GM-CSF enhances lung growth and causes alveolar type II
epithelial cell hyperplasia in transgenic mice
JACQUELYN A. HUFFMAN REED,1 WARD R. RICE,1 ZSUZSANNA K. ZSENGELLÉR,1
SUSAN E. WERT,1 GLENN DRANOFF,2 AND JEFFREY A. WHITSETT1
1Division of Pulmonary Biology, Children’s Hospital Medical Center, Cincinnati, Ohio 45229-3039;
and 2Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115
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 genetargeting experiments demonstrated the vital role of
granulocyte macrophage colony-stimulating factor (GMCSF) in pulmonary surfactant metabolism. Mice deficient in GM-CSF (GM2/2) or the GM-CSF receptor
b-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 GM2/2 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.
proliferation; alveolar macrophage; morphometric analysis;
lung injury and repair; lung growth factor
MATERIALS AND METHODS
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
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 58 to 38 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 (GM1/1) FVB/N ova
fertilized with GM-CSF null mutant (GM2/2) C57Bl/6/129S
sperm, generating founder mice with the GM1/2,SP-CGM1/2 genotype. To determine genotypes, tail DNA was
digested with BamH I and was Southern blotted, using
GM-CSF cDNA to probe for GM1 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. GM1/2,SP-C-GM1/2 founder mice were backcrossed to GM2/2 mice producing GM2/2,SP-C-GM1/2 and
GM1/2,SP-C-GM1/2 progeny, which were then bred to
1040-0605/97 $5.00 Copyright r 1997 the American Physiological Society
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Huffman Reed, Jacquelyn A., Ward R. Rice, Zsuzsanna
K. Zsengellér, Susan E. Wert, Glenn Dranoff, and Jeffrey A. Whitsett. GM-CSF enhances lung growth and causes
alveolar type II epithelial cell hyperplasia in transgenic mice.
Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17): L715–L725,
1997.—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 (GM1/1) or GM-CSF
null mutant (GM2/2) mice. Lung weight and volume were
significantly increased in SP-C-GM mice compared with
GM1/1 or GM2/2 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 GM1/1 and GM2/2 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 a-subunit in both GM1/1
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.
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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 31
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
Vlung 5 SPlung 3 Apoint 3 tinterval
where SPlung is the count of all points falling on the sections,
Apoint the area of one test point at a magnification of one, and
tinterval is the mean thickness of the interval (2).
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 3 104 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 32 magnification and were
counted at 340 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 a-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 a-subunit (GMCSF-Ra) 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-Ra. Slides were washed in PBS and were
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obtain homozygote mice of either the GM1/1,SP-C-GM1/1
or GM2/2,SP-C-GM1/1 genotype. Lines of GM1/1 or
GM2/2 control mice were derived by backcrossing SP-CGM2/2 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 phosphatebuffered saline (PBS), which were pooled, measured for
recovery volume, and stored at 220°C until assay. GM-CSF
was measured using the Endogen GM-CSF Minikit enzymelinked 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 GM1/1 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-proSP-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.
GM-CSF ENHANCES LUNG GROWTH AND TYPE II CELL HYPERPLASIA
RESULTS
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 GM2/2 and GM1/1 backgrounds; line 48 SP-C-GM1/1 homozygote mice were
bred only in the GM2/2 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 GM2/2,SP-C-GM1/1 mice and
line 59 GM1/1,SP-C-GM1/1 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 GM2/2,SP-C-GM1/1 and
GM1/1,SP-C-GM1/1 BAL fluids. In comparison, GMCSF concentrations in the BAL fluid from GM1/1
control mice, although detectable, were near the lower
limit of detection (5 pg/ml) by ELISA and so are
reported as not detectable. GM2/2 mice were completely deficient in GM-CSF.
LPS was administered intratracheally to nontransgenic GM1/1 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
GM1/1 and GM2/2 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 GM1/1 and GM2/2 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
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incubated in goat anti-rabbit secondary antibody (described
above) for 30 min. Anti-GM-CSF-Ra 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-Ra in isolated type II cells.
Type II cells were isolated from GM1/1 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 H2 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-Ra cDNA (Genbank accession no. M85078). The forward primer sequence was 58TGCGGGGCCAGTGCGGTTCCT-38; the reverse primer sequence was 58-CAGTGCTTCATCCTCGTGTCG-38. PCR was
performed according to the PCR SuperMix protocol (GIBCOBRL) 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 3 104 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 3 10211 to 1 3 1027 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 substrateenzyme reaction after 10 min, and absorbance was measured
at 450 nm using a Dynatech plate reader.
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GM-CSF ENHANCES LUNG GROWTH AND TYPE II CELL HYPERPLASIA
of any of the animals. Additionally, four separate
GM1/2,SP-C-GM1/2 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.
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 GM1/1 and GM2/2
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 GM2/2 and GM1/1 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 PCNAstaining alveolar macrophages were also found in the
alveolar spaces and interstitium of SP-C-GM compared
with GM2/2 and GM1/1 lungs.
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 GM2/2 and GM1/1 mice. Fluid-filled
lungs of SP-C-GM mice were 30–40% heavier than
those of GM2/2 and GM1/1 control mice. Correspondingly, the total volume (mm3 ) of inflated SP-C-GM
lungs was 25–40% larger than GM2/2 and GM1/1
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 3 105 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 GM2/2 and GM1/1 mice (Table 1).
GM-CSF receptors on type II cells and alveolar macrophages. Immunohistochemical staining was used to
identify GM-CSF-Ra subunits. The GM-CSF receptor
complex consists of GM-CSF-Ra and a shared bsubunit (bc ) 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-CSFRa, 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).
RT-PCR was used to amplify nucleotides 642–951 of
the GM-CSF-Ra 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
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Fig. 1. Concentrations of granulocyte macrophage colony-stimulating factor (GM-CSF) in bronchoalveolar lavage
(BAL) fluid. A: BAL fluid was collected from lungs, and GM-CSF was measured by enzyme-linked immunosorbent
assay (ELISA). Increased concentrations of GM-CSF were found in BAL fluids from surfactant protein (SP)-C-GMCSF transgene positive mice (SP-C-GM mice) compared with GM-CSF gene-targeted mice (GM2/2 mice) and
wild-type mice homozygous for GM-CSF (GM1/1 mice). GM-CSF was not detected (ND) in GM2/2 mice. GM-CSF
was at the borderline of the limit of detection (#5 pg/ml) in GM1/1 mice, reported as ND. Mice (n 5 20/group)
ranged in age from 2 to 5 mo. Values are means 6 SE. B: LPS was administered intratracheally to nontransgenic
GM1/1 FVB/N mice. BAL fluid was collected, and GM-CSF was measured by ELISA. Concentrations of endogenous
GM-CSF in the BAL fluid at 2, 4, 6, and 8 h posttreatment were similar to those found in SP-C-GM mice. Data points
represent values for individual animals; n 5 2 experiments/time point. LPS, lipopolysaccharide.
GM-CSF ENHANCES LUNG GROWTH AND TYPE II CELL HYPERPLASIA
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Fig. 2. Lung histology. Lung sections were stained with hematoxylin and eosin. At 4–5 mo of age, lungs from line 59
GM-CSF gene-targeted mice also homozygous for the SP-C-GM transgene (GM2/2,SP-C-GM1/1; A) and mice
homozygous for GM-CSF and the SP-C-GM transgene (GM1/1,SP-C-GM1/1; B) had hyperplastic alveolar walls
and increased numbers of alveolar macrophages compared with GM2/2 (C) and GM1/1 (D) mice. A is
representative of marked alveolar hyperplasia found in regions of all SP-C-GM lungs, and B is representative of the
appearance of both hyperplastic and relatively normal regions in all SP-C-GM mice at 2–5 mo. Similar histological
changes were found in mice from line 48 GM2/2,SP-C-GM1/1 and GM1/2,SP-C-GM1/2 founder mice as
described in MATERIALS AND METHODS. Short arrows, alveolar walls; long arrows, alveolar macrophages. Bar 5 50 µm.
band was consistently amplified from type II cell mRNA.
The precise identity of that band is unknown. The
presence of GM-CSF-Ra 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.
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 dosedependent manner, indicating that DNA synthesis was
increased in the presence of GM-CSF (Fig. 7). The number
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Fig. 3. Type II cell hyperplasia in SP-C-GM mice. Pro-SP-C was detected by immunoperoxidase staining. Increased
numbers of type II cells were noted in lungs from line 59 GM2/2,SP-C-GM1/1 (A) and GM1/1,SP-C-GM1/1 (B)
mice compared with GM2/2 (C) and GM1/1 (D) mice. Type II cells from GM2/2,SP-C-GM1/1 and GM1/1,SP-CGM1/1 mice were larger and stained more intensely than type II cells from GM2/2 and GM1/1 mice. Arrows, type
II cells. Bar 5 25 µm.
of cells per well was not increased by the addition of
GM-CSF, as assessed by cell counts (data not shown).
DISCUSSION
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 GM2/2 or GM1/1 control
mice, as assessed by weight or morphometric analysis,
and numbers of type II cells per lung were increased threeto fourfold. Lung epithelial cell-specific GM-CSF expression corrected alveolar proteinosis in the GM2/2 mice,
enhanced lung growth, and caused type II cell hyperplasia
in mice from either GM2/2 or GM1/1 backgrounds.
Lung injury initiates a complex cascade of inflammatory responses that limit tissue damage and enhance
GM-CSF ENHANCES LUNG GROWTH AND TYPE II CELL HYPERPLASIA
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Fig. 4. Colocalization of anti-proliferating cell nuclear antigen (PCNA) and anti-pro-SP-C staining in type II cells.
Anti-pro-SP-C antibodies were identified with a secondary antibody conjugated to Texas red; anti-PCNA was
stained with a secondary antibody conjugated to fluorescein isothiocyanate. Type II cells labeled with anti-pro-SP-C
have orange-red cytoplasm (arrowheads), whereas proliferating type II cells labeled with anti-pro-SP-C and
anti-PCNA have yellow-orange cytoplasm and intense yellow nuclear staining (large arrows). Proliferating cell
types other than type II cells are labeled with green nuclear staining (small arrows). GM2/2,SP-C-GM1/1 (A) and
GM1/1,SP-C-GM1/1 (B) mice have markedly increased numbers of quiescent and PCNA-staining type II cells and
PCNA-staining alveolar macrophages compared with GM2/2 (C) and GM1/1 (D) mice. Bar 5 40 µm.
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 GM2/2 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
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GM-CSF ENHANCES LUNG GROWTH AND TYPE II CELL HYPERPLASIA
Table 1. Lung weights and morphometric analysis
Genotype
Body
Weight, g
Lung Wet
Weight, g
Inflated Lung
Weight, g
Total Lung
Volume, mm3
Type II Cells Per 1 3 105 µm3
Lung Tissue
Type II Cells
Per Lung
GM2/2, SP-C-GM1/1
GM1/1, SP-C-GM1/1
GM2/2
GM1/1
28.2 6 1.1
23.5 6 0.7
25.3 6 0.8
28.5 6 2.1
0.32 6 0.03*
0.29 6 0.02*
0.26 6 0.01
0.23 6 0.01
0.94 6 0.04
0.83 6 0.08
0.61 6 0.02
0.54 6 0.01
764 6 28†
694 6 52†
521 6 3
446 6 24
12.5 6 1.6†
12.7 6 2.0†
4.4 6 0.4
4.7 6 0.2
9.5 6 1.2 3 107 †
8.8 6 1.9 3 107 †
2.3 6 0.2 3 107
2.1 6 0.5 3 107
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 differ-
Fig. 5. Immunohistochemical staining with anti-GMCSF receptor a-subunit (GM-CSF-Ra) antibodies. Type
II cells and bronchiolar epithelial cells in GM1/1 mice
were stained with GM-CSF-Ra antibodies (A). Alveolar
macrophages and endothelial cells were also stained
(not shown). Staining in GM1/1 tissue sections was
blocked when primary (anti-GM-CSF-Ra) antibody was
preincubated with an immunizing peptide (B). Staining
of tissue sections incubated without primary antibody
was indistiguishable from staining seen in B. Staining
in GM2/2,SP-C-GM1/1 lungs (not shown) was similar
to the pattern observed in GM1/1 lungs. Short arrows,
type II cell; long arrows, bronchiolar epithelium. Bar 5
40 µm.
entiation 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
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Values are means 6 SE. Lungs from 4- to 5-mo-old mice (n 5 11) were removed and weighed. * Differences in lung wet weights between
surfactant protein (SP)-C granulocyte macrophage (GM) transgenic mice (SP-C-GM mice) and GM colony-stimulating factor (CSF)
gene-targeted mice (GM2/2) or wild-type mice homozygous for GM-CSF (GM1/1 mice) were statistically significant as determined by
analysis of variance (ANOVA; P , 0.03) and Student-Newman-Keuls test (P , 0.05). Three mice of each genotype were killed at 4–5 months of
age for morphometric analysis. Lungs were inflation-fixed and weighed, and total lung volume (mm3 ) was determined using the Cavalieri
method as described in MATERIALS AND METHODS. Type II cells were counted per 1 3 105 µm3 of lung parenchyma, and total number of type II
cells per lung was calculated. Lungs of SP-C-GM mice were 30–40% larger, and total number of type II cells was increased 3- to 4-fold
compared with controls. † Differences between SP-C-GM mice and GM2/2 or GM1/1 control mice were statistically significant as determined
by ANOVA (P , 0.005) and Student-Newman-Keuls test (P , 0.05). Differences between GM2/2, SP-C-GM1/1 and GM1/1, SP-C-GM1/1
mice or between GM2/2 and GM1/1 mice were not statistically significant.
GM-CSF ENHANCES LUNG GROWTH AND TYPE II CELL HYPERPLASIA
Fig. 6. Reverse transcriptase (RT)-polymerase chain reaction (PCR)
of GM-CSF-Ra from isolated mouse type II cells. RNA was harvested
from a mouse alveolar macrophage cell line (MH-S cells) or primary
type II cells isolated from GM1/1 mice and was used for RT-PCR to
detect GM-CSF-Ra transcripts. A band of the expected size (309
nucleotides) was generated from MH-S and type II cell (TII) RNA. In
addition, a slightly larger band was consistently generated from type
II cell RNA. No band was observed in the reaction mix without
template (Ø). A band of ,0.7 kb was produced in the reaction
containing genomic DNA as template.
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 GM2/2,SP-C-GM1/1 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
Fig. 7. GM-CSF enhanced bromodeoxyuridine (BrdU) uptake by type
II cells in vitro. Primary cultures of rat type II cells were prepared
and treated with GM-CSF at the indicated concentrations for 72 h.
BrdU was added to the cells for the final 24 h, and BrdU incorporation was quantitated by ELISA. GM-CSF significantly enhanced
BrdU incorporation at concentrations .1 3 1029 M, as assessed by
analysis of variance (P , 0.05). Data represent means 6 SE for 5
independent cell preparations; n 5 4 for each preparation.
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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 GM1/1
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 GM1/1 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 SPC-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.
L723
L724
GM-CSF ENHANCES LUNG GROWTH AND TYPE II CELL HYPERPLASIA
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 bc 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.
We acknowledge and thank Dr. Harriet Iwamoto, Dana Fiedeldey,
Kristen Spradlin, and Sherri Profitt for technical assistance.
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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