Am J Physiol Lung Cell Mol Physiol
281: L697–L703, 2001.
SP-D and GM-CSF regulate surfactant
homeostasis via distinct mechanisms
MACHIKO IKEGAMI, WILLIAM M. HULL, MITSUHIRO YOSHIDA,
SUSAN E. WERT, AND JEFFREY A. WHITSETT
Division of Pulmonary Biology, Children’s Hospital Medical Center, Cincinnati, Ohio 45229-3039
Received 8 March 2001; accepted in final form 17 April 2001
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 ac-
cumulate 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 calciumdependent 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 GMCSF 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
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:
[email protected]).
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.
phosphatidylcholine; surfactant protein A; pulmonary alveolar proteinosis; emphysema; surfactant protein D; granulocyte-macrophage colony-stimulating factor
http://www.ajplung.org
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society
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Ikegami, Machiko, William M. Hull, Mitsuhiro Yoshida, Susan E. Wert, and Jeffrey A. Whitsett. SP-D and
GM-CSF regulate surfactant homeostasis via distinct mechanisms. Am J Physiol Lung Cell Mol Physiol 281:
L697–L703, 2001.—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 [SPD(⫺/⫺),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.
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SP-D AND GM-CSF ON SURFACTANT HOMEOSTASIS
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 GMCSF null mutant [SP-D(⫺/⫺),GM(⫺/⫺)] mice and SPD(⫺/⫺),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.
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 doubleknockout 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).
All of the studies were performed with 7- to 9-wk-old mice.
Mice were maintained in cages containing high-efficiency
particulate-filtered air in a barrier facility. There was no
evidence of pathogens in sentinel mice comaintained in the
vivarium. All procedures were conducted under Institutional
Animal Care and Use Committee-approved methods.
Sat PC pool size. Sat PC pools (calculated as ␮mol/kg body
wt) were measured in alveolar lavage fluid and lung tissue
after alveolar lavage as previously described (11) in 22 wildtype 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 ␮g 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 ␮l of saline containing 0.5 ␮Ci of
[3H]choline-labeled dipalmitoylphosphatidylcholine (DPPC;
American Radiolabeled Chemicals, St. Louis, MO), 1.5 ␮g of
DPPC, and 3.3 ␮g 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 ␮l saline/g body wt containing 0.5
␮Ci [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 SPD(⫺/⫺),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.
To assess DPPC degradation, natural surfactant isolated
from normal mouse lung lavage fluid was labeled by mixing
with 1,3-phosphatidyl-N-methyl-[3H]choline,1,2-dipalmitoyl
([3H]DPPC; Amersham, Arlington Heights, IL). The labeled
surfactant was resuspended in medium containing 100 ␮g/ml
phospholipid with 10 ␮Ci/ml [3H]DPPC. To determine degradation of [3H]DPPC, cells were incubated for 5 h at 37°C
with the [3H]DPPC-labeled surfactant in culture medium.
After incubation, the supernatant was collected, and the cells
were lysed with lysis buffer [50 mM Tris 䡠 HCl (pH 8), 150 mM
NaCl, 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate, and
5 mM EDTA]. The lipid and aqueous fractions were extracted
from both supernatants and cells according to Bligh and Dyer
(2). The degradation of [3H]DPPC by alveolar macrophages
was estimated by measuring the generation of radioactive
products partitioning in the water-methanol phase during
the extraction. Background radioactivity in the absence of
the cells was subtracted.
SP-D AND GM-CSF ON SURFACTANT HOMEOSTASIS
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.
RESULTS
Sat PC and SP-A pool size. Surfactant Sat PC (␮mol/
kg) was determined in alveolar lavage fluid, lung tissue
AJP-Lung Cell Mol Physiol • VOL
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 SPD(⫺/⫺),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 GMCSF 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 GMCSF 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
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Fig. 1. Increased saturated phosphatidylcholine (Sat PC) in doubletransgenic surfactant protein (SP) D-deficient [(⫺/⫺)],granulocytemacrophage colony-stimulating factor (GM-CSF)-deficient [GM(⫺/
⫺)] mice. A: Sat PC pool sizes were determined in wild-type, SP-D(⫺/
⫺),GM(⫺/⫺), and SP-D(⫺/⫺),GM(⫺/⫺),SP-C/GM(⫹/⫹) mice in
alveolar lavage fluid (alveolar) and lung tissue after lavage (tissue).
The sum of alveolar and tissue fractions (total) is also shown. Sat PC
was increased markedly in double-transgenic SP-D(⫺/⫺),GM(⫺/⫺)
mice. Repletion of GM-CSF in SP-D(⫺/⫺),GM(⫺/⫺),SP-C/GM(⫹/⫹)
mice partially corrected the Sat PC content seen in double-transgenic mice but not to wild-type levels. B: total Sat PC in lungs of the
transgenic mice was normalized to total Sat PC in wild-type mice
(given the value of 1.0). Relative Sat PC contents in lungs from
SP-D(⫺/⫺); GM(⫺/⫺); SP-D(⫺/⫺),GM(⫺/⫺),SP-C/GM(⫹/⫹); and SPD(⫺/⫺),GM(⫹/⫹),SP-C/GM(⫹/⫹) mice are shown. Lung Sat PC in
SP-D(⫺/⫺) mice and GM(⫺/⫺) mice are included for comparison and
were published previously (10, 11). Total lung Sat PC content in
lungs from SP-D(⫺/⫺),GM(⫺/⫺) mice was significantly higher than
in other genotypes. Increased expression of GM-CSF in SP-D(⫺/
⫺),GM(⫺/⫺),SP-C/GM(⫹/⫹) mice decreased the Sat PC pool size to a
pool size similar to that in SP-D(⫺/⫺) mice. Expressing GM-CSF
(SP-C/GM) did not alter Sat PC pool size in SP-D(⫺/⫺),GM(⫹/⫹)
mice, with pool sizes in SP-D(⫺/⫺),GM(⫹/⫹),SP-C/GM(⫹/⫹) mice
being similar to SP-D(⫺/⫺) mice. aData from Ref. 11. bData from Ref.
10. *P ⬍ 0.001 vs. wild-type mice. tP ⬍ 0.001 vs. other mice.
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SP-D AND GM-CSF ON SURFACTANT HOMEOSTASIS
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 SPD(⫺/⫺),GM(⫺/⫺) mice was ⬃41% lower than that observed in previous studies in GM(⫺/⫺) mice (10), reflecting a modest increase in clearance in the doubleknockout 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 weightadjusted 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
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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
Fig. 4. Recovery of [3H]palmitic acid-labeled Sat PC in alveolar
lavage sample (A) and in total lung (alveolar ⫹ tissue; B). Initial
incorporation (at 2 h) of [3H]palmitic acid in Sat PC was similar for
the 3 groups studied. Secretion rate calculated as %[3H]Sat PC in
alveolar lavage fluid in total lung (at 16 h) was similar for the 3
groups and represents ⬃29% secretion. At 48 h, [3H]Sat PC in the
lung was 4- to 5-fold higher in SP-D(⫺/⫺),GM(⫺/⫺) mice and SPD(⫺/⫺),GM(⫺/⫺),SP-C/GM(⫹/⫹) mice than in wild-type mice. Clearance of synthesized Sat PC from the lungs was altered in transgenic
mice. CPM, counts/min. *P ⬍ 0.05 and **P ⬍ 0.001 vs. wild type.
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Fig. 2. A: SP-A content was determined in alveolar lavage samples
containing 1 ␮g of Sat PC by Western blot. The SP-A-to-Sat PC ratio
was normalized to the quantity of SP-A in alveolar lavage samples
from wild-type mice (which was given the value of 1.0). Relative SP-A
content in SP-D(⫺/⫺) mice and GM(⫺/⫺) mice is provided from
previous studies (10, 11). The relative abundance of SP-A in SPD(⫺/⫺) mice was 0.14 of wild type. Alveolar SP-A normalized to Sat
PC in GM(⫺/⫺) mice was similar to that in wild-type mice. SP-A was
decreased in SP-D(⫺/⫺),GM(⫺/⫺) mice to 0.4 of wild-type mice.
Relative SP-A content was 0.2 in SP-D(⫺/⫺),GM(⫺/⫺),SP-C/
GM(⫹/⫹) mice. B: SP-A pool size/body wt in alveolar lavage fluid.
SP-A pool size in wild-type mice was normalized to a value of 1.0.
SP-D(⫺/⫺),GM(⫺/⫺) mice had 8 times more SP-A than wild-type
mice. SP-A pool size decreased to 0.15 in SP-D(⫺/⫺),GM(⫺/⫺),SP-C/
GM(⫹/⫹) mice, which is similar to SP-D(⫺/⫺) mice. aData from Ref.
11. bData from Ref. 10. *P ⬍ 0.001 vs. wild-type mice. tP ⬍ 0.001 vs.
SP-D(⫺/⫺),GM(⫺/⫺) mice.
Fig. 3. [3H]dipalmitoylphosphatidylcholine (DPPC; 0.5 ␮Ci) was
given with a trace amount of surfactant by intratracheal injection as
described in METHODS. Recovery of [3H]DPPC was measured in alveolar lavage fluid and lung 40 h after injection. Percent recovery of
intratracheal [3H]Sat PC was 5-fold higher in SP-D(⫺/⫺),GM(⫺/⫺)
mice than in wild-type or SP-D(⫺/⫺),GM(⫺/⫺),SP-C/GM(⫹/⫹) mice,
reflecting decreased clearance. *P ⬍ 0.001 vs. others.
SP-D AND GM-CSF ON SURFACTANT HOMEOSTASIS
Fig. 5. Degradation of [3H]DPPC by alveolar macrophages in vitro.
Alveolar macrophages (1 ⫻ 105/well) were isolated from wild-type,
SP-D(⫺/⫺),GM(⫺/⫺); SP-D(⫺/⫺),GM(⫺/⫺),SP-C/GM(⫹/⫹); and SPD(⫺/⫺) mice and incubated with a surfactant suspension containing
[3H]DPPC mouse surfactant for 5 h at 37°C. Degradation of [3H]DPPC
by alveolar macrophages was estimated by measuring the radioactivity recovered in the water-methanol phase after Folch extraction.
Degradation of [3H]DPPC by alveolar macrophages from transgenic
mice was normalized to that from wild-type mice. Identical experiments with alveolar macrophages from GM(⫺/⫺) mice are shown for
comparison (17). DPPC degradation by alveolar macrophages from
SP-D(⫺/⫺) mice and SP-D(⫺/⫺),GM(⫺/⫺),SP-C/GM(⫹/⫹) mice was
rapid, whereas degradation by alveolar macrophages from SP-D(⫺/
⫺),GM(⫺/⫺) mice was decreased and similar to that in GM(⫺/⫺)
mice (17). Expression of GM-CSF corrected surfactant catabolism by
alveolar macrophages from the SP-D(⫺/⫺),GM(⫺/⫺),SP-C/GM(⫹/⫹)
mice. *P ⬍ 0.01 vs. wild type.
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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 GMCSF corrected the alveolar proteinosis typical of the
GM(⫺/⫺) mice but did not correct the structural abnormalities typical of the SP-D(⫺/⫺) mice.
DISCUSSION
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 GMCSF 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
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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 SPD(⫺/⫺),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
L701
L702
SP-D AND GM-CSF ON SURFACTANT HOMEOSTASIS
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 GMCSF play distinct roles in the maintenance of pulmonary morphology. Although emphysema was not restored by increased expression of GM-CSF in the SPD(⫺/⫺) 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.
AJP-Lung Cell Mol Physiol • VOL
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 wildtype 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
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Fig. 6. Histopathology of SP-D(⫺/⫺),GM(⫺/⫺)
mice (A–C) and SP-D(⫺/⫺),GM(⫺/⫺),SP-C/
GM(⫹/⫹) mice (D–F). Histopathology of the SPD(⫺/⫺),GM(⫺/⫺) mice was similar to the abnormalities found in both SP-D(⫺/⫺) and
GM(⫺/⫺) mice, including perivascular lymphocytic infiltrates (arrows); enlarged, malformed
alveoli; eosinophilic, proteinaceous exudates
(arrowheads, A and B); and focal aggregates of
large, foamy macrophages (C). Repletion of the
double-knockout mice with the SP-C/GM(⫹/⫹)
transgene corrected the alveolar proteinosis but
did not correct the airspace abnormalities (D),
perivascular lymphocytic infiltrates (arrows, D
and E), or macrophage abnormalities (F). BR,
bronchiole; V, vessel; L, lymphatic. Original
magnification for A and D ⫽ ⫻5 and for B, C, E,
and F ⫽ ⫻20.
SP-D AND GM-CSF ON SURFACTANT HOMEOSTASIS
This work was supported in part by National Heart, Lung, and
Blood Institute Grants HL-61646, HL-56387, and HL-63329.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
REFERENCES
1. Bartlett GR. Phosphorus assay in column chromatography.
J Biol Chem 234: 466–468, 1959.
2. Bligh EG and Dyer WJ. A rapid method of total lipid extraction
and purification. Can J Biochem Physiol 37: 911–917, 1959.
3. Botas C, Poulain F, Akiyama J, Brown C, Allen L, Goerke
J, Clements J, Carlson E, Gillespie AM, Epstein C, and
Hawgood S. Altered surfactant homeostasis and alveolar type
II cell morphology in mice lacking surfactant protein D. Proc
Natl Acad Sci USA 95: 11869–11874, 1998.
4. Crouch EC. Collectins and pulmonary host defense. Am J
Respir Cell Mol Biol 19: 177–201, 1998.
5. Dranoff G, Crawford AD, Sadelain M, Ream B, Rashid A,
Bronson RT, Dickersin GR, Bachurski CJ, Mark EL, Whitsett JA, and Mulligan RC. Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis.
Science 264: 713–716, 1994.
6. Fisher JH, Sheftelyevich V, Ho YS, Fligiel S, McCormack
FX, Korfhagen TR, Whitsett JA, and Ikegami M. Pulmonary-specific expression of SP-D corrects pulmonary lipid accumulation in SP-D gene-targeted mice. Am J Physiol Lung Cell
Mol Physiol 278: L365–L373, 2000.
7. Gurel O, Ikegami M, Chroneos Z, and Jobe A. Macrophage
and type II cell catabolism of SP-A and saturated phosphatidyl-
AJP-Lung Cell Mol Physiol • VOL
19.
20.
21.
22.
23.
24.
choline in mouse lung. Am J Physiol Lung Cell Mol Physiol 280:
L1266–L1272, 2001.
Huffman JA, Hull WM, Dranoff G, Mulligan RC, and Whitsett JA. Pulmonary epithelial cell expression of GM-CSF corrects the alveolar proteinosis in GM-CSF- deficient mice. J Clin
Invest 97: 649–655, 1996.
Ikegami M, Jobe AH, Huffman-Reed JA, and Whitsett JA.
Surfactant metabolic consequences of overexpression of GM-CSF
in the epithelium of GM-CSF-deficient mice. Am J Physiol Lung
Cell Mol Physiol 273: L709–L714, 1997.
Ikegami M, Ueda T, Hull W, Whitsett JA, Mulligan RC,
Dranoff G, and Jobe AH. Surfactant metabolism in transgenic
mice after granulocyte macrophage-colony stimulating factor
ablation. Am J Physiol Lung Cell Mol Physiol 270: L650–L658,
1996.
Ikegami M, Whitsett JA, Jobe AH, Ross G, Fisher J, and
Korfhagen T. Surfactant metabolism in SP-D gene-targeted
mice. Am J Physiol Lung Cell Mol Physiol 279: L468–L476,
2000.
Jacobs H, Jobe A, Ikegami M, and Conaway D. The significance of reutilization of surfactant phosphatidylcholine. J Biol
Chem 258: 4156–4165, 1983.
Korfhagen TR, Sheftelyevich V, Burhans MS, Bruno MD,
Ross GF, Wert SE, Stahlman MT, Jobe AH, Ikegami M,
Whitsett JA, and Fisher JH. Surfactant protein-D regulates
surfactant phospholipid homeostasis in vivo. J Biol Chem 43:
28438–28443, 1998.
Kramer BW, Jobe A, and Ikegami M. Exogenous surfactant
changes the phenotype without activation of alveolar macrophages in mice. Am J Physiol Lung Cell Mol Physiol 280: L689–
L694, 2001.
LeVine AM, Reed JA, Kurak KE, Cianciolo E, and Whitsett
JA. Granulocyte-macrophage colony stimulating factor (GMCSF) deficient mice are susceptible to pulmonary group B streptococcal infection. J Clin Invest 103: 563–569, 1999.
Mason RJ, Nellenbogen J, and Clements JA. Isolation of
disaturated phosphatidylcholine with osmium tetroxide. J Lipid
Res 17: 281–284, 1976.
McCormack FX, Ikegami M, LeVine AM, Korfhagen TR,
Whitsett JA, and Dienger K. In vivo structure/function analyses of surfactant protein A (Abstract). Am J Repir Crit Care
Med 161: A42, 2000.
Nishinakamura R, Wiler R, Dirksen U, Morikawa Y, Arai
K, Miyajima A, Burdach S, and Murray R. The pulmonary
alveolar proteinosis in granulocyte macrophage colony-stimulating factor/interleukins 3/5 ␤c receptor-deficient mice is reversed
by bone marrow transplantation. J Exp Med 183: 2657–2662,
1996.
Reed J, Ikegami M, Cianciolo E, Lu W, Cho PS, Hull W,
Jobe AH, and Whitsett JA. Aerosolized GM-CSF ameliorates
pulmonary alveolar proteinosis. Am J Physiol Lung Cell Mol
Physiol 276: L563–L566, 1999.
Reed JA, Ikegami M, Robb M, Begley CG, Ross G, and
Whitsett JA. Distinct changes in pulmonary surfactant homeostasis in common ␤ chain- and GM-CSF-deficient mice. Am J
Physiol Lung Cell Mol Physiol 278: L1164–L1171, 2000.
Rider ED, Ikegami M, and Jobe AH. Intrapulmonary catabolism of surfactant-saturated phosphatidylcholine in rabbits.
J Appl Physiol 69: 1856–1862, 1990.
Wert SE, Yoshida M, LeVine AM, Ikegami M, Jones T, Ross
GF, Fisher JH, Korfhagen TR, and Whitsett JA. Increased
metalloproteinase activity, oxidant production, and emphysema
in surfactant protein D gene-inactivated mice. Proc Natl Acad
Sci USA 97: 5972–5977, 2000.
Wright JR and Clements JA. Metabolism and turnover of
lung surfactant. Am Rev Respir Dis 135: 426–444, 1987.
Yoshida M, Ikegami M, Reed JA, Chroneos ZC, and Whitsett JA. GM-CSF regulates protein and lipid catabolism by
alveolar macrophages. Am J Physiol Lung Cell Mol Physiol 280:
L379–L386, 2001.
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Sat PC continued to accumulate in alveolar lavage
fluid in SP-D(⫺/⫺),GM(⫺/⫺),SP-C/GM(⫹/⫹) and SPD(⫺/⫺) 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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