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Am. J. Respir. Cell Mol. Biol., Volume 21, Number 4, October 1999 463-472

Surfactant Protein-B-Deficient Mice Are Susceptible to Hyperoxic Lung Injury

Keisuke Tokieda, Harriet S. Iwamoto, Cindy Bachurski, Susan E. Wert, William M. Hull, Kazushige Ikeda, and Jeffrey A. Whitsett

Division of Neonatology and Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio


    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Surfactant protein-B (SP-B) is a small, hydrophobic peptide that plays a critical role in pulmonary function and surfactant homeostasis. To determine whether SP-B protects mice from oxygen-induced injury, heterozygous SP-B+/- gene-targeted mice and wild-type SP-B+/+ littermates were exposed to hyperoxia (95% oxygen for 3 d) or room air. Although specific lung compliance in room air in SP-B+/- mice was slightly reduced as compared with that in SP-B+/+ mice, it was reduced more markedly during hyperoxia (46% versus 25% decrease, respectively). The larger decrease in lung compliance in SP-B+/- mice was associated with increased severity of pulmonary edema, hemorrhage and inflammation, lung permeability and protein leakage into the alveolar space. Hyperoxia increased SP-B messenger RNA (mRNA) and total protein concentrations by 2-fold in SP-B+/+ and SP-B+/- mice, but decreased the abundance of SP-B protein in lavage fluid relative to total protein only in SP-B+/- mice. Hyperoxia increased SP-B expression, but apparently not enough to maintain SP-B function and lung compliance in the presence of increased protein leakage in SP-B+/- mice. Increased alveolar-capillary leakage and relative deficiency of SP-B may therefore contribute to oxygen-induced pulmonary dysfunction in SP-B+/- mice. These data support the concept that SP-B plays an important protective role in the lung.


    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Surfactant protein-B (SP-B) is a 79-amino-acid amphipathic polypeptide that is essential for postnatal lung function. SP-B interacts strongly with phospholipids, enhancing the surface properties of pulmonary surfactant. The lack of SP-B in gene-targeted mice and in humans with null mutations in the SP-B gene causes lethal respiratory failure at birth (1, 2). SP-B deficiency disrupts lamellar body formation and intracellular routing of surfactant phospholipids and proteins, and blocks tubular myelin formation. Recent findings in SP-B+/- mice demonstrated that SP-B messenger RNA (mRNA) and protein were reduced to approximately 50% of that in wild-type SP-B+/+ mice, demonstrating the importance of gene dosage in controlling SP-B gene expression (3, 4). Although SP-B+/- mice have no clinical symptoms under normal conditions, pulmonary function tests demonstrated a modest decrease in lung compliance in adult SP-B+/- mice as compared with SP-B+/+ littermates (3), raising the possibility that decreased SP-B may enhance the susceptibility to pulmonary dysfunction.

Exposure to hyperoxia causes acute injury that often complicates the clinical management of various pulmonary disorders. Oxygen injury is associated with pulmonary edema, hemorrhage, and increased alveolar-capillary leakage (5). Alveolar-capillary leakage increases the abundance of plasma protein in the alveolar space and interferes with surfactant function. Hyperoxia is associated with increased transcription of a number of genes that are thought to be involved in protection against oxygen injury (10). Synthesis of surfactant proteins-A, -B, and -C (SP-A, SP-B, and SP-C) is altered by hyperoxia (13), suggesting that these proteins may serve protective roles in maintaining lung function during oxygen-induced injury. The present study was designed to test whether decreased SP-B content in the lungs and bronchoalveolar lavage fluid (BALF) of SP-B+/- gene-targeted mice increases their susceptibility to oxygen-induced injury in vivo.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Mice

All procedures were approved by the Institutional Animal Care and Use Committee at the Children's Hospital Research Foundation. SP-B+/+ and SP-B+/- mice were produced by mating SP-B+/- mice. SP-B-deficient mice were generated by ablation of the murine SP-B gene through insertion of the neomycin resistance gene into the fourth exon of the murine gene in embryonic stem cells (D3), as previously described (1). DNA was isolated from tail samples, and the genotype of the resulting mice was determined through amplification with the polymerase chain reaction (PCR). At 12-20 wk of age, SP-B+/- and SP-B+/+ littermates were placed in a Plexiglas chamber in which the O2 concentration was maintained at 95% at 1 atmosphere for 72 h. Control mice breathed room air for the same period. In separate groups of animals, lung compliance and volumes, routine histologic analyses, immunohistochemistry for SP-B, proSP-B and proSP-C, quantitation of SP-B and total protein content in BALF, and changes in surfactant protein mRNA levels in lung tissue were determined as subsequently described.

Lung Compliance

SP-B+/+ and SP-B+/- mice were exposed to 95% O2 for 3 d or to room air (n = 10 in each group). Mice were injected with sodium pentobarbital (200 mg/kg) and placed in a container containing 100% O2 to ensure complete collapse of the alveoli by oxygen absorption. The trachea was cannulated and connected to a syringe and a pressure sensor (X-ducer; Motorola, Phoenix, AZ) via a three-way connector. After the diaphragm was opened, the lungs were inflated in 100-µl increments every 20-30 s to a maximum inflation pressure of 30 cm H2O, and were then deflated. Pressure and volume on inflation and deflation were recorded. Pressure-volume curves were generated for each animal. Specific lung compliance was determined by dividing the slope of the deflation portion of the curve, where pressure was usually less than 10 cm H2O, by the average lung volume in that portion of the curve (16). Maximum lung volume was taken as the volume of the lung at an inflation pressure of 30 cm H2O.

mRNA Analysis

SP-B+/+ (n = 13) and SP-B+/- (n = 12) mice were exposed to O2 or room air and killed. Lungs were frozen immediately in liquid nitrogen and stored at -80°C until analysis. Total lung RNA was extracted with the acid guanidinium- phenol-chloroform technique and the Phase Lock Gel II System (5Prime-3Prime, Boulder, CO). SP-B and ribosomal protein L32 mRNAs were detected by S1 nuclease protection analysis as described previously (17). Briefly, linearized probes for SP-B and L32 were end-labeled with [gamma -32P]adenosine triphosphate ([gamma -32P]ATP), and combined and hybridized overnight at 56°C with 3 µg of total lung RNA. Protected fragments were liberated by digestion with 110 units of S1 nuclease in the presence of excess unlabeled carrier DNA for 1 h at room temperature. The fragments were resolved by electrophoresis in an 8 M urea 6% polyacrylamide gels, visualized by autoradiography, and quantitated by PhosphorImaging (Storm 860°; Molecular Dynamics, Sunnyvale, CA). PhosphorImage units for SP-B mRNA were normalized to units of L32 mRNA for each lane.

SP-B Protein Analysis

Bronchoalveolar lavage was accomplished by tracheal cannulation after intraperitoneal injection of pentobarbital. Lungs were lavaged with three 1-ml aliquots of 0.15 M saline. The SP-B concentration in pooled samples from each animal was determined though enzyme-linked immunosorbent assay (ELISA) analysis, using purified bovine SP-B as the standard (18). Several dilutions of the samples were assessed to ensure linearity of dilutions. Total protein in pooled samples was determined according to the method of Lowry and coworkers, using bovine serum albumin as the standard (38). SP-B concentration was expressed as ng/ml lavage fluid and as ng SP-B/mg protein.

Histology and Immunohistochemistry

Three mice of each genotype were exposed to 95% O2 or room air for 3 d. The mice were anesthetized with sodium pentobarbital and exsanguinated by clipping the inferior vena cava and descending aorta. The trachea was cannulated and the lungs were collapsed by piercing the diaphragm. The lungs were inflation-fixed for 1 min at a pressure of 25 cm H2O with 4% paraformaldehyde in phosphate-buffered saline (PBS). The cannula was then removed, and the trachea was tied off. The isolated lungs were immersed in cold fixative for an additional 24 h, rinsed in PBS, dehydrated, and embedded in paraffin. Deparaffinized, rehydrated, 5-µm sections were stained with hematoxylin and eosin (H&E) for histopathologic analysis or with antisera to SP-B, proSP-B, or proSP-C, using immunohistochemical techniques described previously (19, 20).

Tissue sections were preincubated with normal goat serum and then incubated overnight at 4°C with rabbit polyclonal antibodies generated against mature SP-B (R28031), recombinant proSP-B (R96189), or proSP-C (R68514) peptides (dilution: SP-B = 1:2,000; proSP-B = 1:1,000; and proSP-C = 1:1,000). Antisera were characterized previously by standard immunochemical analyses; the specificity of each antibody was confirmed by competition with its respective peptide (19, 21). The sections were then incubated with biotinylated antirabbit IgG secondary antibody for 30 min. Antibody binding was detected with an avidin-biotin-peroxidase detection system (Vectastain Elite ABC kit; Vector Laboratories, Inc., Burlingame, CA). The enzymatic reaction product was enhanced with nickel cobalt, and the treated tissue sections were counterstained with nuclear fast red.

In preliminary studies, the size of the alveoli in each group was measured using video capture and computer- assisted image analysis (MetaMorph Imaging Software; Universal Imaging Corp., West Chester, PA). Longitudinal and cross-sectional profiles of large alveolar ducts were excluded from the study, and only fields with groups of single, roughly circular alveoli were included in the quantitation. The mean alveolar diameter in the oxygen-injured SP-B+/+ mice increased from 26 µm (range: 23-27 µm) before O2 exposure to 30 µm (range: 27-32 µm) after O2 exposure. In the SP-B+/- mice, the mean alveolar diameter increased from 25 µm (range: 23-27 µm) before O2 exposure to 35 µm (range: 32-37 µm) after O2 exposure. The overall abundance of immunostaining was estimated as the number of alveoli that stained positively per 100 alveoli. An alveolus was defined as a single, roughly circular unit with an estimated diameter of 20-40 µm. If an alveolus was stained anywhere along its circumference, it was considered positive. Three fields in two different lobes from each of three animals per group were analyzed by three independent viewers.

Lung Permeability

Four mice of each genotype were exposed to 95% O2 for 0, 24, 48, or 72 h. Five microcuries of [125I]albumin (4.45 µCi/µg; NEN Life Science Products, Boston, MA) were incubated with Sephadex G-50 resin to remove free [125I] ions; the supernatant was injected intraperitoneally. Two hours later, mice were anesthetized (200 mg/kg sodium pentobarbital intraperitoneally) and weighed. The trachea was cannulated and the lungs were lavaged with five 1-ml aliquots of cold 0.15 M NaCl, with care taken to prevent overinflation of the lungs. This procedure has been shown to recover over 85% of alveolar radioactivity (22). Duplicate aliquots of the injectate and lavage fluid were counted in a gamma counter (Multiprias 4; Packard Instruments, Downers Grove, IL). The total amount of radioactivity in lavage fluid was determined, and the percentage of total counts that was recovered in the lavage fluid was calculated as an estimate of lung permeability.

Statistical Analysis

Differences between SP-B+/+ and SP-B+/- mice were assessed by two-way analysis of variance, and differences between means were assessed by contrast comparisons and the Student-Newman-Keuls test (StatView; Abacus Concepts, Inc., Berkeley, CA). Data are expressed as means ± SD.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

All SP-B+/+ mice (n = 28) and 27 of 29 SP-B+/- mice survived exposure to 95% O2 for 72 h. Exposure to 95% O2 for 3 d significantly decreased body weight and increased lung weight in both SP-B+/+ and SP-B+/- mice (Table 1). Body weights of SP-B+/+ and SP-B+/- mice exposed to O2 were 13.5% and 15.7% less than those of mice exposed to room air (P < 0.01), and lung weights were significantly greater in mice exposed to 95% O2 (P < 0.01). Oxygen exposure increased the lung-to-body weight ratio significantly in SP-B+/+ and SP-B+/- mice (P < 0.001).

                              
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TABLE 1
Hyperoxia alters lung and body weights in SP-B+/+ and SP-B+/- mice

In animals exposed to room air, specific lung compliance in SP-B+/- mice was significantly lower than that of SP-B+/+ mice (Figure 1). Hyperoxia decreased lung compliance by 46% in SP-B+/- mice, but only by 25% in SP-B+/+ mice (Figure 1). Total lung capacity, defined as the lung volume at an inflation pressure of 30 cm H2O, was decreased in hyperoxic SP-B+/- mice as compared with room air controls (711 ± 306 µl versus 1,086 ± 90 µl; P < 0.05) but was not altered in SP-B+/+ mice (Figure 1).


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  		Figure 1.   Hyperoxia decreases lung compliance in SP-B+/+ and SP-B+/- mice. Lung compliance was determined in SP-B+/+ (open bars) and SP-B+/- (closed bars) mice by inflating and deflating the lungs in 100-µl increments to a maximum pressure of 30 cm H2O. Lung compliance was reduced in SP-B+/- mice compared with SP-B+/+ mice after exposure to 95% O2 for 3 d and in room air controls (# P < 0.001). Hyperoxia significantly decreased lung compliance in both SP-B+/+ and SP-B+/- mice (*P < 0.005). Maximum lung volume, defined as lung volume at an inflation pressure of 30 cm H2O, was similar in the room air control groups. Hyperoxia significantly decreased lung volume only in SP-B+/- mice (P < 0.0001; n = 10 in each group).

As previously reported (3), SP-B mRNA in lungs from SP-B+/- mice was approximately 50% of that in SP-B+/+ mice (Figure 2). Hyperoxia increased SP-B mRNA by approximately 2-fold in both SP-B+/+ and SP-B+/- mice, but SP-B mRNA in lungs from SP-B+/- mice remained about half that in SP-B+/+ mice (Figure 2).


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  		Figure 2.   Hyperoxia increases SP-B mRNA synthesis in SP-B+/+ and SP-B+/- mice. On the top is a representative autoradiogram of an S1 nuclease protection assay detecting SP-B mRNA performed on total RNA (3 µg) harvested from the lung. Samples from two mice in each group were assayed. The graph on the bottom shows the summary data of all S1 nuclease assays from SP-B+/+ (open bars) mice exposed to room air (n = 5) or hyperoxia (n = 8), and from SP-B+/- mice (closed bars) exposed to room air (n = 5) or hyperoxia (n = 7). Data are the means ± SD of the SP-B mRNA values normalized to the L32 values (*significantly different from room air group in the same genotype; #significantly different from SP-B+/+ genotype in the same treatment group).

The SP-B concentration in lavage fluid was approximately 2-fold greater in SP-B+/+ than in SP-B+/- mice (P < 0.05) (Figure 3). Hyperoxia increased SP-B concentrations in BALF in both groups of mice, but SP-B concentrations in SP-B+/- mice remained approximately half those in SP-B+/+ mice (P < 0.01). Total protein concentration in lavage fluid was initially the same in both groups but increased to a greater extent in SP-B+/- than in SP-B+/+ mice (P < 0.05). As a result, SP-B protein normalized to total protein increased in SP-B+/+ mice but decreased dramatically in SP-B+/- mice (P < 0.01).


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  		Figure 3.   Hyperoxia increases SP-B and total protein in lavage fluid. Hyperoxia increased the amount of SP-B (measured with an ELISA) and total protein recovered in BALF in SP-B+/+ mice (open bars; n = 5, room air; n = 6, hyperoxia) and SP-B+/- mice (closed bars; n = 4, room air; n = 6, hyperoxia). Hyperoxia increased SP-B normalized to total protein in SP-B+/+ mice but decreased it in SP-B+/- mice (*significantly different from room air values; # significantly different from SP-B+/+ values in the same treatment group).

There were no differences in lung permeability between SP-B+/+ and SP-B+/- mice in room air or after 1 d of oxygen exposure (Figure 4). After 2 d of oxygen exposure, lung permeability increased significantly in SP-B+/+ mice (P < 0.05). After 3 d, lung permeability increased in both groups relative to baseline values, but the increase in permeability in SP-B+/- mice was greater than that in SP-B+/+ mice (P < 0.005).


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  		Figure 4.   Hyperoxia increases lung permeability. Lung permeability was estimated as the percentage of [125I]albumin recovered in lavage fluid 2 h after intraperitoneal injection of 5 µCi of [125I]albumin. Lung permeability increased in SP-B+/+ mice (open bars) with 2 d of hyperoxia. Lung permeability in SP-B+/- mice (closed bars) increased within 3 d to an extent that was significantly greater than in SP-B+/+ mice (P < 0.005) (*significantly different from room air values; # significantly different from SP-B+/+ values in the same treatment group).

Before oxygen exposure, the appearance of the lungs of SP-B+/- and SP-B+/+ mice at the light-microscopic level was similar. After exposure to hyperoxia, diffuse pulmonary injury, including epithelial necrosis, microhemorrhage and congestion, inflammation, and intraalveolar exudates and cellular debris was observed in lungs from both SP-B+/- and SP-B+/+ mice (Figure 5).


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  		Figure 5.   Effect of hyperoxia on pulmonary histology. Lung tissue was harvested from SP-B+/+ (A) and SP-B+/- (B through D) mice after 0 (A and B) or 3 (C and D) d of exposure to 95% O2. Tissue was fixed in 4% paraformaldehyde, embedded in paraffin, cut (5-µm sections), and stained with H&E. In SP-B+/+ and SP-B+/- mice exposed to room air (A and B, respectively), the lungs appeared normal with regularly-shaped alveoli in the peripheral lung. After exposure of SP-B+/- mice to hyperoxia (C and D), diffuse alveolar, vascular, and bronchoepithelial damage was present and was characterized by congestion (increased numbers of erythrocytes) in the alveoli, alveolar septal thickening and disruption, perivascular swelling (C, arrows), and bronchoepithelial erosion and sloughing (D, arrows). Bar = 36 µm.

Although the cellular localization and distribution of SP-B, proSP-B, and proSP-C staining were similar in lungs from SP-B+/+ and SP-B+/- mice (Table 2), cytoplasmic staining for both proSP-B and SP-B was less intense in SP-B+/- mice than in SP-B+/+ mice (Figures 6 and 7). As previously reported, proSP-C was detected in the lung parenchyma, consistent with the distribution of type II cells, whereas SP-B and proSP-B were detected in both bronchiolar and alveolar cells of SP-B+/+ and SP-B+/- mice (Figures 7 and 8). After 3 d of O2 exposure, the overall staining intensity for SP-B and proSP-B in type II cells decreased in SP-B+/+ mice and became nearly undetectable in SP-B+/- mice as compared with mice exposed to room air (Table 2; Figures 6 and 7), whereas staining for SP-B increased in alveolar macrophages of SP-B+/+ mice (Figure 6). Overall staining for pro-SP-C decreased to comparable levels in both groups. Cytoplasmic staining for proSP-B was less intense than that for SP-B in SP-B+/- mice, resulting in a decrease in the overall number of detectable proSP-B-positive cells in hyperoxia-exposed SP-B+/- mice (Table 2). Staining for SP-B in the bronchiolar epithelium was variable from one section to another in both room air- and hyperoxia-exposed mice of both genotypes (not shown). Staining for proSP-B, however, was detected consistently throughout the bronchiolar epithelium of mice of both genotypes (Figure 8). Staining for proSP-B was increased slightly in the bronchiolar epithelium of SP-B+/+ mice exposed to hyperoxia (Figure 8). ProSP-B-positive material was found primarily along the apical cell surface and in sloughed cells and cellular debris lining the bronchiolar epithelium of mice of both genotypes (Figure 8).

                              
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TABLE 2
Hyperoxia alters surfactant protein distribution in the lungs of SP-B+/+ and SP-B+/- mice


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  		Figure 6.   Effect of hyperoxia on SP-B and proSP-B expression in the alveolar epithelium. Immunohistochemical detection of SP-B (A through D) and proSP-B (E through H ) was done in lung tissue harvested from SP-B+/+ (A, B, E, and F ) and SP-B+/- (C, D, G, and H ) mice after 0 (panels on left, room air [RA]) and 3 (panels on right, O2) d of exposure to 95% O2. Immunohistochemistry was performed as described in MATERIALS AND METHODS. The immunoperoxidase reaction product was enhanced with nickel-cobalt to give a black precipitate, and the sections were counterstained with nuclear fast red. In both SP-B+/+ and SP-B+/- mice exposed to RA, SP-B and proSP-B were easily detected in alveolar type II cells (A, C, E, and G). Staining intensity for both SP-B and proSP-B was reduced, however, in SP-B+/- mice (C and G). After exposure to hyperoxia (O2), immunostaining for SP-B was reduced in type II cells (arrows) and increased in alveolar macrophages (arrowheads) of the SP-B+/+ mice (B); staining for proSP-B was not detected (F ). Neither SP-B nor proSP-B was detected in type II cells or in alveolar macrophages of SP-B+/- mice exposed to hyperoxia (D and H). Bar = 50 µm.


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  		Figure 7.   Effect of hyperoxia on SP-B, proSP-B, and proSP-C expression in type II cells. Higher magnification of intracellular staining patterns for SP-B (A through D), proSP-B (E through H ), and proSP-C (I and J ) in type II cells of SP-B+/+ (A, B, E, and F ) and SP-B+/- (C, D, G, and H through J ) mice after 0 (panels on left, room air [RA]) and 3 ( panels on right, O2) d of exposure to 95% O2. Immunostaining for both SP-B and proSP-B in type II cells (arrows) was more intense in SP-B+/+ mice (A and E) than in SP-B+/- mice (C and G) exposed to RA. After exposure to hyperoxia (O2), immunostaining for SP-B was significantly reduced in type II cells (arrows) of the SP-B+/+ mice (B). Immunostaining for proSP-B was not detected in any of the type II cells (arrows) of SP-B+/+ mice exposed to hyperoxia (F ). Neither SP-B nor proSP-B was detected in type II cells of SP-B+/- mice exposed to hyperoxia (D and H ). ProSP-C was easily detected in the type II cells of both SP-B+/+ (not shown) and SP-B+/- mice after 0 (I ) and 3 d (J ) of exposure to 95% O2. Bar = 30 µm.


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  		Figure 8.   Effect of hyperoxia on proSP-B expression in the bronchiolar epithelium. Immunohistochemical detection of proSP-B was done in lung tissue harvested from SP-B+/+ (left panels) and SP-B+/- (right panels) mice after 0 (A through D) and 3 (E and F ) d of exposure to 95% O2. ProSP-B was detected in cells (arrows) of the bronchiolar epithelium in both SP-B+/+ (A and C) and SP-B+/- (B and D) mice exposed to room air (RA). Not all of the cells in the bronchiolar epithelium were positive for proSP-B (A and B), and in the terminal bronchioles, composed primarily of Clara cells, only a subpopulation of cells (arrows) was positive for proSP-B in both SP-B+/+ (C) and SP-B+/- mice (D). Staining intensity for proSP-B was reduced in the bronchiolar cells (arrows) of SP-B+/- mice (B and D). After exposure to hyperoxia (O2), immunostaining for pro- SP-B was detected at the apical cell surface (arrows) and in sloughed cells and cellular debris (arrowheads) lining the bronchiolar epithelium in both SP-B+/+ (E) and SP-B+/- (F ) mice. Bar for A, B = 100 µm; bar for C- F = 50 µm.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The effects of oxygen exposure on the adult mammalian lung include alveolar septal thickening, edema, inflammation, and breakdown of the epithelial and endothelial barriers (5). Progression of these responses during continued exposure to high concentrations of oxygen leads eventually to death. In the present study, we showed that mice heterozygous for SP-B were more susceptible to the deleterious effects of hyperoxia than were their wild-type littermates. Hyperoxia decreased lung compliance significantly in SP-B+/+ and SP-B+/- mice, but the decrease was significantly greater in SP-B+/- mice. Hyperoxia increased SP-B mRNA to a similar extent in both wild-type and SP-B+/- mice, but the levels in SP-B+/- mice were half those in SP-B+/+ mice, and the concentration of immunoreactive SP-B and the ratio of SP-B to total protein in BALF was significantly lower in SP-B+/- mice. This was probably due to the greater increase in permeability with hyperoxia in the SP-B+/- mice. These findings reveal an increased susceptibility of SP-B+/- mice to hyperoxia-induced lung injury and support the concept that alveolar SP-B plays an important role in maintaining lung function during hyperoxia. The findings are consistent with previous in vitro findings demonstrating the protective effects of surfactant proteins on surfactant function during lung injury and alveolar-capillary leakage (8, 23, 24).

SP-B mRNA and protein concentrations show regional differences and are subject to complex regulatory influences at both the transcriptional and posttranscriptional levels. Confirming previous reports, lung SP-B mRNA and SP-B protein in lavage fluid of wild-type mice increased after exposure to 95% O2 for 3 d (13). SP-B mRNA and protein concentrations in SP-B+/- mice, initially one-half as abundant as in wild-type mice, also increased, but only to levels that were about half those of SP-B+/+ mice. These results are consistent with our previous observations that gene dosage influences SP-B expression (1, 3, 4) and further demonstrate that the response of the remaining SP-B allele to hyperoxia was maintained.

Despite the increase in BALF SP-B content, hyperoxia decreased cytoplasmic staining for SP-B, proSP-B, and proSP-C in the lung parenchyma of both SP-B+/+ and SP-B+/- mice. Furthermore, staining for proSP-B and SP-B was virtually undetectable in type II cells of hyperoxia-exposed SP-B+/- mice. In the conducting airways, proSP-B-positive material was found primarily in association with sloughed cells and cellular debris lining injured bronchiolar epithelium of mice of both genotypes. These data suggest that hyperoxia increased SP-B secretion, leading to a depletion of intracellular stores of this surfactant protein.

It is unclear from the immunohistochemical analyses which cells contributed to the increased SP-B concentration in BALF during hyperoxia. Previous studies in our laboratory demonstrated that hyperoxia increased SP-B mRNA in a regionally selective manner, whereby SP-B expression increased in bronchiolar cells but decreased in alveolar type II cells (14). Although cytoplasmic staining for SP-B and proSP-B decreased in type II cells, there was very little increased staining for SP-B and proSP-B in the bronchiolar epithelium of hyperoxia-exposed mice in the current study. Several reasons may exist for this discrepancy. In contrast to type II cells, Clara cells may synthesize, process, and secrete SP-B so rapidly that very little protein is actually stored or accumulates in these cells. Thus, the intracellular stores of SP-B may be too low to be detectable at the dilutions of antiSP-B antiserum used in this study. On the other hand, there is some evidence that Clara cells may not process proSP-B to the mature SP-B peptide. For example, processing of proSP-B in human pulmonary adenocarcinoma cells with Clara cell-like characteristics (H441 cells) is incomplete, resulting in large proteolytic fragments (16 kD and 27-32 kD), with no production of the mature SP-B peptide (25). This is in contrast to processing of proSP-B to the mature SP-B peptide in isolated rat type II cells (26). In addition, SP-B null mutant mice that have been crossed with CCSP-hSP-B transgenic mice and express SP-B mRNA only in Clara cells develop respiratory distress and die shortly after birth (27). Although the full-length proSP-B peptide is synthesized in the Clara cells of these transgenic mice, no mature SP-B peptide can be detected, and both the 42-kD proprotein and 25-kD processing intermediate are detected in the BALF of these mice. Therefore, we cannot say for certain that the increased amount of SP-B appearing in BALF after hyperoxia in the present study was derived from the bronchiolar epithelium.

Despite the observation that hyperoxia increased SP-B protein recovered in lavage fluid from both SP-B+/+ and SP-B+/- mice, there were differences in the relative amounts of SP-B and protein in BALF from the two genotypes of mice. In the SP-B+/- mice, lung permeability and total protein in BALF increased to a greater extent than in SP-B+/+ mice, resulting in a larger decrease in the SP-B/protein ratio in SP-B+/- mice after hyperoxia. Because serum proteins interfere with surfactant function (28), these results indicate that the SP-B+/- mice may be unable to produce SP-B in amounts sufficient to maintain alveolar stability and lung compliance when alveolar-capillary permeability increases during O2 exposure. Differences in susceptibility to hyperoxia have been shown to be associated with differences in lung permeability in different strains of mice (29) and in mice lacking Clara-cell secretory protein (30).

Hyperoxia induces a number of different responses in the production of pulmonary surfactant across species (15). Lipid concentrations decrease in mice (31) and rabbits (23) but increase in hamsters (32) in response to hyperoxia, whereas surfactant protein expression increases in rats (13, 33) and mice (14). In hamsters exposed to hyperoxia, SP-B expression increases whereas SP-A and SP-C expression decreases only after an initial increase (32). Exposure of premature baboons to hyperoxia results in an increase in both SP-B and SP-C mRNA without a significant change in SP-A mRNA (34). The most likely explanation for the results presented here is that the effective concentration of SP-B in hyperoxia-exposed mice decreased as a result of an increase in lung permeability. Increases in lung permeability are accompanied by serum protein leakage into the alveolar compartment, which has been shown to alter the ability of SP-B and of pulmonary surfactant in general to reduce surface tension (28). In addition, exposure to hyperoxia may interfere with surfactant function through the formation of peroxynitrite, free radicals, and their reactive intermediates---all of which are oxidizing agents that may damage lipids and proteins directly (35).

The present findings show that an approximate 50% reduction in BALF SP-B was associated with markedly decreased lung compliance after O2 exposure, providing support for the concept that decreased SP-B may impart susceptibility to pulmonary oxygen toxicity. Of clinical relevance is that the SP-B content in BALF is decreased in a variety of clinical conditions including prematurity, adult respiratory distress syndrome (ARDS), and pneumonia, and after viral infection (38). Decreased SP-B mRNA and protein synthesis are also observed after exposure of pulmonary adenocarcinoma cells to tumor necrosis factor (TNF)-alpha or phorbol esters, these effects being mediated, at least in part, by the destabilization of SP-B mRNA (36, 37). Likewise, intratracheal administration of TNF-alpha or of replication-deficient adenovirus decreases SP-B mRNA or protein (17, 20), suggesting that various forms of lung inflammation may alter SP-B gene expression or SP-B homeostasis, thereby conferring increased susceptibility of the lung to oxygen-induced injury.

Furthermore, although human hereditary SP-B deficiency is lethal in the neonatal period, the finding that SP-B+/- mice are susceptible to oxygen injury raises concerns about the possible susceptibility to such injury of heterozygous SP-B carriers of mutant SP-B alleles, who may also be deficient in SP-B. Genetic or acquired SP-B deficiency may therefore create an increased risk of lung dysfunction after lung inflammation and O2 exposure. The present findings suggest that therapeutic strategies designed to enhance SP-B gene expression or to provide exogenous SP-B as surfactant replacement may be useful therapies for various lung disorders, including respiratory distress syndrome (RDS), ARDS, and viral or bacterial infections. We speculate that the clinical benefits of surfactant replacement during oxygen exposure may be mediated, at least in part, by the protective effects of SP-B on surfactant function.

    Footnotes

Abbreviations: bronchoalveolar lavage fluid, BALF; messenger RNA, mRNA; surfactant protein B, SP-B.

(Received in original form May 27, 1998 and in revised form April 5, 1999).

Acknowledgments: The authors gratefully acknowledge Brenda Wynn, Sherri Profitt and Tim Grimes for expert technical assistance, and Ann Maher for excellent secretarial support. This work was supported by grants HL56387 (J.A.W., S.E.W., H.S.I.), and HL38859 (J.A.W.) from the National Institutes of Health, and by the Cystic Fibrosis Foundation (J.A.W., S.E.W.)
    References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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