Dual Functionality of Myeloperoxidase in Rotenone-Exposed Brain-Resident Immune Cells
2011; Elsevier BV; Volume: 179; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2011.04.033
ISSN1525-2191
AutoresChi Young Chang, Mi Jeon Song, Sae-Bom Jeon, Hee Jung Yoon, Daekee Lee, In-Hoo Kim, Kyungho Suk, Dong‐Kug Choi, Eun Jung Park,
Tópico(s)Immune cells in cancer
ResumoRotenone exposure has emerged as an environmental risk factor for inflammation-associated neurodegenerative diseases. However, the underlying mechanisms responsible for the harmful effects of rotenone in the brain remain poorly understood. Herein, we report that myeloperoxidase (MPO) may have a potential regulatory role in rotenone-exposed brain-resident immune cells. We show that microglia, unlike neurons, do not undergo death; instead, they exhibit distinctive activated properties under rotenone-exposed conditions. Once activated by rotenone, microglia show increased production of reactive oxygen species, particularly HOCl. Notably, MPO, an HOCl-producing enzyme that is undetectable under normal conditions, is significantly increased after exposure to rotenone. MPO-exposed glial cells also display characteristics of activated cells, producing proinflammatory cytokines and increasing their phagocytic activity. Interestingly, our studies with MPO inhibitors and MPO-knockout mice reveal that MPO deficiency potentiates, rather than inhibits, the rotenone-induced activated state of glia and promotes glial cell death. Furthermore, rotenone-triggered neuronal injury was more apparent in co-cultures with glial cells from Mpo−/− mice than in those from wild-type mice. Collectively, our data provide evidence that MPO has dual functionality under rotenone-exposed conditions, playing a critical regulatory role in modulating pathological and protective events in the brain. Rotenone exposure has emerged as an environmental risk factor for inflammation-associated neurodegenerative diseases. However, the underlying mechanisms responsible for the harmful effects of rotenone in the brain remain poorly understood. Herein, we report that myeloperoxidase (MPO) may have a potential regulatory role in rotenone-exposed brain-resident immune cells. We show that microglia, unlike neurons, do not undergo death; instead, they exhibit distinctive activated properties under rotenone-exposed conditions. Once activated by rotenone, microglia show increased production of reactive oxygen species, particularly HOCl. Notably, MPO, an HOCl-producing enzyme that is undetectable under normal conditions, is significantly increased after exposure to rotenone. MPO-exposed glial cells also display characteristics of activated cells, producing proinflammatory cytokines and increasing their phagocytic activity. Interestingly, our studies with MPO inhibitors and MPO-knockout mice reveal that MPO deficiency potentiates, rather than inhibits, the rotenone-induced activated state of glia and promotes glial cell death. Furthermore, rotenone-triggered neuronal injury was more apparent in co-cultures with glial cells from Mpo−/− mice than in those from wild-type mice. Collectively, our data provide evidence that MPO has dual functionality under rotenone-exposed conditions, playing a critical regulatory role in modulating pathological and protective events in the brain. Pesticide exposure has received considerable attention as an environmental risk factor for several diseases, including Parkinson's disease (PD). An increasing number of epidemiological and biochemical studies1Miller R.L. James-Kracke M. Sun G.Y. Sun A.Y. Oxidative and inflammatory pathways in Parkinson's disease.Neurochem Res. 2009; 34: 55-65Crossref PubMed Scopus (257) Google Scholar, 2Drechsel D.A. Patel M. Role of reactive oxygen species in the neurotoxicity of environmental agents implicated in Parkinson's disease.Free Radic Biol Med. 2008; 44: 1873-1886Crossref PubMed Scopus (249) Google Scholar have implicated the oxidative properties of pesticides in the pathogenesis of such diseases. Rotenone, one of the most widely used herbicides, is a ketone compound extracted from the roots of Lonchocarpus species.3Betarbet R. Sherer T.B. MacKenzie G. Garcia-Osuna M. Panov A.V. Greenamyre J.T. Chronic systemic pesticide exposure reproduces features of Parkinson's disease.Nat Neurosci. 2000; 3: 1301-1306Crossref PubMed Scopus (2933) Google Scholar It is a highly lipophilic pesticide that readily crosses the blood-brain barrier and accumulates throughout the brain.4Talpade D.J. Greene J.G. Higgins Jr, D.S. Greenamyre J.T. In vivo labeling of mitochondrial complex I (NADH: ubiquinone oxidoreductase) in rat brain using [(3)H]dihydrorotenone.J Neurochem. 2000; 75: 2611-2621Crossref PubMed Scopus (106) Google Scholar, 5Fleming S.M. Zhu C. Fernagut P.O. Mehta A. DiCarlo C.D. Seaman R.L. Chesselet M.F. 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Myeloperoxidase polymorphism is associated with gender specific risk for Alzheimer's disease.Exp Neurol. 1999; 155: 31-41Crossref PubMed Scopus (270) Google Scholar, 23Maki R.A. Tyurin V.A. Lyon R.C. Hamilton R.L. DeKosky S.T. Kagan V.E. Reynolds W.F. Aberrant expression of myeloperoxidase in astrocytes promotes phospholipid oxidation and memory deficits in a mouse model of Alzheimer disease.J Biol Chem. 2009; 284: 3158-3169Crossref PubMed Scopus (99) Google Scholar However, the precise roles of MPO and the underlying mechanisms responsible for its action have not been determined. Immune and inflammatory responses in the central nervous system (CNS) are mainly coordinated by the interaction of the brain-resident immune cells, microglia, and astrocytes with neurons. Thus, we questioned how glial cells respond to rotenone exposure and whether glial cells play a role in the pathophysiological consequences of rotenone exposure. In the present study, we investigated the responses of glial cells and their potential roles in combating against rotenone-induced damage in the CNS. Intriguingly, we found that MPO may act as an essential modulator, regulating the activation of glia and affecting neuronal injury under rotenone-exposed conditions. Our data provide new insights into the cellular responses associated with MPO in the rotenone-exposed brain and suggest a potential target for the development of a therapeutic intervention in diseases associated with rotenone exposure. Rotenone and human MPO were obtained from Calbiochem (La Jolla, CA); minimal essential medium, Life Technologies, Inc. (Gaithersburg, MD); Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum, Hyclone (Logan, UT); salicyl hydroxamic acid and (D+)-mannose, Sigma-Aldrich (St Louis, MO); and 4-aminobenzoylhydrazide (ABAH), Calbiochem (San Diego, CA). The antibodies used in this study included the following: mouse anti-α-tubulin (Sigma-Aldrich), anti-MPO (Dako, Glostrup, Denmark), anti-glial fibrillary acidic protein (GFAP; Cell Signaling, Beverly, MA), anti-CD11b (Serotec, Oxford, UK), anti-manganese-containing superoxide dismutase (MnSOD) (Upstate, Lake Placid, NY), anti-cyclooxygenase (COX)-2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-inducible nitric oxide synthase (iNOS) (Upstate), anti-interferon-γ receptor (IFNγR) (Santa Cruz Biotechnology), anti-arginase-1 (BD Biosciences, San Jose, CA), anti-CD16/CD32 (Fc Block; BD Biosciences), and anti-tyrosine hydroxylase (TH; Abcam, Cambridge, MA). Fluorophore-conjugated secondary antibodies (Alexa Fluor 488 and 546) were obtained from Molecular Probes (Eugene, OR), and horseradish peroxidase–conjugated secondary antibodies were obtained from Bio-Rad (Hercules, CA). Sprague-Dawley rats were obtained from SamTako Bio Korea (Osan, Korea), and C57BL/6 and B6.129 × 1-Mpotm1Lus/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Adult timed-pregnant Sprague-Dawley rats and CrljOri:CD1 mice were obtained from ORIENT BIO (Sungnam, Korea). All animal procedures were performed according to the National Cancer Center guidelines for the care and use of laboratory animals. Glial cells isolated from the cerebral cortex of 1- to 3-day-old Sprague-Dawley rats were triturated into single-cell suspensions, plated in 75-cm2 T-flasks (0.5 hemispheres per flask), and cultured in minimal essential medium containing 10% fetal bovine serum for 2 weeks.24Du Y. Chen C.P. Tseng C.Y. Eisenberg Y. Firestein B.L. Astroglia-mediated effects of uric acid to protect spinal cord neurons from glutamate toxicity.Glia. 2007; 55: 463-472Crossref PubMed Scopus (89) Google Scholar The microglia were detached from the flasks by mild shaking and applied to a nylon mesh to remove astrocytes and cell clumps. Cells were plated in 60-mm2 dishes (8 × 105 cells per dish) or 100-mm2 dishes (2 × 106 cells per dish). One hour later, the cells were washed to remove unattached cells before being used in experiments. After removal of the microglia, primary astrocytes were prepared using trypsinization.25Jou I. Lee J.H. Park S.Y. Yoon H.J. Joe E.H. Park E.J. Gangliosides trigger inflammatory responses via TLR4 in brain glia.Am J Pathol. 2006; 168: 1619-1630Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar Murine BV2 microglial cells were maintained in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified incubator under 5% CO2. Ventral mesencephalic tissues were dissected from embryonic day 14 or 15 Sprague-Dawley rats or embryonic day 12 CrljOri:CD1 mice and dissociated enzymatically (0.1% trypsin) and mechanically.26Han B.S. Hong H.S. Choi W.S. Markelonis G.J. Oh T.H. Oh Y.J. Caspase-dependent and -independent cell death pathways in primary cultures of mesencephalic dopaminergic neurons after neurotoxin treatment.J Neurosci. 2003; 23: 5069-5078Crossref PubMed Google Scholar Cells were seeded onto six-well plates (2 × 106 cells per well) or 24-well plates (5 × 105 cells per well) precoated with poly-d-lysine (5 mg/mL) and laminin (0.2 mg/mL). Rat neurons were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin (P/S) at 37°C in a humidified 5% CO2 incubator. On the following day, the medium was replaced with a chemically defined serum-free medium containing 50% DMEM; 50% Ham's F12 media; 1% insulin, transferring, selenium; and 1% P/S. Then, it was incubated for 48 hours before treatment. Mouse neurons were resuspended in Neurobasal medium (Invitrogen, Carlsbad, CA) containing 1× B27 supplement (Invitrogen), 0.5 mmol/L glutamine, and 1% P/S at 37°C in a humidified 5% CO2 incubator. During the subsequent 4 to 5 days, cells were refed every 2 days and replaced with Neurobasal medium without B27 for the lactate dehydrogenase (LDH) assay. Microglia were plated in 60-mm2 dishes (2.5 × 104 cells per dish) and then treated or left untreated with rotenone. The phagocytic capacity was measured by incubating cells with fluorescein isothiocyanate (FITC)–conjugated phagocytic beads (8 × 106 beads/mL; FluoSpheres polystyrene microspheres; Molecular Probes) at 37°C for 3 hours. Cells were then washed three times with PBS and then gently removed from the wells using cell scrapers for fluorescence-activated cell sorter (FACS) analysis. To examine Fcγ receptor–mediated phagocytosis, BV2 cells were blocked using 10 μg/mL Fc Block (BD Biosciences) directed against FcγRIII(CD32)/FcγRII(CD16). Fc Block also binds the FcγI receptor (CD64) via its Fc domain. MPO activity was measured using an EnzChek Myeloperoxidase Activity Assay Kit, as described by the manufacturer (Molecular Probes or Invitrogen). Briefly, primary microglia and astrocytes were lysed using a standard freeze-thaw method and suspended in 50 μL of PBS. Lysates (50 μL) and supernatant were incubated for 30 minutes at room temperature in the working solution, according to the manufacturer's instructions. Fluorescence was measured at 590 nm after excitation at 530 nm using a Spectra-Max Gemini fluorometer (Molecular Devices, Sunnyvale, CA) at room temperature. Total intracellular γ-glutamyl-cysteinyl-glycine (GSH) content was measured using a kit from Cayman, according to the manufacturer's instructions. In brief, primary microglia were scraped from 60-mm2 dishes (8 × 105 cells per dish), homogenized in 0.1 mL cold buffer, and then centrifuged at 10,000 × g for 15 minutes at 4°C. The supernatant was collected and deproteinized by mixing with metaphosphoric acid before GSH content measurement. Cells grown on coverslips were fixed in ice-cold methanol and permeabilized in 0.1% Triton X-100/PBS for 10 minutes. Cells were then blocked with 10% bovine serum albumin/0.1% Triton X-100/PBS for 30 minutes at room temperature, and the coverslips were washed twice with 0.1% Triton X-100/PBS. Fluorescent images were acquired with a confocal laser scanning microscopy system (model LSM510 meta; Carl Zeiss, Jena, Germany) and Axio Observer Z1 (Carl Zeiss) using rhodamine, fluorescein, and DAPI filters. The confocal system software and Axiovision software were used to capture and store the images. Total RNA was isolated using Easy-Blue (iNtRON, Daejeon, Korea), and cDNA was synthesized using avian myeloblastosis virus reverse transcriptase (TaKaRa, Dalian City, Japan), according to the manufacturer's instructions. PCRs were performed with 35 cycles of sequential reactions. Oligonucleotide primers were obtained from Bioneer (Seoul, Korea). RT-PCR analysis was performed using previously reported primers27Jeon S.B. Yoon H.J. Park S.H. Kim I.H. Park E.J. Sulfatide, a major lipid component of myelin sheath, activates inflammatory responses as an endogenous stimulator in brain-resident immune cells.J Immunol. 2008; 181: 8077-8087PubMed Google Scholar, 28Jeon S.B. Yoon H.J. Chang C.Y. Koh H.S. Jeon S.H. Park E.J. Galectin-3 exerts cytokine-like regulatory actions through the JAK-STAT pathway.J Immunol. 2010; 185: 7037-7046Crossref PubMed Scopus (133) Google Scholar and the following primers: rat catalase, 5′-TTATGTTACCTCACAGCCTGGT-3′ (forward) and 5′-GTGTTGTGTGTTCTGTGTGTGTAG-3′ (reverse); rat COX-2, 5′-ACACTCTATCACTGGCATCC-3′ (forward) and 5′-GAAGGGACACCCTTTCACAT-3′ (reverse); rat glutathione peroxidase (GPx)-1, 5′-TGAGAAGTGCGAGGTGAATG-3′ (forward) and 5′-AACACCGTCTGGACCTACCA-3′ (reverse); rat GPx-2, 5′-TGCCCTACCCTTATGACGAC-3′ (forward) and 5′-GGAGATTCCTAGGCTGAGCA-3′ (reverse); rat matrix metalloproteinase (MMP)-3, 5′-CTGGAATGGTCTTGGCTCAT-3′ (forward) and 5′-CTGACTGCATCGAAGGACAA-3′ (reverse); rat MnSOD, 5′-AACGCGCAGATCATGCAGCTGC-3′ (forward) and 5′-ACATTCTCCCAGTTGATTACAT-3′ (reverse); rat MPO, 5′-GTATCGAACCATCACTGGAC-3′ (forward) and 5′-AGCTGGTCTCACAGTTGAGT-3′ (reverse); rat neuronal NOS, 5′-GGCACTGGCATCGCACCCTT-3′ (forward) and 5′-CTTTGGCCTGTCCGGTTCCC-3′ (reverse); and mouse tumor necrosis factor (TNF)-α, 5′-ATGAGCACAGAAAGCATGATC-3′ (forward) and 5′-TACAGGCTTGTCACTCGAATT-3′ (reverse). Enzyme-linked immunosorbent assay (ELISA) kits were used according to the manufacturer's protocols. After treatment with stimuli, 100 μL of conditioned media was collected and assayed using ELISA kits for rat TNF-α (eBioscience, San Diego, CA), IL-4, IL-6, and IL-13 (BioSource International, Comarillo, CA). Cells were washed twice with cold PBS and lysed in ice-cold modified radioimmunoprecipitation assay buffer (50 mmol/L Tris-HCl, pH 7.4; 1% Nonidet P-40; 0.25% Na-deoxycholate; 150 mmol/L NaCl; and 10 mmol/L Na2HPO4) containing protease inhibitors (2 mmol/L phenyl-methyl sulfonyl fluoride, 10 μg/mL leupeptin, 10 μg/mL pepstatin, 0.5 mmol/L Na3VO4, 0.5 mol/L NaF, and 2 mmol/L EDTA). The proteins in the medium were further fractionated by using the Rapid-Con Protein concentration kit (Elpis Biotech, Daejeon, Korea), according to the manufacturer's protocol. The lysate was centrifuged for 20 minutes at 13,000 rpm at 4°C, and supernatant proteins were separated by SDS-PAGE on 8% gels and transferred to nitrocellulose membranes. The membranes were incubated with primary antibodies and horseradish peroxidase–conjugated secondary antibodies and then visualized using an enhanced chemiluminescence system. Cells cultured according to standard procedures were dissociated from the culture plates by pipetting and washed twice with PBS. Antibody incubations were performed for 30 minutes at 4°C. Cells were washed by sedimenting at 450 × g for 10 minutes at 4°C. Flow cytometric measurements were performed using a Becton Dickinson FACSCalibur system (Becton Dickinson, Mansfield, MA), and data were analyzed using FlowJo software (Treestar, Inc., San Carlos, CA). Cells were suspended in 5 μmol/L CM-H2DCFDA (DCF; Molecular Probes) or 10 μmol/L aminophenyl fluorescein (APF; Molecular Probes) for 30 minutes at 37°C in the dark. After incubation, the cells were washed twice with PBS and suspended in PBS. The green emission of DCF, APF, and rhodamine-123 was measured using an FACSCalibur flow cytometer (BD Biosciences). DCF detects H2O2, hydroxyl radical, peroxyl radical, and peroxynitrite anion, whereas APF reacts preferentially with HOCl. To evaluate oxidative stress using rhodamine-123 (Molecular Probes), the cells were incubated with 1 μg/mL rhodamine-123 for 10 minutes in culture media at 37°C. LDH released into the supernatant by damaged cells was measured by collecting 50 μL of cell-free supernatant into 96-well plates (n = 3) and then adding 125 μL of NADH solution and 25 μL of pyruvate solution. The LDH level was determined immediately by measuring absorbance at a wavelength of 340 nm in kinetics mode for 5 minutes on a microplate reader (Molecular Devices). The percentage cytotoxicity was calculated as follows (using total cellular LDH as a low control): Cytotoxicity (%) = [(Experimental Value-Low Control)/(High Control-Low Control)] × 100%. Cell viability was determined using the Live/Dead Viability Cytotoxicity Kit (Molecular Probes) or the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan), according to the manufacturer's instruction. For the Live/Dead Viability Cytotoxicity assay, cells were grown in 24-well plates (primary microglia, 8 × 104 cells per well; primary mesencephalic neurons, 5 × 105 cells per well) and incubated with the indicated stimuli. Viability was assessed by staining cells according to the manufacturer's protocol and analyzing by fluorescence microscopy (Axio Observer Z1). The percentage cell viability was defined in each image as the percentage of live and dead cells versus the total number of cells, counting at least 350 cells per image. For the CCK-8 assay, viable cells were counted by absorbance measurements at 450 nm using a Versamax microplate reader (Molecular Devices) at room temperature. The cytotoxic effects of rotenone were assessed by flow cytometry after staining the cells with propidium iodide (PI). Briefly, 1 × 106 cells per sample were washed twice with cold PBS and fixed in ice-cold 75% ethanol at 4°C. The cells were then washed twice with PBS and incubated with 50 μg/mL RNase A and 40 μg/mL PI for 30 minutes at 4°C. For measurement of cell death using annexin V, cells were harvested and washed in binding buffer, then incubated with annexin V–FITC (BD Pharmingen, San Diego, CA) and 7-amino-actinomycin D for 15 minutes at room temperature in the dark. Cells were then immediately analyzed by flow cytometry using an FACSCalibur flow cytometer (Becton Dickinson). Gating was defined using untreated control samples to exclude aggregates and to determine the appropriate quadrants. All data were expressed as the mean ± SEM and analyzed by one-way analysis of variance, followed by post hoc comparisons (Student-Newman-Keuls test) using the Statistical Package for Social Sciences 8.0 (SPSS, Chicago, IL). Studies29Gao H.M. Hong J.S. Zhang W. Liu B. Distinct role for microglia in rotenone-induced degeneration of dopaminergic neurons.J Neurosci. 2002; 22: 782-790Crossref PubMed Google Scholar, 30Gao H.M. Hong J.S. Zhang W. Liu B. Synergistic dopaminergic neurotoxicity of the pesticide rotenone and inflammogen lipopolysaccharide: relevance to the etiology of Parkinson's disease.J Neurosci. 2003; 23: 1228-1236Crossref PubMed Google Scholar have suggested a potential role for microglia in modulating pathophysiological events in the rotenone-exposed brain. In an effort to precisely understand the distinct responses of glial cells to rotenone exposure, we first examined the cell viability of rotenone-treated primary microglia cultures compared with those of mesencephalic neuron-enriched cultures. Rat primary microglia and neurons were mock treated or treated with rotenone for 3 days, after which the degree of cell death was determined using the LDH assay. Interestingly, rotenone (1 to 100 nmol/L) did not significantly affect the viability of primary microglia but did enhance neuronal cell death in mesencephalic neuron-enriched cultures (Figure 1A; see also Supplemental Figure S1A at http://ajp.amjpathol.org). To further determine the effects of rotenone on microglial cell viability in the brain, we separately cultured rat primary microglia and neurons using transwell chambers. The cells were mock treated or treated with 30 nmol/L rotenone for 3 days, and the viability of each cell type was determined by FACS analysis (Figure 1B). As was the case with the microglia alone culture, microglial cell death was not detected, but neuronal cell death was increased compared with mock-treated controls. Similar results as previously described were obtained by fluorescence microscopy using a Live/Dead Viability Cytotoxicity kit and FACS analysis (Figure 1C; see also Supplemental Figure S1, A and B, at http://ajp.amjpathol.org). These findings indicate that rotenone does not trigger microglial cell death in the absence or presence of neurons. Although rotenone did not trigger microglial cell death, rat primary microglia displayed typical activated phenotypes under rotenone-exposed conditions, indicating that microglia could actively respond to rotenone exposure (see Supplemental Figure S1C at http://ajp.amjpathol.org). Therefore, we carefully examined the characteristics of rotenone-exposed microglia by analyzing the expression profile of genes associated with microglial activation. As shown in Figure 2, A and B, rotenone exposure significantly increased the expression of markers representative of classic (inflammatory M1) activation in rat primary microglia.31Colton C.A. Heterogeneity of microglial activation in the innate immune response in the brain.J Neuroimmune Pharmacol. 2009; 4: 399-418Crossref PubMed Scopus (640) Google Scholar, 32Kigerl K.A. Gensel J.C. Ankeny D.P. Alexander J.K. Donnelly D.J. Popovich P.G. Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord.J Neurosci. 2009; 29: 13435-13444Crossref PubMed Scopus (1569) Google Scholar Treatment of microglia with rotenone obviously enhanced the production of several proinflammatory mediators, including TNF-α, IL-1β, IL-6, IL-12p40, iNOS, IFN-γ, and IFNγR. To better characterize the properties of activated microglia under conditions of rotenone exposure, we further examined the expression of anti-inflammatory M2 activation markers.31Colton C.A. Heterogeneity of microglial activation in the innate immune response in the brain.J Neuroimmune Pharmacol. 2009; 4: 399-418Crossref PubMed Scopus (640) Google Scholar, 33Biswas S.K. Mantovani A. Macrophage plasticity and interaction wi
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