Pneumonitis and Emphysema in sp-C Gene Targeted Mice*

Stephan W. GlasserDagger §, Emily A. DetmerDagger , Machiko IkegamiDagger , Cheng-Lun NaDagger , Mildred T. Stahlman, and Jeffrey A. WhitsettDagger

From the Dagger  Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039 and the  Department of Pediatrics/Division of Neonatology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2370

Received for publication, October 25, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SP-C-deficient (SP-C -/-) mice developed a severe pulmonary disorder associated with emphysema, monocytic infiltrates, epithelial cell dysplasia, and atypical accumulations of intracellular lipids in type II epithelial cells and alveolar macrophages. Whereas alveolar and tissue surfactant phospholipid pools were increased, levels of other surfactant proteins were not altered (SP-B) or were modestly increased (SP-A and SP-D). Analysis of pressure-volume curves and forced oscillatory dynamics demonstrated abnormal respiratory mechanics typical of emphysema. Lung disease was progressive, causing weight loss and cardiomegaly. Extensive alveolar remodeling was accompanied by type II cell hyperplasia, obliteration of pulmonary capillaries, and widespread expression of alpha -smooth muscle actin, indicating myofibroblast transformation in the lung parenchyma. Dysplastic epithelial cells lining conducting airways stained intensely for the mucin, MUC5A/C. Tissue concentrations of proinflammatory cytokines were not substantially altered in the SP-C (-/-) mice. Production of matrix metalloproteinases (MMP-2 and MMP-9) was increased in alveolar macrophages from SP-C (-/-) mice. Absence of SP-C caused a severe progressive pulmonary disorder with histologic features consistent with interstitial pneumonitis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SP-C is a 34-35-amino acid peptide expressed selectively in type II epithelial cells in the alveolus of the lung (for review see Refs. 1 and 2). A single sp-C gene is located on human chromosome 8 that is syntenic to that in the mouse located on chromosome 14. The sp-C gene encodes a proprotein of 197 or 191 amino acids (pro-SP-C) that is palmitoylated, proteolytically processed, and routed through the rough endoplasmic reticulum and multivesicular bodies to lamellar bodies in which surfactant is stored. The SP-C peptide is secreted into the airspace where it enhances the stability and spreading of phospholipids. The SP-C peptide is highly hydrophobic and also contains two cysteine residues in an NH2-terminal domain. These cysteines are palmitoylated and located near an extended hydrophobic domain wherein 19 of 23 residues are valine, leucine, or isoleucine. This hydrophobic region forms an alpha -helical structure that spans a lipid bilayer (3). Both the alpha -helical domain and the cysteine-linked palmitoyl groups are tightly associated with phospholipids. SP-C disrupts phospholipid acyl chain packing and enhances recruitment of phospholipids to monolayers and multilayers at the air-liquid interface (4, 5). These features suggest a structural role for SP-C in facilitating the movement of phospholipids between multilayered films. Biological functions of purified SP-C or synthetic SP-C peptides are highly active in vitro and in vivo, enhancing surfactant properties of lipids and restoring lung function in surfactant-deficient animals (6, 7). These results indicate that SP-C plays an important role in the spreading and stabilization of phospholipid films in the alveolus.

An unexpected role for SP-C in pulmonary homeostasis was provided by recent studies (8, 9) demonstrating that a mutation in the sp-C gene was associated with idiopathic interstitial pneumonitis (IIP)1 in humans. Pulmonary disease in these patients was inherited as an autosomal dominant trait. Interstitial pneumonitis includes various pulmonary disorders including desquamating interstitial pneumonitis, usual interstitial pneumonitis, nonspecific interstitial pneumonitis, and other disorders broadly termed idiopathic interstitial pneumonitis (IIP) (10). Individuals with these disorders usually present with progressive lung disease associated with exercise limitation, tachypnea, and shortness of breath. Because mutations in the SP-C proprotein resulted in the production of an abnormal pro-SP-C peptide that was not fully processed, it has been unclear whether the lack of SP-C per se or misfolding of pro-SP-C or SP-C was involved in the pathogenesis of IIP in these patients (5). In general, various forms of IIP are associated with alveolar inflammation, pulmonary infiltration with monocytes-macrophages, progressive loss of alveolar structure, and pulmonary fibrosis (10). The molecular mechanisms involved in the pathogenesis of IIP have been elusive despite well recognized histologic and clinical manifestations.

Targeted disruption of the sp-C gene in outbred Swiss black mice resulted in mild abnormalities in lung function consistent with decreased stability of the surfactant film at low lung volumes (11). In this outbred genetic background, sp-C gene targeted mice had only subtle alterations in lung mechanics that were exacerbated by oxygen-induced injury and an accompanying deficiency in SP-B (12). In the present study, the SP-C (-/-) allele was studied in a congenic 129/Sv strain. SP-C-deficient mice developed severe, progressive pulmonary disease associated with emphysema, alpha -smooth muscle actin staining, monocytic infiltrates, and epithelial cell dysplasia in conducting and peripheral airways.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals-- SP-C (-/-) mice were generated by targeted gene inactivation as described previously (11). Chimeric founder mice were bred to 129/Sv mice, Taconic Farms (Germantown, NY). Offspring were screened for transmission of the targeted SP-C allele by genomic Southern blot analysis. Animals positive for the targeted allele were bred to establish 129/Sv mice that were homozygous for the targeted SP-C allele. Mice were maintained in a barrier containment facility. All animals were handled under aseptic condition and caged in sterilized units with filtered air, water, and autoclaved food. Sentinel mice from this room were negative for common viral, bacterial, or parasitic pathogens. At 12 months of age, lung homogenates prepared under aseptic conditions from SP-C (-/-) and wild type littermates did not contain bacteria or fungus. Serology for 23 mouse viral pathogens was negative.

Morphological Analysis-- Mice were killed by intraperitoneal injection of a mixture of ketamine, xylazine, and acepromazine. Lungs were inflated by intratracheal instillation of 4% paraformaldehyde at a pressure of 25 cm H2O. After overnight fixation, the tissue was processed through conventional paraffin embedding. Six-micron tissue sections were stained with hematoxylin-eosin, Mason's trichrome stain, or orcein stain. Immunohistochemical staining was performed for MAC-3, MUC5A/C, Clara cell secretory protein, SP-B, TTF-1, and alpha -SMA using biotinylated primary or secondary antibodies and avidin-biotin peroxidase (Vector Elite ABC Kit, Vector Laboratories, Inc., Burlingame, CA) or streptavidin (Zymed Laboratories Inc.) using methods described previously (13). Electron microscopy was performed on lung tissue obtained from 9-month-old SP-C (-/-) and age-matched controls after fixation in glutaraldehyde as described previously (14).

Morphometry-- Quantitation of distal airspace was performed from hematoxylin- and eosin-stained lung tissue sections from 12-month-old mice. Three representative fields of lung from 3 mice of each genotype were analyzed. Morphometric measurements of terminal airspace area were made using an Image-1/Metamorph Imaging System (Universal Imaging). Calibration measurements were made from images of a micrometer grid-ruler at ×10 magnification, allowing the computer to calculate pixels/mm at this power. The image analysis program allowed areas of blood vessels or bronchiolar airways to be excluded from area measurements. Measurements were recorded in square micrometers.

Phospholipid and Surfactant Proteins-- Fifteen-month-old mice (n = 5/group) were anesthetized with pentobarbital sodium (100 mg/kg intraperitoneally) and killed by exsanguination. Trachea was cannulated, and five 1-ml aliquots of 0.9% NaCl were flushed into the lungs and withdrawn by syringe three times for each aliquot. The lavaged lung tissue was removed and homogenized in 2 ml of 0.9% NaCl. Saturated phosphatidylcholine (SatPC) in lipid extracts of bronchoalveolar lavage fluid (BALF) and lung tissue were isolated with osmium tetroxide (15) followed by phosphorus measurement (16), as described previously (11). For phospholipid composition analyses, extracted lipids of lung tissue after BAL were used for two-dimensional thin layer chromatography (17). The spots were visualized with iodine vapor, scraped, and assayed for phosphorus content. Surfactant proteins in BALF were analyzed by Western blot after SDS-PAGE (11, 18).

Cytokine Measurements-- Concentrations of tumor necrosis factor-alpha , IL-1beta , IL-13, and IL-6 were measured in BALF and in whole lung homogenates post-lavage. Five animals of each genotype were assessed. Enzyme-linked immunosorbent assay kits were used according to manufacturer's instructions (R & D Systems, Minneapolis, MN).

MMP Activity-- Matrix metalloproteinase (MMP-2 and MMP-9) activity was measured in macrophage-conditioned media collected from 12-month-old SP-C (-/-) or SP-C (+/+) 129/Sv mice, as described previously (19). Macrophages were isolated by sequential lung lavage with 1 ml of phosphate-buffered saline. Lavages were pooled and placed in culture at 5 × 105 cells per well of a 24-well tissue culture dish for 18 h in serum-free RPMI media supplemented with 1% Nutriodoma (Roche Molecular Biochemicals) and 1% antibiotics. Proteinases from the conditioned media were concentrated by incubation of 100 µl of media with 15 µl of gelatin-Sepharose 4-B beads (Amersham Biosciences) for 3 h at 4 °C. The beads were pelleted by gentle centrifugation, washed with phosphate-buffered saline, and the proteinases eluted by incubation of the beads in Laemmli sample buffer without beta -mercaptoethanol for 1 h at 37 °C. Samples were directly analyzed by electrophoresis under nonreducing conditions into 10% zymogram gelatin gels (NOVEX, San Diego, CA). Gels were washed twice with 2.5% Triton X-100 (15 min each) and incubated for 16 h in a developing buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 5 mM CaCl2). Gels were then stained in 0.5% (w/v) Coomassie Blue in 50% methanol, 10% acetic acid followed by partial destaining to reveal clear bands of protease activity.

Lung Mechanics-- Resistance and elastic forces were measured in airways and/or lung parenchyma of 15-month-old wild type and SP-C (-/-) mice (n = 5/group). Mice were anesthetized with 0.1 ml/10 g body weight of a mixture (intraperitoneally) containing 40 mg/ml ketamine and 2 mg/ml xylazine. Mice were tracheostomized, and respiratory impedance was measured by using the forced oscillation technique (0.25-20 Hz) delivered by computerized flexiVent (SCIREQ, Montreal, Canada) (20). Estimated total lung compliance, airway resistance, airway elastance, tissue damping, and tissue elastance for mice at 2 cm H2O positive end expiratory pressure were obtained by fitting a model to each impedance spectrum (21). Hysteresivity was calculated as the ratio of tissue damping to tissue elastance. With this system, the calibration procedure removed the impedance of the equipment and tracheal tube.

Pressure-volume relationships were studied in 10-12-month-old wild type and SP-C (-/-) mice (n = 5/group). Mice were anesthetized with pentobarbital sodium (100 mg/kg intraperitoneally) and placed in a box containing 100% O2 to ensure complete collapse of the alveoli by O2 absorption. After the mice were killed by exsanguination, the cannula was inserted into trachea, connected to a pressure sensor (Mouse Pulmonary Testing System, TSS, Cincinnati, OH), and lung volume/kg body weight was determined at intervals of 5 cm H2O during inflation and deflation (22).

Hydroxyproline Determinations-- The hydroxyproline content of lyophilized tissue was measured using a minor modification of a technique published previously (23). Briefly, 10 mg of lung tissue was hydrolyzed in 6 N HCl at 120 °C overnight. Aliquots were added to citric acetate buffer and reacted with chloramine T, and color was developed with Erlich's reagent and absorbance read at 550 nm. In the final reaction, 85% sulfuric acid was substituted for 70% perchloric acid, and the reaction was incubated at 103 °C instead of 65 °C. Results were compared against standards of hydroxyproline from 0 to 200 µg/ml (Aldrich).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SP-C (-/-) 129/Sv Congenic Mice-- SP-C (+/-) chimeric founders were generated from gene-targeted 129/Sv embryonic stem cells. These chimeras were initially bred into a Swiss black background and were also bred to 129/Sv mice. Because only embryonic stem cell-derived sperm transmit the SP-C mutation from the chimeric male founder, SP-C (-/-) offspring resulting from the chimera × 129/SvEv cross were derived entirely from 129/Sv germ cells. Thus, the SP-C (-/-) offspring represent an inbred 129/Sv strain. Poor health and reduced fecundity were noted in SP-C (-/-) mice by 2 months of age. Few litters were produced by animals older than 6 months of age. Poor grooming and conjunctivitis were noted in all SP-C (-/-) 129/Sv animals beyond 6 months of age. Deterioration of coat condition was observed in most SP-C (-/-) mice after 2 months of age. The average body weight of 12-13-month-old SP-C (-/-) mice was reduced by 24% (25.7 g ± 3.2, n = 7 versus 33.5 g ± 3.0, n = 7) compared with controls. In these older SP-C (-/-) mice, relative heart weight was increased as determined by heart/body weight ratios. Ratios were increased by 30% with the right ventricle being more enlarged than the left 0.00565 ± 0.00026, mean ± S.D., n = 10 (SP-C -/-) versus 0.00431 ± 0.00033, mean ± S.D., n = 7 (SP-C +/+), p < 0.007.

Morphological Changes in the Lungs of SP-C (-/-) Mice-- Whereas lung structure of SP-C (-/-) mice was normal at birth (data not shown), enlargement of alveoli was observed by 2 months of age and thereafter, consistent with the development of emphysema (Fig. 1). Alveolar septation was irregular with absent or shortened alveolar septal tips observed throughout the lung parenchyma. Multifocal cellular infiltrates that generally consisted of alveolar macrophages and other mononuclear cells were detected (Fig. 1B). In lungs from 6-month-old mice, consolidated parenchymal infiltrates were commonly observed. Regions of type II cell hyperplasia and interstitial thickening were observed in the lung parenchyma. The extent and severity of parenchymal abnormalities and cellular infiltrates increased with age, often resulting in regions with complete obliteration of some alveolar spaces at 12 months of age. Areas with epithelial cell hyperplasia and interstitial thickening were observed in alveoli and airways. Extensive perivascular and peribronchiolar monocytic infiltrates were detected in the most severely affected animals (Fig. 1F). The considerable loss of alveolar structures and resulting airspace enlargement is shown in comparative low magnification images of lung from SP-C (+/+) and SP-C (-/-) mice (Fig. 2, A and B, respectively). Eosinic cellular infiltrates are evident in the central and lower regions of the SP-C (-/-) mice in Fig. 2B. The degree of airspace enlargement was evaluated from sections of 12- and 14-month-old mice. The emphysematous alterations were widespread but not uniform. Areas of mild and severe alveolar loss were quantitated. The fractional airspace was increased 5.4 ± 0.24-fold (mean ± S.E., p < 0.01) in lungs from SP-C (-/-) compared with age-matched control mice.


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Fig. 1.   Progression of pulmonary histopathology in SP-C (-/-) mice. Lungs were obtained from wild type littermates (A, C, and E) or SP-C (-/-) (B, D, and F) mice. Lungs were inflation-fixed at 20 cm H2O of pressure and stained with hematoxylin-eosin. Extensive airspace remodeling, stromal thickening, and monocytic infiltration were noted at 2 months (B), 6 months (D), and 12 months (F) of age. Perivascular and peribronchiolar mononuclear infiltrates and epithelial cell dysplasia in conducting airways are shown in F. Micrographs ×625 are representative of at least 3-5 animals of similar ages.


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Fig. 2.   Emphysema in SP-C (-/-) mice. Lung histology in control (A) and SP-C (-/-) mice (B) demonstrate the extensive loss of alveoli at 1 year of age. Macrophage infiltrates are observed in the central and lower regions of B.

Alveolar Remodeling-- Trichrome staining demonstrated regions of abnormal blue staining in the lung parenchyma at sites of thickened alveolar septal structures, consistent with local fibrosis and collagen deposition (Fig. 3). In some regions, abnormal staining was distributed in extended web-like configurations throughout the lung parenchyma. Extensive alpha -SMA staining, indicating myofibroblast transformation, was observed throughout the alveoli of SP-C (-/-) mice. The intensity and extent of alpha -SMA staining was, in general, increased with age but variable within lung sections and among littermates (Fig. 3). Loss of the network of alveolar elastin fibers detected with orcein stain was observed in areas of alveolar disruption in the SP-C (-/-) mice (Fig. 3). Regions with reduced orcein staining colocalized with sites of increased trichrome staining. Areas of interstitial thickening and intense trichrome staining were localized to areas of extensive remodeling and macrophage infiltration. Total lung hydroxyproline content was not altered in the SP-C (-/-) mice. This finding is consistent with the focal nature of the abnormalities seen by trichrome staining.


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Fig. 3.   Remodeling and increased trichrome staining in lungs from SP-C (-/-) mice. Mason trichrome (A and B), orcein (C and D), and alpha -smooth muscle actin immunostaining (E and F) are shown from lung tissue at 6 months of age in wild type (A, C, and E) and SP-C (-/-) (B, D, and F) littermates. Extensive airspace remodeling with monocytic infiltration and dense blue staining was observed (arrows). Orcein staining demonstrated that elastin fibers were absent in many of the remodeled airspaces (D). Smooth muscle actin staining was observed in alveolar regions of the lung parenchyma in SP-C (-/-) (arrows) but not wild type mice.

Electron Microscopic Findings-- At the electron microscopic level, alveoli of the SP-C (-/-) mice were often thickened and lined by hyperplastic type II epithelial cells (Fig. 4A). Increased numbers of cuboidal cells were observed lining alveolar surfaces, and type II cells contained excessive numbers of lamellar bodies. Capillary walls were thickened or obliterated by surrounding stroma and collagen. Bronchi and bronchioles were lined by a highly atypical columnar epithelia. Conducting airways were lined by non-ciliated columnar epithelial cells that contained numerous atypical electron dense organelles, consistent with the atypical mitochondria characteristic of Clara cells (Fig. 4B) (24). Type II cells were hypertrophic, containing increased numbers of lamellar bodies and lipid inclusions. In the alveolus, basement membranes were thickened, containing numerous collagen fibrils. Many capillary lumena were obliterated, and regions of increased collagen fibrils were readily discerned. Basement membranes and endothelial surfaces of larger vessels were disrupted. Abnormal alveolar macrophages contained large accumulations of surfactant-like material with structural features of tubular myelin and lamellar bodies (Fig. 4C).


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Fig. 4.   Ultrastructural abnormalities in lungs of SP-C (-/-) mice. Electron microscopy was performed on SP-C (-/-) mice at 9 months of age. Marked abnormalities were observed in the alveolar walls from the SP-C (-/-) mice (A). Type II cells were hyperplastic, containing numerous lamellar body-like inclusions, and collagen deposition was noted within the alveolar walls. Alveolar capillaries were surrounded by thickened subepithelial stroma. Conducting airways were lined by dysplastic epithelial cells with atypical morphology (B). Numerous cytopathic dense organelles, likely representing atypical mitochondria, were observed in nonciliated columnar epithelial cells. Alveolar macrophages were hyperplastic, some containing dense crystals (top cell, C). Others containing excessive amounts of surfactant lipids, including lamellar bodies and tubular myelin figures, were observed. Pulmonary vascular abnormalities were observed in small vessels in SP-C (-/-) mice. Vessels were occluded or absent in many alveoli. Abnormal membrane blebbing was recurrently observed along the intima of the abnormal vessels (D).

Macrophage Morphology and Abnormal Lipid Accumulations-- Subsets of mononuclear cells in the alveolar spaces of SP-C (-/-) mice stained intensely with the MAC3 antibody, an alveolar macrophage cell marker (Fig. 5). Abnormal intracellular lipid inclusions were observed in alveolar macrophages (Fig. 5D). Likewise, lipid accumulations were also noted in the hyperplastic type II epithelial cells lining residual alveoli (Fig. 5D). At the ultrastructural level, the atypical alveolar macrophages contained abundant surfactant components including lamellar bodies and tubular myelin, extracellular forms of pulmonary surfactant (Fig. 4). Other macrophages contained numerous cytoplasmic crystals consistent with those formed by Ym1, a mammalian lectin (25). Mass spectroscopic analysis confirmed the presence of increased Ym1 in the BALF (data not shown). Accumulation of the intracellular crystals and lipids was not detected in alveolar macrophages from control 129/Sv maintained in this barrier facility. In BALF from 6-month-old SP-C (-/-) mice, the number of alveolar macrophages was increased 4.4-fold, 9021 ± 1017 versus 2039 ± 497 (n = 5), in SP-C (-/-) versus SP-C (+/+), respectively. The percentage of lymphocytes was not altered. Changes in polymorphonuclear cells and eosinophils were not observed.


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Fig. 5.   MAC-3 staining and alveolar macrophage infiltrates in the SP-C (-/-) mice. MAC-3 immunostaining was assessed in wild type (A) and SP-C (-/-) (B) mice at 6 months of age. Extensive infiltration with MAC-3 staining cells was noted in association with severe emphysema (B). Micrograph (×625) is representative of at least 5 SP-C (-/-) mice and controls. Semi-thin sections of wild type (C) and SP-C (-/-) (D) mice were stained with toluidine blue, demonstrating alveolar and alveolar macrophage abnormalities. Extensive lipid inclusions were noted in hyperplastic type II cells lining the alveoli and in the numerous alveolar macrophages accumulating in the airspaces.

Epithelial Cell Dysplasia-- Pronounced changes in conducting airway epithelial cell morphology were observed in SP-C (-/-) mice (Fig. 6). Epithelial cell dysplasia was readily apparent at 6-12 months of age; the conducting airways were lined by hyperplastic and pseudostratified columnar epithelium (Fig. 1F and Fig. 6B). Whereas MUC5A/C staining cells were rarely seen in wild type mice, MUC5A/C-positive cells lined most of the conducting airways of the SP-C (-/-) mice (Fig. 6D). MUC5A/C staining of conducting airways was generally extensive; however, heterogeneity in the pattern of staining occurred. Immunostaining for Clara cell secretory protein and pro-SP-B was detected, but the extent and intensity of staining was decreased in severely affected conducting airways in SP-C (-/-) mice, also consistent with epithelial cell dysplasia (data not shown). In the alveoli, septal thickening and dense monocytic infiltration were noted in the areas of extensive epithelial hyperplasia. However, in some areas with severe airspace remodeling, some alveoli lacked type II cells. In those lesions, web-like strands of squamous cells formed alveoli that were devoid of capillaries.


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Fig. 6.   Epithelial cell dysplasia in conducting airways of 2-month-old SP-C (-/-) mice. Conducting airways from wild type (A and C) or SP-C (-/-) (B and D) are observed after hematoxylin-eosin staining (A and B) or MUC5A/C immunohistochemistry (C and D). Marked epithelial cell dysplasia was observed in large and small conducting airways of SP-C (-/-) mice. The abnormal epithelial cells were hypertrophic with abnormal foci of pseudostratified epithelia. Whereas MUC5A/C staining cells were rarely seen in wild type mice (C), extensive staining for MUC5A/C was observed throughout bronchi and bronchioles (D) and was occasionally observed in the peripheral lung parenchyma in SP-C (-/-) mice (not shown). A and B, ×625 magnification; C and D, ×1250 magnification.

Pulmonary Mechanics-- At higher pressures on the deflation limb of pressure-volume curves, lung volumes were significantly increased in SP-C (-/-) compared with wild type mice (Fig. 7), consistent with the emphysema observed histologically (see Figs. 1 and 2). At lower pressures, lung volumes were normal, and residual lung volumes were maintained at 0 pressure, consistent with normal surfactant function. Similarly, there were no significant differences between SP-C (-/-) and control mice in dynamic lung compliance obtained with ventilation volumes of 7 ml/kg (Table I). Whereas airway and tissue elastance was unaltered, both airway resistance and tissue damping were significantly increased in SP-C (-/-) mice (p < 0.02 and 0.002, respectively). Hysteresivity was significantly increased in the SP-C (-/-) mice (p < 0.01). These findings are consistent with the observed emphysema and with maintenance of surfactant function.


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Fig. 7.   Pressure-volume analysis consistent with emphysema in SP-C (-/-) mice. Pressure-volume curves were performed in tracheotomized wild type and SP-C (-/-) mice at 10-12 months of age, n = 5 per group. Significantly increased lung volumes at higher pressure were observed in SP-C (-/-) mice. *, p < 0.01 as assessed by two-tailed Student t test, mean ± S.E.


                              
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Table I
Lung mechanics
Values are mean ± S.E. as assessed by two-tailed Student's t test, n = 5 per group.

Surfactant Composition-- Tissue and total surfactant phospholipid pool sizes were increased ~2-fold in SP-C (-/-) mice (Fig. 8). The composition of lipids in lung tissue after BAL was unchanged (Table II). SP-A, SP-B, and SP-D were estimated by Western blot analysis of BALF. Whereas surfactant protein B levels were unaltered, SP-A and SP-D were significantly increased in SP-C (-/-) mice.


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Fig. 8.   Phospholipid (SatPC) and surfactant proteins in SP-C (-/-) mice. A, SatPC pool sizes were determined in wild type and SP-C (-/-) mice in BALF, lung tissue after BAL, and the sum of BALF and tissue fractions (total). SatPCs were increased 60% in BALF and 2-fold in tissue and total in SP-C (-/-) mice as compared with wild type mice at 15 months of age. B, amounts of surfactant proteins in BALF were estimated by Western blot relative to the amount of SatPC. Values for wild type mice were normalized to a value of 1. SP-A and SP-D were increased in SP-C (-/-) mice. C, pool sizes/body weight for SP-A, SP-B, and SP-D in BALF were normalized to a value of 1 for wild type mice. Whereas SP-B levels were unaltered, SP-A and SP-D were increased in SP-C (-/-) mice. Mean ± S.E. *, p < 0.05 as assessed by two-tailed Student's t test.


                              
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Table II
Phospholipid content in lung tissue
Values are mean ± S.E.; n = 5 per group. The abbreviations used are: SM, sphingomyelin; PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PG, phosphatidylglycerol.

Cytokine and Metalloproteinase Expression-- Concentrations of proinflammatory cytokines were determined in BALF and lung homogenates from 6-month-old mice. Tumor necrosis factor-alpha , IL-6, MIP-2, and IL-13 were not altered in the SP-C (-/-) mice. The supernatants of cultured alveolar macrophages from SP-C (-/-) and (+/+) were tested for MMP activity by SDS/PAGE zymography at 1 year of age. Gelatinase activity was readily detectable in the conditioned media from SP-C (-/-) macrophages but was undetectable in media from control macrophages (SP-C +/+). Proteinase bands migrated at ~72 and 105 kDa, consistent with MMP-2 and MMP-9, respectively (Fig. 9). A third faint band of ~55 kDa was detected in media from the SP-C (-/-) macrophages. The size of this band is consistent with the latent form of MMP-12. In addition, MMP-12 mRNA was increased 3.58-fold in lung RNA from SP-C (-/-) compared with wild type mice. The elevated expression of MMP activity may contribute to alveolar remodeling seen in the SP-C (-/-) mice.


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Fig. 9.   Increased metalloproteinase activity produced by macrophages from SP-C (-/-) mice. MMP activity was assessed by zymography of conditioned media from alveolar macrophages from SP-C (-/-) (lane 1) and SP-C (+/+) (lane 2). Protease activity 72 (MMP-2) and 105 kDa (MMP-9) was increased in media from SP-C (-/-) mice (arrows). A faint band at 55 kDa, consistent with the size of MMP-12, was also increased in conditional media from SP-C (-/-) mice (arrowhead). Gels are consistent with observations from 4 separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A severe pulmonary disorder characterized by emphysema, epithelial cell dysplasia, monocytic cell infiltration, increased alpha -smooth muscle staining, and abnormal lipid accumulations was caused by targeted deletion of the sp-C gene in a congenic strain of SP-C (-/-)/129/Sv mice. Heterogeneous pulmonary lesions contained the following: 1) thickened alveolar walls that stained for alpha -smooth muscle actin; 2) extensive monocytic infiltrates and increased expression of metalloproteinases; 3) regions of severe emphysema with septal thinning and degeneration of pulmonary capillaries; 4) epithelial cell dysplasia and MUC5A/C expression in conducting airways; and 5) accumulation of intracellular lipids in various cell types. Pathologic findings in the SP-C (-/-) mice were consistent with, but not identical to, those seen in lungs from patients with various conditions termed idiopathic interstitial pneumonitis (IIP). Thus, lack of SP-C or pro-SP-C can be directly linked to the pathogenesis of interstitial lung disease in mice.

A mutation in the sp-C gene was recently associated with familial interstitial pneumonitis that was inherited as an autosomal dominant effect (8, 9). In a sibship with mutation c460 + 1Gly right-arrow Ala, resulting in an exon 4 deletion of the pro-SP-C peptide, misprocessed pro-SP-C accumulated within type II epithelial cells; tissue and lung lavage material lacked the active SP-C peptide (8). Similarly, a single base pair substitution (L188Q) altered subcellular localization of pro-SP-C in an extended family with IIP (9). Therefore, it has been unclear whether the severe pulmonary disease in these patients results from the lack of SP-C or to abnormal accumulations of misfolded mutant SP-C or pro-SP-C proteins. The present studies demonstrate that the lack of SP-C per se can recapitulate many of the pathologic findings consistent with various forms of adult and childhood interstitial pneumonitis.

Although the absence of pro-SP-C and/or SP-C caused severe lung disease in the mouse, the molecular pathogenesis of this disorder remains unclear. At the light microscopic level, lung structure in the SP-C (-/-) mice was normal at E19.5 and postnatal day 1 (data not shown). Abnormalities seen in lung structure increased with advancing age, suggesting that emphysema and remodeling do not arise from abnormalities in lung morphogenesis but from ongoing injury and repair processes. The expression of various pro-inflammatory cytokines that have been associated previously with emphysema and inflammation were not altered in the SP-C (-/-) mice. There was no change in neutrophil number, and there was no evidence of viral or bacterial infection in SP-C (-/-) mice. These findings suggest that the remodeling and inflammation are caused by cellular abnormalities intrinsic to the lung, and dependent upon the functions of SP-C or perhaps the result of selective degradation of extracellular matrix by MMPs elaborated by the macrophages rather than to susceptibility to pathogens. MMP-9 and MMP-2 production by alveolar macrophages and MMP-12 mRNA levels were increased and therefore may play a role in the pathogenesis of the lung disease in the SP-C (-/-) mice. Increased MMP-2, MMP-9, and MMP-12 expression was previously associated with emphysema in sp-D gene targeted mice (26).

Whereas pro-inflammatory cytokines were not increased in the lungs of the SP-C (-/-) mice, the lungs were infiltrated with atypical alveolar macrophages containing numerous lipid inclusions and Ym1 crystals (25). The numbers of the abnormal macrophages were increased 4-5-fold compared with control. Cellular infiltration was associated with discrete sites of alveolar thickening and fibrosis. The myofibroblast transformation and collagen deposition seen at the ultrastructural level were consistent with increased alpha -SMA staining seen throughout the alveolar walls of the SP-C (-/-) mice. Paradoxically, marked epithelial cell dysplasia was observed in conducting airways in the SP-C (-/-) mice, despite the fact that pro-SP-C is not expressed in these cells in wild type mice. Furthermore, high levels of expression of MUC5A/C were observed in the conducting airways at sites in which SP-C mRNA and protein are not normally expressed. MUC5A/C is normally expressed at low levels in the conducting airways of mice but is readily induced by inflammation or inflammatory cytokines, being increased by IL-4, IL-13, and allergens (for review see Ref. 27). These latter findings suggest that the lack of SP-C may influence gene expression outside the alveolus, implying that SP-C plays a role, directly or indirectly, in the conducting airways. However, it is unclear whether cellular abnormalities in the conducting airways of SP-C (-/-) mice are mediated directly by SP-C-dependent signaling events or might be related to SP-C-dependent modulation of surface forces or changes in mucociliary clearance in the absence of SP-C.

The finding that severe lung disease can be caused by either the expression of a dominantly inherited mutant pro-SP-C protein or the deletion of sp-C gene suggests several potential mechanisms by which SP-C may contribute to the pathogenesis of IIP. In the IIP patients described by Nogee et al. (8) and Thomas et al. (9), the mutant pro-SP-C protein accumulated within type II cells, potentially creating cell injury related to the misfolding or misprocessing of the precursor protein. In support of this concept, Conkright et al. (28) recently demonstrated that expression of an SP-C mutant protein caused lethal lung dysfunction in vivo. However, the active SP-C peptide was absent in the SP-C (-/-) mice and in patients with idiopathic pulmonary fibrosis caused by this dominantly inherited SP-C mutation (8). Thus, the lack of SP-C per se may be involved in the pathogenesis of IIP. Amin et al. (29) recently described a sibship in which three individuals were severely affected by IIP, each of whom lacked detectable expression of either pro-SP-C or SP-C in alveolar lavage, despite the failure to find mutations in the coding region of SP-C. Whether the selective lack of pro-SP-C or SP-C directly caused the disorder in these patients is unclear.

Do Abnormalities in Surfactant Function Contribute to IIP?-- The present findings demonstrate unequivocally that the lack of SP-C per se causes inflammation and alveolar remodeling in mice. Because SP-C enhances surface properties of phospholipids in the airspace, it is possible that the lack of SP-C alters surfactant function in time leading to pneumonitis. However, lung phospholipid content was unaltered in SP-C (-/-) mice in the Swiss black strain (11) and was increased 2-fold in SP-C (-/-) mice in 129/Sv background. Surfactant phospholipid composition, structure of lamellar bodies, and tubular myelin were generally preserved in both strains of SP-C (-/-) mice. Changes in lung mechanics and lung histology shown in a previous study of 8-week-old Swiss black SP-C (-/-) mice were distinct from the present study. In SP-C (-/-) Swiss black mice, there was no evidence of inflammation or emphysema. Hysteresivity, which describes the mechanical coupling between tissue resistance and elastace, was decreased, and findings were consistent with a modest abnormality of in vitro surface activities of surfactant. In contrast, the SP-C (-/-) mice in the present study showed more severe abnormalities in airway resistance, tissue damping, and hysteresivity. Taken together, the findings of altered pulmonary mechanics are consistent with similar functional changes detected in a transgenic model that develops emphysema (30). Furthermore, SP-B and surfactant phospholipid pool sizes were normal or increased, consistent with the observed preservation of surfactant function. The modestly increased levels of SP-A and SP-D in the SP-C (-/-) 129/Sv mice may reflect changes related to chronic lung inflammation. Thus, there is no evidence at present that surfactant deficiency accounts for the chronic lung disease in the SP-C (-/-) 129/Sv mice, but it remains possible that subtle differences in sheer forces not discernible in the present studies may contribute to the disruption of lung structure and function in the SP-C (-/-) mice. In vitro studies demonstrate that various growth factors, cytokines, and sheer stress can cause myofibroblast transformation of lung fibroblasts. Consistent with the increased alpha -SMA staining observed in the present study in mice, the extensive fibrosis and myofibroblast transformation is often seen in humans with IIP (10). Whereas abnormal trichrome staining was observed in the lungs of SP-C (-/-) mice, these areas were relatively sparse and heterogeneous. Severe fibrotic lesions characteristic of human patients with IIP or sp-C gene mutations were not observed. It is presently unclear whether these differences reflect species or age-related differences or that the pathological processes are distinct. If lack of SP-C contributes to the pathogenesis of the pulmonary disease, therapy in which exogenous SP-C is administered might be considered for patients with deficiency or mutations in SP-C. On the other hand, if the disorder is caused by misrouting and abnormal accumulations of SP-C or mutant SP-C, the addition or increased expression of normal SP-C may actually contribute to the disorder.

Does SP-C Deficiency Cause a Lipid Storage Disease?-- Surfactant lipids, lamellar bodies, and tubular myelin accumulated in the atypical macrophages, and prominent lipid droplets were observed in the abundant fibroblasts underlying type II cells in the lungs of SP-C (-/-) mice. These pathologic findings suggest the possibility that the absence of SP-C alters the catabolism of surfactant or other cellular constituents, creating a storage disorder. In vitro studies (31) have demonstrated that SP-C enhances surfactant lipid uptake by type II epithelial cells, functioning in a manner distinct from that of SP-A and SP-B, the latter serving to maintain large surfactant aggregates associated with the epithelial surfaces. Thus SP-C may have both intracellular and extracellular roles in surfactant homeostasis.

Strain Influences the Pathologic Finding in the SP-C (-/-) Mice-- The severe lung disease observed in the SP-C (-/-) mice in the 129/Sv strain contrasts sharply with the milder abnormalities seen in SP-C (-/-) mice when maintained in outbred Swiss black background. Whereas the SP-C (-/-) Swiss black mice do not have overt abnormalities in lung structure, these mice are susceptible to lung dysfunction when placed in hyperoxia and reduction of surfactant protein B (12). In recent studies with SP-C (-/-) Swiss black mice, we have observed pneumonitis in mice after 1 year of age.2 The strong strain-dependent influence on the SP-C (-/-) phenotype and the heterogeneity of pulmonary lesions that vary in severity, time, and place are consistent with findings in patients with familial idiopathic fibrosis caused by mutations in the sp-C gene (32). These syndromes are clinically and pathologically distinct from the emphysema associated with alpha 1-antitrypsin deficiency. In IIP, clinical and pathologic findings vary greatly in these sibships, and multiple histopathological diagnoses have been made within the same family. Whereas the nature of the SP-C mutations may influence the disorder, marked heterogeneity in severity, age of presentation, and time of progression of pulmonary disease characterizes this disorder, suggesting that environmental factors or other genes strongly influence its pathogenesis. The observed strain differences in the severity of lung disease caused by SP-C deficiency in the SP-C (-/-) mice suggest that the phenotype associated with SP-C deficiency or SP-C mutations may be strongly influenced by genetic factors. Whereas there is no evidence that infection complicated the interpretation of the present study, lung dysfunction in patients with IIP is exacerbated by infection.

Implications for Diagnosis and Therapy-- The present study and recent human studies (8, 32) provide perhaps the first association between sp-C gene mutations and pulmonary disease. Because the absence of SP-C caused severe lung disease in the SP-C (-/-) mice, it is also possible that deficiency of SP-C, whether genetic or secondary to injury, may contribute to acute and chronic lung disease. The association between mutations in SP-C with IIP in humans makes feasible genetic testing for the risk of the disease. Likewise, histologic diagnosis of the various pathologies caused by mutations in the sp-C gene can be made by immunohistochemistry. Detection of mutant sp-C genes or the presence or absence of SP-C from BALF may provide diagnostic insights into the role of SP-C in patients with complex lung diseases. Finally, it is unclear whether human IIP is caused by the following: 1) the absence of SP-C and pro-SP-C; 2) misfolding and misrouting of either the SP-C proprotein or the active SP-C peptide; or 3) altered routing, processing, or degradation of other cellular components whose homeostasis is dependent upon pro-SP-C and/or SP-C. If protein misfolding in type II or other lung cells contributes to the pathogenesis of lung disease, the misfolding of proteins other than SP-C may be considered in the pathogenesis of interstitial lung disease. Clarification of cellular and molecular mechanisms causing interstitial lung disease related to abnormalities in SP-C may provide a conceptual basis for the development of new therapies for IIP.

    ACKNOWLEDGEMENTS

We thank Danielle Eiseman and Dr. Tim LeCras for technical assistance and Ann Maher for assistance in preparation of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL50046 (to S. W. G.), HL61646 (to J. A. W., S. W. G., M. I., and M. T. S.), HL56387 (to J. A. W. and M. I.), HL63329, and HD11932 (to M. I.).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.

§ To whom correspondence should be addressed: Cincinnati Children's Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039. Tel.: 513-636-7850; Fax: 513-636-7868; E-mail: glass0@chmcc.org.

Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M210909200

2 S. W. Glasser, E. A. Detmer, M. Ikegami, C.-L. Na, M. T. Stahlman, and J. A. Whitsett, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: IIP, idiopathic interstitial pneumonitis; MMP, matrix metalloproteinases; BALF, bronchoalveolar lavage fluid; BAL, bronchoalveolar lavage; IL, interleukin; SatPC, saturated phosphatidylcholine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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