1 Department of Cell Biology, Duke University, Durham, North Carolina 27710; and 2 Department of Pediatrics, Division of Pulmonary Biology, Cincinnati Children's Hospital, Cincinnati, Ohio 45529
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ABSTRACT |
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Previous in vitro studies have suggested
that surfactant protein A (SP-A) may play a role in pulmonary
surfactant homeostasis by mediating surfactant secretion and clearance.
However, mice made deficient in SP-A [SP-A (/
) animals] have
relatively normal levels of surfactant compared with wild-type SP-A
(+/+) animals. We hypothesize that SP-A may play a role in surfactant
homeostasis after acute lung injury. Bacterial lipopolysaccharide was
instilled into the lungs of SP-A (
/
) mice and SP-A (+/+) mice to
induce injury. Surfactant phospholipid levels were increased 1.6-fold in injured SP-A (
/
) animals, although injury did not alter
[3H]choline or [14C]palmitate incorporation
into dipalmitoylphosphatidylcholine (DPPC), suggesting no change in
surfactant synthesis/secretion 12 h after injury. Clearance of
[3H]DPPC from the lungs of injured SP-A (
/
) animals
was decreased by ~40%. Instillation of 50 µg of exogenous SP-A
rescued both the clearance defect and the increased phospholipid defect
in injured SP-A (
/
) animals, suggesting that SP-A may play a role in regulating clearance of surfactant phospholipids after acute lung injury.
lipopolysaccharide; surfactant secretion; surfactant homeostasis; dipalmitoylphosphatidylcholine
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INTRODUCTION |
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PULMONARY SURFACTANT is a mixture of lipids and proteins produced by the alveolar type II cell that serves to reduce surface tension at the air-liquid interface and to protect the host against infection. Surfactant is made up of ~90% lipid and 10% protein by weight. The major phospholipid component of surfactant is dipalmitoylphosphatidylcholine (DPPC), which reduces surface tension, although surfactant also contains smaller amounts of other phospholipids and cholesterol (reviewed in Refs. 20 and 46). Two of the surfactant proteins, surfactant protein (SP)-B and SP-C, are small hydrophobic proteins that play a role in surface tension reduction by facilitating the absorbance of surfactant lipids to the air-liquid interface (39). The two other major surfactant proteins, SP-A and SP-D, are both members of the collectin superfamily (12, 36). Both proteins are large oligomeric molecules that have the ability to recognize and bind carbohydrate moieties present on foreign organisms through a calcium-dependent carbohydrate recognition domain.
Studies using model systems consisting of isolated cells and purified
surfactant proteins as well as mice made deficient in SP-A [SP-A
(/
)] by homologous recombination have demonstrated a role for SP-A
in host defense. SP-A has been shown to bind and/or aggregate a variety
of pulmonary pathogens in vitro (reviewed in Refs. 11 and
22). In some cases this interaction can lead to an
increased response by alveolar macrophages against the pathogens (34, 37, 44). Furthermore, SP-A (
/
) mice were more
susceptible to challenge by group B streptococcus (GBS) (27,
30), Pseudomonas aeruginosa
(28), Haemophilus influenzae
(30), and respiratory syncytial virus (RSV)
(29). Premixing of GBS with SP-A before challenge in the
SP-A (
/
) animals "rescued" the clearance defect (27), and treatment of SP-A (
/
) mice with exogenous
SP-A concurrent with RSV exposure decreased the inflammatory response
(29).
In vitro studies also supported a role for SP-A in surfactant homeostasis. SP-A has been shown to bind receptors on the surface of type II cells (24, 50) and alveolar macrophages (38); these interactions may result in SP-A-mediated stimulation of liposome clearance by both type II cells (51) and alveolar macrophages (2, 52) in vitro. Alveolar macrophages incubated with liposomes and SP-A also degraded more of the internalized lipid (40). SP-A has also been shown to inhibit agonist-stimulated lipid secretion by type II cells (6, 43) and phospholipase A2 activity in lung tissue homogenate (9).
Although the in vitro studies suggest that SP-A regulates surfactant
metabolism, SP-A deficiency did not severely alter surfactant homeostasis in SP-A (/
) mice (reviewed in Ref. 22).
Mice lacking SP-A produce similar levels of surfactant phospholipids
and the other surfactant proteins compared with wild-type [SP-A
(+/+)] mice (21). There were also no differences in the
incorporation of radiolabeled palmitic acid and choline into
disaturated phosphatidylcholine (Sat PC), suggesting that under normal
conditions surfactant synthesis and secretion are not altered by the
SP-A deficiency (17). Clearance of radiolabeled DPPC
instilled into the lungs of SP-A (
/
) mice was similar to that of
SP-A (+/+) animals (17). Also, there were no differences
in surfactant Sat PC levels between SP-A (+/+) and SP-A (
/
) animals
after exercise or hyperoxia, although hyperoxia did result in increased
lavage protein levels in SP-A (
/
) animals (16).
Acute lung injury has been shown to alter the levels of pulmonary surfactant. In isolated perfused rat lungs, exposure to bacterial lipopolysaccharide (LPS) led to increased lavage phospholipid levels 2 h after LPS exposure (8), although studies where LPS was instilled into rats showed decreases in lavage phospholipid levels 4 h after exposure (35). At 72 h, rats exposed to LPS showed an increase in intracellular surfactant phospholipid pools as well as a decrease in extracellular surfactant phospholipid pools (49). Differences in the effects of LPS on phospholipid pool size are likely due to the time point examined, although it is possible that the dose and type of LPS used might also be important. It has been shown that SP-A levels are increased in tissue as early as 6 h after LPS exposure and in lavage and tissue as late as 72 h after LPS exposure (33, 49).
LPS exposure also leads to changes in the cells resident in the
alveolar space, resulting in an influx of neutrophils and an increase
in the overall number of cells that can be isolated by lavage
(41, 49, 53). Neutrophils isolated from LPS-treated lungs
have been shown to internalize and degrade surfactant-like lipids in an
SP-A-dependent manner, and macrophages isolated from these lungs are
activated for phospholipid internalization and degradation
(41). Due to the changes in the populations of cells in
the lung and the alterations in the levels of surfactant components, we
hypothesize that SP-A may play an important role in the maintenance of
surfactant phospholipid levels after acute lung injury and that SP-A
(/
) mice will have defects in surfactant homeostasis under these conditions.
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MATERIALS AND METHODS |
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Materials.
DPPC, dipalmitoylphosphatidylglycerol (DPPG), egg
phosphatidylcholine (egg PC), and cholesterol were purchased from
Avanti Polar Lipids (Birmingham, AL). L--dipalmitoyl
[(2-palmitoyl-9, 10-3H(N))]-phosphatidylcholine (89 Ci/mmol) was obtained from DuPont New England Nuclear (Boston, MA).
Phosphate-buffered saline (PBS) was purchased from GIBCO. Chloroform
and methanol were from Mallinkrodt. Bovine serum albumin (BSA),
ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), ammonium
molybdate, Fiske-Subbarow reducer, and Escherichia coli
026:B6 LPS were obtained from Sigma (St. Louis, MO). BCA protein
assay reagent was purchased from Pierce (Rockford, IL). Hemacolor
differential hematoxylin staining kit was obtained from EM Science
(Gibbstown, NJ).
Animals.
SP-A (/
) mice were generated as previously described
(21) and have been maintained for over six years as
breeding colonies in either the vivarium at Children's Hospital
(Cincinnati, OH) or the vivarium at Duke University (Durham, NC). SP-A
(+/+) mice (129J) were also maintained as breeding colonies in the same
facilities. 129J mice obtained from Jackson Laboratories were used as
SP-A (+/+) controls for some precursor incorporation studies. Results obtained with mice from Jackson Laboratories were not different from
SP-A (+/+) animals bred at Duke University or Children's Hospital.
SP-A preparation. SP-A was purified as previously described (51). Briefly, SP-A was isolated from the lavage fluid of alveolar proteinosis patients by extraction of the sedimented lipids with butanol. Purified protein was treated with polymyxin to lower endotoxin levels (32).
Preparation of liposomes. Small unilamellar liposomes were prepared with a lipid composition similar to that of surfactant: 52% DPPC, 26% egg PC, 15% DPPG, and 7% cholesterol by weight with trace amounts of [3H]DPPC (12 µCi/mg phospholipid) (51). The lipids were resuspended in 0.9% NaCl and extruded from a French pressure cell, resulting in small unilamellar liposomes at a concentration of 1 mg of lipid/ml (13).
LPS injury model system. To initiate LPS injury in mice, the animals were anesthetized with isoflurane, and the trachea was exposed. The dose of LPS (100 µg of E. coli 026:B6 LPS/kg body wt in 50 µl of saline) was delivered by injection into the trachea through a 22.5-gauge needle. Control animals were uninjured, untreated animals, although instillation of saline did not alter the number or type of cells isolated by lavage (average of 2.2 × 105 cells/animal for 2 animals). For rescue animals the instilled dose contained 100 µg of E. coli 026:B6 LPS/kg body wt as well as 50 µg of purified human SP-A in a total volume of 50 µl.
DPPC precursor incorporation. Mice were given an intraperitoneal injection of saline containing 5% BSA, [3H]choline (38 µCi/ml), and [14C]palmitate (7.7 µCi/ml) at a dose of 13 µl/g body wt. In cases where LPS injury was induced, the lipid precursors were injected when the LPS injury was initiated. After 12 h, the animals were euthanized with a lethal dose of pentobarbital followed by exsanguination. The trachea was exposed, and a cannula was inserted. We lavaged the lungs by inflating the lungs to total capacity (~1 ml) with PBS containing 0.2 mM EGTA (pH 7.4), which was maintained at 37°C. Each volume of lavage fluid was recirculated three times. The lavage was repeated five times, and all five washes were pooled. The lungs were homogenized in 4 ml of PBS containing 0.2 mM EGTA (pH 7.4) by a motor-driven Potter-Elvehjem tissue homogenizer. Total phospholipids were extracted from aliquots of lavage and tissue homogenate by the method of Bligh and Dyer (3). Sat PC was then extracted by the method of Mason and coworkers (31). Radioactivity of the samples was analyzed by liquid scintillation counting. A sample of lavage fluid was taken for determination of total protein via BCA assay before Bligh-Dyer extraction.
DPPC clearance in vivo. Mice were instilled with 10 µg of radiolabeled phospholipid in 50 µl of PBS by the same technique as described for instillation of LPS. For rescue experiments, 50 µg of SP-A/animal were instilled in the same volume as the LPS. After incubation of radiolabel in the lungs, the animals were euthanized with a lethal dose of pentobarbital followed by exsanguination. The trachea was exposed by dissection, and a cannula was inserted. The lungs were inflated to total lung capacity with PBS containing 0.2 mM EGTA at 37°C and recycled three times. Lavage was repeated a second time and pooled with the first wash for a total volume of ~2 ml. Alveolar resident cells were separated from the lavage supernatant by centrifugation at 228 g for 10 min. An aliquot of the cells was taken for differential counting, and the remainder was resuspended in lysis buffer. The lungs, excluding the trachea, were removed, placed in PBS, and chopped into large pieces. The lung tissue was homogenized with four to seven passes in a motor-driven Potter-Elvehjem tissue homogenizer. Radioactivity of the lavage supernatant, lavage pellet, and tissue was determined by liquid scintillation counting. An aliquot of the lavage supernatant was used to determine the phospholipid levels.
Phosphorous assay. Inorganic phosphorus was measured by the method of Bartlett (1). Briefly, samples were incubated with concentrated sulfuric acid at 160°C for 3 h. To remove any color, 30% H2O2 was added, and the samples were heated to 160°C for 90 min. Ammonium molybdate and Fiske-Subbarow reducer were added, producing a color that could be detected at 825 nm. Phospholipid samples were assumed to contain 4% inorganic phosphorus by weight.
Data analyses. Data were compared by Student's t-test for unpaired samples or analysis of variance and a Tukey test when appropriate. n refers to individual animals.
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RESULTS |
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SP-A deficiency leads to an increase in the number of alveolar
resident cells after LPS-induced injury.
The number of cells isolated by lavage from the lungs of healthy SP-A
(+/+) animals (2.6 ± 0.6 × 105 cells) and
healthy SP-A (/
) animals (3.9 ± 0.4 × 105
cells) was not significantly different, and both populations contained
>95% macrophages (data not shown). However, after LPS instillation,
the number of cells that could be isolated by lavage from the lungs of
SP-A (
/
) animals (51 ± 4 × 105 cells)
increased significantly over the number of cells that could be isolated
by lavage from SP-A (+/+) animals (32 ± 5 × 105
cells) (Fig. 1A). Instillation
of SP-A at the initiation of injury in SP-A (
/
) animals reduced
cell yields to levels comparable to SP-A (+/+) animals (28 ± 5 × 105 cells). In all three groups, LPS-induced
injury led to an influx of neutrophils in the lung, resulting in a
population of cells that was ~85% neutrophils (Fig. 1B).
Vehicle-instilled SP-A (+/+) animals averaged 2.2 × 105 cells/animal and >95% macrophages (n = 2 animals).
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SP-A deficiency results in increased phospholipid levels, but no
increase in protein levels after LPS-induced injury.
The levels of protein isolated by lavage from the lungs of healthy or
LPS-exposed SP-A (+/+) or SP-A (/
) animals did not vary between
groups. Lung lavage of healthy SP-A (+/+) and SP-A (
/
) animals
recovered 30.6 ± 6.2 mg protein/kg body wt and 27.8 ± 2.6 mg protein/kg body wt, respectively. After LPS exposure, lung lavage of
SP-A (+/+) and SP-A (
/
) animals recovered 33.5 ± 3.5 mg
protein/kg body wt and 34.9 ± 4.6 mg protein/kg body wt,
respectively. LPS instillation resulted in a 1.6-fold increase in
phospholipid levels in the lavage supernatant of SP-A (
/
) mice
(20.9 ± 1.2 mg phospholipid/kg body wt) compared with SP-A (+/+)
mice (13.3 ± 0.7 mg phospholipid/kg body wt) (Fig.
2). Phospholipid levels in animals where
SP-A was instilled with LPS (13.6 ± 1.2 mg phospholipid/kg body
wt) were not significantly different from phospholipid levels in
injured SP-A (+/+) animals. Vehicle-instilled SP-A (+/+) animals
averaged 9.9 mg phospholipid/kg body wt (n = 2 animals).
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SP-A deficiency does not alter Sat PC precursor incorporation after
LPS-induced injury.
Healthy or LPS-exposed SP-A (+/+) and SP-A (/
) mice incorporated
similar amounts of [3H]choline and
[14C]palmitate into Sat PC (Figs.
3A and
4A). The amounts of label appearing in tissue pools, lavage pools, or the total Sat PC pools were
not statistically different among groups for either label. Although there was no difference in the total radioactivity in tissue and lavage among the groups of animals, the proportions of
radioactivity in the two pools changed in some groups. Significantly more [3H]choline counts in Sat PC were isolated from
lavage than from tissue in healthy SP-A (+/+) mice (ratio of lavage
counts-to-tissue counts, 2.5 ± 0.4), LPS-exposed SP-A (+/+) mice
(3.7 ± 0.7), and LPS-exposed SP-A (
/
) (2.4 ± 0.4) (Fig.
3B). In the case of [14C]palmitate, a similar
increase in the ratio of radioactivity found in the lavage compared
with the radioactivity found in the tissue was observed in the
LPS-exposed SP-A (+/+) (2.3 ± 0.6) and LPS-exposed SP-A
(
/
) mice (2.5 ± 0.7), but not in healthy SP-A (+/+)
(1.1 ± 0.3) or healthy SP-A (
/
) mice (1.3 ± 0.3) (Fig. 4B).
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SP-A deficiency leads to altered clearance of surfactant
phospholipid in vivo.
Phospholipid clearance was measured 2 h after the initial 12 h of LPS exposure (14 h in total) in SP-A (+/+) and SP-A (/
) mice
(Fig. 5). LPS-treated SP-A (
/
)
animals cleared significantly less [3H]DPPC from the
alveolar space compared with healthy SP-A (+/+), healthy SP-A (
/
),
or LPS-treated SP-A (+/+) animals. In LPS-treated SP-A (+/+) mice,
66.9 ± 1.0% of the recovered radioactivity appeared in the
lavage supernatant, 8.2 ± 1.0% appeared in the lavage pellet, and 24.9 ± 1.6% appeared in the tissue. SP-A (
/
) mice
treated with LPS had significantly more radioactivity remaining in the lavage supernatant (80.5 ± 2.4%) and tended towards less
appearing in the lavage pellet (4.7 ± 0.9%, P < 0.07) and significantly less in the tissue (14.9 ± 2.1%).
Although dilution of the radiolabel may have contributed to decreased
clearance, phospholipid levels in SP-A (
/
) mice did not increase
sufficiently to completely account for decreased clearance in the
tissue and cell pellet, due to dilution of the radiolabeled lipid.
Phospholipid levels would have had to increase by greater than 1.7-fold
to dilute the label sufficiently to account for the decreased clearance observed in LPS-treated SP-A (
/
) animals. Total recovery of radioactivity (ranging from 30 to 60% and averaging 41 ± 2%)
did not vary between groups, and clearance of radiolabeled phospholipid from the lungs was significantly greater 2 h after instillation than 5 min after instillation (data not shown).
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LPS injury leads to decreased phospholipid clearance by cells
isolated from the lung.
Although the percentage of radioactivity recovered in the lavage cell
pellet was not statistically different among the four treatment groups
(Fig. 5B), the number of cells recovered by lavage increased
with LPS-exposure (Fig. 1A). On a per cell basis, the cells
isolated from the lavage of healthy SP-A (+/+) or healthy SP-A (/
)
animals contained ~10-fold more radiolabeled lipid than did the cells
isolated from the lungs of LPS-treated SP-A (+/+), LPS-treated SP-A
(
/
), or rescue animals (Fig. 5D). There was no
statistical difference in the amount of radioactivity recovered per
cell between healthy SP-A (+/+) and healthy SP-A (
/
) animals, nor
was there a difference in clearance between any of the LPS-treated groups.
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DISCUSSION |
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A great deal of experimental evidence using in vitro methods
supports a role for SP-A in the mediation of surfactant homeostasis by
regulation of both surfactant secretion by type II cells (6, 25) and surfactant clearance by type II cells and alveolar
macrophages (2, 51, 52). However, SP-A (/
) mice
maintain a functional surfactant system with relatively minimal defects
that do not seem to interfere with the gas exchange functions of the
lung (17, 18, 21). It has also been shown that induction
of an acute lung injury alters the levels of both protein and lipid components of surfactant in vivo (8, 33, 35, 49). In this
study we sought to determine whether SP-A regulates surfactant phospholipid levels after acute lung injury by measuring the levels of
phospholipid in lavage in both SP-A (+/+) and SP-A (
/
) animals and
to determine whether any alterations in surfactant phospholipid levels
may be due to changes in SP-A-mediated synthesis and secretion and/or
clearance of surfactant phospholipids. These data suggest a role for
SP-A in regulating the clearance of surfactant phospholipid from the
alveolar space after LPS-induced acute lung injury in vivo.
Intratracheal instillation of E. coli 026:B6 LPS resulted in
an inflammatory response in both SP-A (+/+) and SP-A (/
) animals, although the severity of the injury varied. In both types of animals, LPS instillation resulted in a large influx of neutrophils (Fig. 1B), although the absolute number of cells that could be
isolated by lavage from the alveolar space of SP-A (
/
) animals was
60% greater than the cells isolated from the alveolar space of
LPS-treated SP-A (+/+) animals. However, the levels of protein found in
the lavage were not different among any of the treatment groups. Borron et al. (4) have shown that, at 3 h after LPS
instillation, protein levels and lavage cell number are increased in
both SP-A (+/+) and SP-A (
/
) animals compared with control animals
but are not different from each other. Instillation of 50 µg of
human SP-A at the time of LPS instillation decreased the number of
cells found in the alveolar space to levels similar to those found
in LPS-treated SP-A (+/+) animals (Fig. 1A).
Other studies have also shown a more severe inflammatory response in
SP-A (/
) animals compared with SP-A (+/+) animals. Pulmonary
infiltrates were observed earlier in response to GBS (27)
and persisted longer in response to P. aeruginosa in SP-A (
/
) animals compared with SP-A (+/+) animals (28).
Also, intratracheal inoculation of RSV led to an increased number of
cells and an increased influx of neutrophils into the alveolar space in
the absence of SP-A; rescue with SP-A at the time of RSV inoculation also decreased the number of cells found in the alveolar space of SP-A
(
/
) animals to levels similar to those of inoculated SP-A (+/+)
animals (29). Exposure to LPS (4), GBS,
H. influenzae (30), P. aeruginosa
(28), and RSV (29) resulted in greater increases in inflammatory cytokines in the lungs of SP-A (
/
) mice
compared with SP-A (+/+) mice. It is possible that the changes we
observed in surfactant lipid clearance occur secondarily to the
increased inflammatory response in the absence of SP-A.
In addition to a more severe injury, LPS-treated SP-A (/
) animals
had 60% more lavage phospholipid than LPS-treated SP-A (+/+) animals;
instillation of SP-A at the initiation of injury rescued this defect
(Fig. 2). We hypothesized that increases in phospholipid in the
alveolar space could be due to either increases in surfactant synthesis
and secretion by type II cells or decreases in surfactant clearance by
type II cells and the cells resident in the alveolar space in the
absence of SP-A. We examined the synthesis/secretion of surfactant
phospholipids by measuring the incorporation of radiolabeled precursors
into Sat PC isolated from both the lavage and tissue of LPS-treated
SP-A (+/+) and SP-A (
/
) animals. Previous studies have shown a
slight increase in [3H]choline incorporation in the
lavage Sat PC of healthy SP-A (
/
) mice (17). We did
not observe any significant differences in precursor incorporation into
the Sat PC of lavage or tissue of any of the groups of animals using
either label (Figs. 3 and 4). One difference that we did observe was
the decrease in 3H- and 14C-labeled Sat PC in
the tissue of both SP-A (+/+) and SP-A (
/
) animals after
LPS-induced injury (Figs. 3B and 4B); the same
pattern of decreased tissue [3H]choline labeling was
observed for healthy SP-A (+/+) animals. This suggests that LPS-induced
injury might alter the distribution of Sat PC after acute lung injury,
suggesting that LPS injury may itself be slightly altering surfactant homeostasis.
To determine whether clearance of surfactant phospholipid from the
alveolar space is altered in the absence of SP-A after acute lung
injury, we instilled liposomes containing radiolabeled DPPC into the
lungs of healthy and injured SP-A wild-type and knockout animals and
determined the amount of lipid that was cleared from the extracellular
pool of phospholipids. This lipid was cleared either by the cells that
can be isolated by lavage or by the cells associated with the lung
tissue. Our data show that injured SP-A (/
) animals clear ~40%
less radiolabeled lipid by the cells associated with the tissue than
healthy animals or injured wild-type animals, suggesting that SP-A may
play a role in regulating clearance. The cells isolated by lavage from
LPS-treated SP-A (
/
) animals also tended towards decreased
clearance compared with those from SP-A (+/+) animals, although the
difference was not statistically significant (P < 0.07). Interestingly, the cells isolated by lavage from LPS-treated
SP-A (
/
) mice rescued with exogenous SP-A displayed greater lipid
clearance than any other group (Fig. 5C). This could be due
to the dose and type of SP-A used for rescue. Future studies using
mouse SP-A rather than human proteinosis SP-A at varying doses could
determine the range of SP-A concentration required for rescue.
Estimates of the total amount of phospholipid cleared from the alveolar
space over 2 h also suggest that SP-A stimulates surfactant
clearance after lung injury (Table 1).
Although it is possible that rescue with SP-A occurs through interactions between SP-A and LPS, this is unlikely because it has been shown that SP-A does not bind very avidly to smooth strains of LPS (45). Borron et al. (4) also showed that, in solution, 1 µg of SP-A bound 5.8 ng of FITC-labeled 026:B6 LPS. In our rescue experiments, the 50 µg of SP-A would bind 300 ng of LPS, or ~12% of the LPS dose. It is unlikely that this low level of binding of LPS impacts these studies, although the possibility that it does cannot be completely discounted.
These studies do not take into account any degradative products that might be released into the alveolar space from type II cells, macrophages, or neutrophils. If the cells release these radiolabeled products, it might alter the apparent clearance of radiolabeled lipid from the alveolar space. Thin-layer chromatography analysis could be used to determine if degradative products have been incorporated into other surfactant lipids.
It is also important to note that the LPS injury itself alters the amount of radioactivity recovered per cell, possibly due to the increased number of cells found in the alveolar space (Fig. 5D). In all three conditions where the mice were exposed to LPS, the population of lavage cells is predominantly neutrophils (Fig. 1B). Our previous studies suggest that the neutrophils and macrophages found in the alveolar space after LPS treatment may have increased levels of phospholipid clearance (41) due to activation of alveolar macrophages for phospholipid clearance and degradation and the ability of neutrophils to internalize and degrade liposomes in an SP-A-dependent manner. This does not appear to be the case in vivo. However, it is possible that an increase in the number of cells capable of internalizing lipid would lead to a decrease in the amount of lipid internalized per cell, if the pool available for clearance did not increase. It may also be that only a subpopulation of cells is responsible for lipid clearance and that the large increase in the cells found in the alveolar space would lead to an apparent decrease in the amount of lipid internalized per cell. Alternatively, injury may alter the subtypes of surfactant, thereby altering clearance (47, 48). Also, since these studies measure only cell-associated radioactivity it is conceivable that the cells have actually taken up and degraded DPPC and that the degradation products have been released into the hypophase.
It is also possible that the properties of cells recruited to the lung
during injury in the absence of SP-A may be different from the
properties of similar cell types recruited in the presence of SP-A,
leading to alterations in clearance indirectly mediated by SP-A. A
similar situation exists in the case of granulocyte macrophage-colony
stimulating factor (GM-CSF). For example, alveolar macrophages isolated
from GM-CSF (/
) mice have a decreased ability to clear surfactant
phospholipids compared with macrophages isolated from wild-type mice
(54).
Although it is possible that type II cells may be primarily responsible for the phospholipid clearance that can be attributed to the tissue, other tissue-associated cell types exist that may also contribute to phospholipid clearance. Using a similar model, we previously found that neutrophils isolated from the tissue of LPS-treated rat lungs after lavage are stimulated for clearance of SP-D compared with lavage neutrophils (15). It is possible that a similar population of stimulated neutrophils (or macrophages) exists in the lung tissue and that SP-A mediates lipid uptake by these cells after LPS injury.
A variety of studies in mice have shown that alterations in surfactant
phospholipid levels and/or synthesis and secretion depend on the
stimuli being administered to (or withheld from) the animals. GM-CSF
(/
) mice showed increases in alveolar and total lung Sat PC at ages
as early as 7 days and as late as 56 days after birth
(42). These animals incorporated increased amounts of
radiolabeled precursors into both total lung and lavage Sat PC and also
showed decreased clearance of radiolabeled Sat PC from the alveolar
space. Overexpression of interleukin-4 in lung Clara cells
under the control of the SP-C promoter also leads to increased lavage
phospholipid levels, increased precursor incorporation, and increased
clearance of radiolabeled phospholipid from the alveolar space
(19). Data using purified SP-D in vitro has not supported
a role for SP-D in mediating lipid uptake (14) or surfactant secretion (26), although SP-D (
/
) mice have
alterations in type II cell morphology and increased phospholipid
levels in the alveolar space (5, 23). Overexpression of
SP-D in the Clara cells of SP-D (
/
) mice corrects these defects
(10). Although all of these other stimuli alter surfactant
homeostasis, it is important to note that overexpression of SP-A does
not lead to changes in tissue or lavage phospholipid pools sizes
(7). This suggests either that SP-A does not play a major
role in maintaining surfactant phospholipid levels under normal
conditions or that compensatory mechanisms involving these other
molecules including SP-D may also regulate surfactant phospholipid levels.
By examining only one time point after LPS injury, we may have overlooked earlier changes in surfactant homeostasis that lead to increased lavage phospholipid levels. Using this experimental design, we are unable to differentiate whether changes in precursor incorporation are due to alterations in de novo Sat PC synthesis or reacylation and recycling. To understand more completely the effects of LPS-induced injury on surfactant phospholipid pools, it will be important to analyze a time course of Sat PC synthesis in vivo at different time points after LPS exposure. By examining incorporation of precursor into Sat PC at varying times after LPS injury, one may be able to determine whether transient alterations in surfactant synthesis/secretion or recycling contribute to the observed alterations in lavage phospholipid levels in the absence of SP-A. It is also possible that differences in surfactant clearance at earlier time points contribute to later changes in lavage phospholipid levels; this question could be answered by performing a time course study as well.
In summary, these data support a role for SP-A in the regulation of extracellular surfactant phospholipid pools after LPS-induced acute lung injury in vivo. Lack of SP-A resulted in decreased clearance of surfactant from the alveolar space after LPS-induced injury, which may lead to an increase in phospholipid levels over time. Although this result may demonstrate a role for SP-A in surfactant homeostasis after acute lung injury, it may also relate to a more general characteristic of SP-A deficiency. In the cases of GBS, P. aeruginosa, and radiolabeled liposomes (after LPS treatment), SP-A deficiency resulted in reduced clearance. These data may point toward changes in the metabolism of surfactant by type II cells and tissue-associated phagocytic cells in the lung in the absence of SP-A.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jeffrey A. Whitsett for providing us with SP-A (+/+)
and SP-A (/
) mice. We also thank John Alcorn for his valuable assistance with animal surgery.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-30923, HL-28623, and HL-58795 and a supplement to HL-30923 from the Office of Research on Minority Health.
Address for reprint requests and other correspondence: J. R. Wright, Box 3709, Dept. of Cell Biology, Duke Univ. Medical Center, Durham, NC 27710 (E-mail: j.wright{at}cellbio.duke.edu).
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.
10.1152/ajplung.00418.2001
Received 25 October 2001; accepted in final form 31 January 2002.
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