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Regular Articles |
From the Department of Anatomy, Physiology, and Cell
Biology,*
School of Veterinary Medicine, University of
California–Davis, Davis, California; the Department of
Otorhinolaryngology,
Tampere University
Hospital, Tampere, Finland; and the Departments of Medicine and Cell
Biology and Physiology,
and the Department of
Pediatrics,
Washington University School of
Medicine and Barnes-Jewish Hospital, St. Louis, Missouri
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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vß6 integrin in airway
inflammation,4
the role of gelatinase B in
bleomycin-induced fibrosing alveolitis and
bronchiolization,5
and the role of metalloproteinases
(matrilysin) in tracheal epithelial repair.6
These
transgenic animals, however, are derived from several different strains
of mice. Confounding the use of transgenic and knockout mice is the
lack of unified studies to compare the differences in injury or disease
response between the wild-type animals of the strains in which these
genetic manipulations are made. Some of the most common strains of mice
used for transgenic manipulation include the C57BL/6, 129/TerSv, and
129/SvEv mouse strains. Many of these strains of mice were originally
developed based on their susceptibility to specific disease entities,
such as cancer, or for their differences in sensitivity or metabolic
response to specific xenobiotics. For example, the responsiveness to
acetylcholine after oxidative stress is markedly different between
C57BL/6, 129/J, and DBA/2J mice.7
These types of
strain-related differences raise the question as to the use of
genetically manipulated mice and the wild-type strains from which they
were derived, in defining molecular, cellular, and biochemical
mechanisms of injury and repair. In this study, we compared the injury and repair response of three mouse strains commonly used as platforms for genetic manipulation, C57BL/6, 129/TerSv, and 129/SvEv, to a well-defined model of bronchiolar epithelial injury and repair originally developed in the male Swiss Webster mouse.8-11 The distal airways are frequent sites of epithelial injury because of several factors, including distribution of the toxicant through the branching airway structure, cellular composition of the bronchiolar epithelium, and the capability of cells in this region to activate and/or detoxify chemicals. We, and others, have exploited Clara cell metabolic activation of the simple polycyclic aromatic hydrocarbon, naphthalene, to model distal airway injury and subsequent wound healing.10,12-14 A single intraperitoneal dose of naphthalene is an acute Clara cell toxicant that is both airway level and cell-type-specific.9,15 In Swiss Webster mice, acute Clara cell injury and death are followed by a clearly defined progression of epithelial events: ciliated cell squamation, proliferation, migration, and differentiation that progresses in a proximal to distal direction.8,10 By 14 days after injury, epithelial repair is considered complete. However, the role of the ciliated cell in bronchiolar repair after acute Clara cell injury has not previously been examined in detail.
The purpose of this study was twofold: 1) to characterize the role of the ciliated cell in repair of distal airway Clara cell injury, and 2) to define strain-specific differences in the injury and repair responses of distal airway epithelium between Swiss Webster mice and C57BL/6, 129/TerSv, and 129/SvEv mice. These strains were compared with respect to the extent of injury and the progression and duration of epithelial repair, including the onset and duration of cell proliferation, changes in ciliated cell size and conformation, abundance at various phases of repair, and re-establishment of preinjury steady state epithelial organization and differentiation.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Nine-week-old, male Swiss Webster (Charles River Breeding Laboratory, Wilmington, MA), C57BL/6 (Charles River), 129/SvEv (Washington University, St. Louis, MO), and 129/TerSv (Jackson Laboratories, Bar Harbor, ME) were housed in a HEPA-filtered cage rack and maintained on a 12/12 hour light/dark cycle with free access to food and water for at least 7 days before use.
Airway Epithelial Injury Induction and Tissue Treatment
Naphthalene (Aldrich Chemical Co., Milwaukee, WI) was administered by intraperitoneal injection of 20 mg/ml of naphthalene in corn oil (Mazola, Best Foods/CPC International Inc., Englewood Cliffs, NJ) at a dose of 200 mg/kg body weight. Control animals received an equivalent volume of corn oil carrier. Mice of all strains were killed at each of the following times after naphthalene injection: 1, 2, 7, and 14 days (DPN). All mice were injected with 50 mg/kg of 5-bromo-2'-deoxyuridine (BrdU; Fitzgerald Industries International, Inc., Concord, MA) 1 hour before euthanasia. At the time of necropsy, all animals were anesthetized with an overdose of pentobarbital sodium and exsanguinated. After euthanasia, the trachea was exposed by a ventral midline cervical incision and cannulated at the larynx.
The lungs from three treated and one control animal for each time point were collected for paraffin tissue sections. The diaphragm was punctured and the lungs were infused in the thorax via intratracheal cannula for 1 hour at 30 cm pressure with 1% paraformaldehyde in 0.1 mol/L of phosphate buffer (pH 7.4). Once removed from the thorax, the right cranial lobe was processed for scanning electron microscopy (SEM) and the middle and caudal lobes were processed for paraffin embedding. For paraffin sectioning, the lobes were embedded whole with the mediastinal surface down. Paraffin blocks were sectioned at 5 µm on a Reichert-Jung Supercut microtome and placed on Silane-Prep glass slides (Sigma Diagnostics, St. Louis, MO).
The lungs from three treated and one control animal for each time point were collected for high-resolution light microscopy and SEM. The diaphragm was punctured and the lungs were infused while in the thorax for 1 hour at 30-cm pressure with a mixture of 0.7% paraformaldehyde and 0.9% glutaraldehyde in cacodylate buffer (pH 7.4, 330 mOsm).16 Once removed from the thorax, the left lobe was transected into three equal segments cut perpendicular to the long axis of the lung lobe. The tissues were postfixed in 1% osmium tetroxide in Zetterquist’s buffer, processed by large block methodology, and embedded cut surface down in Araldite 502 (Electron Microscopy Sciences, Fort Washington, PA) epoxy resin.16 Araldite blocks were sectioned at 1 µm on a Sorvall JB-4 Porter-Blum microtome (Dupont Company Biotechnology Systems, Wilmington, DE), and stained with methylene blue/azure II with 0.5% sodium borate.
For SEM, the apical lobe of the right lung was affixed to a 1.2-cm2 Corning cover glass (Corning Glass Works, Palo Alto, CA), mediastinal side down, with cyanoacrylate tissue glue (Nexaband; Veterinary Products, Phoenix, AZ). The lumena of the mediastinal airways extending from the lobar bronchus to the terminal bronchioles were opened by removing the dorsal half of the airway while immersed in phosphate-buffered saline. Microdissection was aided by the use of a Wild Heerbrugg dissecting microscope (Technical Instruments, San Francisco, CA). While affixed to the coverslip, the microdissected lungs were dehydrated in 10-minute washes of a graded ethanol series of 70%, 85%, 95%, and 100%. To remove airway secretions from the epithelial surfaces, the lobes were agitated briefly in a 50/50 solution of 100% alcohol and toluene, then taken to 100% toluene. The process was reversed until the tissue was back in the 100% alcohol. The dehydrated lobes were then bathed in hexamethyldisilizane (Electron Microscopy Sciences, Fort Washington, PA) for 5 minutes at room temperature. The lung lobes were glued to SEM chucks with Nexaband and allowed to air-dry overnight. The lungs were sputter-coated for 2 minutes with gold using a Polaron II ES100 sputter-coater (acceleration voltage 2.5 kV, 10 mA current in argon) (Energy Beam Sciences, Agawan, MA). The microdissected lobes were viewed and imaged with a Philips SEM 501 microscope (FEI Corporation, Hillsboro, OR).
Transmission Electron Microscopy
Thin sections (60 to 90 nm) were cut using a diamond knife on a LKB Nova ultramicrotome (LKB Bromma, Sweden). Sections were stained with uranyl acetate and lead citrate, and visualized with a Zeiss EM10 at 80 kV (Zeiss Microimaging, Thornwood, NY).
Definition of Airway Levels
The microdissected lungs of the four different mouse strains were
examined by SEM (Figure 1)
and the
histological changes are described and quantified by airway level.
Summary data are reported for three animals per group, with more than
10 distal airways examined per animal. The distal airways were defined
as the three most distal generations of bronchioles. All airways were
examined in detail including airway bifurcations and airway segments
between bifurcations.
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The avidin-biotin-peroxidase procedure as outlined by the supplier (Vector Laboratories, Burlingame, CA) was used to identify BrdU antibody-binding sites. In addition, before the peroxidase block, all sections were digested for 10 minutes at 60°C with HCl, followed by a 10-minute neutralization step in borate buffer. Sections were then exposed to 0.05% proteinase K (Sigma Chemical Co.) for 3 minutes followed by a nonspecific protein-blocking step with bovine serum albumin. The sections were incubated with a 1:100 dilution of polyclonal sheep anti-BrdU (Fitzgerald Industries International, Inc.) as the primary antibody in a humidity chamber at 4°C overnight. Secondary biotinylated rabbit anti-sheep IgG (H+L) antibody (Vector Laboratories) was used at 1:500 and 3,3'-diaminobenzidine (Sigma Chemical Co.) was used as the chromagen.
Proliferation
Cross-sections of whole lung, from each of three animals from each time point, including carrier controls were examined for BrdU-labeled nuclei. At least 10 longitudinal cross-sections from the distal airways of each animal of every strain were categorized by the number of BrdU-positive nuclei within the airway: 1, 2, 3, or >4. Distal airways were defined by their proximity to or connection with a terminal bronchiole. The percentage of positive-labeled nuclei per category was calculated for each airway level and for each of the following days after naphthalene: 0, 1, 2, 4, 7, and 14. The data were entered into Cricket Graph III, version 1.0 (Computer Associates International, Inc.) for graphical analysis.
Ciliated Cell Size and Number of Ciliated Cells per Unit Surface Area
Scanning electron microscopic images were collected from one surface of specific airway segments between bifurcations for the distal three airway generations of three animals at each of the following time points: 0 (corn oil control), 2, 7, and 14 DPN. The time points selected represent the following phases of response in Swiss Webster mice: steady state, the phase of maximal squamation, near complete repair, and complete repair. The airways were imaged at x750 magnification and surface area of 10 ciliated cells selected at random was measured using Scion Image (NIH). Omitted from counting were those ciliated cells present on any curved surface. For numeric density calculations, the same SEM images were used. The total airway surface area of each image was kept constant and the total number of ciliated cells per image was counted. The images were counted in random order to decrease bias based on mouse strain, airway level, or days after injury.
Statistics
All data from ciliated cell surface area and number of ciliated cells per unit surface area were imported into Stat View (Abacus Concepts, Berkeley, CA) for analysis of variance and Bonferroni/Dunn post hoc analysis. Differences between values within the same mouse strain and differences between Swiss Webster mice and C57BL/6, 129/TerSv, or 129/SvEv mice were assessed with significance determined by Bonferroni/Dunn at P < 0.05.
All proliferation data were also imported into Stat View for analysis. Association between strains and the number of BrdU-positive cells by airway level were assessed by chi-square analysis.17 For all analyses a P value of <0.05 was considered statistically significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Control Mice All Strains
The epithelium lining distal bronchioles was similar in all four
mouse strains (Figure 2)
. Clara cells
were arranged in slightly irregular longitudinal rows no more than two
cells wide, and oriented parallel to the long axis of the airway (not
shown). The rows of Clara cells were separated by discontinuous rows of
ciliated cells no more than one cell wide. All Clara cells appeared to
have at least one surface contact with a ciliated cell (Figure 1B)
.
Clara cells had large luminal surface areas and prominent apical
projections that protruded into the lumen, whereas ciliated cells were
angular with much smaller surface areas and had short cilia (Figure 2
;
A, B, C, E, and G). There was no phenotypic difference between the
surface epithelium of airway bifurcations and the epithelium of airway
segments between bifurcations (Figure 1B)
.
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Two days after naphthalene treatment (2 DPN), the distal airways
were lined by a diffuse sheet of large polygonal cells with short
surface microvilli and prominent intercellular demarcation. The cells
were attenuated to low cuboidal with intercellular borders depressed
from the luminal surface plasma membrane (Figure 2B)
. The majority of
the cells had central tufts of cilia, whereas a few cells lacked
surface differentiation or had central, slightly raised blebs. Those
cells lacking surface differentiation had short, scattered, individual,
peripheralized cilia or had a central circular area that was smooth and
lacked surface microvilli. The mean ciliated cell surface area at 2 DPN
was 1.5-fold greater than the surface area found in control animals
(Figure 3)
. The mean number of ciliated
cells per unit surface area was half that of steady state (Figure 4)
.
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At 14 DPN, the majority of ciliated cells lining the distal airways were integrated with Clara cells. A few clusters of ciliated cells with prominent, elongate cilia were scattered randomly within the distal airways, and several of the ciliated cells still had a large surface area. The mean ciliated cell surface area decreased two-thirds below that found at 7 DPN and in controls, to an area averaging 24% smaller than the ciliated cell surface area of epithelium at steady state. The mean number of ciliated cells per unit surface area was 1.3-fold greater than the number at steady state.
C57BL/6 Mice
When compared to the mean ciliated cell surface area of Swiss
Webster mice at steady state, the surface area of the C57BL/6 was
smaller (Figure 3)
. In addition, C57BL/6 mice had 1.6-fold more
ciliated cells per unit of airway surface area when compared to Swiss
Webster mice (Figure 4)
. Statistically significant differences in mean
ciliated cell surface area and in number of ciliated cells per unit of
airway surface area in comparison to Swiss Webster mice are summarized
in Tables 1 and 2
. At 2 DPN, the diffuse sheets of
ciliated cells had fewer cells that lacked surface differentiation and
had fewer with reduced or scattered cilia. The majority of ciliated
cells had central membrane invaginations. By high-resolution light
microscopy, foci of epithelial hyperplasia were present primarily at
airway bifurcations and fewer were at airway segments between
bifurcations. Some cells at airway segments between bifurcations had
swollen ciliated cells that were raised above the surrounding
epithelium. The mean ciliated cell surface area increased approximately
threefold compared to the surface area at steady state and 1.8-fold
greater than in Swiss Webster mice at 2 DPN. In addition, the number of
ciliated cells per unit surface area increased compared to control
animals, a difference of more than fourfold between Swiss Webster and
C57BL/6 mouse strains.
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At 14 DPN, the distal airways were lined by integrated ciliated and Clara cells and no ciliated cell cords or clusters were present. The mean ciliated cell surface area was two-thirds of that at 7 DPN, but still one-third larger than the ciliated cells at steady state and twofold greater than in Swiss Webster mice at 14 DPN. The mean number of ciliated cells per unit surface area was greater than at 7 DPN, and similar to the number at steady state. The number of ciliated cells per unit surface area at 14 DPN in C57BL/6 was greater than in Swiss Webster mice at 14 DPN.
129/TerSv Mice
When compared to the mean ciliated cell surface area of Swiss
Webster mice at steady state, ciliated cells in 129/TerSv mice were
smaller (Figure 3)
. In addition, 129/TerSv mice had 1.5-fold more
ciliated cells per unit surface area than Swiss Webster mice at steady
state (Figure 4)
. Statistically significant differences in mean
ciliated cell surface area and in number of ciliated cells per unit of
airway surface area in comparison to Swiss Webster mice are summarized
in Tables 1 and 2
. At 2 DPN, the sheets of ciliated cells were
extremely attenuated (Figure 2F)
and tufts of cilia were extremely
reduced in length. In some areas intercellular borders were poorly
delineated. These areas were identified by high-resolution light
microscopy and transmission electron microscopy as areas of denuded
basement membrane (Figures 5 and 6)
. At some bifurcations, approximately
four cells on either side of the crest between airways were cuboidal
cells that were either ciliated or slightly domed (Figure 5)
. The
ciliated cell surface area increased 12.5-fold over the surface area at
steady state, which was 6.5-fold greater than in Swiss Webster mice at
the same period after injury (Figure 3)
. In addition, the number of
ciliated cells per unit of airway surface area decreased to 20% of the
number of ciliated cells at steady state, resulting in fewer cells when
compared to Swiss Webster mice at the 2 DPN time point (Figure 4)
.
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. By high-resolution light
microscopy the airway epithelium was attenuated, and had multifocal
areas of hyperplasia at airway bifurcations and airway segments between
bifurcations (Figure 5)
. At airway segments between bifurcations, some
areas of basement membrane were covered by thin cytoplasmic extensions
from adjacent cells. The ciliated cell surface area was decreased
slightly from the 2 DPN time point, which was greater than eightfold
larger than the surface area of the ciliated cells of Swiss Webster
mice at 7 DPN. In addition, the number of ciliated cells per unit
airway surface area was low at 7 DPN (similar to 2 DPN) to a number
one-fourth of that in Swiss Webster mice at the same time after injury
(Figure 4)
.
|
. In addition,
the mean number of ciliated cells per unit surface area increased
fivefold, to a number 18% greater than the number of ciliated cells at
steady state and 1.4-fold greater than the number of ciliated cells in
Swiss Webster mice at 14 DPN (Figure 4)
. 129 SvEv Mice
When compared to the mean ciliated cell surface area of Swiss
Webster mice at steady state, ciliated cells of 129/SvEv mice were 50%
smaller (Figure 3)
. In addition, 129/SvEv mice had 1.6-fold more
ciliated cells per unit surface area than Swiss Webster mice at steady
state (Figure 4)
. Statistically significant differences in mean
ciliated cell surface area and in number of ciliated cells per unit of
airway surface area in comparison to Swiss Webster mice are summarized
in Tables 1 and 2
. At 2 DPN, the ciliated cells were extremely
attenuated and there was extensive basement membrane exposure (Figure 2H
and Figure 6
). Tufts of cilia were extremely reduced in length.
Rare, small clusters of epithelial cells at bifurcations were less
squamated and had prominent intercellular borders. The ciliated cell
surface area increased 6.2-fold from steady state surface area, which
was twofold greater than the ciliated cell surface area in Swiss
Webster mice (Figure 3)
. In addition, the number of ciliated cells per
unit airway surface area decreased 4.2-fold over the number of ciliated
cells at steady state, resulting in little difference when compared to
Swiss Webster mice at the same time point (Figure 4)
.
At 7 DPN, the majority of the epithelium was composed of cells lacking
surface differentiation. Clara cells with prominent apical domes were
present multifocally in small clusters and ciliated cells were often
individual and scattered among the undifferentiated cells. By
high-resolution light microscopy, sheets of swollen cells were
multifocal and separated by fine cytoplasmic extensions from adjacent
cells. The ciliated cell surface area decreased four-fifths of the 2
DPN time point to an area 2.5-fold greater than Swiss Webster mice
(Figure 3)
. In addition, ciliated cell numbers decreased to two-thirds
of 2 DPN, to a number that was 20% of the number of ciliated cells in
Swiss Webster mice at the same time after injury (Figure 4)
.
At 14 DPN, the airways were lined by integrated ciliated and Clara
cells and no ciliated cell cords or clusters were present. The mean
ciliated cell surface area decreased more than sixfold, to an area
two-thirds that of Swiss Webster ciliated cells and similar to the
129/SvEv ciliated cell surface area at steady state (Figure 3)
. In
addition, the mean number of ciliated cells per unit airway surface
area increased sixfold, to a number approximately equal to the number
of ciliated cells at steady state and 1.3-fold greater than the number
of ciliated cells in Swiss Webster mice (Figure 4)
.
Proliferative Response
Swiss Webster Mice
In carrier controls, 94% of the distal airways lacked
BrdU-positive nuclei (Figure 8)
. The
remaining 6% had no more than one positive nucleus per airway profile.
At 1 DPN, 38% of the distal airways had BrdU-positive nuclei and 17%
had two or more positive nuclei per airway. At 2 DPN, 100% of the
airways had BrdU-positive nuclei, and 43% had four or more positive
nuclei per airway. BrdU-positive nuclei were distributed throughout the
bronchioles, including airway bifurcations as well as airway segments
between bifurcations. At 4 DPN, only 18% of the distal airways had
BrdU-positive nuclei and 15% had no more than one positive nucleus per
airway. At 7 DPN, 17% of the airways had BrdU-positive nuclei and 2%
had two to three positive nuclei per airway. At 14 DPN, none of the
airways had BrdU-positive nuclei.
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In carrier controls, 94% of the distal airways lacked
BrdU-positive nuclei and the 6% that were positive had no more than
one per airway (Figure 8)
. At 1 DPN, 9% of the airways had
BrdU-positive nuclei and all airways had no more than one positive
nucleus per airway. By 2 DPN, 72% of the airways had positive nuclei,
and 49% had two or more positive nuclei per airway. At 4 DPN, 91% of
the distal airways had BrdU-positive nuclei and 38% had four or more
BrdU-positive nuclei per airway. BrdU-positive nuclei were distributed
throughout the bronchioles, including airway bifurcations as well as
airway segments between bifurcations. By 7 DPN, 40% of the airways had
positive nuclei and 13% had two or more positive nuclei per airway. At
14 DPN, 9% of the airways had BrdU-positive nuclei and none had more
than one positive nucleus per airway. The differences in the extent of
proliferation compared to Swiss Webster mice were statistically
significant at 1, 2, 4, and 7 DPN (Table 3)
.
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In carrier controls, 95% of the distal airways had no
BrdU-positive nuclei and of the 5% that were positive, none had more
than one positive nucleus per airway (Figure 8)
. At 1 DPN, 2% of the
distal airways had BrdU-positive nuclei and none had more than one
positive nucleus per airway. At 2 DPN, 38% of the airways had
BrdU-positive nuclei and 17% had two or more positive nuclei per
airway. At 4 DPN, 58% of the airways had BrdU-positive nuclei, and
32% had two or more positive nuclei per airway. At 7 DPN, 94% of the
distal airways had BrdU-positive nuclei, and 47% of those had more
than two positive nuclei per airway. BrdU-positive nuclei were
distributed throughout the bronchioles, including airway bifurcations
as well as airway segments between bifurcations. At 14 DPN, 34% of the
airways had BrdU-positive nuclei and 7% had two or more positive
nuclei per airway. The differences in the extent of proliferation
compared to Swiss Webster mice were significant at 1, 2, 4, 7, and 14
DPN (Table 3)
.
129/SvEv Mice
In carrier controls, 97% of the distal airways had no
BrdU-positive-labeled nuclei, and the 3% of airways that did, all had
two positive nuclei per airway (Figure 8)
. At 1 DPN, 7% of the airways
had BrdU-positive nuclei, and none had more than one BrdU-positive
nucleus per airway. At 2 DPN, 71% of the distal airways had positive
labeled nuclei and 22% had four or more positive nuclei per airway. At
4 DPN, 88% of the distal airways had BrdU-positive nuclei with 61%
having four or more positive nuclei per airway. BrdU-positive nuclei
were distributed throughout the bronchioles, including airway
bifurcations as well as airway segments between bifurcations. At 7 DPN,
51% of the airways had BrdU-positive nuclei with 28% having four or
more positive nuclei per airway. At 14 DPN, 12% of distal airways had
BrdU-positive nuclei and 10% had no more than one positive labeled
nucleus per airway. The differences in the extent of proliferation
compared to Swiss Webster mice were statistically significant at 1, 2,
4, and 7 DPN (Table 3)
.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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).
We used a well-defined model of acute airway epithelial injury that is
both cell-type- and airway level-specific and has a well-characterized
pattern of repair in Swiss Webster mice that includes cell squamation,
proliferation, migration, and cytodifferentiation.8
The
naphthalene-induced injury/repair model uses a Clara cell-specific
toxicant that targets the distal bronchioles and spares the ciliated
cell. In addition to significant differences in the temporal pattern of
airway epithelial injury and repair between all four strains of mice,
we found commonalities in the sequence of events and in the plasticity
of the ciliated cell response.
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Based on SEM, all Clara cells were removed from the most distal three airway generations, with the exception of branch points, in all four strains of mice. A similar surviving cell population at branch points has also been described by Stripp and colleagues.12 In their study, a population of cells at airway bifurcations expressed CC10 mRNA before proliferation suggesting that these cells were either Clara cells that survived injury or an immature Clara cell population. In our study, we were able to demonstrate by SEM that some of these cells maintain a Clara cell phenotype although most squamate in response to the surrounding cell loss.
Mouse strain-related differences were present in the response of the surviving cell population, ciliated cells, to Clara cell injury and exfoliation. In all four strains of mice, ciliated cells demonstrated dynamic changes in terms of their ability to alter their shape in response to cell loss or increases in cell density, as well as their ability to alter their surface appearance through changes in the plasma membrane, microvilli, and cilia. Surviving ciliated cells in Swiss Webster mice respond to cytotoxic Clara cell injury and exfoliation by squamating and sending cytoplasmic pseudopodia beneath necrotic Clara cells before their exfoliation, leaving the basement membrane undenuded as previously described.8 The response of ciliated cells in C57BL/6 mice was similar to that of Swiss Webster mice, but large areas of basement membrane were left denuded in 129/TerSv and 129/SvEv mice. The amount of basement membrane exposure in both the 129/TerSv and 129/SvEv strains of mice seemed excessive relative to the abundance and size of the ciliated cell population at steady state when compared to the Swiss Webster and C57BL/6 mice where denudation was not apparent. It has been well established that the Clara cell is the cellular target in naphthalene-induced injury,9,15 however it is possible that the loss of Clara cells from the basement membrane was too rapid in 129/TerSv and 129/SvEv mice for the ciliated cells to completely respond and maintain an intact epithelial barrier. In all four strains of mice, ciliated cells demonstrated extreme plasticity in their ability to squamate, especially the ciliated cells of 129/TerSv mice (12.5-fold increase in surface area compared to steady state). Yet the basement membrane was left exposed. A possible explanation is that some ciliated cells were lost as bystanders of acute Clara cell injury. Regardless, it seems that ciliated cells do not have the same cytokinetic ability to squamate and cover the basement membrane in response to Clara cell loss in all strains of mice. Additionally, the increase in number of cells lacking surface differentiation was proportional to the increased abundance of proliferating cells in these strains of mice. These phenotypic changes in the ciliated cell population during repair suggest that the ciliated cell is capable of undergoing biological changes that allow it to proliferate and take an active role in epithelial repair.
In this study ciliated cells demonstrated a remarkable plasticity in their efforts to form an epithelial barrier after acute exfoliation of injured Clara cells. A novel finding in this study, facilitated by the coupling of microdissection with SEM, is that ciliated cell plasticity also includes resorption of cilia to yield a distal airway epithelium that lacks the characteristics of the two predominant differentiated cell types found in the distal airways: apical protrusions and cilia. We observed directly, using SEM, large membrane invaginations in the center of squamating ciliated cells that engulfed the cilia present on the cell surface. Resorption of specialized membrane structures is not a new concept. Several diverse organisms, including protozoa,22, 23 sea urchins,24 and vertebrate rat kangaroo cells25 resorb cilia. Rieder and colleagues,25 using the rat Kangaroo cell line PtK1, documented that resorption of cilia occurs before mitosis and speculated that the ciliary tubules were "re-used" during cell division. When sea urchin embryo cells are disaggregated and then allowed to reaggregate, the cilia are resorbed and the epithelial cells take on the appearance of a less differentiated cell as the epithelium sorts and the embryo reassembles.24 Ciliary resorption is hypothesized to be related to the ability of the sea urchin epithelial cells to migrate. It has been speculated that resorption of the tubulin-rich ciliary structure is necessary before cell division can occur. Presumably the tubulin is recycled for use in spindles. We suggest that a similar phenomenon may be occurring in squamated bronchiolar ciliated cells, perhaps triggered by their change in shape in response to Clara cell injury.
In the adult lung the ciliated cell is regarded as terminally differentiated. This is based primarily on the low labeling index of this cell type in long-term labeling studies and on the pattern of proliferation described in previous injury models that injure primarily ciliated cells (such as NO2 and O3).26-28 Our current study found that the ciliated cell is a much more plastic phenotype than was previously appreciated. When distal airway Clara cells are injured by naphthalene and exfoliate, the remaining ciliated cells resorb their cilia to return to a less-differentiated cell phenotype (dedifferentiation) and may even be capable of transdifferentiation. Dedifferentiation of conducting airway epithelial cells during wound healing in the lung has been previously described in detail by Erjefalt and colleagues.29 Using the tracheal scrape model in guinea pigs, they clearly describe the presence of large flattened epithelial cells that migrate from the edge of the wound and form a barrier as early as 8 hours after wounding. These cells lack markers of cell surface differentiation. Although it is clear that the ciliated cells remaining after Clara cell injury undergo a similar phenotypic shift to an undifferentiated cell type, what is less clear is what role this flattened epithelial cell type plays in repopulation of the distal airways. Erjefalt and colleagues29 were unable to determine whether squamated cells were also the actual proliferating cell population because of the presence of basal cells, which are known to be capable of proliferation. In the distal airways, basal cells are not present and proliferation is not restricted to airway bifurcations where neuroendocrine cells have been shown to congregate.14,30 We hypothesize that the ciliated cell is not terminally differentiated and plays a dynamic role in the repair of airway epithelium through the process of transdifferentiation. A similar switch in phenotype has previously been described for another lung epithelial cell type thought to be terminally differentiated: alveolar type 1 cells that convert to type 2 cells in vitro.31 Indeed, the issue of bronchiolar-ciliated cell transdifferentiation has interesting parallels with alveolar cells in that extracellular factors and changes in cell shape may play a prominent role in signaling phenotype shifts in the bronchiolar cells as suggested by Danto and colleagues31 for alveolar cells. Clearly further studies are needed to clarify the cellular events involved in transdifferentiation of airway epithelium and to identify changes in the composition of the basement membrane in the airways of mice injured by naphthalene.
The onset and duration of proliferation was different for each of the four strains of mice, although the extent of Clara cell exfoliation was the same. In those strains whose ciliated cell response resulted in complete coverage of the basement membrane, proliferation began and ended more rapidly than in those strains whose ciliated cells squamated more or where ciliated cells failed to completely cover the basement membrane. In addition, although 129/TerSv mice exhibited the greatest increase in ciliated cell surface area during squamation, they subsequently exhibited lower numbers of proliferating cells initially, a delayed peak in proliferative rate, and greater numbers of proliferating cells later in repair when compared to 129/SvEv mice that also had exposed basement membrane. Although extensive cell stretch has been shown to have a positive effect on endothelial cell survival,32 our study suggests the opposite effect for airway epithelial cells because the strain whose ciliated cells achieved the greatest surface area (129/TerSv), was also the strain with the greatest delay and prolongation of the proliferation phase. It seems that the greater the extent of cell squamation the greater the delay in onset of cell proliferation. The strain difference in the ability of ciliated cells to squamate has an inverse regulatory effect on the proliferative rate in the injury target zone, the distal airways.
The repair phases of migration and cytodifferentiation were marked by the return of ciliated cell abundance and surface area, as well as their integration with Clara cells, to a phenotypic organization consistent with steady state conditions. In all four strains of mice, an epithelial organization consistent with steady state for each strain was reached by 14 DPN, but for one strain, Swiss Webster, it was earlier. This occurred regardless of the varied responses during squamation and proliferation.
In summary, phenotype-specific acute bronchiolar epithelial injury exposed profound differences in the temporal pattern of injury and repair of four strains of mice: Swiss Webster, C57BL/6, 129/TerSv, and 129/SvEv. These strain-related differences centered on the biology of one cell type, the ciliated cell. This study demonstrates the importance of defining the phases of injury and repair by strain of mouse and in using like strains of mice for defining mechanisms of acute airway epithelial injury and repair. The varied responses in different strains of mice greatly emphasizes the need to base assumptions regarding parameters of lung injury on a clear understanding of the degree of resistance or sensitivity of the specific mouse strain under experimentation. The impact that strain differences have on the pattern of airway epithelial repair after acute injury can have a profound impact on the interpretation of biological responses in genetically modified mice. The potential for redundancies in genes regulating biological processes present significant difficulties in the interpretation of their role in airway epithelial repair when using genetically modified mice. Strain differences in the pattern of responses, such as those demonstrated here will only compound interpretive problems if not taken into account. Further, this study shows that the ciliated cell is capable of remarkable plasticity of cell shape and phenotypic expression and documents that ciliated cells play a prominent but strain-variable role in distal bronchiolar repair. Defining repair parameters for additional mouse strains that have not been previously evaluated will become more important with the broader use of transgenic and knockout mouse variants. Our findings provide support for good laboratory practice: that control animals must be of the same strain, ideally litter mates, when transgenic or knockout mice are used for the study of airway repair processes and mechanisms.
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Supported by grants from the National Institute of Environmental Health Sciences (ES04311, ES04699, ES06700, ES05707, NHLBI, HL07013, and HL47328 to R. M. S.), the American Lung Association Research Training Fellowship Program (to G. W. L.), the National Institute of Environmental Health Sciences Training Fellowship Program (to G. W. L.), a California Tobacco-Related Diseases Research Program (grant 6KT-0306 to L. S. V.), and the Alan W. and Edith L. Wolff Charitable Trust (to R. M. S.).
Accepted for publication September 17, 2001.
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