µg/ml in sterile saline. LPS (100 µl) was instilled intratracheally, as described previously (35). Mice were anesthetized by intraperitoneal injection of pentobarbital sodium and were killed by aortic exsanguination at 2, 4, 6, 8, or 12 h posttreatment. BAL was performed, and GM-CSF was measured as described above.
Processing and staining of tissues for histopathology. Lungs were inflation fixed, as described previously (3). Briefly, animals were anesthetized with pentobarbital sodium injected intraperitoneally and were killed by aortic exsanguination. The trachea was exposed and cannulated; 4% paraformaldehyde-PBS, pH 7.4, was instilled from a height of 23 cm for 1 min, and the trachea was ligated. Lungs and heart were dissected out of the chest cavity, placed in cold 4% paraformaldehyde-PBS, pH 7.4, and stored at 4°C for 24 h. Tissue was then washed in cold PBS and was cryoprotected in 30% sucrose for frozen sections or dehydrated in a series of alcohols and embedded in paraffin. Hematoxylin and eosin and Masson's trichrome staining were used for histological analysis of 5-µm paraffin sections. Cytospin preparations of BAL cells were stained using a Leukocyte Acid Phosphatase kit (Sigma Diagnostics) to identify tartrate-resistant cells.
Immunofluorescence and immunohistochemistry. Paraffin sections (5 µm) were used for immunohistochemical staining of pro-SP-C. Anti-pro-SP-C (68514) is a rabbit polyclonal antibody raised against the NH2-terminal domain of the proprotein, as described previously (31). Tissues were deparaffinized, and endogenous peroxidases were quenched using 3% H2O2-methanol for 1 h. Tissues were rinsed with 0.1 M PBS, pH 7.4, and 2% Triton X-100 (PBS-Triton), blocked with 2% normal goat serum (NGS) in PBS-Triton for 2 h at room temperature, and incubated at 4°C overnight with anti-pro-SP-C diluted 1:1,000. Slides were washed in PBS-Triton. Anti-pro-SP-C was detected using the Vectastain ABC goat anti-rabbit immunohistochemical horseradish peroxidase (HRP) kit (Vector Laboratories, Burlingame, CA) and nickel diaminobenzaldehyde (Ni-DAB) substrate. Tissues were counterstained with tris(hydroxymethyl)aminomethane (Tris)-cobalt and nuclear fast red.
Frozen sections (10 µm) were used for antiproliferating cell nuclear antigen (PCNA) and anti-pro-SP-C immunofluorescent localization studies. Anti-PCNA is a human polyclonal antibody to human PCNA (Immunovision, Springdale, AZ). Tissues were blocked with 2% NGS for 2 h at room temperature, rinsed with PBS-Triton, and incubated overnight at 4°C with anti-PCNA diluted 1:400 in 2% NGS. Sections were washed with PBS-Triton, and goat anti-human fluorescein isothiocyanate-conjugated secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA) was applied for 1 h at room temperature. Slides were washed with PBS-Triton and were incubated in a 1:1,000 dilution of anti-pro-SP-C (68514) at 4°C overnight. Slides were washed in PBS-Triton, and goat anti-rabbit Texas red-conjugated secondary antibody (Jackson Immuno Research Laboratories) was applied for 1 h. Slides were washed in PBS-Triton, placed under a coverslip with Citifluor antiquench mounting medium (University of Kent Chemical Laboratory, Canterbury, Kent, UK), and viewed under ultraviolet illumination.
Morphometric analysis. Lungs of mice 4-5 mo of age were inflation fixed and were processed for paraffin embedding as described above. Heart, trachea, and any other tissues were carefully dissected away, leaving only lung tissue. Total lung volume in cubic millimeters was determined using the Cavalieri method (2). Before embedding, all five lobes were weighed, measured from proximal to distal tips, bisected in the transverse plane, and embedded in blocks containing proximal or distal tissues. Blocks were cut in 5-µm serial sections, and the cross-sectional area of every one-hundredth section (intervals of 0.5 mm) was measured. Slides were viewed using a ×1 objective and were transferred via video camera to a computer screen using Metamorph software (Universal Imaging, Westchester, PA). A computer-generated grid was superimposed on each cross-sectional surface area, and grid points falling over the tissue sections were counted. The distance between grid points was 0.6 mm. Total lung volume (Vlung) in cubic millimeters was calculated using the equation
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Type II cells were counted using paraffin sections (5 µm) stained
with anti-pro-SP-C and avidin-biotin-HRP conjugate, as described above.
Four slides from each pair of blocks were selected to represent distinct distal and proximal regions of all five lobes. Five to eight
consecutive fields of 2 × 104
µm2 contained within a larger
grid were counted in each lobe (100 fields counted/mouse). Regions
for counting type II cells were selected at random at ×2
magnification and were counted at ×40 magnification regardless of
structures present (alveolar, bronchiolar, endothelial, etc.) to
accurately represent the lung parenchyma. Type II cells per lung were
calculated by multiplying the total lung volume
(mm3) by the number of cells
counted per area (2).
Detection of GM-CSF receptor -subunit in tissue
sections. Paraffin sections (5 µm) were cut,
quenched, and blocked as described above. Slides were incubated for 30 min with primary rabbit anti-mouse GM-CSF receptor
-subunit
(GM-CSF-R
) antisera, M-20 (Santa Cruz Biotechnology, Santa Cruz,
CA), 10 µg/ml in 1% bovine serum albumin-PBS. Negative control
slides were incubated in blocking solution without primary antisera or
in primary antisera preincubated with immunizing peptide.
The immunizing peptide contained amino acids 363-382 of the
carboxy terminus of mouse GM-CSF-R
. Slides were washed in PBS and
were incubated in goat anti-rabbit secondary antibody (described above)
for 30 min. Anti-GM-CSF-R
was detected using the Vectastain ABC goat
anti-rabbit immunohistochemical HRP kit (Vector Laboratories) and
Ni-DAB. Tissues were counterstained with Tris-cobalt and nuclear fast
red.
RT-PCR detection of GM-CSF-R in isolated type II
cells. Type II cells were isolated from GM+/+ mice as
described by Corti et al. (7). Because macrophages are known to express
GM-CSF receptors, a mouse alveolar macrophage cell line designated as MH-S (American Type Culture Collection, Rockville, MD) was used as a
positive control. RNA was isolated as described previously (12), and
cDNA was generated using the SuperScript ribonuclease H
reverse transcriptase
(RT) protocol (GIBCO-BRL, Gaithersburg, MD). Specific primers were then
used to amplify a 309-nucleotide (nt) polymerase chain reaction (PCR)
product corresponding to nucleotides 642-951 of GM-CSF-R
cDNA
(Genbank accession no. M85078). The forward primer sequence was
5'-TGCGGGGCCAGTGCGGTTCCT-3'; the reverse primer sequence
was 5'-CAGTGCTTCATCCTCGTGTCG-3'. PCR was performed
according to the PCR SuperMix protocol (GIBCO-BRL) with 35 cycles of 45 s at 94°C, 30 s at 61°C, and 2 min at 72°C.
Rat type II cell isolation and
culture. Type II epithelial cells were isolated from
rat lungs as described previously for bromodeoxyuridine (BrdU) labeling
studies (26). Briefly, animals were anesthetized with pentobarbital
sodium, and the lungs were perfused via the pulmonary artery. Elastase
(Worthington Biochemicals, Freehold, NJ) was used to digest the type II
cells from the basement membrane. Type II cells were obtained by the
panning method described by Dobbs and colleagues (9). Cells were
resuspended in Dulbecco's modified Eagle's medium supplemented with
5% fetal bovine serum and 1% antibiotic-antimycotic (GIBCO-BRL) and
then were plated at a density of 2 × 104 cells per well in 96-well
tissue culture plates. Cultures prepared using this method generally
contained 90-95% type II cells. Type II cells were then treated
with 1 × 1011 to 1 × 10
7 M mouse GM-CSF
(PeproTech, Rocky Hill, NJ) for 48 h; BrdU was added, and cells were
allowed to incubate for an additional 24 h. Control plates received no
GM-CSF treatment. A colorimetric immunoassay (Boehringer Mannheim,
Indianapolis, IN) was used to assess BrdU incorporation. Fixed cells
were incubated with anti-BrdU antibody complex in PBS (1:100) for 90 min. Cells were washed, and colorimetric substrate solution was added.
Sulfuric acid (1 M solution) was added to stop the substrate-enzyme
reaction after 10 min, and absorbance was measured at 450 nm using a
Dynatech plate reader.
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RESULTS |
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Production of SP-C-GM-CSF transgenic
mice. Promoter sequences (3.7SP-C) from the human SP-C
gene were used to construct a chimeric SP-C-GM-CSF gene (SP-C-GM)
directing expression of mouse GM-CSF cDNA in lung epithelial cells, as
reported previously (12). Two separate founder lines, designated 48 and
59, were expanded for this study. Line 59 mice were bred to
homozygosity in both GM/
and GM+/+ backgrounds; line 48 SP-C-GM+/+ homozygote mice were bred only in the GM
/
background. Line 48 and 59 mice appeared healthy, bred normally, and
produced litters of 8-12 pups. Pups grew at a normal rate and
required no special care. The majority of SP-C-GM mice lived a normal
life span, with some surviving at least two years. Three other founder
mice, killed at 12 mo of age for histological analysis, failed to
transmit the SP-C-GM transgene to the
F1 generation. One additional
founder mouse that failed to breed was found moribund and was killed at
4 mo of age. No changes in SP-C-GM mice compared with control mice were
noted in the number or relative cell counts of peripheral blood white cells, peritoneal macrophages, or liver and spleen histology, as
assessed by hematoxylin and eosin staining, which was described previously (12).
GM-CSF protein production. GM-CSF
concentrations were measured in the BAL fluid by ELISA (Fig.
1A).
In line 48 and 59 GM/
,SP-C-GM+/+ mice and line 59 GM+/+,SP-C-GM+/+ mice, GM-CSF concentrations in BAL fluid ranged from
215 to 1,825 pg/ml. GM-CSF levels in BAL fluid from line 48 mice,
although higher on average, were more variable than those in line 59 animals. In mice from line 59, GM-CSF concentrations were similar in
GM
/
,SP-C-GM+/+ and GM+/+,SP-C-GM+/+ BAL fluids. In
comparison, GM-CSF concentrations in the BAL fluid from GM+/+ control
mice, although detectable, were near the lower limit of detection (5 pg/ml) by ELISA and so are reported as not detectable.
GM
/
mice were completely deficient in GM-CSF.
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LPS was administered intratracheally to nontransgenic GM+/+ FVB/N mice, and GM-CSF was measured in the BAL fluid (Fig. 1B). GM-CSF was detected readily in BAL fluid at 2, 4, 6, 8, and 12 h after exposure, demonstrating that GM-CSF is secreted into the alveolar lumen after LPS-mediated lung injury. The concentrations of GM-CSF at 2, 4, 6, and 8 h after LPS exposure were comparable to concentrations measured in BAL obtained from SP-C-GM mice.
Atypical lung histology in SP-C-GM
mice. In both GM+/+ and GM/
backgrounds,
lungs from line 48 and line 59 SP-C-GM mice had sparse regions of
thickened hyperplastic alveolar walls and slightly increased numbers of
alveolar macrophages compared with GM+/+ and GM
/
control
mice as early as 25 days of age. Alveolar wall hyperplasia was noted
first in the peripheral, subpleural regions of the lung parenchyma. By
2-5 mo of age, regions of marked alveolar wall hyperplasia were
noted throughout the lung, although some regions appeared relatively
normal (Fig. 2). The identity of alveolar
macrophages was confirmed by tartrate-resistant acid phosphatase
staining (not shown). Some alveolar macrophages were multinucleated,
consistent with other studies of alveolar macrophage morphology after
GM-CSF treatment (5, 16). In some mice, enlarged alveolar macrophages
filled the alveolar airspaces, with the greatest aggregations noted in
areas most affected by alveolar hyperplasia. By 9-12 mo of age,
hyperplastic alveolar walls were found throughout the lung parenchyma.
Alveolar spaces contained hypertrophic alveolar macrophages that were
frequently multinucleated. At 12 mo of age, alveolar macrophages were
also noted in larger bronchioles in some SP-C-GM mice. Collagen
deposition, as assessed by Masson's trichrome stain, was not increased
in animals examined at any age (not shown). No increase in eosinophils
or granulocytes was observed in airways of any of the animals.
Additionally, four separate GM+/
,SP-C-GM+/
founder mice
that failed to breed or to pass on the SP-C-GM transgene were killed at
4 or 12 mo of age for histological assessment. Hyperplastic alveolar
walls and increased numbers of alveolar macrophages were noted in each
of these animals, similar to the findings in line 48 and 59 mice. Because levels of GM-CSF in the BAL fluid and alveolar wall hyperplasia were most consistent in mice from line 59, subsequent experiments used
mice derived from this line.
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Type II cell hyperplasia in SP-C-GM
mice. Type II cells were identified in the hyperplastic
alveolar walls of SP-C-GM lungs by staining with anti-pro-SP-C, an
antibody specific for the amino terminal domain of pro-SP-C. Pro-SP-C
is synthesized only by type II cells. Immunoperoxidase labeling of
pro-SP-C demonstrated marked type II cell hyperplasia in the SP-C-GM
mice compared with control mice (Fig. 3).
Type II cells in SP-C-GM mice were also larger and stained more
intensely than type II cells of GM+/+ and GM/
mice.
Immunofluorescent anti-pro-SP-C staining also demonstrated increased
numbers of type II cells in the SP-C-GM mice (Fig.
4). Furthermore, colocalization of
anti-pro-SP-C and anti-PCNA immunofluorescent staining was consistently
observed in SP-C-GM mice compared with GM
/
and GM+/+
controls. The colocalization of anti-pro-SP-C and anti-PCNA indicates
increased proliferation of type II cells, which may contribute to the
increased numbers of type II cells seen in SP-C-GM mice. Increased
numbers of PCNA-staining alveolar macrophages were also found in the
alveolar spaces and interstitium of SP-C-GM compared with
GM
/
and GM+/+ lungs.
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Increased lung weight and volume in SP-C-GM
mice. At 4-5 mo of age, lungs from mice of each
genotype were removed and weighed (Table
1). Lung weight was significantly increased
in SP-C-GM mice compared with those from GM/
and GM+/+
mice. Fluid-filled lungs of SP-C-GM mice were 30-40% heavier than
those of GM
/
and GM+/+ control mice. Correspondingly, the
total volume (mm3) of inflated
SP-C-GM lungs was 25-40% larger than GM
/
and GM+/+ lungs. The increased weight and lung size detected in the morphometric analysis were consistent with increased lung DNA and total protein noted in SP-C-GM mice at 2 mo of age [Ikegami et al.
(13a)]. To quantitate the total number of type II cells per lung,
type II cells were counted in representative fields (1 × 105
µm3 of lung parenchyma) from
each lung lobe. The number of type II cells was more than doubled per
parenchymal unit and was increased fourfold per lung in SP-C-GM
mice compared with GM
/
and GM+/+ mice (Table
1).
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GM-CSF receptors on type II cells and alveolar
macrophages. Immunohistochemical staining was used to
identify GM-CSF-R subunits. The GM-CSF receptor complex consists of
GM-CSF-R
and a shared
-subunit
(
c) that also is part of the
interleukin (IL)-3 and IL-5 receptor complexes. Type II cells and
bronchiolar epithelial cells stained with antibodies to GM-CSF-R
,
identifying these cell types as potential targets of GM-CSF in mouse
lung (Fig. 5). Alveolar macrophages
and endothelial cells were also stained (not shown), consistent
with previous studies demonstrating GM-CSF receptors or GM-CSF
responses in these cell types (12, 14).
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RT-PCR was used to amplify nucleotides 642-951 of the GM-CSF-R
subunit mRNA from isolated mouse type II cells (Fig.
6). The amplified band comigrated with a
band generated in a mouse alveolar macrophage cell line but, as
expected, was smaller than the product generated from genomic DNA. An
additional larger band was consistently amplified from type II cell
mRNA. The precise identity of that band is unknown. The presence of
GM-CSF-R
mRNA in mouse type II cells corroborates the
immunohistochemical staining in mouse lung tissue sections and provides
further evidence for GM-CSF receptor expression in type II cells.
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GM-CSF stimulates BrdU uptake by type II cells in vitro. Freshly isolated rat type II cells were incubated with mouse GM-CSF for 72 h. BrdU was added to the cultures for the final 24 h. BrdU uptake increased in a dose-dependent manner, indicating that DNA synthesis was increased in the presence of GM-CSF (Fig. 7). The number of cells per well was not increased by the addition of GM-CSF, as assessed by cell counts (data not shown).
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DISCUSSION |
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Transgenic mice expressing GM-CSF in lung epithelial cells were
generated in GM-CSF null mutant and GM-CSF-replete backgrounds. Expression of GM-CSF had a profound effect on lung growth in the SP-C-GM mice. Lungs from SP-C-GM mice 4-5 mo of age were
30-40% larger than lungs of GM/
or GM+/+ control
mice, as assessed by weight or morphometric analysis, and numbers
of type II cells per lung were increased three- to fourfold. Lung
epithelial cell-specific GM-CSF expression corrected alveolar
proteinosis in the GM
/
mice, enhanced lung growth, and
caused type II cell hyperplasia in mice from either GM
/
or GM+/+ backgrounds.
Lung injury initiates a complex cascade of inflammatory responses that
limit tissue damage and enhance the cell proliferation required to
repair the lung parenchyma (23). Alveolar repair is dependent on both
proliferation and differentiation of type II cells and is thought to be
influenced by various growth factors. For example, hepatocyte growth
factor and keratinocyte growth factor are increased after lung injury
and are known to stimulate type II cell DNA synthesis and proliferation
(17, 22, 23, 30). The present findings suggest that GM-CSF may also
play a role in type II cell proliferation. Because lung size and
alveolar morphology were not perturbed in GM/
mice,
GM-CSF is not required for lung morphogenesis. However, GM-CSF mRNA is
rapidly induced in the lung after endotoxin exposure, suggesting that
GM-CSF may be an early mediator of the repair process after alveolar
injury (24). The enlarged lungs and increased type II cell
proliferation seen in SP-C-GM mice support the hypothesis that GM-CSF
influences postnatal lung growth and/or differentiation and is
consistent with a potential role in repair and remodeling after injury.
GM-CSF is produced in normal lung and was first isolated from lung cell-conditioned media (4). A variety of pulmonary cells express GM-CSF, including mouse and human alveolar macrophages, endothelial cells, and fibroblasts, rat type II cells, and human bronchiolar and tracheal cells (1, 6, 8, 19). However, the normal lung GM-CSF concentrations in BAL fluid from GM+/+ mice were below the limit of detection by ELISA. Although GM-CSF was not readily detected in the normal lung, intratracheal administration of endotoxin in GM+/+ FVB/N mice increased BAL fluid GM-CSF concentrations to levels similar to the those measured in SP-C-GM mice. In recent studies, type II cell hyperplasia was observed in rat lungs 3-7 days after exposure to endotoxin (29). However, it is unknown at present whether the hyperplastic response to LPS was mediated directly by GM-CSF or by other factors influenced by endotoxin. The tightly regulated expression pattern of endogenous GM-CSF mRNA and protein contrasts with the high levels of GM-CSF measured in BAL fluid from the SP-C-GM mice. The continuous high-level production of GM-CSF in the transgenic mice was associated with marked type II cell hyperplasia and infiltration of alveolar macrophages, generating lung histology that is quite distinct from acute changes seen after endotoxin.
GM-CSF caused progressive pulmonary type II cell hyperplasia in the SP-C-GM mice. The hypercellularity of the alveolar walls was largely due to an increase in both size and number of type II cells. Although it is possible that changes in other cell types also contributed to the increased lung size observed in the SP-C-GM mice, type II cell hyperplasia and macrophage infiltration predominated the histological findings. The increase in type II cells per lung was attributed to both increased numbers of type II cells per parenchymal unit and increased lung volume. Immunofluorescent staining for both PCNA and pro-SP-C in SP-C-GM mice was increased, supporting the concept that type II cell proliferation, at least in part, accounts for the histological findings in the lung. Although PCNA staining suggests an increased rate of mitosis, GM-CSF may also influence type II cell numbers by altering differentiation or apoptotic pathways in type II cells. The progressive changes in lung histology suggest that hyperplasia was also related to the prolonged duration of exposure to GM-CSF.
The accumulation of alveolar macrophages observed in SP-C-GM mice is
consistent with the known mitogenic effects of GM-CSF. Metcalf (18)
observed increased numbers of macrophages, eosinophils, and
granulocytes in peritoneal and lung compartments after intraperitoneal injection of GM-CSF and in transgenic mice expressing GM-CSF and particularly noted increased mitosis in macrophages. Increased numbers
of alveolar macrophages were associated with increased PCNA staining in
lungs of SP-C-GM mice, suggesting a proliferative effect of GM-CSF on
local macrophage precursors. Local expansion of the macrophage
population is consistent with in vitro studies that demonstrated that
alveolar macrophages proliferate when stimulated with GM-CSF (5).
Chemotactic properties of GM-CSF may also contribute to the increased
number of alveolar macrophages in lungs of the SP-C-GM mice. The
morphology of the SP-C-GM alveolar macrophages, including enlarged
cytoplasm and multinucleated cells, is consistent with in vitro studies
of morphological changes in alveolar macrophages treated with GM-CSF
(16). No changes were seen in peripheral blood leukocytes, and neither
granulocytes nor eosinophils were increased in lungs of SP-C-GM mice.
These findings support the hypothesis that increased numbers of
alveolar macrophages in the SP-C-GM mice result from local production
of GM-CSF within the lung. Furthermore, increased numbers and activity of macrophages in the SP-C-GM mice likely contribute to the resolution of alveolar proteinosis in the GM/
,SP-C-GM+/+ mice.
Type II cell hyperplasia was consistently observed in SP-C-GM mice and occurred in the absence of pulmonary fibrosis. The present findings therefore are distinct from those in which a recombinant adenovirus was used to direct expression of GM-CSF in the mouse lung (33). GM-CSF concentrations after adenoviral vector delivery may differ from those generated in the SP-C-GM mice. The consistent lack of pulmonary fibrosis in the SP-C-GM model also suggests that GM-CSF is not sufficient to cause fibrosis and that stimulation of additional inflammatory pathways by the adenovirus may have contributed to the fibrosis observed after adenoviral-mediated transfer of GM-CSF.
Whereas marked type II cell hyperplasia was consistently observed in
the SP-C-GM mice, it remains unclear whether direct or indirect
effect(s) of GM-CSF contributed to the changes in lung histology. It is
possible that GM-CSF stimulates the expression of other type II cell
growth regulators. GM-CSF receptors were present in type II epithelial
cells but were also detected on alveolar macrophages and endothelial
and bronchiolar epithelial cells. GM-CSF-R mediates both high- and
low-affinity binding of GM-CSF to its receptor complex. GM-CSF, IL-3,
and IL-5 receptor complexes share the common
c-subunit. The
c and a ligand-specific
-subunit heterodimerize to form a functional high-affinity receptor complex (14). The
c-subunit
neither binds GM-CSF nor determines ligand specificity but contains a
cytoplasmic domain required for signal transduction through the
JAK-STAT pathway (25). The finding that GM-CSF-R
subunits are
present on type II cells suggests that GM-CSF may act directly on type
II cells to regulate proliferation or differentiation or to mediate
other autocrine pathways influencing type II cell proliferation.
The presence of GM-CSF receptors detected with immunohistochemical
staining by GM-CSF-R antibodies was further confirmed by RT-PCR
amplification of GM-CSF-R
mRNA from isolated mouse type II cells. In
addition to the transcript represented by the expected product of 309 nt, a slightly larger transcript was consistently detected in type II
cell RNA preparations. Although the mouse type II cell preparations are
highly purified (~90-95% type II cells), it remains possible
that the GM-CSF-R
309 nt band arises from the presence of other
cells contaminating the type II cell isolate.
The finding that GM-CSF stimulated BrdU uptake in rat type II cells in vitro supports the concept that GM-CSF may play a role in type II cell proliferation in vivo. Although BrdU uptake reflects increased DNA synthesis, GM-CSF alone was not sufficient to increase proliferation of type II cells in culture. The isolated rat type II cell cultures are generally >90% pure. Because contaminating alveolar macrophages are variably present in the cultures, albeit in low numbers, it is also possible that the observed stimulation of BrdU uptake by GM-CSF was mediated by paracrine effects among various cell types present in the culture.
GM-CSF plays an important role in surfactant metabolism.
GM-CSF-deficient mice develop severe alveolar proteinosis related to
decreased catabolism of surfactant lipids and proteins (13). SP and
phospholipid clearance studies of GM/
,SP-C-GM+/+ mice demonstrated that local expression of GM-CSF in respiratory epithelial cells corrected surfactant homeostasis in vivo [Ikegami et al. (13a)]. Mice homozygous for a
c receptor mutation also
developed severe alveolar proteinosis that was substantially corrected
by bone marrow transplantation (21). These studies support an important role for the alveolar macrophage in the pathogenesis of alveolar proteinosis. It remains unclear, however, whether donor macrophages directly corrected impaired surfactant catabolism or compensated for
impaired type II cell function. Furthermore, it is not known if
interactions between donor cells and type II epithelial cells also
contributed to the restoration of surfactant homeostasis seen in the
c mutant mice.
GM-CSF expression in lung epithelial cells enhanced lung growth in association with proliferation of type II cells and alveolar macrophages in the SP-C-GM mice. Biochemical and histological findings in SP-C-GM mice previously demonstrated that GM-CSF was required for regulation of surfactant homeostasis. The increased lung size and type II cell hyperplasia seen in SP-C-GM mice demonstrate an unexpected effect of GM-CSF in the lung and are consistent with a potential role for GM-CSF as a regulator of type II cell proliferation and differentiation after lung injury.
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ACKNOWLEDGEMENTS |
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We acknowledge and thank Dr. Harriet Iwamoto, Dana Fiedeldey, Kristen Spradlin, and Sherri Profitt for technical assistance.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute Specialized Center of Research Grant HL-56387.
Address for reprint requests: J. A. Whitsett, Children's Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039.
Received 30 December 1996; accepted in final form 10 June 1997.
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