Departments of 1 Environmental Medicine and 2 Pediatrics, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642; and 3 Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, California 95616
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ABSTRACT |
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Little is known
about the molecular basis for differential pulmonary oxidant
sensitivity observed between genetically disparate members of the same
species. We have generated mice that are deficient in Clara cell
secretory protein (CCSP /
) and that exhibit an oxidant-sensitive phenotype. We characterized the kinetics and distribution of altered stress-response [interleukin-6 (IL-6) and
metallothionein (MT)] and epithelial cell-specific
[cytochrome P-450 2F2
(CYP2F2)] gene expression to further understand the cellular and
molecular basis for altered oxidant sensitivity in 129 strain
CCSP
/
mice. Increases in IL-6 and MT mRNA abundance were detected by 2 h of exposure to 1 part/million
ozone and preceded reductions in Clara cell CYP2F2 mRNA
expression. Despite being qualitatively similar, increases in
IL-6 and MT mRNA expression were enhanced in CCSP
/
mice with
respect to coexposed 129 strain wild-type mice. Increased MT mRNA
expression, indicative of the stress response, localized to the airway
epithelium, surrounding mesenchyme, and endothelium of blood vessels.
These results demonstrate a protective role for Clara cells and their
secretions and indicate potential genetic mechanisms that may influence
susceptibility to oxidant stress.
uteroglobin; cytokines; metallothionein; ozone; lung injury
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INTRODUCTION |
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ALTHOUGH CLARA CELLS have been identified and investigated in many species, their role in normal lung function remains largely speculative. Clara cells are responsible for secretion of nonmucoid substances into the airway lumen and therefore contribute to the maintenance of extracellular lining fluid (ELF) composition (11, 21, 43). One of the most abundant ELF proteins secreted by Clara cells is a 16-kDa homodimer, referred to as Clara cell 10-kDa secretory protein (CC10, CCSP, or CC16), an ancestral homolog of rabbit uteroglobin (44, 50, 52). The true in vivo function of CCSP is unknown. Insight into potential functions of CCSP has been derived from analysis of its biological and biochemical properties. These properties include the ability to bind progestins and other small lipophilic compounds (8, 36, 49, 50), inhibit secretory phospholipase A2 (31), inhibit phagocyte chemotaxis (56), and bind calcium (3).
The spatial distribution and abundance of Clara cells found within the airway epithelium differ among species. However, Clara cells of all species are considered to be one of the more oxidant-resistant airway cell types. In the normal rodent lung, Clara cells actively participate in regeneration of the epithelium after oxidant-induced damage to ciliated cells (14, 15, 33). The possible involvement of Clara cells and their secretions in protection from pulmonary oxidant stress has been inferred from a number of studies. In the rat lung, adaptation to chronic ozone exposure is associated with airway remodeling, characterized by increases in the number of Clara cells in distal airways (7, 38, 41, 51) and increases in the abundance of CCSP and activity of antioxidant enzymes (13, 42). These data support the hypothesis that tolerance to chronic oxidant stress may be associated with increased secretory capacity of conducting airways, arising from both increased Clara cell numbers and altered Clara cell secretion. Oxidant-induced alterations in the secretory capacity of Clara cells can also be inferred from studies investigating acute responses of conducting airways to inhaled NO2. Acute exposure of rats to NO2 is associated with loss of Clara cell secretory granules, suggesting oxidant-induced secretion (14). It remains to be determined what contribution, if any, constitutive and/or inducible Clara cell secretion makes to antioxidant capacity of the ELF and protection of airways from oxidant injury.
It is well established that genetic factors influence susceptibility to a variety of pneumotoxic agents, including ozone (2, 27-29, 45). Other studies investigating genetic susceptibility to bleomycin-induced lung fibrosis have demonstrated a positive correlation between induction of IL-6 mRNA expression and the sensitivity of mouse strains (6). Tissue injury is a potent stimulus for initiation of the stress response, otherwise known as the acute-phase response. Interleukin-6 (IL-6) is the major cytokine involved in the upregulation of acute-phase proteins in the liver, and this function allows IL-6 to serve as a long-distance messenger of focal injury (18, 47). Metallothionein (MT) is another stress-responsive gene that has been shown to be regulated in the lung in response to a variety of injury stimuli, including oxidant stress (25, 37, 46). It has been suggested that MT may function as an antioxidant (19, 46, 55) and therefore may serve an important protective function during the acute-phase response (34, 39).
To define in vivo roles for CCSP and Clara cell secretions, we have
generated a line of genetically modified mice that are homozygous for a
null allele of the CCSP gene (53). We have shown that CCSP-deficient
(CCSP /
) mice are more sensitive to hyperoxia and exhibit
alterations in inflammatory cytokine gene expression (23). In the
present study, we investigate changes in the expression of IL-6 and MT
mRNAs as biomarkers for the initial phase of the stress response
associated with ozone exposure in wild-type (WT) and CCSP
/
mice. We also examined the expression of cytochrome
P-450 2F2 (CYP2F2), which is only
expressed at high levels within Clara cells of the murine lung (43), to
assess integrity of the airway epithelium, and specifically Clara
cells, after ozone exposure. We demonstrate that CCSP deficiency
enhances the oxidant-induced stress response in CCSP
/
mice. The increase in stress response was characterized by a dramatic
but transient increase in IL-6 mRNA abundance and a more protracted
elevation in the abundance of MT mRNA. Results from in situ
hybridization analysis show that the altered MT mRNA expression of CCSP
/
mice localizes to airway epithelia, surrounding
mesenchyme, and the endothelial cells lining blood vessels. These
results demonstrate a role for Clara cells and their secretions in
protection from ozone-induced lung injury.
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MATERIALS AND METHODS |
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Animals. WT strain 129 (Taconic,
Germantown, NY) and CCSP /
strain 129 mice were maintained as
specific pathogen-free, in-house colonies and were allowed food and
water ad libitum. Male mice between the ages of 2 and 5 mo were used
for all experiments.
Exposures. WT and CCSP /
mice were coexposed to ozone in Rochester chambers (30, 57) for the
indicated times. Ozone was generated by exposing 100%
O2 to ultraviolet light using an OREC model 03V1-0 ozone generator (Ozone Research and Equipment, Phoenix, AZ) and was diluted with filtered room air. Mice were maintained in wire mesh cages with ozone concentration measured at nose
level and continuously recorded using a Dasibi 1003-AH ozone analyzer
(Dasibi Environmental, Glendale, CA) calibrated against a New York
State Department of Environmental Conservation ozone calibrator. Ozone
concentration was maintained at 1.0 ± 0.1 part/million
(ppm) for time-course studies and at either 0.5 ± 0.1, 1.0 ± 0.1, or 2.5 ± 0.1 ppm for dose-response analysis. Control mice were
exposed to filtered room air in an adjacent chamber.
Tissue collection. Mice were killed
immediately after removal from ozone by intraperitoneal injection of
100 mg/kg pentobarbital sodium followed by exsanguination. For time-
course analysis, three to six mice were exposed for any one treatment,
of which two mice from each group were used for both isolation of total RNA and tissue fixation. Total lung RNA was isolated from remaining mice. For RNA isolation and tissue fixation, lungs were exposed, and
the left lobar bronchus was tied using suture silk. Left lobes were
removed, homogenized in guanidine thiocyanate solution (10), and stored
at 20°C until RNA was isolated. Tracheas were then cannulated with an Insyte 20-gauge intravenous catheter.
Right lobes were fixed by inflation with ice-cold 2% glutaraldehyde in
100 mM cacodylate buffer (pH 7.4).
Lungs were inflated to capacity with fixative and then allowed to stand
for 10 min at 10 cmH2O pressure,
followed by immersion in the same fixative for an additional 8 h at
4°C. Fixed lung tissue was washed in 100 mM cacodylate buffer (pH
7.4) at 4°C; lower (caudal) right lobes were removed, dehydrated,
and embedded in paraffin; and 5-µm sections were prepared for in situ
analyses. For dose-response studies, lungs were removed from exposed
mice, and total RNA was isolated.
RNA isolation and analysis. Total lung
RNA was isolated by the method of Chomczynski and Sacchi (10). Cytokine
mRNA expression was determined by RNase protection assay (RPA) using
the RiboQuant mCK-2 multiprobe set (PharMingen, San Diego, CA).
Conditions used have been described previously (23). In brief,
32P-radiolabeled riboprobes were
synthesized based on the protocol by Melton et al. (35), with slight
modifications. Excess probe was hybridized to 5 µg of total RNA at
56°C overnight. Unbound probe and single-stranded RNA
were digested using RNase A and RNase T1 (Boehringer Mannheim,
Indianapolis, IN). Protected probe-mRNA duplexes were denatured and
resolved on 8 M urea, 6% acrylamide sequencing gels. MT and CYP2F2
mRNAs were analyzed using S1 nuclease protection assays (S1) based on
published methods. The CYP2F2 probe (a gift from Dr. J. K. Ritter, National Institute on Child Health and Human Development,
Bethesda, MD) and ribosomal protein L32 probe (used as internal
standard) have been described previously (5, 54). The MT S1 probe was
generated by reverse transcription-PCR, amplifying a 233-nt cDNA
fragment from total lung RNA prepared from ozone-exposed mouse lung
tissue. Oligonucleotide primers were determined from the published
sequence for MT I (16). The oligonucleotides synthesized were
5'-CTTACTCCGTAGCTCCAGCTTCAC-3' as the 5'-primer and
5'-CTGTTCGTCACATCAGGCACAG-3' as the 3'-primer (GIBCO BRL, Grand Island, NY). The resulting PCR product
was purified, ligated into the pCR2.1 vector, and cloned into One Shot
INVF' cells using the TA cloning kit (Invitrogen, San Diego,
CA). PCR amplification using a vector-specific primer and the
3'-MT primer yielded a 606-nt probe fragment capable of
protecting the same 233-nt sequence of mRNA originally PCR amplified
from the MT cDNA. Probes were 5'-end labeled with
32P using T4 polynucleotide kinase
(Promega). Excess probe was denatured by boiling and hybridized with 5 µg of total RNA at 50°C overnight. S1 nuclease (Boehringer
Mannheim) was then added to digest free single-stranded probe.
Protected probe-mRNA duplexes were denatured and resolved on an 8 M
urea, 6% acrylamide denaturing gel, as with RPAs. Both RPA and S1
results were quantitated using a PhosphorImager and ImageQuant software
(Molecular Dynamics, Sunnyvale, CA).
In situ hybridization. In situ hybridization for CYP2F2 and MT mRNA has been described previously (22, 53). Probes were synthesized based on published methods (35). Linearized plasmids containing mouse CYP2F2 cDNA and MT (a gift from G. Andrews, Kansas University) served as templates to generate antisense riboprobes using either SP6 or T7 RNA polymerase in the presence of [33P]UTP. Five-micrometer sections from paraffin-embedded caudal right lobes (see above) were probed for mRNA localization. Tissue section preparation and hybridization conditions followed those described previously (4, 23). After hybridization, slides were RNase treated and washed. Slides were then dehydrated and dipped in Kodak NTB2 photographic emulsion (Kodak, Rochester, NY).
Statistics. Two-way ANOVA was done
using SuperAnova software (Abacus Concepts, Berkeley, CA) to compare
results between coexposed WT and CCSP /
mice. Post hoc
least squares analysis was used to assess significance. After two-way
ANOVA, one-way ANOVA was used to independently compare results from WT
and CCSP
/
mice with their corresponding filtered
air-exposed controls. Post hoc Fisher's protected least significant
difference analysis was used to assess significance. For all analyses,
significance was assessed at P
0.05.
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RESULTS |
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Ozone exposure alters Clara cell gene
expression. To investigate the hypothesis that CCSP
deficiency alters the response of conducting airways to oxidant stress,
we initially examined changes in CYP2F2 mRNA expression in
ozone-exposed WT and CCSP /
mice. Within the mouse lung,
only Clara cells express high levels of CYP2F2 (43). We have previously
demonstrated that CCSP deficiency is not associated with altered
steady-state levels of CYP2F2 mRNA (53). Therefore, we investigated the
kinetics of ozone-induced changes in CYP2F2 as a potential marker of
differential ozone susceptibility. Figure
1 shows the results of S1
nuclease protection assays performed using total lung RNA.
Ozone-induced reductions in CYP2F2 mRNA levels were observed in both WT
and CCSP
/
mice. No significant changes in CYP2F2 mRNA
levels were observed in either WT or CCSP
/
mice until 8 h of ozone exposure, at which time levels were reduced to 69%
(P = 0.105) and 47%
(P = 0.020) of filtered air control
levels, respectively. Regional alterations in CYP2F2 mRNA abundance
were characterized by in situ hybridization of lung tissue sections
using an antisense riboprobe (Fig. 2). Results demonstrated that, within CCSP
/
mice, CYP2F2
mRNA was reduced in Clara cells of terminal bronchioles by 4 h of continuous exposure to 1.0 ppm ozone. These data demonstrate
that, despite regional changes in CYP2F2 mRNA expression, alterations
in total lung CYP2F2 mRNA abundance do not provide a sensitive
measure of the early response to ozone exposure. Because the goal of
the present study was to define early events associated with the
differential oxidant sensitivity of CCSP
/
mice relative to
strain-matched WT mice, further studies involved analysis of
stress-response gene expression.
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Ozone-mediated induction of MT and IL-6 mRNAs is
dramatically enhanced with CCSP deficiency. Unlike
CYP2F2, basal expression of MT and IL-6 mRNAs was low, allowing
increases resulting from oxidant injury to be measured with greater
sensitivity. Analysis of MT mRNA expression by S1 nuclease protection
assay (Fig. 1) and IL-6 mRNA expression by RPA (Fig.
3) revealed similar baseline levels in
filtered air-exposed control WT and CCSP /
mice. Both MT
and IL-6 mRNAs were differentially regulated between WT and CCSP
/
mice after ozone exposure.
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Significant increases in MT mRNA abundance were observed in CCSP
/
mice beginning at 2 h of ozone exposure relative to
filtered air controls (Fig. 1). Levels of MT mRNA remained elevated at ~15-fold over corresponding filtered air controls at both 4- and 8-h
time points in CCSP
/
mice. In contrast, ozone-induced
changes in MT mRNA abundance in WT mice, despite showing an upward
trend at 4 h, were not significantly elevated until 8 h of ozone
exposure. MT mRNA abundance in 8-h ozone-exposed WT mice was comparable to that detected in CCSP
/
mice at the 4-h time point
(14- vs. 14.5-fold, respectively; Fig. 1). Therefore, in ozone-exposed CCSP
/
mice, MT mRNA showed altered kinetics but similar
magnitude of induction relative to similarly exposed WT mice (Fig.
1C).
Ozone-induced increases in IL-6 mRNA were transient and showed peak
mRNA levels at 4 h of ozone exposure in both WT and CCSP /
mice (Fig. 3). However, despite similarities in the
kinetics of induction, ozone-induced increases in IL-6 mRNA showed
quantitative differences between WT and CCSP
/
mice. IL-6
mRNA was elevated 3.9- and 14.1-fold at the 4-h ozone exposure time
point in WT and CCSP
/
mice, respectively. Increases in
IL-6 mRNA were transient, returning to near-control levels in both WT
and CCSP
/
mice by 8 h of ozone exposure (Fig. 3).
Therefore, in contrast to the pattern of differential MT expression
between WT and CCSP
/
mice, ozone-elicited increases in
IL-6 mRNA expression showed similar kinetics but differences in the
magnitude of induction (Fig. 3B).
CCSP /
mice show induction of MT mRNA
expression within airway epithelium and adjacent
tissues. Differential MT and IL-6 mRNA expression
demonstrates increased ozone-induced oxidative stress associated
with CCSP deficiency. These changes in MT mRNA expression were
localized to determine 1) if these
changes occurred in conducting airways (based on the pattern of CCSP
expression in WT mice) and 2) if
other cell types, in addition to those of conducting airways, exhibited
ozone-induced oxidant stress in CCSP
/
mice. Consistent with
results from S1 nuclease protection assays (Fig. 1), robust expression
of MT mRNA was detected in lung tissue of ozone-exposed CCSP
/
mice after 4 h of ozone exposure. At this time, MT mRNA
localized to epithelial cells of conducting airways, mesenchymal tissue
surrounding conducting airways, and endothelial cells of blood vessels
(Fig. 4, C
and D). No specific hybridization
signal was observed in lung tissue from filtered air-exposed CCSP
/
mice (Fig. 4, A and
B) or in lung tissue from either
filtered air- or 4-h ozone-exposed WT mice (data not shown).
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WT mice show similar changes to CCSP /
mice in IL-6 and MT mRNA expression at higher doses of
ozone. Altered kinetics of MT mRNA expression in
ozone-exposed CCSP
/
mice relative to WT mice
demonstrates a shift in the time-course response toward increased
sensitivity. However, because IL-6 mRNA expression differed between WT
and CCSP
/
mice in magnitude but not in kinetics of the
response, dose response rather than time-course analyses were
required to better define the sensitivity relationship. Figure 5 shows the results of dose-response
analysis characterizing the expression of IL-6 and MT mRNA in lungs
of WT and CCSP
/
mice after exposure to 0, 0.5, 1, and
2.5 ppm ozone for 4 h. MT mRNA expression in 2.5 ppm ozone-exposed WT
mouse lungs was similar to that observed in the lungs of CCSP
/
mice after exposure to 1.0 ppm ozone (Fig. 5,
A and
C). Interestingly, MT mRNA abundance showed no further increases in CCSP
/
mice beyond the
1.0-ppm ozone dose. Similar results were obtained for altered IL-6 mRNA expression. As with altered MT mRNA, IL-6 mRNA increased in 2.5 ppm
ozone-exposed WT mouse lungs to similar levels to that observed in
lungs of 1.0 ppm-exposed CCSP
/
mice (Fig. 5,
B and
D). Again, as was the case for the
MT mRNA dose response, IL-6 mRNA abundance showed no further
increases in CCSP
/
mice beyond the 1.0 ppm ozone
dose. These data are consistent with the conclusion from time-course
analysis that ozone sensitivity is enhanced in CCSP
/
mice
relative to strain-matched WT mice.
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DISCUSSION |
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In the present study, we show that CCSP deficiency enhances the
ozone-stimulated pulmonary stress response. Increases in the abundance
of IL-6 and MT mRNAs served as sensitive end points, allowing early
discrimination of differential ozone sensitivity associated with CCSP
deficiency. Both MT and cytokines, such as IL-6, are known to be
regulated by intracellular redox potential, stress resulting from
cell/tissue injury, and inflammation (1, 9, 12, 32). Expression of IL-6
and MT occurs early in the stress response, both before and during
inflammatory cell recruitment (9, 22, 26, 48). Furthermore, the
inflammatory response has the potential to perpetuate cytokine and
stress-response gene expression (20, 32, 34). Increases in
ozone-induced stress-response gene expression in CCSP /
mice
demonstrate a defect in the pulmonary response to oxidant pollutants.
This is further supported by the finding that CCSP deficiency results
in a shift in the dose-response relationship toward increased
sensitivity. In addition, our parallel studies demonstrate increased
epithelial cell necrosis between WT and CCSP
/
mice after
8 h of continuous exposure to 1.0 ppm ozone (Plopper, unpublished
observations). Based on these findings, we speculate that
the increased ozone sensitivity associated with CCSP deficiency is
suggestive of a role for CCSP and/or Clara cells in pulmonary
antioxidant defense.
There are no published studies describing antioxidant activities
associated with CCSP. Interestingly, tolerance associated with chronic
ozone exposure has been associated with elevations in the pulmonary
content of CCSP and antioxidant enzymes (13, 42). Activities associated
with the protein that could contribute to antioxidant defense include
the ability to bind divalent cations and lipophilic compounds (3, 8,
36, 49, 50). It is equally likely that CCSP deficiency has an indirect
influence on antioxidant defenses in the lung. Clara cells have been
shown to lose their secretory granules after oxidant stress (14). Although the contribution that oxidant-induced Clara cell degranulation makes to protection from oxidant stress is unknown, we have previously shown that Clara cells of CCSP /
mice lack secretory granules and
possess other changes to their secretory apparatus, including reduced
cytoplasmic volume of rough endoplasmic reticulum (40, 53). These
ultrastructural alterations, if they translate into functional changes
in Clara cell secretion and/or protein synthesis, could
potentially account for the observed oxidant sensitivity of CCSP
/
mice. This would suggest an important role for Clara cells and
oxidant-induced Clara cell secretion as an immediate protective
response to oxidant stress.
Genetic mechanisms accounting for pollutant sensitivity, particularly
the sensitivity to oxidant pollutants, are poorly defined. Considerable
variability is observed between mouse strains in their sensitivity to
pollutant injury and particularly oxidant stress (17, 24, 27, 58). Much
of this variability has been attributed to genetic background, but the
responsible genes have not been identified (2, 6, 28, 29, 45). Because the CCSP /
mice presented herein were established in an inbred 129 genetic background, we attribute the resulting oxidant sensitivity to this single genetic alteration. However, genetic alterations, such
as the introduction of a null allele of the CCSP gene, have the
potential for pleiotropic effects. Further studies are needed to
determine whether CCSP deficiency per se or changes in either Clara
cell function or the functions of other pulmonary epithelial cells are
the basis for oxidant sensitivity in CCSP
/
mice. In
addition, further investigation is required to determine whether pulmonary changes associated with CCSP deficiency are in any way related to previously characterized strain differences in oxidant sensitivity.
Previously, we have shown that CCSP /
mice are more sensitive to
hyperoxia exposure and displayed altered cytokine gene expression.
Possible explanations for this differential response are altered
sensitivity of the lung to oxidant injury and/or altered regulation of the inflammatory response. Studies presented herein support the concept that CCSP deficiency increases the sensitivity of
the lung to direct oxidant-induced stress. We conclude that the
CCSP-deficient mouse model has utility to define a role of CCSP
and/or Clara cells in protection from oxidant stress. However, due to differences in the sensitivity of pulmonary epithelial cells to
oxidant stress and likely alteration of injury-induced inflammation, we
are unable to draw conclusions regarding a direct role for CCSP in
regulation of pulmonary inflammatory responses.
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ACKNOWLEDGEMENTS |
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We thank Robert Gelein and Alex Lunts for technical assistance, Dr. Günter Oberdörster for advice and critique, Mathew Bowersox for assistance with computer analyses, and Joyce Morgan for administrative services.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HL-51376 and ES-08964 (B.R. Stripp), Training Grant ES-07026 (G. W. Mango), and Grant ES-01247.
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: B. R. Stripp, Dept. of Environmental Medicine, Box EHSC, Univ. of Rochester, 575 Elmwood Ave., Rochester, NY 14642.
Received 13 January 1998; accepted in final form 27 April 1998.
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