1Division of Pulmonary, Allergy and Critical Care Medicine, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213; 3Howard Hughes Medical Institute and Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520; and 4Howard Hughes Medical Institute and Program in Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
Submitted 14 November 2002 ; accepted in final form 17 March 2003
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ABSTRACT |
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hyperoxia; mitogen-activated protein kinase; oxidative stress; stress response; oxygen toxicity; apoptosis
Eukaryotic cells respond to stress conditions, such as hyperoxia, with adaptive genetic responses. The mitogen-activated protein kinase (MAPK) signal transduction pathways, which comprise at least three distinct families, represent ubiquitous inducible mechanisms of eukaryotic cell regulation. Among these, the extracellular signal-regulated kinase (ERK) pathway responds to activation by mitogenic stimuli such as hormones and growth factors (i.e., insulin). The c-Jun NH2-terminal kinase (JNK) or stress-activated protein kinase pathways and the p38 MAPK pathways respond preferentially to environmental stress (i.e., oxidants, ultraviolet radiation) and proinflammatory cytokines (5, 9, 15, 16). The JNKs originate from at least three distinct genes (Jnk1, Jnk2, Jnk3), although multiple isoforms may arise from alternative splicing events (8, 10, 15, 16). Although the functions of the p38 MAPK and JNK pathways remain incompletely understood, these pathways are known to regulate cell growth, apoptosis, and inflammatory response (6, 14, 23, 26).
Previously we have shown that hyperoxia induces apoptotic cell death in a murine macrophage cell line (RAW 264.7), which depends on the activation of the ERK signaling pathway (22). Oxidative stress is known to induce all three major MAPK pathways (ERK, JNK, p38 MAPK) in various experimental models (8, 14, 15, 28). We hypothesized that the inactivation of the hyperoxia-inducible JNK pathway would alter the in vivo response to hyperoxia. In the current study, we show that gene-deleted mice with either Jnk1-/- or Jnk2-/- geno-type display increased susceptibility to the lethal effects of hyperoxia. This enhanced susceptibility is associated with increased epithelial cell apoptosis but, paradoxically, decreased airway leukocyte influx.
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METHODS |
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Hyperoxia exposure in mice. The animals were exposed to >98% O2 at a flow rate of 12 l/min in a 3.7-ft3 glass exposure chamber. The animals were supplied food and water throughout the exposure. For survival studies, animals were monitored hourly, and the time of death was noted. For the measurement of markers of lung injury, separate groups of animals were removed from the chamber at predetermined time points and killed under anesthesia.
Lipopolysaccharide exposure in mice. Mice were placed in a wire mesh cage, which was in turn placed inside an airtight Plexiglas chamber. Lipopolysaccharide (LPS) was nebulized into the chamber continuously for 1 h with a model 40 nebulizer (DeVilbiss, Somerset, PA), which generates 5-µm-diameter particles. The aerosol was directed against a deflector plate at the entrance of the chamber to ensure even dispersal of the LPS. The aerosol was drawn through the chamber at a steady rate by a blower motor (Grainger, Baltimore, MD) connected to the exit port.
Measurement of lung injury markers. The Jnk1-/- mice and their controls were removed from the exposure chamber and killed after 65 h of continuous exposure to hyperoxia. The animals were killed with an overdose of ketamine and transection of the heart. Bronchoalveolar lavage (BAL, 35 ml/kg) was performed four times with PBS (pH 7.4). Cell pellets were pooled from the lavages and resuspended in PBS. The supernatant from the first lavage was saved and frozen for protein determination. Cell counts were performed with a Neubauer hemocytometer (VWR, Boston, MA). For differential analysis, samples were cytocentrifuged and stained with Diff-Quik (Fisher Scientific, Pittsburgh, PA). The protein concentration in each sample was determined by a standard Bradford assay (Sigma, St. Louis, MO) using BSA as the standard. Separate animals were used for myeloperoxidase (MPO) determination (13). After exsanguination, the entire left lung was excised and frozen at -70°C for MPO measurement, whereas the right lung was weighed and desiccated in a 60°C oven to determine the wet-to-dry lung weight. The frozen lung samples were homogenized in phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide, sonicated for 15 s, and further disrupted by a freeze-thaw cycle in liquid nitrogen. The extract was centrifuged at 20,000 g for 20 min at 4°C. We assayed the supernatants for MPO activity by measuring the absorbance at 460 nm, using a 0.167 mg/dl solution of o-dianisidine dihydrochloride in 0.0005% hydrogen peroxide as the substrate. MPO activity is expressed per gram of dry weight.
Western analysis of lung JNK phosphorylation. Assay kits were purchased from New England Biolabs (Beverly, MA) and used per manufacturer's instructions. The protocol is described briefly: following exposure to hyperoxia (65 h), lungs from C57BL/6 mice were excised and homogenized in buffer A [25 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leukopeptin, and 1 µg/ml aprotinin]. Protein concentrations were determined with the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Twenty micrograms of protein were boiled for 5 min and loaded into a 12% polyacrylamide gel. Proteins were separated by SDS-PAGE. After SDS-PAGE, the proteins were transferred to a nitrocellulose membrane. Membranes were incubated with blocking buffer [5% nonfat dry milk in 10% Tween in Tris-buffered saline (TTBS)] for 3 h, washed with TTBS, and then incubated overnight in the corresponding rabbit polyclonal primary antibody directed against phosphorylated JNK. The following day, membranes were washed in TTBS, and proteins were visualized with horseradish peroxidase-conjugated anti-rabbit IgG and the enhanced chemiluminescence assay (Amersham Life Sciences, Arlington Heights, IL). All membranes were subsequently stripped using standard stripping solution (100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris · HCl, pH 6.8) at 50°C. Membranes were reprobed with rabbit polyclonal antibody targeting total nonphosphorylated JNK to assure equal loading.
Northern analysis of lung heme oxygenase-1 mRNA. After exposure to hyperoxia (65 h), lungs from Jnk1-/- and wild-type mice were excised and homogenized. Total RNA was extracted from lung homogenates by solubilization in TRIzol lysis buffer (GIBCO BRL, Carlsbad, CA), followed by chloroform extraction. Total RNA (10 µg) was electrophoresed on a 1% agarose gel and then transferred to GeneScreen Plus membrane (DuPont NEN, Boston, MA). The content and integrity of the RNA were confirmed by ethidium bromide staining of the gel. The membranes were prehybridized and then hybridized at 65°C in Church and Gilbert's buffer containing denatured salmon sperm DNA and a 32P-labeled fragment of rat heme oxygenase (HO)-1 cDNA. The cDNA was labeled with the random primer kit (Amersham, Piscataway, NJ). Nylon membranes were washed 2x 25 min at 65°C in wash buffer A (0.5% BSA, 5% SDS, and 1 mM EDTA in 40 mM phosphate buffer, pH 7.0) and then washed 3x 25 min in wash buffer B (1% SDS, 1.0 mM EDTA, and 40 mM phosphate buffer, pH 7.0), followed by autoradiography.
Microscopic analysis of cell death. After 65-h exposure to hyperoxia, lungs were harvested and inflation fixed with 2% paraformaldehyde in PBS and immersed in the same fixative for 1 h. The lungs were then placed in 30% sucrose in PBS overnight and flash frozen in liquid nitrogen-cooled isopentane. Five-micrometer lung cryosections were cut and washed sequentially in PBS, then in PBS containing 0.5% BSA and 0.15% glycine. The In Situ Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany) was used to detect DNA strand breaks generated during apoptosis. The sections were washed and incubated in a 1:200 dilution of rat anti-mouse macrophage/monocyte antibody (Serotec, Raleigh, NC) for 1 h. After three washes in BSA, the sections were stained with a 1:3,000 dilution of cy3-conjugated AffinPure Goat Anti-Rat IgG (heavy + light chain) (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h, washed, and stained with Hoechst dye (Sigma) for 30 s and mounted in Gelvatol (Monsanto, St Louis, MO). Images were collected with an Olympus Provis microscope (Olympus, Tokyo, Japan) as well as an Olympus Floview scanning confocal microscope.
cDNA array of mouse lung RNA after exposure to hyperoxia. RNA was extracted from the lungs of four Jnk1-/- and four wild-type mice after 65 h of exposure to hyperoxia as described above. The RNA from each group was then pooled. Probe mixtures were synthesized by reverse transcribing both RNA populations with the cDNA primer mix provided by Clontech Laboratories/BD Biosciences (Palo Alto, CA) with the commercially available Atlas cDNA mouse array. The radioactively labeled probe mixes were each hybridized to separate nylon cDNA expression arrays and washed according to the instructions provided. After washing, the hybridization patterns were analyzed by autoradiography. Housekeeping genes included as positive controls on the microarray membrane were used to normalize the hybridization signals. Quantitation of gene expression was performed by Clontech Laboratories/BD Biosciences with proprietary software.
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 difference was accepted at P < 0.05.
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RESULTS |
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Effect of Jnk1 gene deletion on hyperoxia survival time in mice. To determine the effect of Jnk1 on the survival of mice exposed to hyperoxia, we placed mice in an atmosphere of >98% oxygen, and the time of death was recorded. After 80 h of continuous exposure, 86% of wild-type mice (n = 14) remained alive, whereas only 32% of Jnk1-/- were still living (n = 22, Fig. 2, P < 0.05). Jnk2-/- mice demonstrated a similar but less-pronounced susceptibility to hyperoxia (data not shown). The range of survival for Jnk1-/- mice was 6886 h, for Jnk2-/- 6794 h, and for wild-type mice 72125 h.
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Effect of Jnk1 gene deletion on hyperoxia-induced lung injury markers. After 65 h of continuous hyperoxia, Jnk1-/- mice displayed a higher concentration of BAL protein, a marker of lung injury (2), relative to wild-type controls (Fig. 3A) (P < 0.05). The 65-h time point was chosen because this was several hours before the Jnk1-/- mice would begin to succumb to hyperoxia. Again, a similar but less-pronounced trend was seen in Jnk2-/- mice (data not shown). The expression of HO-1, an oxidative stress-responsive gene, was also assayed in lung tissue. After 65 h of continuous exposure to hyperoxia (>98% oxygen), total RNA was extracted from whole lungs and analyzed by Northern blotting. The levels of HO-1 mRNA were substantially higher in three lungs taken from Jnk1-/- mice than in three wild-type controls (Fig. 3B), confirming a higher level of oxidative stress in the JNK1 gene-deleted mice.
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Effect of Jnk1 gene deletion on hyperoxia-induced airway leukocyte content. Jnk1-/- mice, which displayed increased susceptibility to hyperoxia and exhibited higher BAL protein content, had markedly lower levels of leukocytes in BAL than wild-type mice (Fig. 4). The BAL leukocyte counts of Jnk2-/- mice were also significantly reduced relative to wild-type controls, though to a lesser extent than in Jnk1-/- mice (data not shown). The differences in BAL leukocyte counts between the Jnk1-/- or Jnk2-/- mice and control mice were highly statistically significant (P < 0.00001).
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Lung MPO activity. To determine the relationship between BAL leukocyte count and neutrophil content, we measured MPO activity in the lung before and after exposure to hyperoxic conditions. The Jnk1-/- mice had higher basal MPO activity in the lung than wild-type mice (Fig. 5, P < 0.05). Despite lower leukocyte counts in the BAL, Jnk1-/- mice displayed a similar level of MPO activity to control mice after continuous hyperoxia treatment.
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Effect of LPS pretreatment on airway neutrophil influx and hyperoxia-induced mortality in mouse strains. To examine whether the difference in BAL leukocyte count between groups of animals is associated with the abnormal leukocyte trafficking and to test whether the increased susceptibility of Jnk1-/- mice is specific to the hyperoxia model, we used nebulized LPS to stimulate neutrophil migration into the airway. BAL neutrophil content was similar in Jnk1-/- and wild-type mice after LPS treatment (Fig. 6A). Previously, it has been shown that LPS pretreatment confers cross-tolerance against subsequent hyperoxia challenge (3, 28). Pretreatment with LPS prolonged the survival of both wild-type and Jnk1-/- mice to a similar extent during continuous hyperoxia exposure (Fig. 6B).
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Lung apoptosis in mouse strains exposed to hyperoxia. The JNKs participate in the regulation of stress-induced apoptosis (31). Because hyperoxic stress induces apoptosis in the lungs of mice and rats (1820), we examined the effect of hyperoxia on lung apoptosis in Jnk1-/- mice relative to wild-type mice. Lung sections were obtained from mice exposed to either normoxia or hyperoxia and analyzed by an in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay, which labels the 3'-carboxy termini of DNA fragments generated by apoptosis-associated endonuclease activity (29). After hyperoxic exposure, the total number of TUNEL-positive cells was higher in the lungs from Jnk1-/- mice than those of wild-type mice (Fig. 7, A and B). High-powered microscopic analysis of hyperoxia-injured lungs with specific staining for macrophages was undertaken to determine the predominant cell type undergoing apoptosis. Fluorescent images of TUNEL are overlaid with differential-interference-contrast images to show the structure of the underlying lung tissue. From these images (Fig. 7, C and D), it is apparent that most of the TUNEL-positive cells are epithelial cells.
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cDNA array of mouse lung RNA after exposure to hyperoxia. To uncover some of the genes that might contribute to the susceptibility of the Jnk1-/- mice or that might be markers of severity of hyperoxic lung injury, we performed a cDNA microarray. After 65 h of continuous exposure to hyperoxia (>98% oxygen), total RNA was extracted from whole lungs. Microarrays were then probed with cDNA from pooled groups of four Jnk1-/- and four control mice. A total of 79 differentially expressed genes were uncovered; an extract of those data is shown in Fig. 8. Autoradiographs of the hybridized membranes are shown in Fig. 8, bottom.
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DISCUSSION |
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That said, the increased susceptibility of Jnk1-/- or Jnk2-/- mice to hyperoxia suggests that the JNK pathways participate in protective responses to oxidative injury. Hyperoxia-induced lung injury occurs more rapidly and severely in mice lacking the Jnk1 or Jnk2 genes as shown by decreased survival time, increased BAL protein content, and activation of the stress-response gene Ho-1, a general marker of oxidative stress in the lung (1). Deletion of Jnk1, however, does not affect the development of LPS-induced chemotolerance, suggesting that the JNK pathway does not contribute to this process. The mechanisms underlying LPS-induced chemotolerance remain unclear but may involve the upregulation of stress-activated genes such as manganese superoxide dismutase (28).
Although both Jnk1-/- and Jnk2-/- mice proved to be more susceptible to hyperoxia than wild-type mice, this susceptibility was more pronounced in Jnk1-/- mice. This finding is in keeping with the observation by Tournier et al. (30) that Jnk1 gene disruption causes a larger decrease in JNK activity in vitro than that caused by disruption of the Jnk2 gene. Given the more exaggerated response of the Jnk1-/- mice, this strain was used exclusively for many of the above studies.
The discordance between airway leukocyte counts and survival in our model
is intriguing. Hyperoxic lung injury is characterized by leukocyte
accumulation within pulmonary microvessels and subsequent leukocyte
transmigration into the alveolar wall and air spaces
(19). A substantial proportion
of the leukocytes generally found in the microcirculation and alveolar septae
of hyperoxia-exposed lungs are neutrophils
(19). The dramatically
decreased number of leukocytes in the airways of the Jnk gene-deleted
mice in our study was contrary to what would be expected in the setting of
increased injury. The JNK pathway has been shown to play a role in
TNF--induced expression of E-selectin on endothelial cells
(25), which is critical for
leukocyte adhesion and infiltration. The results of the MPO assay indicate
that the total neutrophil content of the Jnk1-/-
mouse lungs is no different from control, supporting the hypothesis that there
is a defect in neutrophil margination or trafficking. With this hypothesis in
mind, we tested the response to inhaled nebulized LPS, a known inducer of
airway neutrophilia. The normal response of
Jnk1-/- mice to nebulized LPS suggests that if
there is a defect in neutrophil trafficking in these mice, it must be specific
to particular stimuli or modes of lung injury.
The JNK pathway is thought to play a major role in cellular apoptosis, but the functional significance of apoptosis in response to hyperoxia remains poorly defined. Mantell et al. (18) reported that the amount of hyperoxia-induced apoptosis in a mouse model correlates with the extent of lung injury. The authors found that a strain of mice resistant to hyperoxia displays a lower level of apoptosis than a hyperoxia-sensitive strain. In contrast, Otterbein et al. (20) reported that older rats display increased tolerance to hyperoxia yet exhibit higher lung apoptotic indexes than rats that were sensitive to hyperoxia. In these studies, the apoptotic index was inversely proportional to the extent of lung injury and correlated with increased survival, opposite to the findings reported here. A major limitation in interpreting such conflicting data arises from the difficulty to determine which specific cell types are responsible for the apoptotic signals in TUNEL assays of lung tissue sections. Our data, which demonstrate that epithelial cells are the predominant cells affected in the hyperoxia-sensitive Jnk1-/- mice, support the notion that increased pneumocyte apoptosis is a consequence of more severe oxidative injury. The baseline elevation of lung MPO activity in Jnk1-/- mice suggests the possibility of impaired neutrophil apoptosis in these mice, but the TUNEL signal from lung leukocytes was low in both Jnk1-/- and control animals.
The broad spectrum of cellular functions that involve JNK signaling gives
rise to many potential future avenues of investigation in this model. One
method for defining future studies is to begin with an assessment of
differentially expressed genes. Gene array technology allows for comparative
analysis of genome-wide patterns of mRNA expression from which inferences
about overall cell function and the function of individual gene components can
be drawn. Using this technique, we have compared the lungs of
Jnk1-/- mice and control mice after exposure to
hyperoxia and identified 79 differentially expressed genes. The particular
commercial array that was used can identify a maximum of 1,176 mouse genes
present at an abundance of 1020 copies per cell or greater. The genes
that were identified belonged to several broad functional classes including
cell signaling, transcription, growth factors, stress response, and matrix
remodeling. Some of the genes that were differentially induced relate to
already identified functions of JNK. For instance, T cell deathassociated
protein (TDAG51) was downregulated in the Jnk1-/-
mouse lungs relative to control. This gene has been shown to play an essential
role in induction of apoptosis by coupling T cell receptor stimulation to Fas
expression (21), and JNK1 is
known to regulate T cell apoptosis and proliferation
(27). Another gene, growth
arrest and DNA damage-inducible protein 45 was highly upregulated in the
Jnk1-/- mouse lungs; this gene has been shown to
mediate the activation of the JNK pathway and cytokine production in effector
TH1 cells (17). Perhaps more
interesting from the point of view of hyperoxic lung injury is the increase in
genes involved in tissue remodeling. Several genes related to transforming
growth factor- were differentially expressed in the
Jnk1-/- mice, as were insulin-like growth factor
binding protein 3, tissue inhibitor of metalloproteinases 2, and several
matrix components. Other genes such as
6-integrin could be
more directly involved in the increased susceptibility of the
Jnk1-/- mice. The abundance of mRNA for this
integrin was fivefold lower in the Jnk1-/-
animals than in wild type; this gene has been implicated in apoptosis, cell
adhesion to the extracellular matrix, and transmigration of neutrophils across
the endothelial cell barrier
(12,
24,
25). The differentially
expressed genes highlighted by the array do not allow for any specific
conclusions to be drawn. Gene array analysis provides a starting point,
however, for the development of hypotheses to explain why mice deficient in
the JNK MAPK signaling pathway are so susceptible to hyperoxic lung
injury.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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