Differential expression of stress proteins in nonhuman primate
lung and conducting airway after ozone exposure
Reen
Wu1,2,3,
Yu Hua
Zhao3,
Charles G.
Plopper2,3,
Mary Mann-Jong
Chang3,
Ken
Chmiel3,
John J.
Cross3,
Alison
Weir2,
Jerold A.
Last1,3, and
Brian
Tarkington3
1 Division of Pulmonary and
Critical Care Medicine;
2 Department of Veterinary
Anatomy, Physiology, and Cell Biology; and
3 Center for Comparative
Respiratory Biology and Medicine, University of California, Davis,
California 95616
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ABSTRACT |
The presence of seven stress proteins including
various heat shock proteins [27-kDa (HSP27), 60-kDa (HSP60),
70-kDa (HSP70) and its constitutive form HSC70, and 90-kDa (HSP90)
HSPs] and two glucose-regulated proteins [75-kDa (GRP75)
and 78-kDa (GRP78) GRPs] in ozone-exposed lungs of nonhuman
primates and in cultured tracheobronchial epithelial cells was examined
immunohistochemically by various monoclonal antibodies. Heat treatment
(42°C) resulted in increased HSP70, HSP60, and HSP27
and slightly increased HSC70 and GRP75 but no increase in GRP78 in
primary cultures of monkey tracheobronchial epithelial cells. Ozone
exposure did not elevate the expression of these HSPs and GRPs. All of
these HSPs including HSP90, which was undetectable in vitro, were
suppressed in vivo in monkey respiratory epithelial cells after ozone
exposure. Both GRP75 and GRP78 were very low in control cells, and
ozone exposure in vivo significantly elevated these proteins. These
results suggest that the stress mechanism exerted on pulmonary
epithelial cells by ozone is quite different from that induced by heat.
Furthermore, differences between in vitro and in vivo with regard to
activation of HSPs and GRPs suggest a secondary mechanism in vivo,
perhaps related to inflammatory response after ozone exposure.
oxidant injury; immunohistochemistry; air pollutants
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INTRODUCTION |
OZONE IS AN EXTREMELY REACTIVE oxidant gas. Its
toxicity is complex because of the large number of biological systems
that can be affected and the variable interactions between ozone and various cellular components (5, 8, 27). Lungs and conducting airways
are primary sites of ozone toxicity. The primary sites of damage
include the trachea, distal bronchioles, centriacinar region, and
alveolar ducts (6, 12, 30). The extent of the damage in various
anatomic sites appears to be more extensive in the trachea and distal
bronchioles than in the bronchi; however, increasing the concentration
of ozone exposure not only results in more severe lesions but also
extends the lesions into the lung (1, 9, 31).
Despite these well-illustrated in vivo lesions, the biochemical
toxicity of ozone and its stress effects on pulmonary cells, especially
on target airway epithelial cells, remain unresolved. Since the
discovery of the phenomenon by Ritossa (34), we have known that cells
from all organisms respond to a variety of stresses by the rapid
transcription and subsequent translation of a group of highly conserved
proteins known as heat shock proteins (HSPs) (20) and glucose-regulated
proteins (GRPs) (19), now collectively called stress proteins. Some of
these stress proteins appear to function protectively in cells in
response to different stress agents. For instance, GRPs are induced due
to the stress related to glucose starvation or defects in glycoprotein
processing (19, 21, 47). These GRPs are found largely at the cytosol of
the stressed cells (21, 26). In contrast, some HSPs are translocated into the nucleus in response to stress. For instance, heat-induced 70-kDa HSP (HSP70) is found in nucleoli immediately after heat treatment (29, 45). This translocation is apparently related to the
reassembly of damaged preribosomal ribonuclear proteins. Thus the
induction of different sets of stress proteins may be related to the
pathway of stress-induced response. In this study, we seek to elucidate
the association between the synthesis of stress proteins and the stress
that is induced by the exposure of airway epithelium to ozone both in
vivo and in vitro. The correlation of the synthesis of some of these
stress proteins with ozone toxicity and the recovery from injury in
target airway epithelial cells may provide a biochemical clue to the
nature of ozone toxicity in vivo.
Cohen et al. (4) have demonstrated an elevated synthesis of various
HSPs in isolated guinea pig airway epithelial cells and alveolar
macrophages induced by heat, sodium arsenite, and acidic gas but not by
ozone or hydrogen peroxide. This result is consistent with the recent
experiment by Sun et al. (39) in which the immortalized
human bronchial epithelial cell line BEAS-2B was exposed to various
concentrations of ozone in vitro. However, we have demonstrated an
induced synthesis of a 45-kDa protein by ozone, and this induction
appears to be dose dependent on ozone from 0.1 to 1.0 part/million
(ppm). In contrast, this induction is not correlated with heat-induced
HSP synthesis. Recently, both Wong et al. (50) and Su and Gordon (38),
using Western blot analysis, have observed an enhanced HSP70 inducible
form level in lung tissue extracts from rats and guinea pigs,
respectively, after ozone exposure. This increase was also observed in
the lavage fluid preparation from guinea pig lungs after ozone exposure
(38). However, neither study revealed the cell type(s) responsible for the increase in HSP70 in the preparation.
Because stress proteins can also be induced by means other than heat
treatment, such as metabolic starvation by glucose deprivation, it is
important to extend the stress protein synthesis study to proteins
other than via the heat-induced mechanism. In this study, we used
monospecific monoclonal antibodies (MAbs) of seven stress proteins,
including those of HSPs and GRPs, to examine the presence of these
stress proteins in monkey airway tissues and primary cultures of airway
epithelial cells after ozone exposure. We have observed a consistently
enhanced presence of GRPs but not of HSPs in airway epithelial cells
after ozone exposure in vivo. In contrast, ozone exposure in vitro had
no effect on the level of HSP and GRP expression despite the observed
enhancement of HSP expression by heat.
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MATERIALS AND METHODS |
Source of Monkeys, Tracheobronchial Tissues, and In Vitro Culture
Conditions
Specific pathogen-free monkeys provided by the California Regional
Primate Research Center at the University of California, Davis were
used for both in vivo and in vitro ozone exposure and in vitro
isolation of tracheobronchial epithelial (TBE) cells for primary
culture. All procedures were approved by the University Animal Protocol
Review Committee. These tissues were immersed in minimal essential
medium and immediately delivered on ice to the laboratory. TBE cell
isolation and culture conditions were carried out as previously
described (23, 35). The isolated TBE cells were plated on a collagen
gel substratum in Millicell inserts at a density of 1 × 104
cells/cm2 as previously described
(39, 41, 49). After 4 days of incubation in the vitamin A (retinol; 1 µM)-supplemented, serum-free hormone-supplemented medium, the primary
TBE cells were maintained biphasically in a well-humidified environment
as previously described (39, 41). Four days later, a confluent culture
was obtained and used subsequently for ozone exposure and heat
(42°C) treatment.
In Vivo Ozone Exposure
Three healthy rhesus monkeys were exposed to chemically and
biologically filtered air for 90 days, while three monkeys were exposed
to 89 days of filtered air plus 1 day of 0.98 ppm ozone (8 h/day) and
an additional three monkeys were exposed to 0.98 ppm ozone 8 h/day for
90 days. The animal housing, exposure, ozone generation, and monitoring
were conducted according to standard procedures at the California
Regional Primate Research Center Inhalation Exposure Facility (17). The
animals were maintained with a 10:14-h light-dark cycle. At the end of
the exposure, the animals were deeply anesthetized and their lungs were
removed and fixed for paraffin block preparation as previously
described (5, 31).
In Vitro Exposure and Heat Treatment
TBE cells cultured between the air phase and liquid medium, with no
medium on the apical phase of epithelial cells, were exposed to either
filtered air or ozone of various concentrations (0.1-1.0 ppm) as
previously described (39-41). Ozone monitoring during exposure was
carried out as previously described (40) with a
computer-based data-acquisition system connected to the ozone analyzer.
For heat treatment, the medium in the TBE cultures was replaced by
freshly prewarmed (42°C) medium, and cultures that were under a
medium-immersed condition were maintained at 42°C for several hours.
Determination of Injury on Cultured Cells by Ozone and Heat
Two colorimetric methods based on the metabolic activity of
mitochondria were used in this study to assess cell injury after ozone
exposure or heat (42°C) treatment. One method, developed by Mosmann
(25), was based on the cleavage of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
which occurs in all living, metabolically active cells but not in cells
that are not viable. The other method is based on the same principle
with a water-soluble dye, Alamar blue, according to the manufacturer's
suggested protocol (Alamar, Sacramento, CA) (39). The amount of color
change in these two methods has been demonstrated to be directly
proportional to the cell number, which was determined by a
hemocytometer or an electronic Coulter counter. The viability assays
based on these two methods are not only simpler but are also compatible
with the traditional trypan blue dye exclusion method (41).
Sources of Antibodies and Immunoanalysis Methods
MAbs specific to seven stress proteins were obtained commercially.
Clone G3.1, obtained from StressGen Biotechnologies, is of
IgG1 class and specific to human 27-kDa HSP (HSP27) (3, 10). Clone
4B9/89, a product of Affinity BioReagents (Neshanic Station, NJ),
is of IgG2a class and specific to human mitochondrial 60-kDa HSP
(HSP60) (37). Clone C92F3A-5, an IgG1 class compound obtained from
StressGen, is specific for the inducible form of human HSP70 (43, 48).
Clone 1B5, obtained from StressGen, is of IgG1a class and specific for
the constitutive form of HSP70, referred to as HSC70 (13, 18, 28).
Clone 9D2, obtained from StressGen, is of IgG2a class and specific to
human 90-kDa HSP (HSP90) (16). Clone 30A5, obtained from StressGen, is
specific for 75-kDa GRP (GRP75) (22). Clone 10C3, obtained from
StressGen, is specific for 78-kDa GRP (GRP78; BiP) and is of IgG2a
class (44). These antibodies were used according to the manufacturers' suggested protocols. The antibodies were also used in both Western blot
analysis and immunocytochemistry according to previous publications (14, 42). A Vectastain Elite ABC kit (Vector Laboratories, Burlingame,
CA) was used for both immunohistochemical staining and Western blot
analysis. In the Western blot analysis, an equal amount of protein (20 µg/lane) was loaded on a SDS-polyacrylamide gel for electrophoresis.
To further verify the uniformity of protein loading, a parallel gel was
stained with Commassie brilliant blue for a visual inspection. Protein
concentration was determined by Bradford's (2) method (Bio-Rad,
Hercules, CA). The control slides were stained in the absence of the
primary antibody or with other unrelated MAbs.
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RESULTS |
Specificity of Antibodies and Induction of Stress Protein Synthesis In
Vitro
Initially, we had to determine the specificity of these MAbs to stress
proteins of monkey TBE cells because these antibodies were raised
against human proteins. Using heat treatment, elevation of some of
these stress proteins could be characterized. As shown by Western blot
analysis (Figs. 1 and
2), six of seven antibodies used in this
study [all except anti-HSP90 (clone 9D2)] reacted in only
one protein band of monkey cell extract compared with the control
cultures (Figs. 1E and
2B) in which the primary antibody was replaced by control mouse serum. The corresponding molecular masses of these single-protein bands were consistent with
the human ones, suggesting the monospecific nature of these antibodies against the monkey stress proteins. Clone 9D2, an HSP90-specific MAb,
did not stain any band over the bands found in control cultures (data
not shown).

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Fig. 1.
Western blot analysis of heat stress proteins (HSPs) in primary
cultures of monkey tracheobronchial epithelial (TBE) cells during heat
treatment. TBE cells were cultured in serum-free hormone-supplemented
medium as described in text. After 8 days of incubation at 37°C,
temperature was raised to 42°C for various lengths of time. Cells
were lysed by SDS lysis buffer, and proteins were separated on
SDS-polyacrylamide gel by electrophoresis. Protein gel was then
electrophoretically blotted to a nitrocellulose membrane (38).
Antibodies to 70-kDa HSP (HSP70; clone C92F3A-5;
A), constitutive form of HSP70
(HSC70; clone 1B5; B), 60-kDa HSP
(HSP60; clone 4B9/89; C), and 27-kDa
HSP (HSP27; clone G3.1; D) and
control mouse serum (E) were used to
stain the membrane as previously described (42). S, standard
molecular-mass proteins (nos. on
left); 1, untreated cultures at
37°C; 2, cultures treated at 42°C for 4 h; 3, cultures treated
at 42°C for 6 h; 4, cultures treated at 42°C for 8 h.
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Fig. 2.
Western blot analysis of glucose-regulated proteins (GRPs) in primary
cultures of monkey TBE cells during heat treatment. Experiments were
carried out as described in Fig. 1. A:
75-kDa GRP (GRP75; clone 30A5). B:
control mouse serum. C: 78-kDa GRP
(GRP78; clone 10C3). Note: a different Vectastain ABC kit from that
used in A and
B was used in
C. S, standard molecular-mass proteins
(nos. on left); 1, untreated
cultures at 37°C; 2, cultures treated at 42°C for 4 h; 3, cultures treated at 42°C for 6 h; 4, cultures treated at 42°C
for 8 h.
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An elevation in the incubation temperature from 37 to 42°C
significantly enhanced the abundance of HSP70 (Fig.
1A), HSP27 (Fig.
1D), and, to a lesser extent, HSP60
(Fig. 1C) as demonstrated by Western
blot analysis. As a comparison, the constitutively expressed HSP70
form, HSC70, was only slightly elevated by the high-temperature
treatment (Fig. 1B). In contrast to
these HSPs, the response of GRPs to high-temperature treatment was
either slight or nil. For GRP75, there was a slight elevation in the cells after heat treatment (Fig.
2A), whereas there was no obvious increase for GRP78 (BiP; Fig. 2C).
In contrast to the heat treatment, exposed cultured cells under the
biphasic culture condition to ozone at 1 ppm for 90 min showed no
significant increase or a decrease in the abundance of HSP27 (data not
shown) or HSP70 (Fig.
3A).
These results are consistent with the previous study (39) in an
immortalized human bronchial epithelial cell line (BEAS-2B), which
demonstrated an elevated synthesis of HSPs as analyzed by a
two-dimensional SDS-PAGE system after heat but not after the ozone
exposure treatment, even though both treatments injured cells
substantially. A similar conclusion was obtained with GRPs. As shown in
Fig. 3B, there was no effect on the
GRP78 level in cultured cells after ozone exposure. This nonresponse
phenomenon was not related to the protein loading because the loading
was verified by Commassie blue staining (Fig.
3C). It is necessary to point out
that these in vitro ozone exposure experiments have been repeated more
than five times and on only one occasion have the levels of GRP75 and
GRP78 been elevated by ozone (data not shown). The reason for this
inconsistency in this particular experiment is not clear. Nevertheless,
most studies have consistently demonstrated no change in the levels of
stress proteins in monkey TBE cells in vitro by ozone.

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Fig. 3.
Western blot analysis of stress proteins in primary cultures of monkey
TBE cells after ozone exposure. Primary cells were cultured
biphasically as described in text. On day
10, cultures with no liquid medium on apical side were
exposed to filtered air (FA) or 1 part/million (ppm) ozone for 90 min.
At various times after incubation, protein extracts were prepared from
these cultures and subjected to Western blot analysis as described in
Fig. 1. A: anti-HSP70 (clone
C92F3A-4). B: anti-GRP78 (clone 10C3).
C: gel stained with Commassie blue to
demonstrate equal input of protein in each well of gel. S, standard
molecular-mass proteins (nos. on
left); 1, cultures before exposure;
2, cultures exposed to FA and 0-h postexposure incubation; 3, cultures
exposed to ozone and 0-h postexposure incubation; 4, cultures exposed
to FA and 24-h postexposure incubation; 5, cultures exposed to ozone
and 24-h postexposure incubation.
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Immunohistochemical Comparison of HSP Distribution In Vivo
Morphological lesions in monkey lungs after 0.98 ppm ozone exposure
have been reported (1, 6, 9, 12, 30). With the use of the same set of
tissue sources, except prepared in paraffin sections, an
immunohistochemical comparison of stress protein distribution was
carried out. For identifying stress protein-related antigens, MAbs
monospecific to stress proteins, as demonstrated in the Western blot
analysis, were used. The following analysis represents a summary from
tissue sections prepared from three monkey lungs. Only representative
pictures are presented.
Anti-HSP27 (clone G3.1). The labeling
was heaviest in the epithelium and smooth muscle of the proximal
bronchi and respiratory bronchioles in filtered-air control monkey
lungs (Fig. 4,
A and B, respectively). Labeling in the
respiratory bronchioles included both the cuboidal and squamous
epithelial populations. There was also focal labeling of epithelial and
interstitial elements in the interalveolar septa. After either
short-term (1-day) or long-term (90-day) exposure to ozone, there was a
marked reduction in the expression of this protein in all compartments
but primarily in the epithelium and interstitial compartments (Fig. 4,
C-F).
In the proximal bronchi, there was a restriction of the protein
expression to the most apical boundaries of ciliated cells and to some
of the low cuboidal cells (basal cells) adjacent to the basal lamina (data not shown). In the respiratory bronchioles, there was a reduction
in the epithelial cells that reacted positively with this antibody and
a reduction in the intensity in labeled cuboidal epithelium (Fig.
4D). This reduction was even more
marked in animals exposed for a long time to ozone, with protein
expression in the epithelium markedly reduced to very focal portions of
some ciliated cells of proximal bronchi and to a very small number of
cells in the respiratory bronchioles (Fig. 4,
E and
F). Binding to the smooth muscle of
both blood vessels and peribronchial areas did not appear to be altered
by ozone exposure.

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Fig. 4.
Immunohistochemical staining of monkey lung with anti-HSP27 (clone
G3.1). Lung tissues after FA (A and
B) and 0.98 ppm ozone exposure
(C-F)
were fixed, and paraffin sections were prepared. Immunostaining on
these paraffin-sectioned slides was carried out as previously described
(14). A: bronchus, FA.
B: distal bronchiolar region, FA.
C: bronchus, 8 h of ozone and 16-h
postexposure incubation. D: distal
bronchiolar region, 8 h of ozone and 16-h postexposure incubation.
E: bronchus, 90 days of ozone (8 h/day) and 16-h postexposure incubation.
F: distal bronchiolar region, 90 days
of ozone (8 h/day) and 16-h postexposure incubation. Bars, 50 µm.
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Anti-HSP60 (clone 4B9/89). In
filtered-air control animals, HSP60 immunoreactivity was observed in
all cellular and interstitial compartments. There was intense labeling
of epithelial cells and interstitial elements, including smooth muscle
and cartilage, and in both the endothelial and smooth muscle
compartments of large vessels (Fig.
5A). All
elements of the respiratory bronchioles and the gas-exchange parenchyma
were also strongly labeled (Fig. 5B). Short-term exposure to ozone
did not alter the distribution of this protein within the lung (Fig. 5,
C and
D). Long-term exposure, in contrast,
significantly reduced the intensity of the labeling in the gas-exchange
compartments, leaving only focal areas of intense staining in what
appeared to be cuboidal cells of the interalveolar septa (Fig. 5,
E and
F). The epithelial and interstitial components of the respiratory bronchioles also had significantly reduced labeling, with the exception of the peribronchiolar smooth muscle. In the proximal bronchi, there was a redistribution of the
antigen to the apical portions of ciliated cells. There was no
alteration of labeling in large blood vessels in long-term exposed
animals.

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Fig. 5.
Immunohistochemical staining of monkey lung with anti-HSP60 (clone
4B9/89). Experiments were carried out as described in Fig. 4.
A: bronchus, FA.
B: distal bronchiolar region, FA.
C: bronchus, 8 h of ozone and 16-h
postexposure incubation. D: distal
bronchiolar region, 8 h of ozone and 16-h postexposure incubation.
E: bronchus, 90 days of ozone (8 h/day) and 16-h postexposure incubation.
F: distal bronchiolar region, 90 days
of ozone (8 h/day) and 16-h postexposure incubation. Bars, 50 µm.
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Anti-HSP70 (clone C92F3A-5). In
filtered-air control animals, a moderate level of reactivity was
observed in the epithelial cells lining the conducting airways and
respiratory bronchioles (Fig. 6,
A and
B, respectively). The predominant
epithelial labeling occurred in the apex of the ciliated cell
population and in the cuboidal cells of the respiratory bronchioles.
There was some expression in the interstitial connective tissues
surrounding the airways and in the chondrocytes of the peribronchial
cartilage. In the distal airways, there was a low level of binding in
most of the connective tissue elements and in what appeared to be the interstitium of the interalveolar septa. After short-term exposure to
ozone, there appeared to be a minimal reduction in the expression of
HSP70 antigen in the epithelium and connective tissue (Fig. 6,
C and
D). In the respiratory bronchioles,
a large number of cuboidal cells that contained labeling for this
protein were present (Fig. 6D).
After long-term exposure to ozone, there was little change in the
expression of this protein except for focal decreases in the labeling
in bronchiolar epithelial cells (Fig. 6,
E and F).

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Fig. 6.
Immunohistochemical staining of monkey lung with anti-HSP70 (clone
C92F3A-5). Experiments were carried out as described in Fig. 4.
A: bronchus, FA.
B: distal bronchiolar region, FA.
C: bronchus, 8 h of ozone and 16-h
postexposure incubation. D: distal
bronchiolar region, 8 h of ozone and 16-h postexposure incubation.
E: bronchus, 90 days of ozone (8 h/day) and 16-h postexposure incubation.
F: distal bronchiolar region, 90 days
of ozone (8 h/day) and 16-h postexposure incubation. Bars, 50 µm.
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Anti-HSC70 (clone 1B5). The
distribution of HSC70 in filtered-air control animals was similar to
that observed for anti-HSP27 (clone G3.1; data not shown). The
reduction and compartmental changes associated with short- and
long-term exposure to ozone with clone G3.1 were also observed with
clone 1B5.
Anti-HSP90 (clone 9D2). In
filtered-air control animals, there was moderate reactivity for this
protein in the epithelial cells of the proximal bronchi, but there was
limited reactivity to the apexes of some ciliated cells, a moderately
intense distribution in smooth muscle and connective tissue elements of
both vessels and airways, and minimal labeling in the interalveolar
septa (Fig. 7,
A and
B). After short-term exposure to
ozone, there was no significant difference in the distribution of this
protein compared with filtered-air control animals. After long-term
exposure to ozone, there was a reduction in the expression of this
protein throughout the parenchymal mass (Fig. 7,
C and
D). Little distribution change
occurred in the proximal conducting airways, in either the epithelium
or interstitial components, or in the large vessels and bronchiolar
components of the respiratory bronchioles.

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Fig. 7.
Immunohistochemical staining of monkey lung with anti-HSP90 (clone
9D2). Experiments were carried out as described in Fig. 4.
A: bronchus, FA.
B: distal bronchiolar region, FA.
C: bronchus, 90 days of ozone (8 h/day) and 16-h postexposure incubation.
D: distal bronchiolar region, 90 days
of ozone (8 h/day) and 16-h postexposure incubation. Bars, 50 µm.
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Immunohistochemical Comparison of GRP Distribution In Vivo
Anti-GRP75 (clone 30A5). In
filtered-air control animals, the intensity of labeling for clone 30A5
was minimal (Fig. 8,
A and
B). There was a light density in the
ciliated epithelial cells and in some cells of the cartilage. In the
distal airways and the gas-exchange area, there was little labeling,
with the exception of a slightly increased intensity in smooth muscle.
After short-term exposure to ozone, there was an elevated intensity of
labeling, primarily in the connective tissue of proximal airways (Fig.
8C). The intensity of labeling in
the bronchial epithelium also increased at the apexes of ciliated
cells, but the distribution of this labeling was not changed from that
in the control animals. In the respiratory bronchioles (Fig.
8D), the epithelium on the
nonalveolarized side showed intense labeling, and there was some
increase in bronchiolar epithelial labeling in other parts of the
respiratory bronchiole. There appeared to be some increase in the
interstitial labeling of the interalveolar septa but not a major
alteration in the peribronchial connective tissue distribution. In
animals exposed long term to ozone, the intensity and distribution of
labeling in the proximal bronchial epithelium did not change markedly
from that of control or short-term exposed animals (Fig. 8,
E and
F). The primary difference was a
decrease in the intensity of the interstitial labeling. In the distal
airways, especially the respiratory bronchioles, there was a reduction
in labeling in all compartments except the smooth muscle of blood
vessels. No clearly definable labeling occurred in epithelial cells in
either the respiratory bronchiole or gas-exchange area.

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Fig. 8.
Immunohistochemical staining of monkey lung with anti-GRP75 (clone
30A5). Experiments were carried out as described in Fig. 4.
A: bronchus, FA.
B: distal bronchiolar region, FA.
C: bronchus, 8 h of ozone and 16-h
postexposure incubation. D: distal
bronchiolar region, 8 h of ozone and 16-h postexposure incubation.
E: bronchus, 90 day of ozone (8 h/day)
and 16-h postexposure incubation. F:
distal bronchiolar region, 90 days of ozone (8 h/day) and 16-h
postexposure incubation. Bars, 50 µm.
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Anti-GRP78/Bip (clone 10C3). In
filtered-air control animals, there was minimal distribution of this
antigen in the lungs (Fig. 9,
A and
B). There were small focal areas of
labeling in the apexes of ciliated cells of the proximal bronchi and
small focal distributions of immunoreactivity in the parenchyma. There was virtually no reactivity with the smooth muscle in either the blood
vessels or airways or in the epithelial cells lining the respiratory
bronchioles. After short-term exposure to ozone, there was a marked
elevation in the intensity of immunoreactivity in both the proximal and
distal airways (Fig. 9, C and
D, respectively). Intense labeling was
found in the apexes of all ciliated cells throughout the airway tree
and what appeared to be the majority of basal cells in the proximal
airways. The smooth muscle surrounding both the vessels and airways was
also intensely labeled. In the respiratory bronchioles, virtually every
epithelial cell expressed this protein whether they were cuboidal cells
of the respiratory bronchioles or alveolar cells lining alveolar
outpocketings. A small increase in density occurred in the
peribronchiolar connective tissue as well, which appeared to be
associated with both smooth muscle and connective tissue elements. No
marked change, however, was found in expression in the parenchyma.
After long-term exposure to ozone, the expression of this protein was
reduced compared with animals exposed for a short term (Fig. 9,
E and
F). The change was most marked in
the proximal bronchi where protein expression resembled that in the
epithelium of filtered-air control animals. The primary difference in
the proximal bronchi and blood vessels was that the labeling in the
peribronchial smooth muscle and vascular smooth muscle resembled that
observed in short-term exposed animals. In the respiratory bronchioles,
there was a reduced intensity in the immunoreactivity in the
peribronchiolar connective tissue and, to a certain extent, in some of
the epithelial populations lining the air space. This was especially
true for alveolar outpockets and for more distal portions of the
respiratory bronchiole. There did not appear to be a significant
difference in the distribution or intensity of this protein in the
parenchyma after long-term exposure compared with either filtered-air
control or acute short-term exposed animals.

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Fig. 9.
Immunohistochemical staining of monkey lung with anti-GRP78 (clone
10C3). Experiments were carried out as described in Fig. 4.
A: bronchus, FA.
B: distal bronchiolar region, FA.
C: bronchus, 8 h of ozone and 16-h
postexposure incubation. D: distal
bronchiolar region, 8 h of ozone and 16-h postexposure incubation.
E: bronchus, 90 days of ozone (8 h/day) and 16-h postexposure incubation.
F: distal bronchiolar region, 90 days
of ozone (8 h/day) and 16-h postexposure incubation. Bars, 50 µm.
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DISCUSSION |
Stress proteins are a set of proteins characterized by the property
that their levels within cells increase under a variety of stress
conditions (24). Some of these stress proteins involve a chaperone
function (7, 11), whereas others are still unresolved. In this study,
seven MAbs specific to human stress proteins were used to illustrate
the effects of ozone on stress protein synthesis in monkey lung tissues
and cultured TBE cells. These antibodies were chosen because of their
IgG class and their monospecificity toward a single stress protein. The
specificities of these antibodies on monkey proteins were confirmed at
the Western blot level except for anti-HSP90 (clone 9D2). We have
observed that most heat-induced stress proteins other than HSP90, such
as HSP27, HSP60, and HSP70, could be induced by heat under in vitro
culture conditions. For HSP90, it was difficult to detect this protein
in cultured cells by Western blot. This problem may be related to the
use of dexamethasone in the culture medium. Because HSP90 is known to
be bound to the glucocorticoid hormone receptor (36), the interactions
between dexamethasone and the glucocorticoid hormone receptor might
increase the turnover of HSP90 in cultured cells (35). In contrast to these HSPs, GRPs were not significantly elevated by heat.
Unfortunately, our experiments with in vitro ozone exposure did not
reveal any change in the levels of these stress proteins in cultured
cells. This result is consistent with recent data by Sun et al. (39) in
which newly synthesized proteins labeled by
[35S]methionine were
analyzed by two-dimensional gel electrophoresis. Using primary guinea
pig tracheal epithelial cultures, Cohen et al. (4) have obtained
similar results suggesting no inducible HSP synthesis by ozone in
vitro. Here, we have further demonstrated no change in GRPs by ozone in culture.
In contrast to these in vitro observations, in vivo exposure with ozone
on nonhuman primates caused significant changes in various stress
proteins in lung tissues. We have observed a paradoxical difference in
the expression of HSPs and GRPs in vivo in monkey lungs after ozone
exposure. These results are best summarized in Table
1. After both short- and
long-term exposures, the levels of various heat-induced proteins,
HSP27, HSP60, and HSP70, decreased significantly, whereas the levels of
heatless inducible stress proteins, GRP75 and GRP78, were obviously
enhanced in lung tissues after ozone exposure. HSC70, the constitutive
form of HSP70, which is not induced by heat but shares a similar
function as HSP70, also decreased in lung tissues after ozone exposure.
HSP90, a stress protein known to be bound to the glucocorticoid hormone receptor (36), was suppressed only under long-term exposure conditions.
These results further support the notion that the regulations between
the HSPs and GRPs are quite different. It is necessary to point out
that the difference between the induction of HSPs and GRPs by different
stress means has been noticed (19-21, 47). The in vivo results
demonstrated in this study further confirm this notion, suggesting that
the stress mechanism exerted by ozone on cells is quite different from
that exerted by the high-temperature treatment.
Because the level of HSPs in airway epithelial cells was not affected
by a direct exposure of airway epithelial cells to ozone in vitro, the
in vivo phenomenon of a decrease in HSPs is likely due to an indirect
mechanism exerted by ozone toxicity. At present, it is still difficult
to elucidate the toxic mechanism of ozone in vivo because ozone is
highly reactive and can react with many biological materials (5, 8,
27). Pryor (32) and Pryor and Church (33) have suggested that ozone
cannot penetrate deep into the lumen of airways. Furthermore, it is
known that most respiratory epithelial cells in vivo are covered by a
layer of extracellular fluids. It is therefore likely that whatever
reaction occurs between ozone and the lining fluids may be responsible for this in vivo HSP decrease phenomenon. In addition to this possibility, neutrophil influx to the airway lumen commonly occurs after ozone exposure (15), and the effect of neutrophils on stress
protein synthesis is not clear. We do know that neutrophils under the
influence of cytokines are able to produce myeloperoxidase, and the
presence of myeloperoxidase in the airway lumen will cause significant
oxidative stress on tissues.
The suppression of HSP-like antigens in monkey lungs and conducting
airways by ozone is quite different from that seen in rat (50) and
guinea pig (38) experiments, which showed an enhanced HSP70 expression
in lung tissue extracts and lavage fluid by ozone exposure. This
difference is difficult to explain because different species and sample
preparations are involved. Because neither rat (50) nor guinea pig (38)
studies revealed the cell type(s) responsible for the enhancement, it
is possible that cell types other than pulmonary epithelial cells are
responsible for the increase in HSP70 in these rodent animals by ozone.
It is interesting to note that HSP70 enhancement in guinea pigs by ozone is more pronounced in the lavage fluid preparation compared with
that in the lung tissue extract (38). This result may further suggest a
significant contribution from the inflammatory cells to the elevated
HSP70 presence in these lung tissue extract preparations. Alternatively, the lung tissue extracts in these rodent animal experiments may include a contribution from the exfoliated lung epithelial cell population, which was not fixed and included in the
paraffin sections of the lung tissue preparation in the present study.
In contrast to HSP, the regulation of GRPs by ozone is quite different
(Table 1). We notice that both GRP75 and GRP78 are very low in vivo in
monkey lung tissues of control animals maintained under filtered-air
conditions but are elevated in animals after ozone exposure. However,
similar to HSP, this in vivo phenomenon could not be duplicated in
culture. This failure could be related either to the difference in the
in vitro and in vivo responses of cells to stress or to a different
stress-induced mechanism as described in the HSP system above. However,
we noticed that cultured TBE cells expressed an unusually high level of
GRPs. This high level of GRP expression in cultured cells may interfere with the inducibility of cells by ozone treatment. Because the cultured
condition is relatively anaerobic, it is possible that the elevated
expression of GRPs may be related to this anoxic condition that
increases glucose depletion by increased glycolysis (19, 21, 47).
It has been observed that there are at least three classes of induced
mechanisms for GRPs (19, 46). The first class, which includes such
inhibitors of glycoprotein processing as tunicamycin, castanospermine,
and glucosamine, can induce expression of GRPs but not of HSPs. The
second class, including amino acid analogs and heavy metals,
simultaneously induces the synthesis of both HSPs and GRPs. The third
class, including the calcium ionophore A-23187 and the glucose analog
2-deoxyglucose, coordinately induces the synthesis of GRPs and
represses HSP expression. For the induction of GRP synthesis, a
transcriptional mechanism is postulated, whereas in the depression of
HSPs, a posttranscriptional mechanism is proposed (46). We have
observed that ozone effects in vivo on stress proteins are similar to
those of the third class of inducer. These results are consistent with
the notion that the modes of induction of stress proteins in airway
epithelial cells are probably complex and interrelated.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Gary Konas for reviewing and editing this manuscript
before its submission.
 |
FOOTNOTES |
The research was supported by National Institute of Environmental
Health Sciences Grants ES-00628, ES-06553, ES-06230, ES-05707, and
ES-09701; National Heart, Lung, and Blood Institute Grant HL-35635; and
California Tobacco Disease-Related Research Program Grant 7RT-0149.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. Wu, Center
for Comparative Respiratory Biology and Medicine, Univ. of
California, Davis, Surge 1, Rm. 1121, One Shields Ave., Davis,
CA 95616 (E-mail: rwu{at}ucdavis.edu).
Received 28 October 1998; accepted in final form 7 May 1999.
 |
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