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Pretreatment with interferon-γ protects microglia from oxidative stress via up-regulation of Mn-SOD
Free Radical Biology & Medicine 46 (2009) 1204–1210
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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f r e e r a d b i o m e d
Original Contribution
Pretreatment with interferon-γ protects microglia from oxidative stress via up-regulation of Mn-SOD Xia Chen a,b, In Young Choi b, Tong-Shin Chang c, You Hyun Noh c, Chan Young Shin d, Chun-Fu Wu a, Kwang Ho Ko e, Won-Ki Kim b,⁎ a
Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China Department of Neuroscience, College of Medicine, Korea University, Seoul, Republic of Korea College of Pharmacy and Division of Life and Pharmaceutical Sciences, Ewha Woman's University, Seoul, Republic of Korea d Department of Pharmacology, School of Medicine, Konkuk University, Seoul, Republic of Korea e Department of Pharmacology, College of Pharmacy, Seoul National University, Seoul, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 23 May 2008 Revised 29 January 2009 Accepted 29 January 2009 Available online 9 February 2009 Keywords: Interferon-γ Microglia Oxidative stress Hydrogen peroxide Free radicals
a b s t r a c t Microglial cells, resident macrophage-like immune cells in the brain, are exposed to intense oxidative stress under various pathophysiological conditions. For self-defense against oxidative injuries, microglial cells must be equipped with antioxidative mechanisms. In this study, we investigated the regulation of antioxidant enzyme systems in microglial cells by interferon-γ (IFN-γ) and found that pretreatment with IFN-γ for 20 h protected microglial cells from the toxicity of various reactive species such as hydrogen peroxide (H2O2), superoxide anion, 4-hydroxy-2(E)-nonenal, and peroxynitrite. The cytoprotective effect of IFN-γ pretreatment was abolished by the protein synthesis inhibitor cycloheximide. In addition, treatment of microglial cells with both IFN-γ and H2O2 together did not protect them from the H2O2-evoked toxicity. These results imply that protein synthesis is required for the protection by IFN-γ. Among various antioxidant enzymes such as manganese or copper/zinc superoxide dismutase (Mn-SOD or Cu/Zn-SOD), catalase, and glutathione peroxidase (GPx), only Mn-SOD was up-regulated in IFN-γ-pretreated microglial cells. Transfection with siRNA of Mn-SOD abolished both up-regulation of Mn-SOD expression and protection from H2O2 toxicity by IFN-γ pretreatment. Furthermore, whereas the activities of Mn-SOD and catalase were up-regulated by IFN-γ pretreatment, those of Cu/Zn-SOD and GPx were not. These results indicate that IFN-γ pretreatment protects microglial cells from oxidative stress via selective up-regulation of the level of Mn-SOD and activity of Mn-SOD and catalase. © 2009 Elsevier Inc. All rights reserved.
Microglial cells are generally considered the “macrophageal cells in the central nervous system” and have important functions, especially after brain injury and during inflammation. In the healthy brain, microglial cells are quiescent and have a ramified morphology; however, they become activated in any pathological condition of the brain [1,2]. Activated microglial cells produce and release a large number of biologically active substances, including reactive oxygen (ROS) and nitrogen species [3]. Microglia-derived reactive species such as hydrogen peroxide (H2O2) and peroxynitrite are thought to play a defensive role in pathological conditions. However, the reactive species may in turn harm microglial cells. For self-protection against oxidative damage, therefore, microglial cells must be equipped with antioxidative defense mechanisms. They are shown to contain a high concentration of glutathione and substantial activities of antioxidative enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, as well ⁎ Corresponding author. Fax: +82 2 953 6095. E-mail address: [email protected] (W.-K. Kim). 0891-5849/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2009.01.027
as NADPH-regenerating enzymes. Their highly effective antioxidative potential may protect microglial cells from oxidative damage that could impair important functions of these cells in defense and repair of the brain. In various neuropathological situations, interferon-γ (IFN-γ) is produced by activated T lymphocytes and natural killer cells [4]. IFNγ modulates immunological responses and stimulates the production of reactive nitrogen and oxygen species and cytokines/ chemokines in microglia [5–7]. Combined treatment of IFN-γ and lipopolysaccharides has previously been shown to increase the expression of manganese superoxide dismutase (Mn-SOD) in macrophages or the macrophage tumor line RAW264.7 [8,9]. Our recent preliminary study showed that microglial cells in pure culture are highly vulnerable to H2O2. In the present study, therefore, we examined whether IFN-γ could reduce the susceptibility of microglial cells to exogenous H2O2 and found that IFN-γ reduced the toxicity of H2O2 as well as other oxidants toward these cells via selective up-regulation of the level of Mn-SOD and activities of MnSOD and catalase.
X. Chen et al. / Free Radical Biology & Medicine 46 (2009) 1204–1210
Materials and methods Reagents H2O2, xanthine, xanthine oxidase, cycloheximide (CHX), rotenone, and 3-amino-1,2,4-triazole (3-AT) were obtained from Sigma–Aldrich (St. Louis, MO, USA). IFN-γ was purchased from Calbiochem (Darmstadt, Germany) and 4-hydroxynonenal from Cayman Chemical Co. (Ann Arbor, MI, USA). Potassium cyanide was obtained from Aldrich Chemical Co. (Milwaukee, WI, USA). Antibodies against catalase (LF-PA0060), Cu/Zn-SOD (LF-PA0013), and Mn-SOD (LFPA0021) were obtained from LabFrontier (Seoul, Korea). Antiglutathione peroxidase (GPx) (ab8850) and anti-β-actin (ab6276) antibodies were purchased from Novus Biologicals (Littleton, CO, USA) and Abcam (Seoul, Korea), respectively.
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ment of the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] (Sigma Chemical Co.). Briefly, cells were incubated with 0.25 mg/ml MTT for 1 h at 37°C and formazan products were dissolved in DMSO. The amount of formazan formed was measured by absorbance change using a microplate reader (Molecular Devices, Palo Alto, CA, USA). The result was expressed as a percentage of control cells (100%). Nitrite assay NO production was determined by measuring nitrite, a stable oxidation product of NO. An aliquot of the conditioned medium was mixed with an equal volume of 0.1% N-1-naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid. Absorbance at 550 nm was determined using a microplate reader (Molecular Devices).
Cell culture Measurement of mitochondrial transmembrane potential (ΔΨm) in cells Rat microglial-enriched cultures were prepared from Sprague– Dawley rats. Cerebral cortices dissected from pups (postnatal day 1 or 2) were freed of meninges, minced manually, and triturated slowly with a pipette in serum-free minimum essential medium (MEM). The cells were plated into poly-D-lysine (1 μg/ml)-coated 75-cm2 T flasks and maintained in MEM containing 10% fetal bovine serum (FBS) (Hyclone) and penicillin and streptomycin 100 mg/ml (37°C, 5% CO2). After 6 or 7 days, the flasks were vigorously agitated on a rotary shaker at 200 rpm for 1 min. The medium was collected in a sterile culture tube and centrifuged at 2000 rpm for 200 s. Cells were seeded onto plates at a density of 3 × 105 cells/ml. After 12 h, microglia were used for experiments.
Microglia cultured on plates were loaded for 20 min at 37°C with JC-1 (1.0 μg/ml) in culture medium. Depolarization of ΔΨm was assessed by measuring the fluorescence intensities at 530- and 590nm emission wavelengths using a fluorescence microplate reader (SpectraMax GeminiEM; Molecular Devices). During measurements, cells were maintained at 37°C and protected from light. Fluorescence intensity was measured every 3 min. All fluorescence measurements were corrected for autofluorescence. Autofluorescence of cells without JC-1 load was constant throughout the experiment. In control experiments, no photobleaching was observed during fluorescence monitoring.
Assessment of cell injury or death Antioxidant enzyme activity assay Cell injury or death was assessed by morphological examination using phase-contrast microscopy or fluorescence microscopy. For fluorescence microscopy, cells were fixed for 15 min with 4% paraformaldehyde at room temperature. Cells were then washed twice with phosphate-buffered saline (PBS) and stained with propidium iodide (PI; 2 μg/ml) for 20 min at room temperature. After three washes with PBS, fluorescence images were acquired using a fluorescence microscope (Leica DMIL) equipped with a Leica camera (DFC 420C). Cell injury or death was quantified by counting the PI-positive cells or measuring the amount of lactate dehydrogenase (LDH) released into the bathing medium, using a diagnostic kit (Sigma Chemical Co., St. Louis, MO, USA). Cell viability was expressed as a percentage of total LDH, which was measured in sister cultures frozen and thawed after the experiments. Cell viability was also quantified by measure-
After treatment with IFN-γ for 20 h, microglia were stimulated with H2O2 for 1 h. For SOD activity assay, microglial cells were washed with warm PBS and collected by centrifugation at 3000 rpm for 10 min. Cell pellets were lysed in cold 20 mM Hepes buffer (pH 7.2) containing 1 mM EGTA, 210 mM mannitol, 70 mM sucrose, and 0.3% Triton X-100. The lysate was recentrifuged at 13,000 rpm for 5 min. Total SOD and Mn-SOD activities in the supernatant were determined using a superoxide dismutase assay kit (Cayman Chemical). For MnSOD activity assay, 2 mM potassium cyanide was added to the lysate to inhibit Cu/Zn-SOD. The activity of Cu/Zn-SOD was calculated as the difference between the total SOD and the Mn-SOD activities. For catalase activity assay, microglia were washed with warm PBS and then lysed in PBS containing 1% Triton X-100. The lysate was centrifuged at 13,000 rpm for 15 min. Catalase activity in the
Fig. 1. Pretreatment of IFN-γ attenuates H2O2 toxicity. Microglial cells (3 × 105 cells/ml) were incubated with IFN-γ (100 U/ml) for 20 h and then treated with H2O2 (100 μM) in the absence of IFN-γ. (A–D) Eight hours after H2O2 treatment, cell morphology was observed with a phase-contrast microscope, and (a–d) necrotic cell death was determined with PI staining. (A and a) Untreated; (B and b) IFN-γ pretreated; (C and c) H2O2 treated; (D and d) IFN-γ pretreated and then H2O2 treated. Microphotographs are representatives of six separate experiments. (E) The cell viability was quantified by counting the PI-positive cells in square millimeters and expressed as percentage of total cell counts. N = 6.
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Fig. 2. Effects of IFN-γ cotreatment and pretreatment on the cytotoxicity induced by H2O2. (A and B) Microglial cells were (A) pretreated with IFN-γ (100 U/ml) for 20 h before or (B) cotreated with H2O2 (50, 100 μM). (C) Cells were pretreated with cycloheximide (CHX; 0.01 μM) for 1 h and then cotreated with IFN-γ (100 U/ml) for 20 h. To eliminate CHX and IFNγ, cells were rinsed with medium and then further incubated in the absence or presence of 100 μM H2O2. Three hours later, cell injury or death was determined by measuring the LDH release. Data are expressed as percentage of total LDH and represent means ± SEM. N = 4. ⁎p b 0.05; significantly different from each untreated group. #p b 0.05; significantly different from the LDH release in the cells treated with IFN-γ and H2O2.
supernatant was determined using a catalase assay kit (Oxis International, Foster City, CA, USA). For GPx activity assay, microglia were washed with warm PBS and lysed in Tris–HCl (50 mM, pH 7.5) containing 5 mM EDTA and 1 mM DTT. The lysate was centrifuged at 10,000 rpm for 15 min, and GPx activity in the supernatant was determined using a GPx assay kit (Oxis International). The results obtained from the antioxidant enzyme activity assay were expressed as the percentage of control. Western blot analysis Microglial cells were washed twice with PBS and then lysed for 10 min at 4°C in a solution containing 25 mM Hepes–NaOH (pH 7.4), 2 mM EGTA, 1% Triton X-100, 10% glycerol, 1 mM Na3VO4, 5 mM NaF, 1 mM 4-(2-aminoethyl)benzene sulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin. The lysate was centrifuged at 15,000 g for 10 min, and the protein concentration of the resulting supernatant was determined using a protein assay (Bio-Rad Laboratories). For SDS–PAGE, the lysate was diluted 1:5 with 5× sample buffer [10% SDS, 12.5% β-mercaptoethanol, 300 mM Tris (pH 6.8), 0.05% bromophenol blue, and 50% glycerol] and heated at 95°C for 2 min before resolution on SDS–PAGE gels. Proteins were transferred onto nitrocellulose membrane. Membranes were blocked with 5% nonfat milk in TBS/T [25 mM Tris (pH 8), 150 mM NaCl, and 0.1% Tween 20] and incubated with primary antibodies diluted 1:1000 in 5% milk in TBS/T. Reactive proteins were visualized by HRP-conjugated secondary antibodies (Amersham Biosciences, Buckinghamshire, England) and chemiluminescence using Western Lightning ECL (Amersham Biosciences).
SiRNA transfection Mn-SOD siRNA constructs were a mixture of three selected siRNA constructs against Mn-SOD (rat SOD II), provided by the Invitrogen Stealth Select RNAi library. The catalog numbers of the constructs are RSS302728, RSS302727, and RSS302729. As control, a mixture of two control siRNA (Cat. Nos. 12935-300 and 12935-200) was used. MnSOD siRNA and control siRNA were transfected into primary microglial cells using Lipofectamine 2000 reagent (Invitrogen Corp., Carlsbad, CA, USA) for 4 h, as recommended by the manufacturer, and incubated in the growth medium containing 10% FBS and penicillin/streptomycin for 48 h before further experiments. Statistical analysis Data are expressed as mean ± SEM and analyzed for statistical significance using ANOVA followed by Scheffe's test for multiple comparison. A p value of b0.05 was considered significant.
Results IFN-γ pretreatment protects microglia from H2O2 cytotoxicity Morphological examination and LDH measurement showed that H2O2 injured microglial cells rapidly, and the cytotoxicity of H2O2 was largely prevented by pretreatment of the cells with IFN-γ for 20 h before H2O2 treatment (Figs. 1A–D and 2A). Staining of injured
Fig. 3. Pretreatment of IFN-γ decreases microglial injury/death induced by various oxidants. Cells were treated with IFN-γ (100 U/ml) for 20 h and then exposed to (A) SIN-1 (500 μM), (B) xanthine/xanthine oxidase (X/XO) (100 μM/2 mU/ml), (C) HNE (0.5 μM), or (D) rotenone (0.1 μg/ml). Three hours later, cell death was determined by measuring the LDH release. N = 4. ⁎p b 0.05; significantly different from each paired untreated group.
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Fig. 4. Pretreatment with IFN-γ prevents H2O2-stimulated depolarization of ΔΨm. Microglial cells were incubated with IFN-γ (100 U/ml) for 20 h and then exposed to H2O2 (100 μM). Mitochondrial potentials were evaluated using JC-1 staining as described under Materials and methods. Cells were loaded with JC-1 (1.0 μg/ml), and fluorescence intensity at 530 nm (J monomer) and 590 nm (J aggregate) was measured every 3 min for 30 min. Time 0 indicates the time at which H2O2 was added. Data represent means ± SEM from four independent experiments and are expressed as the ratio of aggregate fluorescence to monomer fluorescence.
or dead cells with PI revealed that IFN-γ pretreatment markedly protected microglial cells from H2O2 cytotoxicity (Fig. 1E). On the other hand, concomitant treatment of the cells with IFN-γ and H2O2 did not prevent the H2O2-evoked toxicity (Fig. 2B). The protein
Fig. 5. Effects of IFN-γ pretreatment on the expression and activity of SOD. Cells were pretreated with IFN-γ (100 U/ml) for 20 h and then exposed to H2O2 (100 μM). One hour later, cells were collected for SOD assay. (A) The expression of Cu/Zn-SOD and MnSOD was determined by Western blot analysis. (B and C) The density ratios of Cu/ZnSOD/β-actin and Mn-SOD/β-actin are expressed as fold level, compared to untreated group. (D and E) The activities of cytosolic Cu/Zn-SOD and mitochondrial Mn-SOD were measured with a Cayman SOD activity assay kit. N = 4. ⁎p b 0.05; significantly different from control. #p b 0.05; significantly different from H2O2-treated group.
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synthesis inhibitor cycloheximide completely blocked the cytoprotective effect of IFN-γ, suggesting that protein synthesis is required for IFN-γ-mediated protection of microglial cells (Fig. 2C). In addition, IFN-γ pretreatment strongly reduced the toxicity caused by various oxidants such as peroxynitrite [3-morpholinosydnonimine (SIN-1)], superoxide anion (O2·-) released from xanthine/ xanthine oxidase, and a lipid peroxidation product, 4-hydroxy-2nonenal (HNE; Figs. 3A–C). Superoxide anions in the mitochondrial matrix are quickly dismutated by Mn-SOD, whereas those in the intermembranous space are dismutated by Zn/Cu-SOD [11]. Rotenone, a mitochondrial Complex I blocker, is well known to induce a large production of O2·- in mitochondria, resulting in cell death [12]. Thus, we further tested whether IFN-γ pretreatment prevented the toxicity of rotenone. As expected, we found that IFN-γ pretreatment significantly reduced the toxicity of rotenone (Fig. 3D). Mitochondria, which play vital roles in cell survival, are known to be highly vulnerable to oxidative stress, and we recently showed that H2O2 rapidly depolarizes the ΔΨm, resulting in cell injury or death [10]. Fig. 4 shows that pretreatment of microglial cells with IFN-γ inhibited the H2O2-evoked ΔΨm depolarization. Interestingly,
Fig. 6. Effects of IFN-γ pretreatment on the expression and activity of catalase and GPx. Cells were pretreated with IFN-γ (100 U/ml) for 20 h and then exposed to H2O2 (100 μM). One hour later, cells were collected for catalase and GPx assays. (A and B) The expression of catalase and GPx was determined by Western blot analysis, and the density ratios from Western blots for catalase and GPx were expressed as fold level compared with untreated group. (C and D) The activities of catalase and GPx were measured with Oxis activity assay kits. N = 4. ⁎p b 0.05; significantly different from control. (E) Cells were pretreated with IFN-γ (100 U/ml) for 20 h and then exposed to H2O2 (100 μM). 3-AT (2.5 mM) was added 17 h after IFN-γ treatment started. Three hours later, cell injury or death was determined by measuring the LDH release. N = 4. ⁎p b 0.05; significantly different between the groups.
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Transfection of Mn-SOD siRNA reduces the cytoprotection by IFN-γ pretreatment To further confirm whether up-regulation of Mn-SOD by IFN-γ pretreatment is associated with its cytoprotective effect against H2O2 toxicity, we down-regulated Mn-SOD expression by transfection of Mn-SOD siRNA. As seen in Fig. 7A, transfection of Mn-SOD siRNA blocked the increase in Mn-SOD expression that was induced by IFN-γ. In microglial cells transfected with control siRNA, IFN-γ pretreatment significantly inhibited the H2O2-induced cell death (Fig. 7B). In microglial cells transfected with Mn-SOD siRNA, however, IFN-γ pretreatment did not reverse the H2O2-induced cell death significantly, compared with the H2O2 toxicity shown in cells that were not treated with IFN-γ (Fig. 7B). Less toxicity of H2O2 was observed when microglia were transfected with Mn-SOD siRNA (Fig. 7B), most probably because of compensatory up-regulation of other cellular antioxidant systems. To exclude the off-target effect of siRNA experiments, we used an additional control siRNA that differs in only two nucleotides from the target sequence (i.e., 5′-AGAUUCUUCACGUAGGUCACGUGGU-3′). This additional control did not alter the expression level of Mn-SOD (97.3 ± 10.3% of control) or NO production in microglial cells (Fig. 7C). Because control as well as Mn-SOD siRNA-transfected cells showed similar levels of IFN-γinduced NO production, the results of siRNA seem to be specific against Mn-SOD. Cycloheximide does not alter the protein expression and activity of Mn-SOD in microglial cells
Fig. 7. Transfection of Mn-SOD siRNA abolishes the cytoprotective effect of IFN-γ. Twenty-four hours after transient transfection with Mn-SOD siRNA, cells were treated with IFN-γ for 20 h and then exposed to H2O2 (100 μM) for 1 h. (A) The expression of Mn-SOD was determined by Western blot analysis. (B) MTT reduction assay. Data represent means ± SEM from four independent experiments and are expressed as percentage of control. ⁎p b 0.05, ⁎⁎⁎p b 0.001; significantly different from matched groups. (C) The effect of off-target for the siRNA. The control siRNA used here differs in only two nucleotides from the target sequence (5′-AGAUUCUUCACGUAGGUCACGUGGU-3′). The density ratio of Mn-SOD/β-actin was expressed as fold change. The level of NO was assessed by measuring the nitrite formation. N = 4, ⁎p b 0.05; significantly different from control groups.
As shown in Figs. 1 and 2, CHX completely blocked the cytoprotective effect of IFN-γ pretreatment on H2O2 toxicity. However, CHX had previously been reported to increase the expression of MnSOD in certain cell lines such as HeLa, A549, and Kuramochi [13]. Thus, we further examined the protein expression and activity of Mn-SOD in CHX-treated microglial cells. Fig. 8 shows that, regardless of pretreatment with IFN-γ, CHX did not alter the protein expression or activity of Mn-SOD in microglial cells; however, CHX completely inhibited the increased protein expression and activity of Mn-SOD in IFN-γpretreated microglia.
however, pretreatment with IFN-γ alone significantly hyperpolarized the ΔΨm. IFN-γ pretreatment augments the expression of Mn-SOD Microglial cells are equipped with many antioxidant enzyme systems, such as SOD, catalase, and GPx [3]. Of two different SOD isoforms, the protein expression and activity of only mitochondrial Mn-SOD were up-regulated by pretreatment with IFN-γ (Fig. 5). In contrast, however, the protein expression and activity of cellular Cu/ Zn-SOD were significantly decreased by IFN-γ pretreatment as well as H2O2 (Fig. 5). Although the protein expression levels of cytosolic GPx and catalase were not significantly changed by pretreatment with IFN-γ (Figs. 6A and B), the activity of catalase, but not that of GPx, was significantly increased by IFN-γ pretreatment (Figs. 6C and D). Furthermore, 3-AT, a catalase inhibitor, partially reversed the cytoprotective effect of IFN-γ in H2O2-treated microglial cells (Fig. 6E). This implies that up-regulation of catalase activity is also in part involved in the cytoprotective effect of IFN-γ pretreatment on microglial cells.
Fig. 8. Effects of cycloheximide on the protein expression level and activity of Mn-SOD. Cells were pretreated with CHX (0.01 μM) for 1 h and then cotreated with CHX and IFN-γ(100 U/ml) for 20 h. The protein level was assessed by Western blot analysis (top). The activity of Mn-SOD was determined by using the Cayman SOD activity assay kit (bottom). Data represent means ± SEM from four independent experiments. ⁎p b 0.05; significantly different from control group.
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Discussion Many earlier studies showed that pretreatment of microglia with IFN-γ triggers the production of reactive nitrogen and oxygen species. However, our present study demonstrates that the increased oxidative stress caused by IFN-γ pretreatment was ameliorated by an enhancement of the resisting power of microglia. The protective effect of IFN-γ pretreatment was at least partly due to up-regulation of mitochondrial Mn-SOD. Thus, transfection of Mn-SOD siRNA decreased the survival of microglial cells under oxidative stress, induced by IFN-γ pretreatment. Expectedly, IFN-γ pretreatment blocked both exogenously added and endogenously produced reactive species. Hydrogen peroxide, a highly diffusible ROS, has been associated with neuronal cell injury and death in neurodegenerative diseases [14]. Indeed, morphological observation and LDH analysis in this study showed that H2O2 rapidly induced the death of cultured microglial cells: hydrogen peroxide can potentially oxidize proteins and lipids, resulting in injury of cellular microorganelles such as mitochondria [15,16]. Although mitochondria are the predominant source of ROS in most cell types, they are also vulnerable to oxidative stress [17,18], and ΔΨm is an important parameter of mitochondrial function and often used as an indicator of cell health. Oxidative stress depolarizes ΔΨm, and inhibition of depolarization protects cells from oxidant toxicity [10,19]. In this study, the degree of ΔΨ = m depolarization evoked by H2O2 was reduced when microglial cells were pretreated with IFN-γ. This inhibition of ΔΨm depolarization in IFN-γ-treated microglial cells may keep mitochondrial function intact and contribute to the cell survival of H2O2 toxicity. Treatment of microglial cells with cycloheximide, a protein synthesis inhibitor, during IFN-γ pretreatment reduced the cytoprotective effect of IFN-γ on H2O2 toxicity. On the other hand, treatment with IFN-γ alone during exposure to H2O2 failed to protect microglial cells from H2O2 cytotoxicity. These data indicate that newly synthesized proteins are required for the cytoprotective effect of IFN-γ in microglial cells. Increased levels of antioxidant enzymes in microglial cells would decrease the cell damage induced by H2O2 [20,21]. In this study, we found that IFN-γ pretreatment selectively increased the expression and activity of mitochondrial Mn-SOD; IFN-γ pretreatment decreased the expression and activity of even Cu/Zn-SOD. Transfection of Mn-SOD siRNA partly abolished the decrease in H2O2-evoked death of microglial cells after IFN-γ pretreatment. Similar to the present study, Mn-SOD has been shown to be an important antioxidant enzyme associated with many mitochondrial functions [22,23]. Thus, selective up-regulation of Mn-SOD could be of great help for the maintenance of mitochondrial function in oxidative stress. The molecular mechanisms underlying the protective effect of MnSOD have previously been explored by other studies [24,25]. In general, H2O2 itself is not a strong oxidant. In the presence of ferrous iron (Fe2+), however, H2O2 produces toxic hydroxyl radical (OH·) through the Fenton reaction [24]. Hydroxyl radical oxidizes thiol (–SH) groups of the mitochondrial permeability transition pore complex, thus inducing pore opening [26]. The opening of nonspecific channels in the mitochondrial inner membrane may cause a depolarization of ΔΨm. Thus, inhibition of OH· production may help maintain the ΔΨm and inhibit cell death induced by H2O2. Because endogenous production of O2·- is necessary for the oxidation of labile iron–sulfur clusters and the regeneration of Fe2+ from ferric iron (Fe3+), Mn-SOD, which can eliminate O2·- and block the supply of Fe2+, will efficiently decrease the generation of OH·. In addition, catalase and GPx have been shown to be oxidized and inactivated by oxidants, including O2·-. Thus, removal of O2·- by SOD would prevent inactivation of catalase and GPx [25]. Peroxidases such as catalase and GPx can directly detoxify H2O2 by converting it to water and oxygen. In this study, although pretreatment with IFN-γ did not alter the protein level and activity of GPx, it increased the activity
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of catalase with no change of its protein expression. Our experimental results obtained by using the catalase inhibitor 3-AT indicate that the increased catalase activity also contributes in part to the cytoprotection induced by IFN-γ pretreatment. Microglial cells have recently been shown to play either a beneficial or a harmful role in neuronal cell survival in various neurodegenerative diseases [27–30]. Therefore, a better understanding of the responses of microglial cells to certain kinds of cytokines such as IFN-γ may provide a valuable therapeutic clue for the treatment of such neurodegenerative diseases. Acknowledgments This study was supported by a grant (M103KV010010-06K220101010) from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, a grant from the Brain Korea 21 Project (to Dr. I.Y. Choi), and in part by a grant from Korea University, the Republic of Korea. References [1] Kreutzberg, G. W. Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19:312–318; 1996. [2] Garden, G. A.; Moller, T. Microglia biology in health and disease. J. Neuroimmune Pharmacol. 1:127–137; 2006. [3] Dringen, R. Oxidative and antioxidative potential of brain microglial cells. Antioxid. Redox Signal. 7:1223–1233; 2005. [4] Schroder, K.; Hertzog, P. J.; Ravasi, T.; Hume, D. A. Interferon–gamma: an overview of signals, mechanisms and functions. J. Leukoc. Biol. 75:163–189; 2004. [5] Zielasek, J.; Tausch, M.; Toyka, K. V.; Hartung, H. P. Production of nitrite by neonatal rat microglial cells/brain macrophages. Cell. Immunol. 141:111–120; 1992. [6] Pawate, S.; Shen, Q.; Fan, F.; Bhat, N. R. Redox regulation of glial inflammatory response to lipopolysaccharide and interferon-gamma. J. Neurosci. Res. 77:540–551; 2004. [7] Hausler, K. G.; Prinz, M.; Nolte, C.; Weber, J. R.; Schumann, R. R.; Kettenmann, H.; Hanisch, U. K. Interferon-gamma differentially modulates the release of cytokines and chemokines in lipopolysaccharide- and pneumococcal cell wall-stimulated mouse microglia and macrophages. Eur. J. Neurosci. 16:2113–2122; 2002. [8] Ferret, P. J.; Soum, E.; Negre, O.; Fradelizi, D. Auto-protective redox buffering systems in stimulated macrophages. BMC Immunol. 3:3; 2002. [9] Sano, H.; Hirai, M.; Saito, H.; Nakashima, I.; Isobe, K. I. A nitric oxide-releasing reagent, S-nitroso-N-acetylpenicillamine, enhances the expression of superoxide dismutases mRNA in the murine macrophage cell line RAW264-7. Immunology 92:118–122; 1997. [10] Lee, S. J.; Jin, Y.; Yoon, H. Y.; Choi, B. O.; Kim, H. C.; Oh, Y. K.; Kim, H. S.; Kim, W. K. Ciclopirox protects mitochondria from hydrogen peroxide toxicity. Br. J. Pharmacol. 145:469–476; 2005. [11] Turrens, J. F. Mitochondrial formation of reactive oxygen species. J. Physiol. 552:335–344; 2003. [12] Camello-Almaraz, C.; Gomez-Pinilla, P. J.; Pozo, M. J.; Camello, P. J. Mitochondrial reactive oxygen species and Ca2+ signaling. Am. J. Physiol., Cell Physiol. 291: C1082–1088; 2006. [13] Fujii, J.; Nakata, T.; Miyoshi, E.; Ikeda, Y.; Taniguchi, N. Induction of manganese superoxide dismutase mRNA by okadaic acid and protein synthesis inhibitors. Biochem. J. 301:31–34; 1994. [14] Tabner, B. J.; Turnbull, S.; El-Agnaf, O. M.; Allsop, D. Formation of hydrogen peroxide and hydroxyl radicals from A(beta) and alpha-synuclein as a possible mechanism of cell death in Alzheimer's disease and Parkinson's disease. Free Radic. Biol. Med. 32:1076–1083; 2002. [15] Floyd, R. A.; Carney, J. M. Free radical damage to protein and DNA: mechanisms involved and relevant observations on brain undergoing oxidative stress. Ann. Neurol. 32 (Suppl.):S22–27; 1992. [16] Janero, D. R.; Hreniuk, D.; Sharif, H. M. Hydrogen peroxide-induced oxidative stress to the mammalian heart-muscle cell (cardiomyocyte): lethal peroxidative membrane injury. J. Cell. Physiol. 149:347–364; 1991. [17] Nulton-Persson, A. C.; Szweda, L. I. Modulation of mitochondrial function by hydrogen peroxide. J. Biol. Chem. 276:23357–23361; 2001. [18] Sims, N. R.; Anderson, M. F.; Hobbs, L. M.; Kong, J. Y.; Phillips, S.; Powell, J. A.; Zaidan, E. Impairment of brain mitochondrial function by hydrogen peroxide. Brain Res. Mol. Brain Res. 77:176–184; 2000. [19] Medina, S.; Martinez, M.; Hernanz, A. Antioxidants inhibit the human cortical neuron apoptosis induced by hydrogen peroxide, tumor necrosis factor alpha, dopamine and beta-amyloid peptide 1-42. Free Radic. Res. 36:1179–1184; 2002. [20] Huang, Q.; Wu, L. J.; Tashiro, S.; Onodera, S.; Ikejima, T. Elevated levels of DNA repair enzymes and antioxidative enzymes by (+)-catechin in murine microglia cells after oxidative stress. J. Asian Nat. Prod. Res. 8:61–71; 2006. [21] Hou, R. C.; Wu, C. C.; Huang, J. R.; Chen, Y. S.; Jeng, K. C. Oxidative toxicity in BV-2 microglia cells: sesamolin neuroprotection of H2O2 injury involving activation of p38 mitogen-activated protein kinase. Ann. N.Y. Acad. Sci. 1042:279–285; 2005.
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[22] Venkataraman, S.; Wagner, B. A.; Jiang, X.; Wang, H. P.; Schafer, F. Q.; Ritchie, J. M.; Patrick, B. C.; Oberley, L. W.; Buettner, G. R. Overexpression of manganese superoxide dismutase promotes the survival of prostate cancer cells exposed to hyperthermia. Free Radic. Res. 38:1119–1132; 2004. [23] Keller, J. N.; Kindy, M. S.; Holtsberg, F. W.; St Clair, D. K.; Yen, H. C.; Germeyer, A.; Steiner, S. M.; Bruce-Keller, A. J.; Hutchins, J. B.; Mattson, M. P. Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction. J. Neurosci. 18:687–697; 1998. [24] Bruno-Barcena, J. M.; Andrus, J. M.; Libby, S. L.; Klaenhammer, T. R.; Hassan, H. M. Expression of a heterologous manganese superoxide dismutase gene in intestinal lactobacilli provides protection against hydrogen peroxide toxicity. Appl. Environ. Microbiol. 70:4702–4710; 2004.
[25] Chaudiere, J.; Ferrari-Iliou, R. Intracellular antioxidants: from chemical to biochemical mechanisms. Food Chem. Toxicol. 37:949–962; 1999. [26] Kowaltowski, A. J.; Castilho, R. F.; Vercesi, A. E. Mitochondrial permeability transition and oxidative stress. FEBS Lett. 495:12–15; 2001. [27] Streit, W. J. Microglia and neuroprotection: implications for Alzheimer's disease. Brain Res. Brain Res. Rev. 48:234–239; 2005. [28] Kim, Y. S.; Joh, T. H. Microglia, major player in the brain inflammation: their roles in the pathogenesis of Parkinson's disease. Exp. Mol. Med. 38:333–347; 2006. [29] Lai, A. Y.; Todd, K. G. Microglia in cerebral ischemia: molecular actions and interactions. Can. J. Physiol. Pharmacol. 84:49–59; 2006. [30] Streit, W. J.; Conde, J. R.; Fendrick, S. E.; Flanary, B. E.; Mariani, C. L. Role of microglia in the central nervous system's immune response. Neurol. Res. 27:685–691; 2005.
Contents lists available at ScienceDirect
Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f r e e r a d b i o m e d
Original Contribution
Pretreatment with interferon-γ protects microglia from oxidative stress via up-regulation of Mn-SOD Xia Chen a,b, In Young Choi b, Tong-Shin Chang c, You Hyun Noh c, Chan Young Shin d, Chun-Fu Wu a, Kwang Ho Ko e, Won-Ki Kim b,⁎ a
Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China Department of Neuroscience, College of Medicine, Korea University, Seoul, Republic of Korea College of Pharmacy and Division of Life and Pharmaceutical Sciences, Ewha Woman's University, Seoul, Republic of Korea d Department of Pharmacology, School of Medicine, Konkuk University, Seoul, Republic of Korea e Department of Pharmacology, College of Pharmacy, Seoul National University, Seoul, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 23 May 2008 Revised 29 January 2009 Accepted 29 January 2009 Available online 9 February 2009 Keywords: Interferon-γ Microglia Oxidative stress Hydrogen peroxide Free radicals
a b s t r a c t Microglial cells, resident macrophage-like immune cells in the brain, are exposed to intense oxidative stress under various pathophysiological conditions. For self-defense against oxidative injuries, microglial cells must be equipped with antioxidative mechanisms. In this study, we investigated the regulation of antioxidant enzyme systems in microglial cells by interferon-γ (IFN-γ) and found that pretreatment with IFN-γ for 20 h protected microglial cells from the toxicity of various reactive species such as hydrogen peroxide (H2O2), superoxide anion, 4-hydroxy-2(E)-nonenal, and peroxynitrite. The cytoprotective effect of IFN-γ pretreatment was abolished by the protein synthesis inhibitor cycloheximide. In addition, treatment of microglial cells with both IFN-γ and H2O2 together did not protect them from the H2O2-evoked toxicity. These results imply that protein synthesis is required for the protection by IFN-γ. Among various antioxidant enzymes such as manganese or copper/zinc superoxide dismutase (Mn-SOD or Cu/Zn-SOD), catalase, and glutathione peroxidase (GPx), only Mn-SOD was up-regulated in IFN-γ-pretreated microglial cells. Transfection with siRNA of Mn-SOD abolished both up-regulation of Mn-SOD expression and protection from H2O2 toxicity by IFN-γ pretreatment. Furthermore, whereas the activities of Mn-SOD and catalase were up-regulated by IFN-γ pretreatment, those of Cu/Zn-SOD and GPx were not. These results indicate that IFN-γ pretreatment protects microglial cells from oxidative stress via selective up-regulation of the level of Mn-SOD and activity of Mn-SOD and catalase. © 2009 Elsevier Inc. All rights reserved.
Microglial cells are generally considered the “macrophageal cells in the central nervous system” and have important functions, especially after brain injury and during inflammation. In the healthy brain, microglial cells are quiescent and have a ramified morphology; however, they become activated in any pathological condition of the brain [1,2]. Activated microglial cells produce and release a large number of biologically active substances, including reactive oxygen (ROS) and nitrogen species [3]. Microglia-derived reactive species such as hydrogen peroxide (H2O2) and peroxynitrite are thought to play a defensive role in pathological conditions. However, the reactive species may in turn harm microglial cells. For self-protection against oxidative damage, therefore, microglial cells must be equipped with antioxidative defense mechanisms. They are shown to contain a high concentration of glutathione and substantial activities of antioxidative enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, as well ⁎ Corresponding author. Fax: +82 2 953 6095. E-mail address: [email protected] (W.-K. Kim). 0891-5849/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2009.01.027
as NADPH-regenerating enzymes. Their highly effective antioxidative potential may protect microglial cells from oxidative damage that could impair important functions of these cells in defense and repair of the brain. In various neuropathological situations, interferon-γ (IFN-γ) is produced by activated T lymphocytes and natural killer cells [4]. IFNγ modulates immunological responses and stimulates the production of reactive nitrogen and oxygen species and cytokines/ chemokines in microglia [5–7]. Combined treatment of IFN-γ and lipopolysaccharides has previously been shown to increase the expression of manganese superoxide dismutase (Mn-SOD) in macrophages or the macrophage tumor line RAW264.7 [8,9]. Our recent preliminary study showed that microglial cells in pure culture are highly vulnerable to H2O2. In the present study, therefore, we examined whether IFN-γ could reduce the susceptibility of microglial cells to exogenous H2O2 and found that IFN-γ reduced the toxicity of H2O2 as well as other oxidants toward these cells via selective up-regulation of the level of Mn-SOD and activities of MnSOD and catalase.
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Materials and methods Reagents H2O2, xanthine, xanthine oxidase, cycloheximide (CHX), rotenone, and 3-amino-1,2,4-triazole (3-AT) were obtained from Sigma–Aldrich (St. Louis, MO, USA). IFN-γ was purchased from Calbiochem (Darmstadt, Germany) and 4-hydroxynonenal from Cayman Chemical Co. (Ann Arbor, MI, USA). Potassium cyanide was obtained from Aldrich Chemical Co. (Milwaukee, WI, USA). Antibodies against catalase (LF-PA0060), Cu/Zn-SOD (LF-PA0013), and Mn-SOD (LFPA0021) were obtained from LabFrontier (Seoul, Korea). Antiglutathione peroxidase (GPx) (ab8850) and anti-β-actin (ab6276) antibodies were purchased from Novus Biologicals (Littleton, CO, USA) and Abcam (Seoul, Korea), respectively.
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ment of the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] (Sigma Chemical Co.). Briefly, cells were incubated with 0.25 mg/ml MTT for 1 h at 37°C and formazan products were dissolved in DMSO. The amount of formazan formed was measured by absorbance change using a microplate reader (Molecular Devices, Palo Alto, CA, USA). The result was expressed as a percentage of control cells (100%). Nitrite assay NO production was determined by measuring nitrite, a stable oxidation product of NO. An aliquot of the conditioned medium was mixed with an equal volume of 0.1% N-1-naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid. Absorbance at 550 nm was determined using a microplate reader (Molecular Devices).
Cell culture Measurement of mitochondrial transmembrane potential (ΔΨm) in cells Rat microglial-enriched cultures were prepared from Sprague– Dawley rats. Cerebral cortices dissected from pups (postnatal day 1 or 2) were freed of meninges, minced manually, and triturated slowly with a pipette in serum-free minimum essential medium (MEM). The cells were plated into poly-D-lysine (1 μg/ml)-coated 75-cm2 T flasks and maintained in MEM containing 10% fetal bovine serum (FBS) (Hyclone) and penicillin and streptomycin 100 mg/ml (37°C, 5% CO2). After 6 or 7 days, the flasks were vigorously agitated on a rotary shaker at 200 rpm for 1 min. The medium was collected in a sterile culture tube and centrifuged at 2000 rpm for 200 s. Cells were seeded onto plates at a density of 3 × 105 cells/ml. After 12 h, microglia were used for experiments.
Microglia cultured on plates were loaded for 20 min at 37°C with JC-1 (1.0 μg/ml) in culture medium. Depolarization of ΔΨm was assessed by measuring the fluorescence intensities at 530- and 590nm emission wavelengths using a fluorescence microplate reader (SpectraMax GeminiEM; Molecular Devices). During measurements, cells were maintained at 37°C and protected from light. Fluorescence intensity was measured every 3 min. All fluorescence measurements were corrected for autofluorescence. Autofluorescence of cells without JC-1 load was constant throughout the experiment. In control experiments, no photobleaching was observed during fluorescence monitoring.
Assessment of cell injury or death Antioxidant enzyme activity assay Cell injury or death was assessed by morphological examination using phase-contrast microscopy or fluorescence microscopy. For fluorescence microscopy, cells were fixed for 15 min with 4% paraformaldehyde at room temperature. Cells were then washed twice with phosphate-buffered saline (PBS) and stained with propidium iodide (PI; 2 μg/ml) for 20 min at room temperature. After three washes with PBS, fluorescence images were acquired using a fluorescence microscope (Leica DMIL) equipped with a Leica camera (DFC 420C). Cell injury or death was quantified by counting the PI-positive cells or measuring the amount of lactate dehydrogenase (LDH) released into the bathing medium, using a diagnostic kit (Sigma Chemical Co., St. Louis, MO, USA). Cell viability was expressed as a percentage of total LDH, which was measured in sister cultures frozen and thawed after the experiments. Cell viability was also quantified by measure-
After treatment with IFN-γ for 20 h, microglia were stimulated with H2O2 for 1 h. For SOD activity assay, microglial cells were washed with warm PBS and collected by centrifugation at 3000 rpm for 10 min. Cell pellets were lysed in cold 20 mM Hepes buffer (pH 7.2) containing 1 mM EGTA, 210 mM mannitol, 70 mM sucrose, and 0.3% Triton X-100. The lysate was recentrifuged at 13,000 rpm for 5 min. Total SOD and Mn-SOD activities in the supernatant were determined using a superoxide dismutase assay kit (Cayman Chemical). For MnSOD activity assay, 2 mM potassium cyanide was added to the lysate to inhibit Cu/Zn-SOD. The activity of Cu/Zn-SOD was calculated as the difference between the total SOD and the Mn-SOD activities. For catalase activity assay, microglia were washed with warm PBS and then lysed in PBS containing 1% Triton X-100. The lysate was centrifuged at 13,000 rpm for 15 min. Catalase activity in the
Fig. 1. Pretreatment of IFN-γ attenuates H2O2 toxicity. Microglial cells (3 × 105 cells/ml) were incubated with IFN-γ (100 U/ml) for 20 h and then treated with H2O2 (100 μM) in the absence of IFN-γ. (A–D) Eight hours after H2O2 treatment, cell morphology was observed with a phase-contrast microscope, and (a–d) necrotic cell death was determined with PI staining. (A and a) Untreated; (B and b) IFN-γ pretreated; (C and c) H2O2 treated; (D and d) IFN-γ pretreated and then H2O2 treated. Microphotographs are representatives of six separate experiments. (E) The cell viability was quantified by counting the PI-positive cells in square millimeters and expressed as percentage of total cell counts. N = 6.
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Fig. 2. Effects of IFN-γ cotreatment and pretreatment on the cytotoxicity induced by H2O2. (A and B) Microglial cells were (A) pretreated with IFN-γ (100 U/ml) for 20 h before or (B) cotreated with H2O2 (50, 100 μM). (C) Cells were pretreated with cycloheximide (CHX; 0.01 μM) for 1 h and then cotreated with IFN-γ (100 U/ml) for 20 h. To eliminate CHX and IFNγ, cells were rinsed with medium and then further incubated in the absence or presence of 100 μM H2O2. Three hours later, cell injury or death was determined by measuring the LDH release. Data are expressed as percentage of total LDH and represent means ± SEM. N = 4. ⁎p b 0.05; significantly different from each untreated group. #p b 0.05; significantly different from the LDH release in the cells treated with IFN-γ and H2O2.
supernatant was determined using a catalase assay kit (Oxis International, Foster City, CA, USA). For GPx activity assay, microglia were washed with warm PBS and lysed in Tris–HCl (50 mM, pH 7.5) containing 5 mM EDTA and 1 mM DTT. The lysate was centrifuged at 10,000 rpm for 15 min, and GPx activity in the supernatant was determined using a GPx assay kit (Oxis International). The results obtained from the antioxidant enzyme activity assay were expressed as the percentage of control. Western blot analysis Microglial cells were washed twice with PBS and then lysed for 10 min at 4°C in a solution containing 25 mM Hepes–NaOH (pH 7.4), 2 mM EGTA, 1% Triton X-100, 10% glycerol, 1 mM Na3VO4, 5 mM NaF, 1 mM 4-(2-aminoethyl)benzene sulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin. The lysate was centrifuged at 15,000 g for 10 min, and the protein concentration of the resulting supernatant was determined using a protein assay (Bio-Rad Laboratories). For SDS–PAGE, the lysate was diluted 1:5 with 5× sample buffer [10% SDS, 12.5% β-mercaptoethanol, 300 mM Tris (pH 6.8), 0.05% bromophenol blue, and 50% glycerol] and heated at 95°C for 2 min before resolution on SDS–PAGE gels. Proteins were transferred onto nitrocellulose membrane. Membranes were blocked with 5% nonfat milk in TBS/T [25 mM Tris (pH 8), 150 mM NaCl, and 0.1% Tween 20] and incubated with primary antibodies diluted 1:1000 in 5% milk in TBS/T. Reactive proteins were visualized by HRP-conjugated secondary antibodies (Amersham Biosciences, Buckinghamshire, England) and chemiluminescence using Western Lightning ECL (Amersham Biosciences).
SiRNA transfection Mn-SOD siRNA constructs were a mixture of three selected siRNA constructs against Mn-SOD (rat SOD II), provided by the Invitrogen Stealth Select RNAi library. The catalog numbers of the constructs are RSS302728, RSS302727, and RSS302729. As control, a mixture of two control siRNA (Cat. Nos. 12935-300 and 12935-200) was used. MnSOD siRNA and control siRNA were transfected into primary microglial cells using Lipofectamine 2000 reagent (Invitrogen Corp., Carlsbad, CA, USA) for 4 h, as recommended by the manufacturer, and incubated in the growth medium containing 10% FBS and penicillin/streptomycin for 48 h before further experiments. Statistical analysis Data are expressed as mean ± SEM and analyzed for statistical significance using ANOVA followed by Scheffe's test for multiple comparison. A p value of b0.05 was considered significant.
Results IFN-γ pretreatment protects microglia from H2O2 cytotoxicity Morphological examination and LDH measurement showed that H2O2 injured microglial cells rapidly, and the cytotoxicity of H2O2 was largely prevented by pretreatment of the cells with IFN-γ for 20 h before H2O2 treatment (Figs. 1A–D and 2A). Staining of injured
Fig. 3. Pretreatment of IFN-γ decreases microglial injury/death induced by various oxidants. Cells were treated with IFN-γ (100 U/ml) for 20 h and then exposed to (A) SIN-1 (500 μM), (B) xanthine/xanthine oxidase (X/XO) (100 μM/2 mU/ml), (C) HNE (0.5 μM), or (D) rotenone (0.1 μg/ml). Three hours later, cell death was determined by measuring the LDH release. N = 4. ⁎p b 0.05; significantly different from each paired untreated group.
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Fig. 4. Pretreatment with IFN-γ prevents H2O2-stimulated depolarization of ΔΨm. Microglial cells were incubated with IFN-γ (100 U/ml) for 20 h and then exposed to H2O2 (100 μM). Mitochondrial potentials were evaluated using JC-1 staining as described under Materials and methods. Cells were loaded with JC-1 (1.0 μg/ml), and fluorescence intensity at 530 nm (J monomer) and 590 nm (J aggregate) was measured every 3 min for 30 min. Time 0 indicates the time at which H2O2 was added. Data represent means ± SEM from four independent experiments and are expressed as the ratio of aggregate fluorescence to monomer fluorescence.
or dead cells with PI revealed that IFN-γ pretreatment markedly protected microglial cells from H2O2 cytotoxicity (Fig. 1E). On the other hand, concomitant treatment of the cells with IFN-γ and H2O2 did not prevent the H2O2-evoked toxicity (Fig. 2B). The protein
Fig. 5. Effects of IFN-γ pretreatment on the expression and activity of SOD. Cells were pretreated with IFN-γ (100 U/ml) for 20 h and then exposed to H2O2 (100 μM). One hour later, cells were collected for SOD assay. (A) The expression of Cu/Zn-SOD and MnSOD was determined by Western blot analysis. (B and C) The density ratios of Cu/ZnSOD/β-actin and Mn-SOD/β-actin are expressed as fold level, compared to untreated group. (D and E) The activities of cytosolic Cu/Zn-SOD and mitochondrial Mn-SOD were measured with a Cayman SOD activity assay kit. N = 4. ⁎p b 0.05; significantly different from control. #p b 0.05; significantly different from H2O2-treated group.
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synthesis inhibitor cycloheximide completely blocked the cytoprotective effect of IFN-γ, suggesting that protein synthesis is required for IFN-γ-mediated protection of microglial cells (Fig. 2C). In addition, IFN-γ pretreatment strongly reduced the toxicity caused by various oxidants such as peroxynitrite [3-morpholinosydnonimine (SIN-1)], superoxide anion (O2·-) released from xanthine/ xanthine oxidase, and a lipid peroxidation product, 4-hydroxy-2nonenal (HNE; Figs. 3A–C). Superoxide anions in the mitochondrial matrix are quickly dismutated by Mn-SOD, whereas those in the intermembranous space are dismutated by Zn/Cu-SOD [11]. Rotenone, a mitochondrial Complex I blocker, is well known to induce a large production of O2·- in mitochondria, resulting in cell death [12]. Thus, we further tested whether IFN-γ pretreatment prevented the toxicity of rotenone. As expected, we found that IFN-γ pretreatment significantly reduced the toxicity of rotenone (Fig. 3D). Mitochondria, which play vital roles in cell survival, are known to be highly vulnerable to oxidative stress, and we recently showed that H2O2 rapidly depolarizes the ΔΨm, resulting in cell injury or death [10]. Fig. 4 shows that pretreatment of microglial cells with IFN-γ inhibited the H2O2-evoked ΔΨm depolarization. Interestingly,
Fig. 6. Effects of IFN-γ pretreatment on the expression and activity of catalase and GPx. Cells were pretreated with IFN-γ (100 U/ml) for 20 h and then exposed to H2O2 (100 μM). One hour later, cells were collected for catalase and GPx assays. (A and B) The expression of catalase and GPx was determined by Western blot analysis, and the density ratios from Western blots for catalase and GPx were expressed as fold level compared with untreated group. (C and D) The activities of catalase and GPx were measured with Oxis activity assay kits. N = 4. ⁎p b 0.05; significantly different from control. (E) Cells were pretreated with IFN-γ (100 U/ml) for 20 h and then exposed to H2O2 (100 μM). 3-AT (2.5 mM) was added 17 h after IFN-γ treatment started. Three hours later, cell injury or death was determined by measuring the LDH release. N = 4. ⁎p b 0.05; significantly different between the groups.
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Transfection of Mn-SOD siRNA reduces the cytoprotection by IFN-γ pretreatment To further confirm whether up-regulation of Mn-SOD by IFN-γ pretreatment is associated with its cytoprotective effect against H2O2 toxicity, we down-regulated Mn-SOD expression by transfection of Mn-SOD siRNA. As seen in Fig. 7A, transfection of Mn-SOD siRNA blocked the increase in Mn-SOD expression that was induced by IFN-γ. In microglial cells transfected with control siRNA, IFN-γ pretreatment significantly inhibited the H2O2-induced cell death (Fig. 7B). In microglial cells transfected with Mn-SOD siRNA, however, IFN-γ pretreatment did not reverse the H2O2-induced cell death significantly, compared with the H2O2 toxicity shown in cells that were not treated with IFN-γ (Fig. 7B). Less toxicity of H2O2 was observed when microglia were transfected with Mn-SOD siRNA (Fig. 7B), most probably because of compensatory up-regulation of other cellular antioxidant systems. To exclude the off-target effect of siRNA experiments, we used an additional control siRNA that differs in only two nucleotides from the target sequence (i.e., 5′-AGAUUCUUCACGUAGGUCACGUGGU-3′). This additional control did not alter the expression level of Mn-SOD (97.3 ± 10.3% of control) or NO production in microglial cells (Fig. 7C). Because control as well as Mn-SOD siRNA-transfected cells showed similar levels of IFN-γinduced NO production, the results of siRNA seem to be specific against Mn-SOD. Cycloheximide does not alter the protein expression and activity of Mn-SOD in microglial cells
Fig. 7. Transfection of Mn-SOD siRNA abolishes the cytoprotective effect of IFN-γ. Twenty-four hours after transient transfection with Mn-SOD siRNA, cells were treated with IFN-γ for 20 h and then exposed to H2O2 (100 μM) for 1 h. (A) The expression of Mn-SOD was determined by Western blot analysis. (B) MTT reduction assay. Data represent means ± SEM from four independent experiments and are expressed as percentage of control. ⁎p b 0.05, ⁎⁎⁎p b 0.001; significantly different from matched groups. (C) The effect of off-target for the siRNA. The control siRNA used here differs in only two nucleotides from the target sequence (5′-AGAUUCUUCACGUAGGUCACGUGGU-3′). The density ratio of Mn-SOD/β-actin was expressed as fold change. The level of NO was assessed by measuring the nitrite formation. N = 4, ⁎p b 0.05; significantly different from control groups.
As shown in Figs. 1 and 2, CHX completely blocked the cytoprotective effect of IFN-γ pretreatment on H2O2 toxicity. However, CHX had previously been reported to increase the expression of MnSOD in certain cell lines such as HeLa, A549, and Kuramochi [13]. Thus, we further examined the protein expression and activity of Mn-SOD in CHX-treated microglial cells. Fig. 8 shows that, regardless of pretreatment with IFN-γ, CHX did not alter the protein expression or activity of Mn-SOD in microglial cells; however, CHX completely inhibited the increased protein expression and activity of Mn-SOD in IFN-γpretreated microglia.
however, pretreatment with IFN-γ alone significantly hyperpolarized the ΔΨm. IFN-γ pretreatment augments the expression of Mn-SOD Microglial cells are equipped with many antioxidant enzyme systems, such as SOD, catalase, and GPx [3]. Of two different SOD isoforms, the protein expression and activity of only mitochondrial Mn-SOD were up-regulated by pretreatment with IFN-γ (Fig. 5). In contrast, however, the protein expression and activity of cellular Cu/ Zn-SOD were significantly decreased by IFN-γ pretreatment as well as H2O2 (Fig. 5). Although the protein expression levels of cytosolic GPx and catalase were not significantly changed by pretreatment with IFN-γ (Figs. 6A and B), the activity of catalase, but not that of GPx, was significantly increased by IFN-γ pretreatment (Figs. 6C and D). Furthermore, 3-AT, a catalase inhibitor, partially reversed the cytoprotective effect of IFN-γ in H2O2-treated microglial cells (Fig. 6E). This implies that up-regulation of catalase activity is also in part involved in the cytoprotective effect of IFN-γ pretreatment on microglial cells.
Fig. 8. Effects of cycloheximide on the protein expression level and activity of Mn-SOD. Cells were pretreated with CHX (0.01 μM) for 1 h and then cotreated with CHX and IFN-γ(100 U/ml) for 20 h. The protein level was assessed by Western blot analysis (top). The activity of Mn-SOD was determined by using the Cayman SOD activity assay kit (bottom). Data represent means ± SEM from four independent experiments. ⁎p b 0.05; significantly different from control group.
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Discussion Many earlier studies showed that pretreatment of microglia with IFN-γ triggers the production of reactive nitrogen and oxygen species. However, our present study demonstrates that the increased oxidative stress caused by IFN-γ pretreatment was ameliorated by an enhancement of the resisting power of microglia. The protective effect of IFN-γ pretreatment was at least partly due to up-regulation of mitochondrial Mn-SOD. Thus, transfection of Mn-SOD siRNA decreased the survival of microglial cells under oxidative stress, induced by IFN-γ pretreatment. Expectedly, IFN-γ pretreatment blocked both exogenously added and endogenously produced reactive species. Hydrogen peroxide, a highly diffusible ROS, has been associated with neuronal cell injury and death in neurodegenerative diseases [14]. Indeed, morphological observation and LDH analysis in this study showed that H2O2 rapidly induced the death of cultured microglial cells: hydrogen peroxide can potentially oxidize proteins and lipids, resulting in injury of cellular microorganelles such as mitochondria [15,16]. Although mitochondria are the predominant source of ROS in most cell types, they are also vulnerable to oxidative stress [17,18], and ΔΨm is an important parameter of mitochondrial function and often used as an indicator of cell health. Oxidative stress depolarizes ΔΨm, and inhibition of depolarization protects cells from oxidant toxicity [10,19]. In this study, the degree of ΔΨ = m depolarization evoked by H2O2 was reduced when microglial cells were pretreated with IFN-γ. This inhibition of ΔΨm depolarization in IFN-γ-treated microglial cells may keep mitochondrial function intact and contribute to the cell survival of H2O2 toxicity. Treatment of microglial cells with cycloheximide, a protein synthesis inhibitor, during IFN-γ pretreatment reduced the cytoprotective effect of IFN-γ on H2O2 toxicity. On the other hand, treatment with IFN-γ alone during exposure to H2O2 failed to protect microglial cells from H2O2 cytotoxicity. These data indicate that newly synthesized proteins are required for the cytoprotective effect of IFN-γ in microglial cells. Increased levels of antioxidant enzymes in microglial cells would decrease the cell damage induced by H2O2 [20,21]. In this study, we found that IFN-γ pretreatment selectively increased the expression and activity of mitochondrial Mn-SOD; IFN-γ pretreatment decreased the expression and activity of even Cu/Zn-SOD. Transfection of Mn-SOD siRNA partly abolished the decrease in H2O2-evoked death of microglial cells after IFN-γ pretreatment. Similar to the present study, Mn-SOD has been shown to be an important antioxidant enzyme associated with many mitochondrial functions [22,23]. Thus, selective up-regulation of Mn-SOD could be of great help for the maintenance of mitochondrial function in oxidative stress. The molecular mechanisms underlying the protective effect of MnSOD have previously been explored by other studies [24,25]. In general, H2O2 itself is not a strong oxidant. In the presence of ferrous iron (Fe2+), however, H2O2 produces toxic hydroxyl radical (OH·) through the Fenton reaction [24]. Hydroxyl radical oxidizes thiol (–SH) groups of the mitochondrial permeability transition pore complex, thus inducing pore opening [26]. The opening of nonspecific channels in the mitochondrial inner membrane may cause a depolarization of ΔΨm. Thus, inhibition of OH· production may help maintain the ΔΨm and inhibit cell death induced by H2O2. Because endogenous production of O2·- is necessary for the oxidation of labile iron–sulfur clusters and the regeneration of Fe2+ from ferric iron (Fe3+), Mn-SOD, which can eliminate O2·- and block the supply of Fe2+, will efficiently decrease the generation of OH·. In addition, catalase and GPx have been shown to be oxidized and inactivated by oxidants, including O2·-. Thus, removal of O2·- by SOD would prevent inactivation of catalase and GPx [25]. Peroxidases such as catalase and GPx can directly detoxify H2O2 by converting it to water and oxygen. In this study, although pretreatment with IFN-γ did not alter the protein level and activity of GPx, it increased the activity
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of catalase with no change of its protein expression. Our experimental results obtained by using the catalase inhibitor 3-AT indicate that the increased catalase activity also contributes in part to the cytoprotection induced by IFN-γ pretreatment. Microglial cells have recently been shown to play either a beneficial or a harmful role in neuronal cell survival in various neurodegenerative diseases [27–30]. Therefore, a better understanding of the responses of microglial cells to certain kinds of cytokines such as IFN-γ may provide a valuable therapeutic clue for the treatment of such neurodegenerative diseases. Acknowledgments This study was supported by a grant (M103KV010010-06K220101010) from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, a grant from the Brain Korea 21 Project (to Dr. I.Y. Choi), and in part by a grant from Korea University, the Republic of Korea. References [1] Kreutzberg, G. W. Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19:312–318; 1996. [2] Garden, G. A.; Moller, T. Microglia biology in health and disease. J. Neuroimmune Pharmacol. 1:127–137; 2006. [3] Dringen, R. Oxidative and antioxidative potential of brain microglial cells. Antioxid. Redox Signal. 7:1223–1233; 2005. [4] Schroder, K.; Hertzog, P. J.; Ravasi, T.; Hume, D. A. Interferon–gamma: an overview of signals, mechanisms and functions. J. Leukoc. Biol. 75:163–189; 2004. [5] Zielasek, J.; Tausch, M.; Toyka, K. V.; Hartung, H. P. Production of nitrite by neonatal rat microglial cells/brain macrophages. Cell. Immunol. 141:111–120; 1992. [6] Pawate, S.; Shen, Q.; Fan, F.; Bhat, N. R. Redox regulation of glial inflammatory response to lipopolysaccharide and interferon-gamma. J. Neurosci. Res. 77:540–551; 2004. [7] Hausler, K. G.; Prinz, M.; Nolte, C.; Weber, J. R.; Schumann, R. R.; Kettenmann, H.; Hanisch, U. K. Interferon-gamma differentially modulates the release of cytokines and chemokines in lipopolysaccharide- and pneumococcal cell wall-stimulated mouse microglia and macrophages. Eur. J. Neurosci. 16:2113–2122; 2002. [8] Ferret, P. J.; Soum, E.; Negre, O.; Fradelizi, D. Auto-protective redox buffering systems in stimulated macrophages. BMC Immunol. 3:3; 2002. [9] Sano, H.; Hirai, M.; Saito, H.; Nakashima, I.; Isobe, K. I. A nitric oxide-releasing reagent, S-nitroso-N-acetylpenicillamine, enhances the expression of superoxide dismutases mRNA in the murine macrophage cell line RAW264-7. Immunology 92:118–122; 1997. [10] Lee, S. J.; Jin, Y.; Yoon, H. Y.; Choi, B. O.; Kim, H. C.; Oh, Y. K.; Kim, H. S.; Kim, W. K. Ciclopirox protects mitochondria from hydrogen peroxide toxicity. Br. J. Pharmacol. 145:469–476; 2005. [11] Turrens, J. F. Mitochondrial formation of reactive oxygen species. J. Physiol. 552:335–344; 2003. [12] Camello-Almaraz, C.; Gomez-Pinilla, P. J.; Pozo, M. J.; Camello, P. J. Mitochondrial reactive oxygen species and Ca2+ signaling. Am. J. Physiol., Cell Physiol. 291: C1082–1088; 2006. [13] Fujii, J.; Nakata, T.; Miyoshi, E.; Ikeda, Y.; Taniguchi, N. Induction of manganese superoxide dismutase mRNA by okadaic acid and protein synthesis inhibitors. Biochem. J. 301:31–34; 1994. [14] Tabner, B. J.; Turnbull, S.; El-Agnaf, O. M.; Allsop, D. Formation of hydrogen peroxide and hydroxyl radicals from A(beta) and alpha-synuclein as a possible mechanism of cell death in Alzheimer's disease and Parkinson's disease. Free Radic. Biol. Med. 32:1076–1083; 2002. [15] Floyd, R. A.; Carney, J. M. Free radical damage to protein and DNA: mechanisms involved and relevant observations on brain undergoing oxidative stress. Ann. Neurol. 32 (Suppl.):S22–27; 1992. [16] Janero, D. R.; Hreniuk, D.; Sharif, H. M. Hydrogen peroxide-induced oxidative stress to the mammalian heart-muscle cell (cardiomyocyte): lethal peroxidative membrane injury. J. Cell. Physiol. 149:347–364; 1991. [17] Nulton-Persson, A. C.; Szweda, L. I. Modulation of mitochondrial function by hydrogen peroxide. J. Biol. Chem. 276:23357–23361; 2001. [18] Sims, N. R.; Anderson, M. F.; Hobbs, L. M.; Kong, J. Y.; Phillips, S.; Powell, J. A.; Zaidan, E. Impairment of brain mitochondrial function by hydrogen peroxide. Brain Res. Mol. Brain Res. 77:176–184; 2000. [19] Medina, S.; Martinez, M.; Hernanz, A. Antioxidants inhibit the human cortical neuron apoptosis induced by hydrogen peroxide, tumor necrosis factor alpha, dopamine and beta-amyloid peptide 1-42. Free Radic. Res. 36:1179–1184; 2002. [20] Huang, Q.; Wu, L. J.; Tashiro, S.; Onodera, S.; Ikejima, T. Elevated levels of DNA repair enzymes and antioxidative enzymes by (+)-catechin in murine microglia cells after oxidative stress. J. Asian Nat. Prod. Res. 8:61–71; 2006. [21] Hou, R. C.; Wu, C. C.; Huang, J. R.; Chen, Y. S.; Jeng, K. C. Oxidative toxicity in BV-2 microglia cells: sesamolin neuroprotection of H2O2 injury involving activation of p38 mitogen-activated protein kinase. Ann. N.Y. Acad. Sci. 1042:279–285; 2005.
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