Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039
Submitted 13 January 2003 ; accepted in final form 9 March 2003
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ABSTRACT |
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transgenic; surfactant proteins B and C; lung; respiratory distress syndrome
The phospholipid and protein components of pulmonary surfactant are synthesized by alveolar type II epithelial cells and stored as tightly packed lipid bilayers in specialized secretory granules called lamellar bodies (38). Targeted disruption of the SP-B locus in mice profoundly perturbs lipid packaging in lamellar bodies, resulting in secretory granules filled with vesicles and electron-dense material but lacking characteristic membrane lamellae (9). Although the contents of these lamellar bodies are secreted into the air space, newborn mice fail to inflate their lungs and die of respiratory distress syndrome (RDS) shortly after birth (32, 36). Similarly, human infants with mutations leading to SP-B deficiency rapidly develop RDS after birth and invariably die of respiratory failure (25, 26). Targeted disruption of loci encoding the other three surfactant genes, SP-C, SP-A, and SP-D (7, 12, 20, 21) did not alter perinatal survival, indicating that SP-B is the only surfactant protein required for successful transition to air breathing at birth.
SP-B is synthesized as a precursor protein that is processed to the
79-amino acid mature peptide in the multivesicular body during transit to the
lamellar body (39). Expression
of SP-B is tightly linked to the processing of SP-C, which is also synthesized
as a precursor protein. In the absence of SP-B, the propeptide of SP-C is not
completely removed, resulting in accumulation of a misprocessed form of SP-C
(proSP-C), consisting of the mature peptide and an NH2-terminal
extension [relative molecular weight (Mr) 6,000], as
well as a significant decrease in the concentration of the mature SP-C peptide
in alveolar surfactant (9,
37). There is considerable
overlap in the ability of SP-C and SP-B to promote formation of a stable
surface film in vitro and restore lung function in surfactant-deficient
newborn animals and human infants. The decreased concentration of alveolar
SP-C content associated with hereditary SP-B deficiency may, therefore,
exacerbate lung dysfunction at birth.
Since SP-B-/- mice die immediately after birth, the potential role of SP-B in the postnatal or adult lung has not been critically assessed. SP-B concentrations in bronchoalveolar lavage (BAL) fluid were decreased in patients with acute respiratory distress syndrome (ARDS), suggesting a link between SP-B deficiency and lung disease in adults (14, 15). However, ARDS is a complex disease, and it is not clear whether SP-B deficiency contributed to respiratory failure or was secondarily perturbed by lung injury. To assess the role of SP-B in postnatal lung function, mouse SP-B was conditionally replaced in SP-B-/- mice. Selective loss of SP-B in adult mice caused respiratory failure, indicating that SP-B is required for postnatal lung function.
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MATERIALS AND METHODS |
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Compound transgenic mice were generated in which SP-B was conditionally expressed under control of the reverse tetracycline transactivator (rtTA) protein in type II cells of SP-B-/- mice. Briefly, the mouse SP-B cDNA was isolated, cloned under control of the (teto)7 promoter (27), and injected into fertilized FVB/N oocytes at the Cincinnati Children's Hospital Transgenic Core. Potential founder mice (F0) were identified by PCR using transgene-specific primers (upstream primer 5'-TGC TGC CAG GAG CCC TCT TG and downstream primer 5'-AAG GCA CGG GGG AGG GGC AAA); PCR results were confirmed by Southern blot analysis. F0 (teto)7 SP-B mice were bred with transgenic mice expressing rtTA under control of the 3.7-kb human SP-C promoter (SP-C rtTA) (33). Compound transgenic [SP-C rtTA/(teto)7 SP-B] offspring were identified by PCR using transgenespecific primers (SP-C rtTA transgene upstream primer 5'-GAC ACA TAT AA GAC CCT GGT CA and downstream primer 5'-AAA ATC TTG CCA GCT TTC CCC). SP-B transgene expression in compound transgenic mice was assessed by RT-PCR after 72 h of doxycycline (Sigma, St. Louis, MO) treatment (0.5 mg/ml in the drinking water) using RNA isolated from lung tissues and PCR primers specific for the (teto)7 SP-B transgene. Four independent compound transgenic lines expressing the transgenic RNA were subsequently bred with SP-B+/- mice. Compound SP-B+/- offspring were identified by PCR and bred again with SP-B+/- mice to generate compound SP-B-/- mice [SP-C rtTA/(teto)7 SP-B/SP-B-/-]. Compound SP-B+/- dams were given doxycycline in the drinking water from day 0 of gestation to stimulate transcription of the SP-B transgene during fetal lung development. Compound SP-B-/- progeny survived and were maintained on doxycycline; adult animals were crossed with siblings to establish colonies.
Characterization of Compound SP-B-/- Mice
Doxycycline was removed from the drinking water of adult compound SP-B-/- mice at 6-8 wk of age. The animals were closely monitored and killed after the onset of visible respiratory distress, determined by the presence of retractions at rest, decreased respiratory rate, and decreased activity of the animals. Mice were injected intraperitoneally with a lethal dose of pentobarbital sodium and placed in a chamber containing 100% oxygen to completely deflate their lungs. The trachea was then cannulated using an 18-gauge angiocatheter, and pressure-volume curves were obtained. The lungs were inflated with air using 75-µl increments every 10 s to a maximum pressure of 36 cmH2O and deflated in a similar fashion, as previously described (35). Pressure-volume curves were similarly generated for wild-type mice, and compound SP-B-/- mice were maintained on doxycycline. BAL was performed using three 1-ml aliquots of PBS, and the lavage samples were subsequently pooled for analysis. Large aggregate surfactant was isolated from BAL fluid, and surface activity was assessed with a captive bubble surfactometer, as previously described (16). All experiments described in this report were approved by the Institutional Animal Care and Use Committee of Cincinnati Children's Hospital.
Western blot analysis. The protein concentration in BAL fluid isolated from wild-type mice and compound SP-B-/- mice was determined by bicinchoninic acid assay (31). Aliquots of BAL containing equal amounts of protein were subjected to SDS-PAGE under nonreducing electrophoretic conditions for analysis of SP-B mature peptide (Mr = 16,000) and lysozyme (Mr = 14,000) or under reducing electrophoretic conditions for analysis of SP-C mature peptide (Mr = 4,000) and proSP-C (Mr = 6,000). Gels were electrophoretically transferred to nitrocellulose membranes, and Western blotting was performed with polyclonal rabbit antibodies directed against mature SP-B (23), mature SP-C (29), proSP-C (37), and lysozyme (Accurate Chemicals and Scientific, Westbury, NY). SP-B, SP-C, proSP-C, and lysozyme were quantitated by densitometry and normalized to lysozyme, which served as an internal control.
Surfactant phospholipid. Saturated phosphatidylcholine (PC) was recovered from BAL fluid by extracting the pellet with chloroform-methanol (2:1), reacting the lipid extract with OsO4 in carbon tetrachloride, and isolating saturated PC by column chromatography on neutral alumina, according to Mason et al. (24). Phosphorous in saturated PC was measured by the Bartlett assay (3). For analysis of surfactant phospholipid composition, chloroform-methanol extracts of BAL fluid from three animals were pooled and used for two-dimensional thin-layer chromatography (18). Phospholipid spots were visualized with iodine vapor, scraped, and assayed for phosphorous content.
Type II cell isolation. Type II cells were prepared from wild-type and compound SP-B-/- mice by a modification of the method of Corti et al. (10), as recently described (28). Type II cells were resuspended in culture media without doxycycline and cultured for up to 5 days on matrigel (BD Bioscience Franklin Lakes, NJ)/rat tail collagen (70:30). Cells were labeled with 35[S]methonine/cysteine for the last 4 h of culture on days 0, 3, and 5 and immunoprecipitated for SP-B mature peptide, as previously described (23).
Statistics. The data were evaluated using Student's t-test with significance defined as P < 0.05.
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RESULTS |
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Four independent lines of compound transgenic SP-B-/- mice were
generated and bred to SP-B+/- mice. Although
all SP-B-/- mice died after birth,
administration of doxycycline to dams resulted in survival of compound
SP-B-/- progeny (6% of offspring).
Compound SP-B-/- mice from all four
transgenic lines survived beyond 1 yr of age when doxycycline was included in
drinking water. Subsequent studies, therefore, focused to one line of compound
SP-B-/- mice (transgenic line B).
Histological and ultrastructural analyses of lung tissues from 6- to 8-wk-old
mice, the age of all animals used in this study, demonstrated that lung
morphology of compound SP-B-/- mice (line B)
was indistinguishable from age- and strain-matched wild-type FVB/N mice (not
shown). There was no evidence of inflammation or alveolar remodeling; at the
ultrastructural level, lamellar body size and organization in type II cells
was unperturbed. Together, conditional reconstitution of SP-B expression in
SP-B-/- mice protected against respiratory
failure at birth and sustained normal lung structure in adult mice.
The importance of SP-B for postnatal lung function was assessed by removing the mice from doxycycline at 6 wk of age. Compound SP-B-/- mice developed respiratory symptoms, including decreased activity, retractions at rest, and decreased respiratory rate, resulting in death 7.5 ± 3.5 days after withdrawal from doxycycline. The concentration of SP-B in BAL after the onset of respiratory symptoms was 25% of that in BAL fluid isolated from wild-type mice (Fig. 1). SP-B expression in the absence of doxycycline may have contributed to the relatively long survival period and the level of SP-B peptide in the air spaces. To determine whether SP-B protein was actively synthesized in the absence of doxycycline, type II cells were isolated from compound SP-B-/- and wild-type mice and cultured for up to 5 days in media without doxycycline. Immunoprecipitation of 35[S]methionine/cysteine-labeled cell lysates detected newly synthesized SP-B in cells from both wild-type and transgenic mice, although the level of SP-B expression was greatly reduced in the cells from compound SP-B-/- mice (Fig. 2). Compound SP-B-/- mice that did not receive doxycycline in utero died immediately after birth, indicating that the level of SP-B expression in the absence of doxycycline was not sufficient to reverse respiratory failure in these animals. These results indicate that SP-B is required for postnatal lung function and that a decrease in alveolar SP-B content to levels of 25% or less of those in wild-type mice is associated with fatal respiratory distress syndrome.
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Altered Surfactant Function and Composition After Removal from Doxycycline
Postnatal SP-B deficiency led to changes in surfactant composition and function. Compound SP-B-/- mice were removed from doxycycline, and BAL was collected after the onset of respiratory symptoms. The concentration of phosphatidylglycerol (PG) in BAL was decreased, and the concentration of sphingomyelin was increased in control compound SP-B-/- mice; the derangement of these two surfactant phospholipids was increased after removal of mice from doxycycline (Fig. 3). The concentration of PC and other surfactant phospholipids (phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine, not shown) was not affected by SP-B deficiency. SP-C concentration in BAL declined by 15%, and the concentration of proSP-C increased by 30% (Fig. 4). Changes in the concentration of surfactant phospholipids and proteins were accompanied by an increase in total protein in BAL, consistent with alveolocapillary leak (Fig. 5). The impact of altered surfactant composition on surfactant function was assessed by isolating large aggregate surfactant from BAL and testing surface activity in a captive bubble surfactometer. Equilibrium surface tension for surfactant isolated from wild-type or control compound SP-B-/- mice was virtually identical and was significantly elevated for surfactant isolated from SP-B-deficient mice (Fig. 6A). Minimum surface tension was <3 mN/m for wild-type and control compound SP-B-/- mice and was dramatically increased to >17 mN/m for SP-B-deficient mice (Fig. 6B). Altered surfactant activity in vitro was associated with changes in lung function in vivo, including decreased hysteresis, decreased maximum lung volumes, and lower lung volumes at low pressures (Fig. 7).
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DISCUSSION |
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Compound SP-B-/- mice developed severe RDS and died when SP-B content in BAL declined to <25% of that in healthy wild-type mice. Disruption of a single SP-B allele in mice (SP-B+/- mice) resulted in a 50% decrease in lung content of SP-B that was associated with mild air trapping and decreased lung compliance (8). Importantly, SP-B+/- mice were more susceptible to oxygen-induced lung injury, which was prevented by intratracheal administration of SP-B (34, 35). In human patients at risk for ARDS, SP-B levels were decreased in BAL and elevated in plasma, the latter increase likely reflecting alveolocapillary leak (6, 14). SP-B concentration in BAL from patients with established ARDS ranged from 25-55% of normal levels (14, 15). Collectively, these observations suggest that a decrease in alveolar SP-B content to <50% of normal may increase the risk of respiratory failure, whereas SP-B levels below 25% of normal may result in fatal RDS.
Diminished expression of SP-B has been reported following infection with a variety of airway pathogens. SP-B and SP-A content in BAL was decreased in children infected with respiratory syncytial virus (19). Intratracheal infection of mice with adenovirus resulted in decreased immunostaining for SP-B and SP-A in type II cells and focal loss of SP-B and SP-A mRNA expression (42). SP-B, but not SP-A, protein and mRNA were decreased following intratracheal inoculation of mice with Pneumocystis carinii (1, 4). Similarly, when mice were challenged with aerosolized endotoxin, SP-B, but not SP-A or SP-C, protein and mRNA were decreased in conjunction with impaired surfactant function (17). We have confirmed a selective decrease in SP-B protein following endotoxin challenge and have shown that SP-B protects against endotoxin-mediated changes in lung inflammation and surfactant function in adult mice (11). These results suggest that the severity of RDS may be exacerbated by diminished expression of SP-B associated with pulmonary inflammation and that superimposition of pulmonary infection on genetic deficiency of SP-B may further increase the risk of respiratory failure.
Decreased concentrations of SP-B in BAL of compound SP-B-/- mice were associated with changes in surfactant composition and function. The content of PG decreased, and sphingomyelin was increased, while the concentrations of other surfactant phospholipids, including PC, were unchanged. PG, but not PC, was similarly decreased in BAL from SP-B-deficient human infants (5). SP-B interacts specifically with PG head-groups in surfactant (2), but the impact of PG defi-ciency on surfactant function and the mechanisms by which loss of SP-B leads to decreased PG content in alveolar surfactant remains unclear. In addition to altered PG concentration, SP-C content was slightly but significantly decreased in BAL from adult SP-B-deficient mice (85% of wild type). This outcome differed from newborn SP-B-/- mice (9) in which SP-C levels were very low or undetectable, suggesting that the half-life of SP-C in the air spaces of compound SP-B-/- mice was relatively long following withdrawal from doxycycline. However, in spite of the relatively high concentration of SP-C, compound SP-B-/- mice progressed to respiratory failure, indicating that SP-C could not compensate for SP-B deficiency in these animals. Although both SP-C and SP-B are decreased in BAL from ARDS patients (14, 15, 30), these results suggest that correction of SP-B deficiency may be more important for recovery from respiratory failure. This hypothesis is supported by the observation that the complete absence of SP-C in mice was associated with only minor changes in surfactant function (12, 16).
One of the characteristic features of SP-B deficiency in humans is the accumulation of a partially processed form of the SP-C proprotein (proSP-C) in lung tissues (37). Conditional expression of SP-B in SP-B-/- mice restored SP-B peptide concentration in BAL to wild-type levels but failed to completely correct the SP-C-processing defect. This phenomenon was observed in four independent transgenic lines and was likely related to the lack of expression (or low-level expression) of the SP-C-rtTA transgene in some type II cells, leading to SP-C processing defects and secretion of proSP-C by affected cells. We have previously reported that the human 3.7-kb SP-C promoter did not fully restore SP-B expression in all type II cells of SP-B-/- mice, resulting in misprocessing of SP-C in SP-B-deficient cells (22). The physiological consequences of accumulation of proSP-C in the air spaces of compound SP-B-/- mice are not clear. Lung function in adult compound SP-B-/- mice was apparently normal; however, in the absence of doxycycline, the concentration of proSP-C in BAL increased slightly, raising the possibility that accumulation of this peptide may exacerbate surfactant dysfunction in SP-B deficiency.
Withdrawal of adult compound SP-B-/- mice from doxycycline resulted in fatal RDS after 7.5 days. This unexpectedly long survival time was likely related, in part, to the slow release of doxycycline from tissue stores, resulting in continued transcription of the SP-B transgene (27). Analysis of SP-B synthesis in type II cells isolated from compound SP-B-/- mice indicated low-level expression of SP-B during culture in the absence of doxycycline. Transgene expression in the absence of the appropriate inducer drug (i.e., promoter leak) has previously been noted in other conditional expression systems (40, 41). It is, therefore, likely that residual binding of SP-C rtTA to the (teto)7 promoter in the absence of doxycycline contributed to prolonged survival. However, the amount of SP-B produced by promoter leak was well below the 25% threshold required for survival and was, therefore, insufficient to rescue newborn compound SP-B-/- mice deprived of doxycycline during gestation.
In summary, doxycycline-regulated expression of a mouse SP-B transgene in SP-B-/- mice completely reversed the neonatal lethality associated with inherited SP-B deficiency. Withdrawal of adult compound SP-B-/- mice from doxycycline resulted in respiratory failure associated with a decrease of SP-B concentration in BAL. Secondary effects of SP-B deficiency included decreased concentration of PG and increased concentrations of proSP-C and total protein in BAL. Consistent with surfactant dysfunction, adult SP-B-deficient mice exhibited a significant decrement in lung function. These results indicate that SP-B is required for postnatal lung function and that levels at or below 25% of normal lead to surfactant dysfunction and respiratory failure.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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Present address of K. R. Melton: Division of Neonatology, Children's Mercy Hospital, Kansas City, MO 64108.
Present address of J. W. Tichelaar: Department of Environmental Health, Division of Toxicology, University of Cincinnati School of Medicine, Cincinnati, OH 45267-0056.
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FOOTNOTES |
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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.
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REFERENCES |
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