Pulmonary apoptosis in aged and oxygen-tolerant rats exposed
to hyperoxia
Leo E.
Otterbein1,
Beek Yoke
Chin1,
Lin L.
Mantell2,3,
Leah
Stansberry3,
Stuart
Horowitz2, and
Augustine M. K.
Choi1,4
4 Section of Pulmonary and
Critical Care Medicine, Yale University School of Medicine,
New Haven 06250 and Veterans Affairs Connecticut Health System,
West Haven, Connecticut 06516;
1 Division of Pulmonary and
Critical Care Medicine, The Johns Hopkins University School of
Medicine, Baltimore, Maryland 21205;
2 Departments of Pediatrics and
Pulmonary and Critical Care Medicine, The CardioPulmonary Research
Institute, Winthrop-University Hospital, State University of New
York at Stony Brook School of Medicine, Mineola 11501; and
3 Department of Biology, Hofstra
University, Hempstead, New York 11550
 |
ABSTRACT |
Accumulating evidence demonstrates that genotoxic
and oxidant stress can induce programmed cell death or apoptosis in
cultured cells. However, little is known about whether oxidative stress resulting from the deleterious effects of hyperoxia can induce apoptosis in vivo and even less is known regarding the functional significance of apoptosis in vivo in response to hyperoxia. Using hyperoxia as a model of oxidant-induced lung injury in the rat, we show
that hyperoxic stress results in marked apoptotic signals in the lung.
Lung tissue sections obtained from rats exposed to hyperoxia exhibit
increased apoptosis in a time-dependent manner by terminal transferase
dUTP nick end labeling assays. To examine whether hyperoxia-induced
apoptosis in the lung correlated with the extent of lung injury or
tolerance (adaptation) to hyperoxia, we investigated the pattern of
apoptosis with a rat model of age-dependent tolerance to hyperoxia. We
show that apoptosis is associated with increased survival of aged rats
to hyperoxia and with decreased levels of lung injury as measured by
the volume of pleural effusion, wet-to-dry lung weight, and
myeloperoxidase content in aged rats compared with young rats after
hyperoxia. We also examined this relationship in an alternate model of
tolerance to hyperoxia. Lipopolysaccharide (LPS)-treated young rats not
only demonstrated tolerance to hyperoxia but also exhibited a
significantly lower apoptotic index compared with saline-treated rats
after hyperoxia. To further separate the effects of aging and
tolerance, we show that aged rats pretreated with LPS did not exhibit a
significant level of tolerance against hyperoxia. Furthermore, similar
to the hyperoxia-tolerant LPS-pretreated young rats, the nontolerant LPS-pretreated aged rats also exhibited a significantly reduced apoptotic index compared with aged rats exposed to hyperoxia alone. Taken together, our data suggest that hyperoxia-induced apoptosis in
vivo can be modulated by both aging and tolerance effects. We conclude
that there is no overall relationship between apoptosis and tolerance.
programmed cell death; oxidative stress; lung injury
 |
INTRODUCTION |
THE LUNG IS A MAJOR TARGET ORGAN for oxidant injury by
hyperoxia (6, 7). Patients suffering from pulmonary diseases including
adult respiratory distress syndrome and emphysema require supplemental
O2 therapy to maintain lung
function, which further increases the oxidant burden of the lung (9,
14). It is believed that the damaging effects of hyperoxia are mediated
by reactive oxygen species (ROS) including superoxide and hydroxyl
radicals and hydrogen peroxide, which are generated by the incomplete
reduction of O2 (12, 19).
Oxidative stress resulting from these ROS can cause cellular damage by
oxidizing nucleic acids, proteins, and membrane lipids. Cells have
evolved both nonenzymatic and enzymatic antioxidant defenses to
detoxify ROS to defend against the deleterious effects of hyperoxia
(13, 23). Nonenzymatic defenses include vitamins C and E and
sulfhydryl-containing glutathione (3). Enzymatic antioxidant defenses
against hyperoxia include superoxide dismutase, catalase, and
glutathione peroxidase (3). When these antioxidant defenses become
overwhelmed by the oxidative stress of hyperoxia, the resultant cell
injury can lead to cell death.
Cell death from oxidative stress can occur via cell necrosis or
apoptosis (11, 27, 34). Cell necrosis is a mode of cell death that
occurs exclusively in environmental disruption apart from physiological
conditions, resulting in inflammatory reactions caused by cell lysis
and release of intracellular contents into the extracellular space
(11). Cell necrosis is always pathological. Programmed cell death or
apoptosis, on the other hand, is a gene-regulated process in which a
coordinated series of morphological changes such as nucleus and
chromatin condensation, cell membrane blebbing, and fragmentation of
the cell into membrane-bound apoptotic bodies occurs, resulting in cell
death (28, 30). Removal of apoptotic bodies by phagocytosis by
neighboring cells, in particular macrophages, occurs without
disturbance to the tissue architecture and without initiating
inflammation. Apoptosis is often a physiological process, especially important during embryogenesis, organ atrophy, and normal adult tissue turnover (8). However, accumulating evidence suggests that genotoxic and oxidant stress can induce cell death via
apoptosis (8, 10, 18).
A recent study (18) suggests that cultured pulmonary cells can also
undergo apoptosis after oxidant stress, yet little is known about
apoptosis from oxidant stress in the lung in vivo. What is the
functional significance of apoptosis in vivo after hyperoxic stress?
Does apoptosis represent a mere marker of cell and tissue injury after
oxidant injury, or does it play a role in adaptation or defense against
hyperoxic stress in vivo? We attempted to address these questions by
utilizing various in vivo models of tolerance to hyperoxia. We show
that hyperoxia exposure in vivo can indeed cause a significant level(s)
of apoptosis in the rat lung. Using two distinct in vivo models of
tolerance to hyperoxia, we show that apoptosis in the rat lung can be
regulated by both aging and tolerance effects and that there is no
clear relationship between apoptosis and tolerance to
hyperoxia.
 |
MATERIALS AND METHODS |
Animals and O2 exposure.
Virus-free 6- and 24-mo-old male Fischer 344 rats and 2-mo-old
Sprague-Dawley rats were purchased from Harlan Sprague Dawley (Indianapolis, IN) and allowed to acclimate on arrival for 7 days before experimentation. The animals were fed rodent chow and water ad
libitum. All experimental protocols were approved by the Animal Care
and Use Committee, The Johns Hopkins University School of Medicine
(Baltimore, MD). The animals were exposed to hyperoxia (>99%
O2 at a flow rate of 12 l/min) in
a 3.70-ft3 glass exposure chamber.
The chamber was humidified for 10-15 min at the start of the
exposure. Food and water were provided ad libitum during the exposure.
Lipopolysaccharide exposure. The rats
were placed in a wire mesh cage with nine individual slots to keep the
animals separated but free to move around during the aerosol exposure.
This cage was placed inside an airtight Plexiglas chamber, and
lipopolysaccharide (LPS) was nebulized into the chamber continuously
for 1 h with a model 40 nebulizer (DeVilbiss, Somerset, PA). The
nebulizer generates particles 5 µm in diameter. The aerosol that was
generated was directed against a Plexiglas deflector plate immediately
on entering the chamber to ensure even distribution and dispersal of
the LPS. A blower motor (Grainger, Baltimore, MD) was connected to the
exit port to draw the aerosol through the chamber at a steady rate.
This was done to maintain a constant flow of particulate over the
animals. A second deflector plate adjacent to the exit port was
installed to ensure equal amount removal of the aerosol from the
chamber. The pressure in the chamber was maintained at 0.4 cmH2O. After exposure, the animals
were removed and immediately placed into hyperoxia.
Lung tissue preparation. The lungs
were fixed by perfusion of 10% Formalin at
20-cmH2O gauge pressure and
embedded in paraffin. Lung sections of 4-5 µm were mounted onto
slides pretreated with 3-aminopropylethoxysilane (Digene Diagnostics,
Beltsville, MD). The slides were baked for 30 min at 60°C and
washed two times in fresh xylene for 5 min each to remove the paraffin.
The slides were then rehydrated though a series of graded alcohols and
then washed in distilled water for 3 min each.
Apoptosis by terminal transferase dUTP nick end
labeling assay and photomicrography. The terminal
transferase dUTP nick end labeling (TUNEL) method was used for the
apoptosis assay of lung tissue sections as previously described (22).
TUNEL reagents including rhodamine-conjugated anti-digoxigenin Fab
fragment were obtained from Boehringer Mannheim (Indianapolis, IN).
Tissue sections were counterstained with 2 µg/ml of
4',6-diamidine-2-phenylindole dihydrochloride (DAPI; Boehringer
Mannheim) for 10 min at room temperature. Photomicrographs were
recorded on 35-mm film with a Nikon Optiphot microscope and
UFX camera system (Nikon, Melville, NY) and transferred onto a
KodakPhotoCD. The images were digitally adjusted for contrast with
Adobe Photoshop 3.0 (Adobe Systems, Mountain View, CA).
Computer-aided image analysis. To
quantify the extent of apoptosis in the rat lung, samples were studied
by epifluorescence to visualize either TUNEL-positive nuclei (590 nm)
or total DAPI-stained nuclei (420 nm). Images were captured with a
charge-coupled device video camera. The captured images were analyzed
with the Image 1 system (Universal Imaging, West Chester, PA), which is
a personal computer-based software applications package for
quantitative morphometry and image analysis. All images were captured
to a hard disk at identical black level and gain settings of the
charge-coupled device camera. Similarly, images were digitally set at a
threshold with identical settings for each set of either
DAPI- or TUNEL-fluorescent groups. The total number of cells (nuclei)
or the number of TUNEL-positive cells in each field was determined in
the object counting mode. At least 25 fields were analyzed from at
least 2 individual animals at each time point. The apoptotic index was
calculated as the percentage of TUNEL-positive apoptotic nuclei divided
by the DAPI-staining nuclei.
Measurement of injury markers. At
various time points into the hyperoxia exposure, rats were removed and
anesthetized with pentobarbital sodium. The pleural effusion was
collected by inserting an 18-gauge needle and a 10-ml syringe through
the diaphragm and withdrawing all fluid present in the pleural cavity.
After exsanguination, the entire lung was excised and divided. The left
lobe was weighed and frozen for myeloperoxidase (MPO) determination
(20). The right lower lobe was weighed and placed into a 60°C oven
to dry. After 2 days, the tissue was weighed to determine the
wet-to-dry lung weight. Under these conditions, lung samples became
completely desiccated.
Statistical analysis. Data are
expressed as means ± SE. Differences in measured variables between
the experimental and control groups were assessed with Student's
t-test. Statistical calculations were
performed on a Macintosh personal computer with the Statview II
Statistical Package (Abacus Concepts, Berkeley, CA). Statistical difference was accepted at P < 0.05.
 |
RESULTS |
Hyperoxia induces apoptosis in the rat
lung. To determine whether hyperoxia can induce
apoptosis in vivo, lung sections were obtained from rats exposed to
either normoxia or hyperoxia and analyzed for apoptotic signals by in
situ TUNEL assay, which labels the 3'-COOH ends of DNA cut by
endonucleases that are activated during apoptosis (31). Figure
1A
illustrates the lack of apoptotic signals in the lungs from rats
exposed to air alone. In contrast, a marked increase in TUNEL-positive
cells was observed in lungs from rats exposed to hyperoxia (Fig.
1B).

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Fig. 1.
Hyperoxia induces apoptosis in rat lung. Lung tissue sections from
control normoxic (2-mo-old; A) and
hyperoxic (2-mo-old; B) rats (61 h
of exposure) were analyzed for apoptotic signals by terminal
transferase dUTP nick end labeling (TUNEL) assays as described in
MATERIALS AND METHODS. Bar, 5-µm
section.
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|
Effect of acquired (age-induced) tolerance to
hyperoxia on apoptosis. To examine whether
hyperoxia-induced apoptosis in the rat lung correlated with the extent
of lung injury or tolerance (adaptation) in response to hyperoxic
stress, we investigated the pattern of apoptosis using a rat model of
tolerance to hyperoxia. Choi et al. (4) have previously
reported that the rats exhibit increased tolerance (increased survival)
to hyperoxia as they age normally. The young rats (2-6 mo old)
succumbed to continuous hyperoxia exposure by 72 h, whereas the aged
rats (24 mo old) survived up to 92 h of continuous hyperoxia exposure
(P < 0.05) (4). We then used this
model to examine whether apoptosis is modulated by age-induced acquired
tolerance to hyperoxia. Lung sections were obtained from young
(2-mo-old) and aged (24-mo-old) rats exposed to air or hyperoxia and
then analyzed for apoptosis by TUNEL assay. Figure
2 demonstrates the kinetics of increase in
TUNEL-positive cells after 24, 48, and 61 h of hyperoxia exposure in
young (2-mo-old) and aged (24-mo-old) rats. In the young rats, we did
not observe increased TUNEL-positive cells at either 24 or 48 h of
hyperoxia exposure (Fig. 2, E and
F, respectively) but detected a marked
increase in apoptotic cells at 61 h of hyperoxia exposure (Fig.
2G). In the aged rats, we did not
detect any evidence of apoptotic signals at 24 h of hyperoxia exposure
(Fig. 2A) as in the young rats (Fig.
2E). However, we observed a marked
increase in TUNEL-positive cells at 48 h of hyperoxia (Fig.
2B), with a sustained increase at 61 and 96 h of hyperoxia exposure (Fig. 2,
C and
D, respectively). Figure
2H illustrates DAPI fluorescence of
the same lung section shown in Fig. 2D
(TUNEL stain) to demonstrate the total number of nucleated cells.

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Fig. 2.
Kinetics of hyperoxia-induced apoptosis in rat lung. Lung sections from
aged (24-mo-old;
A-D)
and young (2-mo-old;
E-G)
rats were analyzed for apoptotic signals by TUNEL assays as described
in MATERIALS AND METHODS at 24 (A), 48 (B), 61 (C), and 96 (D) h of hyperoxia for aged rats and
24 (E), 48 (F), and 61 (G) h for young rats.
H: 4',6-diamidine-2-phenylindole
dihydrochloride (DAPI)-stained lung sections from aged rats at 96 h of
hyperoxia.
|
|
We then quantitated the level of apoptosis in these lung sections by
determining the apoptotic index (number of TUNEL-positive cells/number
of DAPI-stained cells). As shown in Fig. 3,
the apoptotic index increased with advancing exposure in both exposed
groups but increased more rapidly in the aged (tolerant) rats.

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Fig. 3.
Apoptotic index of rat lung after hyperoxia. Lung tissue sections from
young (2-mo-old) and aged (24-mo-old) rats were analyzed for
TUNEL-positive cells and costained for DAPI stain to determine
apoptotic index (no. of TUNEL-positive cells/no. of DAPI-stained
cells). Solid bars, young rats; open bars, aged rats.
* P < 0.05 compared with young
rats at 48 h.
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|
Hyperoxia-induced apoptosis is associated with
increased survival and decreased lung injury in acquired tolerance to
hyperoxia. Based on our observation that apoptosis was
associated with increased survival in the acquired model of tolerance
to hyperoxia (earlier onset and higher level of apoptosis in the aged
tolerant rats), we then examined whether apoptosis was associated with
a decreased level of lung injury. The young rats (2 mo old) exhibited
increased signs of lung injury as measured by an increased volume of
pleural effusion and wet-to-dry lung weight after 48 h of hyperoxia
(Fig. 4, A and
B, respectively) at a time point where
only negligible levels of apoptotic signals were observed (Figs. 2 and
3). In contrast, the aged rats (24 mo old) exhibited a significantly decreased level of lung injury compared with the young rats as measured
by the volume of pleural effusion, wet-to-dry lung weight, and MPO
content after 48 h of hyperoxia (Fig. 4,
D-F,
respectively). Interestingly, at this time point, the aged rats
exhibited significant levels of apoptotic signals (Figs. 2 and 3).

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Fig. 4.
Indexes of lung injury in young
(A-C)
and old
(D-F)
rats after hyperoxia. Pleural fluid volume
(A and
D), wet-to-dry lung weight
(B and
E), and myeloperoxidase (MPO)
content (C and
F) were determined as described in
MATERIALS AND METHODS after 48 h of
hyperoxia exposure. * P < 0.05.
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Effect on chemical tolerance to hyperoxia on
apoptosis. Based on the observation that apoptosis in
the rat lung was associated with decreased lung injury and increased
survival in a model of acquired tolerance to hyperoxia (aging), we
chose to test this relationship in an alternate model of tolerance to
hyperoxia. We used the established model of LPS-induced tolerance
(chemical tolerance) to hyperoxia (7, 29). Young rats (2 mo old)
pretreated with LPS exhibited a marked increased survival to hyperoxia
(Fig. 5) compared with control
rats pretreated with saline before hyperoxia exposure. Lung sections
were obtained from these tolerant rats and assayed for apoptotic
signals by TUNEL assay. As shown in Fig. 6,
young rats exposed to hyperoxia alone demonstrated a marked increase in
the apoptotic index. However, young rats pretreated with LPS not only
demonstrated a tolerance to hyperoxia but also exhibited a
significantly reduced apoptotic index compared with rats exposed to
hyperoxia alone (Fig. 6). To separate the effects of aging and
tolerance, we performed an identical experiment in the aged rats (24 mo
old). In contrast to the young rats, aged rats pretreated with LPS did
not exhibit a significant level of tolerance against hyperoxia (Fig.
5). Furthermore, lung sections were obtained from these nontolerant
LPS-treated aged rats and assayed for apoptotic signals by TUNEL assay.
As shown in Fig. 6, aged rats exposed to hyperoxia alone
demonstrated a marked increase in the apoptotic index. However, similar
to the tolerant LPS-treated young rats, the nontolerant LPS-treated
aged rats exhibited a significantly reduced apoptotic index compared
with aged rats exposed to hyperoxia alone (Fig. 6). Interestingly, aged
rats treated with LPS alone also exhibited increased apoptosis after
hyperoxia (Fig. 6).

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Fig. 5.
Survival curve of rats to hyperoxia after lipopolysaccharide (LPS)
aerosolization. Young (2-mo-old) and aged (24-mo-old) rats were
aerosolized with LPS as described in MATERIALS AND
METHODS before exposure to 100%
O2. , Young rats pretreated
with saline before hyperoxia exposure; , young rats pretreated with
LPS before hyperoxia exposure; , aged rats pretreated with saline
before hyperoxia exposure; , aged rats pretreated with LPS before
hyperoxia exposure. P < 0.001 for
young LPS-treated vs. young saline-treated rats after hyperoxia.
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Fig. 6.
Apoptotic index of rat lung after hyperoxia with LPS pretreatment. Rats
were pretreated with aerosolized LPS as described in
MATERIALS AND METHODS, and lung tissue
sections from rats were analyzed for TUNEL-positive cells and costained
with DAPI stain to determine apoptotic index (no. of TUNEL-positive
cells/no. of DAPI-stained cells) after 48 h of hyperoxia exposure.
A: young (2-mo-old) rats.
B: aged (24-mo-old) rats.
* P < 0.05 compared with
untreated control group. ** P < 0.05 compared with O2-alone
group.
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 |
DISCUSSION |
Direct exposure of cultured cells to oxidants such as hydrogen
peroxide, ionizing irradiation, various superoxide-generating agents,
and glutathione depletors can cause cell death via the induction of
apoptosis, depending on the dose (8, 18, 26, 32). Although the
biochemical and molecular mechanisms by which oxidants mediate
apoptosis in cells are not clearly understood, recent reports that
Bcl-2 prevents apoptosis by an antioxidant pathway at the sites of
oxygen radical formation (15, 17) and that antioxidants such as
N-acetyl-L-cysteine
and thioredoxin (25) prevent apoptosis strongly suggest that oxidants
may serve as key signaling molecules in the development of apoptosis in cultured cells. Surprisingly, although the damaging effects of hyperoxia in the lung are in part mediated by superoxide and hydroxyl radicals and hydrogen peroxide, the products of which can induce apoptosis in various cell types (8, 18, 32), hyperoxia exposure does
not cause apoptosis in cultured lung cells (18). For instance, Kazzaz
et al. (18) recently reported that hyperoxia causes cell death via
necrosis and not apoptosis in pulmonary A549 epithelial cells. Our
laboratory has also observed that hyperoxia causes cell necrosis and
not apoptosis in other cell types such as human bronchial epithelial
cells (Choi, unpublished data). Little is known regarding
the effects of hyperoxia on apoptosis in the lung in vivo. In contrast
to cultured cells that do not undergo apoptosis in response to
hyperoxia (18), we hypothesized that hyperoxia induces apoptosis in the
lung in vivo and that a clear functional relationship exists between
apoptosis and tolerance to hyperoxia. We decided to investigate this
relationship between hyperoxia and apoptosis in vivo.
This study clearly shows that hyperoxia induces apoptosis in the rat
lung in a time-dependent manner in contrast to absence of apoptosis in
cultured cells exposed to the same concentration of hyperoxia (>95%
O2). Several possibilities exist
to underlie this disparate observation between in vitro lung cultured
cells and in vivo lung tissue. 1)
The lack of hyperoxia-induced apoptosis in cultured lung cells may
reflect cell specificity because there are recent reports of vascular
endothelial cells and lymphoblastoid cells exhibiting susceptibility to
hyperoxia-induced apoptosis (1, 5).
2) The ability of hyperoxia to
induce apoptosis in the lung in vivo may reflect the effect of systemic
mediators released during the inflammatory phase of hyperoxic lung
injury, which may be responsible for inducing apoptosis rather than
hyperoxia itself. For example, tumor necrosis factor-
, a potent
inducer of apoptosis in various cell types, is released in vivo during hyperoxia (16). 3) The
susceptibility of lung cells in vivo to undergo apoptosis may reflect
the effect of the quiescent state of the cells in vivo compared with
the more proliferative state of cultured cells growing in cultured
conditions.
The functional significance of apoptosis in the lung in vivo is not
clearly understood. Mantell et al. (22) reported in a mouse model of
hyperoxia that the level of hyperoxia-induced apoptosis correlated with
the extent of lung injury. Using an inbred (rather than acquired) model
of tolerance, they reported higher levels of apoptosis in a sensitive
strain of mouse and lower apoptosis in a resistant strain. This
suggests that apoptosis may serve as a marker of cell and tissue
injury. Our data, however, do not support our hypothesis that there is
a clear functional relationship between hyperoxia and apoptosis. This
simple relationship, however, was not observed in our age-dependent rat
model of hyperoxia. We observed that the increased apoptotic index of
lungs from the age-dependent tolerant rat is inversely proportional to
the extent of lung injury and is associated with increased survival.
This relationship may reflect the effects of the aging process and not
of tolerance; however, the increased level of apoptosis observed in the
aged tolerant rats was associated with decreased lung injury in the
aged rats as determined by pleural effusion volume, wet-to-dry lung
weight, and MPO determination in this study and decreased albumin
levels in bronchoalveolar lavage fluid (4) after hyperoxia. These
studies may suggest that the apoptosis in the lung may represent a
physiological process that is pivotal in protecting the lung from
oxidant injury. However, a recent study (22) showed that neonatal
rabbits that are tolerant to hyperoxia exhibited significantly less
apoptosis than the more susceptible adult rabbits after hyperoxia. This
observation supports the formal possibility that the hyperoxia-induced apoptosis can also be regulated by the aging process independent of the
tolerance effect.
The complexity of the functional role of apoptosis in lung injury is
further highlighted by our observations that in contrast to the
age-dependent tolerance model, the apoptotic index of the lung is not
associated with survival in the LPS-induced tolerance model of
hyperoxia in young rats. For example, LPS-induced hyperoxia-tolerant young rats exhibited a significantly reduced level of apoptosis than
saline-treated rats after hyperoxia, although baseline levels were
similar. These observations suggest that in some cases apoptosis may
represent a marker of tissue injury rather than a marker of tolerance.
However, we observed similar attenuation of apoptosis in LPS-treated
old rats, which were not tolerant to hyperoxia. These apparently
conflicting results highlight the difficulty of interpreting a simple
role for apoptosis in the rat lung after hyperoxia. It seems likely
that a greater knowledge of the cell types undergoing apoptosis during
lung injury will lead to a better understanding of the role(s) for
apoptosis in lung injury. The antioxidant manganese superoxide
dismutase in response to an LPS stimulus contributes significantly to
the protective effect of LPS against hyperoxia (7, 29) in young rats.
Although no direct evidence exists that manganese superoxide dismutase
can attenuate the apoptosis in the lung, accumulating evidence suggests that antioxidants may play vital roles in attenuating the apoptotic process (15, 25).
Although one cannot yet confidently determine the cell type responsible
for the apoptotic signals in TUNEL assays of lung tissue sections,
increased apoptosis in the aged-tolerant rats coincided with
significantly decreased lung MPO after hyperoxia, suggesting that
neutrophil clearance by apoptosis may represent one major mechanism of
increased survival in the aged rats. Interestingly, a study (24)
involving patients with human adult respiratory distress syndrome
demonstrated that the apoptotic process was more prominent during the
repair phase than the acute phase of this syndrome, suggesting that
apoptosis serves as a beneficial process in the resolution of acute
lung injury.
It is generally thought that apoptosis occurs without eliciting an
inflammtory response, whereas cell or tissue necrosis can cause
inflammation (8, 33). However, our studies challenge this paradigm in
that the inflammatory response (neutrophil and macrophage), as
determined by bronchoalveolar lavage analysis, was similar in both the
young and aged rats after hyperoxia (4). However, we observed marked
differences in the kinetics and extent of apoptosis between the young
and aged rats. Recent reports (2, 32) also suggested that apoptosis
induced by tumor necrosis factor-
and X-irradiation in cultured
cells can be inhibited by the transcription factor nuclear factor-
B
(NF-
B). Interestingly, we did not observe differences in the
induction of NF-
B activation in vivo in the lungs after hyperoxia in
both tolerance models (age and LPS) (4; Choi, unpublished
data) that may reflect differences between the in vivo and
in vitro systems or may represent stimulus specificity. Interestingly,
Li et al. (21) recently reported that apoptosis can occur in the
absence of NF-
B activation in cultured epithelial cells after
hydrogen peroxide treatment and that the hyperoxic induction of NF-
B
fails to protect cultured cells from death.
In summary, our study demonstrates that hyperoxia in vivo can induce
apoptosis in the rat lung. Furthermore, these studies strongly suggest
that apoptosis is not a universal indicator of tolerance to hyperoxia
and that the functional significance of apoptosis in vivo after
hyperoxic oxidant stress largely depends on the mechanism of tolerance,
whether chemically induced or acquired by aging. The complex regulation
of hyperoxia-induced apoptosis by both aging and tolerance poses a
challenge to a straightforward assessment of the physiological function
of apoptosis in lung injury. Further investigations leading to the
identification of cell types undergoing apoptosis in these models of
tolerance to hyperoxia will be helpful in delineating the functional
significance of apoptosis in oxidant-injured lung.
 |
ACKNOWLEDGEMENTS |
A. M. K. Choi was supported by National Heart, Lung, and Blood
Institute First Award R29-HL-55330 and National Institute of Allergy
and Infectious Diseases Grant RO1-AI-42365. S. Horowitz was supported
in part by Basic Research Grant 1-FY96-0752 from the March of Dimes
Birth Defects Foundation and grants from the National Institutes of
Health and Winthrop-University Hospital.
 |
FOOTNOTES |
Address for reprint requests and present address of A. M. K. Choi:
Section of Pulmonary and Critical Care Medicine, Yale Univ. School of
Medicine, 333 Cedar St., LCI 105, New Haven, CT 06520.
Received 17 July 1997; accepted in final form 20 March 1998.
 |
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