Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039
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ABSTRACT |
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Clara cell secretory protein (CCSP) deficiency
in mice is associated with increased susceptibility to pulmonary
inflammation after hyperoxia or viral infection. Because adenoviral
exposure perturbs pulmonary surfactant homeostasis in vivo, we
hypothesized that CCSP deficiency would influence surfactant metabolism
after pulmonary infection. Alveolar and total lung saturated
phosphatidylcholine pool sizes were similar in CCSP-deficient
[CCSP(/
)] and wild-type [CCSP(+/+)] mice before and 7 days after intratracheal
administration of adenovirus. Radiolabeled choline and palmitate
incorporation into saturated phosphatidylcholine was similar, and there
was no alteration by previous infection 7 days before the incorporation measurements. Furthermore, CCSP deficiency did not influence clearance of
[14C]dipalmitoylphosphatidylcholine
and 125I-labeled recombinant
surfactant protein C. Increased persistence of alveolar capillary leak
was observed in CCSP(
/
) mice after adenoviral infection.
Surfactant lipid homeostasis was not influenced by CCSP before or after
administration of adenovirus to the lung. Persistence of alveolar
capillary leak in CCSP(
/
) mice after adenovirus provides
further evidence for the role of CCSP in the regulation of pulmonary inflammation.
phosphatidylcholine; transgenic mice; surfactant protein A; surfactant protein C; Clara cells; Clara cell secretory protein
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INTRODUCTION |
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THE PRESENCE OF PHOSPHOLIPIDS and the autoradiographic
concentration of palmitate into the secretory granules of nonciliated bronchiolar epithelial cells (Clara cells) suggested that these cells
contributed to surfactant homeostasis in the lung (4, 6, 7). Clara
cells also express surfactant protein (SP)-A, SP-B, and SP-D (18, 22).
However, the most abundant secretory product of Clara cells is Clara
cell secretory protein (CCSP), a 10- to 16-kDa protein that binds
xenobiotics and may play a role in the modulation of lung inflammation
(3, 20). CCSP-deficient [CCSP(/
)] mice were
more sensitive to oxygen injury (14), and leukocytic infiltration and
chemokine and cytokine production were increased in
CCSP(
/
) mice after viral infection (8). In normal mice,
adenoviral infection altered SP homeostasis, increasing alveolar
protein concentration and locally inhibiting SP mRNA expression at the
site of inflammation (17, 23). CCSP concentrations are decreased in
chronic lung disease such as chronic bronchiolitis and lung fibrosis,
and these diseases are associated with changes in the amount of
surfactant components in bronchoalveolar lavage (9).
Crystallized CCSP is a dimer with a hydrophobic pocket that binds
phosphatidylcholine (PC) and phosphatidylinositol, and these phospholipids copurify with CCSP (21). CCSP also inhibits the activity
of phospholipase A2 by binding PC
and could modulate surfactant catabolism (19). Given the links of Clara
cells and CCSP to the surfactant system and the potential
anti-inflammatory role of CCSP in the lungs, we hypothesized that
CCSP(/
) mice would have abnormalities in surfactant
metabolism. We also hypothesized that the increased and prolonged
inflammatory response resulting from adenoviral exposure of
CCSP(
/
) mice would further alter surfactant metabolism.
We evaluated metabolic variables of saturated PC (Sat PC) and a
recombinant human SP-C (rSP-C) in CCSP(
/
) mice and in
CCSP(
/
) mice challenged with replication-deficient adenovirus to induce lung injury.
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METHODS |
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Mice. CCSP(/
) (129J
Ola/129J hybrid), generated as previously described (20), and wild-type
[CCSP(+/+); 129J, Taconic Farms, Germantown, NY] mice were
housed in the Children's Hospital (Cincinnati, OH) Research Foundation
vivarium under pathogen-free conditions (8). Groups of 8-10
eight-week-old CCSP(
/
) and CCSP(+/+) mice were used for
all the studies.
Intratracheal administration of
adenovirus. CCSP(/
) and CCSP(+/+) mice
were randomized to intratracheal adenovirus or saline treatment.
Metabolic studies of surfactant were performed 1 day after adenovirus
instillation to evaluate the surfactant system during the peak of the
early inflammatory response (8). The metabolic studies also were
repeated on day 7 because the
CCSP(
/
) mice have a prolonged proinflammatory response to
adenovirus (8). Mice were anesthetized with methoxyflurane and orally
intubated with a 25-gauge animal feeding needle. Each mouse received 50 µl of saline or 50 µl of Av1Luc1 (1 × 109 plaque-forming units), an E1-
to E3-deleted replication-deficient adenoviral vector expressing
firefly luciferase from the Rous sarcoma virus promoter in buffer (10 mM Tris, 1 mM MgCl2, and 10% glycerol, pH 7.4) (8). The characteristics of the
inflammation after exposure of mice to this virus were previously
described (8).
Precursor incorporation into Sat PC and
secretion. Eight hours before alveolar wash, which was
24 h or 7 days after intratracheal administration of adenovirus or
saline, CCSP(+/+) and CCSP(/
) mice were given an 8 µl/g
body wt intraperitoneal injection of 0.1 µCi/g of
[3H]choline chloride
(DuPont-New England Nuclear, Boston, MA) and 0.2 µCi/g of
[14C]palmitic acid
(American Radiolabeled Chemicals, St. Louis, MO) (11). The palmitic
acid was stabilized in solution with 2.5% human serum albumin. An
interval of 8 h between precursor injection and alveolar wash was used
because this time provides an accurate estimate of total precursor
incorporation into Sat PC (13). At 8 h, the ratio of radiolabeled Sat
PC in the alveolar wash to the sum of radiolabeled Sat PC in the
alveolar wash and lung tissue was used to estimate net secretion on the
linear part of the secretion curve (13).
Clearance of dipalmitoylphosphatidylcholine and
rSP-C. CCSP(+/+) and CCSP(/
) mice were
given intratracheal injections of 50 µl of saline that contained 0.3 µCi of
[14C]choline-labeled
dipalmitoylphosphatidylcholine (DPPC), and 0.15 µCi of
125I-rSP-C.
[14C]DPPC was
purchased from Amersham (Arlington Heights, IL). The rSP-C (a gift from
Byk Gulden, Constance, Germany) is the 34-amino acid human sequence
altered by replacement of cystine-3 and -4 with phenylalanine and
methionine-32 with isoleucine (10). The rSP-C, iodinated with the
125I-labeled Bolton-Hunter reagent
as previously reported (10), was metabolized in rabbit and mouse lungs
comparably to the native peptide.
[14C]DPPC and
125I-rSP-C were mixed with a small
amount of a chloroform-methanol extract of natural mouse surfactant in
chloroform, dried under N2, and
resuspended in saline by brief sonication. The 50-µl injection volume
contained a trace dose of 0.1 µmol/kg of Sat PC relative to the
endogenous pool of ~15 µmol Sat PC/kg. The intratracheal injections
were given with 30-gauge needles after exposure of the trachea of each
anesthetized mouse (13). The interval between intratracheal injection
of the radiolabeled surfactant components and alveolar wash was 16 h
based on a previous study (13) of clearance of surfactant components in mice.
Protein permeability. The recovery of 125I-albumin in alveolar washes 2 h after intraperitoneal injection of 5 µCi in 100 µl of iodinated bovine serum albumin was used as a measure of lung injury (12).
Alveolar lavage and tissue processing. Mice were given intraperitoneal pentobarbital sodium to achieve deep anesthesia, and the distal aorta was cut to exsanguinate each animal. The chest of the animal was opened, a 20-gauge blunt needle was tied into the proximal trachea, and five aliquots of 0.9% NaCl were flushed into the lungs to achieve full inflation (~1 ml) and were withdrawn by syringe three times for each aliquot (13). The recovered lavage fluid was pooled, and the volume was measured. The relevant alveolar washes and lung tissue after alveolar wash were counted for 125I to measure recovery of 125I-rSP-C relative to the recovery evaluated in animals killed 10 min after the intratracheal injection. The amount of Sat PC recovered by alveolar wash was measured by extracting the alveolar wash with chloroform-methanol (2:1) followed by treatment of the lipid extract with OsO4 in carbon tetrachloride and silica column chromatography according to Mason et al. (16). Phosphorus in Sat PC was measured with the Bartlett (1) assay. Lung tissue after alveolar wash was homogenized in saline, and an aliquot was extracted and analyzed for Sat PC content.
Data analysis. All values are means ± SE. Differences between groups were tested by two-tailed Student's t-tests. When more than two comparisons were made, analysis of variance followed by the Student-Newman-Keuls multiple comparison procedure was used. Significance was accepted at P < 0.05.
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RESULTS |
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Protein permeability. Radiolabeled
albumin was given by intraperitoneal injection 2 h before the mice were
killed for alveolar washes. Adenovirus or vehicle had been given 24 h
or 7 days before the alveolar washes. The recovery of
125I-albumin in alveolar washes
increased in both CCSP(+/+) and CCSP(/
) mice 24 h
after adenoviral exposure, indicating increased
alveolar-capillary permeability (Fig. 1).
Recovery of the labeled albumin was not increased 7 days after
adenoviral exposure in the CCSP(+/+) mice but remained elevated in the
CCSP(
/
) mice, indicating persistent injury.
|
Sat PC pool sizes. The alveolar pool
sizes of Sat PC were similar in CCSP(+/+) and CCSP(/
)
mice 24 h after saline or adenoviral administration (Fig.
2). In comparison to the values for
saline-instilled animals, both CCSP(+/+) and CCSP (
/
)
mice had small increases in alveolar and total lung (alveolar plus
tissue) Sat PC 7 days after adenoviral exposure.
|
Precursor incorporation into Sat PC.
The CCSP(+/+) and CCSP(/
) mice were given
[3H]choline and
[14C]palmitic acid 8 h
before alveolar wash and 24 h or 7 days after intratracheal challenge
with adenovirus or saline. Choline incorporation into total lung Sat PC
was not increased after adenoviral exposure in the CCSP(+/+) and
CCSP(
/
) mice relative to that in the saline control mice
(Fig. 3). There was more variability in the
measurements for CCSP(
/
) mice after adenoviral exposure,
which did not reach significance except for a higher
[3H]choline-labeled
Sat PC in alveolar lavage in CCSP(
/
) mice 7 days after
adenovirus injection. A similar pattern of responses was observed in
experiments with labeled palmitate (Fig.
4). The [14C]palmitic
acid-labeled Sat PC tended to be higher in CCSP(
/
) mice 7 days after adenoviral exposure. Percent secretion of
[14C]palmitic acid-
and
[3H]choline-labeled
Sat PC in the alveolar washes relative to that in the total lung is
shown in Figs. 3 and 4. Percent secretion was higher 24 h after
adenoviral exposure for
[14C]palmitic
acid-labeled Sat PC. Percent secretion also was higher 7 days after
adenoviral exposure for
[3H]choline-labeled
Sat PC in CCSP(
/
) mice compared with that in the saline
control group and was somewhat lower in CCSP(
/
) mice 24 h
after adenovirus injection. There were no changes in surfactant
secretion in CCSP(+/+) mice after adenovirus infection.
|
|
Recovery of DPPC and rSP-C. The
CCSP(/
) and CCSP(+/+) mice received a trace dose of
surfactant radiolabeled with
[14C]DPPC and
125I-rSP-C by tracheal injection
16 h before alveolar wash and 24 h or 7 days after intratracheal saline
or adenovirus challenge. About 18% of the radiolabeled DPPC was
recovered by alveolar wash, and 24% was recovered in the total lung of
CCSP(+/+) and CCSP(
/
) mice 24 h and 7 days after
intratracheal saline or adenovirus (Fig.
5). The recovery of rSP-C was ~17% in
the alveolar washes, and total lung recovery was ~32% in all groups
(Fig. 6). Although more rSP-C than DPPC was
recovered, there was no effect of CCSP deficiency or adenovirus on the
recovery of the surfactant components.
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DISCUSSION |
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CCSP deficiency did not alter surfactant phospholipid pool sizes,
precursor incorporation, or Sat PC secretion and clearance in vivo.
Furthermore, pulmonary infection with the replication-deficient adenovirus that caused pulmonary inflammation did not alter surfactant metabolism either acutely at 24 h or more chronically after 7 days (8).
Small differences in Sat PC secretion were observed after adenovirus
challenge in CCSP(/
) but not in CCSP(+/+) mice. The
persistence of increased alveolar capillary permeability as indicated
by the increased recovery of
125I-albumin in the alveolar
washes of the CCSP(
/
) mice at 7 days supports an
increased duration of lung inflammation in CCSP(
/
) mice.
Adenoviral exposure also increased alveolar and total lung Sat PC pool
sizes at 7 days in both CCSP(+/+) and CCSP(
/
) mice, indicating the effects of the adenoviral infection on the surfactant system.
The present findings indicate that CCSP does not play a major role in the control of surfactant homeostasis in vivo. Although Clara cells contain SP-A, SP-B, and SP-D mRNAs, it remains unclear whether Clara cells secrete significant amounts of these proteins and whether these SPs play a role in the function of conducting airways. A link between surfactant homeostasis and Clara cell function was suggested by the observations that SP-D deficiency results in large increases in alveolar phospholipid pool sizes (2, 15). The ability of CCSP to inhibit phospholipase A2 activity also suggests a link between CCSP and surfactant (19).
The function(s) of the major secretory product of Clara cells, CCSP,
has remained enigmatic. CCSP deficiency in mice results in increased
sensitivity to oxygen and persistent inflammation after pulmonary
infection with adenovirus (8, 14). Both stresses to the lung result in
elevated levels of proinflammatory mediators in CCSP(/
)
mice relative to those in CCSP(+/+) mice. Because surfactant lipid and
protein concentrations are altered after hyperoxic and viral-induced
lung injury, we hypothesized that CCSP is playing a role in the
regulation of inflammation that, in turn, influences surfactant
homeostasis. This hypothesis is not supported by the experiments that
we performed. The results are of sufficiently high resolution to
exclude large effects of CCSP on the surfactant system. Certainly,
subtle regional effects in small airways could occur that would not be
detected by the techniques used, which emphasize the net metabolic
activities of type II cells and alveolar macrophages.
Although Clara cells produce surfactant-specific products and are
positioned in the airways in locations that require surfactant to
maintain airway patency (5), the major secretory product of these cells
seems to have no role in the regulation of surfactant metabolism. The
persistent injury resulting from adenoviral exposure of
CCSP(/
) mice also had minimal effects on surfactant metabolism.
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ACKNOWLEDGEMENTS |
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This work was funded by National Heart, Lung, and Blood Institute Grants HL-28623 and P01-HL-61646 and the Cystic Fibrosis Foundation Research Development Program Center.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Ikegami, Children's Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: machiko.ikegami{at}chmcc.org).
Received 6 May 1999; accepted in final form 12 July 1999.
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