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1 Children's Hospital Medical Center, Division of Pulmonary Biology, Cincinnati, Ohio 45229-3039; and 2 TVW Telethon Institute for Child Health Research, Division of Clinical Science, University of Western Australia, Perth, Australia 6001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Although
the surface properties of surfactant protein (SP)-B and SP-C are
similar, the contributions that either protein may make to lung
function have not been identified in vivo. Mutations in SP-B cause
lethal respiratory failure at birth; however, SP-B null mice are
deficient in both SP-B and SP-C. To identify potential contributions of
SP-C to lung function in vivo, the following transgenic mice were
generated and exposed to 95% O2 for 3 days: (SP-B+/+,SP-C+/+),
(SP-B+/+, SP-C
/),
(SP-B+/,SP-C+/+),
(SP-B+/,SP-C+/),
and (SP-B+/,SP-C/).
Hyperoxia altered pressure-volume curves in mice that were heterozygous
for SP-B, and these values were further decreased in
(SP-B+/,SP-C/) mice. Likewise, alveolar
interleukin (IL)-6 and IL-1
were maximally increased by
O2 exposure of (SP-B+/,SP-C/)
mice compared with the other genotypes. Lung hysteresivity was lower in
the (SP-B+/,SP-C/) mice. Surfactant
isolated from (SP-B+/+,SP-C/) and
(SP-B+/,SP-C/) mice failed to stabilize
the surface tension of microbubbles, showing that SP-C plays a role in
stabilization or recruitment of phospholipid films at low bubble
radius. Genetically decreased levels of SP-B combined with superimposed
O2-induced injury reveals the distinct contribution of SP-C
to pulmonary function in vivo.
lung injury; transgenic mice; saturated phosphatidylcholine; surfactant protein-B and -C
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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PULMONARY SURFACTANT
is a complex mixture of lipids and proteins that reduces surface
tension at the air-liquid interface in the alveolus. Surfactant
proteins play important roles in surfactant homeostasis
(14), host defense (17), and surface activity (33) in the lung. Surfactant protein (SP)-B and SP-C are
small, hydrophobic proteins that contribute to the surface activity of surfactant phospholipids, enhancing the adsorption, spreading, and
stability at the air-liquid interface to reduce alveolar surface tension. Recent studies with the captive bubble surfactometer demonstrated that both SP-B and SP-C enhanced lipid adsorption and
lowered minimum surface tension. SP-C was more effective in promoting
lipid adsorption whereas SP-B was more effective in lowering surface
tension (8, 28). These results and the unstable microbubbles described for surfactant isolated from SP-C-deficient mice
(8) are consistent with the hypothesis that SP-C helps maintain a surfactant reservoir beneath the surface film and
facilitates transfer of surfactant lipid to the monolayer. Addition of
SP-B and/or SP-C to phospholipid mixtures enhances their surface
properties in vitro (1, 7, 24, 26, 28, 34) and improves
lung function in surfactant-deficient animals in vivo (6, 20, 21,
25). Although both SP-B and SP-C are active in vitro, the
potential unique contributions of either protein to surfactant function
have not been clarified in vivo. Knockout mice have not provided clear
insight into the distinct actions of SP-B (4, 5) and SP-C
(8). SP-B deficiency in mice and humans caused lethal
respiratory failure that was accompanied by failure to fully process
pro-SP-C to its functional mature peptide form (5, 22,
31); in contrast, mature SP-B was produced normally in SP-C/ mice, and pulmonary function was only subtly
altered (8).
Heterozygous SP-B mice (SP-B+/) survive with no
apparent abnormalities in surfactant function (4).
However, strikingly decreased survival and altered pressure-volume
curves were observed in SP-B+/ mice after 3 days of
O2 exposure. These abnormalities were reversed by
pretreatment with a surfactant containing SP-B (30).
To discern potential contributions of SP-C to lung function in vivo,
heterozygous (SP-B+/,SP-C+/) mice were
interbred to generate offspring with reduced levels of both SP-B and
SP-C. These mice were exposed to 95% O2 for 3 days. Lung
inflammation, mechanics, and the surface activity of surfactant were
assessed to identify changes in lung function linked to altered SP-C levels.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Mice.
Heterozygous SP-B+/ mice (FVB) (4) were bred
to heterozygous SP-C-deficient mice SP-C+/ (Swiss Black)
(8) to produce heterozygous
(SP-B+/,SP-C+/) mice.
SP-B+/, SP-C+/ mice were mated to
heterozygous SP-C-deficient littermates
(SP-B+/+,SP-C+/) to produce progeny with the
following genotypes: wild-type mice (SP-B+/+,SP-C+/+); SP-C null mice
(SP-B+/+,SP-C/), heterozygous
SP-B-deficient mice (SP-B+/,SP-C+/+),
heterozygous SP-B and SP-C-deficient mice
(SP-B+/,SP-C+/), and heterozygous
SP-B-deficient and null SP-C mice
(SP-B+/,SP-C/). Mice were identified by
specific PCR of tail DNA with primers that amplified endogenous SP-B
(upstream primer 5'-ATCCTCCCTTCTCTGCTCTCC-3' and downstream primer
5'-TGTGCTTATTGCGTCTGTGG-3') and endogenous SP-C (upstream primer
5'-CAGGATTACTCGGCAGGTCC-3' and downstream primer
5'-GTCTGCTGTGCCTCTTTGCC-3'). The neomycin resistance gene inserted into
the SP-B and SP-C loci was amplified by PCR of tail DNA using SP-B or
SP-C upstream primers described above and a downstream primer
corresponding to the phosphoglycerate kinase promoter of the
neomycin cassette (5'-CACTTGTGTAGCGCCAAGTGCC-3'). Genotype was further
confirmed by Western blot analysis for SP-B and SP-C in alveolar lavage
samples (14). Mice were maintained in hepa-filtered cages
in a barrier facility. There was no evidence of pathogens in sentinel
mice co-maintained in the vivarium. All of the studies were performed
with 7- to 8-wk-old mice. All procedures were conducted under
Institutional Animal Care and Use Committee approved methods.
/),
(SP-B+/+, SP-C+/), and
(SP-B+/,SP-C/) groups. For SP-B
analysis, aliquots containing 0.27 nmol of saturated
phosphatidylcholine (Sat PC) were electrophoresed under nonreducing
conditions. For SP-C, samples containing 2.7 nmol of Sat PC were
electrophoresed in the presence of -mercaptoethanol. SP-B and SP-C
samples were separated on 10-20% SDS-polyacrylamide gels with
tricine buffer (Novex, San Diego, CA). After electrophoresis, proteins
were transferred to polyvinylidene difluoride paper (Bio-Rad, Hercules,
CA). Immunoblot analysis was carried out with rabbit anti-bovine SP-B,
1:10,000, and rabbit antirecombinant human SP-C 1:25,000. Appropriate
peroxidase-conjugated secondary antibodies were used at 1:10,000
dilutions. Immunoreactive bands were detected with enhanced
chemiluminescence reagents (Amersham, Chicago, IL). Protein bands were
quantitated by densitometric analyses with Alpha Imager 2000 documentation and analysis software (Alpha Innotech, San Leandro, CA).
Hyperoxia. Eight transgenic mice from each group were placed in a Plexiglas chamber in which the O2 concentration was maintained at 95% at 1 atmosphere by mixing 100% O2 and air for 72 h. These exposure conditions were similar to those previously reported for SP-A- and SP-B-deficient mice (13, 30). The stability of O2 percentage in the chamber was monitored two times a day and did not require any readjustment of the O2 or airflow.
Pressure-volume curve. Pressure-volume curves were measured after 3 days of exposure to hyperoxia (13). Mice were sedated with pentobarbital sodium (100 mg/kg ip) and placed in a box containing 100% O2 to ensure complete collapse of the alveoli by O2 absorption after spontaneous breathing had stopped. The mice were killed by exsanguination, and the trachea was cannulated and connected by a syringe to a pressure sensor (mouse pulmonary testing system, TSS, Cincinnati, OH) via a three-way connector. After the diaphragm was opened, lungs were inflated in 75-ml increments every 10 s to 36.0 ± 0.4 cmH2O pressure and were similarly deflated.
Sat PC, protein, and cytokine analysis.
One milliliter of 0.9% NaCl at 4°C was flushed into the airway until
the lungs were fully expanded. The fluid was withdrawn and infused by
syringe three times for each lavage. Lavage was repeated five times,
and the samples were pooled. After lavage, lung tissue was homogenized
in 0.9% NaCl and adjusted to the same volume for all the samples.
Aliquots of BALF and lung homogenate were extracted with
chloroform-methanol (2:1), and Sat PC was isolated by the technique of
Mason et al. (18). The amount of Sat PC was measured by
phosphorus assay (11). To measure the increase in protein
leak into the alveolar space caused by hyperoxic lung injury, the
protein in an aliquot of BALF was assessed by the method of Lowry et
al. (16). Protein in total BALF volume was normalized to
body weight of mice in air or before hyperoxia because body weight is
proportionate to lung weight and surface area (23). Total
protein in lung homogenate after lavage was measured (16)
and used to normalize lung air volumes in pressure-volume measurements.
IL-6, IL-1, macrophage inflammatory protein-2 (MIP-2), and
tumor necrosis factor-
(TNF-) in BALF were measured by using Quantikine (R&D Systems, Minneapolis, MN).
Lung mechanics. For lung mechanics, mice were anesthetized with 0.1 ml/10 g of a mixture containing xylazine (2.0 mg/ml) and ketamine (40 mg/ml). Two-thirds of the dose was given to induce anesthesia, with the remaining given when the animals were attached to the ventilator. A tracheotomy was performed, and a polyethylene cannula (1.0 cm, 0.023 cm ID) was inserted. Mice were ventilated with a tidal volume of 8 ml/kg at a rate of 450 breaths per minute, with an end-expiratory pressure of 2 hPa, by using a custom-designed ventilator (flexiVent, Scireq, Montreal, PQ, Canada). This ventilator allowed measurement of lung function using a modification of the low-frequency forced-oscillation technique (8, 9). Respiratory input impedance was measured between 0.25 and 20 Hz by applying a composite signal containing 19 mutually prime sinusoidal waves during pauses in regular ventilation. Measurements were made at mean pressure of 2 and 5 hPa, and small-amplitude oscillatory signal produced pressure oscillations around the chosen pressure. For each measurement, the ventilator was paused to the mean pressure for 1 s to ensure pressure equilibration. The expiratory valve was then closed, and the oscillatory forcing function was applied by the piston. Because the mouse was exposed to a closed system during the measurements, changes in lung volume were avoided. The constant-phase model described by Hantos et al. (9) was used to partition respiratory input impedance into components representing the mechanical properties of the airway resistance, tissue resistance, and tissue elastance. Hysteresivity describes the mechanical coupling between tissue resistance and elastance and is calculated as hysteresivity = tissue resistance/tissue elastance. The calibration procedure removes the impedance of the equipment and tracheal tube. The results reported represent the mechanical properties of the mouse lung alone.
Surface activity. Large-aggregate surfactant was isolated from BALF by centrifugation at 40,000 g for 15 min over a 0.8 M sucrose in 0.9% NaCl cushion. The large-aggregate surfactant was recovered at the sucrose interface. The surface activity of three pools (3 mice/pool) of large-aggregate surfactant was measured from mice of each genotype with a captive bubble surfactometer (27) at 37°C. The concentration of each sample was adjusted to 3 nmol Sat PC/µl, and 3 µl of the surfactant were applied to the air interface of 25.6 ± 0.5 µl bubble by microsyringe. Surface tension was measured every 10 s for 300 s, equilibrium surface tension was measured, and then bubble pulsation was started. The minimum surface tension after 65% bubble volume reduction was measured at the fifth pulsation. To study the stability of smaller bubbles, 3 µl of 0.3 nmol Sat PC/µl isolated large-aggregate surfactant was applied to 1.3 ± 0.1 µl volume bubble, and surface tension were recorded for 12 min (8).
Statistics. Values are means ± SE. Differences between the two groups were determined by a two-tailed Student's t-test. Between-group comparisons were made by ANOVA followed by the Student-Newman-Keuls multiple-comparison procedure.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Alveolar SP-B and SP-C pool sizes.
The amount of the SP-B and SP-C in alveolar lavage samples relative to
Sat PC was estimated by Western blot and normalized to the quantity of
SP-B and SP-C in (SP-B+/+,SP-C+/+) mice, which
was given the value of 1 (Fig. 1).
Alveolar SP-B content in (SP-B+/,SP-C+/+),
(SP-B+/, SP-C+/), and
(SP-B+/,SP-C/) mice was decreased to
~40% compared with wild-type mice (P < 0.01 vs. wild type). In heterozygous SP-C-deficient mice,
(SP-B+/, SP-C+/), the SP-C pool size was
~60% of that for wild-type mice (P < 0.001).
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Hyperoxia. There were no statistical differences in body weight among the different genotypes. The mean body weight for all mice was 25.5 ± 0.4 g. There were no deaths on day 3 of hyperoxia in any group. After 3 days of exposure to 95% O2, body weight was reduced by 20% in all groups (mean body weight = 20.6 ± 0.4 g).
Pressure-volume curve.
The protein content of lung tissue after alveolar lavage was similar in
all the genotype groups, both in air and after 95% O2,
with mean value of 9.5 ± 0.4 mg/mouse. Therefore, lung volumes (ml) at each pressure were normalized to total lung tissue protein (Fig. 2). Lung volumes at maximum
pressure (Vmax) for mice breathing air were similar in all five
genotype groups when compared by ANOVA. The Vmax for three groups in
air with heterozygous SP-B alleles
[(SP-B+/,SP-C+/+),
(SP-B+/,SP-C+/), and
(SP-B+/,SP-C/)] was larger than that for
wild-type mice when analyzed by t-test (P < 0.05). As previously demonstrated in heterozygous SP-B mice (5,
30), air volumes at 5 and 0 cmH2O on the deflation
limb for (SP-B+/,SP-C+/+) mice were higher
than for the groups with wild-type SP-B alleles, (SP-B+/+,SP-C/) and
(SP-B+/+,SP-C+/+) mice (P < 0.05 by ANOVA). Similarly, (SP-B+/,SP-C+/)
and (SP-B+/,SP-C/) mice showed higher air
volume than wild-type mice at 5 cmH2O on the deflation limb
(P < 0.05 by t-test). After exposure to 95% O2 for 3 days, the pressure-volume curves were not
altered in (SP-B+/+,SP-C+/+) and
(SP-B+/+,SP-C/) mice. Hyperoxia altered
lung pressure-volume curves in all groups, with heterozygous
SP-B+/ alleles resulting in lowered lung volumes and
reduced hysteresis after 3 days in 95% O2. The percent
reductions in lung volumes at Vmax were 40% in
(SP-B+/,SP-C/), 37% in
(SP-B+/,SP-C+/), and 19% in
(SP-B+/,SP-C+/+) mice. In these three groups
of mice in which SP-B was reduced, the decrease in Vmax and hysteresis
caused by hyperoxia tended to be more severe when SP-C content was
decreased [P < 0.05 (SP-B+/,SP-C/) vs.
(SP-B+/,SP-C+/+) mice].
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Protein in alveolar lavage.
Total protein in BALF was measured as an indicator of proteinaceous
alveolar edema (Fig. 3) after hyperoxia.
For 95% O2 groups, body weight before exposure to
hyperoxia was used to normalize the total amount of protein in BALF.
Total protein in alveolar lavage samples was low for all the groups in
air and was increased almost twofold by hyperoxia (P < 0.01 vs. air). After 3 days in 95% O2, the highest
alveolar protein levels (P < 0.05) were observed in
(SP-B+/,SP-C/) mice.
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, SP-C+/) and
(SP-B+/,SP-C/) mice had subtle but
significantly higher amounts of alveolar protein than the two groups
with wild-type SP-B alleles,
(SP-B+/+, SP-C+/+) and
(SP-B+/+,SP-C/) mice (P < 0.05).
Alveolar cytokines.
IL-6, IL-1, MIP-2, and TNF- were measured in BALF from
O2-exposed mice (Fig. 4).
TNF- was not detectable in any sample (data not shown). IL-6 was
significantly increased after hyperoxia in
(SP-B+/+,SP-C+/+),
(SP-B+/+,SP-C/), and
(SP-B+/,SP-C/) mice and was significantly
higher in (SP-B+/,SP-C/) mice. IL-1
was significantly increased in BALF from
(SP-B+/,SP-C/) mice only. Mean MIP-2
concentrations were highest in
(SP-B+/, SP-C/) mice, although the
difference among the groups was not statistically significant. During
O2 exposure, BALF cytokines were increased most markedly in
(SP-B+/,SP-C/) mice, revealing a possible
protective role for SP-C during hyperoxia.
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Sat PC content.
Sat PC in alveolar lavage and total lung (alveolar lavage plus lung
tissue after lavage) are shown in Fig. 5.
In air, alveolar and lung Sat PC pool sizes were similar in all the
genotypes. Sat PC content was decreased in alveolar lavage samples
after 3 days of exposure to 95% O2 (P < 0.05) except in (SP-B+/+, SP-C/) mice. A
modest decrease (P < 0.05) in total lung Sat PC was observed in (SP-B+/+,SP-C+/+) and
(SP-B+/+,SP-C/) mice after hyperoxia.
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Baseline lung mechanics.
Mice were sent from Cincinnati to Perth, Western Australia for
measurement of lung mechanics. Numbers of
(SP-B+/+,SP-C/) mice were inadequate for
study at the time of shipment; thus, it was not possible to measure
lung mechanics in this group. However, lung mechanics in this genotype
(SP-C-deficient mice) was studied previously (8),
demonstrating lower hysteresivity at low mean pressure compared with
wild-type mice. Measurements made at increasing mean pressures at 2 and
5 hPa showed no significant changes in airway resistance, tissue
resistance, and tissue elastance between the groups studied. Airway
resistance was 0.37 ± 0.04 for
(SP-B+/+, SP-C+/+), 0.32 ± 0.03 for
(SP-B+/,SP-C/), and 0.34 ± 0.03 for
(SP-B+/,SP-C/) mice. Baseline
hysteresivity at 2 hPa was similar in all the groups studied (Fig.
6). As expected, hysteresivity was
increased as pressure was increased from 2 to 5 hPa. Interestingly,
hysteresivity was significantly decreased in
(SP-B+/, SP-C/) mice at mean pressure
of 5 hPa compared with (SP-B+/+,SP-C+/+) mice
(P < 0.05), indicating reduced viscoelasticity of the
lung at lower lung volumes.
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Surface activity.
Using a captive bubble surfactometer with a bubble size of 26 µl, we
produced higher equilibrium surface tension from surfactant from
(SP-B+/,SP-C/) mice compared with
other groups (Table 1). There were no
statistical differences in minimum surface tension among the groups,
but marked variations were seen in three groups with reduced SP-B. The
observed difference in altered hysteresivity of lung mechanics was
considered to reflect altered surface activity in
(SP-B+/,SP-C/) mice at low mean pressure
or small alveolar volume. Therefore, surfactant was applied to a bubble
of minimum size (1.3 µl), and the stability of the microbubble was
recorded over time. Surfactant from SP-C+/+ and
SP-C+/ mice stabilized the microbubble (Fig.
7). Microbubbles made with surfactant
from SP-C-deficient mice, whether
(SP-B+/+,SP-C/) or
(SP-B+/,SP-C/), were unstable, with rapid
changes in bubble shape and surface tension occurring during the 14-min
study period. More experiment-to-experiment variations were seen in the
SP-C/ surfactant samples.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In SP-B+/ mice, pressure-volume curves and
inflammatory cytokine, alveolar protein, and Sat PC content responses
to lung injury caused by hyperoxia were markedly increased in the
absence of SP-C. Mechanical coupling between tissue resistance and
elastance (hysteresivity) was decreased at low lung volumes, and
surfactant function (the ability to stabilize micro bubbles) was
altered in SP-C/ compared with SP-C-replete mice. Thus
decreased levels of SP-B revealed a physiological role of SP-C in
maintenance of lung function during hyperoxia and the role of SP-C in
stabilizing the alveolus at low lung volume.
SP-C and SP-B are hydrophobic peptides that enhance the surface activity of surfactant phospholipids. SP-C and/or SP-B increased the surface activity of phospholipid mixtures in vitro (1, 24, 29, 34) and restored lung function in surfactant-deficient immature lungs (10, 25) and adult lungs with inactivated surfactant function (15). Commercially available surfactant used for surfactant treatment (Survanta), containing phospholipids and bovine SP-C with lesser amounts of SP-B, are highly useful as surfactant replacement for treatment of respiratory distress syndrome. Likewise, synthetic surfactants made with recombinant human SP-C and phospholipid mixtures (Venticute) are highly functional in surfactant-deficient lungs in vivo (6, 20, 21). These surfactants dramatically improve lung function of patients with respiratory distress syndrome or acute respiratory distress syndrome as well as animal models with surfactant deficiency or altered surfactant function. The improvement in lung function with SP-C rich surfactant is similar to the activity of surfactants containing physiological levels of SP-C and SP-B (e.g., CLSE, Infasurf).
Although in vitro studies and the successful use of SP-C-containing
surfactants for replacement therapy supported an important role for
SP-C in surfactant function, the distinct contribution of each protein
has not been understood. Initial analyses of lung function of SP-B
(5) and SP-C (8) knockout mice did not provide clear insight into the role of SP-C for lung function in vivo.
SP-C knockout mice grow normally without apparent pulmonary abnormalities. Concentrations of other surfactant proteins, including SP-B, were not altered in BALF. Because SP-B-deficient mice die of
respiratory failure immediately after birth, it has not been possible
to study SP-C in the absence of SP-B. Furthermore, the processing of
SP-C precursor is altered in SP-B/ mice and infants. An
aberrant form of pro-SP-C is produced, SP-B/ mice and
individuals being deficient in both SP-B and SP-C in the alveolus. We
hypothesized that SP-B alone was sufficient for surfactant function but
that transgenic mice with ~50% reduced amounts of SP-B in the
alveolar lavage (SP-B+/) might reveal the contribution of
SP-C in response to hyperoxic stress.
As previously reported for (SP-B+/,SP-C+/+)
mice (4), in air, lung volumes on the deflation limb of
the pressure-volume curves were higher in all the mice with reduced
levels of SP-B [(SP-B+/,SP-C+/+),
(SP-B+/,SP-C+/), and
(SP-B+/,SP-C/)] compared with the mice
with normal amounts of SP-B
[(SP-B+/+, SP-C+/+) and
(SP-B+/+,SP-C/) mice]. After exposure to
95% O2 for 3 days, lung volumes on the pressure-volume
curve were decreased in all SP-B+/ mice. In contrast,
lung volumes were unchanged by O2 exposure in the
SP-B+/+ mice, with either SP-C+/ or
SP-C/. In the SP-B+/ mice,
decreased levels of SP-C (both SP-C+/ and
SP-C/) further perturbed pressure-volume relationships,
which were worsened after hyperoxia. During O2 exposure,
the increase in total protein and cytokines in BALF was most severe in
SP-B+/ mice in the absence of SP-C compared with all
other genotypes studied. The current study provides important
information regarding the role of SP-C during hyperoxic stress in mice
with genetic deficiency of SP-B. These results suggest that other
stresses known to reduce SP-B levels in BALF may lead to altered lung
function and that the outcome may well be affected by SP-C levels.
To assess tolerance to hyperoxia, we exposed mice to 95%
O2 for 3 days. The 3-day exposure period was chosen because
recent studies in mice indicated that SP-B+/ mice
(30), SP-A/ mice (13), and
wild-type mice tolerated 3 days of hyperoxia with increased total
protein in BALF and increased protein permeability. Pressure-volume
curves were altered in SP-B+/ mice, but no changes in
lung function were seen in SP-A/ mice. Sat PC was
reduced during exposure to O2 in the present study, and
reduced amounts of surfactant phospholipid after hyperoxia were shown
in other species (2, 12, 19). Likewise, decreased surfactant secretion was noted after 48-h exposure to 95%
O2 in vitro (32) and in our present study.
Reduction in Sat PC was more marked in BALF than in the lung. After
hyperoxia, alveolar macrophages were activated and expressed cytokine
mRNAs (3). Each cytokine has a different time course of
response to inflammation. TNF- is a fast responder and returns to
baseline more rapidly than other cytokines (3). BALF was
collected at 72 h of hyperoxia, in the present study, and TNF-
was not increased at that late time point. IL-6 has been reported to
increase in BALF after hyperoxia (3), and IL-6 was
significantly increased by hyperoxic exposure of
(SP-B+/,SP-C/) mice. We did not
characterize inflammatory cells in BALF, but increased cytokines and
total protein strongly suggest a larger inflammatory response in the
(SP-B+/,SP-C/) mice than in the other groups.
SP-B and SP-C play important roles in the formation, maintenance, and
function of the surface film. The results of in vitro studies suggest
that SP-B is more effective in lowering minimum surface tension whereas
SP-C is more effective in stabilizing the surface film (24,
26). The mechanisms underlying these functions are not clear but
may include SP-C-mediated recruitment of lipids into the expanding
surface film from a surfactant reservoir in the subphase (7, 26,
28). Surfactant from mice with normal levels of SP-C and 50% of
the normal amount of SP-B exhibited stable surface tensions consistent
with rapid transport of Sat PC from the reservoir to the gap in the
surface film resulting from lipid squeeze out; in contrast, surfactant
lacking SP-C produced an unstable surface film in which surface tension
fluctuated. Surface film instability was only detected when the bubble
radius was very small and packing of phospholipids was consequently
very high. This outcome, coupled with lowered hysteresivity in lung of
SP-C/ mice at low mean airway pressure, suggests that
SP-C stabilizes surfactant activity at low lung volumes. Under normal
conditions, SP-B plays a dominant role in surfactant function; however,
under conditions of stress and decreased levels of SP-B, SP-C can help maintain lung volumes during lung injury when alveolar capillary leak
inhibits surfactant activity and levels of surfactant lipids are decreased.
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ACKNOWLEDGEMENTS |
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This research was supported by National Heart, Lung, and Blood Institute Grants HL-61646 and HL-38859.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Ikegami, Professor of Pediatrics, Children's Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: machiko.ikegami{at}chmcc.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/japplphysiol.00459.2001
Received 10 May 2001; accepted in final form 19 September 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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