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Institute of Environmental Medicine, New York University Medical Center, Tuxedo, New York 10987
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Su, Wei Yi, and Terry Gordon. In vivo exposure to ozone
produces an increase in a 72-kDa heat shock protein in guinea pigs.
J. Appl. Physiol. 83(3): 707-711, 1997.
Although several lines of evidence have suggested that oxidizing
agents can induce heat shock proteins (HSPs) in vitro, little is known
about the induction of HSPs during in vivo exposure to oxidants. Guinea pigs were exposed to ozone for 6 h and euthanized up to 72 h later. Proteins from lavage cells and lung tissue were characterized by
immunoblotting with 72- and 73/72-kDa HSP monoclonal antibodies. Although 73-kDa HSP was expressed constituitively in lung tissue, it
was not affected by ozone. In contrast, 72-kDa HSP was significantly increased in lavage cells and lung tissue of animals exposed to 0.4 and
0.66 parts/million of ozone. Both heat treatment and arsenite induced
72-kDa HSP in cultured alveolar macrophages. The increase in 72-kDa HSP
in the lavage cell pellet peaked at 24 h after ozone, whereas the
influx of polymorphonuclear leukocytes peaked at 4 h. Examination of
the induction of HSPs by ozone may provide clues to the development of
ozone tolerance in humans and animals.
tolerance; adaptation; lung; macrophage
INTRODUCTION THE HEAT SHOCK or stress response is a conserved
physiological response of cells to injury. This response is
characterized by the de novo synthesis of so-called heat shock/stress
proteins (HSPs), which are categorized by their apparent molecular
weight on polyacrylamide electrophoresis gels (20, 23).
The term HSP 70 family is often used in the literature to collectively address a group of gene products that is induced by heat and other stress-inducing agents and has been widely studied (20, 22, 26). In
most mammals, 73-kDa HSP is constituitively expressed, whereas 72-kDa
HSP, an inducible form, is present at a much lower level in primates
under normal conditions. A variety of stress-inducing agents, including
heat, transition metals, and amino acid analogs, has been reported to
activate 72-kDa HSP expression (3). Another broadly defined inducer of
72-kDa HSP is oxidative stress, which encompasses many chemical and
physical agents such as hydrogen peroxide (15), hydroxyquinoline (3),
ischemia-reperfusion (6), anoxia-reoxygenation (6), and gamma
irradiation (13).
A function for 72-kDa HSP has been proposed under both physiological
and pathological conditions. Transient expression during development
suggests a role in cell differentiation and proliferation (9). There is
also in vitro evidence that 72-kDa HSP is involved in the onset of
adaptation or tolerance to heat and other environmental stresses. The
phenomenon of HSPs in adaptation/tolerance is defined originally from
the finding that the treatment of organisms with a sublethal dose of
heat confers on them a resistance to a subsequent exposure to a
normally lethal temperature. It is now understood that 72-kDa HSP binds
to proteins to prevent denaturation and to facilitate their recovery
after stress (5). More recent studies with transgenic animal models and
transfection of cells in vitro have demonstrated the
effects that overexpression of 72-kDa HSP confers on
animal or cell resistance to subsequent stress condition (14, 19).
Similarly, inhibition of 72-kDa HSP results in an increased sensitivity
to heat (8). Therefore, these studies suggest a clear role for HSP 70 as a protective mechanism in cells responding to environmental
stresses.
Ozone is a ubiquitous environmental pollutant, and acute exposure to
this oxidant gas causes a number of concentration-dependent alterations
in the mammalian respiratory tract. The sequelae of the oxidative
stress produced by ozone include lipid peroxidation, generation of free
radicals, and alterations in antioxidants (10). Pryor and Church (16,
17) have demonstrated that aldehydes, hydrogen peroxide, organic
radicals, and hydroxyl radical are among the toxic mediators generated
by ozone. Interestingly, some of these reactive oxygen species have
been shown to elicit a heat shock response in cells and animals. More
specifically, hydrogen peroxide and the hydroxyl radical are strong
inducers of 72-kDa HSP in cells and animals. Given that hydrogen
peroxide and the hydroxyl radical are present in the process of
ozone-initiated oxidative stress, it is reasonable to propose that in
vivo ozone exposure may also elicit 72-kDa HSP expression in the lung.
On the basis of the understanding of the role of HSPs in the
development of tolerance/adaptation in a spectrum ranging
from bacteria to mammal, we speculate that HSP 70 could play an
important role in ozone-induced tolerance. To explore these possible
mechanisms, we examined whether in vivo ozone exposure induces HSP
72/73 in the respiratory tract of the guinea pig.
MATERIALS AND METHODS
70°C. For
each animal, both pieces of lung tissue were pooled together and
homogenized.
Gel electrophoresis and Western blotting of 72- and 73-kDa HSP.
Lavaged cells (n = 4 at each time
point and dose) and lung tissue (n = 4 at each time point and dose, except n = 2 at the 48- and 72-h time points) were lysed in Laemmli
buffer (tissue sections were first homogenized manually in
a glass-Teflon homogenizer) and solubilized by sonicating and boiling
for 5 min. The cell and tissue lysates were centrifuged at 2,000 g for 5 min, and the supernatants were
stored at 70°C. Protein concentrations of all samples were
determined by the Lowry microprotein assay kit (Sigma Chemical) with
bovine serum albumin as the standard. An equal amount of protein (40 µg/lane) for each sample was separated by a discontinuous gel
electrophoresis system (Bio-Rad, Richmond, CA) with 4% stacking and
7.5% sodium dodecyl sulfate-polyacrylamide gel elctrophoresis
(SDS-PAGE) gels by using piperazine diacrylamide. Prestained protein
markers were used to monitor the efficiency of transfer of proteins
from the polyacrylamide gel to a nitrocellulose membrane (Bio-Rad). The
membranes were immunoblotted for 1 h with monoclonal antibodies C92 or
N27 (1:1,000). The monoclonal antibodies recognizing the inducible
72-kDa HSP (C92) or the HSP 72/73 (N27) were a gift (Dr. W. J. Welch,
University of California, San Francisco; Ref. 24) or purchased from
StressGen (Vancouver, BC). The HSP immunoreactive antibodies were
visualized by using a goat anti-mouse immunoglobulin G as secondary
antibody conjugated with alkaline phosphatase (Organon Teknika, West
Chester, PA) by using 5-bromo-4-chloro-3-indoyl phosphate/nitroblue
tetrazolium (Sigma Chemical) as the substrate. To quantify the relative
HSP expression, blots were scanned (HP ScanJet IIcx, Hewlett-Packard)
and quantified with imaging software (BandLeader 2.0, Tel Aviv,
Israel). Band densities of protein samples from air-exposed animals
were defined as 100%. At least three samples were assessed for each
group.
In vitro treatment of alveolar macrophages.
Alveolar macrophages were collected by lung lavage (as described above)
and purified by adherence for 1 h, followed by washing with sterile,
pyrogen-free phosphate-buffered saline (GIBCO). Macrophages (2 × 106) were cultured with
RPMI-1640 (GIBCO) in 37-mm petri dishes and treated with 5, 20, 60, 100, or 200 µM sodium arsenite (Sigma Chemical) for 3 h or heated for
20 min at 44°C. After treatment, the cells were incubated at
37°C for 18 h (arsenite-treated cells were first washed). Cells
were lysed in Laemmli buffer, and proteins from cell lysates were
separated by SDS-PAGE and immunoblotted by using the anti-HSP 72 antibody as described above. Total cell protein was also visualized by
silver staining of an identical SDS-PAGE gel that was run in parallel
with the gel used for Western blotting.
Statistics.
Data are means ± SE. Statistical differences among groups were
analyzed with a one-factor analysis of variance. A Dunnett's two-tailed test was used in a post hoc analysis comparing values from
ozone groups to the values from the air group. A
P value 
0.05 was considered
statistically significant.
RESULTS
The effect of ozone on the in vivo expression of 72- and 73-kDa HSP was
assessed in lavage cells and in lung tissue. Western blot analysis
demonstrated that 72-kDa HSP protein expression was barely detectable
in the lavage cell pellet or lung tissue of air-exposed animals.
Exposure to ozone had a dose-dependent and time-dependent effect on
72-kDa HSP expression in both the lavage cell pellet and lung tissue.
At 0 and 4 h, 72-kDa HSP was increased (50 and 245%,
respectively, P < 0.05) in the lavage cell lysate of
animals exposed to 0.66 ppm ozone for 6 h (Fig. 1). Induction of 72-kDa HSP in the lavage
cells reached a peak at 24 h (366% increase) and returned to
near-control values by 48 h after ozone exposure. Exposure to 0.4 and
0.66 ppm, but not 0.2 ppm, ozone induced significant increases in the
72-kDa HSP in the cell lysate (Fig. 2). In
lung tissue, 72-kDa HSP was also significantly increased at 24 h
(192%) after exposure to 0.66 ppm ozone, and this enhanced 72-kDa
HSP expression in lung tissue appeared to last longer than that in cell
lysates (Fig. 3). The level of 72-kDa HSP
in lung tissue of ozone-exposed animals was still higher than control
values at 72 h after exposure. As in the lavage cell lysates, a
concentration-response relationship was observed for 72-kDa HSP in lung
tissue (Fig. 4). Unlike the effect of ozone
on 72-kDa HSP, there were no significant changes in 73-kDa HSP in
either lavaged cell lysates or lung tissue at any postexposure time or
ozone concentration (data not shown).
0.05.
0.05.
0.05.
0.05.
Guinea pig alveolar macrophages treated in vitro with sodium arsenite
showed a concentration-dependent induction of 72-kDa HSP in the range
of 5 to 200 µM (Fig. 5,
bottom). Heat treatment of alveolar
macrophages at 44°C for 20 min also produced an increase in 72-kDa
HSP expression. The induction of a 72-kDa protein by arsenite in
macrophages was even detectable on SDS-PAGE gels visualized with a
silver stain (Fig. 5, top).
As shown in Fig. 6, polymorphonuclear
leukocytes (PMNs) in lavage fluid peaked at 4 h and returned to near
control levels at 24 and 48 h after ozone exposure.
Monocytes/macrophages were the major cell population and comprised
>84% of the total lavage cells at all time points except at 4 h
after exposure (42% of total lavage cells).
0.05.
DISCUSSION
The present study has shown that exposure to ozone can induce the expression of a member of the HSP 70 family in an in vivo animal model. Levels of the 72-kDa HSP form were significantly induced by exposure to ozone in a dose- and time-dependent manner. Although 73-kDa HSP was constituitively expressed in all guinea pig lungs examined, it was not altered by exposure to ozone.
The mechanism underlying the induction of 72-kDa HSP by in vivo exposure to ozone is not known, but we speculate that oxidative stress may play a role. Hydrogen peroxide and hydroxy radical have been reported to induce members of the HSP 70 family in macrophages and other cells (15). Other oxidative stressors such as hydroxyquinoline (3), ischemia-reperfusion (14), anoxia-reoxygenation (6), and gamma irradiation (13) have also been reported to induce the expression of HSPs in vivo or in vitro. Thus states of imbalance in oxidants/antioxidants may underlie the observed inductions in the HSP 70 family of gene products. We speculate that oxidative stress produced by ozone may be responsible for the observed 72-kDa HSP induction in guinea pig lung. Ozone is thought to exert its toxic effects either by interacting directly with cell membranes or generating secondary products that are often more reactive than ozone itself. Moreover, hydrogen peroxide, hydroxy radical, superoxide radical, and an array of ozonides are among the secondary products generated by resident and transient respiratory cells activated by ozone exposure (16, 17). Thus oxidative stress originated from activated pulmonary inflammatory cells may participate in the induction of HSPs. This indirect induction of 72-kDa HSP by ozone-initiated inflammation may be critical because direct exposure of alveolar macrophages and transformed human airway epithelial cells to ozone in vitro does not elicit HSP induction (4, 21).
On repeated exposure, the mammalian respiratory tract is known to acquire tolerance to some of the adverse effects typically observed after a single exposure to ozone. Although changes in levels of antioxidants occurring after ozone exposure may contribute to the development of ozone-induced adaptation, we hypothesize that 72-kDa HSP may also play a role. In addition to the lines of research that have provided strong evidence that members of the HSP 70 gene family are essential for homeostasis of cellular environment (5), many in vitro studies suggest that HSP 70 may be important for protection of cellular function during environmental stress. For example, expression of HSP 70 by transfection of rat fibroblasts or by microinjection into Chinese hamster ovary cells confers heat resistance to these cells (12). Competitive inhibition of HSP 70 gene products also causes thermosensitivity in Chinese hamster ovary cells (8). Comparatively few studies have examined the role of HSPs in the development of tolerance in vivo. Ribeiro et al. (18) have reported that induction of HSP 70 by sodium arsenite in rat lung protects rats against sepsis. Plumier et al. (14) have demonstrated that overexpression of human HSP 70 in transgenic mice significantly improves the recovery from ischemic myocardial injury. These observations provide convincing evidence that an increased level of HSP 70 expression is correlated with the acquisition of resistance to environmental stress. Thus the increased level of 72-kDa HSP expression observed in the present study may be indicative of an adaptive response to protect the respiratory tract from repeated exposure to ozone.
The quick induction and the pattern of 72-kDa HSP production in the lavage cells of ozone-exposed animals are compatible with the features of development of adaptation to ozone, which have been characterized as being quick in onset and short lasting (2, 7, 25). The peak induction of 72-kDa HSP in the lavage cell pellet occurred at 24 h after ozone exposure. Expression of 72-kDa HSP in the lavage cell pellet returned to near-baseline values by 72 h after exposure. The finding of a small elevation in 72-kDa HSP in lung tissue at 72 h, but not in the lavage cell pellet, suggests a difference in kinetics in these two compartments. The rapid decline of HSP expression in the cell pellet may have resulted from a more rapid turnover of 72-kDa HSP in that cell population. The macrophages, PMNs, lymphocytes, and eosinophils present as free cells in the alveolar spaces have a shorter half-life than epithelial lining cells and vascular endothelial cells in the lung. The transient nature of these free cells may be responsible for the more rapid decline in the lavage cell pellet.
This study did not identify the cell type(s) responsible for the increase in 72-kDa HSP in the lavage cell pellet. Although PMNs were significantly increased after exposure, the major infiltration of neutrophils was observed at 4 h after exposure, whereas the peak induction in 72-kDa HSP occurred at 24 h. Despite the fact that this temporal difference does not exclude the possible contribution of PMNs, it is likely that other lavage cells are involved in the induction of 72-kDa HSP by ozone. As visualized on silver-stained gels, in vitro treatment of alveolar macrophages with heat or sodium arsenite confirmed that 72-kDa HSP is the major stress protein produced by macrophages. Although this may provide indirect evidence that macrophages play an important role in the increased expression of 72-kDa HSP in the lavage cell pellet of ozone-exposed animals, immunohistochemical studies at the light or electron microscopic level would be more appropriate in determining the relative contribution of macrophages, PMNs, and eosinophils.
As demonstrated in primates by Welch and Suhan (24), a low-level expression of 72-kDa HSP, the inducible form of the HSP 70 gene family, was observed in guinea pig lung. Blake et al. (1) have speculated that HSP 70 might play an important role in the homeostasis of lung cells. An alternate explanation of its low-level expression in lung tissue may be that the respiratory system is under constant "stress" during its direct interaction with the outside environment, even though this interaction is part of its normal physiological condition. In the present study, the additional environmental stress produced by the in vivo exposure to an oxidant gas such as ozone led to an induction of 72-kDa HSP in lavage cells (4.6-fold) and in lung tissue (2.9-fold). Examination of the induction of HSPs by in vivo ozone exposure may provide clues to the development of tolerance in ozone-exposed human subjects and animals.
ACKNOWLEDGEMENTS
The authors thank Dr. W. J. Welsh of the University of California, San Francisco, for the generous gift of antibodies and Dr. C. Miller for expert advice in protein electrophoresis.
FOOTNOTES
Address for reprint requests: T. Gordon, Dept. of Environmental Medicine, New York Univ. School of Medicine, Long Meadow Rd., Tuxedo, NY 10987.
Received 26 July 1996; accepted in final form 23 April 1997.
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