Mitogen-activated protein kinase pathway mediates hyperoxia-induced apoptosis in cultured macrophage cells

Irina Petrache1, Mary E. Choi2,3, Leo E. Otterbein4, Beek Yoke Chin4, Lin L. Mantell5, Stuart Horowitz5, and Augustine M. K. Choi2,6

6 Section of Pulmonary and Critical Care Medicine and 3 Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine, New Haven 06520; 2 Connecticut Veterans Affairs HealthCare System, West Haven, Connecticut 06516; 5 Departments of Thoracic Cardiovascular Surgery and Pediatrics, The CardioPulmonary Research Institute, Winthrop-University Hospital, State University of New York at Stony Brook School of Medicine, Mineola, New York 11501; and 1 Division of Pulmonary and Critical Care Medicine and 4 Department of Environmental Health Sciences, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have previously demonstrated that the lungs of mice can exhibit increased programmed cell death or apoptosis after hyperoxic exposure in vivo. In this report, we show that hyperoxic exposure in vitro can also induce apoptosis in cultured murine macrophage cells (RAW 264.7) as assessed by DNA-laddering, terminal deoxynucleotidyltransferase dUTP nick end-labeling, and nucleosomal assays. To further delineate the signaling pathway of hyperoxia-induced apoptosis in RAW 264.7 macrophages, we first show that hyperoxia can activate the mitogen-activated protein kinase (MAPK) pathway, the extracellular signal-regulated kinases (ERKs) p42/p44, in a time-dependent manner as assessed by increased phosphorylation of ERK1/ERK2 by Western blot analyses. Neither the c-Jun NH2-terminal kinase/stress-activated protein kinase nor the p38 MAPK was activated by hyperoxia in these cells. Chemical or genetic inhibition of the ERK p42/p44 MAPK pathway by PD-98059, a selective inhibitor of MAPK kinase, and dominant negative mutants of ERK, respectively, attenuated hyperoxia-induced apoptosis as assessed by DNA laddering and nucleosomal ELISAs. Taken together, our data suggest that hyperoxia can induce apoptosis in cultured murine macrophages and that the MAPK pathway mediates hyperoxia-induced apoptosis.

programmed cell death; oxygen; signal transduction; extracellular signal-regulated kinase; cell death


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

EXPOSURE TO HYPEROXIA, or supraphysiological concentrations of O2, is associated at the cellular level with an accumulation of reactive oxygen species (ROS) such as superoxide, hydroxyl radicals, and hydrogen peroxide (H2O2), with resultant damage of proteins, lipids, and DNA (10, 11). When the oxidant insult is no longer compensated by the host's antioxidant defense mechanisms, cell injury and death ensue (6, 8, 22, 26). The lung in particular is a major target of oxidative injury in a variety of disease states including acute respiratory distress syndrome, lung fibrosis, and transplant rejection (8, 11, 23). Moreover, the supplemental O2 administered to patients with such lung injury can in itself add to the oxidative burden already present.

Experimentally, the exposure of animals to hyperoxia causes a form of acute lung injury similar to that seen in human acute respiratory distress syndrome, a pathological process characterized by edema and influx of inflammatory cells into the lung where the cells release toxic ROS capable of initiating or amplifying lung injury (6, 8, 23). Recent studies by our laboratories (26, 29) have also suggested that a major histological feature of hyperoxia-induced lung injury in vivo is programmed cell death or apoptosis. Apoptosis is an active form of cellular demise characterized by cell shrinkage, DNA fragmentation, and nuclear condensation; unlike necrosis, apoptosis is not associated with architectural distortion of the tissues and does not trigger an inflammatory response (31). Kazzaz et al. (22) have demonstrated that hyperoxia induces alveolar epithelial cell necrosis and not apoptosis in human lung A549 epithelial cells, whereas oxidants such as H2O2 trigger apoptosis of these cells. We show in this study that hyperoxia can induce apoptosis in vitro depending on the cell type. We demonstrate in murine macrophages that hyperoxic toxicity might be responsible for this phenomenon.

The signal transduction pathways of hyperoxia-induced cell necrosis, or hyperoxia-induced apoptosis in particular, are not yet fully elucidated. It is known that hyperoxia induces the activation of transcription factors including activator protein-1 and nuclear factor-kappa B (23, 24). Both of these transcription factors have been implicated in the signaling pathways in programmed cell death (16, 25, 27, 35). Moreover, extracellular stimuli such as growth factors and cellular stresses including DNA damage, hyperosmolarity, oxidative stress, and mechanical stress are known to activate cascades of protein kinases that participate in signal transduction; of these, mitogen-activated protein kinase (MAPK) family members play an important role in cellular growth and differentiation (33, 36, 37). Recent data (13, 15, 20) have demonstrated that the MAPK pathway plays an important role in mediating apoptosis in various in vitro models. Based on these observations, we hypothesized that hyperoxia activates the MAPK pathway and that this activation, in turn, is important in mediating hyperoxia-induced apoptosis. We demonstrate that hyperoxia activates the extracellular signal-regulated kinase (ERK) p42/p44 MAPK pathway preferentially and that the ERK MAPK pathway is necessary for mediating hyperoxia-induced apoptosis in murine RAW 264.7 macrophage cells.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell culture and hyperoxic exposures. Murine peritoneal macrophages (RAW 264.7 cells), alveolar macrophages (MHS cells), and L929 fibroblasts were cultured in Dulbecco's modified Eagle's medium and RPMI medium containing 10% fetal bovine serum (FBS; GIBCO BRL, Life Technologies, Grand Island, NY), 100 U/ml of penicillin, and 100 µg/ml of streptomycin. Rat alveolar macrophages (NR 8383) and human pulmonary epithelial (A549) cells were maintained in Ham's F-12 medium containing 10% FBS (GIBCO BRL). The cultures were maintained at 37°C in a humidified atmosphere of 5% CO2-95% air. The cells were exposed to hyperoxia (95% O2-5% CO2) in a tightly sealed modular chamber (Billup-Rothberg, Del Mar, CA) at 37°C.

Cell transfections. RAW 264.7 cells were transfected with a dominant negative mutant of the ERK MAPKs (9) (provided by Dr. Andrew Larner, Food and Drug Administration, Bethesda, MD) as previously described in our laboratory (22). Two dominant negative mutant constructs were used: TEYE, where Thr183 and Tyr185 residues required for phosphorylation were replaced with glutamic acid, and TAYE, where the same residues were replaced with alanine and phenylalanine, respectively (9). The wild-type (WT) ERK MAPK was obtained from Dr. Larner (9). RAW 264.7 cells transfected with the neomycin gene or WT ERK MAPK as previously described (22) were used as controls.

Genomic DNA isolation DNA-laddering assay. Genomic DNA was isolated from cultured cells with the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). Briefly, the cells were lysed after medium removal with lysis buffer followed by a 1-h incubation with RNase A. The cell lysates were precipitated for proteins and spun at 2,000 g for 15 min. The supernatant was precipitated with isopropanol for isolation of DNA. After an alcohol wash, DNA was hydrated and quantified. Equal amounts (20 µg) of DNA were electrophoresed on a 1.5% agarose gel (with incorporated ethidium bromide) in 1× Tris-acetate buffer. The gel was then photographed under ultraviolet (UV) luminescence.

Nucleosomal ELISA. Cell lysates were isolated, and ELISAs were performed according to the manufacturer's protocol (Calbiochem, San Diego, CA).

Terminal deoxynucleotidyltransferase dUTP nick end-labeling method in cells. A two-step binding assay was used to label the 3'-hydroxyl ends of the DNA breaks, resulting in fluorescent labeling of the apoptotic cells. An APO-BRDU kit (Phoenix Flow Systems, San Diego, CA) combines terminal deoxynucleotidyltransferase to catalyze extensions of the 3'-hydroxyl ends and Br-dUTP to substitute for thymidine. FITC-conjugated anti-5-bromo-2'-deoxyuridine results in the green labeling. The cells were resuspended in RNase and propidium iodide to hydrolyze the double-stranded RNA and to stoichiometrically label total DNA, respectively. Apoptosis was correlated with cell cycle by generating two independent fluorescent signals on the FACStarplus flow cytometer.

MAPK activation assays. The various MAPK assays were performed according to the manufacturer's instructions (New England Biolabs, Beverly, MA). Briefly, cells were lysed in a buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta -glycerophosphate, 1 mM Na3VO4, 1 µg/ml of leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and sheared by passage through a 25-gauge needle. Protein concentrations of the cell lysates were determined by Coomassie blue dye-binding assay (Bio-Rad, Hercules, CA). Total protein (200-µg) samples were incubated with phospho-specific p42/44 MAPK antibody (1:50) overnight on a rocker at 4°C. The p42/44 MAPK antibodies detect only the Tyr204-phosphorylated forms of ERK1 and ERK2 and thus select for the activated (phosphorylated) MAPK. The samples were analyzed by 12% SDS-PAGE and electroblotted by Western blot as per the manufacturer's protocol. After overnight incubation with the primary antibody at 4°C, the membrane was incubated for 1 h with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2,000) at room temperature with gentle rocking. The proteins were subsequently detected with LumiGLO (New England Biolabs) and exposed to X-ray film. p38 MAPK activity was measured with a PhosphoPlus p38 MAPK (Thr180/Tyr182) antibody kit (New England Biolabs) according to the manufacturer's instructions. A PhosphoPlus stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK; Thr183/Tyr185) antibody kit (New England Biolabs) was used to measure JNK phosphorylation or activity. The MAPK kinase (MEK) inhibitor PD-98059 chemical was purchased from New England Biolabs.

O2 exposure of animals. Eight-week-old pathogen-free male C57BL/6 mice were purchased from Jackson Laboratories and allowed to acclimate on arrival for 7 days before experimentation. The animals were fed rodent chow and water ad libitum. The animals were exposed to hyperoxia (>99% O2) at a flow rate of 12 l/min in a 3.70-cubic foot Plexiglas exposure chamber. The mice were supplied with rodent chow and water ad libitum during the exposure. At the start of the exposure, the chamber was humidified for 10-15 min. These experiments were carried out according to the animal protocol approved by the Animal Care and Use Committee.

Lung tissue preparation. The lungs were fixed by perfusion with 10% Formalin at 20 cmH2O 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 twice in fresh xylene for 5 min each to remove the paraffin. The slides were rehydrated though a series of graded alcohols and then washed in distilled water for 3 min each.

Terminal deoxynucleotidyltransferase dUTP nick end-labeling assay in tissue sections and photomicrography. The terminal deoxynucleotidyltransferase dUTP nick end-labeling (TUNEL) method was used for the apoptosis assay of lung tissue sections as previously described (26).

TUNEL reagents, including a rhodamine-conjugated anti-digoxigenin Fab fragment, were obtained from Boehringer Mannheim (Indianapolis, IN). The nonspecific signals or autofluorescence was excluded by subtracting the signals generated from the TUNEL assay with signals generated by all reagents except the terminal deoxynucleotidyltransferase in the assay. Tissue sections were counterstained with 2 µg/ml of 4',6-diamidine-2-phenylindole dihydrochloride (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). The images were captured with a charge-coupled device video camera. Uniform camera control settings were used for image capture, and image threshholding was identical for all images. The captured images were analyzed with the Image 1 system (Universal Imaging, West Chester, PA) running on a personal computer. At least 25 fields were analyzed from at least two animals at each time point.

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). Significant difference was accepted at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hyperoxia induces apoptosis in macrophages in vitro. To determine whether hyperoxia induces cell death via programmed cell death (apoptosis), genomic DNA was isolated from RAW 264.7 cells after hyperoxic (>95% O2) treatment and analyzed for DNA laddering. Figure 1A shows marked DNA laddering at 24 h of hyperoxic treatment (lane 2) in contrast to cells exposed to normoxia (lane 1). Agents such as endotoxin, ATP, or H2O2, which have been shown to induce apoptosis in other cell types, did not induce apoptosis in RAW 264.7 cells (Fig. 1A). We also observed that hyperoxic exposure induced apoptosis in another macrophage cell line, MHS cells, as assessed by DNA-laddering assay (Fig. 1B) and nucleosomal assay (control, 0.05 nucleosomal U/mg protein; hyperoxia for 16 h, 0.39 nucleosomal U/mg protein). To further complement our DNA-laddering assays and to better quantify the effect of hyperoxia-induced apoptosis, identical experiments were performed with the TUNEL and nucleosomal assays. Both assays further demonstrate evidence of apoptosis in RAW 264.7 cells after hyperoxia (Fig. 2).



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Fig. 1.   Hyperoxia induces apoptosis in murine macrophage cells. A: genomic DNA was isolated from RAW 264.7 cells exposed to hyperoxia for 24 h and fractionated with 1.5% agarose gel electrophoresis as described in EXPERIMENTAL PROCEDURES. Lane 1, control normoxia; lane 2, 95% O2 for 24 h; lane 3, lipopolysaccharide (LPS; 10 µg/ml) for 24 h; lane 4, 5 mM ATP for 6 h; lane 5, 600 µM H2O2 for 6 h. B: genomic DNA was isolated from MHS cells exposed to hyperoxia and fractionated with 1.5% agarose gel electrophoresis as described in EXPERIMENTAL PROCEDURES. Lane 1, control normoxia; lane 2, 95% O2 for 4 h; lane 3, 95% O2 for 8 h; lane 4, 95% O2 for 16 h.



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Fig. 2.   A: terminal deoxynucleotidyltransferase dUTP nick end-labeling (TUNEL) analysis of RAW 264.7 cells exposed to hyperoxia. Cells were analyzed for positive TUNEL staining as described in EXPERIMENTAL PROCEDURES. 1, Control normoxia; 2, 95% O2 for 24 h. Data are means ± SE of 3 independent experiments. * P < 0.001 compared with normoxic control cells. B: nucleosomal ELISA of RAW 264.7 cells exposed to hyperoxia. Cells lysates were isolated after 24 h of hyperoxic exposure, and ELISA was performed as described in EXPERIMENTAL PROCEDURES. 1, Control normoxia; 2, 95% O2 for 24 h. Data are means ± SE of 3 independent experiments. * P < 0.006 compared with normoxic control cells.

Hyperoxia induces apoptosis in mouse lungs. Our laboratories (26, 29) have previously reported that hyperoxia increases the total apoptotic index in the lungs of both rats and mice. To examine whether lung alveolar macrophages contributed to the increased total apoptotic index in the lungs after hyperoxic exposure in vivo, lung sections were obtained from mice exposed to hyperoxia and analyzed for apoptotic signals by an in situ TUNEL assay, which labels the 3'-OH ends of DNA cut by endonucleases that are activated during apoptosis (26). A marked increase in TUNEL staining was observed in cells resembling macrophages in the alveoli of lungs from mice exposed to hyperoxia compared with mice exposed to normoxia alone (Fig. 3).


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Fig. 3.   Apoptosis of alveolar macrophages and lung parenchyma. Normoxic (A) and hyperoxic (B) mouse lungs were isolated after 72 h of 100% O2 exposure and fixed and embedded as described in EXPERIMENTAL PROCEDURES. Lung tissue sections were processed for TUNEL assay to identify apoptotic cells (TUNEL stain) and dual labeled with 4',6-diamidine-2-phenylindole dihydrochloride (DAPI) to visualize all nuclei in the field. Differential interference contrast (DIC) image was digitally enhanced to emphasize surface topology. Arrows, presumptive alveolar macrophage in lumen of alveolus; arrowheads, epithelial cells of alveolar wall. Bar, 15 µm.

Hyperoxia activates the ERK p42/p44 MAPK signal transduction pathway in murine macrophages. Extracellular stimuli such as growth factors, cellular stresses including DNA damage, hyperosmolarity, oxidative stress, and mechanical stress are known to activate cascades of protein kinases that participate in signal transduction. The MAPK pathway represents one major signaling pathway of these cellular stressors (33, 36). We hypothesized that hyperoxia activates the MAPK pathway in cultured macrophages. To determine the ERK p42/p44 MAPK activity in response to hyperoxia, we selectively immunoblotted cellular proteins isolated from RAW 264.7 cells after hyperoxic treatment with a monoclonal phospho-specific antibody to p42/p44 MAPK (Thr202 and Tyr204). Increased ERK p42/p44 MAPK activity was observed as early as 30 min, which returns to baseline at 8-24 h of hyperoxic exposure (Fig. 4). In contrast, exposure of RAW 264.7 cells to hyperoxia did not result in activation of the JNK or p38 MAPK pathway (Fig. 5).


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Fig. 4.   Kinetics of extracellular signal-regulated kinase [ERK; p42/p44 mitogen-activated protein kinase (MAPK)] activity of RAW 264.7 cells exposed to hyperoxia. Lane 1, control normoxia; lane 2, 30 min of hyperoxia; lane 3, 1 h of hyperoxia; lane 4, 2 h of hyperoxia; lane 5, 4 h of hyperoxia; lane 6, 8 h of hyperoxia; lane 7, 24 h of hyperoxia. Blot is representative of 4 independent experiments. Nos. on right, molecular mass.



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Fig. 5.   A: kinetics of stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) activity of RAW 264.7 cells exposed to hyperoxia. Lane 1, control normoxia; lane 2, 30 min of hyperoxia; lane 3, 1 h of hyperoxia; lane 4, 2 h of hyperoxia; lane 5, 4 h of hyperoxia; lane 6, 8 h of hyperoxia; lane 7, 30 min of ultraviolet irradiation as positive control. Blot is representative of 3 independent experiments. B: kinetics of p38 MAPK activity of RAW 264.7 cells exposed to hyperoxia. Lane 1, control normoxia; lane 2, 15 min of hyperoxia; lane 3, 30 min of hyperoxia; lane 4, 4 h of hyperoxia; lane 5, 4 h of 1 µg/ml of LPS as positive control. Blot is representative of 3 independent experiments.

Requirement of ERK p42/p44 MAPK signaling pathway for hyperoxia-induced apoptosis in macrophages. Based on our observations that hyperoxia induces apoptosis and activates the ERK p42/p44 MAPK pathway in RAW 264.7 cells, we hypothesized that the ERK p42/p44 MAPK pathway may mediate hyperoxia-induced apoptosis in RAW 264.7 cells. To test this hypothesis, our strategy was to inhibit ERK MAPK genetically and chemically and then examine whether inhibition of the ERK MAPK pathway modulates hyperoxia-induced apoptosis. First, RAW 264.7 cells were transfected with dominant negative mutants of ERK MAPK (9) and then were exposed to hyperoxia. DNA laddering was detected in the WT ERK MAPK RAW 264.7 cells after hyperoxic treatment but not in the TEYE dominant negative mutant cells (Thr183 and Tyr185 residues required for phosphorylation were replaced with glutamic acid), suggesting that the ERK pathway plays an important role in mediating hyperoxia-induced apoptosis (Fig. 6). Identical results were obtained with the TAYE dominant negative mutant cells (Thr183 and Tyr185 residues were replaced with alanine and phenylalanine, respectively; data not shown). To further confirm the role of the ERK p42/p44 MAPK pathway in hyperoxia-induced apoptosis, we treated RAW 264.7 cells with PD-98059, a selective inhibitor of MEK (located upstream from ERK) before the hyperoxic exposure and then examined the cells for DNA laddering. As seen in Fig. 7, RAW 264.7 cells when exposed to hyperoxia alone (lane 3) exhibited marked DNA laddering, whereas cells exposed to hyperoxia in the presence of PD-98059 exhibited attenuation of DNA laddering (lane 4). To better quantify the effect of inhibition of ERK MAPK on hyperoxia-induced apoptosis, we performed nucleosomal ELISA assays in TEYE dominant negative mutant cells in the presence of hyperoxia. As shown in Fig. 8, a marked increase in nucleosomal units (index of apoptosis) were observed in RAW 264.7 neo control cells after hyperoxia (lane 2; P < 0.03 compared with normoxic control cells in lane 1). In contrast, no significant increase in nucleosomal units was observed in TEYE dominant negative mutant cells after hyperoxia (Fig. 8, lane 4) compared with that after normoxia (Fig. 8, lane 3; P = not significant). Statistical analysis of nucleosomal units (index of apoptosis) between RAW 264.7 neo control cells and TEYE dominant negative mutant cells after hyperoxia demonstrated significant differences between these two groups (P < 0.06). We observed similar attenuation of hyperoxia-induced nucleosomal units (index of apoptosis) in RAW 264.7 cells exposed to hyperoxia in the presence of the chemical inhibitor PD-98059 (data not shown).


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Fig. 6.   Effect of dominant negative mutants of ERK on hyperoxia-induced apoptosis. Genomic DNA was isolated from cells exposed to hyperoxia for 24 h and then fractionated with 1.5% agarose gel electrophoresis as described in EXPERIMENTAL PROCEDURES. Left: lane 1, wild-type (WT) ERK MAPK RAW 264.7 cells in normoxia; lane 2, WT ERK MAPK RAW 264.7 cells in hyperoxia. Right: lane 1, TEYE dominant negative mutant RAW 264.7 cells in normoxia; lane 2, TEYE dominant negative mutant RAW 264.7 cells in hyperoxia.



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Fig. 7.   Effect of MAPK kinase (MEK) inhibitor on hyperoxia-induced apoptosis. Genomic DNA was isolated from cells exposed to hyperoxia for 24 h in absence and presence of PD-98059 (10 µM), a specific MEK inhibitor, and then fractionated with 1.5% agarose gel electrophoresis as described in EXPERIMENTAL PROCEDURES. Lane 1, normoxia; lane 2, PD-98059 alone; lane 3, hyperoxia; lane 4, PD-98059 plus hyperoxia.



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Fig. 8.   Effect of dominant negative mutants of ERK on hyperoxia-induced apoptosis by nucleosomal ELISA. Cells lysates were obtained at 24 h of hyperoxic exposure, and nucleosomal ELISA was performed as described in EXPERIMENTAL PROCEDURES. 1, RAW 264.7 neo control cells in normoxia; 2, RAW 264.7 neo control cells after 24 h in hyperoxia; 3, TEYE dominant negative mutant RAW 264.7 cells in normoxia; 4, TEYE dominant negative mutant RAW 264.7 cells after 24 h of hyperoxia. Data are means ± SE of 5 independent experiments. * P < 0.03 compared with control cell in normoxia; ** P < 0.006 compared with control cells after 24 h of hyperoxia.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It is well established that direct exposure of cultured cells to ROS such as H2O2, superoxide-generating agents, and glutathione depletors induce cell death via induction of programmed cell death or apoptosis in many model systems. Although hyperoxia is a form of oxidant stress, being associated with an accumulation of ROS and triggering common antioxidant responses, recent data (22) suggest that hyperoxia to a large extent induces cell death via cell necrosis rather than via programmed cell death. For example, lung epithelial cells (A549) die by cell necrosis when exposed to hyperoxia (22). Human bronchial epithelial cells, rat alveolar macrophages (NR 8383 cells), and murine fibroblasts (L929 cells) do not exhibit genomic DNA fragmentation (laddering) when exposed to hyperoxia (data not shown). We show in this study that murine macrophages (RAW 264.7 and MHS cells), in contrast to rat macrophages (NR 8383 cells), have the capacity to undergo programmed cell death or apoptosis after hyperoxic exposure, suggesting species specificity of the hyperoxic insult. Moreover, the complexity of programmed cell death is further highlighted by recent observations (1, 19, 21, 34) demonstrating cell-type specificity in that hyperoxia can cause apoptosis in cells such as human small-airway epithelial cells, retinal capillary endothelial cells, cerebral endothelial cells, and pheochromocytoma cells.

Given the fact that in vivo exposure to hyperoxia is associated with significant apoptosis in the lungs of mice and rats (26, 29), it is possible that the alveolar macrophage is the predominant cell population that contributes to this effect (Fig. 3), although other cell types may also be contributing to the increased apoptotic index of mouse lungs exposed to hyperoxia. Rigorous studies are needed in the future to accurately determine the type of cells undergoing apoptosis both quantitatively and qualitatively.

During the apoptotic process, macrophages assume the role of scavengers, "silently" engulfing adjacent apoptotic cells and possibly contributing to the resolution of acute pulmonary inflammation (7, 31). On the other hand, macrophages possess the ability to undergo programmed cell death or apoptosis themselves as observed in this study in response to hyperoxia and to other cellular stressors such as asbestos, bleomycin, nitric oxide and peroxynitrite, endotoxin, and interferon-gamma (2, 3, 14, 17, 30). The functional significance of macrophage apoptosis to these stimuli, and hyperoxic injury in particular, is not well established. Based on the known observations that macrophages can undergo apoptosis to many cellular stimuli that are potent activators of macrophages (3, 14, 17, 30), one can speculate that macrophage cell death via apoptosis may represent a mechanism by which the host attempts to minimize ongoing inflammatory process during macrophage activation.

To date, there have been three main branches of the MAPK pathway described, although there is considerable cross talk among them. The ERK p42/p44 MAPK pathway is activated by cell growth and mitogenic signals, DNA damage, or oxidative stress (33, 37, 38). SAPK/JNK and p38 protein kinase represent the two other branches of the MAPK pathway, being activated by cellular stimuli such as cytokines, oxidant stresses, and osmotic shock (13, 15, 28, 36). These three branches, ERK p42/p44, JNK, and p38, of the MAPK pathway have been implicated in the regulation of the signaling pathways leading to apoptosis depending on the various models. In our model, with early activation of the ERK p42/p44 MAPK pathway relative to the unaffected JNK and p38 MAPK pathways, it appears that the ERK p42/p44 MAPK pathway mediates hyperoxia-induced apoptosis. Interruption of the ERK p42/p44 branch of the MAPK signaling pathway, either genetically or chemically, blocked hyperoxia-induced apoptosis. The importance of the ERK p42/p44 MAPK pathway in mediating apoptosis has also been recently observed in other models of oxidant-induced apoptosis including asbestosis, UV irradiation, and tumor necrosis factor-alpha (12, 17, 18). However, the complexity of the MAPK pathway in mediating apoptosis is further highlighted by studies demonstrating the importance of either the JNK or p38 branch of the MAPK pathway in regulating the signaling pathways in the apoptotic process depending on cell type and cell stimulus. For example, UV irradiation-induced apoptosis in small cell lung cancer cells appears to be mediated by the JNK MAPK pathway (4, 5), whereas aspirin-induced apoptosis in fibroblasts is mediated by the p38 MAPK pathway (32). Additionally, both the JNK and p38 MAPK pathways have been shown to mediate nerve growth factor removal-induced apoptosis in PC-12 pheochromocytoma cells (38).

In summary, our study demonstrates that hyperoxia causes apoptosis in cultured murine macrophages and that among the three branches of the MAPK pathway, the ERK p42/p44 pathway appears to play an important role in mediating hyperoxia-induced apoptosis. Murine macrophage cell lines will serve as a useful cell culture model to investigate the signaling pathway(s) upstream from the ERK p42/p44 MAPK pathway mediating hyperoxia-induced apoptosis and to further understand the functional significance of hyperoxia-induced apoptosis.


    ACKNOWLEDGEMENTS

A. M. K. Choi was supported by National Heart, Lung, and Blood Institute Grants R01-HL-55330 and R01-HL-60234; National Institute of Allergy and Infectious Diseases Grant R01-AI-42365; and an American Heart Association Established Investigator Award. 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. L. L. Mantell was supported in part by grants from the American Lung Association and the Stony Wold-Herbert Fund. M. E. Choi was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-01298-09 and by a Veterans Affairs Career Development Award.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: 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 (E-mail: augustine.choi{at}yale.edu).

Received 17 September 1998; accepted in final form 7 April 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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