Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, California 95616-8732
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ABSTRACT |
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Nonciliated bronchiolar (Clara) cells metabolize environmental toxicants, are progenitor cells during development, and differentiate postnatally. Because differentiating Clara cells of neonatal rabbits are injured at lower doses by the cytochrome P-450-activated cytotoxicant 4-ipomeanol than are those of adults, the impact of early injury on the bronchiolar epithelial organization of adults was defined by treating neonates (3-21 days) and examining them at 4-6 wk. Bronchiolar epithelium of 6-wk-old animals treated on day 7 was most altered from that of control animals. Almost 100% of the bronchioles were lined by zones of squamous epithelial cells. Compared with control animals, the distal bronchiolar epithelium of 4-ipomeanol-treated animals had more squamous cells (70-90 vs. 0%) with a reduced overall epithelial thickness (25% of control value), fewer ciliated cells (0 vs. 10-20%), a reduced expression of Clara cell markers of differentiation (cytochrome P-4502B, NADPH reductase, and 10-kDa protein), and undifferentiated nonciliated cuboidal cell ultrastructure. We conclude that early injury to differentiating rabbit Clara cells by a cytochrome P-450-mediated toxicant inhibits bronchiolar epithelial differentiation and greatly affects repair.
cell differentiation; lung development; age sensitivity; cytochrome P-450 metabolism
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INTRODUCTION |
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THE RESPIRATORY SYSTEM is a primary target for a wide range of environmental toxicants and potential carcinogens, the potency of which depends on metabolic activation by the cytochrome P-450 monooxygenase system. Examples include aromatic hydrocarbons such as benzo(a)pyrene (6), nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (6), and chlorinated ethylenes such as vinyl chloride (18). Not all of the more than 40 differentiated lung cell phenotypes found in adults are equally susceptible to injury by these cytotoxicants and carcinogens. In the lungs of adult mammals, the increased sensitivity of the Clara cell to cytochrome P-450-activated cytotoxicants is accounted for by its high levels of activation enzymes. In fact, Clara cells are thought to have the greatest cytochrome P-450 level of any lung cell (15). Well-recognized cytochrome P-450-mediated Clara cell toxicants include naphthalene, 3-methylindole, and 4-ipomeanol (Ipo) (2, 33).
In a recent study, Plopper et al. (24) found that the relationship between high levels of cytochrome P-450 monooxygenase activity and elevated Clara cell susceptibility to the cytochrome P-450-mediated cytotoxicant Ipo does not apply to Clara cells undergoing differentiation in postnatal animals. Differentiating Clara cells characteristically have low levels of cytochrome P-450 activity that increase with cellular maturity (23, 27). They also begin the process of differentiation with less smooth endoplasmic reticulum, secretory granules, and Clara cell secretory (10-kDa) protein (CC10) and more glycogen compared with mature Clara cells (1, 14, 21, 23, 28).
In adult rabbits, Ipo is preferentially metabolized by pulmonary cytochrome P-450 isozymes 2B4 and 4B1 (26, 32), initially referred to as cytochromes P-450I and P-450II, which make up ~80-90% of the adult rabbit Clara cell cytochrome P-450 isozymes (4, 26). Immature Clara cells of neonatal rabbits have very low levels of these two isozymes at birth. Adult levels are reached after 4 wk of age (23). Despite possessing limited isozyme levels, immature Clara cells were found to be more susceptible to injury at lower doses of Ipo compared with mature Clara cells. Furthermore, 1 wk postinjury, both cytochrome P-450 activity (as measured by pentoxyresorufin O-dealkylation) and expression of cytochrome P-450 proteins in neonatal rabbit lungs were reduced compared with age-matched control lungs (24).
The present study was designed to extend the postinjury recovery time and address two questions: 1) does injury to differentiating Clara cells in early postnatal animals alter the differentiation and development of bronchiolar epithelium, and 2) does the impact on the developmental process vary by the stage of Clara cell differentiation during which injury occurs?
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MATERIALS AND METHODS |
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Experimental protocol. Male and female New Zealand White rabbits, free of chronic respiratory disease as judged by gross examination and histopathology, were obtained from respiratory disease-free colonies maintained by the University of California (Davis) or Dutchland Laboratories (Denver, PA). Postnatal animals 2, 3, 5, 7, 9, and 21 days of age were given a single intraperitoneal injection of 5 mg/kg of Ipo (a generous gift from Dr. M. R. Boyd, National Cancer Institute, Bethesda, MD) dissolved in 20% propylene glycol. Age-matched control rabbits from the same litter were injected with propylene glycol carrier only (see Table 1 for summary of treatments). The animals were killed at 4 or 6 wk of age. They were anesthetized with pentobarbital sodium (1 mg/kg), tracheotomized, and killed by exsanguination. The lungs were removed and then split in half for morphological and immunohistochemical analysis.
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High-resolution microscopy. The right lung was infused with glutaraldehyde-paraformaldehyde in cacodylate buffer (adjusted to pH 7.4, 330 mosmol) via the main stem bronchus at 30 cmH2O pressure for 1 h. The lung remained in the fixative until the beginning of the embedding process when the right caudal lung lobe was sliced into 2- to 4-mm-thick pieces and postfixed for 2 h in 1% osmium tetroxide. The pieces were embedded in Araldite-502 after a process that allowed selection of specific areas from large tissue faces (20). One-micrometer-thick sections were cut with glass knives on a Sorvall JB4 microtome and stained with toluidine blue for light-microscope examination. Areas of the block faces containing terminal bronchiole-alveolar duct junctions were excised, remounted, and thin sectioned (30-50 nm) with a diamond knife on a Sorvall MT5000. Sections were stained with uranyl acetate-lead citrate and examined with a Zeiss 10 transmission electron microscope at 60 kV.
Sampling for quantitative evaluation.
Initial histopathological screening established that one of the primary
lesions in the Ipo-treated animals was the replacement of cuboidal
epithelium with squamous epithelium at the distal end of the terminal
bronchiole matrix architecture. This could be related by position to
the distal pulmonary arteriole. Whereas the cuboidal epithelium in control animals was found to end 114.5 ± 74.6 (SD) µm
(n = 49 bronchioles) distal to the
distal edge of the pulmonary arteriole (Fig.
1A),
the cuboidal epithelium in treated animals was found to end more
proximally (213.6 ± 167.6 µm; n = 32 bronchioles) from the distal edge of the arteriole (Fig.
1B). For quantitative histopathological assessment, bronchioles in 1-µm sections were blindly evaluated for the presence or absence of squamous epithelium associated with terminal bronchiolar architecture
(n = 11 animals) and in relation to
the distal pulmonary arteriole (n = 14 animals). Morphometric comparisons of the bronchiolar epithelium of
treated animals were conducted in two fashions to quantitatively
characterize the composition of the epithelium:
1) based on distances along the
bronchiole in relation to the pulmonary arteriole and
2) based on distances from the
cuboidal epithelial end point. In control animals, the end point of the
cuboidal epithelium and its junction with the squamous epithelial cells
of the alveolar duct were designated distance 0 µm (Fig.
1A). Distances were marked in a
proximal direction at 100-µm intervals. In Ipo-treated animals, the
cuboidal epithelium ended ~100 µm proximal to the distal edge of
the pulmonary arteriole and ~200 µm proximal to the alveolar duct
junction (Fig. 1B). To ensure that
the composition of the epithelium in treated animals was evaluated at
sites anatomically comparable to those of the control animals, sites
100 and 200 µm distal from the cuboidal epithelial end point in
Ipo-treated animals were identified as distances 100 and
200 µm, respectively (Fig.
1B).
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Morphometry. Selected areas from 1-µm sections were imaged with the use of a Sony digital photo camera DKC 5000 connected to an Olympus Provis AX70 microscope interfaced to a Power Macintosh 7200/90. An oil-immersion ×60 objective (×2 zoom) was used. Images were captured in Adobe Photoshop 3.0.5 and converted to gray scale.
The size and abundance of epithelial cells were analyzed with morphometric procedures our laboratory previously used to define changes in bronchiolar epithelium as a result of oxidant injury (7, 16) and during development (11). These procedures are discussed in detail elsewhere (9, 10). The volume density (Vv) of the cytoplasm and nucleus of three categories of cells (nonciliated, ciliated, and squamous) was determined by point counting with a 130-point cycloid grid and was calculated with the formula Vv = Pp = Pn/Pt, where Pp is the point fraction of the number of test points (Pn) hitting the structure of interest and Pt is the total points hitting the reference space (epithelium or interstitium). The surface area of the epithelial basement membrane per reference volume (Sv) was determined by point and intercept counting with a cycloid grid and calculated with the formula Sv = 2Io /Lr, where Io is the number of intersections with the object (epithelial basal lamina) and Lr is the length of the test line in the reference volume (epithelium or interstitium). The calculations included a correction factor for the plane of orientation depending on whether the plane of the section was determined to be a cross section or a longitudinal section (9, 10). The thickness of the epithelium, or volume per unit area of basal lamina (in µm3/µm2), was then calculated with the formula for arithmetic mean thickness (Vv /Sv).
Each side of the bronchiole, the side adjacent to the pulmonary arteriole and the side opposite it, was evaluated. Bronchioles were selected that exhibited profiles in longitudinal section and that were adjoining to and contiguous with alveolar ducts. The first bronchioles encountered on a section meeting this criteria were chosen from each animal: 11 bronchioles from six 6-wk-old control animals and 13 bronchioles from seven 6-wk old animals treated with Ipo on day 7.
Immunohistochemistry. The left lung was infused for 1 h with 1% paraformaldehyde in 0.1 M phosphate buffer via the left main stem bronchus. The left cranial and caudal lobes were sliced into 1- to 4-mm-thick slices, washed in 0.2 M phosphate buffer for 30 min, embedded in paraffin, and sectioned at 6 µm.
Immunohistochemistry was done on sections from 11 animals (4 control, 3 treated with Ipo on days 2-3, and 4 treated with Ipo on day 7) that were killed at 4 wk of age. The following antibodies were used: goat anti-rabbit pulmonary cytochrome P-450 monooxygenase isozyme cytochrome P-4502B (CYP2B) antibody diluted 1:20,000 (25), goat anti-rabbit pulmonary cytochrome P-450 NADPH reductase antibody diluted 1:20,000 (25), and goat anti-rabbit Clara cell secretory protein antibody diluted 1:10,000 (19). All antibodies were diluted in phosphate-buffered saline (PBS; pH 7.4).
Sections were deparaffinized, hydrated, and treated with 3% hydrogen peroxide in deionized water for 30 min to block endogenous peroxidase. A 20-min wash in PBS and a 30-min incubation with 5% bovine serum albumin to block nonspecific reactivity followed. The slides were incubated with the primary antibody overnight at 4°C in humidified chambers. Antibodies were detected with avidin-biotin peroxidase reagents that were purchased from Vector Laboratories (Burlingame, CA) and the peroxidase substrate diaminobenzidine (Sigma). The procedure outlined by Vector Laboratories was followed. Control sections included the substitution of primary antibody and/or the secondary antibody with PBS.
Statistics. Tissue mass, or arithmetic mean thickness (in µm), cell volume fraction (in percent), and cell volume per unit area of basal lamina (in µm3/µm2) were calculated per animal and used to calculate the mean ± SD for each group at each position and by orientation in relation to the blood vessel. Differences between groups and sites were determined by analysis of variance and one-way regression analysis. Determination of significance was based on Bonferroni-Dunn and Scheffé's F tests as P < 0.05 (8).
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RESULTS |
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Histopathology. In carrier-treated rabbits at 4 and 6 wk of age, the epithelium lining the most distal generations of bronchioles down to the alveolar duct junction was composed of variable numbers of ciliated cells and cuboidal epithelium containing nonciliated (Clara) cells, the apices of which projected into the airway lumen (Fig. 2A). In animals treated with Ipo on postnatal days 2-3, a variable number of bronchioles had the most distal aspects of their matrix lined by squamous cells. This condition was also present in the bronchioles of animals treated on day 5. In animals treated with Ipo on day 7 or day 9, virtually all of the distal bronchioles were lined primarily by squamous and very low cuboidal epithelium (Fig. 2B). In contrast, it was difficult to differentiate the bronchioles from 4-wk-old animals treated on day 21 from those of age-matched control animals.
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Quantitation. Lung sections from rabbits treated with Ipo or carrier on days 2-3 or day 7 and killed at 4 or 6 wk were blindly evaluated for the percentage of bronchioles that contained large areas of squamous epithelium. There was a significant, but age-dependent, increase in the number of terminal bronchioles with extensive areas of squamous epithelium in animals treated with Ipo (Fig. 3, A and B). Less than 10% of the bronchioles of 4-wk-old carrier-treated control animals and none of the bronchioles of 6-wk-old carrier-treated animals had zones lined by squamous epithelium. In contrast, animals treated with Ipo on day 2 or 3 had between 25 and 45% of their bronchioles lined by squamous epithelial cells, and animals treated on day 7 had almost 100% of their bronchioles predominantly lined by squamous epithelial cells at 4-6 wk of age. Lung sections from 6-wk-old rabbits treated with Ipo or carrier on day 7 were blindly evaluated for the percentage of distal pulmonary arterioles associated with squamous epithelium in the adjacent bronchiole. Distal pulmonary arterioles were associated with squamous epithelium in 14.5% of the carrier-treated animals and 80% of the Ipo-treated animals (Fig. 3C).
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The total thickness of the epithelium lining the terminal bronchioles
was evaluated morphometrically in control animals. No significant
differences in thickness were found between the blood vessel and
non-blood vessel sides of the bronchiole. When the total epithelial
thickness of control and Ipo-treated animals was compared based on the
same position on the bronchiolar wall, Ipo-treated animals had
approximately one-fourth of the epithelial thickness of the control
animals in the most distal end of the terminal bronchiole (Ipo
distances 200 and
100 µm vs. carrier distances 0 and
100 µm; P < 0.05; Tables
2 and 3).
However, at the cuboidal epithelium end point of Ipo-treated animals,
the epithelial thickness was nearly identical in both carrier- and Ipo-treated animals (Ipo distance 0 µm vs. carrier distance 200 µm;
Tables 2 and 3). More proximally along the bronchiole, the cuboidal
epithelium in the Ipo-treated animals (Ipo distances 100 and 200 µm)
varied from being equal in thickness to being more than double in
thickness compared with the control animals (carrier distances 300 and
400 µm). When the epithelial thickness was compared based on the
distance from the two cuboidal epithelium end points (Ipo and carrier
distances 0 µm), the epithelial thickness of the control animals
averaged two-thirds of that of the Ipo-treated animals at the end
point. At more proximal sites, the epithelial thickness of the control
animals was also less and, in some cases, less than one-half compared
with the Ipo-treated animals.
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The organization of the epithelium varied in terms of the abundance of
nonciliated, ciliated, and squamous cells (Fig.
4). In the control animals, nonciliated
cells averaged 60-80% of the epithelial volume at all measured
positions. In the Ipo-treated animals, nonciliated cells proximal to
the cuboidal epithelium end point averaged 70-90% of the
epithelial volume, but distal to the cuboidal epithelium end point (at
100 and
200 µm), nonciliated cuboidal cells averaged
only 10-20% of the epithelial volume. In the control animals, the
epithelial volume of ciliated cells ranged from 15 to 40% (Fig. 4). In
the Ipo-treated animals, ciliated cells were not observed distal to the
cuboidal epithelium end point, and in proximal areas, their volume was
a smaller percentage of the population compared with the control cells
(the maximum measured was 25% at Ipo distance 200 µm; Table 3).
Squamous cells were not found in the cuboidal epithelium lining the
terminal bronchioles in the control animals (Fig. 4). In the
Ipo-treated animals, squamous cells were the predominant epithelial
cell type in the most distal part of the terminal bronchiole, making up 70-90% of epithelial cell volume (Ipo distances
200 and
100 µm). A small percentage of squamous cells was also found
proximal to the cuboidal epithelium end point in the Ipo-treated
animals.
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The mass per surface area of nonciliated, ciliated, and squamous cells
varied from that of the control cells. Based on the same position on
the bronchiolar wall, the mass per surface area of nonciliated cells in
the Ipo-treated animals was significantly decreased compared with the
control cells in the most distal ends of the bronchiole (Ipo distances
200 and
100 µm vs. control distances 0 and 100 µm;
Fig. 5). However, at the cuboidal
epithelium end point and proximal to it, the mass per surface area of
nonciliated cells in the Ipo-treated animals was generally greater
compared with that in the control animals (carrier distances 200 and
300 µm vs. Ipo distances 0 and 100 µm; Fig. 5). When the
nonciliated cell populations were compared in relation to the two
cuboidal epithelium end points, there was a significantly larger mass
of nonciliated cells in the Ipo-treated animals compared with that in
the control animals at the two positions on the non-blood vessel side
of the airway (Ipo distance 0 µm vs. carrier distance 0 µm and Ipo
distance 200 µm vs. carrier distance 200 µm;
P < 0.05; Table 3).
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At the most distal ends of the bronchiole (Ipo distances 200 and
100 µm and carrier distances 0 and 100 µm), there was a significantly larger mass of nonciliated cell nuclei on both sides of
the bronchiole in the Ipo-treated animals compared with that in the
control animals. The same was true for the cytoplasm. For example, at
the end points of the cuboidal population on the blood vessel side of
the airway, the mass of nonciliated cell cytoplasm was larger in the
epithelium of the Ipo-treated animals (6.71 ± 2.74 µm3/µm2 at distance 0 µm for
Ipo-treated animals vs. 3.37 ± 0.66 µm3/µm2 for control animals), and this
continued more proximally (11.24 ± 6.46 µm3/µm2 at distance 300 µm for
Ipo-treated animals vs. 3.45 ± 1.56 µm3/µm2 at 300 µm for control animals;
P < 0.05).
When calculated as mass per surface area based on the same position on
the bronchiolar wall, the mass per surface area of ciliated cells of
the Ipo-treated animals was significantly decreased compared with that
in the control cells in the most distal ends of the bronchiole (Ipo
distances 200 and
100 µm vs. control distances 0 and
100 µm; Fig. 5, Tables 2 and 3). In the Ipo-treated animals, ciliated
cells had a significantly larger mass per surface area on the non-blood
vessel side than on the blood vessel side 200 µm proximal to the end
point of the cuboidal epithelium (Ipo distance 200 µm). No
significant differences were found based on distances from the two
cuboidal epithelium end points.
In contrast to nonciliated and ciliated cells, squamous cell mass per
surface area in the Ipo-treated animals was significantly increased
compared with that in the control animals in the most distal ends of
the bronchiole (Ipo distances 200 and
100 µm vs.
control distances 0 and 100 µm; Fig. 5, Tables 2 and 3) when
comparisons were based on the same position on the bronchiolar wall.
Squamous cells were more widely distributed on the blood vessel side of
the bronchiole than on the non-blood vessel side (Fig.
6). No significant differences were found
based on distances from the two cuboidal epithelium end points.
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Expression of differentiation markers. Immunoreactive CYP2B was distributed throughout the nonciliated cells in the terminal bronchioles of 4-wk-old carrier control rabbits. Staining intensity varied by bronchiole and by position within a bronchiole in the control animals (Fig. 7A). In animals treated with Ipo on day 7 and examined at 4 wk of age, a substantial portion of the bronchiolar matrix extending to the junction with the alveolar duct was free of epithelial cells containing immunoreactive CYP2B (Fig. 7B). In most cases, CYP2B immunoreactive cells were observed one generation proximal to the terminal bronchiole-alveolar duct junction. Animals treated with Ipo on day 2 or 3 and evaluated at 4 wk of age were similar to control animals except that in many bronchioles small portions of the bronchiolar epithelium adjacent to the alveolar duct junction were free of epithelial cells containing CYP2B immunoreactivity.
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Four-week-old rabbits treated with carrier on day 7 had bronchiolar airways lined by cuboidal epithelial cells that were strongly immunoreactive for NADPH reductase (Fig. 8A). Nonciliated cells were immunopositive and extended down to the terminal bronchiole-alveolar duct junction. In 4-wk-old animals treated with Ipo on day 7, the majority of the terminal bronchiolar matrix in virtually all of the bronchioles was free of epithelial cells that were immunoreactive for NADPH reductase (Fig. 8B). When reductase-positive cells were present, they were found one generation proximal to the terminal bronchiole-alveolar duct junction and were found as single cells or in small clumps. In 4-wk-old rabbits treated with Ipo on day 2 or 3, the bronchiolar epithelium was poorly reactive or nonreactive for NADPH reductase only just proximal to or at the alveolar duct junction.
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In carrier-treated control animals at 4 wk of age, CC10 expression was found in the apices and perinuclear cytoplasm of nonciliated bronchiolar cells lining the distal generations of bronchioles down to the terminal bronchiole-alveolar duct junction (Fig. 9A). Treatment with Ipo on day 7 markedly reduced the extent and intensity of immunoreactive CC10 expression within the terminal bronchioles of 4-wk-old rabbits (Fig. 9B). Significant portions of the matrix in the distal bronchioles were lined by cells that were free of CC10. Animals treated with Ipo on day 2 or 3 had areas of epithelium with reduced immunoreactivity for CC10 but not to the extent of animals treated on day 7.
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Epithelial ultrastructure. The bronchiolar epithelium of carrier-treated control animals was composed of ciliated and nonciliated cuboidal epithelial cells in airways one to two generations proximal to the bronchiole-alveolar duct junction. Similar cell composition was observed regardless of location within the bronchiole. Nonciliated cells contained abundant smooth endoplasmic reticulum, large mitochondria, and secretory granules (Fig. 10A). Nonciliated cells found near the bronchiole-alveolar duct junction had apical projections into the airway lumen, the lateral surfaces of which extended out from the apical surfaces to a greater extent than those of the more proximal nonciliated cuboidal cells (Fig. 10B). At the terminal bronchiole-alveolar duct junction, nonciliated cells formed tight junctions with squamous cells, ciliated cells, and alveolar type II cells.
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In 4- to 6-wk-old animals treated with Ipo on day 7, the overall ultrastructure of the nonciliated bronchiolar cells in proximal generations of bronchioles was similar to that in control animals except that there appeared to be less smooth endoplasmic reticulum and fewer apical projections into the airway lumen, whereas large mitochondria, often characteristic of nonciliated bronchiolar cells in adult rabbits, were not present. In distal areas of the terminal bronchioles, cuboidal epithelium contained irregular-shaped cells, ciliated cells, and nonciliated cells. The nonciliated cells contained little smooth endoplasmic reticulum, large areas of glycogen, and an irregular cuboidal shape. Cuboidal cells immediately adjacent to the squamous epithelial cells had a similar appearance (Fig. 11A). The squamous epithelial cells had long narrow cytoplasmic extensions and irregularities on their luminal aspects (Fig. 11B). In many areas close to the terminal bronchiole-alveolar duct junction, groups of irregular cuboidal epithelial cells were clumped on top of each other in a nodular configuration (Fig. 12). These cells contained secretory granules and, in some cases, whorls of electron-dense membranes, little other smooth endoplasmic reticulum, and abundant glycogen. Ciliated cells in irregular configurations were also present.
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DISCUSSION |
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This study demonstrates that acute injury to differentiating Clara cells by a cytochrome P-450-mediated cytotoxicant, Ipo, impedes the normal progress of Clara cell differentiation and bronchiolar epithelial repair. Approximately 3-6 wk post-Ipo-induced injury in neonatal animals, Clara cells have failed to repopulate the distal terminal bronchioles and are replaced by squamous cells. This effect does not appear to be reversible over a long period of time and seems to be related to the age at injury during the early postnatal period. Four- or six-week-old animals treated with Ipo on postnatal day 7 had twice as many bronchioles lined by squamous epithelial cells as animals treated on postnatal day 2 or 3, whereas bronchioles from animals treated on day 21 were virtually indistinguishable from those of age-matched control animals.
Because the end point of the bronchiolar cuboidal epithelium is not the same in carrier- and Ipo-treated animals, morphometric comparisons to characterize epithelial organization were conducted two ways: 1) based on distances along the bronchiole in relation to the pulmonary arteriole and 2) based on the distance from the cuboidal epithelial end point. The first method of comparison revealed that the epithelial thickness in the Ipo-treated animals was 25% of that in the control animals in the most distal areas of the terminal bronchioles. The second method of comparison revealed that the Ipo-treated animals have a thicker epithelium compared with the control animals along the entire length of bronchiole that was measured. Analysis of epithelial cellular abundance indicated that in the Ipo-treated animals the most distal areas of the terminal bronchioles were predominantly populated with squamous cells versus nonciliated and ciliated cells in the control animals. Squamous cells were absent in the terminal bronchioles of control animals. In the Ipo-treated animals, ciliated cells were only found at and proximal to the cuboidal epithelium end point, but their percentage and mass per surface area were reduced compared with that in the control animals.
Animals treated with Ipo and examined at 4-6 wk of age had irregular epithelium in the terminal bronchioles. Electron microscopy revealed simple squamous epithelial cells; abnormally configured ciliated cells; and groups of nonciliated cuboidal epithelial cells containing secretory granules, little smooth endoplasmic reticulum, and much cytoplasmic glycogen. The epithelial ultrastructure was generally similar to that seen in the control animals in more proximal bronchioles.
The irregularities observed in the Ipo-treated rabbits may be due to an alteration in either the epithelial repair process and/or the normal pattern of epithelial development of neonatal rabbits. Both processes share many essential steps. Repair after bronchiolar epithelial injury appears to have three components: migration, proliferation, and differentiation. With severe injury, surviving cells migrate to cover denuded areas, flatten, and become squamous (5, 29). In the present study, the source of the squamous cells residing long term in the bronchioles could be from resident progenitor cells, migrated and dedifferentiated type II cells, or dedifferentiated ciliated or nonciliated cells. After migration, studies of epithelial repair found that epithelial proliferation ensues ~12-48 h after the injury, and with time (1-3 wk), the epithelium differentiates into the normal cell population (5, 30).
In adult animals, repair after Ipo injury proceeds along this classic injury-repair pattern. In a study by Newton et al. (17), adult rats were given 10 or 25 mg/kg of Ipo intraperitoneally, and the repair process was evaluated with time. One day after injury, necrosis and degeneration of the terminal bronchiolar epithelium ensued, specifically, nonciliated and ciliated cells. The bronchiolar basement membrane was exposed in some areas, and acute pulmonary perivascularitis and peribronchiolitis were present. Plopper et al. (24) observed the same pattern of acute injury in neonatal rabbits 2 days post-Ipo injection. As an extension of this previous study of acute Ipo toxicity in neonatal rabbits, we have compared the acute injury for neonates at all the treatment ages used in the present study. We found that, at all postnatal ages from 2-3 to 21 days, substantial bronchiolar injury occurs at a dose that does not affect adults (5 mg/kg of Ipo). In adult rats, the extent of injury appeared diminished 3 days after Ipo treatment, and the previously denuded areas of basement membrane were populated with simple squamous epithelium. Epithelial regeneration was nearly complete by 1 wk, and by day 10, the epithelium of Ipo-treated animals was the same as the epithelium of control animals (17). This pattern also occurs in calves given 5 mg/kg of Ipo at an age when bronchiolar epithelium is differentiated and metabolically active cytochrome P-450s are present (13). Clearly, our study of Ipo-treated neonatal rabbits documents that the bronchiolar epithelium does not follow the normal time course of repair or pattern characterized for mature animals.
Many of the cuboidal cells found in the distal bronchioles of Ipo-treated rabbits have characteristics of cells undergoing differentiation during postnatal lung development, such as large volumes of glycogen and small amounts of smooth endoplasmic reticulum. Detailed studies have shown that the bronchioles of perinatal rabbits are lined primarily with glycogen-filled nonciliated cells. These nonciliated cells differentiate into Clara cells, with a dramatic loss of glycogen and rough endoplasmic reticulum correlated with a large gain in smooth endoplasmic reticulum and secretory granules (up to 40% of cell volume) (1, 21). The Ipo-treated animals also have a bronchiolar epithelium expressing little CC10, CYP2B, and NADPH reductase. These proteins are usually found at low levels soon after birth but increase to adult levels within 4 wk (1, 23).
The increased susceptibility of immature Clara cells to Ipo injury and their consequent inability to repair may be related to the high level of cell proliferation normally occurring during the neonatal period. In rats, the highest levels of epithelial proliferation in the terminal bronchioles occur after birth, and the labeling index decreases with age. The lowest levels of cell proliferation in the terminal bronchioles are found in adults (12). Replication can render a cell more prone to injury due to a shortened repair time. Because nonciliated bronchiolar cells of rabbits are believed to be the progenitors of both themselves and ciliated cells (22), early injury to these cells may severely compromise population expansion and therefore affect the normal development of the epithelium. A severely compromised epithelial population may also subsequently be unable to proliferate rapidly enough to populate the increasing surface area of the bronchioles as the animal grows in size. This may be related to time-dependent changes in the extracellular matrix critical for proper cell migration and differentiation. These time-dependent factors could be responsible for the apparent failure of epithelial repair in rabbits treated with Ipo on day 7 compared with rabbits treated on day 3 or 21.
Precisely what makes the undifferentiated Clara cell initially more susceptible to Ipo injury is unclear. That developing systems can have an increased susceptibility to certain toxicants is not a new observation. Examples include fetal alcohol syndrome, childhood lead poisoning, and reduced lung function in children and young adults exposed prenatally to environmental tobacco smoke (3, 31). The present study defines an animal model of neonatal lung injury that will permit identification of mechanisms responsible for the elevated lung toxicity experienced by neonates as well as the failure to adequately repair this injury, which may occur in chronic human diseases in which the etiology lies in childhood.
In conclusion, this study has shown that, based on the established patterns of Clara cell differentiation, 1) injury by a cytochrome P-450-activated cytotoxicant (Ipo) during Clara cell differentiation in early postnatal animals inhibits subsequent differentiation and development of bronchiolar epithelium and severely affects its repair and 2) developmental and repair processes depend on modification of the stage of bronchiolar epithelium differentiation at which injury occurs.
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ACKNOWLEDGEMENTS |
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We thank Dr. A. R. Buckpitt for advice concerning this study.
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FOOTNOTES |
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This study was supported in part by National Institute of Environmental Health Sciences Grants ES-06700 and ES-05707.
Address for reprint requests: S. M. Smiley-Jewell, Dept. of Anatomy, Physiology and Cell Biology, Univ. of California, Davis, CA 95616-8732.
Received 10 June 1997; accepted in final form 22 December 1997.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Cardoso, W. V.,
L. G. Stewart,
K. E. Pinkerton,
C. Ji,
G. E. Hook,
G. Singh,
S. L. Katyal,
W. M. Thurlbeck,
and
C. G. Plopper.
Secretory product expression during Clara cell differentiation in the rabbit and rat.
Am. J. Physiol.
264 (Lung Cell. Mol. Physiol. 8):
L543-L552,
1993
2.
Cho, M.,
C. Chichester,
C. Plopper,
and
A. Buckpitt.
Biochemical factors important in Clara cell selective toxicity in the lung.
Drug Metab. Rev.
27:
369-386,
1995[Medline].
3.
Cunningham, J.,
D. W. Dockery,
and
F. E. Speizer.
Maternal smoking during pregnancy as a predictor of lung function in children.
Am. J. Epidemiol.
139:
1139-1152,
1994[Abstract].
4.
Domin, B. A.,
T. R. Devereux,
and
R. M. Philpot.
The cytochrome P-450 monooxygenase system of rabbit lung enzyme components, activities, and induction in the nonciliated bronchiolar epithelial (Clara) cell, alveolar type II cell, and alveolar macrophage.
Mol. Pharmacol.
30:
296-303,
1986[Abstract].
5.
Evans, M. J.,
and
S. G. Shami.
Lung cell kinetics.
In: Lung Cell Biology, edited by D. Massaro. New York: Dekker, 1989, vol. 41, p. 1-36. (Lung Biol. Health Dis. Ser.)
6.
Foth, H.
Role of the lung in accumulation and metabolism of xenobiotic compoundsimplications for chemically induced toxicity.
Crit. Rev. Toxicol.
25:
165-205,
1995[Medline].
7.
Fujinaka, L. E.,
D. M. Hyde,
C. G. Plopper,
W. S. Tyler,
D. L. Dungworth,
and
L. O. Lollini.
Respiratory bronchiolitis following long-term ozone exposure in bonnet monkeys: a morphometric study.
Exp. Lung Res.
8:
167-190,
1985[Medline].
8.
Glantz, S.
Primer of Biostatistics. New York: McGraw-Hill, 1992.
9.
Hyde, D.,
C. Plopper,
J. St. George,
and
J. Harkema.
Morphometric cell biology of air space epithelium.
In: Electron Microscopy of the Lung, edited by D. Schraufnagel. New York: Dekker, 1990, vol. 48, p. 71-120. (Lung Biol. Health Dis. Ser.)
10.
Hyde, D. M.,
D. J. Magliano,
and
C. G. Plopper.
Morphometric assessment of pulmonary toxicity in the rodent lung.
Toxicol. Pathol.
19:
428-446,
1991[Medline].
11.
Hyde, D. M.,
C. G. Plopper,
P. H. Kass,
and
J. L. Alley.
Estimation of cell numbers and volumes of bronchiolar epithelium during rabbit lung maturation.
Am. J. Anat.
167:
359-370,
1983[Medline].
12.
Ji, C. M.,
C. G. Plopper,
and
K. E. Pinkerton.
Clara cell heterogeneity in differentiation: correlation with proliferation, ultrastructural composition, and cell position in the rat bronchiole.
Am. J. Respir. Cell Mol. Biol.
13:
144-151,
1995[Abstract].
13.
Li, X.,
and
W. L. Castleman.
Ultrastructural morphogenesis of 4-ipomeanol-induced bronchiolitis and interstitial pneumonia in calves.
Vet. Pathol.
27:
141-149,
1990[Abstract].
14.
Massaro, G. D.,
L. Davis,
and
D. Massaro.
Postnatal development of the bronchiolar Clara cell in rats.
Am. J. Physiol.
247 (Cell Physiol. 16):
C197-C203,
1984[Abstract].
15.
Massaro, G. D.,
G. Singh,
R. Mason,
C. G. Plopper,
A. M. Malkinson,
and
D. B. Gail.
Biology of the Clara cell.
Am. J. Physiol.
266 (Lung Cell. Mol. Physiol. 10):
L101-L106,
1994
16.
Moffatt, R. K.,
D. M. Hyde,
C. G. Plopper,
W. S. Tyler,
and
L. F. Putney.
Ozone-induced adaptive and reactive cellular changes in respiratory bronchioles of bonnet monkeys.
Exp. Lung Res.
12:
57-74,
1987[Medline].
17.
Newton, P. E.,
J. R. D. Latendresse,
D. R. Mattie,
and
C. Pfledderer.
Alterations in alveolar clearance after 4-ipomeanol-induced necrosis of Clara and ciliated cells in the terminal bronchiole of the rat.
Toxicol. Appl. Pharmacol.
80:
534-541,
1985[Medline].
18.
Okine, L. K.,
and
T. E. Gram.
In vitro studies on the metabolism and covalent binding of [14C]1,1-dichloroethylene by mouse liver, kidney and lung.
Biochem. Pharmacol.
35:
2789-2795,
1986[Medline].
19.
Patton, S. E.,
R. P. Gupta,
S. Nishio,
E. M. Eddy,
A. M. Jetten,
C. G. Plopper,
P. Nettesheim,
and
G. E. Hook.
Ultrastructural immunohistochemical localization of Clara cell secretory protein in pulmonary epithelium of rabbits.
Environ. Health Perspect.
93:
225-232,
1991[Medline].
20.
Plopper, C. G.
Structural methods of studying bronchiolar epithelial cells.
In: Models of Lung Disease, edited by J. Gil. New York: Dekker, 1990, vol. 47, p. 537-559. (Lung Biol. Health Dis. Ser.)
21.
Plopper, C. G.,
J. L. Alley,
C. J. Serabjitsingh,
and
R. M. Philpot.
Cytodifferentiation of the nonciliated bronchiolar epithelial (Clara) cell during rabbit lung maturation: an ultrastructural and morphometric study.
Am. J. Anat.
167:
329-357,
1983[Medline].
22.
Plopper, C. G.,
S. J. Nishio,
J. L. Alley,
P. Kass,
and
D. M. Hyde.
The role of the nonciliated bronchiolar epithelial (Clara) cell as the progenitor cell during bronchiolar epithelial differentiation in the perinatal rabbit lung.
Am. J. Respir. Cell Mol. Biol.
7:
606-613,
1992[Medline].
23.
Plopper, C. G.,
A. J. Weir,
D. Morin,
A. Chang,
R. M. Philpot,
and
A. R. Buckpitt.
Postnatal changes in the expression and distribution of pulmonary cytochrome P450 monooxygenases during Clara cell differentiation in rabbits.
Mol. Pharmacol.
44:
51-61,
1993[Abstract].
24.
Plopper, C. G.,
A. J. Weir,
S. J. Nishio,
A. Chang,
M. Voit,
R. M. Philpot,
and
A. R. Buckpitt.
Elevated susceptibility to 4-ipomeanol cytotoxicity in immature Clara cells of neonatal rabbits.
J. Pharmacol. Exp. Ther.
269:
867-880,
1994[Abstract].
25.
Serabjit-Singh, C. J.,
C. R. Wolf,
and
R. M. Philpot.
The rabbit pulmonary monooxygenase system. Immunochemical and biochemical characterization of enzyme components.
J. Biol. Chem.
254:
9901-9907,
1979[Abstract].
26.
Smith, P. B.,
H. F. Tiano,
S. Nesnow,
M. R. Boyd,
R. M. Philpot,
and
R. Langenbach.
4-Ipomeanol and 2-aminoanthracene cytotoxicity in C3H/10T1/2 cells expressing rabbit cytochrome P450 4B1.
Biochem. Pharmacol.
50:
1567-1575,
1995[Medline].
27.
Strum, J. M.,
T. Ito,
R. M. Philpot,
A. M. Desanti,
and
E. M. McDowell.
The immunocytochemical detection of cytochrome P-450 monooxygenase in the lungs of fetal, neonatal, and adult hamsters.
Am. J. Respir. Cell Mol. Biol.
2:
493-501,
1990[Medline].
28.
Ten Have-Opbroek, A. A.,
and
E. C. De Vries.
Clara cell differentiation in the mouse: ultrastructural morphology and cytochemistry for surfactant protein A and Clara cell 10 kD protein.
Microsc. Res. Tech.
26:
400-411,
1993[Medline].
29.
Van Winkle, L. S.,
A. R. Buckpitt,
S. J. Nishio,
J. M. Isaac,
and
C. G. Plopper.
Cellular response in naphthalene-induced Clara cell injury and bronchiolar epithelial repair in mice.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L800-L818,
1995
30.
Van Winkle, L. S.,
J. M. Isaac,
and
C. G. Plopper.
Repair of naphthalene-injured microdissected airways in vitro.
Am. J. Respir. Cell Mol. Biol.
15:
1-8,
1996[Abstract].
31.
Wang, X.,
D. Wypij,
D. R. Gold,
F. E. Speizer,
J. H. Ware,
B. G. Ferris, Jr.,
and
D. W. Dockery.
A longitudinal study of the effects of parental smoking on pulmonary function in children 6-18 years.
Am. J. Respir. Crit. Care Med.
149:
1420-1425,
1994[Abstract].
32.
Wolf, C. R.,
C. N. Statham,
M. G. McMenamin,
J. R. Bend,
M. R. Boyd,
and
R. M. Philpot.
The relationship between the catalytic activities of rabbit pulmonary cytochrome P-450 isozymes and the lung-specific toxicity of the furan derivative, 4-ipomeanol.
Mol. Pharmacol.
22:
738-744,
1982[Abstract].
33.
Yost, G. S.,
A. R. Buckpitt,
R. A. Roth,
and
T. L. McLemore.
Mechanisms of lung injury by systemically administered chemicals.
Toxicol. Appl. Pharmacol.
101:
179-195,
1989[Medline].