Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229
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ABSTRACT |
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Surfactant
proteins and phospholipids accumulate in the alveolar spaces and lung
tissues of mice deficient in granulocyte-macrophage colony-stimulating
factor (GM-CSF), with pathological findings resembling the histology
seen in the human disease pulmonary alveolar proteinosis (PAP).
Previous metabolic studies in GM-CSF-deficient [GM(/
)] mice indicated that defects in
surfactant clearance cause the surfactant accumulation in PAP. In the
present study, GM(
/
) mice were treated daily or weekly
with recombinant mouse GM-CSF by aerosol inhalation or intraperitoneal
injection for 4-5 wk. Lung histology, alveolar macrophage
differentiation, and surfactant protein B immunostaining returned
toward normal levels in the GM-CSF aerosol-treated mice. Alveolar and
lung tissue saturated phosphatidylcholine and surfactant protein B
concentrations were significantly decreased after treatment with
aerosolized GM-CSF. Cessation of aerosolized GM-CSF for 5 wk resulted
in increased saturated phosphatidylcholine pool sizes that returned to
pretreatment levels. In contrast, PAP did not improve in
GM(
/
) mice treated daily for 5 wk with larger doses of
systemic GM-CSF. Aerosolized GM-CSF improved PAP in the
GM(
/
) mice, demonstrating that surfactant homeostasis can
be influenced by local administration of GM-CSF to the respiratory tract.
granulocyte-macrophage colony-stimulating factor; surfactant; surfactant proteins; alveolar macrophage
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INTRODUCTION |
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GRANULOCYTE-MACROPHAGE colony-stimulating factor
(GM-CSF) is a hematopoietic growth factor required for pulmonary
surfactant homeostasis (5, 26). Although gene-targeted disruption of either the GM-CSF or the GM-CSF-receptor common -subunit (
c) locus in mice failed to perturb hematopoiesis, surfactant proteins (SPs) and phospholipids accumulated in the alveolar spaces (5, 20, 24,
26). Lung histopathology in GM-CSF-deficient
[GM(
/
)] and
c-deficient mice resembled
that of the human disease known as pulmonary alveolar proteinosis
(PAP). Pulmonary surfactant is a complex mixture of phospholipids and
SP-A, -B, -C, and -D, which are synthesized and secreted primarily by
alveolar type II epithelial cells (29). Approximately 80% of alveolar
surfactant is cleared by type II cells, which recycle or catabolize SPs
and lipids; most of the remaining 20% of surfactant is catabolized by
macrophages (23, 30). Metabolic studies demonstrated that decreased
surfactant clearance in GM(
/
) mice contributed to a
progressive accumulation of surfactant (15, 22).
GM-CSF was selectively expressed in the lungs of the
GM(/
) mouse with pulmonary epithelial cell-specific
promoter sequences of the human
SP-C gene (11).
Expression of the SP-C-GM-CSF (SPC-GM) transgene restored surfactant lipid and protein clearance and corrected
the PAP typical of GM(
/
) mice (11, 14). Correction of PAP
with the SPC-GM transgene was associated
with increased numbers and altered histology of alveolar macrophages
and type II cells, consistent with the concept that local production of GM-CSF influences differentiation and/or function of these pulmonary cell types (12).
Alteration of GM-CSF production or response has been recently associated with PAP in humans. Alveolar macrophages from a patient with PAP synthesized GM-CSF mRNA when stimulated with lipopolysaccharide in vitro but, unlike normal alveolar macrophages, did not release GM-CSF into the medium (27). Seymour et al. (25) demonstrated that a daily subcutaneous injection of GM-CSF improved oxygen saturation and exercise tolerance in one patient with acquired PAP, suggesting that therapy with GM-CSF may be useful for treatment of PAP.
In the present study, GM(/
) mice were treated with
injected or inhaled recombinant mouse GM-CSF. Analyses of lung tissues and bronchoalveolar lavage (BAL) fluid demonstrated that aerosolized GM-CSF significantly inhibited the progression of PAP in
GM(
/
) after 4-5 wk of daily treatment, whereas
neither weekly aerosol treatments nor daily intraperitoneal injections
of GM-CSF altered PAP.
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METHODS |
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Animals. The GM(/
) mice
used were generated by targeted ablation of the
GM-CSF gene locus as previously described
(5). The GM(
/
) mutation was maintained in a C57Bl/6/FVB/N
background. Mice were bred and housed in microisolator cages and
required no special care. Equal numbers of male and female mice were
randomly divided into groups for aerosol inhalation or intraperitoneal injection of GM-CSF (Table 1). Mice were 6 wk old when treatments were initiated.
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Aerosol treatment. Mice were placed in 50-ml conical centrifuge tubes with a hole in the tip and ventilation holes along the sides of each tube. A 4-liter container with lid was modified by drilling a 1.25-inch-diameter hole in one end to serve as an inlet and four 0.75-inch-diameter holes in the rear of the box for an outlet. Mice were positioned in the container to ensure exposure of the nasal area to the aerosol. A Puritan Bennett model US-1 clinical ultrasonic nebulizer was used to generate the aerosol at a flow rate of 1.5 l/min. Treatment time for each group was ~45 min.
Administration of aerosolized GM-CSF.
To determine the volume of aerosol reaching the lungs of
GM(/
) mice, 100 µCi of a technetium-sulfur colloid in
12 ml of saline were nebulized to a group of five mice. Recovery of
radioactivity was measured in the lung parenchyma to estimate the
dosage of GM-CSF delivered by the aerosol. The volume of solution
deposited in the lungs was ~1.5 µl in 45 min. Recombinant mouse
GM-CSF was kindly provided by Immunex (Seattle, WA). The vehicle
solution contained 0.1% mouse albumin fraction V from Sigma
Diagnostics (St. Louis, MO) in 0.9% saline. GM-CSF was diluted in
vehicle in a total volume of 12 ml immediately before nebulization.
Mice treated for 4 wk inhaled an aerosol solution containing 1.3 µg/ml of GM-CSF, thus receiving an estimated dose of ~2 ng/mouse
(equivalent to 0.07 ± 0.01 µg/kg), administered 5 days/wk. Mice
treated for 5 wk inhaled an aerosol solution containing 2.6 µg/ml of
GM-CSF, receiving an estimated dose of ~4 ng/mouse (equivalent to
0.14 ± 0.02 µg/kg), administered either 1 or 5 days/wk for 5 wk.
Control mice received aerosolized vehicle 5 days/wk for 5 wk. All mice
described above were killed for analysis 24 h after the final
treatment. Three groups of mice treated with aerosolized GM-CSF or
vehicle for 5 wk were analyzed 5 wk after completion of the aerosol treatments.
Intraperitoneal injection of GM-CSF.
GM-CSF was diluted in 1% serum-saline vehicle to a final concentration
of 2 µg/ml. The serum used to prepare the vehicle was collected from
untreated GM(/
) mice and pooled. Mice were injected
intraperitoneally with 100 µl of GM-CSF solution (200 ng of GM-CSF,
equivalent to a dose of ~8 µg/kg body wt) or vehicle 5 days/wk for
5 wk.
Intratracheal injection of GM-CSF.
GM(/
) and GM-CSF-positive
[GM(+/+)] mice were briefly anesthetized with inhaled
methoxyflurane (Metofane, Pitman-Moore, Mundelein, IL) and
intratracheally intubated with a 24-gauge animal feeding needle (Popper
& Sons, New Hyde Park, NY). Three daily treatments of 15 ng of GM-CSF
(equivalent to 6.3 µg/kg body wt) in 100 µl of saline or saline
alone were instilled into the lungs via the intratracheal needle. On
the third day of treatment, 0.1 µCi of
125I-labeled SP-C was added to the
GM-CSF injection solution. Recombinant SP-C (Byk Gulden, Constance,
Germany) was iodinated with
125I-labeled Bolton-Hunter reagent
(ICN, Irvine, CA) as previously described (13). All mice were killed at
40 h after the final intratracheal injection. The lungs were lavaged
and homogenized. 125I-SP-C content
in BAL and lung homogenate samples was counted, and saturated
phosphatidylcholine (Sat PC) pool sizes were measured as previously
described (13, 15).
Processing and staining of tissues for histopathology. Mice were anesthetized with intraperitoneal pentobarbital sodium. The lungs were inflation fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, for 24 h as previously described (11). The tissues were then washed in PBS, dehydrated in a series of alcohols, and embedded in paraffin. Hematoxylin and eosin staining was used for histological analysis of paraffin sections of the lung, liver, and spleen.
Immunohistochemistry. Paraffin sections (5 µm) sampling all five lobes of lung tissue were stained for SP-B in each mouse. Anti-SP-B, a rabbit anti-bovine polyclonal antibody that recognizes mature SP-B, was detected with the Vectastain ABC anti-rabbit immunohistochemical horseradish peroxidase kit from Vector Laboratories (Burlingame, CA) as previously described (1). The tissues were counterstained with Tris-cobalt and nuclear fast red and qualitatively assessed for relative SP-B immunostaining. In addition, 5 random fields in 3 vehicle-treated and 5 GM-CSF-treated mice were examined under high magnification (×40), and 31-56 alveoli were counted per field. Alveoli with and without immunostained proteinosis material were counted to determine the percentage of alveoli containing stained material.
BAL. The trachea was cannulated, and the lungs were washed three times with five 1-ml aliquots of PBS (15). BAL fluids for each animal were pooled, measured for volume, and divided into aliquots for phospholipid or protein measurement. The lung tissue was homogenized for Sat PC measurement as previously described (15). SP-B was measured by enzyme-linked immunosorbent assay (ELISA) with bovine SP-B as a standard as previously described (17). The total alveolar pool size was calculated and divided by body weight. Replicate SP-B concentrations for each sample are expressed as a percentage of the control values measured on the same ELISA plate to control for plate-to-plate variability.
-Naphthyl acetate esterase
detection. Cytospins of BAL cells were prepared from an
aliquot of BAL fluid and stained for
-naphthyl acetate esterase
(
-NAE) activity with an
-NAE staining kit (Sigma Diagnostics, St.
Louis, MO) according to the protocol provided in the package insert.
Differential cell counts of at least 250 cells/slide were performed to
determine the percentages of stained and unstained alveolar macrophages.
Statistical analysis. Values are
expressed as means ± SE of n
observations. Statistical analyses of the data were performed by ANOVA
or 2 test;
P < 0.01 was considered significant.
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RESULTS |
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Aerosolized GM-CSF ameliorated PAP.
Histological examination of the lungs from GM(/
) mice
treated with aerosolized GM-CSF for 4 or 5 wk demonstrated a marked
improvement in PAP compared with that in vehicle-treated mice (Fig.
1). Improvement in PAP was observed after
treatment for 4 wk with 2 ng or 5 wk with 4 ng of GM-CSF daily.
However, aerosol treatments were consistently more effective in the
mice receiving the higher concentration of GM-CSF daily for 5 wk.
Perivascular and peribronchiolar mononuclear cell infiltrates were also
decreased in mice treated daily with aerosolized GM-CSF. PAP was not
improved in GM(
/
) mice treated 1 day/wk with 4 ng of
GM-CSF for 5 wk; histology of these lungs resembled that in
vehicle-treated or untreated GM(
/
) control mice (data not
shown).
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The alveolar spaces of untreated GM(/
) mice contained
abundant SP-B staining of PAP material as previously described (5, 11,
12). SP-B staining in GM(
/
) mice treated daily with aerosolized vehicle did not differ from that in untreated
GM(
/
) mice. In contrast, SP-B staining was markedly
decreased in the alveolar spaces of lung tissue sections from
GM(
/
) mice receiving daily aerosolized GM-CSF (Fig.
2). Only scant, scattered
SP-B staining was observed in the alveolar spaces of aerosolized
GM-CSF-treated GM(
/
) mice, whereas cell-associated
staining for SP-B appeared to be increased. The number of alveoli
containing SP-B-immunostained proteinosis material was counted in lung
sections from mice treated with daily aerosolized GM-CSF or vehicle;
the percentage of alveoli containing immunostaining was 9.6 ± 1.5%
in GM-CSF-treated mice compared with 42.3 ± 2.6% in
vehicle-treated mice (data not shown; P < 0.001). Consistent with the
histological assessment of PAP by hematoxylin and eosin staining, mice
treated for 5 wk with 4 ng of GM-CSF had less alveolar SP-B staining
than mice treated for 4 wk with 2 ng of GM-CSF. Intense SP-B staining
was observed in the alveolar spaces of GM(
/
) mice treated
1 day/wk with aerosolized GM-CSF (data not shown), these lungs being
indistinguishable from those of vehicle-treated or untreated
GM(
/
) mice.
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SP-B concentrations decreased significantly in mice treated with daily
aerosolized GM-CSF (Fig.
3). The SP-B concentration in BAL fluid from mice treated daily for 4 wk with aerosolized GM-CSF
(2 ng) was 63 ± 5% (SE) of that from mice treated with vehicle.
SP-B from mice treated daily for 5 wk with GM-CSF (4 ng) was 23 ± 3% of that from mice treated with vehicle. BAL fluid SP-B
concentrations in mice treated weekly with aerosolized GM-CSF were
unchanged, being 105 ± 5% of that in GM(/
)
mice treated with vehicle alone. SP-B concentrations in BAL fluid from
GM-CSF-treated mice remained approximately four- to fivefold higher
than those recovered from untreated wild-type mice.
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Sat PC, the major component of surfactant, was measured in mice treated
with GM-CSF for 5 wk (Fig.
4). Daily aerosolized
GM-CSF (4 ng) significantly decreased alveolar and lung tissue Sat PC pool sizes in GM(/
) mice compared with that in
GM(
/
) mice treated daily with vehicle. However, alveolar
and lung Sat PC pool sizes in GM-CSF-treated mice remained
approximately fivefold and twofold higher, respectively, than levels
measured in untreated wild-type mice (data not shown). Treatment with
aerosolized GM-CSF 1 day/wk did not improve Sat PC pool sizes in the
GM(
/
) mice.
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To investigate the effects of GM-CSF treatment on alveolar macrophage
morphology, cytospins of BAL cells were prepared from mice treated with
aerosolized GM-CSF for 5 wk. The number of cells from GM-CSF-treated
mice did not differ from that recovered from vehicle-treated or
untreated GM(/
) mice (data not shown). The cells were
stained for fluoride-resistant
-NAE activity, an indicator of tissue
macrophage differentiation (18, 31). In untreated or vehicle-treated
GM(
/
) mice, the majority of alveolar macrophages were
enlarged, containing a foamy cytoplasm with abundant lipid-filled vacuoles (Fig. 5). Fluoride-resistant
-NAE staining was observed in only 11.3 ± 2.5% (SE) of alveolar
macrophages from untreated or vehicle-treated GM(
/
) mice.
In contrast, significantly fewer of the alveolar macrophages recovered
from GM-CSF-treated mice had the characteristic foamy, lipid-laden
cytoplasm observed in vehicle-treated or untreated GM(
/
)
mice, and 60.0 ± 4.6% (SE) of alveolar macrophages stained
positively for fluoride-resistant
-NAE activity.
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Recurrence of PAP after discontinuing
GM-CSF. To investigate the reversibility of the effects
of GM-CSF on PAP, mice were treated with GM-CSF (4 ng) aerosol for 5 wk
and analyzed 5 wk after cessation of treatment. SP-B concentrations
were higher than those measured immediately after 5 wk of aerosolized
GM-CSF but remained lower than those in vehicle-treated or untreated age-matched control GM(/
) mice (Fig.
6A).
Immunostaining of SP-B in the alveolar spaces increased in
GM(
/
) mice after the cessation of treatment, consistent
with a recurrence of PAP (data not shown). Alveolar, tissue, and total
lung Sat PC pool sizes increased in the lungs of GM(
/
)
mice 5 wk after treatment with aerosolized GM-CSF was discontinued,
reaching levels similar to those in vehicle-treated mice (Fig.
6B).
|
Systemic GM-CSF did not improve PAP.
Systemic administration of GM-CSF had no effect on PAP in
GM(/
) mice. Histological analyses and anti-SP-B
immunostaining demonstrated widespread PAP in GM(
/
) mice
injected intraperitoneally with 200 ng of GM-CSF daily for 5 wk,
similar to findings in vehicle-treated or untreated GM(
/
)
mice (Fig. 7). Likewise, SP-B
concentrations measured by ELISA in BAL fluid and Sat PC pool sizes of
GM(
/
) mice treated with injected GM-CSF did not differ
from vehicle-treated or untreated GM(
/
) mice (Fig.
8).
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|
High doses of intratracheal GM-CSF for 3 days did not
alter PAP in GM(/
) mice. GM-CSF (15 ng)
delivered directly to the lungs daily for 3 days did not alter Sat PC
or the clearance of exogenous SP-C. At 40 h after the third and final
treatment, Sat PC pool sizes in GM(
/
) and GM(+/+) mice
treated with GM-CSF remained similar to those in mice treated with
saline (Fig.
9A). Likewise, the 125I-SP-C recovery
from treated and untreated GM(
/
) mice was unchanged (Fig.
9B).
125I-SP-C recovery from treated
and untreated GM(+/+) control mice also did not differ.
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DISCUSSION |
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Surfactant phospholipids and SPs, accompanied by enlarged, foamy
alveolar macrophages and perivascular or peribronchiolar mononuclear
cell infiltrates, accumulate in the lungs of GM(/
) mice.
Histological findings in GM(
/
) mice resemble the lung pathology seen in humans with PAP. The present study demonstrated the
efficacy of inhaled GM-CSF for treatment of PAP. Mice were treated with
aerosolized recombinant mouse GM-CSF daily for 4 or 5 wk or with
systemic GM-CSF daily for 5 wk. PAP was improved in GM(
/
)
mice receiving daily GM-CSF aerosol treatments but not in mice
receiving larger doses of systemic GM-CSF. Additionally, intratracheal
treatment of GM(
/
) mice for 3 days with 15 ng of GM-CSF
daily did not alter phospholipid or SP-C clearance from the lungs.
The finding that aerosolized GM-CSF improves PAP in GM(/
)
mice supports the concept that GM-CSF influences surfactant homeostasis through local signaling pathways within the lung. Previous studies (3,
19) demonstrated that GM-CSF signaling in hematopoietic cells occurs
through both the JAK-STAT and Ras-Raf pathways. STAT5A plays a role in
GM-CSF-induced proliferation and gene expression in hematopoietic
cells; however, PAP was not evident in STAT5A-deficient mice (7). The
precise identity of the pathway(s) involved in the resolution of PAP
remains unclear, but it may regulate expression levels of genes
encoding proteases or phospholipases required for surfactant catabolism
in alveolar macrophages and type II cells. Clearance of surfactant from
the alveolar spaces was markedly impaired in GM(
/
) mice
and corrected by gene replacement in which GM-CSF was expressed at high
levels in respiratory epithelial cells of the lung (14, 15). Alveolar
macrophages and type II cells clear the majority of surfactant from the
alveolar spaces and are known to express GM-CSF receptors (12). Thus
GM-CSF appears to have a role in modulating catabolic pathways in
alveolar macrophages and/or type II cells that contribute to the
disordered surfactant homeostasis seen in PAP.
In previous studies of GM(/
) mice (12, 32), type II cell
proliferation was increased in response to continuous exposure to
GM-CSF, suggesting that increases in type II cell number may have
contributed to resolution of PAP. However, in the present study, type
II cell proliferation or number, as assessed by proliferating cell
nuclear antigen staining and morphology, did not appear to increase
(data not shown). GM-CSF levels are likely increased only transiently
after aerosolized GM-CSF, in contrast to the continuous levels of
GM-CSF produced throughout development in SPC-GM transgenic mice, and
may account for the absence of type II cell proliferation in the
present study (11, 12). With aerosolized GM-CSF, improvement in PAP
occurred in the absence of changes in the numbers of type II cells,
suggesting that the growth stimulatory effects of GM-CSF were not
required for resolution of PAP in GM(
/
) mice.
Alveolar macrophages from untreated GM(/
) mice have a
characteristic morphology, with enlarged, foamy cytoplasm and
lipid-filled vacuoles thought to result from a defect in surfactant
catabolism (5, 15, 26). Similar alterations in alveolar macrophage morphology and function have been observed in alveolar macrophages from
humans with PAP (9, 10). It is unclear, however, whether the
alterations in macrophage morphology are directly related to a lack of
GM-CSF during differentiation in the lung per se. Morphological changes
seen in alveolar macrophages in PAP may also represent changes
secondary to the increased surfactant concentrations. In the present
study, alveolar macrophage morphology was improved by aerosolized
GM-CSF. In addition, perivascular and peribronchiolar mononuclear cell
infiltrates were reduced. It is not known whether mature alveolar
macrophages obtained from the GM-CSF-treated mice were derived by
correction of the foamy alveolar macrophages or by recruitment and
differentiation of precursor cells, although the latter hypothesis
seems more likely.
Induction of alveolar macrophage maturation is likely an important
factor in the resolution of PAP by aerosolized GM-CSF. Paucity of
fluoride-resistant -NAE staining in alveolar macrophages from
untreated GM(
/
) mice suggests impaired or delayed
maturation of these cells. Although the number of macrophages did not
change with GM-CSF, the proportion of these cells staining for
fluoride-resistant
-NAE was increased by aerosolized GM-CSF.
Previous studies (2, 21, 32) associated alveolar macrophage
differentiation or function with resolution of PAP in
mice. The length of time (4-5 wk) required for
improvement in PAP in response to GM-CSF is consistent with the time
for immature monocytic cells to be recruited to the lung and/or
stimulated to differentiate. Monocytic precursor cells generally
require 2-3 wk of GM-CSF stimulation in vitro to promote alveolar
macrophage differentiation (6, 16). This time requirement for cell
differentiation and restoration of macrophage morphology and activity
in the lungs of GM(
/
) mice is also consistent with the
estimated 21- to 28-day turnover of alveolar macrophages in mouse lungs
(8, 28). The finding that acute intratracheal treatment with a
relatively high concentration of GM-CSF (15 ng) did not alter lung Sat
PC or SP-C clearance supports the concept that an extended period of
treatment, perhaps dependent on alveolar macrophage differentiation, is
necessary for the improvement in PAP in this model.
Improvement in PAP after aerosolized GM-CSF was associated with
macrophage maturation, supporting the concept that GM-CSF plays an
important role in restoring alveolar macrophage function in
GM(/
) mice. However, changes in alveolar macrophage
function after daily aerosolized GM-CSF may not completely explain the improvement in PAP. Because type II cells clear ~80% of alveolar Sat
PC in the rabbit, it is reasonable to predict that disordered type II
cell function may play a role in PAP (23, 30). In the present study,
aerosolized GM-CSF was more effective in restoring tissue rather than
alveolar pools of surfactant lipid. The alveolar Sat PC pool decreased
by ~50% after GM-CSF treatment but remained eightfold higher than
that seen in wild-type mice. In contrast, lung tissue Sat PC was
decreased to ~30% of that in the vehicle-treated GM(
/
)
mice, remaining twofold higher than in wild-type mice. Of interest,
staining of type II cells for SP-B was more intense after aerosolized
GM-CSF treatment. Taken together, these results suggest that the
aerosolized GM-CSF increased surfactant reuptake or catabolism in type
II cells in addition to its effects on macrophage-mediated clearance.
Histological similarities in the pathology of GM(/
) mice
and humans with PAP suggest that findings in GM(
/
) mice
may be relevant to human disease. Impairment of expression or function of GM-CSF was recently associated with PAP in humans (25, 27). The
present study demonstrating that exogenous GM-CSF delivered to the lung
improves PAP in GM(
/
) mice supports the use of GM-CSF as
a potential therapeutic agent for PAP in humans. PAP has also been
associated with decreased GM-CSF-receptor binding and a point mutation
in the GM-CSF-receptor
c subunit (4). However, it is unlikely that
GM-CSF will be effective unless the GM-CSF receptor and its signaling
pathways are functional, thus limiting its application to those cases
where the putative cause of PAP is in the GM-CSF ligand rather than in
the receptor or signal transduction pathway.
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ACKNOWLEDGEMENTS |
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Recombinant mouse granulocyte-macrophage colony-stimulating factor was provided by Immunex Corporation (Seattle, WA).
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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.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. A. Whitsett, Div. of Pulmonary Biology, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229 (E-mail: whitj0{at}chmcc.org).
Received 13 May 1998; accepted in final form 20 December 1998.
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