1 Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039; and 2 Pulmonary Section/Critical Care, Denver Health Medical Center, University of Colorado Health Sciences Center, Denver, Colorado 80204-4507
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ABSTRACT |
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Mice with
surfactant protein (SP)-D deficiency have three to four times more
surfactant lipids in air spaces and lung tissue than control mice. We
measured multiple aspects of surfactant metabolism and function to
identify abnormalities resulting from SP-D deficiency. Relative to
saturated phosphatidylcholine (Sat PC), SP-A and SP-C were decreased in
the alveolar surfactant and the large-aggregate surfactant fraction.
Although large-aggregate surfactant from SP-D gene-targeted [(/
)]
mice converted to small-aggregate surfactant more rapidly,
surface tension values were comparable to values for surfactant from
SP-D wild-type [(+/+)] mice. 125I-SP-D was cleared with a
half-life of 7 h from SP-D(
/
) mice vs. 13 h in SP-D(+/+)
mice. Although initial incorporation and secretion rates for
[3H]palmitic acid and [14C]choline into Sat
PC were similar, the labeled Sat PC was lost from the lungs of
SP-D(+/+) mice more rapidly than from SP-D(
/
) mice. Clearance rates
of intratracheal [3H]dipalmitoylphosphatidylcholine were
used to estimate net clearances of Sat PC, which were approximately
threefold higher for alveolar and total lung Sat PC in SP-D(
/
) mice
than in SP-D(+/+) mice. SP-D deficiency results in multiple
abnormalities in surfactant forms and metabolism that cannot be
attributed to a single mechanism.
dipalmitoylphosphatidylcholine; surfactant protein A; surfactant protein B; surfactant protein C; surfactant protein D
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INTRODUCTION |
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SURFACTANT PROTEIN
(SP) D is a 43-kDa member of the collectin family of proteins that
shares structural homology with SP-A and mannose binding protein
(3). In contrast to SP-A, SP-D does not interact with the
major surfactant phospholipids and is not associated with lamellar
bodies or tubular myelin. Based on in vitro studies, SP-D was thought
to be primarily a host defense protein, and there was no direct
evidence that SP-D contributed to surfactant function or homeostasis
(19). SP-D gene-targeted [(/
)] mice survive and
breed normally under laboratory conditions (2,
18); however, their lungs have marked increases in tissue and alveolar surfactant phospholipids. SP-D(
/
) mice also have increased numbers of enlarged foamy alveolar macrophages, changes in
the structure of extracellular surfactant, enlarged type II cells with
increased lamellar body number and size, and enlarged distal air spaces.
The pool sizes of surfactant lipids and proteins are regulated by the
net contributions from synthesis, secretion, uptake and catabolism by
macrophages, and reuptake by type II cells that recycle or catabolize
surfactant components (35). The factors that influence
alveolar and tissue pools of surfactant components are poorly
understood. The important role of granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling was demonstrated in
GM-CSF- and common -chain receptor-deficient mice that had an
alveolar proteinosis characterized by increases in surfactant lipids
and proteins, with a selective increase in SP-D (26). Likewise, increased expression of interleukin-4 (IL-4) caused increased
surfactant lipid and SP-A, SP-B, and SP-C content in transgenic mice.
The largest change in the IL-4 mice was a 90-fold increase in SP-D
(10). In contrast, surfactant lipids were markedly increased in the lungs of SP-D(
/
) mice without substantial changes in the amounts of SPs relative to those in wild-type mice
(18). To better define the role of SP-D in surfactant
homeostasis, we measured SP-A, SP-B, and SP-C, evaluated surfactant
forms, and characterized the metabolism of Sat PC and SP-D in SP-D
wild-type [(+/+)] and SP-D(
/
) mice in vivo.
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MATERIALS AND METHODS |
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Mice.
NIH Swiss black SP-D(+/+) and SP-D(/
) mice were generated as
previously described (18) and carried as breeding colonies in the vivarium at Children's Hospital (Cincinnati, OH) according to
protocols approved by the Institutional Animal Care and Use Committee.
Genotyping was performed by DNA blot analysis. All of the studies were
performed with 7- to 9-wk-old mice except as indicated for selected
experiments. Processing of tissue and biochemical determinations in
SP-D(+/+) and SP-D(
/
) mice were performed simultaneously.
Sat PC pool sizes.
Total lung Sat PC pools (calculated as µmol/kg body wt) were measured
in 7 SP-D(+/+) and 11 SP-D(/
) mice fetuses 16.5 days after
conception. Measurements of Sat PC pool sizes were also taken in 27 SP-D(+/+) and 35 SP-D(
/
) mice from 4 litters/genotype at 2 days of
age and in separate groups of 8 mice aged 7-140 days. Sat
PC in alveolar lavage fluid and lung tissue (after alveolar lavage) was measured at 21, 56, and 140 days of age. Each mouse was
deeply anesthetized with intraperitoneal pentobarbital sodium, and the
distal aorta was cut to exsanguinate the animal (11). After the chest was opened, a 20-gauge blunt needle was tied into the
trachea, and 0.9% NaCl was flushed into the airway until the lungs
were fully expanded. The fluid was then withdrawn by syringe three
times for each aliquot. The saline lavage was repeated five times and
the samples were pooled, and the volume was measured. 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), and Sat PC was isolated with the technique
of Mason et al. (20). The amount of Sat PC was measured by
phosphorus assay (1).
SP pool sizes.
The SPs in fluid from the alveolar washes and large-aggregate
surfactant were analyzed by Western blot in six mice from each genotype. Samples containing 1.35 nmol of Sat PC were used for analysis
of SP-A, and samples containing 2.7 nmol of Sat PC were used for
analysis of SP-C. Proteins were separated by SDS-PAGE in the presence
of -mercaptoethanol. For SP-B analysis, aliquots containing 0.27 nmol of Sat PC were electrophoresed under nonreducing conditions. SP-A
was separated on 8-16% acrylamide gels with Tris-glycine buffer.
SP-B and SP-C samples were separated on 10-20% acrylamide gels
with Tricine buffer (Novex, San Diego, CA). After electrophoresis, proteins were transferred to nitrocellulose paper (Schleicher & Schuell, Keene, NH) for SP-A or to polyvinylidene difluoride paper
(Bio-Rad, Hercules, CA) for SP-B and SP-C. Immunoblot analysis was
carried out with the following dilutions of antisera: 1:25,000 for
guinea pig anti-rat SP-A; 1:10,000 for rabbit anti-bovine SP-B; and
1:25,000 for rabbit anti-recombinant human SP-C (21, 30, 34). Appropriate peroxidase-conjugated
secondary antibodies were used at 1:10,000 dilutions. Immunoreactive
bands were detected with enhanced chemiluminescence (ECL) reagents
(Amersham, Chicago, IL). Protein bands were quantitated by
densitometric analyses with Alpha Imager 2000 documentation and
analysis software (Alpha Innotech, San Leandro, CA).
Precursor incorporation into Sat PC and secretion.
SP-D(+/+) and SP-D(/
) mice were given intraperitoneal
injections of 8 µl saline/g body wt containing 0.5 µCi
[3H]palmitic acid/g body wt or 0.3 µCi
[14C]choline chloride/g body wt (American Radiolabeled
Chemicals, St. Louis, MO) (11). The palmitic acid was
stabilized in solution with 5% human serum albumin (17).
Groups of six to nine mice were killed at preselected times, and
alveolar lavage fluid was recovered from each animal. Lung tissue was
placed in 0.9% NaCl. Sat PC was isolated from the alveolar lavage
fluid and homogenized lung tissue as described in Sat PC pool
sizes, and radioactivity was measured. The percent
secretion of labeled Sat PC was calculated as the percent of
radioactivity in alveolar Sat PC relative to the total radioactivity in
the alveolar lavage fluid plus lung tissue.
Clearance of dipalmitoylphosphatidylcholine.
Mice 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]palmitoyl-labeled
dipalmitoylphosphatidylcholine (DPPC; American Radiolabeled Chemicals),
1.5 µg of DPPC, and 3.3 µg of lipid-extracted sheep surfactant
suspended by the use of glass beads (11). The phospholipids given by intratracheal injection were 2% of the alveolar
pool size for SP-D(+/+) mice and 0.5% of the alveolar pool size for
SP-D(/
) mice. Groups of four to six SP-D(+/+) and SP-D(
/
) mice
were killed 10 min and 3, 8, 16, 24, 48, and 72 h after
intratracheal injection, and 3H was measured in the lipid
extracts and cell pellets of the alveolar lavage fluid and in the lung
tissue. To recover the cell pellets, alveolar lavage fluid was
layered over a 0.8 M sucrose in 0.9% NaCl cushion and centrifuged at
500 g for 15 min. More than 90% of the cell pellets were macrophages.
Clearance of SP-D.
Mice deficient for both GM-CSF and SP-A accumulate large amounts of
SP-D in their air spaces, providing a source for the isolation of SP-D
that is not contaminated with SP-A. Mouse SP-D was isolated from
alveolar lavage fluid of the GM-CSF- and SP-A-deficient mice by the
methods described by Persson et al. (22), with the use of
an affinity column of mannose-Sepharose 6B in the presence of
Ca2+. Mouse SP-D was iodinated with the
Bolton-Hunter reagent (Amersham Life Science, Arlington Heights, IL)
with the same techniques that were used for SP-A (11).
125I-SP-D was injected intratracheally as described in
Clearance of dipalmitoylphosphatidylcholine. Groups of five
to seven SP-D(+/+) and SP-D(/
) mice were killed 0.5, 2, 8, 24, and
48 h after injection, and 125I was measured in the
alveolar lavage fluid and lung tissues.
Aggregate forms of alveolar surfactant.
Large- and small-aggregate surfactants were isolated from the alveolar
lavage fluid of eight SP-D(+/+) and six SP-D(/
) mice by
centrifugation at 40,000 g for 15 min over a 0.8 M sucrose in 0.9% NaCl cushion (13). The large-aggregate surfactant
was collected from the 0.8 M sucrose interface. The supernatant
contained small-aggregate surfactant. The amount of Sat PC was measured for both large- and small-aggregate surfactant, and the percent of Sat
PC in large-aggregate surfactant in the total alveolar Sat PC was
calculated. The Sat PC was also measured in the cell pellets at the
bottom of the tubes under the sucrose step gradient.
Conversion of large-aggregate to small-aggregate surfactant.
Large-aggregate surfactant (0.1 µmol of Sat PC) was mixed with 2 ml
of Tris buffer in a capped 12 × 75-mm polystyrene tube, and
surface area cycling was performed as previously described (7, 12). Briefly, tubes were attached to the
disk of Rototorque rotators (Cole-Parmer Instruments, Chicago, IL) in a
37°C incubator. Five tubes each for SP-D(+/+) and SP-D(/
) mice
were rotated for 6 h at 40 rotations/min to change the surface
area from 1.1 to 9.0 cm2 two times per cycle. One tube for
each group was incubated for the same time period without rotation. The
large- and small-aggregate surfactants were isolated by centrifugation
at 40,000 g for 15 min. The large-aggregate surfactant in
the pellet and the residual small-aggregate surfactant in the
supernatants were measured by phosphorus assay (1).
Surface activity.
The surface properties of three pools (3 mice/pool) of large-aggregate
surfactant isolated from SP-D(+/+) and SP-D(/
) mice were measured
with the captive bubble surfactometer (31). Aliquots of
the surfactant pools were organic solvent extracted, and Sat PC
concentrations were measured as described in Sat PC pool
sizes. The concentration of each pool was adjusted to 3 µmol Sat
PC/µl, and 3 µl of the native surfactant were applied to the air
interface of a 25-µl bubble. The surface tension was measured every
second for 5 s and at 10, 20, 30, and 60 s, and bubble
pulsation was started. The minimum surface tensions after 65% volume
reductions of the bubbles were measured for 5th and 10th pulsations.
Data analysis. All values are given as means ± SE. Differences between the two groups were determined by a two-tailed Student's t-test. The between-group comparisons were made by analysis of variance followed by the Student-Newman-Keuls multiple comparison procedure. Curves were fit with the use of linear regression.
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RESULTS |
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Sat PC pool sizes.
Sat PC (µmol Sat PC/kg) was similar in lungs from fetal (16.5 days
after conception; term = 18 days) SP-D(/
) and SP-D(+/+) mice
(Fig. 1A). In SP-D(+/+) mice,
Sat PC decreased with advancing age after birth. In contrast, Sat PC
content did not decrease with age in SP-D(
/
) mice. The amount of
Sat PC in lungs from SP-D(
/
) mice increased three- to fourfold
compared with SP-D(+/+) mice after 21 days of age. Alveolar Sat PC pool
sizes were measured in SP-D(+/+) and SP-D(
/
) mice at 21, 56, and
140 days of age (Fig. 1B), and alveolar Sat PC was increased
approximately fourfold in SP-D(
/
) mice compared with levels in
SP-D(+/+) mice.
|
SP pool sizes.
The amounts of the SPs in the fluid from the alveolar washes from
SP-D(/
) mice were estimated by Western blot (Fig.
2A). In SP-D(
/
)
mice, SP-A content was decreased to 12% of normal values. SP-B content
was increased twofold, and SP-C was unchanged. Because alveolar Sat PC
was increased 2.3-fold in the SP-D(
/
) mice used for these
measurements, the contents of the SPs are also expressed relative to
Sat PC (Fig. 2B). In SP-D(
/
) mice, the amount of SP-A
was decreased to 6 ± 3% of the normal value, the amount of SP-B
was unchanged, and there was 52 ± 7% of the normal amount of
SP-C relative to Sat PC.
|
Precursor incorporation into Sat PC.
[3H]palmitic acid labeling of Sat PC was measured for
72 h after precursor injection into SP-D(+/+) and SP-D(/
) mice
(Fig. 3). Three and eight hours after
administration of the radiolabel, [3H]palmitic acid
incorporation into Sat PC was similar in SP-D(
/
) and SP-D(+/+)
mice. These time points best indicate net incorporation of the
precursor into Sat PC. As anticipated, the amount of radiolabeled Sat
PC decreased to 58% of the average of the 3- and 8-h values in the
lungs of SP-D(+/+) mice by 72 h, indicating the anticipated catabolism of the labeled Sat PC synthesized by the lung. There was
only a 36% decrease in labeled Sat PC in total lungs in SP-D(
/
) mice between the average of the 3- and 8-h values and the 72-h value,
demonstrating a slower turnover of labeled Sat PC and suggesting less
catabolism of endogenously synthesized Sat PC in SP-D(
/
) mice.
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Clearance of DPPC.
Percent recoveries of labeled DPPC after intratracheal injection are
shown in Fig. 6 for alveolar lavage
fluid, alveolar cell pellets (primarily macrophages), lung tissues
after lavage, and total lungs (the sums of all the fractions). There
was exponential loss of labeled Sat PC from the alveolar washes, with a
lower recovery of 0.7% at 40 h in SP-D(+/+) mice compared with
the 7.7% recovery measured for SP-D(/
) mice. However, because
alveolar pool sizes were fourfold larger in SP-D(
/
) than in
SP-D(+/+) mice, net losses were 0.2 µmol/h for SP-D(+/+) and 0.6 µmol/h for SP-D(
/
) mice. The cell pellet, containing primarily
macrophages, initially contained ~5% of the labeled Sat PC for both
strains of mice, and the percent decreased exponentially, with more
rapid loss in SP-D(+/+) than in SP-D(
/
) mice. However, the ratio of macrophage-labeled to alveolar-labeled Sat PC recovered was constant at
~0.08, demonstrating no accumulation of labeled Sat PC in
macrophages. The percent of the radiolabel associated with the lung
tissue after lavage was remarkably similar for the SP-D(+/+) and
SP-D(
/
) mice. The exponential loss of labeled Sat PC from the total
lungs measured net catabolic activity within the lungs. Although the loss of labeled Sat PC was somewhat slower in SP-D(
/
) mice, the
increased Sat PC pool sizes in these animals resulted in net losses of
0.6 µmol/h for SP-D(+/+) and 1.9 µmol/h for SP-D(
/
) mice.
|
Clearance of SP-D.
The clearance of mouse 125I-SP-D from alveolar lavage fluid
and total lungs of SP-D(/
) mice was slower than in their SP-D(+/+) counterparts (Fig. 7). By 48 h,
21.7% of the labeled SP-D was recovered from the SP-D(
/
) lungs, a
value higher than the 11.8% recovered from SP-D(+/+) mice
(P < 0.01). The half-life of 17 h for SP-D in the
total lungs of SP-D(
/
) mice was somewhat longer than the half-life
of 13 h for SP-D(+/+) mice.
|
Surfactant aggregates and conversion rates.
The percent Sat PC in large-aggregate form relative to the total Sat PC
in alveolar lavage fluid was significantly lower in lungs from
SP-D(/
) mice than in those from SP-D(+/+) mice (Fig. 8A). Because the Sat PC
alveolar pool sizes were three- to fourfold higher in SP-D(
/
) than
in SP-D(+/+) mice, the amount of Sat PC in large-aggregate surfactant
of SP-D(
/
) mice was increased about twofold compared with that in
SP-D(+/+) animals. The cell pellets from the alveolar washes recovered
after centrifugation over 0.8 M sucrose contained 1.0 ± 0.2% of
the alveolar Sat PC in SP-D(+/+) and 1.2 ± 0.3% of the Sat PC in
SP-D(
/
) mice. Although the amounts were small, the pellets from
lavages of SP-D(
/
) mice contained about four times more Sat PC than
did the pellets of SP-D(+/+) mice.
|
Surface activity.
Large-aggregate surfactants from SP-D(+/+) and SP-D(/
) mice reached
equilibrium surface tension within 10 s. The equilibrium surface
tension was 21.6 ± 0.30 mN/m (n = 3 pools of
surfactant) for SP-D(+/+) mice and 22.8 ± 0.2 mN/m
(n = 3 pools) for SP-D(
/
) mice. The minimum surface
tensions on the 5th pulsation were 4.9 ± 2.6 mN/m for surfactant
from SP-D(+/+)and 4.0 ± 1.0 mN/m for surfactant from SP-D(
/
)
mice. Minimum surface tensions for the 10th pulsations were similar to
those for the 5th pulsations. Therefore, surfactants isolated from both
SP-D(
/
) and SP-D(+/+) mice were highly surface active. The lower
concentrations of SP-A and SP-C did not alter the surface activity of
the surfactant isolated from SP-D(
/
) mice.
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DISCUSSION |
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We found a number of changes in alveolar surfactant that may
contribute to the altered homeostasis in SP-D(/
) mice. The age-independent three- to fourfold increase in both alveolar and total
lung Sat PC is associated with an increased relative amount of
small-aggregate surfactant. The large-aggregate surfactant fraction
converts more rapidly to small-aggregate surfactant when the surface
area is cycled. The ratios of SP-A and SP-C to Sat PC are decreased,
and the ratio of SP-B to Sat PC is not changed in the large-aggregate
surfactant of SP-D(
/
) relative to that in SP-D(+/+) control mice.
Lower SP-A might result in increased small-aggregate surfactant, which
is the form preferentially taken up by type II cells (9).
We interpret the metabolic data based on the assumption that precursor
pools for SP-D(+/+) and SP-D(
/
) mice are the same. The
incorporation of palmitic acid and choline into Sat PC and the initial
secretion of endogenously synthesized radiolabeled Sat PC in SP-D(+/+)
and SP-D(
/
) mice are similar, indicating comparable synthetic and
secretory rates for Sat PC. There is less alveolar clearance and
degradation of the endogenously synthesized radiolabeled Sat PC. The
biological half-lives of exogenously administered DPPC and SP-D
are longer in SP-D(
/
) mice than in their SP-D(+/+) counterparts;
however, the pool sizes are larger, resulting in a net increased
catabolism of Sat PC.
These results extend the descriptions of the first two reports
(2, 18) of separately generated
SP-D-deficient mice with the use of comparable transgenic technologies.
The only substantive difference between the mouse described by Botas et
al. (2) and the mouse that was reported by Korfhagen et
al. (18) was the content of SPs in alveolar
lavage fluid. They (18) found a modest decrease in SP-A
mRNA and decreased total SP-A in fluid from alveolar washes. Botas et
al. (2) reported a 2.4-fold increase in SP-A, a 6-fold
increase in SP-B, and no change in mRNAs for the SPs in animals of a
similar age of 8 wk. Therefore, we further evaluated the amounts of the
SPs in alveolar lavage fluid and related those amounts to the amount of
Sat PC. Relative to Sat PC, SP-A in lavage fluid of SP-D(/
) mice
was only 6% of the normal amount, SP-B was present in normal amounts,
and SP-C was decreased by 50%. This analysis emphasizes the ratio of
the SPs to Sat PC because surfactant function is presumably dependent
on the SP-to-lipid ratios (25, 32). Because
the small-aggregate surfactant does not contain SPs in appreciable amounts and the small-aggregate pool is increased in SP-D(
/
) mice
relative to that in SP-D(+/+) mice, we further evaluated the ratio of
SPs to Sat PC in the large-aggregate surfactant. The large-aggregate
surfactant is the biophysically active fraction of surfactant and the
presumed precursor pool for the surface film (36). In
large-aggregate surfactant, the SP-A- and SP-C-to-Sat PC ratios are 25 and 30%, respectively, of the control values in SP-D(
/
) mice. The
ratio is unchanged for SP-B.
The structure of the large-aggregate surfactant from SP-D(/
) mice
was previously described as having enlarged and dense phospholipid
arrays with less tubular myelin compared with that from SP-D(+/+) mice
(18). These structures are similar to those described for
SP-A(
/
) mice except that those mice have no tubular myelin
(12). Although SP-D can cause the formation of tubular myelin-like structures with surfactant lipids in vitro
(24), the abnormal structures probably result from the low
SP-A-to-Sat PC ratios. The functional consequences are an increased
rate of conversion from large-aggregate to small-aggregate surfactant with surface area cycling. Conversion rates increased in surfactant that lacked SP-A (12), and conversion rates decreased when
excess SP-A was added to rabbit surfactant (8). Therefore,
it is reasonable to attribute the increased conversion from
large-aggregate to small-aggregate surfactant to the very low SP-A
content of large-aggregate surfactant from SP-D(
/
) mice. Despite
the low SP-A- and SP-C-to-Sat PC ratios, surface activity of
large-aggregate surfactant isolated from SP-D(
/
) mice was similar
to surfactant from normal SP-D(+/+) mice. The normal SP-B-to-Sat PC
ratio in large-aggregate surfactant from SP-D(
/
) mice was
sufficient for normal surface activity. SP-C is not required for
surface activity if SP-B is present in normal amounts
(29).
Our studies of the metabolism of Sat PC demonstrate very complex
alterations in SP-D(/
) mice. On the anabolic side of the equation,
type II cell numbers are probably similar in SP-D(
/
) and SP-D(+/+)
mice, although formal morphometrics have not been performed. The mice
that we studied at ~8 wk of age do not have much of the emphysema
that develops markedly after 12 wk of age (33).
Incorporation of the precursors palmitic acid and choline into lung Sat
PC over the initial hours after precursor administration was similar
for SP-D(
/
) and SP-D(+/+) animals. If it is assumed that precursor
pools are similar, this measurement is the in vivo equivalent of
comparable synthetic rates for Sat PC. Similarly, the initial percent
secretion of the newly synthesized radiolabeled Sat PC is comparable
for the mice despite the description of enlarged lamellar bodies in
some type II cells in SP-D(
/
) mice (2). The
incorporation and secretion data for the first 16 h after precursor administration indicate comparable anabolism in SP-D(+/+) and
SP-D(
/
) mice.
As anticipated, palmitic acid-labeled Sat PC is lost from the alveolar
lavage fluid and total lungs, indicating catabolism of endogenously
synthesized Sat PC in SP-D(+/+) mice. This result has been replicated
by multiple measurements (10, 13). In contrast, palmitic acid-labeled Sat PC continues to accumulate in
alveolar washes in SP-D(/
) mice. There also is less loss of
palmitic acid from the lungs of SP-D(
/
) mice. This abnormality is
magnified with the choline label, where choline labeling of Sat PC in
both alveolar lavage fluid and total lungs continues to increase for up
to 72 h. The mice were given the precursor by intraperitoneal
injection because we found more uniform labeling between animals
injected in this fashion than those measured with intravascular
precursor injections (13), and intraperitoneal labeling
was used previously for similar metabolic studies in mice by Gross et
al. (6). However, intraperitoneal labeling will
result in less rapid clearance of the intravascular label and
preferential labeling of the liver. Some label will enter the lungs at
later times, and the stability and pool sizes of choline precursors in
the lung are not known. In wild-type mice, catabolism of the initially
synthesized Sat PC is in excess of any new label entering the lungs,
and net catabolic activity is measured. If it is assumed that new label
entering the lungs is comparable in SP-D(+/+) and SP-D(
/
) mice, the
conclusion is that there is less catabolism of endogenously synthesized
Sat PC in SP-D(
/
) than in SP-D(+/+) mice because labeled Sat PC continues to accumulate. The persistence of the radiolabel in Sat PC
suggests that endogenously synthesized Sat PC is degraded more slowly
in SP-D(
/
) mice than in their SP-D(+/+) counterparts. The caveat is
that the total lung Sat PC pool size is three to four times higher in
SP-D(
/
) than in SP-D(+/+) mice. The larger pool sizes may promote
an increased catabolic rate by mass action. If this endogenously
synthesized pool mixes uniformly with the total pool, the net catabolic
rate (in µmol Sat PC/kg) may not be very different.
Alveolar clearance and catabolism also were measured after
intratracheal injection of radiolabeled DPPC. These measurements avoided the problem of the radiolabel entering the lungs at late times
but depended on the tracers mixing with the alveolar pool (14). In normal animals, metabolic variables estimated by
precursor measurements and from intratracheal injections of labeled
DPPC are similar (15). Alveolar clearance of DPPC was
slower in SP-D(/
) mice than in SP-D(+/+) mice, with biological
half-life values of 5.1 and 2.8 h, respectively. If it is assumed
that the clearance of the tracer and of the total alveolar pool are the
same, discounting recycling, and noting that the alveolar Sat
PC pool size is three to four times larger in SP-D(
/
) mice, the
amount of Sat PC cleared would be 0.2 µmol/h for SP-D(+/+) and 0.6 µmol/h for SP-D(
/
) mice. Therefore, the SP-D(
/
) mice clear
more Sat PC per unit time than SP-D(+/+) mice. Loss of the radiolabel
to macrophages is a relatively constant percentage of the
alveolar pool, demonstrating no accumulation of DPPC in macrophages.
This labeling pattern was described previously in rabbits and provides
no information about the catabolic activity of the macrophages
(28). Alveolar clearance would appear delayed if a high
percentage of the DPPC were recycled. The recycling efficiency in
wild-type mice was estimated to be ~50% (6).
Total lung catabolic activity can be estimated by the loss of DPPC from
the lungs. Intact DPPC does not leave the lungs in appreciable amounts
(23). The biological half-life of 15 h for SP-D(/
) mice was somewhat longer than the 9 h measured for
SP-D(+/+) mice. However, the three- to fourfold larger Sat PC pool
sizes in SP-D(
/
) compared with SP-D(+/+) mice resulted in catabolic rates of 1.9 and 0.6 µmol/h, respectively, demonstrating threefold higher catabolic activity in the SP-D(
/
) group.
These metabolic studies resulted in a conundrum because anabolism
(incorporation and secretion) is comparable in SP-D(+/+) and
SP-D(/
) mice, and endogenously synthesized Sat PC likely is
catabolized more slowly in SP-D(
/
) than in SP-D(+/+) animals. In
contrast, the intratracheal clearance studies indicate more rapid net
catabolism of the larger pools in the SP-D(
/
) mice. These
measurements have not considered recycling. The accumulation of lipids
in the air spaces of SP-D(
/
) mice is not uniform, based on
histopathology, nor is the distribution of cells with large lamellar
bodies (2, 18). It is possible that the
intravascular precursors are selectively evaluating different cell
populations and that intratracheal DPPC is measuring clearance and
catabolism for only part of the alveolar pool.
The contribution of recycling to the increased surfactant pool sizes is not known, but increased recycling might occur because of the increased alveolar small-aggregate surfactant pool. The small-aggregate surfactant thought to be the catabolic form for the phospholipids is preferentially taken up by type II cells in vitro (9). Further studies will be required to sort out the relationship between the endogenous incorporation data and the enlarged lamellar bodies in type II cells. The increased numbers of lipid-laden macrophages will also no doubt contribute to the overall metabolic abnormalities in these mice. Alveolar macrophages contribute ~20% to the overall catabolic rate of surfactant in adult rabbit lungs (27), but the macrophage contribution in mice is not known.
Animal models of increased surfactant phospholipid and increased SP
pool sizes include silica and mineral dust exposure (4), the severe-combined immunodeficient mouse (16),
gene-targeted disruption of the GM-CSF or GM-CSF receptor /C gene
(13, 26), and overexpression of IL-4
(10). Increased surfactant phospholipid in SP-D(
/
)
mice was not associated with increased amounts of SPs, distinguishing
this phenotype from the alveolar lipoproteinosis syndromes.
Each of the models of alveolar proteinosis has different alterations in
metabolic pathways that yield a similar phenotype. For example, GM-CSF
deficiency and GM-CSF receptor inactivation result primarily from a
decrease in phospholipid and SP catabolism (13,
26). Mice with defective GM-CSF signaling also accumulate large amounts of SP-D in the air spaces. In contrast, SP-D was cleared
relatively rapidly in SP-D(
/
) mice. Those that overexpress IL-4
have alveolar proteinosis primarily because of increased precursor
incorporation and secretion rates (10). In these models, the ratio of SPs to Sat PC is normal or increased. The SP-D(
/
) phenotype is unique because of the decreased SP-A and SP-C relative to
Sat PC.
An absolute SP-D(/
) deficiency results in multiple abnormalities in
the surfactant system. In contrast, SP-D(+/-) mice have normal
surfactant lipid pool sizes, and mice that overexpress SP-D also have
normal surfactant pool sizes, indicating that the phenotype is
sensitive to the amount of SP-D (5). SP-D deficiency alters surfactant
lipid, SP-A, and SP-C pools and metabolism by unknown mechanisms.
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ACKNOWLEDGEMENTS |
---|
This work was supported by National Heart, Lung, and Blood Institute Grants HL-63329-01 and HL-61646.
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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. §1734 solely to indicate this fact.
Received 16 February 2000; accepted in final form 12 April 2000.
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