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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

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.

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.

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. black-square, Young rats pretreated with saline before hyperoxia exposure; square , young rats pretreated with LPS before hyperoxia exposure; bullet , aged rats pretreated with saline before hyperoxia exposure; open circle , 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.

    DISCUSSION
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Materials & Methods
Results
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References

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-alpha , 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-alpha and X-irradiation in cultured cells can be inhibited by the transcription factor nuclear factor-kappa B (NF-kappa B). Interestingly, we did not observe differences in the induction of NF-kappa 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-kappa B activation in cultured epithelial cells after hydrogen peroxide treatment and that the hyperoxic induction of NF-kappa 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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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 275(1):L14-L20
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