Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039
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ABSTRACT |
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Mice that are surfactant protein (SP) A
deficient [SP-A(/
)] have no apparent
abnormalities in lung function. To understand the contributions of SP-A
to surfactant, the biophysical properties and functional
characteristics of surfactant from normal [SP-A(+/+)] and
SP-A(
/
) mice were evaluated. SP-A-deficient surfactant
had a lower buoyant density, a lower percentage of large-aggregate forms, an increased rate of conversion from large-aggregate to small-aggregate forms with surface area cycling, increased sensitivity to inhibition of minimum surface tension by plasma protein, and no
tubular myelin by electron microscopy. Nevertheless, large-aggregate surfactants from SP-A(
/
) and SP-A(+/+) mice had similar
adsorption rates and improved the lung volume of surfactant-deficient
preterm rabbits similarly. Pulmonary edema and death caused by
N-nitroso-N-methylurethane-induced lung injury were not different in SP-A(
/
) and SP-A(+/+)
mice. The clearance of
125I-labeled SP-A from lungs of
SP-A(
/
) mice was slightly slower than from SP-A(+/+)
mice. Although the absence of SP-A changed the structure and in vitro
properties of surfactant, the in vivo function of surfactant in
SP-A(
/
) mice was not changed under the conditions of
these experiments.
surfactant protein A; transgenic mice; tubular myelin; surface tension; adsorption rate; surfactant treatment
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INTRODUCTION |
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PULMONARY SURFACTANT is a highly conserved mixture of
phospholipids, neutral lipids, and proteins that is essential for
normal lung function in all mammals. The generally accepted sequence of
the biogenesis of these complexes begins with the cosecretion of
surfactant lipid and the lipophilic surfactant protein (SP) B and SP-C
into the alveolus in the form of lamellar bodies (31). De novo
synthesized SP-A is secreted via separate pathways by type II cells and
perhaps by Clara cells (13). In the hypophase of air spaces, lamellar
bodies unravel, and SP-A associates with the lipoprotein complexes to
form tubular myelin and loose membranous arrays (22, 27). Tubular
myelin forms of surfactant are highly surface active and contribute to
the pool in the hypophase from which the surface film is generated and
maintained. Loss of material from the surface film likely occurs by
formation of small vesicles containing lipids of the same phospholipid
composition as tubular myelin and lipid arrays but that is depleted of
SP-A, SP-B, and SP-C (1, 32). Several functions of SP-A thought to be
important for the maintenance of surfactant homeostasis were identified by in vitro studies. SP-A was essential for tubular myelin formation (22, 27), and SP-A acted cooperatively with SP-B and SP-C to increase
the rate of surface adsorption of the lipids (7, 24, 35). The
resistance of surfactant lipid mixtures to inactivation by proteins in
edema fluid was also increased by SP-A (4, 30). These effects of SP-A
were identified in experiments where SP-A was added to mixtures of
lipids and the other surfactant proteins. The recently described
SP-A-deficient [SP-A(/
)] mouse made possible the evaluation of the properties of a native surfactant that is SP-A
deficient, avoiding potential artifacts resulting from in vitro
recombination of purified lipids and proteins (16). The SP-A(
/
) mouse reproduces normally and has normal lung
anatomy and function. Normal tubular myelin was not seen within alveoli of SP-A(
/
) mice, and at physiologically relevant
concentrations, surfactant from alveolar washes of
SP-A(
/
) mice had minimal surface tension values
comparable to surfactant from normal [SP-A(+/+)] mice. In
the present study, we have further characterized the surfactant from
SP-A(
/
) mice by assessing the physical, biophysical, and
functional properties in comparison to surfactant from SP-A(+/+) mice.
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METHODS |
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Mice. SP-A knockout mice were
generated from embryonic stem cells in which the mouse SP-A gene was
disrupted by homologous recombination (16). SP-A(/
) mice
have been maintained for >3 yr and are carried as breeding colonies
in the vivarium at Children's Hospital (Cincinnati, OH) according to
protocols approved by the Institutional Animal Use and Care Committee.
The SP-A(
/
) mice were bred into the Black Swiss genetic
background for nine generations and have been maintained in filtered
cages under strict isolation conditions. SP-A(+/+) mice were from
National Institutes of Health Black Swiss stock. Genotyping was
performed by Southern blot of total DNA as previously described (16).
Surfactant isolation. For surfactant
studies, ~130 mice from each genotype [SP-A(/
)
and SP-A(+/+)] were injected intraperitoneally with pentobarbital
sodium to achieve deep anesthesia. The distal aorta was cut to
exsanguinate each animal. The chest of the animal was opened, a
20-gauge blunt needle was tied into the proximal trachea, five aliquots
of 0.15 M NaCl were infused into the lungs until they were inflated,
and then the aliquots were withdrawn by syringe three times for each
aliquot (12). Large-aggregate surfactant was isolated from the lavage
fluid by centrifugation (12). Either pooled alveolar washes or alveolar
washes from a single mouse were centrifuged at 40,000 g over a 0.8 M sucrose in 0.15 M NaCl
cushion for 15 min. The large-aggregate surfactant then was collected
from the interface, diluted with 0.15 M NaCl, and centrifuged again at
40,000 g for 15 min. The pellet was
suspended in normal saline and used as large-aggregate surfactant.
The supernatant from the first
40,000-g centrifugation that contained
small-aggregate surfactant was concentrated at 4°C by ultrafiltration with a 300,000 molecular-weight retention filter (Minitan, Millipore, Bedford, MA) (34). The small-aggregate surfactant
was diluted three times with 50 ml of 0.15 M NaCl and ultrafiltered
three times in a stirred cell with an XM300 membrane (Amicon, Beverly,
MA) to remove soluble proteins. Surfactant fractions were stored at
20°C until used. In separate measurements, alveolar washes
were recovered for measurement of the number and distribution of cell
types in the lavage fluid.
Surfactant function in preterm
rabbits. A surfactant-deficient premature rabbit model
was used to test the functional properties of the large- and
small-aggregate surfactant fractions (34). Preterm rabbits at 27 days ± 2 h gestational age were sequentially delivered, weighed, and
anesthetized with an intraperitoneal injection of 10 mg/kg of ketamine
and 0.1 mg/kg of acepromazine. The trachea of each rabbit was
cannulated, and 7.5 µmol saturated phosphatidylcholine (Sat PC)/kg
body weight of large-aggregate or small-aggregate surfactant from
SP-A(/
) or SP-A(+/+) mice was randomly given via the
tracheal tube. A control group was untreated. The rabbits were
ventilated in a series of 37°C temperature-controlled
plethysmographs for 15 min with 100% oxygen at a rate of 30 breaths/min, with an inspiratory time of 1 s and a positive
end-expiratory pressure of 3 cmH2O
(23, 34). Peak inspiratory pressures were individually regulated to
adjust the tidal volume to ~8 ml/kg. Dynamic compliance was
calculated by dividing tidal volume per kilogram body weight by (peak
inspiratory pressure
positive end-expiratory pressure). After
15 min of ventilation, the endotracheal tube was plugged for 5 min to
allow absorption atelectasis to occur. Pressure-volume curves at
37°C were measured by inflating the lungs in
5-cmH2O increments to 35 cmH2O, with the volume measured
after 30 s at each pressure. The lungs were then deflated with the same
5-cmH2O increments, with the
volumes recorded. The lung volume was corrected for the compression
volume of the system and is expressed as milliliters per kilogram body
weight.
Adsorption rate of surfactant.
Isolated large-aggregate surfactants from pooled alveolar washes from
SP-A(+/+) and SP-A(/
) mouse lungs
(n = 3 for each genotype group) were
mixed with saline such that the final concentration of Sat PC was 5 µmol/ml. The large-aggregate surfactant (1.2 µmol) was injected
into the subphase of 40 ml of 0.15 M NaCl that was being continuously
stirred at 37°C in a 5-cm-diameter and 4-cm-deep Teflon well. The
time from the addition of surfactant to the establishment of an
equilibrium surface tension was measured with a platinum dipping plate
connected to a force transducer (15).
Sensitivity of surfactant to plasma protein inhibition. Minimum surface tensions were measured with a Wilhelmy balance using a platinum dipping plate, with 3-min area cycling from 64 to 12.8 cm2 at a temperature of 37°C (11). Adult sheep plasma was added to a surfactant suspension containing 0.025 µmol/ml of Sat PC in 35 ml of 0.15 M NaCl to achieve each plasma protein concentration, and the solutions were mixed well by stirring with a glass rod followed by immediate surface tension measurements. The surface tension-area loops overlapped by the third or fourth compression cycle, and minimum surface tension of the fourth cycle is reported.
Density of large-aggregate surfactant.
The densities of large-aggregate surfactants from SP-A(/
)
and SP-A(+/+) mice were measured by layering 1 µmol Sat PC over
0.1-0.75 M linear sucrose gradients in Tris buffer (0.15 M NaCl,
0.01 M Tris · HCl, 1 mM CaCl2, 1 mM
MgSO4, and 0.1 mM EDTA at pH 7.4)
(8). The sucrose gradients were then centrifuged at 74,000 g for 48 h at 4°C with a Sorvall
AH629 swinging bucket rotor. Each gradient was fractionated into 33 fractions, sucrose concentration was measured with an ABBE-3L
refractometer (Bausch and Lomb Analytical Systems, Rochester, NY), and
the sucrose density was calculated. Phosphorus content of each fraction
was determined (2).
Conversion of large-aggregate surfactant. Large-aggregate surfactant (0.1 µmol Sat PC) was mixed with 2 ml of Tris buffer in a capped 12 × 75-mm polystyrene tube, and surface-area cycling was then performed as previously described (6, 8, 12). Briefly, the tubes were attached to the disk of a Rototorque rotator (Cole-Parmer Instruments, Chicago, IL) in an incubator at 37°C, and four tubes 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. Two other tubes were incubated at 37°C for 6 h without rotation. The large- and small-aggregate surfactants were isolated by centrifugation at 40,000 g for 15 min. The quantity of large-aggregate surfactant in the pellets and the residual small-aggregate surfactant in the supernatants were measured by phosphorus assay.
Electron microscopy. Fresh isolates of
large-aggregate surfactant from three separate pooled alveolar washes
from SP-A(/
) and SP-A(+/+) mice, each containing lavages
from seven mice for each pool, were pelleted and fixed overnight at
4°C with 2.5% glutaraldehyde-2% paraformaldehyde in 0.1 M sodium
cacodylate buffer at pH 7.3. The pellets were then washed in the buffer
and postfixed in 1% potassium-ferrocyanide-reduced osmium tetroxide in
0.1 M sodium cacodylate buffer for 1 h at 4°C. The aggregates were
washed in buffer, resuspended, pelleted in 1% low-temperature-gelling agarose (Sigma, St. Louis, MO), and allowed to solidify overnight in
the cold. The agarose blocks were then rinsed in 70% ethanol, stained
en bloc with 0.5% uranyl acetate in 70% ethanol for 10 min at room
temperature, dehydrated through 70 and 100% ethanol, and embedded in
Eponate 12 resin (Ted Pella, Redding, CA). Ultrathin sections from each
pool were stained with lead citrate and uranyl acetate and examined in
triplicate with a JEOL 100CX electron microscope.
Lung injury with
N-nitroso-N-methylurethane. Acute lung injury was
induced in SP-A(+/+) and SP-A(/
) mice with a subcutaneous injection of
N-nitroso-N-methylurethane
(NMU; Kings Laboratory, Greenville, SC) at a dose of 12 mg/kg. Previous
studies (17, 18) using this dose in rabbits, rats, and dogs
demonstrated a progressive, primarily epithelial lung injury over
several days. Measurement of protein and percentage of large-aggregate
surfactant in alveolar washes were made 1 and 2 days after NMU
injection. The protein permeability of the lung was evaluated with
125I-labeled albumin 2 days after
NMU treatment. Mice were given 10 µCi of
125I-albumin by intraperitoneal
injection, and the recovery of
125I from the alveolar washes was
measured 2 h later. The
125I-albumin was made from monomer
standard bovine serum albumin and carrier-free
125I (ICN, Irvine, CA) using
chloramine T. Labeled albumin was extensively dialyzed before use, and
tetrachloroacetic acid precipitation verified that >97% of the label
was associated with albumin (17).
Clearance of SP-A. SP-A was isolated
from the alveolar washes from granulocyte-macrophage colony-stimulating
factor-deficient transgenic mice that have elevated alveolar pools of
surfactant lipids and proteins (12). The SP-A was purified with
octylglucopyranoside according to Hawgood et al. (7) and iodinated with
Bolton-Hunter reagent (Amersham, Arlington Heights, IL) as before (12).
The iodinated SP-A was mixed with liposomes of
dipalmitoylphosphatidylcholine (DPC) to yield a final suspension
containing 0.01 µmol DPC/ml in 0.15 M NaCl. SP-A(+/+) or
SP-A(/
) mice (7-9 wk old) were randomized to groups
of four to six animals for measurement of the clearance of
125I-SP-A. Each mouse was given 50 µl of saline containing 0.1 µCi of
125I-SP-A by intratracheal
injection. For the tracheal injection, mice were sedated with
intraperitoneal ketamine (50 mg/kg). The trachea was exposed through a
0.5-cm midline skin incision in the neck, and the isotope mixture was
injected with a 30-gauge needle. Alveolar washes were performed as
described in Surfactant isolation, and the five washes for each
lung were pooled (10). The recovered
125I radioactivity was measured
from the alveolar wash and the lung tissue after alveolar wash.
Analytic techniques. Lipids were extracted with chloroform-methanol (2:1 vol/vol), and Sat PC was recovered from the lipid extracts after exposure to osmium tetroxide by neutral alumina column chromatography (20). Sat PC was quantified by phosphorus assay (2). Protein quantification in alveolar washes was by the method of Lowry et al. (19) with a bovine serum albumin standard.
Data analysis. All values are given as means ± SE. Differences between two groups were tested by 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.
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RESULTS |
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Surfactant function in preterm
rabbits. Preterm rabbits at 27 days gestational age
were treated with ~50 mg/kg of the large- and small-aggregate
fractions of surfactant from the alveolar washes of the SP-A(+/+) and
SP-A(/
) mice. Body weights of the rabbits were similar,
and the rabbits were ventilated with a mean tidal volume of 7.9 ml/kg
(Table 1). Ventilatory pressures were significantly lower for the rabbits treated with large-aggregate surfactant from both SP-A(
/
) and SP-A(+/+) mice compared
with the untreated control rabbits. Total thoracic compliance
increased, with large-aggregate surfactant recovered from SP-A(+/+)
mice (Fig.
1A).
Although large-aggregate surfactant from SP-A(+/+) mice tended to
result in higher compliances in the premature rabbit lungs, the
differences between surfactant from SP-A(
/
) and SP-A(+/+) mice were not significant. Maximal lung volumes measured at 30 cmH2O for surfactant from
SP-A(
/
) and SP-A(+/+) mice were not different
(P = 0.09; Fig.
1B). Lung volumes measured at 30 and 10 cmH2O pressure were increased
by large-aggregate surfactants from both SP-A(+/+) and
SP-A(
/
) mice relative to the untreated group (Fig. 1,
B and
C). The small-aggregate surfactant
fractions from either genotype did not improve lung function.
|
|
Adsorption rate. There were no
differences in adsorption rate for the surfactants from SP-A(+/+) and
SP-A(/
) mice. Even at high dilutions, the surfactants
rapidly adsorbed to the surface, and equilibrium surface tensions were
achieved in 48 ± 3 s for SP-A(+/+) mice and in 30 ± 8 s for SP-A(
/
) mice. There were no differences in the
shape of the adsorption curves for the surfactants from the two
genotypes.
Sensitivity to plasma protein
inhibition. Three large-aggregate surfactant pools
isolated from 10 mice for each pool from each genotype were used for
these measurements. The large-aggregate surfactants from both SP-A(+/+)
and SP-A(/
) mice had low minimum surface tensions when
measured at 0.025 µmol Sat PC/ml in the absence of plasma (Fig.
2). The surfactant from
SP-A(
/
) mice was more sensitive to inactivation by plasma
than was the surfactant from SP-A(+/+) mice at protein
concentrations > 0.6 mg/ml (P < 0.01).
|
Surfactant density and form
conversion. Two samples of pooled large-aggregate
surfactants from eight mice of both genotypes had similar densities on
the linear sucrose density gradients (Fig.
3A). The
peak sucrose density of large-aggregate surfactant from
SP-A(/
) mice was lower (1.042 and 1.044) than that of the surfactant from SP-A(+/+) mice (1.059 and 1.060). The percentages of
small-aggregate surfactant derived from large-aggregate surfactant after 6 h of surface area cycling or no cycling are shown in Fig. 3B. The conversion was low for both
genotypes without surface area cycling. After 6 h of cycling, the
conversion from large aggregates to small aggregates was 72.9 ± 5.2% for surfactant from SP-A(
/
) mice and 37.0 ± 2.6% for surfactant from SP-A(+/+) mice
(P < 0.001). The percentages of the
aggregate forms isolated from the alveolar washes of the mice also were
different. In lavages from SP-A(+/+) mice, 48 ± 1% of Sat PC was
in the large-aggregate fraction, and only 15 ± 2% of lavage Sat PC
from SP-A(
/
) mice was in the large-aggregate fraction
(P < 0.01).
|
Electron microscopy of surfactant.
Tubular myelin was easily identified in sections from pellets of
large-aggregate surfactant from SP-A(+/+) mice (Fig.
4A). In
contrast, no tubular myelin was seen in multiple sections of the
large-aggregate surfactant from SP-A(/
) mice (Fig.
4B). Although mixtures of densely
packed and loose lipid arrays were observed in both genotypes, the
lipid arrays were larger in samples from SP-A(
/
) mice
(Fig. 4, C and D).
|
Lung injury by NMU. NMU caused a
severe injury in the mice, resulting in the death of four of eight
SP-A(+/+) mice and two of eight SP-A(/
) mice on
day 3. Total protein in the alveolar washes increased 1 day after NMU treatment and increased further 2 days
after NMU treatment, and the increases were similar in both genotypes
(Fig.
5A). The
recovery of 125I-albumin also was
increased on day 2 after NMU
treatment, and there were no differences between the two genotypes
(Fig. 5B). NMU did not alter the
percentage of large-aggregate forms in the SP-A(+/+) and
SP-A(
/
) mice, although the percentages were different for
the two genotypes (Fig. 5C)
|
Clearance of SP-A. The curves for the
clearance of 125I-SP-A from the
air spaces of SP-A(+/+) and SP-A(/
) mice were
similar (Fig. 6). The loss of
125I-SP-A from the lung was best
fit by semilog curves, with half-life values of 10.2 h for the
SP-A(+/+) mice (r = 0.95)
and 14.5 h for the SP-A(
/
) mice
(r = 0.94). Although these values were not very different, the recoveries of SP-A at 24 and 40 h were significantly different (P < 0.05).
|
Cell content of alveolar washes. The
total cells recovered by alveolar wash from the lungs of three 1-mo-old
and three 2-mo-old SP-A(+/+) mice were 9.7 ± 0.3 × 105 and 7.9 ± 1.1 × 105 cells, respectively. Parallel
measurements in SP-A(/
) mice yielded 7.4 ± 0.2 × 105 and 6.8 ± 0.9 × 105 cells in alveolar
washes at 1 and 2 mo of age, respectively. More than 96% of the cells
in all samples were macrophages, with the residual cells being a small
number of lymphocytes and a few granulocytes.
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DISCUSSION |
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Although pulmonary function in SP-A(/
) mice is unaltered
under normal conditions, distinct structural and functional properties of SP-A-deficient surfactant were noted in vitro. SP-A-deficient surfactant lacks tubular myelin, is more easily inhibited by plasma protein, and forms less dense lipid aggregates. Despite these structural and functional differences, SP-A-deficient surfactant is as
effective as normal surfactant at restoring lung volume in
surfactant-deficient premature rabbits, and the lack of SP-A did not
increase the lung injury caused by NMU. Therefore, the effects of SP-A
on surfactant structure, form, and some biophysical properties do not
result in abnormalities in surfactant function in vivo that are
sufficient to interfere with lung function.
The transgenic mice with an absolute SP-A deficiency have surfactant
that differs only in this major surfactant glycoprotein. There are no
differences in SP-B mRNA, SP-C mRNA, and surfactant phospholipid
composition in SP-A(/
) mice compared with SP-A(+/+) mice
(16). In a clean environment, SP-A(
/
) mice grow,
reproduce normally, and maintain surfactant function and lung function
(16). Therefore, SP-A is not essential for survival. This result is in
contrast to SP-B deficiency, which results in lethal respiratory failure shortly after birth in mice and humans (3). SP-A also does not
regulate the metabolism of Sat PC or SP-B in SP-A(
/
) mice
(11). Surfactant pool sizes were similar in SP-A(
/
) mice and SP-A(+/+) mice, as were precursor incorporation rates into Sat PC,
secretion of Sat PC, and clearances of Sat PC and SP-B. In the present
study, there were small but significant decreases in alveolar and total
lung clearances of SP-A from SP-A(
/
) mice compared with SP-A(+/+) mice. SP-A is known to be cleared and catabolized by both macrophages and type II cells (28, 33). The
explanation for this small difference in SP-A clearance may be a change
in the number of SP-A receptors on type II cells and macrophages. The
effects of SP-A deficiency on overall surfactant metabolism is minimal
in the SP-A(
/
) mouse (10).
The biophysical function of the SP-A-deficient surfactant was very
similar to that of normal surfactant. Minimal surface tensions were
previously reported to be similar for SP-A-deficient and normal
surfactants except at high dilution in the absence of
Ca2+ (16). Adsorption rates also
were similar in SP-A-deficient and normal surfactants. This result
contrasts with previous findings (7, 24, 35) that SP-A enhanced the
adsorption rates of lipid extract surfactants containing SP-B and SP-C.
This discrepancy may result from the organic solvent extraction steps
used for reconstruction experiments that remove SP-A and disrupt the
associations of SP-B and SP-C with phospholipids. Surfactant that
contains no tubular myelin can have adsorption rates and minimal
surface tension values similar to values for normal surfactant when
measured at high dilutions. However, in vivo alveolar surfactant is at a much higher concentration. The Sat PC pool size is ~10 µmol/kg in
a 30-g mouse (12), and if it is assumed that alveolar fluid volume in
the mouse is similar to estimates for other mammals (26), the alveolar
surfactant concentration should be ~25 mg/ml. This concentration is
three orders of magnitude higher than the concentrations generally used
for biophysical studies of surfactant function (4, 35). Therefore,
under normal conditions, the biophysical properties of the
large-aggregate surfactant in the SP-A(/
) mouse should
result in normal lung function. Tubular myelin is not needed for normal
lung function, a result consistent with the good physiological
responses achieved after the treatment of infants with respiratory
distress syndrome with surfactants that do not contain SP-A or tubular
myelin (14). However, the interpretation of the normal function of
surfactant from SP-A(
/
) mice when used to treat preterm
rabbits must be tempered in that the function was tested at a dose
known to give maximal treatment responses (25), and differences between
normal and SP-A-deficient surfactant might be apparent at lower
treatment doses. The preterm rabbit lung could contribute some
endogenous SP-A to the SP-A-deficient surfactant, although SP-A mRNA is
low in preterm rabbit lungs at 27 days gestational age (5, 21).
Although the alveolar pool size of Sat PC was similar for SP-A(+/+) and
SP-A(/
) mice (10), the amount of large-aggregate surfactant was decreased in alveolar washes from SP-A(
/
)
mice to 15% of the pool, in contrast to the value of 48%
large-aggregate forms for the SP-A(+/+) mice. Korfhagen et al.
(16) previously reported that there were no differences in
the amount of large-aggregate forms from SP-A(+/+) and
SP-A(
/
) mice. For the measurements reported
here, a sucrose step gradient was used to separate large-aggregate forms from alveolar cells and other debris (12), although no differences in cell number or cell type were found. Our measurement of
a decrease in large-aggregate forms in SP-A(
/
) mice was
repeated multiple times for this report, and the result is consistent
with the increased rate of conversion of large-aggregate surfactant to
small-aggregate surfactant with surface area cycling and with previous
reports (8, 29) that SP-A decreases the rate of form conversion.
SP-A-deficient surfactant was more sensitive to inhibition by plasma, a
result consistent with the increased resistance of surfactant mixtures
to protein inactivation when SP-A is added (4, 30). The increased
inactivation of SP-A-deficient surfactant by plasma protein suggests
that the surfactant of the SP-A(/
) mouse might be more
sensitive to inactivation with injury. Yukitake et al. (36)
demonstrated that SP-A could modify the inhibitory effects of plasma
proteins in surfactant-treated preterm rabbits. The decreased
percentage of large-aggregate surfactant in the alveolar pool and the
increased rate of conversion to small-aggregate surfactant for
surfactant from SP-A(
/
) mice might also be expected to
compromise surfactant function with lung injury (8, 17). In lung
injuries such as acute respiratory distress syndrome, the amount of
large-aggregate surfactant is decreased, and respiratory dysfunction
correlates with decreased large-aggregate pools in animal models of
lung injury. In previous studies (17, 18), NMU caused a slowly
progressive but severe pulmonary edema in rabbits, rats, and dogs.
Therefore, we used NMU-induced lung injury to test the surfactant
system in the SP-A(
/
) mouse. Although NMU caused
pulmonary edema in both SP-A(+/+) and SP-A(
/
) mice, the
death rates after NMU treatment were similar. Inhibition of surfactant
by edema depends on the absolute concentration of surfactant more than
on the amount of protein inhibitors (9). At high surfactant
concentrations, plasma proteins do not inhibit surfactant very
effectively. The SP-A(
/
) mouse may not be more sensitive to NMU-induced lung injury than the SP-A(+/+) mouse because of the high
concentrations of surfactant that are present in vivo or
because of adaptations to the injury. The unique characteristic of the
injury caused by NMU is the progression of the injury to death over
3-4 days. Perhaps a more acute injury with endotoxin or tumor
necrosis factor-
would identify the effects of SP-A deficiency.
These results demonstrate that the surfactant from the
SP-A(/
) mouse is different in structure, density, and
several other properties from normal surfactant. However, these
differences do not result in abnormal lung function in
SP-A(
/
) mice or in the treatment responses of
surfactant-deficient preterm rabbits. It must be stressed that although
surfactant function was evaluated with NMU-induced injury, other
injuries to the surfactant system may reveal abnormalities of
surfactant function. Compensatory mechanisms such as the adaptation of
SP-D for some SP-A functions in SP-A(
/
) mice also need to
be evaluated.
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ACKNOWLEDGEMENTS |
---|
This work was supported by National Institute of Child Health and Human Development Grants HD-11932 and HD-20748.
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FOOTNOTES |
---|
Address for reprint requests: M. Ikegami, Children's Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039.
Received 14 October 1997; accepted in final form 7 April 1998.
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