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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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Clara cell secretory protein (CCSP) is an abundant component of the extracellular lining fluid of airways.
Even though the in vivo function of CCSP is unknown, in vitro studies support a potential role of CCSP in
the control of inflammatory responses. CCSP-deficient mice (CCSP 
/) were generated to investigate
the in vivo function of this protein (13). In this study, we used hyperoxia exposure as a model to investigate
phenotypic consequences of CCSP deficiency following acute lung injury. The pathologic response of the
mouse lung to hyperoxia, and recovery of the lung, include inflammatory cell infiltrate and edema. Continuous exposure to > 95% O2 was associated with significantly reduced survival time among CCSP /
mice as compared with strain-, age-, and sex-matched wild-type control mice. Differences in survival were
associated with early onset of lung edema in CCSP / mice as compared with wild-type controls. To
further investigate these differences in response, mice were exposed to > 95% O2 for either 48 h or 68 h
with one group receiving 68 h of hyperoxia followed by room-air recovery. Lung RNA was characterized
for changes in the abundance of cytokine messenger RNA (mRNA) using a ribonuclease (RNase) protection assay. After 68 h of hyperoxia, interleukin-6 (IL-6), IL-1
, and IL-3 mRNAs were 14-, 3-, and 2.5-fold higher, respectively, in CCSP / mice than in similarly exposed wild-type control mice. Increased
expression of IL-1 mRNA in hyperoxia-exposed CCSP / mice was localized principally within the
lung parenchyma, suggesting that the effects of CCSP deficiency were not confined to the airway epithelium. We conclude that CCSP deficiency results in increased sensitivity to hyperoxia-induced lung injury
as measured by increased mortality, early onset of lung edema, and induction of proinflammatory cytokine
mRNAs.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Clara cell secretory protein (CCSP) is a 16-kD protein first described in the lungs of rodents, but has since been detected in other mammalian species including humans (1- 7). CCSP is one of the major secretory products of Clara cells and is the most abundant soluble protein within lavage fluid of humans, accounting for 0.4% of total protein (8). Immunocytochemical studies of human and rodent lung have shown that intracellular stores of CCSP are localized within the endoplasmic reticulum and secretory granules of Clara cells (3, 9).
Insight into potential in vivo functions of CCSP comes
from analysis of its biologic and biochemical properties
both in vitro and in vivo. CCSP has been shown to bind
and mediate the in vivo accumulation of methylsulfonyl-polychlorinated biphenyl pollutants (12, 13). Other studies
have demonstrated that CCSP is a potent inhibitor of pancreatic PLA2 (14). However, mechanisms of CCSP inhibition of pancreatic phospholipase A2 (PLA2), which involve Ca2+ sequestration and not a direct inhibitory effect,
call into question earlier speculation that CCSP is a physiologically relevant regulator of PLA2 in vivo (15). More
recently, a study by Dieryck and coworkers suggested that
CCSP may influence interferon-
(IFN-) production and
activity, which has led to speculation that CCSP may function as an endogenous anticytokine (16). Collectively, these studies suggest a potential role for CCSP in the metabolism or clearance of lipophilic xenobiotic pollutants,
or as a regulator of inflammation. However, more studies
are clearly needed to fully understand the in vivo function
of CCSP in the lung.
Oxidant lung injury is an attractive model with which to investigate the function of in vivo regulators of pulmonary inflammation. Acute exposure to hyperoxia results in a well-described pathophysiologic response in the lungs of virtually all animals studied. This response begins with subtle subcellular changes resulting from the generation of highly reactive oxidant species such as superoxide and hydroxyl radicals, and is perpetuated through generation of secondary oxidant species, such as aldehydes and peroxides of lipids or proteins (17). Airway epithelial cell injury associated with acute exposure to oxidant gases involves necrosis and desquamation of ciliated cells and flattening of nonciliated cells (18). Secondary effects include edema and influx of inflammatory cells (25). Neutrophil aggregation develops by 48 h of exposure (26). Increased inflammatory cell content of the interstitium is the most prominent feature of oxygen injury between 48 h and 72 h of exposure (27). At sufficient doses and sufficiently long exposures, hyperoxic injury can result in mortality rates approaching 100% in most animal models. However, important variables in sensitivity of the lungs to hyperoxic injury, including age, gender, nutritional status, infection, genetic background, and species differences have been reported (25).
A line of mice deficient in CCSP expression (CCSP /)
were developed to determine in vivo functions for the protein. We hypothesize that CCSP participates in the defense of pulmonary airways against oxidant injury, and in
the regulation of ensuing inflammatory responses. CCSP
/ mice develop normally, and are apparently healthy
and fertile (13). The only phenotypic abnormalities identified in CCSP / mice housed under normal pathogen-free conditions were ultrastructural changes in Clara cells
(13, 28). Here we report the findings of acute oxidant challenge of CCSP / mice. Results indicate that CCSP-deficient mice are more sensitive to oxidant lung injury through
a mechanism that culminates in early onset of pulmonary
edema and expression of proinflammatory cytokine messenger RNAs (mRNAs).
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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Animals and Hyperoxia Exposures
Adult (2 to 6 mo of age) male strain-129 mice were purchased from Taconic Farms (Germantown, NY) animal
breeders and housed for a minimum of 1 week for acclimatization prior to experimentation. Adult (2 to 6 mo of age)
male strain 129 CCSP / mice (13) were maintained as
an in-house colony. All mice were maintained in strict specific-pathogen-free animal housing and were provided food and water ad libitum. Mice were coexposed to hyperoxia in Plexiglas cages with delivery of 100% O2 at a flow
rate of 4 liters/min. CCSP / and corresponding wild-type controls for each experiment were always exposed at
the same time using the same exposure apparatus and O2
supply. To determine survival time in > 95% O2, 10 strain-129 wild-type and 10 CCSP / mice were exposed, and
the time of death was recorded, with statistical difference
determined by Fisher's probability of least significant difference (PLSD). For analyses of the time course of changes
in proinflammatory cytokine mRNA expression, groups of
three wild-type and three CCSP / mice were removed
after 48 h or 68 h of exposure, or exposed for 68 h to O2
and returned to room air for 24 h. Mice were killed by sodium pentobarbital overdose (100 mg/kg injected intraperitoneally) and tissues removed for isolation of total
lung RNA and histological analysis.
Analysis of lung wet weight:dry weight ratios were performed after continuous exposure of mice (n = 4) to hyperoxia for the indicated times, followed by euthanasia of
mice and surgical removal of lung tissue. Wet lung weight
was determined in preweighed tubes that were reweighed
after drying in a vacuum oven at 80°C. Dry weight was
considered to be the weight of dessicated lung tissue measured after weight ceased to decrease with continued drying in the vacuum oven. Wet weight:dry weight values are
presented as the mean ± 1 SD with statistical significance
determined through analysis of variance (ANOVA) for independent measures with P 
0.05.
Ribonuclease Protection Assay
Whole lungs were flash frozen in liquid N2 and RNA isolated by the acid phenol method (29). Total RNA was resuspended in ribonuclease (RNase)-free water, and RNA
concentrations were determined by absorption at 260 nm.
RNase protection assays were performed with riboprobe
templates for interleukin-1
, (IL-1), IL-1, IL-2, IL-3, IL-4,
IL-5, IL-6, IFN-, tumor necrosis factor- (TNF-) TNF-,
and L32 (mouse ribosomal protein L32; a gift from Dr. M. Hobbs, Scripps Research Institute, San Diego, CA) (30).
Radiolabeled riboprobes were synthesized according to
previously published methods (31), with some modifications with respect to the final concentrations of the synthesis reaction: 40 mM Tris-Cl, pH 7.5, 6 mM MgCl2; 10 mM
dithiothreitol (DDT), 0.5 mM unlabeled nucleoside triphosphates (NTP); 5 µm (33P-UTP and 20 µm unlabeled P-UTP);
2 mM Spermidine; 100 µg/ml bovine serum albumin (BSA);
250 µg/ml yeast transfer RNA (tRNA); 1,000 U/ml RNase
inhibitor (Promega, Madison, WI); 100 µg/ml template DNA; and 1,500 U/ml RNA polymerase. These were incubated at 38°C for 90 min. The DNA template was digested with RNase-free deoxyribonuclease I (DNase I)
(Promega), after which the probe was extracted with an
equal volume of phenol/chloroform 1:1 and precipitated
from ethanol. The hybridization reaction followed published methods (30), with some modification. Dried probes
were dissolved (3 × 105 dpm/µl) in hybridization buffer
(80% formamide, 0.4 M NaCl, 1 mM ethylene diamine tetraacetic acid [EDTA], 40 mM 1,4-piperazine diethanesulfonic acid [PIPES], pH 6.7) and added (2 µl) to tubes containing RNA dissolved in 8 µl of hybridization buffer.
All samples were heated to 90°C for 3 min and incubated
at 56°C for 16 h. Single stranded RNA (ssRNA) was then
digested by addition (100 µl) of a solution of RNase A (0.2 µg/ml, BRL) and RNase TI (600 µg/ml; BRL, Gaithersburg,
MD) in 10 mM Tris, 300 mM NaCl, and 5 mM EDTA, pH
7.5. After incubation (30 min at 37°C), the samples were
treated (30 min at 37°C) with 18 µl of a mixture of proteinase K (0.5 mg/ml; BRL) sodium dodecyl sulfate (SDS) (3.5%), and yeast tRNA (100 µg/ml). RNA duplexes were
isolated by extraction/precipitation as described earlier,
dissolved in 80% formamide and dyes, and electrophoresed in standard 6% acrylamide-8 M urea sequencing gels.
Dried gels were placed on XAR film (Kodak, Rochester,
NY) with intensifying screens at 70°C overnight.
Data Analysis
Quantitative analysis was done with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and each of the cytokine mRNAs was normalized to the abundance of L32 mRNA. The data were evaluated on an Apple Macintosh computer (Apple, Inc., Cupertino, CA), using ANOVA for independent measures, which includes tests for Fisher's PLSD. The two-tailed level of significance was set at P < 0.05. The minimum number of mice per group was three.
Subcloning and Probe Preparation
The mouse clonal DNA (cDNA) for IL-1 was subcloned
into a plasmid vector (pBluescript SK +; Stratagene, La
Jolla, CA) for the in vitro transcription of RNA (31). Linearized plasmid DNA of the homologous cDNA clone for
IL-1 was used as a template for the preparation of cRNA
probes. Sense and antisense orientations were confirmed
on Northern blots. The cRNA synthesis reaction followed
modified published methods (31). Final concentrations of
the synthesis reaction were 40 mM Tris-Cl (pH 7.5), 6 mM
MgCl2, 10 mM DTT, 0.5 mM unlabeled nucleoside triphosphates (NTP), 5 µM 32P-UTP, 20 µM unlabled UTP, 2 mM
spermidine, 100 µg/ml BSA, 250 µg/ml yeast tRNA, 1,000 U/ml RNase inhibitor (Promega), 100 µg/ml template DNA,
and 1,500 U/ml RNA polymerase. These were incubated at 38°C for 90 min. The DNA template was digested with
RNase-free DNase I (Promega), and the probe was then
extracted with an equal volume of phenol/chloroform (1:1),
precipitated from ethanol, and resuspended in diethylpyrocarbonate-treated water. Full-length transcripts were
approximately 1.4 kb prior to hybridization; limited alkaline hydrolysis of RNA probes was done to reduce transcript length to 0.1 to 0.3 kb. Partially hydrolyzed transcripts
were sized by denaturing agarose gel electrophoresis.
Tissue Processing and In Situ Hybridization
Briefly, lung tissue was fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) instilled through a tracheal cannula at 10 cm H2O pressure for 15 min. Lungs were excised from the thorax, fixed in the same buffer for an additional 12 h and then dehydrated through graded alcohols, and embedded in paraffin, from which 5-µm sections were prepared. The tissue sections were treated according to a modified method (32). Briefly, tissue sections were treated for 30 min at 37°C with 1 µg/ml proteinase K, washed, and dipped in fresh 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min. After dehydration through a series of ethanol washes, the sections were dried and hybridized overnight at 56°C in 50% formamide, 0.3 M NaCl, 10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1 X Denhardt's solution, 10% dextran sulfate, 0.5 mg/ml yeast tRNA, and 0.3 µg/ml probe. After hybridization, the slides were washed in 0.1 X standard sodium chloride (SSC) (sodium chloride, sodium citrate), twice for 10 min and once for 40 min. Sections were treated with RNase A (20 µg/ml) in RNase buffer (0.5 M NaCl, 10 mM Tris-Cl, and 1 mM EDTA, pH 7.5) for 30 min at 37°C. Slides were passed through 30-min washes of 37° RNase buffer and 0.1 X SSC at room temperature, 0.1 X SSC at 68°C, and 0.1 X SSC room temperature. The slides were then passed through a series of graded ethanol washes and dried. Autoradiography was then performed.
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Results |
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Materials & Methods
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Discussion
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Continous exposure of mice to hyperoxia was associated
with significant reduction in the survival time of CCSP /
mice relative to wild-type control mice (Figure 1). Mean
survival times were 93 h ± 13.58 h versus 114 ± 18.56 h for
CCSP / and wild-type +/+ mice, respectively (means ± SD; P = 0.01 according to Fisher's PLSD). To monitor the
progress of the edema in the two groups of mice, the lung
wet weight:dry weight ratios were examined after 62 h, 72 h,
and 82 h of oxygen exposure (Figure 2). The wet weight:dry weight ratios were statistically different in the CCSP null
mutant mice as compared with the wild-type mice after 82 h
of exposure, indicating that the mutant suffered more edema
at this time point (a statistically significant difference according to ANOVA for independent measures).
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Figure 3 shows the results of RNase protection assays
for detecting proinflammatory cytokine mRNAs within
representative total lung RNA samples of hyperoxia- or
room-air-exposed wild-type and CCSP / mice. Inflammatory cytokine mRNA abundance was examined at 48 h
and 68 h of exposure to > 95% O2, as well as at 24 h in room air recovery after 68 h of O2 exposure. The results
show increases in the abundance of mRNAs for IL-1,
IL-3, and IL-6 from CCSP / mice, with a maximal response at the 68-h hyperoxia time point. No significant
changes in these cytokine mRNAs were observed in wild-type mice at these hyperoxia exposure time points. Quantitative measurements of these changes in larger numbers
of exposed mice are shown in Figures 4A through 4C). By
68 h of O2 exposure, messages encoding IL-1, IL-3, and
IL-6 were increased 3-, 2.5- and 14-fold, respectively, in
CCSP / mice, whereas these messages remained unaltered in wild-type mice. These changes in cytokine mRNAs
were still detectable after 24 h of recovery in room air, at
which time messages encoding IL-1 and IL-6 remained increased 4- and 5-fold, respectively. Wild-type mice demonstrated no alterations from controls at this time point
(Figure 4).
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The observed differences in proinflammatory cytokine
mRNA expression could result from alterations in the severity of hyperoxia-induced lung injury or in the regulation of injury-induced inflammatory responses. To ensure
that both wild-type and CCSP / mice exhibited similar
inflammatory cytokine mRNA expression at a similar time in their injury response, cytokine mRNAs were measured
at the time of death. The results shown in Figure 5 demonstrate that at the time of death, messages encoding TNF-,
IL-6, IL-1, and IL-3 were increased to similar levels in
both wild-type and CCSP / mice. Messenger RNAs for
TNF-, IL-1, IL-2, IL-4, IL-5, and IFN- were not detected at this or any of the earlier time points.
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To further study the cell-specific expression of IL-1 in
the lungs of hyperoxia-exposed mice, the tissue localization of its mRNA was determined by in situ hybridization.
mRNA for IL-1 was undetectable in the lungs of untreated wild-type (not shown) or CCSP / (Figures 6A
and 6B) mice. By 68 h of > 95% O2 exposure, IL-1 transcripts were abundant within diffusely distributed cells of
the lung parenchyma in CCSP / mice (Figures 6C and
6D). No expression of IL-1 was detected above background levels in the lung tissue of wild-type mice exposed
for 68 h to > 95% O2 (Figures 6E and 6F). These results
show that the altered pulmonary response of CCSP-deficient mice to oxidant injury does not appear to be restricted to epithelia of conducting airways.
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The present study demonstrates differential responses of
wild-type and CCSP-deficient mice to hyperoxia. CCSP-deficient mice show reduced survival time and early onset
of increases in mRNAs for the proinflammatory cytokines
IL-1, IL-3, and IL-6. Increases in the expression of IL-1
mRNA were localized to parenchymal regions of the lung,
suggesting that altered responses to oxidant stress are not
exclusively localized to conducting airways, the normal
site of CCSP expression. These findings suggest an important role for CCSP and/or Clara cells in the pulmonary response to oxidant exposure.
The mechanistic basis for the observed increase in sensitivity of CCSP-deficient mice to hyperoxia remains to be determined. Strain and species differences in the response to environmental pollutants can be clearly demonstrated in many cases, and have the potential to significantly affect pollutant responses in humans. Kleeberger and colleagues (34), investigated strain differences in response to oxidant lung injury among inbred mice, and were able to show increased susceptibility to ozone-induced pulmonary inflammation in C57BL/6 mice relative to C3H/HeJ animals. Further studies with this model have shown that C3H/HeJ mice have a delayed increase in hyperoxia-induced lung permeability as compared with C57BL/6 mice (35, 36). Differences in the response of C57BL/6 and C3H/HeJ mice can also be observed in response to radiation-induced lung injury, suggesting that the basis for these strain differences is related to a general injury/repair pathway (37, 38).
The advent of transgenic mouse technologies has provided a means with which to investigate the roles of individual genes in complex biologic processes. Carlsson (33)
demonstrated that extracellular superoxide dismutase null
mutant (EC-SOD /) mice are more sensitive to hyperoxia-induced injury. EC-SOD / mice showed reduced
survival and earlier onset of lung edema than did wild-type
mice. However, one potential problem with the interpretation of hyperoxia sensitivity in Carlsson's study is the use
of F3 generation 129Sv/C57Bl/6 hybrids. Since strain-129
mice and C57Bl/6 mice show differential responses to oxidant injury (C. J. Johnston, J. F. Finkelstein, and B. R. Stripp, unpublished manuscript), the response of F3 generation hybrids may be very unpredictable. Experiments
in the present study were performed on CCSP / mice and wild-type control mice that were all inbred strain-129
mice.
The observed differences in response of CCSP / and
wild-type mice to hyperoxia-induced injury could potentially be explained either by altered regulation of pulmonary inflammatory responses or altered sensitivity to lung
injury. The potential for altered regulation of pulmonary
inflammation following hyperoxia-induced injury is supported by a number of studies in the literature. CCSP has
been suggested to act as an immunosuppressant as a result of its ability to inhibit calcium-dependent PLA2 in vitro
(39, 40). Other in vitro studies have shown that CCSP can
interfere with IFN--mediated actions of the cytokine network (16). Based on these activities, it has been speculated
that CCSP may regulate inflammatory responses within
the lung and urogenital tract (16). However, studies by
Andersson and coworkers (15) of the mechanism responsible for in vitro inhibition of calcium-dependent PLA2 suggest that the inhihibitory effect is indirect and results
from calcium binding and sequestration. These studies call
into question the physiologic relevance of the in vitro
PLA2-inhibitory activity of CCSP. The alternative explanation for the differential responsiveness of CCSP /
and wild-type mice to hyperoxia is increased sensitivity to
injury. Unlike EC-SOD, CCSP is not known to be an oxidant-protective molecule either through catalytic activities
or direct scavenging of free radicals. Mechanisms for increased oxidant injury in CCSP / mice may be related
to the indirect consequences of CCSP deficiency. CCSP-deficient mice have alterations in the secretory apparatus of Clara cells (13, 28). However, it is unknown how alterations in Clara cell secretory function could affect antioxidant defense in the lung. It is possible that CCSP / mice
have alterations in secretion of extracellular antioxidant components normally present within the airway lining fluid. This
could help explain similar responses of CCSP / mice and
EC-SOD / mice to hyperoxia exposure. Further studies
are needed to understand the phenotypic consequences of
CCSP deficiency and the role that Clara cells play in protection against oxidant lung injury.
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Footnotes |
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Address correspondence to: Dr. Barry R. Stripp, Department of Environmental Medicine, University of Rochester, 575 Elmwood Ave., Box EHSC, Rochester, NY 14642.
(Received in original form June 17, 1996 and in revised form October 14, 1996).
Acknowledgments: This study was supported by grants HL51376, HL36543, CA27791. Gregory W. Mango is supported by Toxicology Training grant ES07026. Dr. Barry Stripp is supported by the Parker B. Francis Foundation. The study was performed using core facilities supplied through the Environmental Health Sciences Center at Rochester under Grant ES0124. The authors also would like to acknowledge the support of the Strong Childrens Research Center of the Department of Pediatrics, University of Rochester.
Abbreviations
CCSP, Clara cell secretory protein;
EC-SOD, extracellular superoxide dismutase;
IL-1, interleukin-1;
mRNA, messenger ribonucleic
acid;
PLA2, phospholipase A2;
tRNA, transfer ribonucleic acid.
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References |
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References
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