From the 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
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ABSTRACT |
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SP-C-deficient (SP-C 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 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 ( Animals--
SP-C ( 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 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- MMP Activity--
Matrix metalloproteinase (MMP-2 and MMP-9)
activity was measured in macrophage-conditioned media collected from
12-month-old SP-C ( Lung Mechanics--
Resistance and elastic forces were measured
in airways and/or lung parenchyma of 15-month-old wild type and SP-C
(
Pressure-volume relationships were studied in 10-12-month-old wild
type and SP-C ( 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).
SP-C ( Morphological Changes in the Lungs of SP-C ( 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 Electron Microscopic Findings--
At the electron microscopic
level, alveoli of the SP-C ( Macrophage Morphology and Abnormal Lipid
Accumulations--
Subsets of mononuclear cells in the alveolar spaces
of SP-C ( Epithelial Cell Dysplasia--
Pronounced changes in conducting
airway epithelial cell morphology were observed in SP-C ( Pulmonary Mechanics--
At higher pressures on the deflation limb
of pressure-volume curves, lung volumes were significantly increased in
SP-C ( Surfactant Composition--
Tissue and total surfactant
phospholipid pool sizes were increased ~2-fold in SP-C ( Cytokine and Metalloproteinase Expression--
Concentrations of
proinflammatory cytokines were determined in BALF and lung homogenates
from 6-month-old mice. Tumor necrosis factor- A severe pulmonary disorder characterized by emphysema, epithelial
cell dysplasia, monocytic cell infiltration, increased 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 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
( Whereas pro-inflammatory cytokines were not increased in the lungs of
the 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
( 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 ( 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 ( Strain Influences the Pathologic Finding in the SP-C
( 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 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
-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
-helical structure that
spans a lipid bilayer (3). Both the
-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.
/
) allele was studied in a congenic
129/Sv strain. SP-C-deficient mice developed severe, progressive
pulmonary disease associated with emphysema,
-smooth muscle actin
staining, monocytic infiltrates, and epithelial cell dysplasia in
conducting and peripheral airways.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
) 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.
-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).
, IL-1
, 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).
/
) 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
-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.
/
) 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.
/
) 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).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
) 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.
/
)
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.
-SMA staining, indicating
myofibroblast transformation, was observed throughout the alveoli of
SP-C (
/
) mice. The intensity and extent of
-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
-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.
/
) 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).
/
) 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.
/
) 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.
/
) 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.
Lung mechanics
/
) 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.
Phospholipid content in lung tissue
, 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.
View larger version (102K):
[in a new window]
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
-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
-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.
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.
/
) 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).
/
) 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
-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.
/
) 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.
/
) 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
-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.
/
) 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.
/
) 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
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.
/
)
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.
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ABBREVIATIONS |
---|
The abbreviations used are: IIP, idiopathic interstitial pneumonitis; MMP, matrix metalloproteinases; BALF, bronchoalveolar lavage fluid; BAL, bronchoalveolar lavage; IL, interleukin; SatPC, saturated phosphatidylcholine.
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