Relationship of Inhaled Ozone Concentration to Acute Tracheobronchial
Epithelial Injury, Site-specific Ozone Dose, and Glutathione Depletion
in Rhesus Monkeys
Charles G. Plopper, Gary E. Hatch, Viviana Wong, Xiuchen Duan, Alison J. Weir, Brian K. Tarkington,
Robert B. Devlin, Suzanne Becker, and A. R. Buckpitt
Departments of Anatomy, Physiology and Cell Biology, and Molecular Biosciences, School of Veterinary Medicine;
California Regional Primate Research Center, University of California, Davis, California; and National Health and
Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park,
North Carolina
Acute pulmonary epithelial injury produced by short-term exposure to ozone varies by site within the tracheobronchial tree. To test whether this variability is related to the local dose of ozone at the tissue site or
to local concentrations of glutathione, we exposed adult male rhesus monkeys for 2 h to filtered air or to
0.4 or 1.0 ppm ozone generated from 18O2. Following exposure, lungs were split into lobes and specimens
were selected by microdissection so that measurements could be made on airway tissue of similar branching history, including trachea, proximal (generation one or two) and distal (generation six or seven) intrapulmonary bronchi, and proximal respiratory bronchioles. One half of the lung was lavaged for analysis of
extracellular components. In monkeys exposed to filtered air, the concentration of reduced glutathione
(GSH) varied throughout the airway tree, with the proximal intrapulmonary bronchus having the lowest
concentration and the parenchyma having the highest concentration. Exposure to 1.0 ppm ozone significantly reduced GSH only in the respiratory bronchiole, whereas exposure to 0.4 ppm increased GSH only
in the proximal intrapulmonary bronchus. Local ozone dose (measured as excess 18O) varied by as much
as a factor of three in different airways of monkeys exposed to 1.0 ppm, with respiratory bronchioles having the highest concentration and the parenchyma the lowest concentration. In monkeys exposed to 0.4
ppm, the ozone dose was 60% to 70% less than in the same site in monkeys exposed to 1.0 ppm. Epithelial
disruption was present to some degree in all airway sites, but not in the parenchyma, in animals exposed to
1.0 ppm ozone. The mass of mucous and ciliated cells decreased in all airways, and necrotic and inflammatory cells increased. At 0.4 ppm, epithelial injury was minimal, except in the respiratory bronchiole,
where cell loss and necrosis occurred, and was 50% that found in monkeys exposed to 1.0 ppm ozone. We
conclude that there is a close association between site-specific O3 dose, the degree of epithelial injury, and
glutathione depletion at local sites in the tracheobronchial tree. Plopper, C. G., G. E. Hatch, V. Wong, X.
Duan, A. J. Weir, B. K. Tarkington, R. B. Devlin, S. Becker, and A. R. Buckpitt. 1998. Relationship
of inhaled ozone concentration to acute tracheobronchial epithelial injury, site-specific ozone dose,
and glutathione depletion in rhesus monkeys. Am. J. Respir. Cell Mol. Biol. 19:387–399.
(Received in original form September 22, 1997 and in revised form April
22, 1998)
The research described in this article has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and
approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of
trade names or commercial products constitute endorsements or recommendation for use.
Address correspondence to: C. G. Plopper, Ph.D., VM-APC, University of
California, Davis, CA 95616.
Abbreviations: reduced glutathione, GSH; high-pressure liquid chromatography, HPLC; interleukin-8, IL-8; leukotriene B4, LTB4; prostaglandin
E2, PGE2; thromboxane B2, TXB2.
Am. J. Respir. Cell Mol. Biol. Vol. 19, pp. 387–399, 1998
Internet address: www.atsjournals.org
A characteristic feature of the response of the respiratory
system to inhalation of reactive oxidant gases, such as the
air pollutant ozone, is the lack of uniformity in the response throughout the lower respiratory tree. In humans,
the acute response to initial exposure includes marked inflammation, detected as marked increases in inflammatory
cells and a variety of proteins and mediators in bronchoalveolar lavage fluid (BALF) (1–4). In rhesus monkeys, the
degree of cellular injury and the nature and amount of the
inflammatory-cell influx into the airway tree vary markedly by position (5). The differences in susceptibility to injury by region are even more marked when consideration
is given to the cellular response to long-term exposure.
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The primary sites for injury from ozone exposure are the
anterior nasal cavity, trachea, and the terminal bronchiole/
alveolar duct junction, where ciliated cells and alveolar
type I cells are most affected (6–12). After long-term exposure, these sites in the respiratory system are also modified in a concentration-dependent fashion, even at inhaled
concentrations of ozone that reflect ambient levels (11–17).
The basis for this wide variation in sensitivity to ozone
injury is poorly understood. Among the potential mechanisms for the variability in cellular response in different regions of the respiratory system are differences in local concentrations of the toxicant and differences in the ability of
target versus nontarget cells to modulate oxidant stress. To
test this latter hypothesis, we recently evaluated the activity of four antioxidant enzymes (glutathione S-transferase,
glutathione peroxidase, catalase, and superoxide dismutase
[SOD]) within defined regions of the respiratory tract (18).
We found substantial differences in the activities of glutathione S-transferase and glutathione peroxidase, but not
in those of superoxide dismutase or catalase. Although this
variability in some of the antioxidant enzymes on a site-bysite basis may account for some of the differences in susceptibility to ozone-induced injury, it is not likely to account for all of the differences in susceptibility, because in
some sites there were no differences in antioxidant enzyme
activities. The current study was designed to test the hypothesis that the differences in susceptibility to acute injury
resulting from inhalation of ozone are the result of differences in the local concentrations of ozone reaction products at specific sites. We tested this hypothesis by defining
the acute cellular response to injury at multiple sites in the
respiratory tree, and measured the concentration of ozone
reaction products (estimated as excess 18O in tissue) and
reduced glutathione concentrations at these sites. Changes
at specific sites were compared with alterations in markers
of inflammation in BALF from the same animals.
330 mOsm) at 30 cm fluid pressure for at least 1 h, and was
then ligated and stored in a similar fixative for up to 2 wk
prior to microdissection. The bronchus to the right caudal
lobe was cannulated and the lobe was inflated with fluorodichloromethane and then frozen by exposure to liquid
nitrogen vapor. The frozen lobes were wrapped in aluminum foil and lyophilized to absolute dryness. The right
middle lobe was inflated with ice-cold Waymouth’s medium (Life Technologies, Grand Island, NY) and the airway compartments were microdissected while still fresh
(within 1 h) for glutathione measurements (18).
Animals and Lung Preparation
Ozone Exposure and Monitoring
Rhesus monkeys were exposed individually in exposure
chambers of 4.2-m3 capacity that were ventilated at a rate
of 30 changes per hour with CBR (chemical, biological and
radiologic filtered air at 24 6 28C at 40 to 50% relative humidity) (5). Ozone concentrations were measured with a
UV ozone monitor (Model 1003-AH; Dasibi Environmental Corporation, Glendale, CA) and reported with respect
to the UV photometric standard. Additional details of exposure and monitoring have been reported previously (9).
At the time of exposure, the animals were divided into
three groups, which were reasonably matched with regard
to body weight and age. Eight monkeys were exposed to
filtered air, four monkeys were exposed to 0.4 ppm ozone
for 2 h, and seven monkeys were exposed to 1.0 ppm ozone
for 2 h. The ozone was generated by substituting isotopically pure 18O2 (97%; Isotech Inc., Miamisburg, OH) for
normal oxygen (which contains only 0.2% 18O2) in a silent
arc ozone generator (Orec Inc., Phoenix, AZ). The 18O2
was diluted to 3% in argon to allow better control of flow
rates. Although the conversion of 18O2 to 18O3 was not
complete and produced some excess 18O2 in the exposure
chamber (about 70 ppm over a background of 420 ppm),
this excess is not sufficient to produce a detectable increase
in tissue 18O (19). The concentration of 18O3 was monitored
continuously. Chamber concentrations deviated by less
than 6% from the target levels over the course of the exposure.
Nineteen colony-reared, young adult male rhesus monkeys
(Macaca mulatta) (age range: 3 yr and 2 mo to 3 yr and 10
mo; body weight: 4.4 to 7.7 kg) were used in this study. On
the basis of physical and radiographic examinations and
complete blood count (CBC) analysis, all monkeys were
found to be free of clinical respiratory disease. The monkeys were housed outdoors until 2 wk prior to their exposure to ozone. Food and water were provided ad libitum.
Following exposure, the animals were deeply anesthetized
and euthanized by exsanguination, and the trachea, lungs,
and mediastinal contents were removed en bloc by thoracotomy. The trachea was divided into three pieces; the right
distal third was frozen immediately in liquid nitrogen, and
the remainder was split into two portions, one of which
was fixed for histopathology and the other prepared for
glutathione measurements. The lungs were separated and
the left primary bronchus was cannulated. The left cranial
and caudal lobes were used for bronchial pulmonary lavage. The right lung was divided by lobe. The bronchus to
the right cranial lobe was cannulated and fixed by airway
inflation with glutaraldehyde/paraformaldehyde (pH 7.4,
Quantitative Histopathology
The fixed right cranial lobe was used for histopathologic
characterization. The tissue was selected by microdissection
of the airway tree down the axial pathway of the major
daughter branch as described previously (5, 17, 20). One tissue sample per airway level was obtained from the first generation of intrapulmonary bronchus, the sixth or seventh
generation of intrapulmonary bronchus, and the proximal
two generations of respiratory bronchioles in the same path.
Samples of the airway were cut perpendicular to the long
axis, and included at least 50% of the airway circumference
plus a large portion of adjacent parenchyma. Of the respiratory bronchioles, one-half was embedded in a longitudinal
section and the other half cut perpendicular to the long axis
in the first-generation respiratory bronchiole. Both tissue
pieces included large amounts of parenchyma. Samples
were also taken perpendicular to the long axis from each
trachea. All samples were postfixed in 1% osmium tetroxide, dehydrated in a series of ethanol concentrations, and
embedded in Araldite. One-micron-thick sections were cut
Materials and Methods
Plopper, Hatch, Wong, et al.: Tracheobronchial Ozone Dose and Epithelial Injury in Monkeys
from each airway such that the epithelium was sectioned
perpendicular to the basal lamina, and were then stained
with toluidine blue. The thickness and relative abundance
of conducting airway epithelial cells were evaluated only on
the cartilaginous side of each airway, through procedures
discussed in detail elsewhere (20, 21). All measurements
were made under high-resolution light microscopy ( 340
and 363 objective and 0.5- to 1.0-mm sections). Measurements were made from video images captured by a DAGE
MTI video camera (Michigan City, IN) mounted on an
Olympus BH-2 microscope (Olympus Corp., Tokyo, Japan), which was interfaced with a Macintosh IIci computer
(Apple Computer, Cupertino, CA) running NIH Image
software. The analysis was performed with a cycloid grid
overlay and software for counting points and intercepts
(Stereology Toolbox, Davis, CA). Volume densities (Vv)
were determined for the following airway epithelial cells:
ciliated, mucous globlet, basal, and necrotic cells, on the basis of criteria previously described (5). Epithelial cells in respiratory bronchioles were counted as either necrotic or viable. Inflammatory cells were counted if they were in direct
contact with epithelial cells either in the epithelial intercellular space or on the apical surface. The volume densities
for each category of cell were determined through point
counting and calculated with the formula: Vv 5 Pp 5 Pn/Pt,
where Pp is the point fraction of P n, the number of test
points hitting the structure of interest, divided by Pt, the total points hitting the reference space (epithelium). The surface area of epithelial basement membrane per reference
volume (Sv) was determined by point and intercept counting, and was calculated with the formula: Sv 5 2 Io/Lr, where
Io is the number of intercepts with the object (epithelial
basal lamina) and Lr is the length of test line in the reference volume (epithelium). To determine thickness of the
epithelium, a volume per unit area of basal lamina (mm3/
mm2) was then calculated, using the formula for arithmetic
mean thickness (t): t 5 Vv/Sv. For each bronchus and the
trachea, four fields were evaluated. Fields were selected at
random from areas of the sections that included transverse
profiles of all epithelial components. In the centriacinar region, fields were selected on the basis of their position in the
proximal respiratory bronchiole.
Glutathione Determination
Procedures for obtaining defined specimens of the tracheobronchial airways by blunt dissection have been described
in detail for rats and monkeys in a previous publication
(18). All monkeys were killed between 10:30 A.M. and 2:30
P.M. A small sample of liver tissue was removed to ensure
that there were no large variations in reduced glutathione
(GSH) concentrations as a result of variations in the time
of death. No statistically significant differences were noted
in hepatic glutathione levels in these animals. The right
middle lobe was inflated with Waymouth’s medium and
stored in the same medium for 30 min at 48C. The intrapulmonary airway tree was removed, beginning at the hilum,
by blunt section under a dissecting microscope. The proximal three generations of intrapulmonary bronchi (termed
major daughter bronchi), the small-diameter distal bronchi (six to 10 generations, termed minor daughter bronchi), and respiratory bronchioles were removed. Following
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microdissection, portions of the parenchymal tissue that
were free of all identifiable blood vessels and airways were
also used. GSH was measured after derivatization with
monobromobimane through high-pressure liquid chromatography (HPLC) with fluorescence detection (22). All
samples were homogenized in 50 mM ice-cold phosphate
buffer (pH 7.4) in glass homogenizers. An aliquot (75 ml)
of each sample was transferred immediately to 25 ml of
ice-cold 800 mM methanesulfonic acid, 20 mM diethylenetriamine pentaacetic acid (DTPA), and 100 ml of 4 M sodium methanesulfonic acid. Samples were stored at 2808C
for up to 1 wk. Defrosted samples were centrifuged at
9,000 3 g for 10 min, and the supernates were derivatized
with monobromobimane at pH 8.0. The monobromobimane derivative was injected into a C18 reverse-phase column (Novapak-0.5 3 10 cm; Waters Associates, Milford,
MA) with a mobile phase of 13% methanol in 0.25% acetic acid (pH 3.5) at a flow rate of 1 ml/min. Glutathione
was monitored with a fluorescence detector, using excitation and emission wavelengths of 360 and 460 nm, respectively. After glutathione was eluted from the column, the
column was washed with 100% methanol (solvent B). Glutathione standards were run with each sample set, and the
response was linear over the range of sample values. Glutathione standards run with each set of samples were linear
from 20 pmol to 2 nmol. Precipitated protein was dissolved
in 1 N NaOH and the amount of protein was determined
by the Lowry method (23), with bovine serum albumin
(BSA) as a standard.
Analysis of Dried Tissues for 18O Content
The frozen right and left caudal lobes, which had been lyophilized to dryness, were desiccated and stored at 48C until
samples were microdissected. Airways samples for analysis were selected by microdissection. Using scalpels and razor blades, we exposed the axial path of the intrapulmonary airway tree in each lobe down to the respiratory
bronchiole. Three levels of intrapulmonary conducting airway were selected: one to three generations (proximal intrapulmonary bronchus), six to 10 generations (distal intrapulmonary bronchus), and 14 to 18 generations (proximal
respiratory bronchiole). The airways were trimmed free
from the lung tissue and divided into small samples, whose
surface area was measured with a micrometer on a Wild M8
Dissecting Microscope (Heerbrugg, Switzerland). The epithelium and submucosa were trimmed free of surrounding
tissue and placed in silver-foil cups, and their dry weight
was determined. The ratio of weight to surface area (mg/
mm2) to be used to convert to surface area for each airway
selected was as follows: trachea, 0.133 (1.0 and 0.4 ppm);
proximal bronchus, 0.162 (0.4 ppm), 0.143 (1.0 ppm); distal
bronchus, 0.084 (0.4 ppm), 0.142 (1.0 ppm) (24). The average weight for respiratory bronchioles was 0.8 6 0.73 mg,
and that for parenchyma free of all airway and blood vascular tissue was 0.99 6 0.50 mg. Assays of lyophilized tissues for 18O measurement were done with a modification of
a method of Santrock and Hayes (25), which uses an elemental analyzer to convert oxygen in dry tissues to carbon
monoxide (the oxygen percentage by dry weight was obtained from this CO content), a column filled with I2O5 to
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convert CO to CO2, and an isotope-ratio mass spectrometer for analysis of the resulting CO2 (24). This procedure
was modified by use of an online injection system between
the elemental analyzer and the mass spectrometer. The
CO2 mass ratios (46/44 D) were initially expressed as a
delta value with respect to a known CO 2 standard. This
delta value was corrected for time-dependent drift within
a sample run. Statistical analyses relating exposed and unexposed tissues were done on these drift-corrected samples. The delta values were then converted to 18O/16O ratios by use of a standard curve generated with standards
included in each sample run. To determine the excess 18O
in the exposed tissue, the mean 18O/16O ratios of unexposed tissues were subtracted from those for the 18O3exposed tissue. The unexposed (background) tissues were
included in each mass-spectrometer run with tissues from
the same site in exposed animals. Data was expressed as
micrograms of 18O per gram of dry weight.
Figure 1. Light microscopic comparison of epithelium lining airway compartments of the tracheobronchial airway tree of rhesus
monkeys exposed to 1.0 ppm
ozone (B, D, F, and H) or filtered
air (A, C, E, and G) for 2 h. (A
and B) Trachea. (C and D) Proximal bronchus (intrapulmonary generation 1 or 2). (E and F) Distal
bronchus (intrapulmonary generations 6 or 7). (G and H) Proximal
respiratory bronchiole. Animals
exposed to 1.0 ppm ozone for 2 h.
Bar for all micrographs equals 50
mm. Araldite sections. Toluidine
blue. Arrow 5 necrotic ciliated
cell; arrowhead 5 mucous goblet
cell.
Analysis of BALF
The left lung was lavaged (50 ml per lavage) with phosphate-buffered saline (PBS) via the bronchial cannula, and
the lavage fluid was evaluated for constituents previously
used to assess either the degree of acute cellular injury (total protein, fibronectin, lactate dehydrogenase) or inflammation (interleukin-8 [IL-8], eicosanoids, differential cell
count) in BALF from humans exposed to ozone (2, 3). Lavage samples were kept on ice during the procedure. Immediately after lavage, samples were centrifuged at 1,100
rpm for 10 min at 48C. To avoid unnecessary dilution of
molecules to be analyzed, supernatants from only the first
and second aliquots recovered from the lavaged lobes
were pooled for immediate analysis or frozen at 2708C.
The total protein was determined with a Bio-Rad Protein
Assay Kit (Bio-Rad, Richmond, CA), using BSA as a standard and following the manufacturer’s instructions. Fibro-
Plopper, Hatch, Wong, et al.: Tracheobronchial Ozone Dose and Epithelial Injury in Monkeys
nectin was quantified with a competitive enzyme-linked immunosorbent assay (ELISA) as described previously (3).
The antibody used in this kit was purified from a rabbit antihuman fibronectin serum and shown to have a single band
by rocket electrophoresis. The elastase activity in lavage
fluid was measured with the chromogenic substrate methoxy-Suc-Ala-Ala-Pro-Val pNa (M-4765; Sigma Chemical
391
Co., St. Louis, MO), as previously described (3). Lactate
dehydrogenase (LDH) activity was measured with a kit
purchased from Sigma. The kit was modified to allow 0.75
ml BALF to be assayed by using a more concentrated substrate solution. IL-8 concentrations in lavage fluid were
quantified with an ELISA kit purchased from R&D Systems, Inc. (Minneapolis, MN).
Figure 2. Morphometric comparison of the epithelial composition of four airway generations in the tracheobronchial airways of rhesus
monkeys exposed to ozone for 2 h. Trachea, proximal bronchus, distal bronchus, and respiratory bronchiole. The mass or volume per
unit surface area is compared for four cell populations (ciliated cells, mucous goblet cells, basal cells, and necrotic/degenerating cells)
from animals exposed to filtered air (FA), 0.4 ppm ozone, or 1.0 ppm ozone. *P , 0.05 compared with FA; **P , 0.05 compared with
FA and 0.4 ppm ozone.
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Eicosanoids present in lavage fluid were quantified by
radioimmunoassay (RIA) of eluates from C18 Sep Pak cartridges (Waters Associates) and processed by previously
established methods (3). Leukotriene CDE4 was assayed
with a kit purchased from New England Nuclear (Boston,
MA), using 3H-leukotriene C4 (3H-LTC4) as a tracer, as
previously described (3). LTB4, thromboxane B2 (TXB2),
and prostaglandin E2 (PGE2) were assayed by RIA as previously described (3). Cells from all recovered lavage aliquots were pooled and washed twice in RPMI 1640 supplemented with 0.025 gentamicin. Cells were counted and
viability determined by trypan blue exclusion; viability exceeded 85% in all samples, and there was no difference between samples from monkeys exposed to air or ozone. Cell
differential counts were done on cytocentrifuged slides
prepared at 700 rpm for 5 min and stained with a modified
Wright stain (Leukostat Solution; Fisher Scientific, Fair
Lawn, NJ). At least 300 cells per slide were counted and
evaluated.
Statistical Methods
Results of morphometric measurements, glutathione determinations, and measurements of 18O content are presented as means 6 1 SD; data from BAL are presented as
means 6 1 SE. Comparisons of exposure groups were
done with analysis of variance (ANOVA). Post hoc tests
were done with Dunnett’s method to test for significant
differences between control and exposed groups. Tests for
concentration-dependent differences and relationships between 18O content and cell death were done by regression
analysis (26). A value of P , 0.05 was considered statistically significant.
Results
Quantitative Histopathology
Figure 1 compares the histologic appearance of epithelium
lining the four sites in the tracheobronchial airways from
animals exposed to filtered air and those exposed to ozone
(1 ppm). The epithelium lining the proximal conducting
airways (trachea, bronchi) of animals exposed to filtered
air was pseudostratified, with three readily apparent cell
types: (1) ciliated cells with apical cilia and a light-staining
cytoplasm that tapered toward the basal lamina; (2) goblet
cells with abundant apical, lucent secretory granules and a
basal nucleus; and (3) basal cells with a low cuboidal to triangular shape, dense cytoplasm, and an extensive basal attachment to the basal lamina. In the three airway levels we
examined, the epithelium of more distal airways varied
from that of the trachea by decreases in overall height (or
thickness), in abundance of all cell types, and in the proportion of basal cells. Following 2 h of ozone exposure, the
epithelium in airways of all three levels differed from that
of controls by the presence of large numbers of ciliated
Figure 3. Morphometric comparison of the thickness of the luminal ciliary layer in three airway generations in the tracheobronchial airway tree of rhesus monkeys exposed to ozone for 2 h.
Trachea, proximal bronchus, and distal bronchus. Rhesus monkeys exposed to filtered air (FA), 0.4 ppm ozone, or 1.0 ppm
ozone. *P , 0.05 compared with FA.
Plopper, Hatch, Wong, et al.: Tracheobronchial Ozone Dose and Epithelial Injury in Monkeys
cells with pyknotic nuclei and dense compressed or vacuolated cytoplasm, and little evidence of cilia loss. In exposed animals, the remainder of the epithelium was disorganized, with focal areas of cell loss and reduced granule
abundance in goblet cells. In the trachea, little difference
was detectable between animals exposed to filtered air
and to 0.4 ppm ozone, but marked differences were
present after 1.0 ppm ozone exposure. In the bronchi, no
difference was evident in the degree of injury between animals exposed to ozone at 0.4 and 1.0 ppm. On the alveolarized side of the respiratory bronchiole, both the alveolar
393
epithelium and simple cuboidal epithelium lining the
bronchiolar wall were injured by ozone exposure at both
concentrations. Alveolar type I cells and cuboidal bronchiolar cells were disrupted or compressed, and were associated with large areas of epithelial denudation and luminal
inflammatory exudates. Figures 2, 3, and 4 summarize the
differences quantitatively.
Trachea. The epithelium of the trachea in control animals (Figure 1A) contained a mixture of basal cells, mucous goblet cells, and ciliated cells. Most of the goblet cells
appeared to be filled with secretory product. The ciliated
Figure 4. Morphometric comparison of inflammatory-cell abundance in four airway generations in the tracheobronchial airways of
rhesus monkeys exposed to ozone for 2 h. Trachea, proximal bronchus, distal bronchus, and respiratory bronchiole. Rhesus monkeys
were exposed to filtered air (FA), 0.4 ppm ozone, or 1.0 ppm ozone. Both neutrophils and eosinophils were counted as the same population in the epithelial mass. In the respiratory bronchiole, neutrophils and eosinophils were distinguished from alveolar macrophages.
*P , 0.05 compared with FA.
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cells had a relatively regular apical surface, which was
lined by distinct cilia. In animals exposed to 1.0 ppm ozone
(Figure 1B) the tracheal epithelium differed from that of
controls in a number of characteristics. There were large
numbers of densely staining necrotic or degenerating ciliated cells. The mucous goblet cells generally had less
secretory product, which appeared highly variable and irregular in amount. There was a rearrangement in the
basal-cell population associated with the basal lamina. Inflammatory cells (neutrophils and eosinophils) were also
present in the epithelium. The cartilaginous portion of the
trachea differed little from the noncartilaginous side. Ciliated cell volume dropped from an average of 22 mm3/mm2
of basal lamina in control animals to 10.8 mm3/mm2 following 1.0 ppm ozone exposure (Figure 2). There were no obvious necrotic cells in control animals, but necrotic cell
mass averaged 8.2 mm3/mm2 after 2 h at 1.0 ppm ozone exposure. Goblet cell mass also decreased in exposed animals, but this change was not statistically significant.
There was an increase in the mass of basal cells in animals
exposed to 1.0 ppm ozone, but this increase also was not
statistically significant. There were no significant changes
in the masses of ciliated, mucous globlet, basal, or necrotic
cells in animals exposed to 0.4 ppm ozone for 2 h. The
thickness of the ciliary layer was not significantly altered
by ozone exposure (Figure 3), and we did not detect a significant increase in the abundance of intraepithelial neutrophils or eosinophils (Figure 4).
Intrapulmonary bronchi. As compared with controls exposed to filtered air, the epithelium of the proximal (one
to two generations intrapulmonary) bronchi and the distal
(six to seven generations intrapulmonary) bronchi of animals exposed to ozone showed similar disarrangements. In
animals exposed to ozone, there was a smaller mass of ciliated, mucous globlet, and basal cells than observed in the
tracheas of control animals (Figure 2). Exposure to ozone
Figure 5. Comparison of reduced
glutathione (GSH) concentration
in five compartments of the tracheobronchial airway tree in
rhesus monkeys exposed to ozone
for 2 h. Distal trachea, proximal
bronchus, distal bronchus, respiratory bronchiole, and lung parenchyma free of all conducting
airway and blood-vascular tissue.
Animals were exposed to filtered
air (FA), 0.4 ppm ozone, or 1.0
ppm ozone. *P , 0.05 compared
with FA.
at either concentration caused marked rearrangement of
the epithelial cellular population (Figure 1). There was a
significant reduction in the abundance of ciliated cells following ozone exposure (Figure 2), but little change in the
mass of mucous goblet cells or basal cells (Figure 2). The
increase in necrotic cells produced by ozone exposure did
not show a dose response in these two airway generations;
similar increases were observed at both 0.4 ppm and 1.0
ppm ozone. The thickness of the ciliary layer was significantly reduced in the proximal bronchus in animals exposed to 1.0 ppm ozone (Figure 3). There was a trend toward an increase in intraepithelial inflammatory cells
(eosinophils and neutrophils) in the proximal bronchus,
but little change with exposure in the distal bronchus (Figure 4).
Respiratory bronchiole. In the proximal respiratory
bronchiole, there were marked changes in the epithelial
cellular population and in the quantity of inflammatory
cells in animals exposed to both 0.4 and 1.0 ppm ozone. In
control animals there was an admixture of simple cuboidal
epithelial cells and alveolar epithelium throughout the
proximal respiratory bronchiole (Figure 1C). Exposure to
ozone reduced by 50% or more the volume per surface
area of intact epithelial cells in the respiratory bronchiole
(Figure 2). There was also a significant increase in the
mass of necrotic epithelial cells found on the respiratory
bronchiolar surface (Figure 2). A highly significant increase in the mass of inflammatory cells was observed
both in the alveolar spaces and along the bronchiolar epithelial surface in animals exposed to 0.4 ppm and 1.0 ppm
ozone (Figure 4).
Glutathione Content
In control animals, glutathione content in tracheobronchial airways ranged from a low of 5.0 nmol/mg protein
(6 2.0) in the proximal intrapulmonary bronchus to a high
Plopper, Hatch, Wong, et al.: Tracheobronchial Ozone Dose and Epithelial Injury in Monkeys
of 10.9 nmol/mg protein (6 3.0) in the respiratory bronchiole (Figure 5). Parenchyma was higher than in any other
compartment of the lung (12.1 6 4.3 nmol/mg). The GSH
content in control animals for other sites was as follows:
trachea (7.1 6 1.6 nmol/mg protein); distal intrapulmonary
bronchus (9.1 6 2.7 nmol/mg protein); and distal bronchus (10.2 6 1.8 nmol/mg protein). Glutathione content in
most of the sites evaluated did not change significantly
with ozone exposure. GSH content of the proximal bronchus increased by twofold (10.4 nmol/mg) in monkeys exposed to 0.4 ppm ozone, but not in animals exposed to 1.0
ppm. In animals exposed to 1.0 ppm ozone the glutathione
content of the respiratory bronchiole decreased (5.7 nmol/
mg protein) significantly from control values (10.2 nmol/
mg). There was a decrease in the GSH content in the distal
bronchus at 1.0 ppm ozone, but this change was not significant.
18
Excess O in Lung Subcompartments
Figure 6 summarizes the differences in 18O content in portions of the tracheobronchial airways from animals exposed to 0.4 and 1.0 ppm ozone as compared with controls
exposed to filtered air. In animals exposed to 1.0 ppm
ozone, excess 18O ranged from a low of 7.8 mg/g dry weight
in the parenchyma to a high of 32.2 mg/g dry weight in respiratory bronchioles. There was less 18O in the trachea,
proximal bronchus, and distal bronchus than was observed
in the respiratory bronchiole. Levels of 18O in the distal
bronchus were higher than in the proximal bronchus or
trachea. No significant excess 18O could be detected in the
tracheas of animals exposed to 0.4 ppm ozone. In other
portions of the respiratory system, excess 18O was approximately 50% that observed in animals exposed to 1.0 ppm
ozone. The lowest detectable concentration of 18O in animals exposed to 0.4 ppm ozone was in the parenchyma
(0.8 mg/g dry weight), and the highest was in the respira-
395
tory bronchiole (13.6 mg/g dry weight). When the mass of
necrotic cells identified at a specific airway level was analyzed by regression against the 18O content at that airway
level, there was a significant correlation in the trachea, distal bronchi, and respiratory bronchioles, but not in the
proximal bronchi (Figure 7). When there was a greater
abundance of necrotic cells, there was a higher concentration of 18O. When the same type of analysis was used to
compare mass of necrotic cells with GSH content by airway level, there was a significant correlation only in respiratory bronchioles (R2 5 0.792, P , 0.001), with fewer necrotic cells closely associated with higher GSH levels.
When GSH concentration was compared with 18O content
by airway level, there were no significant correlations at
any airway level.
Cellular Content of BALF
Table 1 summarizes the cellular content of BALF from
ozone-exposed and control monkeys. There was a decrease in total cells recovered with increasing ozone concentration, but this was not statistically significant. The
presence of recovered cells identified as macrophages decreased significantly below control levels in animals exposed to 1.0 ppm ozone. In animals exposed to 1.0 ppm
ozone there was a significant increase in the percentage of
recovered cells identified as either neutrophils or eosinophils. There was a decrease in the percentage of recovered
cells identified as lymphocytes in animals exposed to 1.0
ppm ozone, but the difference from the control value was
not statistically significant. There was no change in the
percentage of recovered cells identified as monocytes.
Soluble Mediator Content of BALF
Table 2 summarizes the protein content of BALF from
lungs of rhesus monkeys exposed to ozone. The total pro-
Figure 6. Comparison of excess
18
O in the five compartments of
the tracheobronchial airway tree
of rhesus monkeys exposed to
ozone for 2 h. Trachea, proximal
bronchus, distal bronchus, respiratory bronchiole, and parenchyma free of all conducting airways and large blood vessels.
Monkeys were exposed to ozone
generated from 18O2. *P , 0.05
compared with other compartments at the same ozone concentration. **P , 0.05 compared with
different ozone concentrations in
same compartment.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 19 1998
tein in lavage fluid in animals exposed to 1.0 ppm ozone
was more than twice that in controls exposed to filtered
air, but there was no significant difference from animals
exposed to 0.4 ppm ozone. Compared with that from control animals exposed to filtered air, BALF from lungs of
monkeys exposed to 1.0 ppm ozone had more than a fourfold greater amount of detectable IL-8, twice the amount
of LDH, and more than a 10-fold greater amount of
elastase. There was an increase in the fibronectin content,
but this was not significantly different. There was no statistically significant change in the amount of any of these
proteins in animals exposed to 0.4 ppm ozone as compared
with controls exposed to filtered air. There was also no significant change in the concentrations of the following mediators in animals exposed to either 0.4 ppm or 1.0 ppm
ozone as compared with control animals exposed to filtered air: leukotriene DE or E/4 (LTCDE4), LTB4, TXB2
or PGE2 (Table 2).
Discussion
This study was designed to test the hypothesis that the
site-specific differences in acute epithelial injury associated with exposure to ozone are the result of higher local
doses of ozone at these sites, and that loss of the GSH pool
is a significant factor in areas with heightened susceptibil-
Figure 7. Regression analysis of
relationship between abundance
of necrotic cells and excess 18O
concentration in the tracheobronchial airway tree of rhesus monkeys exposed to ozone for 2 h.
Trachea (y 5 20.452 1 0.823x,
R2 5 0.523, P , 0.01), proximal
bronchus (y 5 0.891 1 0.168x,
R2 5 0.361, P 5 0.07), distal bronchus (y 5 0.6 1 0.086x, R2 5
0.484, P , 0.01), and respiratory
bronchioles (y 5 0.19 1 0.51x,
R2 5 0.948, P , 0.0001).
ity to injury. For the present study, we used an exposure of
short duration (2 h) and compared two concentrations of
ozone (0.4 ppm and 1.0 ppm), the lower of which matches
a regime often used in human clinical exposure studies.
We found significant cellular injury at all four of the sites
evaluated (trachea, proximal bronchi, distal bronchi, and
proximal respiratory bronchioles). The greatest cellular injury occurred in the proximal respiratory bronchiole and
was concentration dependent. The response in the more
proximal tracheobronchial airways varied by site, with the
trachea being affected only at the higher ozone concentration and injury in the proximal and distal bronchi being increased at both ozone concentrations. Inflammatory cells
in the epithelium or airway lumen were increased only in
the respiratory bronchiole. There was as much as a twofold variation in the concentration of glutathione at local
sites, but the only significant alterations produced by
ozone exposure were a reduction of GSH in the respiratory bronchiole at the high ozone concentration (1.0 ppm)
and an increase in GSH at 0.4 ppm in the proximal bronchus. The quantity of ozone reaction products at specific
sites varied by a factor of as much as three between the
least affected sites (trachea and proximal bronchus) and
the most affected site (respiratory bronchiole). The 18O
content at each site was dependent on the inhaled 18O3
concentration, with animals exposed to 1.0 ppm ozone
Plopper, Hatch, Wong, et al.: Tracheobronchial Ozone Dose and Epithelial Injury in Monkeys
TABLE 1
Cellular content of bronchoalveolar lavage fluid from
lungs of rhesus monkeys exposed to ozone
for 2 h (mean 6 1 SE)
Total cells, 3 10
% Macrophages
% Polymorphonuclear
leukocytes
% Eosinophils
% Lymphocytes
% Monocytes
6
Filtered Air
0.4 ppm O3
1.0 ppm O3
41.7 6 17.3
87.5 6 2.4
0.6 6 0.6
36.0 6 2.0
88.6 6 2.6
2.1 6 1.0
29.0 6 12.1
70.0 6 2.5*
10.3 6 3.4*
2.2 6 3.8
5.4 6 2.0
2.7 6 0.9
2.0 6 0.8
5.2 6 1.5
2.0 6 0.4
10.3 6 3.2*
2.8 6 0.8
2.8 6 0.8
* P , 0.05 compared with filtered air.
having at least twice the 18O content of animals exposed
to 0.4 ppm. Components of BALF were significantly altered from those of controls exposed to filtered air only after exposure to the highest ozone concentration. The alterations included a decrease in the percentage of macrophages
with an increase in the percentages of both neutrophils and
eosinophils, and a doubling of total lavage protein, IL-8,
LDH, and elastase. There were no alterations in arachidonic acid metabolites.
One purpose of this study was to establish whether inhalation of ozone at a concentration (0.4 ppm) and duration (2 h) within the range to which humans are exposed
under experimental conditions (1–4, 27) will produce epithelial injury within the tracheobronchial airways. Previous studies with rhesus monkeys (9) and rats (8) have
shown that at ozone concentrations as low as 0.8 ppm for
the monkey and 0.5 ppm for the rat, substantial ciliated
cell and type I cell injury and necrosis occur in the centriacinar region within 4 h (rhesus monkey) and 2 h (rat).
The present study shows that at even lower ozone concentrations (0.4 ppm) for very short periods (2 h), there is severe necrosis in respiratory bronchioles. Injury occurs in
the trachea with concentrations in this range after exposure for 3 d or longer in nonhuman primates (7, 28), sheep
(29, 30), and rats (6). In a previous study that mapped the
acute injury response after exposure to a dose of ozone
similar to the higher of the two doses used in this study
(0.96 ppm), we observed the presence of necrotic cells in
the trachea, in a conducting airway comparable to proxi-
397
mal bronchus in the present study, and in the respiratory
bronchiole after a longer exposure (8 h) (5). What the
present study also shows is that necrosis in the trachea and
proximal airways occurs much earlier than at 8 h of exposure. Comparison of the cellular density measurements in
the present study with those of the previous study (5) indicates that there is a substantial loss of epithelium in proximal airways between 2 and 8 h of exposure. The substantial necrosis (25% of the cells in the conducting airways)
after 2 h of exposure is significantly altered after 8 h of exposure, when necrotic cells represent 5% or less of the total cells in either the trachea or conducting airways (5). After 2 h of exposure, the density of the necrotic and ciliated
cells together is approximately the density of the ciliated
cells in control animals exposed to filtered air, suggesting
that most of the necrotic cells are ciliated and that little
exfoliation occurs within 2 h of exposure. There is also a
clear difference in susceptibility to injury within the conducting-airway tree. The trachea does not appear to be
susceptible to injury at 0.4 ppm ozone (2 h), whereas both
the proximal and the distal airways are equally susceptible
at that low dose. The degree of necrosis of epithelial cells
at the various airway levels suggests that the respiratory
bronchiole is the most sensitive airway at low doses and
high doses, even after very short exposures. If the pattern
of dose for rhesus monkeys resembles that for humans,
our study suggests that the protocols used for human exposure (2–4, 27) probably produce substantial cellular necrosis in this time frame.
A short duration of exposure to ozone produces a significant increase in hallmarks of inflammation in the conducting airways of rhesus monkeys. As the current study
illustrates, this response, when evaluated in terms of contents of BALF, is decidedly dose-dependent. In our previous study with rhesus monkeys, after 8 h of exposure to
0.96 ppm ozone, there was an almost threefold increase in
the neutrophil content of lavage fluid, a doubling in the
number of total cells recovered, and little change in the
percentage of macrophages or lymphocytes (5). The same
pattern of change has been observed in humans after the
initial 1 to 2 h of exposure (2–4). At the lower dose (0.4
ppm), we did not see changes comparable to those observed in humans, but in the human studies cited here, the
subjects were exercising and probably had a higher expo-
TABLE 2
Soluble proteins in bronchoalveolar lavage fluid from lungs of rhesus
monkeys exposed to ozone for 2 h (mean 6 1 SE)
Total protein, mg/ml
IL-8, pg/ml
Fibronectin, ng/ml
Lactate dehydrogenase, mmol/ml
Elastase, nm pNa/h
LTCDE4, pg/ml
LTB4, pg/ml
TXB2, pg/ml
PGE2, pg/ml
Filtered Air
0.4 ppm O3
1.0 ppm O3
361.0 6 97.7
8.3 6 3.2
41.6 6 23.3
20.5 6 4.7
0.43 6 0.08
639.8 6 323.0
43.5 6 22.7
26.6 6 4.2
11.1 6 2.1
474.3 6 305.7
7.0 6 1.3
10.8 6 3.9
12.2 6 2.0
0.72 6 0.3
162.4 6 21.7
67.7 6 36.4
67.7 6 44.1
21.1 6 10.0
736.6 6 235.9*
50.3 6 19.5*
77.4 6 31.9
57.7 6 11.8*
5.07 6 2.28*
515.4 6 195.5
29.5 6 5.7
39.0 6 12.2
16.7 6 5.9
Definition of abbreviations: IL-8 5 interleukin-8; LTB4 5 leukotriene B4; LTCDE4 5 leukotriene CDE4; TXB2 5 thromboxane B2; PGE2 5 prostaglandin E2.
* P , 0.05 compared with filtered air.
398
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 19 1998
sure than did the nonexercising monkeys used in our
study. In our previous study with rhesus monkeys, we
found that intraepithelial inflammatory cells were significantly increased at 12 h after the cessation of exposure but
not at 1 h after exposure in the trachea and bronchus,
whereas they were significantly increased in the respiratory bronchiole (5). The current study shows that this increase in inflammatory cells in the respiratory bronchiole
starts within the first 2 h of exposure, and that as a consequence, a majority of the cells observed in the BALF at
that time come primarily from respiratory bronchioles.
This confirms and extends our previous observations regarding the airway-specific pattern of the inflammatory response to acute ozone injury, in terms of the timing of migration of cells and also the degree of response. Moreover,
even concentrations of ozone as low as 0.4 ppm (2 h) are
still above the threshold necessary to induce inflammation
in the respiratory bronchioles.
Other components of the BALF that could have been
indicative of or associated with acute airway injury or inflammation, including total protein, elastase, and fibronectin, were consistently changed in association with the cellular necrosis observed in the present study. Human
exposure for the short times used in the study produces at
least a twofold increase in lavage protein content and at
least a fourfold increase in elastase content (3), which we
observed with the higher-concentration exposure in the
present study. We did not see significant changes in any of
these components or in LDH, a marker of cell injury, at
the lower dose of ozone. Eight hours of exposure to 0.96
ppm ozone produces significant increases in a number of
prostaglandins, including PGF2a, PGD2, and PGE2, but
not in TXB2 or PGF2a in rhesus monkeys (5). Our data indicate that the release of these substances occurs much
later than the initial 2-h response, and that their synthesis
and release are not part of the early phases of the injury
process. Certainly at the lower concentration (0.4 ppm) of
ozone used in our study this response requires a much
longer time frame than it does at the higher concentration.
This fits well with the findings in a previous study in humans lavaged at 18 h after exposure, in which PGE2 was
doubled in concentration after a similar level of exposure
to ozone (3). In rhesus monkeys, secretion of these compounds has returned to near steady-state levels by 12 h after an 8-h exposure, an equivalent time frame to that in
our study (5).
One of the purposes of the present study was to establish whether the airway-selective nature of the pathologic
response to acute ozone exposure may be related to the
concentration of ozone to which an individual is exposed
or to the local amount of ozone found reacting with the tissues. To estimate local dose, we used the concentration of
ozone reaction products following exposure to 18O3 (24).
When the amount of 18O present in dried tissues was measured throughout the conducting-airway tree, we found
significant variability in the amount by airway level. We do
not believe that movement of 18O3 within the airways contributes significantly to these differences, because the halflife of 18O3 label appears to be much longer (about 6 h in
the rat) than the exposure period (2 h), and the lungs of
our animals were rapidly frozen within a short time after
removal from the animals. In all of the airways evaluated,
the amount of 18O detected in monkeys exposed to the
higher concentration of ozone was at least double that observed in animals exposed to the lower concentration. The
pattern of distribution showed the highest concentration
of 18O in the centriacinar region, which is the respiratory
bronchiole in nonhuman primates, and the lowest concentration in the parenchyma. Modeling studies of local ozone
dose based on computer simulations suggest a similar pattern of local dose, with a progressive increase to the centriacinar region and a significant diminution in the parenchymal areas (31, 32). When the local concentration of tissue
18
O is compared with the degree of injury in a specific airway level, it appears that each airway may have its own
pattern of response, and that the threshold for the production of injury is airway-specific. For example, in the trachea and proximal bronchus, similar amounts of 18O were
detected; however, the proximal bronchus had greater injury, particularly at the lower exposure level. A similar
pattern occurred with the more distal of the intrapulmonary bronchi evaluated, but the thresholds for injury were
slightly higher. In the respiratory bronchiole, both inhaled
ozone concentrations produced an exposure-related increase in 18O concentrations to levels higher than observed
in the trachea, and the degree of injury appeared to be correlated with the tissue 18O level.
Because the mechanisms by which ozone injures cells
are still unclear, the variability in the response of GSH to
ozone exposure is difficult to explain. The explanation
may lie in the potential differences of cells in different airways in utilizing, replenishing, and balancing the intracellular glutathione pool in response to oxidant stress. Comparison of the activity of antioxidant enzymes with potential
for protecting against the reaction products of ozone exposure, such as glutathione S-transferases and glutathione peroxidase, indicate that at least for the rhesus monkey, there
is little difference in the activity levels of these two enzymes at the airway levels evaluated in the present study
(18). These two enzyme systems have substantially more
heterogeneity in activity by airway level in the rat (18), but
respond in that species to long-term exposure by increasing only in the distal airways equivalent to the respiratory
bronchioles in the current study (33). Another factor that
may account in part for the variability in site-specific thresholds for ozone-induced injury is the ability of cells lining
different portions of the airway tree to maintain and utilize their glutathione pools. Comparison of the ability of
the trachea, distal bronchi, respiratory bronchioles, and parenchyma to maintain intracellular glutathione in the absence of extracellular glutathione and sulphur-containing
amino acids suggests that maintenance and replenishment
of these pools following depletion varies considerably (34).
It appears that the parenchyma replenishes GSH at a more
rapid rate than does the trachea, which shows the slowest
replenishment, or the minor daughter/respiratory bronchiole zone which appears to be somewhat faster than the trachea but much slower than the parenchyma in replenishing glutathione. These differences in metabolic potential
for regulating intracellular glutathione pools could also account for the differences in the degree of depletion of glutathione observed at different airway levels at different in-
Plopper, Hatch, Wong, et al.: Tracheobronchial Ozone Dose and Epithelial Injury in Monkeys
haled concentrations of ozone. One conclusion to be drawn
from the present study is that injury at each airway level
varies significantly with the ability to regulate the GSH
pool, but that the steady-state pool may be only a partial
determinant of the threshold at which injury occurs.
In summary, we have shown that the local dose of
ozone to tissues, based on the binding to cellular constituents of 18O generated from 18O3, varies by site within the
airway tree, and that this variability is related to the inhaled concentration of ozone. There is a close correlation
between the amount of ozone bound and the degree of
cellular injury at a particular site, but this relationship varies depending on the site. The protocol for studies of human exposure produces significant epithelial necrosis in
very short time frames in distal conducting airways of nonhuman primates. This injury at its greatest extent is commensurate with detectable changes in inflammatory components in BALF at the higher inhaled concentration of
ozone used in the present study but not at the lower concentration. There is a significant difference in the threshold for injury at different airway levels, which could be explained at least in part by differences in the ability of
epithelium at specific sites to utilize glutathione in neutralizing reactive intermediates produced by ozone exposure
and in replenishing the glutathione pool as it is used.
Acknowledgments: The authors thank John McKee for assistance with inhalation exposures to 18O3, and Linda Harris for tissue analyses of 18O. This study
was supported in part by funds from the USEPA-supported Duke University
Extrapolation Modelling Center, and grants ES00628, ES04311 and DRR
00169 from the National Institutes of Health. The University of California at
Davis is a National Institute for Environmental and Health Sciences Center for
Environmental Health Sciences (ES05700).
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