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J Biol Chem, Vol. 274, Issue 27, 19168-19174, July 2, 1999
From the Division of Pulmonary Biology, Children's Hospital
Medical Center, Cincinnati, Ohio 45229-3039
Surfactant protein B (SP-B) mRNA and protein
are restricted to alveolar Type II and Clara cells in the respiratory
epithelium. In order to investigate the function of SP-B in these
distinct cell types, transgenic mice were generated in which SP-B
expression was selectively restored in Type II cells or Clara cells of
SP-B Surfactant protein B
(SP-B)1 is a critical
component of pulmonary surfactant, a lipid-protein mixture which forms
a film along the surface of the alveolar epithelium, and is absolutely
required for maintenance of alveolar stability at low lung volumes.
Surfactant phospholipid mixtures lacking the hydrophobic proteins SP-B
and SP-C have poor surface film forming properties, whereas surfactants containing SP-B as the sole protein component rapidly form a stable phospholipid film in vitro and restore lung function in
surfactant-deficient preterm animals (1-3). Genetic ablation of the
murine SP-B locus leads to acute respiratory distress syndrome at birth
resulting in death within minutes (4). Likewise, mutations resulting in
SP-B deficiency in human infants lead to severe respiratory distress
and death in the neonatal period (5, 6). Intratracheal administration
of exogenous SP-B to infants with hereditary SP-B deficiency failed to
restore lung function, suggesting that SP-B may have functions in
addition to promoting formation of a stable surface film in the
alveolus (7). Consistent with this hypothesis, SP-B deficiency in mice
and human infants was associated with failure to form lamellar bodies
and altered pro-SP-C processing (4, 8).
Expression of SP-B is restricted to alveolar Type II cells and
nonciliated bronchiolar epithelial (Clara) cells of the pulmonary epithelium (9, 10). In Type II cells, proteolytic processing of the
SP-B proprotein is initiated in the multivesicular body with cleavage
of an NH2-terminal propeptide to generate a processing intermediate of 25 kDa (11, 12). Subsequent cleavage of a COOH-terminal
propeptide results in liberation of the hydrophobic mature peptide
which forms homodimers of 18 kDa. Mature SP-B is stored with surfactant
phospholipids in lamellar bodies, the contents of which are released
into the alveolar airspace via basal and stimulus-induced secretion
(reviewed in (13)).
In contrast to the well characterized SP-B biosynthetic pathway in Type
II cells, little is known about synthesis and processing of SP-B in
Clara cells. Technical problems in the isolation of pure populations of
these cells has made study of SP-B processing by Clara cells difficult.
Clara cells are the most abundant cell type within the conducting
airways of the murine lung, comprising more than 50% of the epithelial
cells lining the terminal bronchioles (14, 15). Expression of SP-B in
cells at the terminal airway/alveolar junction suggests that under
certain circumstances, Clara cells may contribute SP-B to the alveolar
surfactant pool. In addition to a putative alveolar surfactant
function, Clara cell SP-B may also promote formation of a surfactant
film in the terminal bronchioles which may be important for maintaining
the patency of small conducting airways. Finally, given the widespread
distribution of Clara cells in the murine airway, it is possible that
SP-B has function(s) that are independent of surfactant activity. In
order to better understand the role of SP-B in Type II cells and Clara
cells, we have generated transgenic mouse lines in which SP-B
expression was ablated and then restored in a cell-specific manner.
Targeted Expression of Human SP-B in Clara Cells of Transgenic
Mice--
The mCCSP/hSP-B transgene, comprised of a 2.4-kb
HindIII/XhoI fragment of the murine CCSP promoter
(16) (a kind gift from Dr. Barry Stripp, Departments of Environmental
Medicine, Pediatrics and Oncology, University of Rochester, Rochester,
NY) ligated to a 1.6-kb XhoI/EcoRI fragment of
the full-length human SP-B cDNA (17) and a 400-bp
EcoRI/NotI fragment containing the SV40 small t
intron and polyadenylation signal, was cloned into the pET21a vector
(Novagen, Madison, WI). Prior to injection, the 4.4-kb transgene was
released from the vector by SalI/NotI digestion and gel-purified using QIAEX II Gel Extraction Kit (Qiagen, Chatsworth, CA). Purified DNA was extensively dialyzed against 5 mM
Tris (pH 7.4) and 0.1 mM EDTA and subsequently
microinjected into fertilized eggs of the FVB/N mouse strain by the
Children's Hospital Transgenic Core facility (Cincinnati, OH). Founder
mice were identified by polymerase chain reaction (PCR) amplification
of mouse tail DNA using a 5'-primer (5'-CAATCACTGCCCTCTACCTCTT-3')
specific to murine CCSP promoter and a 3'-primer
(5'-GCACCCTTGGGAATCATGGCTTGGATC-3') specific to human SP-B to generate
an 800-bp product. Genomic DNA isolated from mouse tail was amplified
in a 35-cycle PCR with 0.5 µM of each primer, 400 µM dNTPs, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 1 µl of
master mixture buffer (10 µM Tris-HCl, pH 8.0, 5 nM EDTA) (Stratagene, La Jolla, CA). PCR results were
confirmed by Southern blotting of BamHI-digested tail DNA with the 400-bp 32P-labeled SV40 small t intron/poly(A)
probe. Transgene expression was identified by S1 nuclease protection
assay with a human SP-B probe (18) and confirmed by Western blot (19).
The two independent lines of mCCSP/hSP-B mice which had detectable
levels of transgene expression were subsequently interbred to increase
SP-B expression. In order to achieve selective expression of SP-B in
Clara cells of SP-B Targeted Expression of Human SP-B in Type II Cells of Transgenic
Mice--
The mSP-C/hSP-B transgene, composed of a 4.8-kb
XbaI/XhoI murine SP-C promoter (a kind gift from
Dr. Stephan Glasser, Division of Pulmonary Biology, Children's
Hospital Medical Center, Cincinnati, OH) followed by a 1.6-kb
XhoI/EcoRI fragment of the full-length human SP-B
cDNA and a 400-bp EcoRI/NotI cassette
containing the SV40 small t intron and polyadenylation signal, was
cloned into the pcDNA3 vector (Invitrogen, San Diego, CA). The
6.8-kb transgene was released from the vector by
XbaI/NotI digestion, gel-purified with QIAEX
resin (Qiagen), extensively dialyzed against 5 mM Tris-HCl (pH 7.4) and 0.1 mM EDTA, and subsequently injected into
fertilized eggs of the FVB/N mouse strain. Founder mice were identified
by an 800-bp PCR product with a 5'-primer
(5'-CTGAAAAGCCAGGAACAAACAAGC-3') specific to the murine SP-C promoter
and a 3'-primer (5'-GCACCCTTGGGAATCATGGCTTGGATC-3') specific to human
SP-B. Transgenic mice were identified by PCR and Southern blotting and
transgene expression verified as described above. Three independent
mSP-C/hSP-B lines were subsequently established. To achieve selective
expression of SP-B in Type II cells of SP-B In Situ Hybridization--
The 1.5-kb murine SP-B cDNA clone
(20) in pBluescript II SK (Stratagene) and the 2-kb human SP-B cDNA
clone (17) in pGEM3Z(f+) (Promega, Madison, WI) were linearized and
used as template to generate sense and antisense riboprobes using
bacteriophage T3 or T7 polymerase and the Riboprobe Gemini Core System
II Transcription Kit (Promega) in the presence of
[35S]UTP (specific activity: 1000-1500 Ci/mmol, NEN Life
Science Products Inc.). In situ hybridization of
paraffin-embedded embryonic and adult lungs was performed using both
human and murine SP-B cRNA probes under stringent conditions followed
by extensive post-hybridization washing to minimize cross-reactivity
between the murine and human SP-B probes, as described previously (21,
22).
Analysis of SP-B Expression (Western Blot)--
To assess the
processing and secretion of SP-B in transgenic animals, 5 to 100 µg
of protein from total lung homogenate or 30 µg of protein from
bronchoalveolar lavage (BAL) were subjected to SDS-PAGE and transferred
to nitrocellulose. Western blotting was performed with antiserum 28031, directed against the mature SP-B peptide, at a dilution of 1:500,
antiserum 96189, directed against recombinant COOH-terminal propeptide
of human SP-B, at a dilution of 1:5000, or antiserum 55522, directed
against recombinant human SP-B proprotein, at a dilution of 1:5000
(23).
Immunohistochemistry--
Immunostaining for SP-B proprotein was
performed with antisera 96189, 55522, or 55019 as described previously
(24). Serial 5-µm paraffin sections from lung tissues were loaded
onto polylysine-coated slides (Fisher, Atlanta, GA). Slides were
deparaffinized, rehydrated in a series of graded alcohols, and treated
with 3% H2O2 in methanol for 15 min to quench
endogenous peroxidase. Nonspecific staining was blocked with 2% normal
goat serum in phosphate-buffered saline with 0.2% Triton X-100 for
2 h at room temperature. Slides were then incubated overnight at
4 °C with antiserum diluted 1:500, 1:1000, or 1:2000 in blocking
solution (24). After washing, the sections were incubated for 30 min at
room temperature with biotinylated goat anti-rabbit secondary antibody
diluted 1:200 in the blocking solution, followed by incubation for 30 min with the avidin-biotin-peroxidase complex using Vectastain Elite
ABC Kit (Vector Laboratories, Inc., Burlingame, CA). The slides were color developed using 3,3'-diaminobenzidine-HCl and further enhanced with nickel cobalt followed by counterstaining with nuclear fast red.
Sections were photographed on a Microphot-FXA Nikon fluorescence microscope.
Electron Microscopy--
Fetuses were collected by cesarean
section at 18 days of gestation and the torso fixed in ice-cold 2%
paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium
cacodylate buffer (SCB) at pH 7.3 for 2 h. The fixed lungs were
removed, cut into 1-2-mm3 blocks, post-fixed in fresh
fixative overnight at 4 °C, washed in 0.1 M SCB, and
incubated with 1% osmium tetroxide (Electron Microscopy Sciences, Ft.
Washington, PA) in 0.1 M SCB for 2 h at room
temperature. Tissues were dehydrated in a series of graded alcohol
solutions and embedded in EMbed 812 (Electron Microscopy Sciences).
Ultrathin sections were cut at 100 nm of thickness using a Reichert
Ultracut E ultramicrotome (Reichert, Austria), and post-stained with
2% uranyl acetate and lead citrate. Alveolar Type II epithelial cells
and Clara cells were photographed at ×8,000 using a JEOL 100-CXII
electron microscope at 80 keV of accelerating voltage. Immunogold
labeling of ultrathin cryosections was performed precisely as we have
previously described (25).
Hyperoxic Challenge--
hSP-C/hSP-B Generation and Characterization of Transgenic Mice Expressing Human
SP-B--
SP-B is synthesized in both alveolar Type II cells and
bronchiolar Clara cells. To define the functions of SP-B in each cell type, transgenic mice were generated in which the full-length human
SP-B cDNA was targeted to the bronchiolar Clara cell (mCCSP/hSP-B) or the alveolar Type II cell (mSP-C/hSP-B) using either the murine CCSP
or the murine SP-C promoter. Of 49 offspring from mCCSP/hSP-B injections, 12 (24%) were transgene positive, identified by both PCR
and Southern blot analyses of tail DNA, whereas 5 out of 24 progeny
(21%) were positive for the mSP-C/hSP-B transgene (not shown). Among
these founders, two lines of mCCSP/hSP-B and three lines of mSP-C/hSP-B
which expressed detectable levels of human SP-B were identified by S1
nuclease protection analyses and Western blotting (not shown). The two
lines of mCCSP/hSP-B mice were subsequently interbred to increase SP-B
production in Clara cells. To target SP-B mRNA to either Type II
cells or Clara cells in SP-B
Previous studies have suggested that the 2.3-kb mouse CCSP (16, 29) and
the 4.8-kb mouse SP-C (30) promoters direct gene expression specific to
Clara cells or alveolar Type II cells, respectively. To assess the
specificity of hSP-B transgene expression from the murine CCSP and
murine SP-C promoters, in situ hybridization was performed
using human and murine SP-B probes (Fig.
1). In the adult wild type animal, murine
SP-B mRNA was detected in alveolar Type II cells and bronchiolar
Clara cells (Fig. 1A). In day 18 fetal mice expressing human
SP-B under control of the murine CCSP promoter in the wild type
background (mCCSP/hSP-B), human SP-B mRNA was detected in Clara
cells but not in Type II cells (Fig. 1D), whereas murine
SP-B mRNA was detected in both alveolar and bronchiolar cells (Fig.
1C). In the mCCSP/hSP-B mice, human SP-B mRNA was
detected at higher levels in the proximal bronchiolar epithelium than
in distal bronchioles on day 18 of gestation (not shown). When these
mice were crossed into the SP-B
In mSP-C/hSP-B(mSP-B Expression of Human SP-B in Clara Cells--
Expression of both
human and murine SP-B RNA was observed in Clara cells from mCCSP/hSP-B
transgenic mice in the SP-B +/+ background. mCCSP/hSP-B transgenic mice
survived without overt evidence of respiratory pathophysiology and had
normal body weight, lung weight, reproductive function, longevity, and
normal gross lung structure, suggesting that expression of human SP-B
in Clara cells did not significantly alter lung function (not shown).
To determine the fate of SP-B synthesized by Clara cells,
bronchoalveolar lavage from wild type and mCCSP/hSP-B transgenic mice
was immunoblotted with antibody directed against the COOH-terminal
propeptide of human SP-B (antibody 96189) (Fig.
5) or antibody (55522) directed against
the full-length human SP-B (not shown). Both the 42-kDa SP-B proprotein
and 25-kDa processing intermediate were detected in increased
concentrations in lavage from mCCSP/hSP-B mice relative to wild type
littermates, consistent with secretion and accumulation of human SP-B
precursor in the airway of transgenic animals. These results also
suggest that partial processing of the SP-B proprotein normally occurs
in Clara cells.
Clara Cell-restricted Expression of Human SP-B--
In order to
restrict SP-B synthesis to Clara cells, both mCCSP/hSP-B transgenic
lines were crossed with hemizygous SP-B +/ Type II Cell-restricted Expression of Human SP-B--
The mSP-C
promoter was used to generate three mSP-C/hSP-B transgenic lines
expressing both endogenous murine SP-B and human SP-B mRNA in Type
II epithelial cells. Expression of human SP-B mRNA and protein in
Type II cells did not alter lung structure or function in any of the
three transgenic lines, consistent with a previous study in which a
truncated human SP-B proprotein was expressed in Type II cells of
transgenic mice (26). In the mSP-C/hSP-B transgenic mice, human SP-B
proprotein was processed to the mature peptide and formed heterodimers
with endogenous mouse SP-B mature peptide (Fig.
8); lung morphology in these mice was
normal (not shown). To generate mice in which SP-B was synthesized only
in Type II cells, mSP-C/hSP-B transgenic mice were crossed into SP-B Role of Clara Cell SP-B during Hyperoxic Lung Injury--
SP-B
mRNA is markedly increased in Clara cells in response to hyperoxia
(28) and SP-B +/ SP-B is expressed in both Type II epithelial cells and
non-ciliated Clara cells of the pulmonary epithelium. Type II cells are
restricted to the alveoli, whereas Clara cells are widely distributed
in conducting airways of the mouse but are absent in alveoli. The
increased abundance of Clara cells in terminal bronchioles suggests
that Clara cells in this region may contribute SP-B to the alveolar
surfactant pool. This hypothesis was tested by selectively replacing
SP-B expression in either Clara cells or Type II cells of SP-B The failure of selective expression of human SP-B in Clara cells to
correct respiratory failure in SP-B In SP-B +/ It has been suggested that the patency of terminal bronchioles is
maintained by the presence of a surfactant film similar to that in
alveoli (33). Given the large numbers of Clara cells present in
terminal bronchioles, it is possible that SP-B produced by these cells
contributes to formation of a surface film. Consistent with this
hypothesis, SP-B +/ Although the role of SP-B in Clara cells remains uncertain, it is clear
that expression of SP-B in Type II cells is absolutely required for
lung function in vivo. Expression of a full-length (this
study) or truncated (26) human SP-B proprotein in Type II cells of SP-B
In summary, selective restoration of SP-B synthesis in Type II cells
completely rescued the respiratory failure following birth of SP-B
*
This work was supported by National Institutes of Health
Grants HL36055 and HL56285 (to T. E. W.), HL56285-S (to
T. E. W. and H. T. A.), and HL38859 (to J. A. W.).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.
The abbreviations used are:
SP-B, surfactant
protein B;
kb, kilobase pair(s);
CCSP, Clara cell secretory protein;
mCCSP/hSP-B, murine 2.3-kb CCSP promoter and human SP-B cDNA;
PCR, polymerase chain reaction;
mSP-C/hSP-B, murine 4.8-kb SP-C promoter and
human SP-B cDNA;
BAL, bronchoalveolar lavage;
H441 cell, human lung
adenocarcinoma cell;
PAGE, polyacrylamide gel electrophoresis.
Surfactant Protein B (SP-B) 
/ Mice Are Rescued by Restoration
of SP-B Expression in Alveolar Type II Cells but Not Clara Cells*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/ mice. The 4.8-kilobase murine SP-C promoter was used to
generate 3 transgenic lines which expressed human SP-B in Type II cells (mSP-C/hSP-B). Likewise, the 2.3-kilobase murine CCSP promoter was used
to generate two transgenic lines which expressed human SP-B in Clara
cells (mCCSP/hSP-B). mSP-C/hSP-B and mCCSP/hSP-B transgenic mice were
subsequently bred to SP-B +/ mice in order to selectively express
SP-B in Type II cells or Clara cells of SP-B / mice. Selective
restoration of SP-B expression in Type II cells completely rescued the
neonatal lethal phenotype in SP-B / mice. Expression of SP-B in
some, but not all Type II cells of SP-B / mice, allowed postnatal
survival, but resulted in significantly altered lung architecture and
function. Selective restoration of SP-B expression in Clara cells of
SP-B / mice resulted in respiratory dysfunction and invariable
neonatal death, related to the complete absence of mature SP-B peptide
in these mice. These results indicate that expression and processing of the SP-B proprotein to the mature peptide in Type II cells is absolutely required for lung function in vivo and that SP-B
expression in Clara cells cannot substitute for this function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/ mice, mCCSP/hSP-B transgenic mice were
crossed with SP-B +/ mice to produce mCCSP/hSP-B(mSP-B /) offspring.
/ mice, mSP-C/hSP-B
transgenic mice were bred with murine SP-B+/ hemizygous mice to
produce SP-C/hSP-B(mSP-B /) offspring.
c(mSP-B
/) transgenic mice express a truncated human SP-B cDNA,
encoding residues 1-279 of the proprotein (i.e. deletion of
the 102 residue COOH-terminal propeptide), under control of the 3.7-kb
human SP-C promoter (26). We have previously demonstrated that SP-B RNA
is not detected in Clara cells of this transgenic line and alveolar
surfactant pool size and phospholipid composition are not different
from wild type littermates (26). Three 6-week-old
hSP-C/hSPBc(mSP-B /) mice and 3 wild type mice in
the same genetic background (FVB/N) were exposed to 95% oxygen for 48 or 72 h as described previously (27, 28). Following exposure, mice
were immediately sacrificed by a lethal injection of pentobarbital,
rapidly exsanguinated, and the lungs perfused with 4% paraformaldehyde
in phosphate-buffered saline. The lungs were excised, post-fixed in 4%
paraformaldehyde overnight, and prepared for either hematoxylin and
eosin staining or immunostaining with antibodies directed against the
mature SP-B or proprotein. Control mice consisted of 3 room air-exposed
littermates of the same genotype. For semiquantitative analysis,
MetaMorph software (Universal Imaging Corporation, West Chester, PA)
was used to acquire and store images of hematoxylin/eosin-stained
sections from the right upper, right middle, right lower, left upper,
and left lower lobes. Five sections from each lung were visualized with
a ×20 objective lens and MetaMorph software used to assess total
number of Type II cells, average Type II cell size, average septal wall
thickness, total alveolar space, and average alveolar space. These
measurements were scored by 3 masked observers and compared
statistically by two-way ANOVA. Each observer also noted the degree of
microhemorrhages and exudation/pulmonary edema (none, mild/minimal, severe).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/ mice, the mCCSP/hSP-B and
mSP-C/hSP-B transgenic mice were crossed with murine SP-B +/
hemizygous mice. Offspring carrying both the human SP-B transgene and a
single copy of the murine SP-B gene were bred to achieve cell-specific
hSP-B expression in the SP-B / background.
/ background to generate
mCCSP/hSP-B(mSP-B /) mice, SP-B mRNA (not shown) and protein
(Fig. 2) were detected in presumptive
Clara cells but not in alveolar epithelial cells of the fetal lung,
consistent with Clara cell restricted expression of SP-B protein.
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Fig. 1.
Cellular localization of human SP-B RNA in
mCCSP/hSP-B transgenic mice. In situ hybridization was
performed on lung sections from 6-week-old wild type (A and
B) and fetal day 18 mCCSP/hSP-B transgenic mice
(C and D) with [35S]UTP-labeled
mouse and human probes specific for murine SP-B (A and
C) or human SP-B transgene (B and D).
Murine SP-B was detected in alveolar Type II cells
(arrowhead), and bronchiolar Clara cells (arrow),
of both wild type (A) and mCCSP/hSP-B transgenic mice
(C). The human SP-B transgene was detected in Clara cells of
mCCSP/hSP-B transgenic mice (arrow, D), but not in Type II
cells. Bar, 100 µM.
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Fig. 2.
Cellular localization of SP-B proprotein in
mCCSP/hSP-B(mSP-B /) transgenic mice. Fetal lungs were
collected from wild type (A) and mCCSP/hSP-B(mSP-B /)
(B) mice on day 18 of gestation and stained for SP-B
proprotein with polyclonal antibody directed against COOH-terminal
propeptide of human SP-B (antibody 96189). Antibody 96189 detected
mouse SP-B very poorly (A) but readily detected human SP-B
(B) in presumptive Clara cells (arrow) and
intra-alveolar inclusions (arrowheads). Counterstained
nuclei were visible in alveolar epithelial cells of mCCSP/hSP-B(mSP-B
/) mice but SP-B immunoreactivity was not detected. Bar,
40 µM.
/) transgenic mice (mice expressing the human
SP-B transgene under control of the mouse SP-C promoter in the SP-B
/ background), human SP-B mRNA was detected only in Type II
cells (Fig. 3B); murine SP-B mRNA was
not detected (Fig. 3A). However, in contrast to the results
for in situ hybridization analyses, SP-B protein was
detected in some Clara cells. In mSP-C/hSP-B(mSP-B /) transgenic
line 1, staining for SP-B in Clara cells was extensive and only
slightly less than that in Clara cells from wild type littermates (not
shown); however, in a second mSP-C/hSP-B(mSP-B /) transgenic line
(line 2), SP-B staining was largely restricted to alveolar Type II
epithelial cells and only a few Clara cells expressing SP-B protein
were detected (Fig. 4).
Immunohistochemical analysis of a third mSP-C/hSP-B(mSP-B /)
transgenic line (line 3) indicated that SP-B protein was detected in
some, but not all, Type II cells (see below, "Type II Cell-restricted
Expression" and Fig. 9). These results suggest that the site of
transgene integration strongly influences expression from the 4.8-kb
murine SP-C promoter.
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Fig. 3.
Cellular localization of human SP-B RNA in
mSP-C/hSP-B(mSP-B /) transgenic mice. In situ
hybridization was performed on lung sections from 6-week-old
mSP-C/hSP-B(mSP-B /) mice with [35S]UTP-labeled human
SP-B riboprobe. Human SP-B RNA was detected in Type II cells
(arrowhead) but not in Clara cells of transgenic mice
(B); human SP-B RNA was not detected in lung tissues of wild
type mice (A). Bar, 100 µM.
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Fig. 4.
Cellular localization of SP-B proprotein in
mSP-C/hSP-B(mSP-B /) transgenic mice. Immunohistochemistry was
performed on lung sections from wild type mice (A) and
mSP-C/hSP-B(mSP-B /) transgenic line 2 (B) using
antibody 55522 which detects both mouse and human SP-B. SP-B
immunoreactivity was detected in Type II cells (arrowheads)
of wild type and transgenic mice. Counterstained nuclei were visible in
Clara cells (arrows) of mSP-C/hSP-B(mSP-B /) mice, but
there was little if any detectable SP-B immunoreactivity in Clara
cells. Magnification is ×95.
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Fig. 5.
SP-B proprotein is detected in BAL from
mCCSP/hSP-B transgenic mice. BAL (30 µg of protein) was
subjected to SDS-PAGE and subsequently analyzed by Western blot with
antibody 96189 or 55522 (not shown). SP-B proprotein,
Mr = 42,000, and processing intermediate,
Mr = 25,000, were readily detected in BAL from
transgenic animals (lanes 1, 3, and 5) but were
detected at much lower levels in BAL from wild type littermates
(lanes 2 and 4). Results with antibody 55522 were
identical. Five litters were analyzed; one representative litter is
shown.
mice to produce
mCCSP/hSP-B(mSP-B /) mice. Newborn mCCSP/hSP-B(mSP-B /) pups
from both mCCSP/hSP-B parental transgenic lines died within minutes of
birth, similar to findings in SP-B / mice (4). To further increase
SP-B protein levels, the two mCCSP/hSP-B lines were interbred prior to
crossing into the SP-B / background. mCCSP/hSP-B(mSP-B /) pups
carrying multiple copies of the mCCSP/hSP-B transgene and expressing
increased levels of SP-B protein (Fig. 6A) also died of acute
respiratory dysfunction within minutes of birth. Western blots of lung
homogenates from newborn mCCSP/hSP-B(mSP-B /) pups with antibody
96189 detected both the 42- and 25-kDa forms of SP-B (Fig.
6A); however, mature SP-B peptide (18 kDa) was not detected,
even when 10-fold more protein was loaded on the gel (Fig.
6B), strongly suggesting that these pups died from the lack
of mature SP-B peptide and that Clara cells did not completely process
pro-SP-B to the active peptide. Mature SP-B peptide was readily
detected in lung homogenates from wild type littermates (Fig.
6B). Consistent with the neonatal death of mCCSP/hSP-B(mSP-B /) mice, ultrastructural analyses of lungs from pups on day 18 of
gestation indicated that Clara cell-restricted expression of hSP-B was
associated with severe disruption of the surfactant biosynthetic
pathway in Type II cells (Fig. 7).
Lamellar bodies, the intracellular storage form of pulmonary
surfactant, were never detected; instead, similar to SP-B / mice,
numerous multivesicular bodies and composite bodies, containing
disorganized membrane lamellae and vesicular elements, were observed in
Type II cells. The ultrastructure of Clara cells was unremarkable (not
shown). Overall, restricted expression of SP-B in Clara cells was not associated with postnatal survival, formation of mature SP-B peptide, or normal lamellar body biogenesis.
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Fig. 6.
SP-B proprotein is not processed to mature
peptide in Clara cells of mCCSP/hSP-B(mSP-B /) transgenic
mice. A, Western blot analysis of SP-B proprotein in
offspring from crosses of two independent mCCSP/hSP-B transgenic lines
bred into the SP-B / background. Lung homogenates (100 µg of
protein) from newborn mCCSP/hSP-B(mSP-B /) pups were subjected to
SDS-PAGE and analyzed by Western blotting with antibody 96189. The SP-B
proprotein (Mr = 42,000) and processing
intermediate (Mr = 25,000) were detected at low
levels in offspring carrying one copy of the transgene (lanes
2-4) and were increased in offspring carrying multiple copies of
the transgene (lanes 1, 5, and 6). SP-B
proprotein and processing intermediate in fetal lung homogenates from
wild type fetuses were below the level of detection (not shown). Three
litters were analyzed; one representative litter is shown.
B, Western blot analysis of SP-B mature peptide in
mCCSP/hSP-B(mSP-B /) mice. 5 µg of lung homogenate from a wild
type newborn mouse (lane 1) or 50 µg of lung homogenate
from mCCSP/hSP-B(mSP-B /) transgenic mice (lanes 2-7)
were subjected to SDS-PAGE under nonreducing conditions and analyzed by
Western blotting with antibody directed against mature SP-B peptide
(antibody 28031). Lane 1, wild type mouse; lanes
2-7, fetal lung homogenates from mCCSP/hSP-B(mSP-B /) mice in
panel A. SP-B homodimer (Mr = 16,000)
was detected in wild type mice but not in mCCSP/hSP-B(mSP-B /)
mice. Three litters were analyzed; one representative litter is
shown.
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Fig. 7.
Ultrastructure of type II cells in
mCCSP/hSP-B(mSP-B /) and mSP-C/hSP-B(mSP-B /) transgenic
mice. Electron microscopy was performed on ultrathin sections from
lungs of mCCSP/hSP-B(mSP-B /) mice (A) and
mSP-C/hSP-B(mSP-B /) mice (B) on day 18 of gestation.
mSP-C/hSP-B(mSP-B /) transgenic line 2, in which expression of SP-B
was largely restricted to Type II cells, was selected for these
studies. Multivesicular bodies (arrow) and composite bodies
(arrowhead) containing multivesicular bodies and membrane
lamellae were detected in alveolar Type II epithelial cells of
mCCSP/hSP-B(mSP-B /) fetuses. Mature lamellar bodies were never
detected in mCCSP/hSP-B(mSP-B /) fetal lung, but were always
detected in mSP-C/hSP-B(mSP-B /) mice (B, arrowhead).
Bar, 1 µM.
/ background. Offspring from mSP-C/hSP-B(mSP-B /) transgenic line 2, in which SP-B expression was largely restricted to Type II
cells, were normal in terms of body weight, lung weight, reproductive function, and longevity, indicating that decreased expression of SP-B
in Clara cells was not associated with any overt pathophysiology (not
shown); furthermore, lamellar body structure was indistinguishable from
wild type littermates and there were no detectable ultrastructural abnormalities in Type II cells (not shown). The ultrastructure of Clara
cells in mSP-C/hSP-B(mSP-B /) mice was also indistinguishable from
that of SP-B +/+ littermates (not shown). Although Western blotting
indicated equivalent levels of SP-B in all three transgenic lines (not
shown), fewer alveolar cells stained for pro-SP-B than for pro-SP-C in
mSP-C/hSP-B(mSP-B /) transgenic line 3, consistent with the lack of
SP-B expression in some Type II cells of mice from this transgenic line
(Fig. 9). Both enlarged and collapsed alveoli were observed, consistent with heterogeneity of alveolar inflation (Fig. 9). While ultrastructural analyses revealed normal Type
II cell morphology, some Type II cells had abnormal lamellar bodies
with morphology similar to those from SP-B / mice, Fig. 7. These
results underscore the importance of SP-B in Type II cells and suggest
that regional deficiency of SP-B may lead to heterogeneous
inflation.
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Fig. 8.
SP-B proprotein is processed to the mature
peptide in mSP-C/hSP-B(mSP-B /) mice. Five µg of lung
homogenate from 6-week-old mice were subjected to SDS-PAGE under
nonreducing electrophoretic conditions and analyzed by Western blotting
with antibody 28031. Lane 1, wild type mice; lane
2, mSP-C/hSP-B transgenic mice; lane 3,
mSP-C/hSP-B(mSP-B /) mice. Mouse SP-B homodimer,
Mr = 16,000, lane 1 migrates more
quickly than human SP-B homodimer, Mr = 18,000 (lane 3). Mouse/human heterodimers were detected when the
mSP-C/hSP-B transgene was expressed in the wild type background
(lane 2).
View larger version (117K):
[in a new window]
Fig. 9.
Failure to restore SP-B proprotein expression
in all Type II cells is associated with microatelectasis and alveolar
dilation. Immunohistochemistry was performed on serial lung
sections from 6-week-old wild type mice (A and B)
and mSP-C/hSP-B(mSP-B /) transgenic line 3 (C and
D) using antibody 28031 to detect SP-B (A and
C), or antibody 68514, to detect SP-C (B and
D). Approximately equal numbers of SP-B and SP-C
immunoreactive cells (arrowheads) were detected in alveoli
of wild type mice (A and B), whereas the number
of SP-B immunoreactive cells was notably decreased in mSP-C/hSP-B(mSP-B
/) transgenic mice (C and D). In transgenic
mice (D), clusters of SP-C immunoreactive Type II cells were
detected, consistent with alveolar collapse (arrows).
Enlarged alveolar space(S) were common in mSP-C/hSP-B(mSP-B /) mice
(C and D). Magnification is ×95 for all
panels.
mice are highly susceptible to oxygen induced injury
in vivo (31). In order to test the hypothesis that SP-B
production by Clara cells is critical for recovery from oxygen-induced
lung injury, 6-week-old hSP-C/hSP-Bc(mSP-B /) mice
were exposed to 95% O2. By immunohistochemistry, SP-B protein was detected in Type II cells but not in Clara cells of hSP-C/hSP-Bc(mSP-B /) mice (not shown) consistent
with the results of previous in situ hybridization studies
(26). There was no difference in gross lung structure between
transgenic mice and wild type littermates with respect to septal
thickening, microhemorrhages, alveolar inflammatory cell infiltration,
or Type II cell hyperplasia or hypertrophy after 48 or 72 h of
O2 exposure. Mean survival times for wild type mice (7.5 days ± 0.5, n = 10) and transgenic mice (7.9 days ± 0.7, n = 10) were not significantly
different (p = 0.79) after 10 days of hyperoxic challenge and mortality was not detected in either group prior to
96 h of exposure. These results suggest that lack of SP-B protein in Clara cells did not increase susceptibility to oxygen-induced lung injury.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
mice. Restoration of SP-B in Clara cells was not sufficient to correct
pulmonary dysfunction or neonatal lethality in SP-B / mice, whereas
selective expression in Type II cells completely restored lung function
in vivo. These results indicate that the neonatal lethality
in SP-B / mice is due to the absence of SP-B expression in Type II
cells and that expression of SP-B in Clara cells cannot replace this function.
/ mice is likely caused by the
lack of mature SP-B peptide in these animals. SP-B proprotein and
processing intermediate (25 kDa) were detected by Western blotting and
immunohistochemistry and were dramatically increased in mice carrying
multiple copies of the transgene; however, complete processing to the
mature SP-B peptide by Clara cells was not observed. These results are
similar to those in the H441 pulmonary adenocarcinoma cell line, and
provide further support for the Clara cell origin of this cell line
(32). The most likely explanation for incomplete processing of SP-B in
H441 cells and Clara cells of mCCSP/hSP-B(mSP-B /) mice is that
these cells lack an endoprotease that cleaves the 25 kDa processing
intermediate to generate the mature SP-B peptide. Given that lamellar
bodies were never detected in mCCSP/hSP-B(mSP-B /) mice, it is
unlikely that SP-B proprotein or the 25-kDa form secreted by Clara
cells is internalized and processed by Type II cells. Incomplete
processing of SP-B proprotein, complete absence of mature SP-B peptide,
and consistent neonatal respiratory failure in mCCSP/hSP-B(mSP-B /) mice strongly suggest that, under resting conditions, Clara cells do
not contribute SP-B to the alveolar surfactant pool.
mice exposed to 95% oxygen for 3 days lung compliance was
markedly reduced relative to wild type littermates (31). Altered lung
function was associated with increased severity of pulmonary edema,
hemorrhage, and inflammation, lung permeability, and protein leakage
into the alveolar space supporting the concept that SP-B deficiency
exacerbates hyperoxic-induced lung injury. In SP-B +/+ mice similarly
exposed to hyperoxia, SP-B RNA was dramatically increased in
bronchiolar cells and decreased in alveolar cells suggesting that
production of SP-B in Clara cells may compensate for diminished
expression in Type II cells during hyperoxic challenge (28). This
hypothesis predicts that loss of SP-B expression in Clara cells would
increase susceptibility to hyperoxic-induced lung injury; however, the
complete loss of SP-B in Clara cells of hSP-C/hSP-B(mSP-B /) mice
did not exacerbate hyperoxic-induced changes in mortality or lung
structure relative to wild type littermates. These results suggest that
expression of SP-B in Clara cells is not required for surfactant
homeostasis in unchallenged mice or for recovery from or adaptation to hyperoxia.
mice, in which SP-B RNA and protein levels are
decreased 50% compared with wild type littermates, exhibit air
trapping related to collapse of small airways (34). However, the
ability of SP-B to promote rapid formation of a stable phospholipid
film is likely exclusive to the mature peptide which is not synthesized
in Clara cells. Since Type II cells are the sole source of mature SP-B,
any SP-B peptide in the terminal airways is likely derived from the
alveolar surfactant pool; however, to date, there is no direct evidence
for movement of SP-B from alveoli to conducting airways.
/ mice completely reversed neonatal respiratory dysfunction and
lethality in these mice. The importance of SP-B expression in Type II
cells is further supported by results in mSP-C/hSP-B(mSP-B /)
transgenic line 3 in which SP-B expression was restored in some, but
not all, Type II cells. These mice had distended alveoli and septal
thickening consistent with focal atelectasis. This outcome is similar
to that in ventilated preterm lambs in which surfactant deficiency
resulted in dilation of alveolar ducts with collapse of some adjacent
alveoli (35). Taken together, these results suggest a model in which
Type II cells lacking SP-B secrete surfactant with poor surface
activity leading to collapse of the involved alveolus at the end
expiration. The collapsed alveolus contributes to thickening of the
alveolar wall, while the expelled air enters an adjacent alveolus
resulting in distension (36). Although distended, this alveolus remains
open at the end expiration because Type II cells in this unit express
SP-B and, consequently, secrete a functional surfactant.
/ mice. Expression of SP-B in some, but not all Type II cells of
SP-B / mice, allowed postnatal survival, but resulted in
significantly altered lung architecture and function. Synthesis of
pro-SP-B in Clara cells of SP-B / mice resulted in respiratory
dysfunction and invariable neonatal death, associated with the complete
absence of mature SP-B peptide in the lungs of these mice. These
results indicate that expression and processing of the SP-B proprotein
to the mature peptide in Type II cells is absolutely required for lung
function in vivo and that synthesis of pro-SP-B in Clara
cells cannot substitute for this function.
FOOTNOTES
To whom correspondence should be addressed: Children's Hospital
Medical Center, Div. of Pulmonary Biology, TCHRF, 3333 Burnet Ave.,
Cincinnati, OH 45229-3039. Tel.: 513-636-7223; Fax: 513-636-7868; E-mail: Tim.Weaver{at}chmcc.org.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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