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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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 chi 2 analysis.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

                              
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Table 1.   Fetal body weights

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.

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.

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.

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%.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 275(5):L870-L876