1 Divisions of Neonatology and Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039; and 2 Department of Pediatrics, Harbor-University of California Los Angeles Medical Center, Torrance, California 90509
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ABSTRACT |
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Pulmonary function
was assessed in newborn wild-type and homozygous and heterozygous
surfactant protein B (SP-B)-deficient mice after birth. SP-B+/+ and
SP-B+/ mice became well oxygenated and survived
postnatally. Although lung compliance was decreased slightly in the SP-B+/
mice, lung volumes and compliances were decreased markedly in homozygous SP-B
/
mice. They died
rapidly after birth, failing to inflate their lungs or oxygenate. SP-B proprotein was absent in the SP-B
/
mice and was reduced
in the SP-B+/
mice, as assessed by Western analysis. Surfactant
protein A, surfactant proprotein C, surfactant protein D, and
surfactant phospholipid content in lungs from SP-B+/
and
SP-B
/
mice were not altered. Lung saturated
phosphatidylcholine and precursor incorporation into saturated
phosphatidylcholine were not influenced by SP-B genotype. Intratracheal
administration of perfluorocarbon resulted in lung expansion,
oxygenation, and prolonged survival of SP-B
/
mice and in
reduced lung compliance in SP-B+/+ and SP-B+/
mice. Lack of SP-B
caused respiratory failure at birth, and decreased SP-B protein was
associated with reduced lung compliance. These findings demonstrate the
critical role of SP-B in perinatal adaptation to air breathing.
lung function; perfluorocarbon; surfactant protein; respiratory distress syndrome; lung compliance
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INTRODUCTION |
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SURFACTANT PROTEIN (SP) B is a 79-amino acid amphipathic polypeptide associated with surfactant phospholipids in the alveolus of the lung (6). SP-B is encoded by a single gene located on chromosome 2 in humans and at a syntenic site on chromosome 6 in the mouse (15). The SP-B gene encodes a 40- to 42-kDa glycosylated preproprotein that is proteolytically processed by type II epithelial cells to produce the active peptide detected within the alveolus (22). The recent observations that the genetic ablation of murine SP-B causes lethal respiratory failure at birth and that mutations in the human SP-B gene cause lethal respiratory distress in full-term newborn infants provide strong support for the critical role of SP-B in lung function (4, 17). SP-B binds phospholipids, causes lipid fusion, and contributes to the formation of multilayer lipid membranes. SP-B also plays a critical role in the intracellular processing of surfactant, being required for the formation of lamellar bodies and the proteolytic processing of surfactant proprotein C (proSP-C). The addition of SP-B and other surfactant proteins, SP-A and SP-C, to phospholipid mixtures enhances adsorption and development of low surface tension at an air-liquid interface (Ref. 9, for review see Ref. 23).
The complete lack of SP-B in newborn mice and humans causes lethal
respiratory failure at birth (4, 17, 18). Infants with hereditary SP-B
deficiency can be temporarily supported by mechanical ventilation,
extracorporeal membrane oxygenation, and exogenous surfactant
replacement (8). Lethal respiratory failure in these infants has been
corrected only by lung transplantation. Partial defects in SP-B can
also cause respiratory failure in full-term infants, and decreased
amounts of SP-B occur in preterm infants with respiratory distress
syndrome (19) and in adults with acute respiratory distress
syndrome (7). Developmental, humoral, inflammatory, and spatial
regulatory factors influence SP-B gene expression (23). Because
inactivation of a single SP-B allele decreased SP-B protein in adult
SP-B+/ mice (3), the present study was designed to evaluate
aspects of lung mechanics, lung function, and phospholipid metabolism
in newborn heterozygous and homozygous SP-B-deficient mice.
Furthermore, because there are no effective pharmacological therapies
for SP-B deficiency in human neonates, the present study also evaluated
the effects of perfluorocarbon on survival and lung function in newborn
SP-B-deficient mice.
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MATERIALS AND METHODS |
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Ablation of the murine SP-B gene by insertion of the neomycin
resistance gene into the fourth exon of the murine gene was accomplished in embryonic stem cells (D3), as previously described (4).
Newborn pups were obtained by mating SP-B+/ parents.
Cesarean section and resuscitation.
Pregnant mice were anesthetized (2-3% isoflurane) at 18-19
days of gestation, and pups were delivered within 3 min. Dams were then
killed by exsanguination. After delivery, pups were placed in an
incubator maintained at 33-35°C with an inspired
O2 fraction of >40% and were
observed for at least 30 min after delivery. Pups were monitored for
breathing activity and skin color. A clinical score based on breathing
activity and skin color was recorded every 10 min for 30 min
[clinical score = breathing activity + skin color; breathing
activity was scored as 0 (none), 1 (gasping), or 2 (continuous
breathing); skin color was scored as 0 (blue), 1 (dusky), or 2 (pink)]. One group of pups (20 SP-B+/+, 23 SP-B+/, and 12 SP-B
/
) received no treatment. A second group of pups (10 SP-B+/+, 15 SP-B+/
, and 13 SP-B
/
) received
perfluorocarbon (FC-75, 3 M) immediately after delivery.
Perfluorocarbon (50 µl) was administered into the oropharynx with a
pipette. The respiratory efforts of the mice facilitated delivery of
perfluorocarbon to the lungs.
Pressure-volume analysis.
Breathing mice were killed with an overdose of pentobarbital sodium
(200 mg/kg ip) and were placed in 100%
O2 to permit the complete collapse
of the alveolar space by O2
absorption. The trachea was cannulated transorally (24-gauge Angiocath;
Becton-Dickinson, Rutherford, NJ) and was connected to a syringe and a
pressure sensor (X-ducer; Motorola, Phoenix, AZ) via a three-way
connecter. Air leaks around the catheter were prevented by application
of glue (Quick tight; Loctite) around the catheter. Lungs were inflated in 10-µl increments to a maximum inflation pressure of ~20
cmH2O and were deflated to
negative pressure. Pressure and volume on inflation and deflation were
recorded and were normalized to the weight of the animal. Lung
compliance was determined from the slope of the deflation curve, where
the curve approximated a straight line between 10 and +10
cmH2O. The hysteresis ratio was
calculated as the area bound by the inflation and deflation curves
divided by the total area bound by the maximum and minimum values for pressure and volume area using software kindly provided by Dr. Gary S. Huvard (Huvard Research and Consulting, Chesterfield, VA). Similar
measurements were made in SP-B
/
mice that failed to
inflate their lungs within 40 min after delivery. These animals had
severe respiratory failure (low clinical scores at 30 min). Lung
compliance measurements were completed by 40-50 min after delivery
in these animals. In animals without respiratory failure (high clinical
scores at 30 min), lung compliance measurements were performed within
2-3 h after delivery.
Histology.
Lungs from neonatal SP-B+/+, SP-B+/, and SP-B
/
mice were fixed after passive deflation by immersion in 4%
paraformaldehyde, embedded in paraffin, cut into 5-µm sections, and
stained with hematoxylin and eosin. A total of 32 lungs were studied
histologically. All lobes of each lung were examined at two different
levels (~25 µm apart). Nine SP-B+/+, thirteen SP-B+/
, and
ten SP-B
/
mice were studied. At least three animals of
each genotype were exposed to room air,
O2, or
O2 + perfluorocarbon for 30 min
before euthanasia. Lung sections were examined and were scored by three
individuals who did not know the source of the histological samples.
Epithelial, vascular, and alveolar portions of the lungs were scored on
the basis of 0% (0),
50% (1),
75% (2), or 100% (3) evidence of
damage or presence of alveolar exudate. These scores were averaged and
analyzed by nested analysis of variance and contrast comparisons.
Genotyping. DNA was isolated from tail or liver tissue. Genotypes were determined using polymerase chain reaction amplification, as described previously (3). Briefly, oligonucleotide primers specific for the neomycin resistance gene and intron 4 of the SP-B gene were used to identify a 1.3-kb product from the mutated allele. Oligonucleotide primers to introns 3 and 4 of the SP-B gene identified a 260-base pair product from the wild-type SP-B allele. Mutated and wild-type alleles were assessed in all mice.
SP-B protein analysis. For Western blot analysis, fetal lung tissues were collected on day 18 of gestation. Tissue was homogenized in 10 mM tris(hydroxymethyl)aminomethane (Tris, pH 7.5), 0.25 M sucrose, 1 mM EDTA, 5 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of pepstatin A, aprotinin, antipain, leupeptin, and chymostatin, and the supernatant was centrifuged at 140 g for 10 min (4°C). Equal amounts of protein from the supernatant were dissolved in electrophoresis sample buffer and were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electrophoretic transfer to nitrocellulose membrane, as previously described (4). The membrane was blocked with 5% milk in Tris-buffered saline and was incubated with rabbit anti-human SP-B antibody overnight at room temperature. Subsequently, the membrane was washed with Tris-buffered saline containing 0.05% Tween 20 and 0.05% Nonidet P-40, incubated with a 1:20,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Calbiochem, La Jolla, CA) for 2 h at room temperature, washed again, and developed with a chemiluminescent substrate [enhanced chemiluminescence (ECL); Amersham, Arlington Heights, IL].
Lung saturated phosphatidylcholine. On day 17 of gestation, three pregnant mice were injected intraperitoneally with 0.5 ml of saline containing 0.5 mCi of [3H]palmitic acid ([9,10-3H]palmitic acid, 60 Ci/mmol; American Radiochemicals, St. Louis, MO) and 42 µCi of [methyl-14C]choline chloride (40 Ci/mol; New England Nuclear, Boston MA). Mice were sedated with an intraperitoneal injection of pentobarbital sodium 6 h later, the fetuses were delivered and chilled on ice, and the livers and lungs were removed and homogenized. The lipids were extracted with 2:1 chloroform-methanol. Phosphatidylcholine was isolated from the liver extract by one-dimensional thin-layer chromatography, and equivalent spots were used for quantification of radioactivity and phosphorus (10). Specific activity, defined as radioactivity divided by micromoles of phosphatidylcholine, was calculated for each fetal liver. Saturated phosphatidylcholine (Sat PC) was isolated from the lung extract after treatment with OsO4 by aluminum column chromatography, according to Mason et al. (13). Specific activities for Sat PC from the fetal lung were calculated from radioactivity and phosphorus measurements. Incorporation of the radiolabels into liver phosphatidylcholine was used to estimate isotope transfer to each fetus.
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Statistical analysis.
To compare specific activities for Sat PC among litters and among
genotypes, the ratio of specific activities of lung Sat PC to liver
phosphatidylcholine was calculated for each mouse. The mean of the
ratio for all mice in each litter was calculated, and the ratio for
each mouse was expressed relative to the mean. The normalized ratios
for SP-B+/+, SP-B+/, and SP-B
/
mice were grouped,
and the group mean ± SE was calculated for each radiolabeled precursor. Differences in other variables among the three genotypes (SP-B+/+, SP-B+/
, and SP-B
/
) were assessed by
nested or two-way analysis of variance, and differences between means
were assessed by contrast comparisons and the Student-Newman-Keuls
test.
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RESULTS |
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Surfactant proteins.
Genotyping of the SP-B+/+, SP-B+/, and SP-B
/
fetal
mice demonstrated the expected frequency of inheritance of the SP-B allele as an autosomal recessive gene. As previously described for
full-term newborn mice (4), SP-B protein, assessed by Western analysis
of lung tissue homogenates, was not detected in newborn homozygous mice
(data not shown). Lung SP-B protein was reduced in SP-B+/
compared with wild-type littermates, whereas SP-A, proSP-C, and SP-D
were not altered in the SP-B+/
or SP-B
/
mice. The proSP-C fragment (10-12 kDa)
and decreased SP-C active peptide (4 kDa) were observed in
SP-B
/
pups but not in SP-B+/+ or SP-B+/
neonates.
The abnormal proSP-C fragment characteristic of SP-B
/
mice was also detected by Western blot analysis of amniotic fluid but
was not detected in amniotic fluid from SP-B+/+ or SP-B+/
mice
(data not shown).
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Lung Sat PC.
In mice from three separate litters, there were similar amounts of Sat
PC in the lungs of SP-B+/+, SP-B+/, and SP-B
/
mice: 82.5 ± 8.5 (n = 5), 75.3 ± 3.5 (n = 19), and 66.4 ± 4.7 µmol/kg (n = 9),
respectively (F = 1.8, P = 0.2 by analysis of variance). There were no differences in lung Sat PC among the genotypes. The trend
toward lower lung Sat PC amounts for the SP-B
/
mice is
explained by more mice being from the litter with the lowest amounts of
lung Sat PC than from the other litters. Incorporation of the
radiolabeled precursors
[3H]palmitic acid and
[14C]choline into lung
Sat PC also was not different for the mice, differing only in SP-B
genotype (Fig. 1).
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Clinical features after delivery.
Most of the SP-B+/+ and SP-B+/ mice survived postnatally
(survival rates were 97.7 and 94%, respectively) and were oxygenated within 20 min in association with visible lung inflation. In contrast, all SP-B
/
mice died of respiratory failure with poor
clinical scores (Fig. 2) within 30 min after birth,
despite vigorous respiratory efforts. Perfluorocarbon was easily
delivered by the oropharyngeal route. All mice survived for at least 30 min after perfluorocarbon treatment. The clinical scores of
SP-B
/
mice at 30 min after perfluorocarbon treatment were
significantly improved (P < 0.05) compared with those of untreated SP-B
/
mice (Fig. 2).
Although SP-B
/
mice were rescued by perfluorocarbon, the
clinical improvement did not achieve normal clinical scores
characteristic of SP-B+/+ or SP-B+/
mice. Lungs of
SP-B
/
mice receiving perfluorocarbon became visually
inflated, but unlike SP-B+/+ and SP-B+/
mice, lungs of the
SP-B
/
mice virtually collapsed at the end of expiration.
Pulmonary function and morphology.
Pressure-volume curves of SP-B/
mice were typical of
surfactant deficiency, with decreased compliance, no hysteresis, and low residual and total lung volumes (Figs. 3 and
4). Data from individual animals are shown in Fig. 3,
and group data are shown in Fig. 4. Lung volumes at 18 cmH2O pressure were markedly
decreased in SP-B
/
mice (Figs. 3 and 4). Although
SP-B+/
mice successfully initiated ventilation, lung compliance
was significantly, although modestly, reduced in SP-B+/
mice
compared with the SP-B+/+ littermates (Fig. 4). Consistent with
pressure-volume analysis, lungs of the SP-B
/
mice
remained atelectatic with low hysteresis ratios (Fig. 4), whereas lungs
of SP-B+/
and SP-B+/+ mice were inflated and indistinguishable
from each other at the light-microscopic level (Fig. 5).
Perfluorocarbon treatment markedly improved the pressure-volume curve
of SP-B
/
mice, restoring lung volume and compliance and increasing the hysteresis ratio (Fig. 4). In contrast, perfluorocarbon significantly decreased the lung compliance of SP-B+/+ and
SP-B+/
mice (Fig. 4). Pressure-volume curves, lung compliance,
and hysteresis after perfluorocarbon treatment were not significantly
different among SP-B+/+, SP-B+/
, and SP-B
/
mice
(Figs. 3 and 4).
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DISCUSSION |
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Mutation of the SP-B gene by homologous recombination completely
ablated SP-B mRNA and protein in homozygous transgenic mice and reduced
the levels of SP-B mRNA and protein in heterozygous SP-B+/ mice
(Refs. 3 and 4 and present study). In the present study, absence of
SP-B caused lethal respiratory failure that was ameliorated by the
administration of perfluorocarbon, which enhanced oxygenation,
increased lung volume and compliance, and increased the time of
survival. Although heterozygous SP-B-deficient mice survived cesarean
delivery, lung compliance was decreased slightly compared with
wild-type littermates, supporting the concept that normal amounts of
SP-B are required for normal perinatal lung function. The finding that
decreased SP-B is associated with pulmonary dysfunction in
SP-B+/
and SP-B
/
newborn mice is consistent with
the increased lung function and compliance observed after treatment of
surfactant-deficient preterm animals with exogenous surfactant
containing SP-B (9). Although SP-B is subject to a variety of temporal
and humoral influences in the neonatal period, gene dosage appears to
play an important role in the control of steady-state SP-B production.
Whereas SP-B peptide was completely absent in the SP-B
/
newborn mice and was reduced in the SP-B+/
newborn mice, the
amount of lung Sat PC and the incorporation of choline and palmitate
into Sat PC were not influenced by the presence or level of SP-B.
SP-B is essential for the formation of lamellar bodies within type II
cells and for the normal processing of SP-C (4, 18). Reduction of SP-B
in heterozygous mice is apparently sufficient to result in normal
lamellar body formation and proSP-C processing in newborn or adult
SP-B+/ mice. Complete reduction of SP-B by gene knockout
surprisingly does not affect lung Sat PC pool sizes and precursor
incorporation by type II cells. Because of the small size of the
newborn pups, we were unable to assess Sat PC or other lipids in
bronchoalveolar lavage fluid. The location, processing, and fate of Sat
PC is unknown but apparently is not affected by the absence of SP-B
protein. Normal incorporation of radiolabeled precursors into
phosphatidylcholine in lung tissue explants from an infant with SP-B
deficiency was recently reported (2).
Reduction in SP-B content in the lungs of neonatal SP-B+/ mice
results in a modest reduction in lung compliance without changes in
lung volume. The reduction in total lung SP-B content in newborn SP-B+/
mice was comparable to that observed in adult
SP-B+/
mice and was consistent with an ~50% decrease in SP-B
content in bronchoalveolar lavage previously observed in the adult
SP-B+/
mice (3). The small decrease in lung compliance in the
neonatal SP-B+/
mice was similar to that recently observed in
adult SP-B+/
mice (3). However, other differences between
SP-B+/+ and SP-B+/
mice, such as the air trapping consistently
observed in the adult SP-B+/
mice, were not apparent in the
neonatal animals. Whether the difference in air trapping in neonatal
compared with adult SP-B+/
mice is related to developmental
differences in local levels of SP-B expression in the respiratory
epithelium or to intrinsic anatomic or mechanical differences between
neonatal and adult mouse lungs is unclear.
Pulmonary function in the SP-B/
mice was markedly
impaired with decreased lung volume and compliance typical of
surfactant dysfunction seen in premature infants with severe
respiratory distress syndrome. Despite gasping activity, administration
of O2, and vigorous stimulation,
none of the SP-B
/
mice survived beyond 30 min of age. The
acute disruption in lung function observed in the SP-B
/
mice is apparently due to the absence of SP-B protein and not to the
presence of an aberrant form of SP-C propeptide. Recent cross-breeding
studies demonstrated that SP-B
/
mice survive to adulthood
if they express the NH2-terminal
portion of human SP-B in their lungs (1). These transgenic mice produce
SP-B and the aberrant form of SP-C propeptide and survive normally.
Administration of perfluorocarbon in the immediate postnatal period was
associated with prolongation of survival, improved oxygenation, and
visual inflation of the lung of SP-B/
mice. Pressure-volume curves demonstrated markedly improved compliance and
increased lung volumes after perfluorocarbon treatment of the
SP-B
/
mice. Perfluorocarbon also altered lung function
and mechanics in SP-B+/+ and SP-B+/
mice, likely related to its
intrinsic surface tension-lowering properties, viscosity, and ablating
effects of endogenous surfactant on lung function (21). All lung
compliance curves, lung volumes, and hysteresis ratios were similar
after perfluorocarbon treatment, indicating that differences among
genotypes were not related to tissue structure. Breathing patterns in
the treated SP-B
/
mice were distinct from those in the
SP-B+/+ and SP-B+/
mice: the lungs of SP-B
/
mice
collapsed at end expiration. These findings suggest that the minimum
surface tension of perfluorocarbon is not sufficient to maintain
complete alveolar stability at end expiration. In the
surfactant-sufficient lung, minimum surface tension of surfactant is
believed to be <1 dyn/cm2 and is
much lower than that of the perfluorocarbon (FC-75), which is ~15
dyn/cm2 (11). Whereas
perfluorocarbon improved lung function, the treatment caused
histological abnormalities of the lung, affecting the bronchial and
bronchiolar epithelium, vascular structures, and presence of alveolar
exudate. Histological abnormalities were most severe in
SP-B
/
mice and were not observed in untreated animals of any genotype. Whether histological abnormalities resulted from perfluorocarbon or lack of endogenous surfactant is unclear.
In the present study, modest reductions in SP-B were associated with
modest changes in pulmonary mechanics in newborn SP-B+/ mice.
Because the levels of SP-B are reduced in premature infants and SP-B
expression is decreased in association with lung injury, infection, and
O2 exposure (23), decreased SP-B
may play a role in the pathogenesis of clinical lung disease. Likewise,
human heterozygous SP-B gene carriers may be susceptible to pulmonary dysfunction at birth, especially if born prematurely.
Perfluorocarbon dramatically improved lung function in
SP-B/
mice, similar to findings in premature animals and
infants (12). This surfactant-like activity of perfluorocarbon has been described when administered as partial liquid ventilation (5, 12, 14,
20, 21). Long-term survival of homozygous SP-B-deficient patients
requires lung transplantation. Because exogenous surfactant therapy has
not been successful in the treatment of neonatal SP-B deficiency in
humans (8), the present findings support the concept that
perfluorocarbon may be useful in stabilizing critically ill
SP-B
/
infants during the early phase of their therapy.
The cytopathology observed after perfluorocarbon treatment suggests that such therapy may not be successful for long-term therapy of
hereditary SP-B deficiency.
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ACKNOWLEDGEMENTS |
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We thank Dr. Susan E. Wert for assistance with the histological analyses. We acknowledge the assistance of Ann C. Maher, the technical assistance of Sherri A. Profitt with histology, and the help of Dr. Mark Kurtzman and Jennifer Melzer with anesthesia.
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FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-51832.
Address for reprint requests: H. S. Iwamoto, Div. of Neonatology, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039.
Received 2 July 1996; accepted in final form 17 June 1997.
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