Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039
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ABSTRACT |
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Both surfactant protein (SP) D and
granulocyte-macrophage colony-stimulating factor (GM-CSF) influence
pulmonary surfactant homeostasis, with the deficiency of either protein
causing marked accumulation of surfactant phospholipids in lung tissues
and in the alveoli. To assess whether the effects of each gene were
mediated by distinct or shared mechanisms, surfactant homeostasis and
lung morphology were assessed in 1) double-transgenic mice
in which both SP-D and GM-CSF genes were ablated
[SP-D(/
),GM(
/
)] and 2) transgenic mice deficient
in both SP-D and GM-CSF in which the expression of GM-CSF was increased
in the lung. Saturated phosphatidylcholine (Sat PC) pool sizes were
markedly increased in SP-D(
/
),GM(
/
) mice, with the effects of
each gene deletion on surfactant Sat PC pool sizes being approximately
additive. Expression of GM-CSF in lungs of SP-D(
/
),GM(
/
) mice
corrected GM-CSF-dependent abnormalities in surfactant catabolism but
did not correct lung pathology characteristic of SP-D deletion. In contrast to findings in GM(
/
) mice, degradation of
[3H]dipalmitoylphosphatidylcholine by alveolar
macrophages from the SP-D(
/
) mice was normal. The emphysema and
foamy macrophage infiltrates characteristic of SP-D(
/
) mice were
similar in the presence or absence of GM-CSF. Taken together, these
findings demonstrate the distinct roles of SP-D and GM-CSF in the
regulation of surfactant homeostasis and lung structure.
phosphatidylcholine; surfactant protein A; pulmonary alveolar proteinosis; emphysema; surfactant protein D; granulocyte-macrophage colony-stimulating factor
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INTRODUCTION |
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IN THE HEALTHY LUNG, alveolar and tissue surfactant pool sizes are tightly regulated. However, the precise mechanisms mediating surfactant homeostasis remain poorly defined. Surfactant homeostasis is maintained by net contributions from synthesis, secretion, uptake, and catabolism by type II cells and alveolar macrophages (23). The metabolism of surfactant components is influenced in complex ways by both surfactant protein (SP) D and granulocyte-macrophage colony-stimulating factor (GM-CSF). Surfactant phospholipids accumulate in lung tissue and airspaces in both GM-CSF (5)- and SP-D-deficient (3, 13) mice. Whether SP-D and GM-CSF regulate distinct or shared pathways to regulate surfactant homeostasis is unknown.
SP-D is a member of the collectin family of calcium-dependent lectins
(4). Although SP-D binds relatively weakly to surfactant phospholipids, null mutations in the SP-D gene [SP-D(/
)] caused marked increases in tissue and alveolar surfactant saturated
phosphatidylcholine (Sat PC) in vivo (3, 13). A number of
distinct changes in surfactant homeostasis are characteristic of
SP-D(
/
) mice, including increased surfactant phospholipids,
markedly decreased SP-A and SP-C relative to Sat PC, rapid conversion
from large-aggregate to small-aggregate surfactant forms, and decreased
catabolism of endogenously synthesized Sat PC (11).
Inactivation of the SP-D gene in mice also caused emphysema and
increased numbers of lipid-laden, foamy alveolar macrophages
(22). Surfactant abnormalities were corrected by genetic
replacement of SP-D in the respiratory epithelium of the SP-D(
/
)
mice (6). Thus SP-D deficiency caused abnormalities in
lung structure and surfactant metabolism.
Targeted disruption of the GM-CSF gene (5, 10) or the
GM-CSF receptor common -chain genes in mice (18, 20)
caused pulmonary alveolar proteinosis associated with a marked increase in tissue and alveolar Sat PC pool sizes. The increase in surfactant phospholipids associated with deficiencies in GM-CSF signaling were
similar in extent to those observed in the SP-D(
/
) mice. However,
unlike the SP-D(
/
) mice, SPs were also markedly increased in the
GM-CSF or GM-CSF receptor
-chain-deficient mice (20). In contrast to findings in SP-D(
/
) mice, catabolism of both surfactant lipid and proteins by alveolar macrophages was markedly decreased in the absence of GM-CSF signaling in vitro (24)
and in vivo (10). Local replacement of GM-CSF in lungs of
GM-CSF-deficient [GM(
/
)] mice corrected alveolar proteinosis
(9, 19) and host defense abnormalities typical of GM-CSF
deficiency (15).
Although both genes are important determinants of surfactant
phospholipid homeostasis, the precise mechanisms by which GM-CSF and
SP-D influence surfactant pools remain unclear. The present studies
were undertaken to identify potential relationships or interactions
between GM-CSF- and SP-D-dependent pathways in the regulation of
surfactant homeostasis. Surfactant metabolism was studied in SP-D and
GM-CSF null mutant [SP-D(/
),GM(
/
)] mice and
SP-D(
/
),GM(
/
) mice in which GM-CSF expression was increased in
the respiratory epithelium by using SP-C as a promoter
[SP-D(
/
),GM(
/
),SP-C/GM(+/+) mice]. Data presented support the
concept that SP-D and GM-CSF regulate surfactant phospholipid
homeostasis by distinct mechanisms.
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METHODS |
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Mice.
Generation of SP-D(/
) (13), GM(
/
)
(5), and GM(
/
),SP-C/GM(+/+) (8) mice has
been described previously. GM(
/
) and SP-C/GM(+/+) mice were mated
to produce heterozygous offspring that were mated to produce
GM(
/
),SP-C/GM(+/+) mice in the C57BL/6 background. GM-CSF is
selectively expressed in the lungs of SP-C/GM(+/+) mice and absent from
other tissues in the GM(
/
) background. Subsequent matings produced
litters of mice with the SP-C/GM(+/+) genotype, confirming homozygosity
of the offspring. These mice were mated to SP-D(
/
) mice, the latter
in the Black Swiss background. Siblings were mated through at least
three generations to produce double-knockout mice
[SP-D(
/
),GM(
/
)] that were also heterozygous or homozygous for
the SP-C/GM transgene. Comparisons were made among control mice (wild
type) that were F2 littermates of Black Swiss C57BL/6
crosses and compared with identical crosses between SP-D(
/
)/Black
Swiss and GM(
/
)/C57BL/6 mice to control for potential
strain-dependent influences. Genotyping was performed on tail DNA by
PCR and/or Southern blot analysis as previously described (5, 8,
13).
Sat PC pool size.
Sat PC pools (calculated as µmol/kg body wt) were measured in
alveolar lavage fluid and lung tissue after alveolar lavage as
previously described (11) in 22 wild-type mice; 40 SP-D(/
),GM(
/
) mice; and 36 SP-D(
/
),GM(
/
),SP-C/GM(+/+)
mice. Each mouse was deeply anesthetized with intraperitoneal
pentobarbital sodium, and the distal aorta was cut to exsanguinate the
animal. After the chest was opened, a 20-gauge blunt needle was tied to
the trachea, and 0.9% NaCl at 4°C was flushed in the airway until the lungs were fully expanded. The fluid was withdrawn by syringe three
times for each aliquot. The saline lavage was repeated five times, and
the samples were pooled. The lavaged lung tissue was homogenized in
0.9% NaCl. Aliquots of alveolar lavage fluid and the lung homogenates
were extracted with chloroform-methanol (2:1; see Ref. 2),
and Sat PC was isolated with the technique of Mason et al.
(16). The amount of Sat PC was measured by phosphorus assay (1).
SP-A content.
The SP-A in alveolar lavage fluid was analyzed by Western blot in six
mice from each genotype (11, 20). Samples containing 1 µg of Sat PC were used for analyses of SP-A. Proteins were separated by SDS-PAGE in the presence of -mercaptoethanol. After
electrophoresis, SP-A was transferred to nitrocellulose paper
(Schleicher and Schnell, Keene, NH), and immunoblot analysis was
carried out with dilution of 1:5,000 guinea pig anti-mouse SP-A.
Appropriate peroxidase-conjugated secondary antibodies were used at
1:5,000 dilutions. Immunoreactive bands were detected with enhanced
chemiluminescence reagents (Amersham, Chicago, IL). Protein bands were
quantitated by densitometric analyses with Alpha Imager 2000 documentation and analysis software (Alpha Innotech, San Leandro, CA).
Linearity of the assay was confirmed for the range of 10-200 ng of
mouse SP-A (R2 = 0.95).
Clearance of dipalmitoylphosphatidylcholine. Eight mice from each group were anesthetized with methoxyflurane and orally intubated with a 25-gauge animal-feeding needle. Each mouse received 60 µl of saline containing 0.5 µCi of [3H]choline-labeled dipalmitoylphosphatidylcholine (DPPC; American Radiolabeled Chemicals, St. Louis, MO), 1.5 µg of DPPC, and 3.3 µg of lipid-extracted mouse surfactant suspended by the use of glass beads (11). The phospholipids given by intratracheal injection were 2% of the alveolar pool size for wild-type mice. After injection (40 h), mice were killed. Radioactivity was then measured in Sat PC isolated from alveolar lavage fluid and lung homogenate.
Precursor incorporation in Sat PC. Mice were given intraperitoneal injections of 10 µl saline/g body wt containing 0.5 µCi [3H]palmitic acid/g body wt (American Radiolabeled Chemicals; see Ref. 10). The palmitic acid was stabilized in solution with 5% human serum albumin. Groups of eight mice were killed at 2, 16, and 48 h after precursor injection, and alveolar lavage fluid was recovered from each animal. Lung tissue was homogenized in 0.9% NaCl. Sat PC was isolated from alveolar lavage fluids and lung homogenates as described for Sat PC pool sizes, and radioactivity was measured.
DPPC degradation by alveolar macrophage.
Alveolar macrophages were isolated from the alveolar lavage fluid, and
the degradation of radiolabeled DPPC was studied as described
previously (24). Alveolar macrophages from wild-type mice,
SP-D(/
) mice, SP-D(
/
),GM(
/
) mice, and
SP-D(
/
),GM(
/
),SP-C/GM(+/+) mice were studied. Alveolar lavage
was performed with PBS containing 0.5 mM EDTA. Lavage fluid was
centrifuged at 1,000 g for 5 min. Recovered cells were then
resuspended in DMEM containing 0.1% BSA and were cultured at a density
of 1 × 105 cells/well in flat-bottom, 96-well tissue
culture plates. Cells were allowed to adhere for 1 h at 37°C.
Nonadherent cells were removed, and the adherent alveolar macrophages
were washed three times with DMEM containing 0.1% BSA. With this
method, >90% of cells recovered were macrophages.
Lung histology. Lungs from each genotype (n = 2/genotype) were inflation fixed at 25 cmH2O with 4% paraformaldehyde. Tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin.
Data analysis. All values are given as means ± SE. The between-group comparisons were made by ANOVA followed by the Student-Newman-Keuls multiple-comparison procedure.
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RESULTS |
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Sat PC and SP-A pool size.
Surfactant Sat PC (µmol/kg) was determined in alveolar lavage fluid,
lung tissue after lavage, and total lung (lavage fluid + tissue;
Fig. 1A). Relative changes in
Sat PC were compared with wild-type mice, which were given the value of
one (Fig. 1B). Total surfactant Sat PC was 14.5-fold higher
in SP-D(/
),GM(
/
) mice than in wild-type mice and was
considerably higher than the Sat PC in either GM(
/
) or SP-D(
/
)
mice previously reported (10, 11). Thus the increases in
Sat PC pools in the SP- D(
/
),GM(
/
) mice were approximately
additive. Repletion of GM-CSF in the SP-D(
/
),GM(
/
) double-knockout mice with the SP-C/GM(+/+) transgene substantially decreased the Sat PC pool size in total lung and lavage fluid to levels
consistent with that of the SP-D(
/
) mice (11). Thus local expression of GM-CSF in the lung partially corrected the defect
in the GM(
/
),SP-D(
/
) mice to that typical of SP-D(
/
) mice.
Comparisons were made among littermates derived from F2 crosses between Black Swiss and C57BL/6 to control for potential strain
differences. Thus combined deficiency of both GM-CSF and SP-D resulted
in increased Sat PC pools, consistent with an approximately additive
effect of each gene on surfactant pool size. These results demonstrated
that expression of GM-CSF in the lung restored surfactant homeostasis
related to GM-CSF deficiency but did not correct SP-D-dependent differences.
|
SP pool sizes.
The content of alveolar SP-A relative to Sat PC was estimated by
Western blot analysis and normalized to the quantity of SP-A in
wild-type mice, which were given the value of one. The concentration of
SP-A in both SP-D(/
) and GM(
/
) mice relative to the wild type
was also included for comparison (Fig.
2A). In GM(
/
),SP-D(
/
) mice, the SP-A content was 0.4 of that in wild-type mice, and in
SP-D(
/
),GM(
/
),SP-C/GM(+/+) mice, SP-A was further decreased to
0.2 of the normal value. In previous studies, SP-A content relative to
Sat PC was unchanged in GM(
/
) mice, whereas SP-A was decreased to
0.1 of the normal value in SP-D(
/
) mice. SP-A content in
SP-D(
/
),GM(
/
),SP-C/GM(+/+) mice was similar to that in
SP-D(
/
) mice, whereas SP-D(
/
),GM(
/
) mice had a SP-A content
of an intermediate value between that of SP-D(
/
) mice and GM(
/
)
mice. Because alveolar Sat PC was increased in the SP-D(
/
),GM(
/
) mice, net alveolar SP-A pool size per kilogram of
body weight normalized to that in wild-type mice was also expressed in
Fig. 2B. Total lung SP-A content in SP-D(
/
),GM(
/
)
mice was eightfold higher than in wild-type mice, likely reflecting the
deficiency of SP-A catabolism characteristic of GM-CSF mice. Furthermore, repletion of GM-CSF did not correct the decreased SP-A
pool size in SP-D(
/
),GM(
/
) mice. Thus the marked reduction in
SP-A characteristic of SP-D(
/
) mice was independent of GM-CSF, and
the lack of GM-CSF increased SP-A levels independently of the SP-D
mutation.
|
Clearance of DPPC.
[3H]DPPC was given by intratracheal injection in mice of
each genotype. Labeled DPPC recovered in the Sat PC pool decreased exponentially from the alveolar lavage fluids of the wild-type mice and
SP-D(/
),GM(
/
),SP-C/GM(+/+) mice 40 h after injection (Fig.
3). Thus the expression of GM-CSF in the
lung corrected the defect in surfactant catabolism characteristics of
SP-D(
/
),GM(
/
) mice. Percent recovery of [3H]DPPC
in SP-D(
/
),GM(
/
) double-knockout mice was increased sevenfold
compared with that in wild-type and SP-D(
/
),GM(
/
),SP-C/GM(+/+) mice, reflecting a marked decrease in DPPC clearance in the
SP-D(
/
),GM(
/
) mice. After administration (40 h), percent
recovery of labeled DPPC in lungs from wild-type mice was ~10 and
20% in SP-D(
/
) mice, reflecting rapid clearance of DPPC. Labeled
DPPC recovered in SP-D(
/
),GM(
/
) mice was ~41% lower than
that observed in previous studies in GM(
/
) mice (10),
reflecting a modest increase in clearance in the double-knockout
SP-D(
/
),GM(
/
) mice compared with the GM(
/
) mice. Thus SP-D
deficiency appears to improve [3H]DPPC clearance in the
lungs of GM(
/
) mice and may have an effect on surfactant clearance
that is independent of degradation by the alveolar macrophage.
|
Precursor incorporation.
Mice were given weight-adjusted doses of [3H]palmitic
acid to measure net incorporation in Sat PC at 2 h, secretion of
the labeled Sat PC to the alveoli at 16 h, and loss of labeled Sat PC from the lung 48 h after precursor injection (Fig.
4). After injection (2 h), no significant
differences in initial incorporation were observed among the three
groups of mice. Likewise, percent secretion in alveolar lavage fluid,
calculated at 16 h as percent of total lung incorporation, was
similar in all groups of mice as follows: 32 ± 2% in wild-type,
29 ± 4% in SP-D(/
),GM(
/
), and 27 ± 6% in
SP-D(
/
),GM(
/
),SP-C/GM(+/+) mice. At 48 h after injection, labeled Sat PC was similar and significantly higher in
SP-D(
/
),GM(
/
) and SP-D(
/
),GM(
/
),SP-C/GM(+/+) mice
compared with wild-type mice, consistent with the increased precursor
incorporation seen at later times after precursor injection typical of
SP-D(
/
) mice, as previously described (11).
|
DPPC degradation by alveolar macrophages.
In vitro surfactant catabolism by alveolar macrophages from SP-D(/
)
and SP-D(
/
),GM(
/
),SP-C/GM(+/+) mice was similar to that from
wild-type mice (Fig. 5). The marked
decrease in [3H]DPPC catabolism by alveolar macrophages
from GM(
/
) mice was demonstrated previously (Fig. 5; see Ref.
24). Degradation of [3H]DPPC by alveolar
macrophages was markedly and similarly decreased in GM(
/
) and
SP-D(
/
), GM(
/
) mice, reflecting the known GM-CSF dependency
for DPPC degradation by alveolar macrophages.
|
Lung histology.
Lung histology was assessed by light microscopy in wild-type,
SP-D(/
),GM(
/
), and SP-D(
/
),GM(
/
),SP-C/GM(+/+) mice. Abnormalities in the SP-D(
/
),GM(
/
) mice consisted of
heterogeneous airspace enlargement, increased perivascular lymphocytic
infiltrates, enlarged foamy macrophages, and alveolar proteinosis,
consistent with the histopathology seen in both SP-D(
/
) and
GM(
/
) mice (Fig. 6; see Refs.
5 and 13). Repletion of GM-CSF with the SP-C/GM(+/+)
transgene corrected the alveolar proteinosis in SP-D(
/
),GM(
/
) mice but did not correct the airspace abnormalities, foamy macrophages, or perivascular lymphocytic infiltrates characteristic of the SP-D(
/
) mice. Thus repletion of GM-CSF corrected the alveolar proteinosis typical of the GM(
/
) mice but did not correct the structural abnormalities typical of the SP-D(
/
) mice.
|
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DISCUSSION |
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Surfactant metabolism in SP-D(/
),GM(
/
) mouse lung was
disrupted in a nearly additive way, with the double-knockout mice sharing characteristics of both GM(
/
) and SP-D(
/
) mice.
Expression of excess GM-CSF under control of human SP-C in the lung of
SP-D(
/
),GM(
/
) mice decreased Sat PC pool size, enhanced the
clearance of surfactant from the lung, and corrected the histological
abnormalities typical of alveolar proteinosis, demonstrating that local
expression of GM-CSF corrected the GM-CSF-dependent effects or
surfactant homeostasis. However, repletion of GM-CSF did not correct
emphysema and inflammatory changes typical of SP-D deficiency. These
results support the concept that SP-D and GM-CSF influence surfactant
homeostasis by distinct pathways.
The precise mechanisms by which surfactant homeostasis is maintained in
normal lung remain unknown. Surfactant lipids are synthesized,
catabolized, or recycled (12) by type II epithelial cells.
Alveolar macrophages play an important role in surfactant catabolism
(21). Precise contribution of the respiratory epithelium and alveolar macrophages in catabolism of surfactant remains unclear. Recent studies in the mouse suggest that ~50% of DPPC is catabolized by alveolar- and tissue-associated macrophages, and ~50% is
catabolized by type II epithelial cells (7). Deficits in
surfactant catabolism account for the increased surfactant pool sizes
characteristic of GM(/
) mice (10, 20, 24). In
contrast, tissue and alveolar pool sizes are increased in SP-D(
/
)
mice without associated abnormalities in surfactant clearance
(11).
Multiple abnormalities in surfactant structure and surfactant
homeostasis were caused by targeted disruption of the mouse SP-D gene.
However, the mechanisms by which lung surfactant homeostasis is altered
in SP-D(/
) mice are complex and not related to defects in
surfactant lipid clearance. Although the increases in surfactant pool
sizes are similar in both GM(
/
) and SP-D(
/
) mice, clearance of
surfactant by the alveolar macrophage is deficient in GM(
/
) but not
in SP-D(
/
) mice. Foamy macrophages accumulate in the lungs of
SP-D(
/
) mice; however, the present in vitro studies demonstrate
that alveolar macrophages from SP-D(
/
) mice degrade phospholipids
normally. Increasing the surfactant pool size by exogenous surfactant
also induces a transient foamy macrophage population but does not alter
surfactant catabolism in mice (14). Thus the abnormalities
in surfactant homeostasis seen in SP-D(
/
) mice do not appear to be
caused by foam cell production or ablation of alveolar macrophage
function. In contrast, catabolism of surfactant by the foamy
macrophages from GM(
/
) mice is markedly decreased, demonstrating
that SP-D and GM-CSF deficiency have distinct effects in surfactant
clearance by the alveolar macrophages. Deletion of the SP-D gene in the
GM(
/
) mice did not improve defective DPPC degradation
characteristic of GM(
/
) mice, resulting in a clearance rate typical
of SP-D deficiency. Conversely, increased GM-CSF did not correct the
abnormalities in macrophage morphology and emphysema typical of the
SP-D(
/
) mice.
The present study demonstrates that SP-D and GM-CSF play distinct roles
in the maintenance of pulmonary morphology. Although emphysema was not
restored by increased expression of GM-CSF in the SP-D(/
) mice, the
increased surfactant pool size seen in double-knockout
SP-D(
/
),GM(
/
) mice was substantively corrected, consistent with
the requirement of GM-CSF for SP clearance. This result may reflect
enhanced macrophage function associated with increased GM-CSF that
improves surfactant catabolism in the SP-D(
/
),GM(
/
) mice.
Findings from kinetic experiments assessing surfactant clearance also
support the findings at steady state wherein Sat PC pool sizes were
increased in SP-D(/
),GM(
/
) mice in an approximately additive
way. The percent recovery of DPPC in SP-D(
/
),GM(
/
),SP-C/GM(+/+) mice at 40 h after intratracheal injection of
[3H]DPPC was similar to that in wild-type mice and to
that in SP-D(
/
) mice (11), reflecting restoration of
clearance of catabolic activity by local production of GM-CSF. In
contrast, in SP-D(
/
),GM(
/
) mice, significantly higher
concentrations of [3H]DPPC were recovered, reflecting
decreased clearance typical of GM-CSF deficiency. However, because
alveolar Sat PC pool sizes were 4-fold higher in
SP-D(
/
),GM(
/
),SP-C/GM(+/+) mice and 14-fold higher in
SP-D(
/
),GM(
/
) mice, the net recovery of [3H]DPPC
was increased markedly in lungs of both of these transgenic mice
compared with that in wild-type mice. Rapid catabolism of endogenously
labeled surfactant was absent in GM-CSF-repleted SP-D(
/
),GM(
/
),SP-C/GM(+/+) mice, and both were similar to that
in SP-D(
/
) mice, consistent with the activity of GM-CSF. In
contrast, palmitic acid-labeled Sat PC continued to accumulate in
alveolar lavage fluid in SP-D(
/
),GM(
/
),SP-C/GM(+/+) and SP-D(
/
) mice, consistent with altered surfactant pools seen previously in SP-D(
/
) mice (11). In the present study,
we compared surfactant metabolism under identical experimental
conditions in mice with distinct genotypes. However, the methodology
used to estimate surfactant metabolism requires a number of
assumptions, such as similar Sat PC precursor pools in all genotypes.
The times chosen for kinetic studies were based on our previous studies of Sat PC synthesis secretion and clearance but are not complete enough
to accurately determine turnover time of Sat PC. Likewise, we assumed
that catabolism of DPPC in the lung was minimal and that labeled DPPC
was uniformly distributed in the alveoli after intratracheal administration.
In vitro degradation of DPPC by alveolar macrophages isolated from
GM(/
) mice was slow and was not influenced by the lack of SP-D.
Thus effects of GM-CSF on surfactant homeostasis are related primarily
to its effects on surfactant catabolism dependent on alveolar
macrophage function and are independent of SP-D. The altered surfactant
homeostasis seen in SP-D(
/
) mice is not directly related to
surfactant catabolism by the alveolar macrophage but is probably
related to changes in surfactant metabolism by type II epithelial cells
and the altered surfactant Sat PC pool in both alveolar and cellular
components of the lung. Effects of SP-D deficiency are not mediated
primarily by effects on surfactant catabolism by alveolar macrophages
and were mediated relatively independently of GM-CSF.
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ACKNOWLEDGEMENTS |
---|
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-61646, HL-56387, and HL-63329.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: M. Ikegami, Children's Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: machiko.ikegami{at}chmcc.org).
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. Section 1734 solely to indicate this fact.
Received 8 March 2001; accepted in final form 17 April 2001.
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