Myeloperoxidase (MPO) is a heme-containing protein that catalyzes the formation of the potent oxidant HOCl and other chlorinating species derived from H
2O
2. MPO and MPO-derived oxidants could mediate inflammatory responses at sites of inflammation, thereby contributing to the defense system against pathogens.
10- Zhang R.
- Brennan M.L.
- Shen Z.
- MacPherson J.C.
- Schmitt D.
- Molenda C.E.
- Hazen S.L.
Myeloperoxidase functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites of inflammation.
Reports
11- Matthijsen R.A.
- Huugen D.
- Hoebers N.T.
- de Vries B.
- Peutz-Kootstra C.J.
- Aratani Y.
- Daha M.R.
- Tervaert J.W.
- Buurman W.A.
- Heeringa P.
Myeloperoxidase is critically involved in the induction of organ damage after renal ischemia reperfusion.
, 12- Sawayama Y.
- Miyazaki Y.
- Ando K.
- Horio K.
- Tsutsumi C.
- Imanishi D.
- Tsushima H.
- Imaizumi Y.
- Hata T.
- Fukushima T.
- Yoshida S.
- Onimaru Y.
- Iwanaga M.
- Taguchi J.
- Kuriyama K.
- Tomonaga M.
Expression of myeloperoxidase enhances the chemosensitivity of leukemia cells through the generation of reactive oxygen species and the nitration of protein.
indicate that MPO levels are significantly increased in various disease states, such as infection, ischemia, atherosclerosis, and acute myeloid leukemia. Increased MPO levels are widely considered characteristic of systemic inflammatory diseases. Recently, several interesting reports
13- Lau D.
- Mollnau H.
- Eiserich J.P.
- Freeman B.A.
- Daiber A.
- Gehling U.M.
- Brummer J.
- Rudolph V.
- Munzel T.
- Heitzer T.
- Meinertz T.
- Baldus S.
Myeloperoxidase mediates neutrophil activation by association with CD11b/CD18 integrins.
, 14- Eiserich J.P.
- Baldus S.
- Brennan M.L.
- Ma W.
- Zhang C.
- Tousson A.
- Castro L.
- Lusis A.J.
- Nauseef W.M.
- White C.R.
- Freeman B.A.
Myeloperoxidase, a leukocyte-derived vascular NO oxidase.
have revealed that MPO has catalytic activity and exhibits cytokine-like properties, activating and modulating inflammatory signaling cascades. MPO has been closely involved in stimulating mitogen-activated protein kinase activity, cell growth, and protease activity, thereby influencing the immune responses and the progression of several inflammation-associated diseases.
10- Zhang R.
- Brennan M.L.
- Shen Z.
- MacPherson J.C.
- Schmitt D.
- Molenda C.E.
- Hazen S.L.
Myeloperoxidase functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites of inflammation.
, 15- El Kebir D.
- Jozsef L.
- Pan W.
- Filep J.G.
Myeloperoxidase delays neutrophil apoptosis through CD11b/CD18 integrins and prolongs inflammation.
, 16- Hirche T.O.
- Gaut J.P.
- Heinecke J.W.
- Belaaouaj A.
Myeloperoxidase plays critical roles in killing Klebsiella pneumoniae and inactivating neutrophil elastase: effects on host defense.
, 17- Daugherty A.
- Dunn J.L.
- Rateri D.L.
- Heinecke J.W.
Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions.
, 18- Choi D.K.
- Pennathur S.
- Perier C.
- Tieu K.
- Teismann P.
- Wu D.C.
- Jackson-Lewis V.
- Vila M.
- Vonsattel J.P.
- Heinecke J.W.
- Przedborski S.
Ablation of the inflammatory enzyme myeloperoxidase mitigates features of Parkinson's disease in mice.
, 19- Nagra R.M.
- Becher B.
- Tourtellotte W.W.
- Antel J.P.
- Gold D.
- Paladino T.
- Smith R.A.
- Nelson J.R.
- Reynolds W.F.
Immunohistochemical and genetic evidence of myeloperoxidase involvement in multiple sclerosis.
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.
Materials and Methods
Reagents and Antibodies
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).
Animals
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 Cell Culture
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-cm
2 T-flasks (0.5 hemispheres per flask), and cultured in minimal essential medium containing 10% fetal bovine serum for 2 weeks.
24- Du 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.
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-mm
2 dishes (8 × 10
5 cells per dish) or 100-mm
2 dishes (2 × 10
6 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.
25- Jou 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.
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% CO
2.
Neuron-Enriched Mesencephalic Cultures
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.
26- Han 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.
Cells were seeded onto six-well plates (2 × 10
6 cells per well) or 24-well plates (5 × 10
5 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% CO
2 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% CO
2 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.
Phagocytosis 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 Assay
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.
GSH Measurement
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.
Confocal Microscopy
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.
RT-PCR Analysis
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 primers
27- Jeon 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.
, 28- Jeon 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.
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).
ELISA
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).
Western Blot Analysis
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.
Flow Cytometric Analysis
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).
Measurement of ROS
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 Assay
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 Assay
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.
Measurement of Cell Death by PI and Annexin V Staining
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.
Data Analysis
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).
Discussion
Microglia and astrocytes are major immune cells that serve as the first line of defense against tissue injury or pathogens in the CNS. They are sensitive to even subtle alterations in the CNS and rapidly undergo a variety of changes in immunological function that prevent neuronal damage from pathological stimuli.
25- Jou 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.
, 27- Jeon 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.
, 44- Park E.J.
- Ji K.A.
- Jeon S.B.
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Rac1 contributes to maximal activation of STAT1 and STAT3 in IFN-gamma-stimulated rat astrocytes.
, 45- Nimmerjahn A.
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Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.
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Microglia in neurodegenerative disease.
Accumulating evidence has revealed that glial cells perform distinct responses in different pathological states, thereby setting in motion an appropriate defense system. However, inappropriate or excessive responses of glia can cause additional damage that contributes to diverse disease in the CNS.
Environmental toxins, such as pesticides, have emerged as an important underlying risk factor for the development of various diseases. Rotenone, a common herbicide, specifically may provoke critical neuronal damage that ultimately leads to neurodegenerative diseases.
6- Betarbet R.
- Porter R.H.
- Greenamyre J.T.
GluR1 glutamate receptor subunit is regulated differentially in the primate basal ganglia following nigrostriatal dopamine denervation.
, 7- Gao H.M.
- Liu B.
- Hong J.S.
Critical role for microglial NADPH oxidase in rotenone-induced degeneration of dopaminergic neurons.
, 8Rotenone destroys dopaminergic neurons and induces parkinsonian symptoms in rats.
In particular, rotenone exposure has been implicated as a potential risk factor for PD. Studies have shown that mice and rats given various concentrations of rotenone, from 2 to 30 mg/kg, exhibited clinical and pathological features of PD, such as loss of TH-positive dopaminergic neurons and/or motor dysfunction. In addition, several reports have described the toxic effects of rotenone on neurons. Notably, Gao et al
29- Gao H.M.
- Hong J.S.
- Zhang W.
- Liu B.
Distinct role for microglia in rotenone-induced degeneration of dopaminergic neurons.
reported that rotenone-induced neurotoxicity is greater in the presence of microglia than in neurons cultured alone, highlighting the potential capacity of microglia to have a role in the pathophysiological consequences of rotenone exposure. These reports raise the question of what events might be mediated by microglia under rotenone-exposed conditions. In the present study, we investigated the response of glial cells and their potential roles in combating rotenone-induced damage in the CNS.
Once rotenone enters the body, it easily crosses the blood-brain barrier and is distributed throughout the brain.
1- Miller R.L.
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- Sun A.Y.
Oxidative and inflammatory pathways in Parkinson's disease.
After systemic administration of 2 to 3 mg/kg per day, the concentration of free rotenone in the brain is approximately 20 to 30 nmol/L.
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- Higgins Jr, D.S.
- Greenamyre J.T.
In vivo labeling of mitochondrial complex I (NADH: ubiquinone oxidoreductase) in rat brain using [(3)H]dihydrorotenone.
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Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone.
In addition, Talpade et al
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- Higgins Jr, D.S.
- Greenamyre J.T.
In vivo labeling of mitochondrial complex I (NADH: ubiquinone oxidoreductase) in rat brain using [(3)H]dihydrorotenone.
suggested that 1 to 30 nmol/L of rotenone may be a reasonable concentration in the living rat brain under rotenone-triggered pathological conditions. Based on these reports, we used 1 to 30 nmol/L of rotenone in our present study. Neurons have been shown to undergo death after rotenone exposure; therefore, we first examined the effects of rotenone on the viability of microglia as part of our efforts to assess the glial responses to rotenone exposure in the brain. In our experiments, neuronal death was detected in mesencephalic neurons cultured with physiologically relevant concentrations of rotenone, consistent with previous reports (
Figure 1). Unlike neurons, exposure to rotenone was not toxic to microglia, in either the presence or the absence of neurons; however, rotenone-treated microglia adopted a distinct activated form, indicating that microglia could actively respond to rotenone exposure. Studies
37- Dringen R.
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Peroxide detoxification by brain cells.
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Role of microglial redox balance in modulation of neuroinflammation.
have shown that activated microglia release numerous inflammatory mediators, such as NO and superoxide, and produce antioxidants to successfully defend the CNS against threats to the brain. Microglia are equipped with efficient antioxidative defense mechanisms to prevent oxidative damage that would compromise their function.
37- Dringen R.
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Peroxide detoxification by brain cells.
In this experiment, we found that rotenone exposure enhanced the levels of pro-oxidative and antioxidative enzymes, including SOD, catalase, and GPxs, showing that the response to rotenone-triggered oxidative stress is active (
Figure 5). In addition, GSH levels were higher in rotenone-exposed microglia than in mock-treated cells. Based on previous reports and on our findings, it is likely that rotenone exposure microglial antioxidant systems protect the cells from self-damage potentially caused by rotenone and microglia-released oxidants, contributing to the efficacy of microglia as effector cells under rotenone-exposed conditions.
MPO is an enzyme that functions as a key molecular component of the host defense reaction against inflammatory stimulators.
38Myeloperoxidase: friend and foe.
, 48Human myeloperoxidase in innate and acquired immunity.
Inflammatory sites, as the result of microorganism infections or injured tissues, are characterized by increased levels of MPO. Indeed, studies have increasingly suggested potential links between MPO and the development of diverse diseases. Interestingly, we found that a distinguishing feature of rotenone-exposed microglia is an increase in the expression level of MPO (
Figure 4). Therefore, we hypothesized that MPO might be associated with pathophysiological events in the brain under rotenone-exposed conditions. MPO is mainly present in neutrophils, monocytes, and macrophages; thus, it has been used as a marker for infiltrated neutrophils at sites of injury.
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Correlation between myeloperoxidase-quantified neutrophil accumulation and ischemic brain injury in the rat: effects of neutrophil depletion.
Recently, MPO was reported to be present in brain-resident cells under pathological conditions.
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Ablation of the inflammatory enzyme myeloperoxidase mitigates features of Parkinson's disease in mice.
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Aberrant expression of myeloperoxidase in astrocytes promotes phospholipid oxidation and memory deficits in a mouse model of Alzheimer disease.
The results of the present study using primary glial cell cultures strongly support the hypothesis that glial cells are among the MPO-expressing cells of the brain. The distinct increase in MPO levels in microglia by rotenone exposure led us to focus our research on the characteristics of MPO in rotenone-exposed glia. Therefore, we further examined the effects of MPO on glial cells using purified MPO. Several reports
15- El Kebir D.
- Jozsef L.
- Pan W.
- Filep J.G.
Myeloperoxidase delays neutrophil apoptosis through CD11b/CD18 integrins and prolongs inflammation.
have shown that purified MPO has its activity when added to cultured cells or animals. Although relatively little is known of the amounts of MPO in the CNS, the plasma levels of MPO in patients with vascular diseases have been described.
51- Baldus S.
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Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes.
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In addition, we detected approximately 25 ng/mL MPO in rotenone-exposed primary microglia (data not shown). On the basis of these reports and our own results, we chose to use 1 to 1000 ng/mL MPO in the present study. In microglia and astrocytes, MPO did not cause cell death at any concentration tested; instead, it exhibited cytokinelike properties. MPO significantly increased the expression of several inflammation-associated molecules, including TNF-α, and promoted the production of ROS. Notably, MPO also augmented its own expression at the mRNA and protein levels, resulting in increased MPO secretion into the extracellular space (
Figure 6,
Figure 7). These observations indicate that MPO may regulate its own expression and activity in an autocrine and paracrine manner in the brain.
Our previous findings lend credence to the interesting notion that MPO could take an active part in cellular events that respond against rotenone in the CNS. To evaluate the influence of MPO on the rotenone-exposed brain, we used MPO-knockout mice and pharmacological inhibitors of MPO. Intriguingly, treatment of glia with MPO inhibitors further enhanced, rather than reduced, the rotenone-stimulated production of ROS. Similarly, rotenone-induced generation of ROS was greater in microglia from
Mpo−/− mice than in those from WT mice (
Figure 8). These unexpected results indicate that MPO deficiency could impair modulation of rotenone-stimulated ROS generation. Therefore, we further investigated the characteristics of glial cells from
Mpo−/− mice after exposure to rotenone. In primary astrocytes and microglia from
Mpo−/− mice, rotenone induced increased levels of several inflammatory mediators compared with normal WT control. In addition, cell viability was significantly reduced in glia from
Mpo−/− mice but not in those from healthy WT control mice. These observations show that MPO deficiency exacerbates rotenone-induced inflammatory responses and causes dysregulation of ROS generation, thus leading to abnormal outcomes in glial cells.
To better assess the action of MPO in the rotenone-exposed CNS, we examined the neuronal response to rotenone in the presence of glial cells with or without MPO. More interestingly, rotenone-induced neuronal cell death in co-cultures with glia from
Mpo−/− mice was significantly enhanced compared with that seen in cultures with glia from healthy mice (
Figure 11). We obtained similar results using MPO inhibitors. These findings indicate that MPO is required for appropriate responses of the glial defense system against rotenone exposure in the brain. There are several reports that MPO deficiency can contribute to pathological conditions in animal models and patients. Mice lacking MPO have a significantly increased incidence of experimental autoimmune encephalomyelitis
53- Brennan M.
- Gaur A.
- Pahuja A.
- Lusis A.J.
- Reynolds W.F.
Mice lacking myeloperoxidase are more susceptible to experimental autoimmune encephalomyelitis.
and show increased atherosclerosis and enhanced lung inflammation.
54- Brennan M.L.
- Anderson M.M.
- Shih D.M.
- Qu X.D.
- Wang X.
- Mehta A.C.
- Lim L.L.
- Shi W.
- Hazen S.L.
- Jacob J.S.
- Crowley J.R.
- Heinecke J.W.
- Lusis A.J.
Increased atherosclerosis in myeloperoxidase-deficient mice.
, 55- Milla C.
- Yang S.
- Cornfield D.N.
- Brennan M.L.
- Hazen S.L.
- Panoskaltsis-Mortari A.
- Blazar B.R.
- Haddad I.Y.
Myeloperoxidase deficiency enhances inflammation after allogeneic marrow transplantation.
Similarly, there is a greater occurrence of severe infections and chronic inflammatory processes among MPO-deficient patients.
56- Kutter D.
- Devaquet P.
- Vanderstocken G.
- Paulus J.M.
- Marchal V.
- Gothot A.
Consequences of total and subtotal myeloperoxidase deficiency: risk or benefit.
In contrast, blockade of MPO activity in mouse models and in humans can reduce the pathological response in diseases such as PD and in pathological conditions such as renal ischemia.
11- Matthijsen R.A.
- Huugen D.
- Hoebers N.T.
- de Vries B.
- Peutz-Kootstra C.J.
- Aratani Y.
- Daha M.R.
- Tervaert J.W.
- Buurman W.A.
- Heeringa P.
Myeloperoxidase is critically involved in the induction of organ damage after renal ischemia reperfusion.
, 18- Choi D.K.
- Pennathur S.
- Perier C.
- Tieu K.
- Teismann P.
- Wu D.C.
- Jackson-Lewis V.
- Vila M.
- Vonsattel J.P.
- Heinecke J.W.
- Przedborski S.
Ablation of the inflammatory enzyme myeloperoxidase mitigates features of Parkinson's disease in mice.
Considering our results in the context of these conflicting reports, it seems that MPO may act with dual functionality, having both pathological and protective functions under abnormal conditions.
MPO has been shown to act as a direct and significant mediator of decreased NO bioavailability. MPO can oxidize NO, thereby inhibiting NO-dependent signaling and modulating reduction-oxidation–sensitive signaling cascades during inflammation.
14- Eiserich J.P.
- Baldus S.
- Brennan M.L.
- Ma W.
- Zhang C.
- Tousson A.
- Castro L.
- Lusis A.J.
- Nauseef W.M.
- White C.R.
- Freeman B.A.
Myeloperoxidase, a leukocyte-derived vascular NO oxidase.
, 57- Eiserich J.P.
- Hristova M.
- Cross C.E.
- Jones A.D.
- Freeman B.A.
- Halliwell B.
- van der Vliet A.
Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils.
One possible explanation for our findings is that MPO-deficient glial cells inappropriately regulate ROS and reactive nitrogen species under rotenone-exposed conditions, perhaps because of increased NO bioavailability (
Figure 8,
Figure 9). Several lines of evidences indicate that MPO may be a metabolic “sink” for several types of ROS, including superoxide and H
2O
2, and may, therefore, compromise NO bioavailability.
13- Lau D.
- Mollnau H.
- Eiserich J.P.
- Freeman B.A.
- Daiber A.
- Gehling U.M.
- Brummer J.
- Rudolph V.
- Munzel T.
- Heitzer T.
- Meinertz T.
- Baldus S.
Myeloperoxidase mediates neutrophil activation by association with CD11b/CD18 integrins.
, 58- Baldus S.
- Heitzer T.
- Eiserich J.P.
- Lau D.
- Mollnau H.
- Ortak M.
- Petri S.
- Goldmann B.
- Duchstein H.J.
- Berger J.
- Helmchen U.
- Freeman B.A.
- Meinertz T.
- Munzel T.
Myeloperoxidase enhances nitric oxide catabolism during myocardial ischemia and reperfusion.
Previous reports
58- Baldus S.
- Heitzer T.
- Eiserich J.P.
- Lau D.
- Mollnau H.
- Ortak M.
- Petri S.
- Goldmann B.
- Duchstein H.J.
- Berger J.
- Helmchen U.
- Freeman B.A.
- Meinertz T.
- Munzel T.
Myeloperoxidase enhances nitric oxide catabolism during myocardial ischemia and reperfusion.
, 59- Brovkovych V.
- Gao X.P.
- Ong E.
- Brovkovych S.
- Brennan M.L.
- Su X.
- Hazen S.L.
- Malik A.B.
- Skidgel R.A.
Augmented inducible nitric oxide synthase expression and increased NO production reduce sepsis-induced lung injury and mortality in myeloperoxidase-null mice.
have associated MPO deficiency with increased levels of pulmonary iNOS expression and NO production. Also, iNOS expression is considerably greater in
Mpo−/− mice than in healthy mice (
Figure 8). Based on these previous reports and our findings, it is likely that MPO may have a regulatory influence over the activation state of immune and inflammatory cells by affecting the production of ROS and the expression of inflammatory mediators. MPO has been shown to act as a physiologically relevant regulator of inflammatory response by oxidatively limiting tissue-degrading protease activity.
16- Hirche T.O.
- Gaut J.P.
- Heinecke J.W.
- Belaaouaj A.
Myeloperoxidase plays critical roles in killing Klebsiella pneumoniae and inactivating neutrophil elastase: effects on host defense.
, 60- Clark R.A.
- Stone P.J.
- El Hag A.
- Calore J.D.
- Franzblau C.
Myeloperoxidase-catalyzed inactivation of alpha 1-protease inhibitor by human neutrophils.
These reports raise the possibility that MPO might contribute to protection of the host from pathogens via diverse mechanisms associated with the activity of inflammatory mediators. Such a regulatory effect of MPO on inflammatory mediators might also help to explain our observations in
Mpo−/− mice. To definitely address these possibilities, we are undertaking a thorough characterization of MPO-deficient mice and investigating the underlying basis of MPO actions under rotenone-exposed conditions.
Appropriate inflammatory responses and efficient resolution are essential attributes of an effective defense system against pathogens and environmental stimuli. MPO is a characteristic of inflammation, and its aberrant expression is presumed to be a detrimental factor in disease. Our present findings suggest that MPO may be associated with pathological outcomes and with protective events in the brain-resident immune cells under rotenone-exposed conditions. We have shown that MPO deficiency exacerbates rotenone-induced generation of ROS in glia and have demonstrated that MPO-null mice display inflammatory responses that are distinct from those of WT mice. Although the origin of adult microglia and microglial progenitors still remains controversial, microglia are widely considered as myeloid lineage–derived cells.
61- Ginhoux F.
- Greter M.
- Leboeuf M.
- Nandi S.
- See P.
- Gokhan S.
- Mehler M.F.
- Conway S.J.
- Ng L.G.
- Stanley E.R.
- Samokhvalov I.M.
- Merad M.
Fate mapping analysis reveals that adult microglia derive from primitive macrophages.
, 62- Hailer N.P.
- Heppner F.L.
- Haas D.
- Nitsch R.
Fluorescent dye prelabelled microglial cells migrate into organotypic hippocampal slice cultures and ramify.
, 63- Schmitz G.
- Leuthauser-Jaschinski K.
- Orso E.
Are circulating monocytes as microglia orthologues appropriate biomarker targets for neuronal diseases.
Recently, we observed that rotenone exposure led to increased MPO levels in peripheral blood mononuclear cells and peritoneal macrophages, as well as in microglia (unpublished data). These results further support the notion that rotenone exposure affects levels of MPO. The results of this study further expand our current understanding of the characteristics and roles of MPO under inflammatory conditions and indicate that the balance of MPO activity could be decisive for efficient resolution of the rotenone-induced pathological state in the brain. MPO has emerged as a regulatory target for the treatment of diverse diseases. Our results provide new insights into the responses associated with MPO and rotenone in the CNS and should inform the development of novel targeted therapies for reducing inflammation.