Maternal exposure to environmental tobacco smoke alters Clara
cell secretory protein expression in fetal rat lung
Chun-Mei
Ji1,
Fred H.
Royce2,
Uyen
Truong1,
Charles G.
Plopper1,
Gurmukh
Singh3, and
Kent E.
Pinkerton1
1 Department of Anatomy,
Physiology, and Cell Biology, School of Veterinary Medicine, and
2 Department of Pediatrics, School
of Medicine, University of California, Davis, California 95616; and
3 Department of Veterans Affairs
Medical Center and Department of Pathology, University of
Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
15240
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ABSTRACT |
We have previously
demonstrated that aged and diluted sidestream cigarette smoke (ADSS)
alters the development of bronchiolar epithelial cells in postnatal
animals (C. M. Ji, C. G. Plopper, H. P. Witschi, and K. E. Pinkerton. Am. J. Respir. Cell Mol.
Biol. 11: 312-320, 1994). This study was designed
to examine the effects of maternal exposure to ADSS on the development
of fetal Clara cells in rats with Clara cell 10-kDa protein (CC10; also
designated Clara cell secretory protein) and CC10 mRNA as
differentiation markers. Immunohistochemistry, Northern blots, and in
situ hybridization were used to determine the abundance and
distribution of CC10 at gestational days
14, 18, and
21. CC10 and CC10 mRNA were absent at
gestational day 14 but were detectable
at gestational day 18 and further
increased by gestational day 21.
Maternal exposure to ADSS was found to significantly increase fetal
expression of CC10 and CC10 mRNA by gestational day
21 but not by gestational day
14 or 18. These
findings demonstrate that in utero exposure to ADSS alters the normal
developmental expression of CC10 in the fetal rat lung.
fetal lung development; Clara cell 10-kilodalton protein; immunohistochemistry; Northern blots; in situ
hybridization
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INTRODUCTION |
NONCILIATED BRONCHIOLAR epithelial cells, or Clara
cells, are a heterogeneous (5, 11) and multifunctional (18) cell type
found in all mammalian lungs. Clara cells are the progenitor cell of
the distal airway epithelium, exhibit cytochrome
P-450 monooxygenase metabolism (10),
and secrete surfactant apoproteins (18). Three proteins with molecular
masses of 6-12, 45-60, and 200 kDa have been
identified in bronchoalveolar lavage (23, 28). The Clara cell 10-kDa
protein (CC10; also designated Clara cell secretory protein) is one of
the principal proteins secreted by the Clara cell (9) and is associated
with the electron-dense secretory granules of these cells (4, 22). A
number of studies have suggested that CC10 may provide an important
mechanism for defense in the distal airways. For example, CC10 has been
shown to bind polychlorinated biphenyls (16), inhibit phospholipase A2, and exhibit antiproteinase
activity (19, 20). Recent studies (7, 21) have shown that increased
cytoplasmic stores of CC10 are associated with the development of
tolerance to oxidant injury. Clara cells, which are initially injured
by ozone, undergo hypertrophy and increases in secretory granule
content (3). Associated with these changes are the cessation of
vacuolarization, cellular edema, and sloughing of Clara cells that
continue to be noted in other epithelial cell types exposed to ozone.
These observations raise the question of whether CC10 could be
important in protecting the delicate developing airways and alveoli
from placentally transmitted tobacco smoke constituents. Human
clinical, epidemiological, and physiological studies document an
important relationship between maternal tobacco smoke exposure and
infant lung function (17) and respiratory illness (30). It is possible
that unborn infants exposed via the mother to environmental tobacco
smoke (ETS) containing a variety of toxic compounds could cause
developmental alterations in Clara cell function and/or
metabolism.
This study was designed to determine whether maternal exposure to
ambient levels of aged and diluted sidestream cigarette smoke (ADSS),
as a surrogate for ETS, would alter the normal developmental expression
of CC10 and CC10 mRNA in the developing fetal rat lung. CC10 was chosen
for two reasons. First, CC10 can be detected in the rat lung before
birth, although at low levels (5). Second, CC10 is an excellent marker
of Clara cell differentiation and maturation (5) and would allow us to
potentially determine subtle changes occurring in the fetal lungs
associated with maternal exposure to ADSS. Although Ji et al.
(12) showed in an earlier study that postnatal exposure to
ADSS leads to an elevation in pulmonary cytochrome
P-4501A1 found in Clara cells, this
change was not apparent until 7 days postnatal age. Therefore, in this study, we opted to use CC10 rather than cytochrome
P-4501A1 as a more appropriate marker
of Clara cell differentiation in the fetal lung with maternal exposure
to ADSS.
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MATERIALS AND METHODS |
Animals. Fourteen timed-pregnant,
specific pathogen-free Sprague-Dawley rats mated during an 8-h window
(4 PM to midnight) were obtained from Zivic-Miller Laboratories
(Zelienople, PA). The day of mating was considered as gestational
day 0. Rats were shipped overnight to
the University of California, Davis for arrival on gestational
day 3 and acclimated for 2 days.
Beginning on gestational day 5, seven
rats were exposed to ADSS for 6 h/day, 7 days/wk, by whole body
exposure until the day before samples were taken. The remaining seven
rats were housed in a chamber and exposed to filtered air only. The
majority of dams were housed in pairs in both the exposure and control
chambers. Room lighting was on a 12:12-h on-off cycle. The rats
received food and water ad libitum. Two dams from the control group and
two dams from the ADSS-exposed group were killed on gestational
days 14 and
18, and three dams from each group
were killed on gestational day 21. All
tissues were prepared during the morning hours (i.e., 8 AM to noon).
Exposure system. The sidestream smoke
exposure system used in this study has been previously described (29).
Briefly, ADSS was generated by burning temperature- and
humidity-conditioned 1RF4 reference cigarettes (purchased from the
Tobacco and Health Research Institute, University of Kentucky,
Lexington, KY) in a smoking machine. Each cigarette was smoked under
rigid conditions of 1 puff (of 35-ml volume for 2-s duration)/min over
a period of 8 min. ADSS was collected from the smoldering end of each
cigarette in a dilution chimney and drawn into a glass and stainless
steel Hinners-type conditioning chamber to be aged and diluted with filtered air for a mean resident time of 2 min. A portion of the air
and smoke from the conditioning chamber was further mixed with fresh
filtered air before entering the
0.44-m3 animal exposure chambers.
Daily measurements of total suspended particulates, nicotine, and
carbon monoxide were performed. Mean (±SD) concentrations over the
course of the study were 1.00 ± 0.07 mg/m3 of total suspended
particulates, 344 ± 85 µg/m3
of nicotine, and 4.9 ± 0.7 parts/million of carbon monoxide. This
concentration of sidestream cigarette smoke was selected because it
represents a high ambient level that individuals could encounter at
home or in other settings where smoking occurs (8).
Tissue preparation. Fetal rats were
obtained by cesarean section from timed-pregnant rats anesthetized with
1 mg/kg body weight of pentobarbital sodium. The tissues were either
flash-frozen in liquid nitrogen for RNA extraction or fixed for
immunohistochemistry and in situ hybridization. For gestational
days 14 and
18, the entire thorax was immersed in
either liquid nitrogen or 4% paraformaldehyde. For gestational
day 21, whole lungs were frozen in
liquid nitrogen or the trachea was instilled with 4% paraformaldehyde
for 1 h before the lungs were removed from the thorax and embedded in paraffin (Paraplast Plus). Sections 5 µm thick were cut with a rotary
microtome and placed on Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA) for immunohistochemistry or on
gelatin-poly-L-lysine-coated slides for in situ hybridization and baked overnight at 37°C. Sections were stained with hematoxylin and eosin for
histopathological evaluation. Glassware was baked at 250°C, and
aqueous solutions were prepared from water treated with diethyl
pyrocarbonate.
Immunohistochemistry. A polyclonal
antibody against purified rat CC10 (26) produced in rabbit was used to
detect CC10 in rat lung by the avidin-biotin complex method with
commercially supplied reagents (Vector Laboratories, Burlingame, CA).
The sections were deparaffinized in three changes of xylene for 5 min each. The sections were rehydrated in decreasing concentrations of
ethanol and treated with 3% hydrogen peroxide to eliminate endogenous peroxidase activity and to unmask antigenic sites. Ten percent bovine
serum albumin (BSA) in phosphate-buffered saline (PBS; pH 7.2-7.4)
was subsequently added to block nonspecific binding sites. The tissue
was incubated with the antibody at 4°C overnight. The
appropriate dilution of antibody was determined to be 1:5,000 in the
adult rat lung. A control with substitution of the primary antibody for
CC10 with serum was included in each run to examine tissues for
nonspecific reactions.
RNA extraction. Lung tissue frozen at
80°C was placed in ice-cold solution
D (4 M guanidinium thiocyanate, 25 mM sodium acetate, pH 7.0, 0.5% sarkosyl, and 0.1 M 2-mercaptoethanol) and homogenized with a glass tissue dounce (6). After phenol-chloroform extraction, the
RNA was precipitated from the aqueous phase in the presence of sodium
acetate and isopropyl alcohol and resuspended in Tris-EDTA buffer
(10 mM Tris · HCl and 1 mM EDTA, pH 8.0). RNA
concentration was determined by measuring ultraviolet light
absorbance at 260 nm, and purity was confirmed by an absorbance ratio
at 260 to 280 nm of 1.7-2.0. RNA integrity was confirmed on
ethidium bromide-stained formaldehyde-agarose gels on the basis
of the presence of sharp 28S and 18S ribosomal bands.
Northern blots. Northern analysis was
performed by electrophoresis of 10 µg of RNA in 1.0% agarose and 2.2 M formaldehyde followed by capillary blot transfer and ultraviolet
cross-linking to Gene Screen (DuPont, Wilmington, DE) nylon membrane
(25). The membranes were prehybridized for 3-6 h in 50%
formamide, 0.04% polyvinylpyrrolidone (mol wt 40,000), 0.04% BSA,
0.04% Ficoll (mol wt 400,000), 5× saline-sodium citrate (SSC;
0.75 M NaCl and 0.075 M sodium citrate, pH 7.0), 1% SDS, and 100 µg/ml of denatured salmon sperm DNA at 42°C in heat-sealed bags.
Complementary DNA for rat CC10 (15) and mouse 18S rRNA (2) were labeled
with [32P]dCTP (NEN)
with random hexamers (Pharmacia) to achieve a specific activity of
1-2 × 109
counts · min
1 · µg
1
and were added to the blot along with fresh hybridization solution and
incubated overnight at 42°C. The blots were washed in 2× SSC two times for 5 min, CC10 was washed in 0.5× SSC-0.1% SDS at
55°C, and 18S rRNA was washed in 0.2× SSC-0.1% SDS at
65°C. The blots were placed against Kodak X-OMAT film backed with an
intensifying screen (DuPont Cornex) at
80°C and developed
after 1-3 days. Probe was stripped from the blots at 70°C with
agitation for 3 h in 0.005 M Tris · HCl (pH 8.0),
0.0002 M Na2EDTA, 0.05% sodium pyrophosphate, 0.002% polyvinylpyrrolidone (mol wt 40,000), 0.002% BSA, and 0.002% Ficoll (mol wt 400,000) and reprobed with 18S rRNA by
the same procedure. Bands corresponding to the hybridized CC10 cDNA or
18S rRNA on autoradiograms were scanned with a digital photo imager
consisting of a high-resolution camera interfaced with a Macintosh IIci
computer with NIH Image 1.4 (National Institutes of Health, Bethesda,
MD) software.
In situ hybridization.
35S-labeled riboprobes were
synthesized from Sma I (antisense) and
Bst XI (sense) linearized rat 0.452-kb CC10 cDNA subcloned between the SP6 and T7 RNA polymerase promoters into pGEM4z (15).
35S-riboprobes were synthesized
with the Gemini Core System II transcription kit (Promega) with 250 µCi (1,367 Ci/mmol) of
-35S-labeled UTP concentrated
in a speed-vacuum; 4 µl of 5× transcription buffer
containing spermidine; 2 µl of 100 mM dithiothreitol; 4 µl of a
mixture of 2.5 mM ATP, CTP, and GTP; 2.4 µl of 100 mM UTP; 20 U of
RNasin; 1 µg of linearized template DNA; 1 µl (15-20 U) of SP6
or T7 RNA polymerase; and nuclease-free water to a final volume of 20 µl. The reaction mixture was incubated for 1 h at 37°C, and
template DNA was removed by RQ1 RNase-free DNase. The labeled
riboprobes were purified by precipitation in the presence of 5 M
ammonium acetate and 75% ethanol and suspended in 20 µl of Tris-EDTA
buffer. Hybridization buffer consisted of 50% deionized formamide, 300 mM NaCl, 20 mM Tris · HCl, pH 8.0, 5 mM EDTA,
10% dextran sulfate, 1× Denhardt's solution, 20 mM
dithiothreitol, and 100 mg/ml of yeast tRNA with 3,000 counts · min
1 · µl
1
of 35S-labeled riboprobe.
In situ hybridization was performed as described by Armstrong et al.
(1). Lung sections were deparaffinized in xylene, rehydrated in
ethanol, and postfixed in 4% paraformaldehyde in PBS. Sections were
permeabilized with 0.3% Triton X-100 for 15 min and then treated with
1 µg/ml of freshly made proteinase K for 30 min at 37°C and
immersed in glycine-PBS two times for 30 s each. Tissues were
acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine
(pH 8.0). The sections were left in 50% formamide-2× SSC (SSC
buffer: 150 mM NaCl and 15 mM sodium citrate, pH 7.0) until
hybridization.
Hybridization buffer containing
35S-riboprobes was applied to the
sections, and a coverslip was applied for 16-18 h at 55°C in
RNase-free humidified chambers. After incubation, the coverslips were
removed, and the slides were washed in several changes of 4× SSC,
30 s each, in 70, 95, and 100% ethanol concentrations containing 0.3 M
ammonium acetate. The following washes were performed at 37°C with
gentle rotary shaking unless otherwise specified: hybridization buffer
for 30 min at 65°C, 2× SSC for 15 min, 40 µg/ml of RNase A in
RNase buffer (0.3 M NaCl, 10 mM Tris · HCl, pH 7.5, and 5 mM EDTA) for 30 min, RNase buffer for 30 min, 2× SSC for 30 min, and 0.1× SSC two times for 30 min each. The slides were
subsequently dehydrated through 70, 95, and 100% ethanol containing
0.3 M ammonium acetate and air-dried.
The slides were dipped in 50% Kodak NTB-2 emulsion prewarmed to
40°C and transferred to autoradiography slide boxes containing fresh desiccant. After exposure for 3 days at 4°C, the slides were
warmed to 15°C, developed for 4 min with Kodak D19 developer at
15°C, and fixed with Kodak fixer for 4 min. The slides were counterstained with hematoxylin for 30 s and eosin for 2 min, covered
with a coverslip, and photographed with a Zeiss light microscope.
Histological distribution of CC10
mRNA. The cellular distribution of CC10 mRNA was
determined by examining the density of silver grains in the distal
airways. Briefly, sections were examined by light microscopy
(×10) to identify the terminal bronchioles opening into the
alveolar ducts. Only terminal bronchioles opening directly into
channels lined by alveolar outpocketings and demonstrating a single
epithelial cell layer in the plane of the tissue section were included
in this study. From the lungs of each fetus, 17-24 images of
terminal bronchioles were captured as digital images and viewed
on a computer screen through a video camera.
With image-analysis software (NIH Image 1.4), the area covered by
silver grains was quantified in each terminal bronchiole. The
measurement of labeled epithelial cells within each terminal bronchiole
was defined as that proportion of the bronchiolar profile area covered
by silver grains that became highlighted by setting the threshold
sensitivity to eliminate all parenchymal background. The threshold
sensitivity was maintained constant throughout the experiment for the
measurement of all terminal bronchioles. Data for terminal bronchioles
was collected in two ways: 1) by
digitizing the entire length of the basal lamina (13) for selected
terminal bronchioles and 2) by
measuring the entire epithelial area of each terminal bronchiole. With
these approaches, the area covered by silver grains could be expressed
as the percentage (or fraction) of total epithelial area of the
terminal bronchiole or as the labeled epithelial area per length of
terminal bronchiole basal lamina. Routine histology of random paraffin
sections demonstrated that the sections used in these studies were of
uniform thickness based on the measurement of section folds, nuclear
profile frequency, and epithelial profile area. Therefore, we could, in
an unbiased manner, make valid comparisons of the degree of labeling
between terminal bronchioles, between animals, and between groups.
Statistical analysis. All data were
compared with an unpaired t-test
(StatView 4.1) and are expressed as means ± SE. Significance was
considered for all comparisons at P < 0.05. The distribution of mRNA CC10 labeling of the epithelium in
terminal bronchioles of control and ADSS-exposed fetal lungs was
evaluated by
2 analysis.
 |
RESULTS |
Fetal body weights. Body weight at
gestational days 14,
18, and
21 is shown in Table
1. Maternal exposure to ADSS did not significantly affect fetal body weight at any gestational age.
Immunohistochemistry. No
immunoreactive CC10 was detected at 14 days gestational age. By 18 days
gestational age, CC10 was present to a limited degree in some cells,
forming the epithelial tubules of the lungs. Maternal exposure to ADSS
did not significantly affect the distribution or staining intensity of
CC10 within the fetal lung by this gestational age. By gestational
day 21, CC10 was clearly evident in
bronchiolar cells of control fetal lungs (Fig.
1A).
Maternal exposure to ADSS was associated with a marked increase in
immunostaining of CC10 compared with fetal lungs from dams exposed to
filtered air only (Fig. 1B).

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Fig. 1.
Cellular expression and distribution of Clara cell 10-kDa protein
(CC10; Clara cell secretory protein) in terminal bronchioles of fetuses
at 21 days gestational age from dams exposed to filtered air
(A) or to aged and diluted
sidestream cigarette smoke (ADSS;
B). Bar, 100 µm.
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Northern blot. CC10 transcripts were
not detectable at gestational day 14.
By gestational day 18, CC10
transcripts were present and were further increased at gestational
day 21. Maternal exposure to ADSS did
not significantly increase the relative level of CC10 mRNA in the fetal
lungs by gestational day 18 or
21 compared with filtered air-exposed
control fetuses of the same age (Fig. 2).

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Fig. 2.
Northern analysis of CC10 expression in whole fetal lung homogenates at
gestational days 14,
18 (n = 7 from filtered air-exposed control and 7 from ADSS-exposed dams on
each day), and 21 (n = 12 from filtered-air control and
12 ADSS-exposed dams). ND, no message was detectable at 14 days
gestational age. Although mean values for CC10/18S rRNA were different
at gestational day 18 or
21 in lungs of fetuses from dams
exposed to filtered air compared with those from dams exposed to ADSS,
they did not attain a level of significance.
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In situ hybridization. CC10 mRNA was
not detected at 14 days gestational age in the fetal lungs after
maternal exposure to filtered air or ADSS. At 18 days gestational age,
airway epithelial cells four generations or more from the respiratory
bud expressed CC10 mRNA. In the three most distal airway generations
directly giving rise to the respiratory bud, CC10 mRNA was not detected in any epithelial cells (Fig. 3). This
pattern was consistently demonstrated at 18 days gestational age, with
CC10 mRNA being highest in the most proximal airway epithelium of the
lungs. Maternal exposure to ADSS did not change the pattern or
intensity of airway epithelial CC10 mRNA labeling by 18 days
gestational age (Fig. 3).

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Fig. 3.
Cellular mRNA expression and distribution of CC10 in terminal
bronchioles of 18-day gestational age fetuses from control dams
(A, bright field, and
B, dark field) and fetuses from dams
exposed to ADSS (C, bright field, and
D, dark field). CC10 mRNA was found
only in airways ~4 generations or more from respiratory bud. Bar, 100 µm.
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CC10 mRNA at 21 days gestational age was evident to a much higher
degree in individual cells of the terminal bronchioles (Fig. 4) compared with gestational
day 18. Maternal exposure to ADSS was
associated with an increase in the number of grains found in individual
airway epithelial cells in the fetal lung, although the number of
airways labeled remained the same regardless of exposure condition. The
numerical density of epithelial cells lining the terminal bronchiole,
as well as the volume of the epithelium lining these airways, was also
unchanged regardless of exposure condition (Fig. 1). In contrast to 18 days gestational age, a threefold increase in labeled bronchiolar
profile area was observed with ADSS exposure compared with control
cells (60 vs. 20%; P < 0.001; Fig.
5). When normalized to the length of the
basal lamina (Fig. 6), this difference
remained significant. There was substantial variation at gestational
day 21 in the expression of CC10 mRNA in terminal bronchioles (Figs. 7 and
8). Within the same fetal lung, it was
observed that some terminal bronchioles had 100% of the epithelial
area labeled, whereas in others the labeling was only 20% of the total
epithelial area.

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Fig. 4.
Cellular mRNA expression and distribution of CC10 at 21 days
gestational age in terminal bronchioles of fetuses from filtered
air-exposed control (A, bright field,
and B, dark field) and ADSS-exposed
dams (C, bright field, and D, dark
field). Bar, 100 µm.
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Fig. 5.
Percentage of labeled silver grain area in fetuses from filtered
air-exposed control and ADSS-exposed dams of 21 days gestational age.
Data were collected from a total of 30 terminal bronchioles (TBs) from
4 different animals/group. * P < 0.05.
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Fig. 6.
Labeling area per basal lamina length of TBs in filtered air-exposed
control and ADSS-exposed fetal lungs at 21 days gestational age. Data
were collected from a total of 30 TBs from 4 different animals/group.
* P < 0.05.
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Fig. 7.
Distribution of TBs with varying percentages of epithelial surface area
labeled by in situ hybridization. TBs were grouped in 11 classes on
basis of labeling area. Each class represents a difference of 10%.
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Fig. 8.
Comparison of TBs expressing different levels of mRNA. TBs were grouped
in 11 classes on basis of labeling area per length of basal lamina.
Each class represents an increase of 10%.
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 |
DISCUSSION |
This is the first study to examine the effects of maternal exposure to
ETS on the developmental expression of CC10 in the fetal lung. The
concentration of ADSS used in this study, although at a high ambient
level (8), is readily attained in enclosed areas (e.g., rooms or cars)
where smoking occurs. We found that CC10 and CC10 mRNA were not
detectable in fetal lungs at gestational day
14, but both were found by gestational
day 18 and increased as parturition
approached. Our intent in the present study was to compare the effects
of maternal exposure to ADSS on the fetal lungs with the effects of
postnatal exposure to ADSS (12) on Clara cell maturation. In an earlier
study, Ji et al. (12) found that exposure to ADSS from
birth did not change CC10 expression at 7 days postnatal age or later
in life. In contrast, this study demonstrated that maternal exposure to
the same concentration of ADSS significantly increased CC10 as well as
CC10 mRNA expression. These findings suggest that the Clara cell is
profoundly affected by environmental contaminants that cross the
placental barrier.
A quantitative assessment of in situ hybridization helped to identify
that greater numbers of epithelial cells in terminal bronchioles of
21-day gestational age fetuses had increased the mRNA signal for CC10
after maternal exposure to ETS. The higher sensitivity of the fetus to
ETS may result from ETS metabolites formed by the mother.
Transplacental delivery of bloodborne compounds may also present a
different toxic profile to the lung than the postnatal inhalation
route. If that is the case, then the timing of exposure (fetal exposure
vs. postnatal exposure) or the route of exposure (transplacental vs.
inhalation) may have important implications for the normal development
of Clara cells. One may further speculate that changes in Clara cell
development may alter airway defense in early life.
Previous studies with tobacco smoke (14) and oxidant gases such as
ozone (3) have shown that adult Clara cells respond to toxicants by
increasing their granule size. Why cytoplasmic CC10 stores do not
change in postnatal animals in response to ETS exposure (12) is not
known. Although it is possible that the increase in the cellular
content of CC10 may represent a difference in the rate of secretion or
protein translation, increased expression of CC10 mRNA in the terminal
bronchioles suggests a redistribution of CC10 mRNA expression to the
terminal bronchioles.
This is the first study to examine the heterogeneity of CC10 mRNA
distribution through the use of in situ hybridization and digital
imaging in the lung. In this experiment, precise quantifying by
counting individual grains was not possible because of the abundant
amounts of message present in the lung for this protein. The
approach taken was to estimate the proportion of the epithelium that
was labeled in terminal bronchioles. Two methods were used: 1) CC10 mRNA-positive epithelial
area per basal lamina length and
2) the percentage of total
epithelial area labeled for CC10 mRNA.
There are two technical aspects of in situ hybridization with paraffin
sections and 35S-labeled
riboprobes that could potentially contribute to artifactual measurements and the differences observed between the two groups. These
are emulsion and section thicknesses. We examined emulsion thickness on
each slide with NIH Image software by measuring the dye density
gradient taken up by the emulsion. Calibration of all sections was made
on those areas of each section with an emulsion break. Emulsion
thickness was found to be highly consistent for all sections based on
the similarity of the dye density measurement. Therefore, it is not
likely that emulsion thickness was a major factor in the observed
differences between exposed and control lung tissues in this study.
Differences in section thickness were determined by measuring
epithelial nuclear frequency of the airways. Thicker sections (compared
with thinner sections) cut perpendicularly through a single layer of
cells (i.e., the epithelium of the terminal bronchiole) would contain
more nuclear profiles per length of basal lamina. We found nuclear
frequency to be highly consistent for all sections. The rationale to
use labeling area per epithelial area as a measure of relative mRNA
levels was valid because epithelial area per basal lamina length was
not changed by maternal exposure to ADSS compared with fetal lungs
where maternal exposure was to filtered air only.
This study demonstrated a redistribution in the cells expressing CC10.
It is not surprising that Northern blot analysis of the whole lung did
not show a significant elevation in mRNA level after exposure to ADSS
because this technique cannot account for shifts in the pattern of CC10
expression in specific subcompartments of the lung such as the terminal
bronchiole. It is likely that if Northern blot analysis were done in
terminal bronchiole subcompartments, significant quantitative
changes in mRNA expression would be observed (25). However, this was
not as feasible to perform in the fetal rat lung. It is likely,
therefore, that the signal was simply too dilute in whole lung
homogenates that contain predominantly nonbronchiolar tissues.
The elevation of CC10 expression in the fetal lung after maternal
exposure to ADSS may be associated with a number of potential physiological effects. These could include an alteration in the fluid
composition and dynamics of the lower airways and a difference in the
growth and/or cellular proliferation characteristics of the
airways. Alteration in Clara cell expression during the late fetal
period may also have a possible implication on the immune response of
the airway epithelium to inhaled pathogens postnatally. It is known
that children exposed to cigarette smoke are more likely to have
increased symptoms of wheeze, cough, and infection (30). On the other
hand, we know from a previous study from our laboratory (5) that
postnatal CC10 expression in the Clara cell rapidly reaches adult
levels by 7-10 days of age. Therefore, the accelerated expression
of CC10 during the late gestational period may have only a transient
effect in the postnatal lung.
In summary, CC10 and CC10 mRNA are present in fetal lungs by
gestational day 18 and increase in
abundance with gestational age. Maternal exposure to ADSS significantly
shifts the cellular expression of CC10 and CC10 mRNA to the terminal
bronchioles just before birth. Whether these alterations in the
terminal bronchiole persist during postnatal development or into
adulthood merits further investigation.
 |
ACKNOWLEDGEMENTS |
We gratefully appreciate the expert technical assistance of Jie Yin
during the course of this study.
 |
FOOTNOTES |
This work was supported by the Center for Indoor Air Research and the
California Tobacco-Related Disease Research Program. The University of
California, Davis is a Center for Environmental Health Sciences
supported by National Institute of Environmental Health Sciences Grant
ES-05707. We acknowledge support by the Center through the core
facilities that were used to perform this work.
Present address of C.-M. Ji: Dept. of Biochemistry and Molecular
Biology, Mayo Clinic Scottsdale, Scottsdale, AZ 85259.
Address for reprint requests: K. E. Pinkerton, VM: Anatomy, Physiology,
and Cell Biology, Univ. of California, Davis, CA 95616.
Received 13 May 1997; accepted in final form 28 July 1998.
 |
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