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Divisions of Neonatology and Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
Submitted 22 October 2004 ; accepted in final form 14 February 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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transgenic mice; surfactant protein B; surfactant protein C
To assess the requirement of SP-B for maintenance of alveolar structure and function in the postnatal lung, we previously generated compound transgenic mice in which expression of the mouse SP-B cDNA (SP-B transgene) was regulated by the tetracycline analog doxycycline (17). The reverse tetracycline transactivator under control of the human SP-C promoter (SP-C-rtTA) transgene and the (tetO)7SP-B transgene were expressed in SFTPB–/– mice [SFTPB–/–, SP-C-rtTAtg, (tetO)7SFTPBtg], herein referred to as compound transgenic mice. The SP-C promoter was used to target expression of rtTA to distal respiratory epithelial cells. In the presence of doxycycline (administered to mice in drinking water or food), the rtTA transcription factor binds to (tetO)7 elements and induces expression of the SP-B transgene in distal respiratory epithelial cells of SFTPB–/– mice. Maternal administration of doxycycline induced expression of the SP-B transgene in fetal lung and completely reversed neonatal lethality in compound transgenic offspring. Postnatal administration of doxycycline was essential for survival: withdrawal of mice from doxycycline resulted in respiratory failure after 7–8 days, indicating that SP-B is required for postnatal lung function. At the time of respiratory failure, SP-B concentration in the alveolar spaces was estimated to be 
25% of that in wild-type mice.
Compound transgenic mice were maintained on doxycycline for more than one year without overt evidence of respiratory dysfunction or alteration in lung structure. Doxycycline-regulated expression of the SP-C-rtTA transgene in compound transgenic mice resulted in alveolar SP-B concentration similar to that in wild-type mice. In the present study, we generated compound transgenic mice in which the rtTA transgene was placed under control of the rat Clara cell secretory protein (CCSP) promoter, which directs expression to both nonciliated bronchiolar cells and type II cells of the respiratory epithelium (21). The overall concentration of SP-B in the air spaces of these compound transgenic mice was significantly reduced compared with wild-type mice; furthermore, the nonuniform expression of SP-B in type II cells of transgenic mice was associated with changes in lung structure and function.
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MATERIALS AND METHODS |
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Analysis of lung structure. Lungs were inflation fixed with 4% paraformaldehyde in PBS at 25 cmH2O and immersed in the same fixative. Tissue was fixed overnight, washed with PBS, dehydrated in a series of alcohols, and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin for histology. One section from each lung lobe of five 6-wk-old compound transgenic mice, four wild-type mice treated with doxycycline from day 1 of gestation through 6 wk of age, and four 6-wk-old single transgenic (CCSP-rtTAtg) mice were examined for air space abnormalities. Focal air space enlargement was defined as two or more contiguous air spaces with diameters larger than that of the average alveolar duct found in the section.
For analysis of lamellar body structure, lung tissues from embryonic (E) day 18.5, postnatal day 1, and 10-wk-old mice were prepared for electron microscopy as previously described in detail (14). Type II cells (100 cells from each animal) were viewed at a magnification of 5,000 and scored based on the predominant lamellar body structure, i.e., wild-type, SP-B knockout, or an intermediate (hybrid) phenotype.
For cell proliferation studies, animals were injected with bromodeoxyuridine (BrdU) labeling reagent (1 ml/100 g body wt; Zymed Laboratories, South San Francisco, CA) 2 h before death. Animals were anesthetized with a 4:1:1 mixture of ketamine, acepromazine, and xylazine, and were then exsanguinated by severing the inferior vena cava and descending aorta. The trachea was cannulated, and the lungs were collapsed by piercing the diaphragm. The lungs were then inflation fixed at 25 cm of water pressure with 4% paraformaldehyde in PBS. The trachea was ligated, and the lungs were excised and immersed in fixative for an additional 16–24 h. After fixation was complete, the lungs were washed three times in PBS, dehydrated through a series of alcohol solutions and xylene, infiltrated with paraffin, and embedded for sectioning. Cell proliferation was examined in wild-type and compound transgenic mice at postnatal day (pnd)5, pnd15, pnd21, and 6 wk (n = 3–5 animals/genotype for each time point). Immunohistochemistry for BrdU and proliferating cell nuclear antigen (PCNA; biotinylated mouse anti-PCNA, clone PC10; Zymed Laboratories) was performed according to the manufacturer's recommendations. Immunohistochemistry for phosphorylated histone H3, a mitotic cell marker (rabbit polyclonal antibody to amino acids 7–20 of human histone H3, phospho-Ser10; United States Biological, Swampscott, MA), was performed overnight at a dilution of 1:100 after quenching the endogenous peroxidase and blocking for 2 h in 2% normal goat serum. The antigen:antibody complexes were detected by using a biotinylated goat anti-rabbit IgG and an avidin-biotin-peroxidase technique (1:200; Vectastain ABC Elite peroxidase kit, Vector Laboratories). The reaction product was enhanced with nickel/cobalt and counterstained with nuclear fast red. Mouse intestine (pnd21 and 6 wk) and/or esophagus (pnd5) harvested from each BrdU-injected animal were used as internal positive controls for BrdU incorporation and immunoreactivity. Mouse embryonic day (E) 14.5 lung, esophagus, and intestine were used as positive controls for the phosphohistone-H3 immunoreaction. Elimination of the primary antibody from the reaction served as a negative control for nonspecific binding of biotinylated secondary antibody and/or other kit components. At least three sections for each of three to five lobes per animal (n = 3–5 mice/genotype for each of 4 postnatal time points or 27–75 sections/genotype per time point) were examined for BrdU-, PCNA-, or phosphohistone-H3-positive cells.
Lung mechanics. Pressure-volume curves were generated for wild-type and compound transgenic mice. A lethal dose of pentobarbital sodium was injected intraperitoneally before the mice were placed in a chamber containing 100% oxygen to completely deflate the lungs. The trachea was cannulated using an 18-gauge angiocatheter, and the lungs were inflated using 75-µl increments every 10 s to a maximum pressure of 36 cmH2O and deflated in a similar fashion, as previously described (25). Lung mechanics in compound transgenic and wild-type mice were assessed with a modified ventilator (flexiVent; Scireq, Montreal, Canada), as previously described. Large aggregate surfactant was isolated from BALF, and surfactant function was assessed by captive bubble surfactometry, as previously described (9).
Analyses of SP-B, surfactant phospholipid, and cell counts in BALF. BAL was performed with five 1-ml aliquots of normal saline containing proteinase inhibitors, and the recovered fractions were pooled for analysis. SP-B concentration in BALF was estimated by ELISA. Sodium bicarbonate (0.1 M; 100 µl) was added to each well, and the plate was incubated at 4°C overnight. The plates were washed with 82b buffer (0.15 M NaCl, 0.01 M Tris, pH 7.4, 5 mg/ml BSA) containing 5% human albumin. The buffer was removed after 15 min, and 100 µl of human SP-B were added at selected concentrations [diluted in PBS with 0.5% Nonidet P-40 (NP-40)] to generate a standard curve. Aliquots of BALF were centrifuged at 500 g for 10 min, and the supernatant was then centrifuged at 10,000 g for 10 min. The surfactant pellet was resuspended in acidified ethanol (pH 3.5) followed by dilution in PBS containing NP-40. After 1–2 h at 37°C, the wells were washed three times with wash buffer (0.01 M Tris, pH 8.0, and 0.05% Tween 20), and 100 µl of bovine SP-B antibody #28031 that cross-reacts with human and mouse SP-B (15, 17) were diluted 1:1,000 in 82b containing 5% human albumin and added to each well. Following incubation for 1 h at 37°C, the wells were washed three times, and 100 µl of goat anti-rabbit horseradish peroxidase conjugate (diluted 1:1,000 in PBS containing 0.05% Tween 20 and 5% human albumin) were added. After 1 h at 37°C, the plates were washed, and 100 µl of substrate solution (0.03% H2O2, 0.037 M o-phenylenediamine in 0.065 M phosphate buffer, pH 6.3, 0.017 M citric acid) were added to each well. The reaction was stopped by adding 100 µl of 10% sulfuric acid, and the absorbance was read at 492 nm.
Surfactant phospholipids were recovered from BALF by extracting the pellet with chloroform-methanol (2:1). Phospholipid composition was assessed by two-dimensional thin-layer chromatography, as previously described (17). Total and differential cell counts in BALF were assessed as previously described (16).
Type II cell analysis. Type II cells were prepared from 6- to 8-wk-old wild-type and compound transgenic mice as recently described (22). Type II cells were resuspended in culture media in the presence or absence of doxycycline and cultured for up to 5 days on Engelbreth-Holm-Swarm/rat tail collagen (70:30). Cells were labeled with 35[S]methionine/cysteine for the last 4 h of culture on days 0, 3, and 5 and were immunoprecipitated for SP-B mature peptide (antibody #28031), as previously described (15, 22).
For fluorescence-activated cell sorting (FACS) analyses, freshly isolated type II cells were permeabilized and incubated with antibody #28031 (BD Cytofix/Cytoperm kit). The cells were washed and incubated with chicken anti-rabbit secondary antibody conjugated to Alexa Fluor 594 (Molecular Probes, Eugene, OR). FACS analyses were carried out on a BD FACSCalibur analytic flow cytometer with FL4-H gating.
Western blot analysis. Lung homogenates were prepared from wild-type and compound transgenic mice, and the total protein concentration was determined by bicinchoninic acid assay. Samples containing equal amounts of protein were subjected to SDS-PAGE under nonreducing electrophoretic conditions for analysis of SP-B mature peptide (Mr = 16,000) or under reducing electrophoretic conditions for analysis of pro-SP-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 or pro-SP-C, as previously described (15, 27).
Statistics. Compound transgenic mice from lines D and E were evaluated for pulmonary histology, type II ultrastructure, and lung function. Initial results confirmed that both transgenic lines had similar phenotypes, and subsequent studies were therefore restricted to line E. Data analysis and results are expressed as means ± SD and evaluated using Student's t-test (Figs. 1, 6, and 8) or ANOVA (Fig. 7) with significance defined as P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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To determine whether expression of SP-B in compound transgenic mice carrying the CCSP-rtTA transgene was completely dependent on doxycycline, type II cells were isolated and cultured in the absence or presence of the drug, as previously described (17, 22). Mature SP-B peptide, Mr = 16,000, was not detected in the absence of doxycycline; addition of doxycycline to the culture medium maintained SP-B expression (Fig. 2). Consistent with these findings, withdrawal of compound transgenic mice from doxycycline resulted in respiratory failure within 3–4 days (not shown). These results indicate that little or no expression of SP-B occurred in the absence of doxycycline.
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6,000) is never detected by Western blotting of lung tissues or BALF from healthy wild-type mice but is readily detected in SFTPB–/– mice; therefore, increased immunostaining for pro-SP-C in compound transgenic mice suggested that there was a substantial population of type II cells that expressed little or no SP-B. Consistent with this hypothesis, Western blot analysis of lung tissue from pnd1 pups detected similar concentrations of pro-SP-C, Mr = 6,000, in compound transgenic mice and newborn SFTPB–/– mice (Fig. 5); mature SP-C peptide concentration was not decreased in lung homogenates from compound transgenic mice (not shown). FACS analysis was therefore performed to sort freshly isolated type II cells based on SP-B content (Fig. 6). A single population of type II cells was detected in wild-type mice, suggesting that all cells contained similar amounts of SP-B (mean fluorescence index = 19.7 ± 3.3). In contrast, at least three populations of cells were identified in compound transgenic mice, including a relatively large population of cells containing less SP-B than wild-type cells (mean fluorescence index = 8.8 ± 0.4; P < 0.05 vs. wild-type cells), a population with SP-B concentration similar to wild-type cells, and a small population with elevated SP-B protein. Thus the results of immunohistochemical and FACS analysis indicated that, unlike wild-type mice, SP-B expression in type II cells of compound transgenic mice was nonuniform.
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Pressure-volume curves and lung mechanics. The effect of chronic, nonuniform SP-B deficiency on lung function was evaluated by pressure-volume curves. There were no differences in maximal tidal volume between wild-type and transgenic mice; however, there was a modest decrease in hysteresis, and lung volume at 0 pressure was significantly decreased (67% of wild type) in compound transgenic mice (Fig. 8). Assessment of lung mechanics indicated that compliance was significantly decreased in compound transgenic mice (1.67 ± 0.08 vs. 2.03 ± 0.07, P = 0.02). Airway resistance, airway elastance, tissue damping, tissue elastance, and hysteresivity in compound transgenic mice were not significantly different from wild-type mice. Together, these data suggest that chronic, nonuniform SP-B deficiency in compound transgenic mice was associated with minor alterations in lung function.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Conditional expression of SP-B in compound transgenic mice was sufficient to completely reverse neonatal lethality that is the hallmark of severe SP-B deficiency. However, the concentration of SP-B in the alveolar spaces of compound transgenic mice was only 60–70% of that in wild-type mice of the same strain. Decreased alveolar SP-B content in compound transgenic mice was due in large part to the nonuniform expression of the transgene in type II cells. The results of FACS analyses indicated that the concentration of mature SP-B peptide was similar in all type II cells of wild-type mice, whereas SP-B concentration varied widely in type II cells of compound transgenic mice. Many type II cells in compound transgenic mice contained lower levels of SP-B than wild-type cells leading to chronic deficiency of SP-B in the air spaces.
Chronic, nonuniform SP-B deficiency was associated with focal air space enlargement in two separate transgenic lines, suggesting that this phenotype was unrelated to the site of transgene integration. In SFTPB+/– mice in which alveolar SP-B content was 50% of that in wild-type mice, collapse of small airways was detected at low-deflation pressures; however, alveolar structure was normal (5). Because alveolar SP-B concentration in compound transgenic mice was higher than that in SFTPB+/– mice, the nonuniform expression of SP-B likely accounted for the alteration in lung structure. SP-B deficiency is characterized by accumulation of an incompletely processed form of pro-SP-C (Mr = 6,000) that has very poor surface activity (13, 27). Staining for pro-SP-C was clearly increased in areas of air space enlargement, indicating the presence of SP-B-deficient type II cells. Air space enlargement was not accompanied by lung inflammation or cell injury/turnover. It is therefore likely that focal SP-B deficiency resulted in instability of affected alveoli, leading to microatelectasis that was detected as a decrease in residual lung volume in pressure-volume curves. Focal alveolar collapse would, in turn, contribute to the air space enlargement detected in histological sections. Thus, chronic, nonuniform SP-B deficiency was associated with alterations in both lung structure and function.
Chronic SP-B deficiency was also associated with a progressive change in lamellar body ultrastructure. The fraction of type II cells containing lamellar bodies with a hybrid phenotype (i.e., lamellar and vesicular content) increased dramatically during the postnatal period at the expense of lamellar bodies with a wild-type or SP-B knockout phenotype. Because the absolute concentration of alveolar SP-B did not change, this outcome likely reflects the redistribution of SP-B among type II cells via recycling of the peptide from the air spaces (4, 12). The uptake of SP-B by type II cells with a knockout phenotype could result in partial correction of phospholipid packaging, leading to a hybrid lamellar body phenotype; in contrast, the overall lower amount of alveolar SP-B available for recycling may result in transition of lamellar bodies with wild-type phenotype to those with a hybrid phenotype.
SP-B was previously shown to be expressed in a nonuniform pattern in compound transgenic mice carrying the SP-C-rtTA transgene in place of the CCSP-rtTA transgene (17). However, in contrast to the results of the current study, alveolar structure, lamellar body morphology, and lung mechanics were normal in the presence of the SP-C-rtTA transgene. This outcome may reflect the fact that SP-B expression in SP-C-rtTA compound transgenic mice was similar to that in wild-type mice, making more SP-B available for recycling. Normal alveolar SP-B content was associated with complete correction of lung structure and function in SP-C-rtTA compound transgenic mice, whereas only partial correction of the phenotype was achieved in compound transgenic mice with chronic SP-B deficiency.
Full or partial correction of lung structure and function in compound transgenic mice suggested that it might be possible to treat inherited SP-B deficiency in human patients with SP-B containing surfactant replacement mixtures. However, an SP-B-deficient infant treated with surfactant at delivery developed progressive respiratory failure and died at 54 days of age despite continued postnatal treatment with an SP-B-containing surfactant (8). It is possible that lung injury related to mechanical ventilation and the initial administration of a surfactant containing very low levels of SP-B reduced the effectiveness of subsequent treatments with an SP-B-containing surfactant. It is also possible that the mature SP-B peptide is not sufficient to restore and maintain lung function in inherited SP-B deficiency. We have previously demonstrated that expression of a truncated SP-B proprotein (lacking the entire 102-amino acid COOH-terminal domain) completely reversed neonatal lethality in SFTPB–/– mice (1). Thus it is possible that the NH2-terminal propeptide of SP-B plays an as yet unidentified role in promoting lung structure and function.
Although SP-B is expressed in both nonciliated bronchiolar (Clara) cells and alveolar type II epithelial cells, synthesis and processing of the proprotein to the mature peptide occur only in type II cells (14). Restoration of SP-B protein in the alveolar spaces of compound transgenic mice therefore requires expression of both the rtTA and SP-B transgenes in the type II cell. In situ hybridization of lung sections from adult compound transgenic mice indicated that the CCSP-rtTA transgene was not expressed in all type II cells (not shown), likely accounting for SP-B-deficient cells detected by FACS. Nonuniform transgene expression was also previously observed with the SP-C promoter and varied considerably among transgenic lines (14). Although the site of transgene integration may contribute to this variation, the precise molecular basis for this phenomenon is not known.
Decreased expression of SP-B has been associated with infection by a variety of airway pathogens. SP-B content was decreased in BALF from children infected with respiratory syncytial virus (11). Intratracheal infection of mice with adenovirus resulted in diminished immunostaining for SP-B and focal loss of in SP-B mRNA expression (29). Likewise, intratracheal administration of Pneumocystis carinii to mice resulted in lower SP-B protein and mRNA (2, 3). Airway administration of endotoxin was also associated with decreased SP-B protein and mRNA in mice (7, 10). Collectively, these reports suggest that airway infection may lead to focal decreases in SP-B mRNA similar to that observed in compound transgenic mice in the current study. Chronic infection may therefore contribute to changes in lung structure and function in part through diminished SP-B expression.
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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Present address of L. L. Nesslein: St. Vincent Children's, Neonatology, 2001 W. 86th St., Indianapolis, IN 46260.
Present address of K. R. Melton: Children's Mercy Hospital, Neonatology Department, 2401 Gillham Rd., Kansas City, MO 64108.
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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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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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