1 Children's Hospital Medical Center, Cincinnati, Ohio 43229-3039; and 2 Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria 3050 Australia
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ABSTRACT |
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Pulmonary alveolar proteinosis (PAP) is caused by
inactivation of either granulocyte-macrophage colony-stimulating factor (GM-CSF) or GM receptor common -chain (
c) genes in
mice [GM(
/
),
c(
/
)], demonstrating a critical role
of GM-CSF signaling in surfactant homeostasis. To distinguish possible
phenotypic differences in GM(
/
) and
c(
/
) mice, surfactant metabolism was
compared in
c(
/
), GM(
/
), and
wild-type mice. Although lung histology in
c(
/
) and GM(
/
) mice was
indistinguishable, distinct differences were observed in
surfactant phospholipid and surfactant protein concentrations and
clearance from lungs of
c(
/
) and
GM(
/
) mice. At 1-2 days of age, lung saturated
phosphatidylcholine (Sat PC) pool sizes were higher in wild-type,
c(
/
), and GM(
/
) mice
compared with wild-type adult mice. In wild-type mice, Sat PC pool
sizes decreased to adult levels by 7 days of age; however, Sat PC
increased with advancing age in
c(
/
) and
GM(
/
) mice. Postnatal changes in Sat PC pool sizes were
different in GM(
/
) compared with
c(
/
) mice. After 7 days of age, the
increased lung Sat PC pool sizes remained constant in
c(
/
) mice but continued to increase in
GM(
/
) mice, so that by 56 days of age, lung Sat PC
pools were increased three- and sixfold, respectively, compared with wild-type controls. After intratracheal injection, the percent recovery of [3H]dipalmitoylphosphatidylcholine
and 125I-recombinant surfactant protein (SP) C was higher
in
c(
/
) compared with wild-type mice,
reflecting decreased clearance in the receptor-deficient mice. The
defect in clearance was significantly more severe in
GM(
/
) than in
c(
/
) mice. The
ratio of SP Sat PC to SP-A, -B, and -C was similar in bronchoalveolar
lavage fluid (BALF) from adult mice of all genotypes, but the ratio of
SP-D to Sat PC was markedly increased in
c(
/
) and GM(
/
) mice (10- and
5-fold, respectively) compared with wild-type mice. GM-CSF concentrations were increased in BALF but not in serum of
c(
/
) mice, consistent with a pulmonary
response to the lack of GM-CSF signaling. The observed
differences in surfactant metabolism suggest the presence of
alternative clearance mechanisms regulating surfactant homeostasis in
c(
/
) and
GM(
/
) mice and may provide a molecular basis for the
range in severity of PAP symptoms.
surfactant metabolism; alveolar
macrophage; granulocyte-macrophage colony-stimulating factor
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INTRODUCTION |
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INACTIVATION OF THE granulocyte-macrophage
colony-stimulating factor (GM-CSF) gene in mice caused alveolar
proteinosis with pathological findings similar to the human disorder
known as pulmonary alveolar proteinosis (PAP) (36). Histological
findings in the lungs of GM-CSF-deficient
[GM(/
)] mice included eosinophilic material
in the alveolar spaces, perivascular and peribronchiolar mononuclear
cell infiltrates, and foamy, lipid-laden alveolar macrophages (9, 15,
16, 32). Metabolic studies demonstrated that the four- to fivefold
increase in phospholipid pool size in GM(
/
) mice was due
at least in part to a defect in catabolism or clearance of surfactant,
demonstrating the critical role that GM-CSF signaling plays in
surfactant clearance (19, 20).
Gene targeting of the c-chain of the GM-CSF receptor
also caused alveolar proteinosis in mice, although the biochemical and physiological mechanisms underlying the histological abnormalities have
not been clarified in the
c-deficient
[
c(
/
)] mice (26, 28).
Histological findings in the lungs of
c(
/
)
mice were similar to those observed independently in
GM(
/
) mice (9, 32). The
c-chain is shared
with interleukin-3 (IL-3) and IL-5 receptor complexes in which
heterodimers of
- and
c-subunits form high-affinity
binding sites (31). The
-subunit of each receptor complex confers
ligand specificity and constitutes a low-affinity binding site. The
-subunit is converted to a high-affinity binding site when bound to
the
c-chain. In the mouse, two distinct
-subunits,
originally known as AIC2A and AIC2B, have been identified. AIC2B, or
mouse
c, is the homolog of the human
-chain and
mediates IL-3, IL-5, and GM-CSF binding and signaling (13). AIC2A, or mouse
IL3, shares 96% homology with mouse
c but is specific for binding the IL-3 receptor
-subunit (IL-3R
) only. The two mouse
-subunits
(
c and
IL3) are transcribed from separate
genes and interact with the IL-3R
with equal affinities. The
IL-3R
-
IL3 receptor complex cross-competes with
IL-3R
-
c complexes for IL-3 binding but has no effect
on GM-CSF or IL-5 binding. Thus GM-CSF and IL-5 signaling are disrupted
in
c(
/
) mice, whereas IL-3 signaling is intact.
There is considerable heterogeneity in the severity and clinical course
of PAP in humans, and the role of GM-CSF signaling in the pathogenesis
of PAP remains poorly understood. An association between hematological
malignancies, including acute myelogenous leukemia and PAP, has been
observed clinically (2, 14). Recent studies have identified patients
diagnosed with PAP in association with loss of GM-CSF production or
inhibition of GM-CSF activity. Tchou-Wong et al. (34) isolated alveolar
macrophages from a PAP patient and treated them with
lipopolysaccharide. Lipopolysaccharide treatment
stimulated transcription of GM-CSF mRNA, but, in contrast to alveolar
macrophages from normal controls, no GM-CSF was secreted into the
culture medium. Tanaka and colleagues (33) isolated a GM-CSF-binding
factor from bronchoalveolar lavage fluid (BALF) samples obtained from
11 PAP patients. The factor specifically bound GM-CSF and neutralized
its growth-promoting activity. These latter studies support the
hypothesis that the GM-CSF signaling pathway is intact in some
individuals but that GM-CSF production or availability is impaired,
causing PAP. Impaired function of the common -chain was observed in
several patients with PAP, suggesting that lesions affecting the GM-CSF
receptor or signaling pathway are involved in the pathogenesis of PAP
in a subset of individuals (8). Studies of PAP patients insensitive to
GM-CSF stimulation were reported by Seymour and colleagues (30). More recently, Dirksen et al. (7) demonstrated a loss of GM-CSF receptor on
alveolar macrophages obtained from several patients diagnosed with both
acute myelogenous leukemia and PAP. After a course of chemotherapy
eliminating leukemic cells, GM-CSF receptor expression was restored in
alveolar macrophages, and PAP symptoms were resolved. Findings of PAP
in association with impairment of GM-CSF receptor signaling provide a
basis for further characterizing surfactant homeostasis in
c(
/
) mice. Earlier studies of
c(
/
) and GM(
/
) mice reported
similar lung morphology, suggesting that the phenotypes were
indistinguishable (9, 26, 28, 32). However, in the present study,
differences in surfactant phospholipid content and metabolism were
noted in
c(
/
) and GM(
/
) mice.
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MATERIALS AND METHODS |
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Animals. c(
/
) mice were generated
by Robb et al. (28) by targeted insertion of the neomycin-resistant
gene into the
c gene locus, as described previously.
GM(
/
) mice used were generated by targeted ablation of
the GM-CSF gene locus, kindly provided by Dranoff et al. (9). The
c(
/
) mutation was maintained in a
C57BL/ 6-129Sv strain background and GM(
/
)
in C57BL/6-FVB/N. Both colonies of mice were bred and housed in
microisolator cages at the Cincinnati Children's Hospital Research
Foundation animal facility and required no special care. C57BL/6
wild-type control mice were obtained from Harlan (Indianapolis, IN).
Processing and staining of tissues for immunohistochemistry.
Mice were anesthetized with intraperitoneal pentobarbital sodium. Lungs
were inflation fixed with 4% paraformaldehyde in PBS, pH 7.4, for 24 h
as described previously (12). Tissues were then washed in PBS,
dehydrated in a series of alcohols, and embedded in paraffin. Paraffin
sections (5 µm) sampling all five lobes of lung tissue were stained
for surfactant protein-B (SP-B) and SP-D. SP-B was detected using an
anti-SP-B (number 28031) rabbit anti-bovine polyclonal antibody that
recognizes mature SP-B described previously (12). SP-D was detected
using a rabbit anti-rat SP-D antiserum, which was a kind gift from Dr.
Frances X. McCormack (University of Cincinnati) (10). Primary antibody
was detected with the Vectastain ABC goat anti-rabbit
immunohistochemical horseradish peroxidase kit from Vector Laboratories
(Burlingame, CA). Tissues were counterstained with
Tris-cobalt and nuclear fast red and qualitatively assessed for
relative SP-B immunostaining. Type II cells were counted with paraffin
sections stained with anti-proSP-C rabbit serum (number 68514) and goat
anti-rabbit immunohistochemical horseradish peroxidase, as previously
described (35). Sections were cut from distinct distal and proximal
regions of all five lobes. Eight consecutive fields of 2 × 104 µm2 contained within a larger grid were
counted in each tissue section (100 fields counted/mouse) and counted
at a magnification of ×40.
Bronchoalveolar lavage, tissue processing, and saturated phosphatidylcholine measurement. Mice were given intraperitoneal pentobarbital sodium to achieve deep anesthesia, and the distal aorta was cut to exsanguinate each animal. The chest of the animal was opened, a blunt needle was tied into the proximal trachea, and five aliquots of 0.9% NaCl were flushed into the lungs to achieve full inflation (about 1 ml) and withdrawn by syringe three times for each aliquot (20). A 22-gauge needle was used to lavage 7- and 15-day-old animals, and a 20-gauge needle was used for older animals. Total BALF for each animal was pooled, measured for volume, and divided into aliquots for analyses of saturated phosphatidylcholine (Sat PC) as described previously (20) or proteins as described in Western blotting. Lung tissue was weighed and homogenized in 0.9% NaCl for Sat PC measurement as described previously (20). Lungs of mice 1- or 2 days old were not lavaged before homogenization.
Phospholipid precursor incorporation into Sat PC. Mice were given an intraperitoneal injection with 8 µl/g body wt containing 0.45 µCi/g [3H]choline chloride (DuPont-NEN; Boston, MA) and 0.27 µCi/g [14C]palmitic acid (American Radiolabeled Chemicals; St. Louis, MO). The palmitic acid was stabilized in solution with 2.5% human serum albumin. Mice were killed with intraperitoneal pentobarbital sodium at 3, 8, 15, 24, or 48 h after isotope injection. Bronchoalveolar lavage, lung homogenization, and Sat PC measurement were performed as described in Bronchoalveolar lavage, tissue processing, and saturated phosphatidylcholine measurement.
Clearance of DPPC and SP-C. Mice were given intratracheal injections of 50 µl of saline that contained 0.3 µCi of [3H]choline-labeled dipalmitoylphosphatidylcholine (DPPC, Amersham; Arlington Heights, IL) and 0.15 µCi 125I-labeled recombinant human SP-C (rSP-C). The rSP-C (a gift from Byk Gulden; Constance, Germany) is a peptide consisting of the 34-amino acid human sequence. The rSP-C was iodinated with 125I-labeled Bolton-Hunter reagent as previously reported (17). Previous studies demonstrated that metabolism of 125I-rSP-C in rabbit and mouse lungs was similar to that of the native SP-C peptide (17). The [3H]DPPC and 125I-rSP-C were mixed with a chloroform-methanol extract of mouse surfactant, dried under N2, and resuspended in 0.9% NaCl by brief sonication. The tracer dose represents a phospholipid dose of 0.1 µmol Sat PC/kg body wt and therefore is unlikely to perturb endogenous pools (15 µmol Sat PC/kg) in the mice. Mice were sedated with isofluorane, and the trachea was exposed through a 0.5-cm midline skin incision. Radiolabeled surfactant was injected using a 1-ml syringe with a 30-gauge needle. At 3 min, 24 h, or 48 h after injection, four to six mice from each genotype group were killed with intraperitoneal pentobarbital sodium. Lungs were lavaged and homogenized for analysis of Sat PC or recovery of [3H]DPPC and 125I-rSP-C. Values for mice killed 3 min after the intratracheal injections were set at 100% and used to calculate the percentage recovery at 24 and 48 h.
Western blotting. Bronchoalveolar lavage samples containing 1 µg of Sat PC were subjected to SDS-PAGE in the presence of
-mercaptoethanol for analysis of SP-A, -C, and -D. For
SP-B analysis, aliquots containing 0.2 µg of Sat PC were
electrophoresed under nonreducing conditions. SP-A and -D were
separated on 8-16% acrylamide gel with Tris-glycine buffer; SP-B
and -C samples were separated on 10-20% acrylamide gel with
Tricine buffer (Novex; San Diego, CA). After electrophoresis, proteins
were transferred to nitrocellulose paper (Schleicher & Schuell; Keene,
NH) for SP-A and -D or to polyvinylidene difluoride paper (Bio-Rad;
Hercules, CA) for SP-B and -C. Immunoblot analysis was
carried out with the following dilutions of antisera: SP-A, 1:25,000
guinea pig anti-rat SP-A; SP-B, 1:10,000 rabbit anti-bovine SP-B; SP-C,
1:25,000 rabbit anti-recombinant human SP-C; and SP-D, 1:10,000 rabbit
anti-rat SP-D (10, 11, 17, 35). The rabbit anti-rat SP-D antiserum was
a kind gift from Dr. Frances X. McCormack (University of Cincinnati). Appropriate peroxidase-conjugated secondary antibodies were used at
1:10,000 dilutions. Immunoreactive bands were detected using enhanced
chemiluminescence reagents (Amersham; Chicago, IL). Semiquantitation of
protein bands was determined using a Molecular Dynamics phosphorimaging system and ImageQuant analysis software.
RNA isolation. RNA was isolated by a modification of the
guanidinium thiocyanate method described by Chomczynski and Sacchi (3).
Briefly, tissues were homogenized in guanidinium thiocyanate. A series
of phenol-chloroform extractions was followed by precipitation in
isopropanol as described in the RNA isolation protocol package insert
for Phase-Lock Gel IIA Heavy Tubes (5 Prime 3 Prime; Boulder, CO).
S1 nuclease protection analysis. Surfactant protein mRNAs were subjected to S1 nuclease protection analysis as described previously (9). Two micrograms of total lung RNA were used for analysis of SP-A, -B, and -C, and 10 µg were used for SP-D. Plasmids containing probe sequences for SP-A, -B, -C, and -D, and L32 were described previously (9, 21). Bands were quantitated using a Molecular Dynamics phosphorimaging system and ImageQuant analysis software.
Measurement of GM-CSF and IL-5 by ELISA. GM-CSF was measured in BALF and serum using Endogen GM-CSF Minikit ELISA (Endogen; Cambridge, MA) as described previously (15). The lower limit of detection is <5 pg/ml. IL-5 was measured by ELISA using a mouse IL-5 kit (Endogen; Cambridge, MA) following the manufacturer's protocol.
Data analysis. All values are reported as means ± SE. Differences between groups were tested by two-tailed Student's t-test. When more than two comparisons were made, ANOVA followed by the Student-Newman-Keuls multiple comparison procedure was used. Significance was accepted at P < 0.05.
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RESULTS |
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Wild-type, c(
/
), and GM(
/
)
mice had similar body weights at all ages analyzed. Mice of all three
genotypes produced litters with similar numbers and survival rates. No
abnormalities were observed in the lungs or other organs of the
animals. There was no evidence of concurrent infection in the colonies
as assessed by serology and necropsy of sentinel mice.
Surfactant protein immunostaining of lung sections from
c(
/
)
and GM(
/
) mice.
Although anti-SP-B staining was observed only in alveolar type II and
bronchiolar epithelial cells in sections from wild-type mice, intense
staining was noted in the alveolar spaces of lungs from
c(
/
) and GM(
/
) mice at
7-9 wk of age (Fig. 1). SP-B staining
was increased in alveolar type II cells and macrophages in lung
sections from
c(
/
) and GM(
/
)
mice compared with wild-type mice. SP-D immunostaining was similar to
that of SP-B, with intense staining in the alveolar spaces of lungs
from
c(
/
) and GM(
/
) mice but
not in those of wild-type mice (data not shown).
|
To determine whether numbers of type II cells were altered in
c(
/
) mice, sections from wild-type,
c(
/
), and GM(
/
) mice were immunostained with antibodies to pro SP-C (data not shown).
Numbers of type II cells within a grid area of 2 × 104 µm2 were counted in each group of animals
and compared with wild-type mice. The average number of type II cells
in lungs from
c(
/
) (7.1 ± 0.2) and
GM(
/
) (6.7 ± 0.3) mice did not differ from wild-type control mice (7.3 ± 0.2, n = 4).
Total lung and alveolar phospholipid pool sizes. Total lung Sat
PC pool sizes were highest (148 ± 8 µmol/kg body wt) in wild-type mice at birth and decreased by 7 days of age to the levels found in
older mice (Fig. 2A). The total
lung Sat PC in c(
/
) mice (151 ± 10 µmol/kg) was not different from that of wild-type mice at 1-2
days but was increased threefold by day 7 and thereafter. In
GM(
/
) mice, total lung Sat PC was somewhat lower (112 ± 3 µmol/kg) than in wild-type mice at birth but subsequently increased so that by 56 days of age it was 4- to 6-fold that of wild-type and
~50% higher than that of
c(
/
) mice
(P < 0.001). The total lung Sat PC was similar in all animals
at 1-2 days of age, suggesting that the PAP seen in older
c(
/
) and GM(
/
) mice is
caused by postnatal changes in surfactant homeostasis.
|
Alveolar Sat PC pool sizes were also measured in the wild-type,
c(
/
), and GM(
/
) animals at
7, 15, 28, and 56 days old (Fig. 2B). Alveolar Sat PC pool
sizes of 40.1 ± 6.1 and 45.4 ± 7.0 µmol/kg in
c(
/
) and GM(
/
) mice at 7 days of age, respectively, were approximately 10-fold higher than those
in wild-type mice, 4.4 ± 0.3 µmol/kg. By 15 days of age, alveolar
Sat PC pool in the
c(
/
) mice was increased
sevenfold compared with wild-type controls and did not change
thereafter. In contrast to findings in
c(
/
) mice, Sat PC continued to increase in
the airways of GM(
/
) mice so that it was twice that of
c(
/
) mice by 28 days of age.
Precursor incorporation into Sat PC. More labeled palmitic acid
and choline were incorporated into the total lung Sat PC of c(
/
) mice than of wild-type mice at all
time points (Fig. 3, A and
B). The differences between labeled Sat PC recovered in the
alveolar wash from
c(
/
) mice and wild-type
mice increased with time. Accumulation of
[14C]palmitate- and
[3H]choline-labeled Sat PC in the alveolar
compartment of
c(
/
) mice was consistent
with decreased catabolism of Sat PC, increased recycling, or both.
|
Previous studies of [14C]palmitate and
[3H]choline incorporation in lungs of
GM(/
) mice demonstrated patterns similar to those presently observed in
c(
/
) mice (19, 20).
To directly compare findings in
c(
/
) and
GM(
/
) mice, [3H]choline was
administered to
c(
/
) and
GM(
/
) mice (n = 8). Alveolar tissue and total
lung 3H-labeled Sat PC were measured at 8 h, and no
significant differences in recovery of radiolabeled Sat PC were found
between
c(
/
) and GM(
/
) mice,
suggesting that precursor incorporation into lung Sat PC was similar in
mice of these two genotypes.
Clearance of [14C]DPPC and
125I-rSP-C. Recovery of intratracheally
instilled [14C]DPPC and 125I-rSP-C
was measured in lung tissues and alveolar wash from wild-type, c(
/
), and GM(
/
) mice (Figs.
4, A and B). Most of the
[14C]DPPC was cleared from the alveolar
compartment of control mice by 24 and 40 h, with only ~18 and ~5%
recovered from the alveoli at these time points, respectively. Recovery
of [14C]DPPC from airways of
c(
/
) mice (~30%) did not differ
significantly from wild-type controls at 24 h. However, the amount of
radiolabel recovered from alveoli of
c(
/
)
mice at 40 h was still ~30%, significantly higher than in the
control mice at 40 h and consistent with decreased clearance of the
rSP-C. Recovery of [14C]DPPC from lungs of
GM(
/
) mice (~52% alveolar) was significantly higher at
24 h compared with wild-type mice. The pattern of
[14C]DPPC recovered in total lung was similar
to that of the alveolar recovery in each genotype. Recoveries of
125I-rSP-C from alveolar wash and total lung in each
genotype group were similar to those of
[14C]DPPC. Previous studies demonstrated
increased recovery of [14C]DPPC,
125I-SP-A, and 125I-SP-B in alveolar wash and
total lungs of GM(
/
) mice (19, 20). Thus in
c(
/
) and GM(
/
) mice,
clearance of both [14C]DPPC and
125I-rSP-C from the airways was impaired.
c(
/
) and GM(
/
) mice accumulated more label in both alveolar and lung tissue compartments than did wild-type mice; however, accumulation of labeled lipids in the
alveolar compartment was greater in GM(
/
) than in
c(
/
) mice, consistent with the greater
increase in alveolar and Sat PC pool sizes in the GM(
/
)
mice.
|
Selective increase in SP-D in both
c(
/
)
and GM(
/
) mice.
Surfactant proteins were analyzed by Western blotting after
normalization to Sat PC content (Fig. 5).
Relative amounts of proteins were obtained by phosphorimaging. The
ratios of SP-A, -B, or -C to Sat PC were similar in all animals,
although the total amount of each surfactant protein was markedly
increased in the airways of
c(
/
) and
GM(
/
) mice compared with wild-type controls. In contrast,
SP-D was selectively increased in
c(
/
) and
GM(
/
) mice compared with controls, with the ratio of SP-D
to Sat PC increasing 10- and 5-fold, respectively. Because alveolar Sat PC pools in
c(
/
) and GM(
/
)
mice were increased three- and sixfold, respectively, the total
alveolar SP-D content in both
c(
/
) and
GM(
/
) mice was increased ~30-fold overall compared with
wild-type control mice.
|
Surfactant protein mRNA concentrations in total lung. Total
lung RNA from wild-type, c(
/
), and
GM(
/
) mice at 7-9 wk of age was analyzed by S1
nuclease protection assay (Fig. 6). SP-A, -B, and -C mRNA content was similar in wild-type and
c(
/
) mice, consistent with previous
studies demonstrating that SP-A, -B, and -C mRNA levels in
GM(
/
) mice did not differ from those in wild-type mice
(9, 15). SP-D mRNA was slightly increased in
c(
/
) mice but was indistinguishable in
GM(
/
) and wild-type mice. Thus changes in SP-A, -B, -C,
or -D mRNA synthesis or accumulation were negligible and not likely to
account for the increased surfactant proteins in
c(
/
) and GM(
/
) mice.
|
GM-CSF and IL-5 in BALF. GM-CSF and IL-5 were measured by ELISA
in the BALF from wild-type, c(
/
), and
GM(
/
) mice. IL-5 was not detectable in BALF from any
mice. GM-CSF concentrations in BALF from wild-type mice were below the
limits of detection by ELISA (<5 pg/ml). As expected, GM-CSF was not
detected in BALF from GM(
/
) mice. However, GM-CSF was
markedly increased in BALF from
c(
/
) mice
(161 ± 30 pg/ml BALF, n = 6). Despite the increased concentration of GM-CSF in the pulmonary compartment, GM-CSF was undetectable in the serum of
c(
/
) mice.
![]() |
DISCUSSION |
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---|
Inactivation of the common -chain
[
c(
/
)] or GM-CSF
[GM(
/
)] gene in mice disrupted pulmonary
surfactant homeostasis, resulting in surfactant accumulation in lung
tissue and airways. Lung histology in
c(
/
)
and GM(
/
) adult mice was perturbed in a like manner;
however, surfactant Sat PC pool sizes and clearance were different in
the two mutant genotypes. Although lung Sat PC pool sizes were
increased in both
c(
/
) and
GM(
/
) mice, the increase was consistently greater in
GM(
/
) than in
c(
/
) mice.
Likewise, whereas decreased clearance of DPPC was seen in both
c(
/
) and GM(
/
) mice, the
metabolic abnormality was more severe in GM(
/
) than
c(
/
) mice. GM-CSF was increased in the BALF of
c(
/
) mice but not in serum,
suggesting a lung-selective response to inactivation of
c. Decreased surfactant clearance and increased
surfactant pool sizes were observed with both gene-targeted mice;
however, differences in severity of the accumulation in GM(
/
) and
c(
/
) mice suggest
the presence of alternative metabolic pathways that influence
surfactant metabolism in these two models.
Lung histology of c(
/
) and
GM(
/
) mice was indistinguishable, consistent with earlier
findings in several laboratories (9, 15, 16, 26, 28, 32). However, the
present study demonstrated differences in the pattern of lung Sat PC
pool sizes in
c(
/
) and GM(
/
)
mice from birth to adulthood, suggesting that distinct pathological
alterations in surfactant homeostasis occurred postnatally. Total lung
Sat PC pool size was similarly increased in all newborn animals of
c(
/
), GM(
/
), and wild-type genotypes compared with wild-type adults. In control mice, lung Sat PC
pool size was decreased to adult levels by 7 days of age. This rapid
decrease in Sat PC pool size from the neonatal period to adulthood is
consistent with earlier studies in rabbits, sheep, and humans (22). In
contrast, Sat PC pool sizes increased in
c(
/
) and GM(
/
) mice by 7 days of age, suggesting that GM-CSF signaling is required for
establishment of normal surfactant homeostasis in the early postnatal
period. The patterns of Sat PC pool sizes differed in
GM(
/
) and
c(
/
) mice with
subsequent development. The increased total lung and alveolar Sat PC
pool sizes in
c(
/
) mice remained stable
from 7 through 56 days. In contrast, total and alveolar Sat PC
progressively accumulated in lungs of GM(
/
) mice over the
same time period. The intermediate level of Sat PC accumulation seen in
c(
/
) mice suggests that Sat PC
concentrations in
c(
/
) mice were
influenced in a manner distinct from that in GM(
/
) mice.
Exogenous radiolabeled DPPC and rSP-C were rapidly removed from the
airways of wild-type mice. In contrast, clearance of radiolabeled DPPC
and rSP-C from the airways was delayed in
c(
/
) and GM(
/
) mice, and the
defect in clearance was more severe in the GM(
/
) mice.
Previous studies demonstrated that little exogenous radiolabeled DPPC,
SP-A, or SP-B was cleared from the airways of GM(
/
) mice by 40 h. In contrast, the half-life for surfactant proteins and lipids
in the normal mouse lung was ~12 h (18-20). Clearance of Sat PC
in
c(
/
) mice was intermediate compared
with wild-type and GM(
/
) mice, consistent with the
greater increase in steady-state Sat PC pools seen in
GM(
/
) mice.
Phospholipid precursor incorporation and accumulation were increased
similarly in both c(
/
) and
GM(
/
) mice (19, 20). The pattern of accumulation in the
airways of
c(
/
) and GM(
/
) mice reflects a decreased loss of labeled Sat PC compared with wild-type control mice, consistent with impaired degradation and/or increased recycling. Accumulation of Sat PC in the alveoli of
c(
/
) mice and GM(
/
) mice is
also consistent with the observed decreased clearance of radiolabeled
DPPC and rSP-C.
The amounts of SP-A, -B, and -C increased in proportion to the
increased Sat PC pool sizes in BALF from
c(
/
) and GM(
/
) mice. This
observation is consistent with previous studies in which SP-A, SP-B,
and phospholipids increased in a similar manner in sheep with
silica-induced alveolar proteinosis (24). Despite the marked increase
in protein, SP-A, -B, and -C mRNA levels were similar in wild-type,
c(
/
), and GM(
/
) mice. Thus
impaired clearance rather than increased surfactant synthesis likely
caused the accumulations of surfactant proteins in lungs of
c(
/
) and GM(
/
) mice.
The ratio of SP-D protein to Sat PC was markedly increased in BALF from
both c(
/
) and GM(
/
) mice.
These findings were consistent with earlier observations in humans,
where SP-D was disproportionately elevated in PAP compared with other
surfactant proteins (5). Alveolar and tissue Sat PC pool sizes were
markedly increased in SP-D gene-targeted mice in the absence of
significant changes in SP-A, -B, or -C concentrations, suggesting that
SP-D plays an important and selective role in surfactant phospholipid metabolism (1, 23). SP-D mRNA levels were similar in
GM(
/
) and wild-type mice and increased only slightly in
c(
/
) mice. The slight (~25%) increase
in SP-D mRNA in lungs from
c(
/
) mice was
statistically significant but is unlikely to account for the ~30-fold
increase in overall SP-D concentration seen in both GM(
/
)
and
c(
/
) mice. The disproportionately high
SP-D concentrations in the lungs of
c(
/
)
and GM(
/
) mice may reflect a compensatory response to the
high-surfactant lipid concentrations.
Increased GM-CSF was measured in BALF but not in serum from
c(
/
) mice, suggesting a local response of
pulmonary tissues to the lack of
c signaling in the
lung. Metcalf and colleagues (25) observed that the histological
findings of alveolar proteinosis were not altered in double-transgenic
c(
/
) mice expressing high levels of
systemic GM-CSF. Although there is no direct evidence that the
increased concentrations of GM-CSF in the airways had a direct effect
on metabolism or steady-state concentrations of surfactant in
c(
/
) mice, we speculate that distinct
clearance pathways exist in the
c(
/
)
compared with GM(
/
) mice. Although it is generally
accepted that the
-subunit of the GM-CSF receptor (GMR
) does not
directly participate in GM-CSF signaling, previous studies in
Xenopus oocytes demonstrated that the human GMR
activates glucose transport in the absence of the
-subunit (6). Ding et al.
(6) hypothesized that multiple intracellular signaling pathways are
activated by the GM-CSF receptor, with specific events, such as some
types of transport signaling machinery, being modulated by the GMR
alone. This was in contrast to findings by Scott et al. (29), in which
hematopoietic cells from
c(
/
) mice did not
take up 2-[3H]deoxy-D-glucose when
stimulated with GM-CSF but did so in control experiments with IL-3
(29).
Nishinakamura et al. (27) transplanted normal
[c(+/+)] mouse bone marrow into
lethally irradiated
c(
/
) mice to study the
role of alveolar macrophages in PAP. Significant numbers of donor
macrophages derived from the engrafted
c(+/+) bone
marrow were found in the lungs of the
c(
/
)
mice within 8-12 wk after transplantation. Alveolar proteinosis
was substantially improved in
c(
/
) mice
receiving
c(+/+) bone marrow cells, although peribronchiolar or perivascular mononuclear cell infiltrates and mildly elevated SP-B levels in the BALF persisted (4). The residual
infiltrates and excess SP-B concentrations suggested that engrafted
alveolar macrophages increased catabolic functions in a compensatory
manner in the lungs of
c(
/
) mice but that the contribution of other cell types, i.e., type II cells, may also
influence surfactant homeostasis.
The present study shows that surfactant clearance is impaired in
c(
/
) and GM(
/
) mice.
However, biochemical and metabolic differences in
c(
/
) and GM(
/
) mice were
observed, and similar pathological mechanisms may underlie the varying
severity of PAP seen in humans and the individual responses to
therapeutic lavage. Studies demonstrating both similar and distinct
features of surfactant metabolism in
c(
/
)
and GM(
/
) mice provide a rationale to more precisely
define the pathogenesis of various forms of PAP in humans.
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ACKNOWLEDGEMENTS |
---|
We thank Wei Lu, Dr. Cindy Bachurski, Dr. Susan Wert, and Sherri Profitt for technical assistance and Dr. Alan Jobe for helpful discussions of this work.
![]() |
FOOTNOTES |
---|
This work was supported in part by National Heart, Lung, and Blood Institute (NHLBI) Grant POI-HL-61646 (M. Ikegami), NHLBI Specialized Center of Research Grant HL-56387 (J. A. Whitsett and M. Ikegami), and NHLBI Training Grant HL-07752 (J. A. Reed).
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 Neonatology and Pulmonary Biology, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229 (E-mail: whitj0{at}chmcc.org).
Received 8 September 1999; accepted in final form 7 January 2000.
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