http://informahealthcare.com/imt ISSN: 1547-691X (print), 1547-6901 (electronic) J Immunotoxicol, 2015; 12(1): 98–103 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/1547691X.2014.902877
RESEARCH ARTICLE
Journal of Immunotoxicology Downloaded from informahealthcare.com by Astellas Pharma Japan on 02/11/15 For personal use only.
Oxidative induction of pro-inflammatory cytokine formation by human monocyte-derived macrophages following exposure to manganese in vitro Matlou I. Mokgobu1,2, Moloko C. Cholo1, Ronald Anderson1, Helen C. Steel1, Maraki P. Motheo1, Thembani N. Hlatshwayo1,3, Gregory R. Tintinger1,4, and Annette J. Theron1,3 1
Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa, 2Department of Environmental Health, Tshwane University of Technology, Pretoria, South Africa, 3Tshwane Academic Division of the National Health Laboratory Service, Pretoria, South Africa, and 4Department of Internal Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa Abstract
Keywords
Manganese (as Mn2+), a superoxide dismutase mimetic, catalyzes the formation of the relatively stable membrane-permeable reactive oxygen species (ROS) hydrogen peroxide (H2O2), a mediator of intracellular redox signaling in immune and inflammatory cells. The goal of this study was to investigate the potential for Mn2+, via its pro-oxidative properties, to activate production of pro-inflammatory cytokines/chemokines IL-1b, IL-6, IL-8, IFNg, TNFa, and G-CSF by human monocyte-derived macrophages in vitro. For these studies, the cells were isolated from peripheral blood mononuclear leukocytes and matured to generate a population of large CD14/CD16 co-expressing cells. The monocyte-derived macrophages were then exposed to bacterial lipopolysaccharide (LPS, 1 mg/ml) or MnCl2 (25–100 mM)—alone or in combination— for 24 h at 37 C, after which cell-free supernatants were analyzed using a multiplex cytokine assay procedure. Exposure of the cells to LPS caused modest statistically insignificant increases in cytokine production; MnCl2 caused dose-related increases in production of all six cytokines (achieving statistical significance of p50.0171–50.0005 for IL-1b, IL-6, IL-8, IFNg, and TNFa). In the case of LPS and MnCl2 combinations, the observed increases in production of IL-1b, IL-6, IL-8, IFNg, and G-CSF were greater than those seen with cells exposed to the individual agents. The Mn2+-mediated induction of cytokine production was associated with increased production of H2O2 and completely attenuated by inclusion of the H2O2-scavenger dithiothreitol, and partially by inhibitors of NF-kB and p38MAP kinase. The findings from the studies here help to further characterize the pro-inflammatory mechanisms that may underpin clinical disorders associated with excess exposure to Mn2+, particularly those disorders seen in the central nervous and respiratory systems.
Cytokines, H2O2, manganese, macrophages, NF-kB
Introduction Manganese is an essential nutrient required in trace amounts for human health; however, excessive exposure to the metal, usually via inhalation in adults exposed occupationally, can be harmful to various organs and tissues, especially the central nervous and respiratory systems (Boojar and Goodarzi, 2002; Environmental Protection Agency, 2007; Wang et al., 1989). Manganese neurotoxicity results from an accumulation of the metal in brain tissue, leading to the development of the neurodegenerative disorder manganism (Dobson et al., 2004). These toxic effects have been most commonly associated with occupations like manganese mining and smelting, battery manufacturing, and steel production (Hochberg et al., 1996; Roels et al., 1992; Santamaria, 2008; Wennberg et al., 1991). Occupational exposure to the metal may, however, also occur in other industries such as production Address for Correspondence: Dr Annette J. Theron, Department of Immunology, PO Box 2034, Pretoria 0001, South Africa. Tel: 27123192623. Fax: 27123230732. E-mail: [email protected]
History Received 15 October 2013 Revised 17 December 2013 Accepted 27 February 2014 Published online 7 May 2014
(and use) of agricultural products like fertilizers and manganesecontaining pesticides (Meco et al., 1994). Concern has also been raised about possible environmental exposure via combustion of methylcyclopentadienyl manganese tricarbonyl (MMT) used as a fuel additive in some countries (Frumkin & Solomon, 1997). Although monocytes and macrophages have an essential role in host defense, these cells may contribute to tissue destruction during acute and chronic inflammatory diseases of both infective and non-infective origin via the excessive release of an array of mediators, including reactive oxygen species (ROS), reactive nitrogen species, proteases, bioactive lipids, cytokines, and chemokines (Laskin et al., 2011; Shi & Pamer, 2011). Proinflammatory cytokines include interleukin (IL)-1, IL-6, IL-8, tumor necrosis factor (TNF)-a, interferon (IFN)-g, as well as colony stimulating factors that promote mobilization, recruitment, and activation of monocytes and neutrophils. In a previous study, we noted that manganese chloride (MnCl2) increased production of hydrogen peroxide (H2O2) by neutrophils and macrophages by acting as a superoxide dismutase mimetic (Mokgobu et al., 2012). Given the involvement of H2O2 as a
DOI: 10.3109/1547691X.2014.902877
Pro-inflammatory effects of MnCl2 on human macrophages
mediator of intracellular redox signaling processes and inflammatory gene transcription, the aim of the current study was to investigate effects of MnCl2 on production of pro-inflammatory cytokines by human monocyte-derived macrophages, as a potential mechanism of MnCl2 toxicity.
glycol-bis (2-aminoethylene)-N,N,N,N-tetracetic acid (EGTA, 2 mM, final) to prevent formation of aggregates. The flasks were then placed on ice with gentle agitation every 10 min (for at least 30 min) to promote detachment of the cells, which were then dislodged by scraping the surface of the flask with a sterile Cell Scraper (Adcock Ingram, Scientific Group, Johannesburg). The cells were then pelleted by centrifugation, the supernatant discarded, and the cell pellet re-suspended in 3 ml of Ca2+-free HBSS containing 2 mM EGTA. The cell suspension (100 ml) was then stained (simultaneously) with 10 ml of CD14-PE and CD16FITC monoclonal antibodies and incubated on ice for 10 min. Analyses were then performed on the FC500 flow cytometer: a minimum of 10 000 events/sample was acquired. In general, purity of the isolated monocyte-macrophage suspensions (large CD14/CD16 dual-expressing cells) was 83 [± 2]%. Remaining populations consisted of undifferentiated monocytes (7 [± 1]% expressing CD14 only) and double-negative cells (9 [± 1]%, probably lymphocytes). Viability of the total cell population measured by propidium iodide exclusion (described below) was 78 [± 3]% (range ¼ 62–94%).
Materials and methods
Journal of Immunotoxicology Downloaded from informahealthcare.com by Astellas Pharma Japan on 02/11/15 For personal use only.
Chemicals and reagents Manganese chloride (MnCl2) was purchased from Sigma (St Louis, MO), dissolved in distilled water to a stock concentration of 10 mM, and used in the assays described below at a final concentration range of 25–100 mM, levels that are pathologically relevant (Bird et al., 1984; Sistrunk et al., 2007; Suzuki et al., 1975). Unless indicated, all chemicals and reagents were purchased from Sigma. Monocyte/macrophage isolation and culture Peripheral blood was obtained from healthy non-smoking male and female (3:7 ratio), mostly Caucasian donors (92%), with a mean age of 42 ± 2 (range ¼ 23–64) years. All donors were medical/medical laboratory personnel, none of who had any history of occupational exposure to manganese. Permission to draw blood was granted by the Research Ethics Committee of the Faculty of Health Sciences of the University of Pretoria (Protocol #43/2006/2009). Subsequent to obtaining informed consent from the donors, mononuclear cells (MNL) were prepared from heparinized (5 U preservative-free heparin/ml) venous blood. Blood was slowly layered atop Histopaque (Sigma) and centrifuged for 25 min (400 x g, room temperature). Following centrifugation, the MNL fraction at the plasma/Histopaque-1077 interface was aspirated, diluted 1:4 with phosphate-buffered saline (PBS, pH 7.4), and the cells pelleted by centrifugation; thereafter, contaminating erythrocytes were removed by hypotonic lysis. Subsequent to another centrifugation step and discarding of the supernatant, the cells were re-suspended in sterile Hanks’ balanced salt solution (HBSS, indicator-free, containing 1.25 mM CaCl2 [pH 7.4], Highveld Biological, Johannesburg, South Africa). The cell preparation was then analyzed of total T-cells, monocytes, granulocytes, and B-cells by flow cytometry. Samples of the cells (100 ml) were simultaneously incubated on ice for 10 min with 10 ml each (according to manufacturer instructions) of the following fluorochrome-labeled monoclonal antibodies (all Beckman Coulter, Miami, FL): anti-CD3 (FITC), anti-CD14 (PE), anti-CD15 (FITC), and anti-CD19 (PE) for tagging of, respectively, T-cells, monocytes, granulocytes, and B-cells. Analysis/enumeration was then performed on a FC500 flow cytometer (Beckman Coulter) using CXP software. A minimum of 10 000 events/sample was acquired. Differential adherence to plastic was used to separate monocytes from other types of MNL and to promote their differentiation. MNL (30 ml of a 3 107/ml suspension in HBSS) were seeded onto sterile 75 cm3 tissue culture flasks and incubated for 2 h at 37 C/5% CO2 to promote adherence of monocytes. Following incubation, each flask was gently rinsed with 50 ml of pre-warmed PBS to remove non-adherent cells. Ten milliliters of tissue culture medium RPMI 1640 (Bio Whittaker, Walkersville, MD) supplemented with antibiotics (penicillin/ streptomycin/amphotericin B, 0.1/0.25/0.1 mg/ml) and 5% autologous serum were then added to each flask and the flasks were then incubated for 7 days at 37 C. Following the incubation, the tissue culture medium was discarded and each flask rinsed once with 10 ml pre-warmed PBS, followed by 10 ml PBS containing Ca2+-chelating ethylene
99
Cytokine production Monocyte-derived macrophages (105/ml in 200 ml Ca2+-free HBSS) were added to wells of 48-well micro-tissue plates. The plates were incubated for 2 h at 37 C to promote cell adherence, after which the supernatant was removed and replaced with 500 ml RPMI 1640 supplemented with antibiotics and 5% autologous serum. The plates were then incubated overnight at 37 C. The well supernatants were then removed and the cells washed twice with 500 ml of pre-warmed PBS, followed by addition of serumsupplemented RPMI plus antibiotics to all wells. MnCl2 (50 ml) at final concentrations of 25–100 mM was then added to designated wells; control wells received 50 ml HBSS, the plates were then incubated a further 30 min before the cells were treated with 50 ml lipopolysaccharide (LPS, from Escherichia coli 0127:B8; 1 mg/well [final]) and the plates incubated overnight at 37 C. After 24 h, well supernatants were collected and frozen at 20 C. The supernatants were ultimately assayed for IL-1b, IL-6, IL-8, G-CSF, IFNg, and TNFa using a magnetic bead-based multiplex assay (Bio-Rad Laboratories, Hercules, CA). This cytokine profile was selected as being representative of an M1 inflammatory macrophage phenotype (high level expression of CD14/CD16, nitric oxide synthase, IL-6, TNFa; a propensity to drive T-helper [TH]-1 cell development; and low-level expression of IL-10). The procedure for cytokine analyses followed manufacturer specifications. A 4-fold serial dilution was made of the pre-mixed standards supplied with the assay kit (concentration range ¼ 1.48–77 755.00 pg/ml). All supernatants were assayed undiluted; the concentration of each cytokine (pg/ml) was extrapolated from the standard curve generated in parallel. In a more limited series of experiments, effects of select agents on the production of IL-6 and IL-8 by LPS/MnCl2 (100 mM)treated monocyte-derived macrophages were investigated. The agents were: (i) NF-kB activation inhibitor (1 mM, Calbiochem); (ii) p38 MAP (mitogen activated protein) kinase inhibitor SB202190 (20 mM, Sigma); and (iii) dithiothreitol (DTT, 5 mM, Sigma), a cell-penetrating scavenger of H2O2. Propidium iodide exclusion assay To test for potential cytotoxic effects of NF-kB activation inhibitor (1 mM), SB202190 (20 mM), and dithiothreitol (5 mM), cell viability assays were performed on mononuclear cells (2 106/ml) using the flow cytometric propidium iodide exclusion assay after a 24-h exposure to the test agents. The cells
100
M. I. Mokgobu et al.
J Immunotoxicol, 2015; 12(1): 98–103
(2 105) were incubated for 5 min with propidium iodide (DNA Prep-Stain, Beckman Coulter, 50 mg/ml) and then cell viability assessed using the flow cytometer. All data were expressed as a percentage of viable cells.
Journal of Immunotoxicology Downloaded from informahealthcare.com by Astellas Pharma Japan on 02/11/15 For personal use only.
Intracellular H2O2 production Intracellular H2O2 was measured using a 20 ,70 -dichlorodihydrofluorescein diacetate (DCF-DA)-based spectrofluorimetric procedure. This agent is hydrolyzed by cellular esterases to 20 ,70 -dichlorodihydrofluorescein which is, in turn, oxidized to 20 ,70 -dichlorofluorescein, primarily by H2O2. Here, DCF-DA (10 mM) was added to a reaction mixture containing 105 monocyte-derived macrophages/ml in the absence and presence of MnCl2 (100 mM) and/or LPS (1 mg/ml) in a total volume of 3 ml HBSS. The samples were then incubated for 10 min in a 37 C waterbath and then transferred to the thermo-regulated cuvette holder of a Hitachi 650 10S fluorescence spectrophotometer with excitation and emission wavelengths at 500 and 530 nm, respectively. Fluorescence responses were then monitored continually for 10 min. These experiments were performed in the presence and absence of diphenyleneiodonium chloride (DPI, 20 mM; inhibitor of NADPH oxidase; O’Donnell et al., 1993) or the ROS scavenger, dithiothreitol (DTT, 100 mM). The latter agent was used at a lower concentration than that used in assays of cytokine production due to interference with the DCF-DA-based assay when used at higher levels. These results are shown as traces of a representative experiment.
Figure 1. Effects of MnCl2 on spontaneous and LPS-activated monocytederived macrophage IL-1b and IL-6 production. Results shown are mean ( ± SEM) pg/ml (n ¼ 6). *p50.05 vs control (0). +p50.05 vs LPS-only control.
Expression and statistical analysis of data Results of each series of experiments are presented as mean ( ± SEM) of absolute values, where n is the number of different donors used in each series of experiments. Levels of statistical significance were determined by comparing the absolute values for each metal-treated system with the corresponding values for the relevant metal-free control systems for each assay using the Wilcoxon matched pairs test. This option was given in the InStat software statistics program (GraphPad, La Jolla, CA) when selecting for a paired non-parametric test.
Results The effects of MnCl2 and LPS alone and in combination on cytokine production by monocyte-derived macrophages are shown in Figures 1–3. Exposure of the cells to the metal alone resulted in statistically significant increases in production of IL-1b, IL-6, IL-8, TNFa, and IFNg (all at 100 mM MnCl2, with the exception of IL-6 for which the effects of 50 mM also achieved statistical significance), but not G-CSF. Treatment with LPS alone caused modest increases in production of all the cytokines/chemokine, none of which achieved statistical significance. In the case of cells treated with combinations of MnCl2 and LPS, the observed increases in generation of IL-1b, IL-6, IL-8, IFNg, and G-CSF were greater than those by cells exposed to the individual agents, achieving statistical significance in the case of IL-6, IL-8, and IFNg when compared with the corresponding MnCl2-free LPS-treated control systems. Although the combination of MnCl2 and LPS treatment caused impressive dose-related increases in production of IL-1b and G-CSF, these did not achieve statistical significance, probably as a consequence of considerable variation in magnitudes of increases between experiments (ranging from 42–374% and from 18–1020% increase in comparison with corresponding LPS-treated control systems for IL-1b and G-CSF, respectively). In the case of TNFa, values for the LPS and MnCl2 combination were comparable to those of the metal alone.
Figure 2. Effects of MnCl2 on spontaneous and LPS-activated monocytederived macrophage IL-8 and TNFa production. Results shown are mean ( ± SEM) pg/ml (n ¼ 6). *p50.05 vs control (0). +p50.05 vs LPS-only control.
The effects of the NF-kB activation inhibitor (1 mM), SB202190 (20 mM), or dithiothreitol (DTT, 5 mM) on MnCl2mediated increases in IL-6 and IL-8 production by macrophages are shown in Figure 4. Exposure of cells to DTT completely abolished the augmentative effects of MnCl2 on generation of both cytokines by LPS-activated cells. In four of the five experiments, NF-kB activation inhibitor and SB202190 caused partial inhibition, ranging from 21–45% and 41–85%, respectively, for IL-6; corresponding values for IL-8 ranged from 25–41% and 24–65%. Propidium iodide exclusion assay Using the propidium iodide exclusion assay, the percentage of viable cells for the control system and for cells treated with
Journal of Immunotoxicology Downloaded from informahealthcare.com by Astellas Pharma Japan on 02/11/15 For personal use only.
DOI: 10.3109/1547691X.2014.902877
Figure 3. Effects of MnCl2 on spontaneous and LPS-activated monocytederived macrophage G-CSF and IFNg production. Results shown are mean ( ± SEM) pg/ml (n ¼ 6). *p50.05 vs control (0). +p50.05 vs LPS-only control.
Pro-inflammatory effects of MnCl2 on human macrophages
101
Figure 5. Effects of MnCl2 (at 100 mM) on unstimulated monocytederived macrophage dichlorofluorescein diacetate fluorescence responses. Traces from a single representative experiment (from n ¼ 4–5) are shown. Experiments were performed in the absence/presence of NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI, 20 mM) or the ROS scavenger dithiothreitol (DTT, 100 mM).
Discussion
Figure 4. Effects of SB202190 (SB, 20 mM), NF-kB activation inhibitor (NF-kBi, 1 mM), or dithiothreitol (DTT, 5 mM) on Mn-mediated increases in monocyte-derived macrophage IL-6 and IL-8 production. Results are mean ( ± SEM) pg/ml (n ¼ 5). *p50.05 vs LPS/MnCl2-treated (inhibitorfree) controls.
NF-kB activation inhibitor, SB202190, or dithiothreitol was 97.5 [± 0.4]%, 97.2 [± 0.4], 97.2 [± 0.7], and 97.2 [± 0.5]%, thus clearly demonstrating a lack of cytotoxicity. Intracellular H2O2 production The results shown in Figure 5 are typical traces from one representative experiment (n ¼ 4–5) that depict the effects of MnCl2 (100 mM) on DCF-DA fluorescence responses of unstimulated monocyte-derived macrophages. Treatment of the cells with MnCl2 caused a dose-dependent increase in fluorescence intensity, compatible with stimulated intracellular H2O2 production by the macrophages. These effects were attenuated by inclusion of DPI (20 mM) and by DTT (100 mM). In contrast, LPS (1 mg/ml) had no effect on the cell responses (data not shown).
Occupational exposure to manganese has been linked to manganism, a specific neuro-pathology characterized by clinical signs and lesions similar to Parkinson’s disease (Meco et al., 1994). According to Littlejohn et al., (2011), cytokines are increasingly implicated in acute and chronic neuronal demise, with a number of clinical studies having demonstrated increased levels of pro-inflammatory cytokines (e.g., TNFa, IL-6, IL-1b, and IFNg) in post-mortem brain, as well as blood or cerebral spinal fluid, of patients with stroke, head injury, Alzheimer’s disease, and Parkinson’s disease (Basic Kes et al., 2008; Brodacki et al., 2008; Goodman et al., 2008; Glass et al., 2010). Moreover, levels of IFNg, IL-1b, and TNFa, as well as of the chemokines monocyte chemoattractant protein (MCP)-1, RANTES, macrophage inflammatory protein (MIP)-1a, and IL-8 were significantly correlated with the severity of Parkinsonism (Reale et al., 2009). These reports underscore the involvement of pro-inflammatory cytokines and chemokines in the development of neurotoxicity in manganese-exposed workers. The current study demonstrated that Mn2+ (25–100 mM) alone or in combination with LPS potentiated production of proinflammatory cytokines IL-1b, IL-6, IL-8, IFNg, TNFa, and G-CSF by human monocyte-derived macrophages. The trends observed in all experiments were consistent, although the standard error bars were large due to variability between experiments, possibly due to extensive multi-step procedures involved in monocyte isolation and maturation. The concentrations of the metal used in the current study are potentially attainable in cells and tissues, particularly those of the brain. In this context, primates exposed to occupationally relevant concentrations of manganese exhibited metal levels between 10–350 mM in the brain (Bird et al., 1984; Sistrunk et al., 2007; Suzuki et al., 1975). With respect to previously published studies of pro-inflammatory effects of Mn2+ upon cells of the innate immune system, the current study differs in that: (i) the focus here was on human blood monocyte-derived macrophages as opposed to microglia/ astrocytes of animal origin and/or cell lines (Dodd & Filipov, 2011; Liu et al., 2006, 2013; Spranger et al., 1998; Zhang et al., 2010); (ii) the relevance of the findings of the current study are related to inflammation-associated organ dysfunction in general, as opposed specifically to any neurotoxicity; and (iii) the
102
M. I. Mokgobu et al.
J Immunotoxicol, 2015; 12(1): 98–103
ineffective. Specific measures of NF-kB and p38 MAPK expression, while of interest, were therefore likely to be of limited value due to redundancy and a convergence of intracellular redox signaling mechanisms. In addition, optimal inflammatory gene expression is accomplished by complementary interactions of several different transcription factors that converge on large nuclear proteins known as co-activator molecules, of which cAMP response element-binding protein (CBP) is a prototype. Following binding of transcription factors to CBP, the co-activator molecule acquires histone acetyl-transferase (HAT) activity, thereby facilitating interaction of transcription factors with their response elements on target genes (Barnes et al., 2005).
Journal of Immunotoxicology Downloaded from informahealthcare.com by Astellas Pharma Japan on 02/11/15 For personal use only.
Conclusions
Figure 6. Summary of potential mechanisms by which manganese causes increased monocyte-derived macrophage pro-inflammatory cytokine production. Macrophage membrane-associated NADPH oxidase gener2+ ates superoxide anions (O (a SOD 2 ) that, in the presence of Mn mimetic), are converted to hydrogen peroxide (H2O2). H2O2 then acts as an intracellular messenger causing the activation of redox-sensitive signaling proteins and downstream transcription factors. This, in turn, leads to activation of genes encoding a range of pro-inflammatory cytokines, prolonged production of which may cause inflammation and tissue injury.
collection of pro-inflammatory cytokines tested here were of particular relevance to the processes of neutrophil/monocyte/ macrophage mobilization and activation. In the case of molecular mechanisms underpinning the induction of cytokine production by MnCl2, we previously reported that the metal potentiates production of H2O2 by activated macrophages, apparently by acting as a superoxide dismutase mimetic (Mokgobu et al., 2012), implying an association between these events. This contention was reinforced by the findings of the current study that exposure of the monocytes/macrophages to MnCl2, but not LPS, caused a spontaneous increase in formation of H2O2 that was attenuated by inclusion of DPI (an inhibitor of NADPH oxidase) as well as by the ROS scavenger DTT. Importantly, H2O2 acts as an intracellular second messenger, thereby modulating several major signal cascades including the ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), MAPK (mitogenactivated protein kinase), and PI3K/Akt (phosphatidylinositol 3-Kinase/Akt) pathways, as well as downstream transcription factors, by targeting cysteine and methionine residues on these proteins (Rhee et al., 2003; Wittmann et al., 2012). Of the ROS commonly produced in tissues, H2O2 appears to be most important in terms of signaling as it is relatively stable and readily crosses cell membranes (Fisher, 2009). The efficacy of DTT in abolishing Mn2+-mediated production of pro-inflammatory cytokines by monocyte-derived macrophages, taken together with the observed attenuation of formation of H2O2, clearly underscores the involvement of H2O2 in the activation of gene expression. The more limited efficacy of the pharmacological inhibitors of NF-kB and p38 MAPK probably reflects multiple redox-signaling mechanisms activated by H2O2, rendering selective inhibitors of a single pathway relatively
The results of the current study are compatible with a prooxidative mechanism whereby MnCl2, as a consequence of its superoxide dismutase mimetic activity, potentiates the generation of H2O2 by monocyte-derived macrophages. In turn, H2O2, via its redox intracellular signaling activities, promotes the activation of genes encoding a range of pro-inflammatory cytokines. A summary of the potential mechanisms by which Mn2+ increases production of pro-inflammatory cytokines by macrophages is shown in Figure 6. Although remaining to be conclusively proven, this pro-oxidative pro-inflammatory mechanism is likely to be implicated in the (immuno)- pathogenesis of Mn-associated neurologic and respiratory dysfunction.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References Barnes, P. J., Adcock, I. M., and Ito, K. 2005. Histone acetylation and deacetylation: Importance in inflammatory lung diseases. Eur. Respir. J. 25:552–563. Basic Kes, V., Simundic, A. M., Nicolac, N., et al. 2008. Pro-inflammatory and anti-inflammatory cytokines in acute ischemic stroke and their relation to early neurological deficit and stroke outcome. Clin. Biochem. 41:1330–1334. Bird, E. D., Anton, A. H., and Bullock, B. 1984. The effect of manganese inhalation on basal ganglia dopamine concentrations in rhesus monkey. Neurotoxicology 5:59–65. Boojar, M. M., and Goodarzi, F. 2002. A longitudinal follow-up of pulmonary function and respiratory symptoms in workers exposed to manganese. J. Occup. Environ. Med. 44:282–290. Brodacki, B., Staszewski, J., Toczylowska, B., et al. 2008. Serum interleukin (IL-2, IL-10, IL-6, IL-4), TNFa, and INFg concentrations are elevated in patients with atypical and idiopathic Parkinsonism. Neurosci. Lett. 441:158–162. Dobson, A. W., Erikson, K. M., and Aschner, M. 2004. Manganese neurotoxicity. Ann. N.Y. Acad. Sci. 1012:115–128. Dodd, C. A., and Filipov, N. M. 2011. Manganese potentiates LPSinduced heme oxygenase-1 in microglia but not dopaminergic cells: Role in controlling microglial hydrogen peroxide and inflammatory cytokine output. Neurotoxicology 32:683–692. Environmental Protection Agency (EPA). 2007. Manganese TEACH Chemical Summary Revised 2007. Available online at: http:// www.epa.gov/teach/chem_summ/manganese summary.pdf [last accessed 11 Jul 2013]. Fisher, A. B. 2009. Redox signaling across cell membranes. Antiox. Redox Signal. 11:1349–1356. Frumkin, H., and Solomon, G. 1997. Manganese in the U.S. Gasoline Supply. Am. J. Ind. Med. 31:107–115. Glass, C. K., Saijo, K., Winner, B., et al. 2010. Mechanisms underlying inflammation in neuro-degeneration. Cell 140:918–934. Goodman, J. C., Van, M., Gopinath, S. P., and Robertson, C. S. 2008. Proinflammatory and pro-apoptotic elements of the neuro-inflammatory response are activated in traumatic brain injury. Acta. Neurochir. S102: 437–439.
Journal of Immunotoxicology Downloaded from informahealthcare.com by Astellas Pharma Japan on 02/11/15 For personal use only.
DOI: 10.3109/1547691X.2014.902877
Hochberg, F., Miller, G., Valenzuela, R., et al. 1996. Late motor deficits of Chilean manganese miners: A blinded control study. Neurology 47: 788–795. Laskin, D. L., Sunil, V. R., Gardner, C. R., and Laskin, J. D. 2011. Macrophages and tissue injury: Agents of defense or destruction? Annu. Rev. Pharmacol. Toxicol. 81:267–288. Littlejohn, D., Mangano, E., Clarke, E., et al. 2011. Inflammatory mechanisms of neurodegeneration in toxin-based models of Parkinson’s Disease. Parkinson’s Dis. 2011:713517. Liu, X., Sullivan, K. A., Madl, J. E., et al. 2006. Manganese-induced neurotoxicity: The role of astroglial-derived nitric oxide in striatal interneuron degeneration. Toxicol. Sci. 91:521–531. Liu, Y., Barber, D. S., Zhang, P., and Liu, B. 2013. Complex II of the mitochondrial respiratory chain is the key mediator of divalent manganese-induced hydrogen peroxide production in microglia. Toxicol. Sci. 132:298–306. Meco, G., Bonifati, V., Vanacore, N., and Fabrizio, E. 1994. Parkinsonism after chronic exposure to the fungicide Maneb (manganese ethylenebis-dithiocarbamate). Scand. J. Work. Environ. Health 20:301–305. Mokgobu, M. I., Anderson, R., Steel, H. C., et al. 2012. Manganese promotes increased formation of hydrogen peroxide by activated human macrophages and neutrophils in vitro. Inhal. Toxicol. 24: 634–644. O’Donnell, B. V., Tew, D. G., Jones, O. T., and England, P. J. 1993. Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase. Biochem. J. 290: 41–49. Reale, M., Iarlori, C., Thomas, A., Gambi, D., et al. 2009. Peripheral cytokines profile in Parkinson’s disease. Brain Behav. Immun. 23: 55–63.
Pro-inflammatory effects of MnCl2 on human macrophages
103
Rhee, S. G., Chang, T. S., Bae, Y. S., et al. 2003. Cellular regulation by hydrogen peroxide. J. Am. Soc. Nephrol. 14:S211–215. Roels, H. A., Ghyselen, P., Buchet, J. P., et al. 1992. Assessment of permissible exposure level to manganese in workers exposed to manganese dioxide dust. Br. J. Ind. Med. 49:25–34. Santamaria, A. B. 2008. Manganese: Exposure, essentiality, and toxicity. Indian J. Med. Res. 128:484–500. Shi, C., and Pamer, E. G. 2011. Monocyte recruitment during infection and inflammation. Nat. Rev. Immunol. 11:762–774. Sistrunk, S. C., Ross, M. K., and Filipov, N. M. 2007. Direct effects of manganese compounds on dopamine and its metabolite Dopac: An in vitro study. Environ. Toxicol. Pharmacol. 23:286–296. Spranger, M., Schwab, S., Desiderato, S., et al. 1998. Manganese augments nitric oxide synthesis in murine astrocytes: A new pathogenetic mechanism in manganism? Exp. Neurol. 149:277–283. Suzuki, Y., Mouri, T., Nishiyama, K., and Fujii, N. 1975. Study of subacute toxicity of manganese dioxide in monkeys. Tokashima J. Exp. Med. 22:5–10. Wang, J. D., Huang, C. C., Hwang, Y. H., et al. 1989. Manganese induced Parkinsonism: An outbreak due to an unrepaired ventilation control system in a ferromanganese smelter. Br. J. Ind. Med. 46:856–859. Wennberg, A., Iregren, A., Struwe, G., et al. 1991. Manganese exposure in steel smelters a health hazard to the nervous system. Scand. J. Work Environ. Health 17:255–262. Wittmann, C., Chockley, P., Singh, S. K., et al. 2012. Hydrogen peroxide in inflammation: Messenger, guide, and assassin. Adv. Hematol. 2012:541471. Zhang, P., Lokuta, K. M., Turner, D. E., and Liu, B. 2010. Synergistic dopaminergic neuro-toxicity of manganese and lipopolysaccharide: Differential involvement of microglia and astroglia. J. Neurochem. 112:434–443.
RESEARCH ARTICLE
Journal of Immunotoxicology Downloaded from informahealthcare.com by Astellas Pharma Japan on 02/11/15 For personal use only.
Oxidative induction of pro-inflammatory cytokine formation by human monocyte-derived macrophages following exposure to manganese in vitro Matlou I. Mokgobu1,2, Moloko C. Cholo1, Ronald Anderson1, Helen C. Steel1, Maraki P. Motheo1, Thembani N. Hlatshwayo1,3, Gregory R. Tintinger1,4, and Annette J. Theron1,3 1
Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa, 2Department of Environmental Health, Tshwane University of Technology, Pretoria, South Africa, 3Tshwane Academic Division of the National Health Laboratory Service, Pretoria, South Africa, and 4Department of Internal Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa Abstract
Keywords
Manganese (as Mn2+), a superoxide dismutase mimetic, catalyzes the formation of the relatively stable membrane-permeable reactive oxygen species (ROS) hydrogen peroxide (H2O2), a mediator of intracellular redox signaling in immune and inflammatory cells. The goal of this study was to investigate the potential for Mn2+, via its pro-oxidative properties, to activate production of pro-inflammatory cytokines/chemokines IL-1b, IL-6, IL-8, IFNg, TNFa, and G-CSF by human monocyte-derived macrophages in vitro. For these studies, the cells were isolated from peripheral blood mononuclear leukocytes and matured to generate a population of large CD14/CD16 co-expressing cells. The monocyte-derived macrophages were then exposed to bacterial lipopolysaccharide (LPS, 1 mg/ml) or MnCl2 (25–100 mM)—alone or in combination— for 24 h at 37 C, after which cell-free supernatants were analyzed using a multiplex cytokine assay procedure. Exposure of the cells to LPS caused modest statistically insignificant increases in cytokine production; MnCl2 caused dose-related increases in production of all six cytokines (achieving statistical significance of p50.0171–50.0005 for IL-1b, IL-6, IL-8, IFNg, and TNFa). In the case of LPS and MnCl2 combinations, the observed increases in production of IL-1b, IL-6, IL-8, IFNg, and G-CSF were greater than those seen with cells exposed to the individual agents. The Mn2+-mediated induction of cytokine production was associated with increased production of H2O2 and completely attenuated by inclusion of the H2O2-scavenger dithiothreitol, and partially by inhibitors of NF-kB and p38MAP kinase. The findings from the studies here help to further characterize the pro-inflammatory mechanisms that may underpin clinical disorders associated with excess exposure to Mn2+, particularly those disorders seen in the central nervous and respiratory systems.
Cytokines, H2O2, manganese, macrophages, NF-kB
Introduction Manganese is an essential nutrient required in trace amounts for human health; however, excessive exposure to the metal, usually via inhalation in adults exposed occupationally, can be harmful to various organs and tissues, especially the central nervous and respiratory systems (Boojar and Goodarzi, 2002; Environmental Protection Agency, 2007; Wang et al., 1989). Manganese neurotoxicity results from an accumulation of the metal in brain tissue, leading to the development of the neurodegenerative disorder manganism (Dobson et al., 2004). These toxic effects have been most commonly associated with occupations like manganese mining and smelting, battery manufacturing, and steel production (Hochberg et al., 1996; Roels et al., 1992; Santamaria, 2008; Wennberg et al., 1991). Occupational exposure to the metal may, however, also occur in other industries such as production Address for Correspondence: Dr Annette J. Theron, Department of Immunology, PO Box 2034, Pretoria 0001, South Africa. Tel: 27123192623. Fax: 27123230732. E-mail: [email protected]
History Received 15 October 2013 Revised 17 December 2013 Accepted 27 February 2014 Published online 7 May 2014
(and use) of agricultural products like fertilizers and manganesecontaining pesticides (Meco et al., 1994). Concern has also been raised about possible environmental exposure via combustion of methylcyclopentadienyl manganese tricarbonyl (MMT) used as a fuel additive in some countries (Frumkin & Solomon, 1997). Although monocytes and macrophages have an essential role in host defense, these cells may contribute to tissue destruction during acute and chronic inflammatory diseases of both infective and non-infective origin via the excessive release of an array of mediators, including reactive oxygen species (ROS), reactive nitrogen species, proteases, bioactive lipids, cytokines, and chemokines (Laskin et al., 2011; Shi & Pamer, 2011). Proinflammatory cytokines include interleukin (IL)-1, IL-6, IL-8, tumor necrosis factor (TNF)-a, interferon (IFN)-g, as well as colony stimulating factors that promote mobilization, recruitment, and activation of monocytes and neutrophils. In a previous study, we noted that manganese chloride (MnCl2) increased production of hydrogen peroxide (H2O2) by neutrophils and macrophages by acting as a superoxide dismutase mimetic (Mokgobu et al., 2012). Given the involvement of H2O2 as a
DOI: 10.3109/1547691X.2014.902877
Pro-inflammatory effects of MnCl2 on human macrophages
mediator of intracellular redox signaling processes and inflammatory gene transcription, the aim of the current study was to investigate effects of MnCl2 on production of pro-inflammatory cytokines by human monocyte-derived macrophages, as a potential mechanism of MnCl2 toxicity.
glycol-bis (2-aminoethylene)-N,N,N,N-tetracetic acid (EGTA, 2 mM, final) to prevent formation of aggregates. The flasks were then placed on ice with gentle agitation every 10 min (for at least 30 min) to promote detachment of the cells, which were then dislodged by scraping the surface of the flask with a sterile Cell Scraper (Adcock Ingram, Scientific Group, Johannesburg). The cells were then pelleted by centrifugation, the supernatant discarded, and the cell pellet re-suspended in 3 ml of Ca2+-free HBSS containing 2 mM EGTA. The cell suspension (100 ml) was then stained (simultaneously) with 10 ml of CD14-PE and CD16FITC monoclonal antibodies and incubated on ice for 10 min. Analyses were then performed on the FC500 flow cytometer: a minimum of 10 000 events/sample was acquired. In general, purity of the isolated monocyte-macrophage suspensions (large CD14/CD16 dual-expressing cells) was 83 [± 2]%. Remaining populations consisted of undifferentiated monocytes (7 [± 1]% expressing CD14 only) and double-negative cells (9 [± 1]%, probably lymphocytes). Viability of the total cell population measured by propidium iodide exclusion (described below) was 78 [± 3]% (range ¼ 62–94%).
Materials and methods
Journal of Immunotoxicology Downloaded from informahealthcare.com by Astellas Pharma Japan on 02/11/15 For personal use only.
Chemicals and reagents Manganese chloride (MnCl2) was purchased from Sigma (St Louis, MO), dissolved in distilled water to a stock concentration of 10 mM, and used in the assays described below at a final concentration range of 25–100 mM, levels that are pathologically relevant (Bird et al., 1984; Sistrunk et al., 2007; Suzuki et al., 1975). Unless indicated, all chemicals and reagents were purchased from Sigma. Monocyte/macrophage isolation and culture Peripheral blood was obtained from healthy non-smoking male and female (3:7 ratio), mostly Caucasian donors (92%), with a mean age of 42 ± 2 (range ¼ 23–64) years. All donors were medical/medical laboratory personnel, none of who had any history of occupational exposure to manganese. Permission to draw blood was granted by the Research Ethics Committee of the Faculty of Health Sciences of the University of Pretoria (Protocol #43/2006/2009). Subsequent to obtaining informed consent from the donors, mononuclear cells (MNL) were prepared from heparinized (5 U preservative-free heparin/ml) venous blood. Blood was slowly layered atop Histopaque (Sigma) and centrifuged for 25 min (400 x g, room temperature). Following centrifugation, the MNL fraction at the plasma/Histopaque-1077 interface was aspirated, diluted 1:4 with phosphate-buffered saline (PBS, pH 7.4), and the cells pelleted by centrifugation; thereafter, contaminating erythrocytes were removed by hypotonic lysis. Subsequent to another centrifugation step and discarding of the supernatant, the cells were re-suspended in sterile Hanks’ balanced salt solution (HBSS, indicator-free, containing 1.25 mM CaCl2 [pH 7.4], Highveld Biological, Johannesburg, South Africa). The cell preparation was then analyzed of total T-cells, monocytes, granulocytes, and B-cells by flow cytometry. Samples of the cells (100 ml) were simultaneously incubated on ice for 10 min with 10 ml each (according to manufacturer instructions) of the following fluorochrome-labeled monoclonal antibodies (all Beckman Coulter, Miami, FL): anti-CD3 (FITC), anti-CD14 (PE), anti-CD15 (FITC), and anti-CD19 (PE) for tagging of, respectively, T-cells, monocytes, granulocytes, and B-cells. Analysis/enumeration was then performed on a FC500 flow cytometer (Beckman Coulter) using CXP software. A minimum of 10 000 events/sample was acquired. Differential adherence to plastic was used to separate monocytes from other types of MNL and to promote their differentiation. MNL (30 ml of a 3 107/ml suspension in HBSS) were seeded onto sterile 75 cm3 tissue culture flasks and incubated for 2 h at 37 C/5% CO2 to promote adherence of monocytes. Following incubation, each flask was gently rinsed with 50 ml of pre-warmed PBS to remove non-adherent cells. Ten milliliters of tissue culture medium RPMI 1640 (Bio Whittaker, Walkersville, MD) supplemented with antibiotics (penicillin/ streptomycin/amphotericin B, 0.1/0.25/0.1 mg/ml) and 5% autologous serum were then added to each flask and the flasks were then incubated for 7 days at 37 C. Following the incubation, the tissue culture medium was discarded and each flask rinsed once with 10 ml pre-warmed PBS, followed by 10 ml PBS containing Ca2+-chelating ethylene
99
Cytokine production Monocyte-derived macrophages (105/ml in 200 ml Ca2+-free HBSS) were added to wells of 48-well micro-tissue plates. The plates were incubated for 2 h at 37 C to promote cell adherence, after which the supernatant was removed and replaced with 500 ml RPMI 1640 supplemented with antibiotics and 5% autologous serum. The plates were then incubated overnight at 37 C. The well supernatants were then removed and the cells washed twice with 500 ml of pre-warmed PBS, followed by addition of serumsupplemented RPMI plus antibiotics to all wells. MnCl2 (50 ml) at final concentrations of 25–100 mM was then added to designated wells; control wells received 50 ml HBSS, the plates were then incubated a further 30 min before the cells were treated with 50 ml lipopolysaccharide (LPS, from Escherichia coli 0127:B8; 1 mg/well [final]) and the plates incubated overnight at 37 C. After 24 h, well supernatants were collected and frozen at 20 C. The supernatants were ultimately assayed for IL-1b, IL-6, IL-8, G-CSF, IFNg, and TNFa using a magnetic bead-based multiplex assay (Bio-Rad Laboratories, Hercules, CA). This cytokine profile was selected as being representative of an M1 inflammatory macrophage phenotype (high level expression of CD14/CD16, nitric oxide synthase, IL-6, TNFa; a propensity to drive T-helper [TH]-1 cell development; and low-level expression of IL-10). The procedure for cytokine analyses followed manufacturer specifications. A 4-fold serial dilution was made of the pre-mixed standards supplied with the assay kit (concentration range ¼ 1.48–77 755.00 pg/ml). All supernatants were assayed undiluted; the concentration of each cytokine (pg/ml) was extrapolated from the standard curve generated in parallel. In a more limited series of experiments, effects of select agents on the production of IL-6 and IL-8 by LPS/MnCl2 (100 mM)treated monocyte-derived macrophages were investigated. The agents were: (i) NF-kB activation inhibitor (1 mM, Calbiochem); (ii) p38 MAP (mitogen activated protein) kinase inhibitor SB202190 (20 mM, Sigma); and (iii) dithiothreitol (DTT, 5 mM, Sigma), a cell-penetrating scavenger of H2O2. Propidium iodide exclusion assay To test for potential cytotoxic effects of NF-kB activation inhibitor (1 mM), SB202190 (20 mM), and dithiothreitol (5 mM), cell viability assays were performed on mononuclear cells (2 106/ml) using the flow cytometric propidium iodide exclusion assay after a 24-h exposure to the test agents. The cells
100
M. I. Mokgobu et al.
J Immunotoxicol, 2015; 12(1): 98–103
(2 105) were incubated for 5 min with propidium iodide (DNA Prep-Stain, Beckman Coulter, 50 mg/ml) and then cell viability assessed using the flow cytometer. All data were expressed as a percentage of viable cells.
Journal of Immunotoxicology Downloaded from informahealthcare.com by Astellas Pharma Japan on 02/11/15 For personal use only.
Intracellular H2O2 production Intracellular H2O2 was measured using a 20 ,70 -dichlorodihydrofluorescein diacetate (DCF-DA)-based spectrofluorimetric procedure. This agent is hydrolyzed by cellular esterases to 20 ,70 -dichlorodihydrofluorescein which is, in turn, oxidized to 20 ,70 -dichlorofluorescein, primarily by H2O2. Here, DCF-DA (10 mM) was added to a reaction mixture containing 105 monocyte-derived macrophages/ml in the absence and presence of MnCl2 (100 mM) and/or LPS (1 mg/ml) in a total volume of 3 ml HBSS. The samples were then incubated for 10 min in a 37 C waterbath and then transferred to the thermo-regulated cuvette holder of a Hitachi 650 10S fluorescence spectrophotometer with excitation and emission wavelengths at 500 and 530 nm, respectively. Fluorescence responses were then monitored continually for 10 min. These experiments were performed in the presence and absence of diphenyleneiodonium chloride (DPI, 20 mM; inhibitor of NADPH oxidase; O’Donnell et al., 1993) or the ROS scavenger, dithiothreitol (DTT, 100 mM). The latter agent was used at a lower concentration than that used in assays of cytokine production due to interference with the DCF-DA-based assay when used at higher levels. These results are shown as traces of a representative experiment.
Figure 1. Effects of MnCl2 on spontaneous and LPS-activated monocytederived macrophage IL-1b and IL-6 production. Results shown are mean ( ± SEM) pg/ml (n ¼ 6). *p50.05 vs control (0). +p50.05 vs LPS-only control.
Expression and statistical analysis of data Results of each series of experiments are presented as mean ( ± SEM) of absolute values, where n is the number of different donors used in each series of experiments. Levels of statistical significance were determined by comparing the absolute values for each metal-treated system with the corresponding values for the relevant metal-free control systems for each assay using the Wilcoxon matched pairs test. This option was given in the InStat software statistics program (GraphPad, La Jolla, CA) when selecting for a paired non-parametric test.
Results The effects of MnCl2 and LPS alone and in combination on cytokine production by monocyte-derived macrophages are shown in Figures 1–3. Exposure of the cells to the metal alone resulted in statistically significant increases in production of IL-1b, IL-6, IL-8, TNFa, and IFNg (all at 100 mM MnCl2, with the exception of IL-6 for which the effects of 50 mM also achieved statistical significance), but not G-CSF. Treatment with LPS alone caused modest increases in production of all the cytokines/chemokine, none of which achieved statistical significance. In the case of cells treated with combinations of MnCl2 and LPS, the observed increases in generation of IL-1b, IL-6, IL-8, IFNg, and G-CSF were greater than those by cells exposed to the individual agents, achieving statistical significance in the case of IL-6, IL-8, and IFNg when compared with the corresponding MnCl2-free LPS-treated control systems. Although the combination of MnCl2 and LPS treatment caused impressive dose-related increases in production of IL-1b and G-CSF, these did not achieve statistical significance, probably as a consequence of considerable variation in magnitudes of increases between experiments (ranging from 42–374% and from 18–1020% increase in comparison with corresponding LPS-treated control systems for IL-1b and G-CSF, respectively). In the case of TNFa, values for the LPS and MnCl2 combination were comparable to those of the metal alone.
Figure 2. Effects of MnCl2 on spontaneous and LPS-activated monocytederived macrophage IL-8 and TNFa production. Results shown are mean ( ± SEM) pg/ml (n ¼ 6). *p50.05 vs control (0). +p50.05 vs LPS-only control.
The effects of the NF-kB activation inhibitor (1 mM), SB202190 (20 mM), or dithiothreitol (DTT, 5 mM) on MnCl2mediated increases in IL-6 and IL-8 production by macrophages are shown in Figure 4. Exposure of cells to DTT completely abolished the augmentative effects of MnCl2 on generation of both cytokines by LPS-activated cells. In four of the five experiments, NF-kB activation inhibitor and SB202190 caused partial inhibition, ranging from 21–45% and 41–85%, respectively, for IL-6; corresponding values for IL-8 ranged from 25–41% and 24–65%. Propidium iodide exclusion assay Using the propidium iodide exclusion assay, the percentage of viable cells for the control system and for cells treated with
Journal of Immunotoxicology Downloaded from informahealthcare.com by Astellas Pharma Japan on 02/11/15 For personal use only.
DOI: 10.3109/1547691X.2014.902877
Figure 3. Effects of MnCl2 on spontaneous and LPS-activated monocytederived macrophage G-CSF and IFNg production. Results shown are mean ( ± SEM) pg/ml (n ¼ 6). *p50.05 vs control (0). +p50.05 vs LPS-only control.
Pro-inflammatory effects of MnCl2 on human macrophages
101
Figure 5. Effects of MnCl2 (at 100 mM) on unstimulated monocytederived macrophage dichlorofluorescein diacetate fluorescence responses. Traces from a single representative experiment (from n ¼ 4–5) are shown. Experiments were performed in the absence/presence of NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI, 20 mM) or the ROS scavenger dithiothreitol (DTT, 100 mM).
Discussion
Figure 4. Effects of SB202190 (SB, 20 mM), NF-kB activation inhibitor (NF-kBi, 1 mM), or dithiothreitol (DTT, 5 mM) on Mn-mediated increases in monocyte-derived macrophage IL-6 and IL-8 production. Results are mean ( ± SEM) pg/ml (n ¼ 5). *p50.05 vs LPS/MnCl2-treated (inhibitorfree) controls.
NF-kB activation inhibitor, SB202190, or dithiothreitol was 97.5 [± 0.4]%, 97.2 [± 0.4], 97.2 [± 0.7], and 97.2 [± 0.5]%, thus clearly demonstrating a lack of cytotoxicity. Intracellular H2O2 production The results shown in Figure 5 are typical traces from one representative experiment (n ¼ 4–5) that depict the effects of MnCl2 (100 mM) on DCF-DA fluorescence responses of unstimulated monocyte-derived macrophages. Treatment of the cells with MnCl2 caused a dose-dependent increase in fluorescence intensity, compatible with stimulated intracellular H2O2 production by the macrophages. These effects were attenuated by inclusion of DPI (20 mM) and by DTT (100 mM). In contrast, LPS (1 mg/ml) had no effect on the cell responses (data not shown).
Occupational exposure to manganese has been linked to manganism, a specific neuro-pathology characterized by clinical signs and lesions similar to Parkinson’s disease (Meco et al., 1994). According to Littlejohn et al., (2011), cytokines are increasingly implicated in acute and chronic neuronal demise, with a number of clinical studies having demonstrated increased levels of pro-inflammatory cytokines (e.g., TNFa, IL-6, IL-1b, and IFNg) in post-mortem brain, as well as blood or cerebral spinal fluid, of patients with stroke, head injury, Alzheimer’s disease, and Parkinson’s disease (Basic Kes et al., 2008; Brodacki et al., 2008; Goodman et al., 2008; Glass et al., 2010). Moreover, levels of IFNg, IL-1b, and TNFa, as well as of the chemokines monocyte chemoattractant protein (MCP)-1, RANTES, macrophage inflammatory protein (MIP)-1a, and IL-8 were significantly correlated with the severity of Parkinsonism (Reale et al., 2009). These reports underscore the involvement of pro-inflammatory cytokines and chemokines in the development of neurotoxicity in manganese-exposed workers. The current study demonstrated that Mn2+ (25–100 mM) alone or in combination with LPS potentiated production of proinflammatory cytokines IL-1b, IL-6, IL-8, IFNg, TNFa, and G-CSF by human monocyte-derived macrophages. The trends observed in all experiments were consistent, although the standard error bars were large due to variability between experiments, possibly due to extensive multi-step procedures involved in monocyte isolation and maturation. The concentrations of the metal used in the current study are potentially attainable in cells and tissues, particularly those of the brain. In this context, primates exposed to occupationally relevant concentrations of manganese exhibited metal levels between 10–350 mM in the brain (Bird et al., 1984; Sistrunk et al., 2007; Suzuki et al., 1975). With respect to previously published studies of pro-inflammatory effects of Mn2+ upon cells of the innate immune system, the current study differs in that: (i) the focus here was on human blood monocyte-derived macrophages as opposed to microglia/ astrocytes of animal origin and/or cell lines (Dodd & Filipov, 2011; Liu et al., 2006, 2013; Spranger et al., 1998; Zhang et al., 2010); (ii) the relevance of the findings of the current study are related to inflammation-associated organ dysfunction in general, as opposed specifically to any neurotoxicity; and (iii) the
102
M. I. Mokgobu et al.
J Immunotoxicol, 2015; 12(1): 98–103
ineffective. Specific measures of NF-kB and p38 MAPK expression, while of interest, were therefore likely to be of limited value due to redundancy and a convergence of intracellular redox signaling mechanisms. In addition, optimal inflammatory gene expression is accomplished by complementary interactions of several different transcription factors that converge on large nuclear proteins known as co-activator molecules, of which cAMP response element-binding protein (CBP) is a prototype. Following binding of transcription factors to CBP, the co-activator molecule acquires histone acetyl-transferase (HAT) activity, thereby facilitating interaction of transcription factors with their response elements on target genes (Barnes et al., 2005).
Journal of Immunotoxicology Downloaded from informahealthcare.com by Astellas Pharma Japan on 02/11/15 For personal use only.
Conclusions
Figure 6. Summary of potential mechanisms by which manganese causes increased monocyte-derived macrophage pro-inflammatory cytokine production. Macrophage membrane-associated NADPH oxidase gener2+ ates superoxide anions (O (a SOD 2 ) that, in the presence of Mn mimetic), are converted to hydrogen peroxide (H2O2). H2O2 then acts as an intracellular messenger causing the activation of redox-sensitive signaling proteins and downstream transcription factors. This, in turn, leads to activation of genes encoding a range of pro-inflammatory cytokines, prolonged production of which may cause inflammation and tissue injury.
collection of pro-inflammatory cytokines tested here were of particular relevance to the processes of neutrophil/monocyte/ macrophage mobilization and activation. In the case of molecular mechanisms underpinning the induction of cytokine production by MnCl2, we previously reported that the metal potentiates production of H2O2 by activated macrophages, apparently by acting as a superoxide dismutase mimetic (Mokgobu et al., 2012), implying an association between these events. This contention was reinforced by the findings of the current study that exposure of the monocytes/macrophages to MnCl2, but not LPS, caused a spontaneous increase in formation of H2O2 that was attenuated by inclusion of DPI (an inhibitor of NADPH oxidase) as well as by the ROS scavenger DTT. Importantly, H2O2 acts as an intracellular second messenger, thereby modulating several major signal cascades including the ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), MAPK (mitogenactivated protein kinase), and PI3K/Akt (phosphatidylinositol 3-Kinase/Akt) pathways, as well as downstream transcription factors, by targeting cysteine and methionine residues on these proteins (Rhee et al., 2003; Wittmann et al., 2012). Of the ROS commonly produced in tissues, H2O2 appears to be most important in terms of signaling as it is relatively stable and readily crosses cell membranes (Fisher, 2009). The efficacy of DTT in abolishing Mn2+-mediated production of pro-inflammatory cytokines by monocyte-derived macrophages, taken together with the observed attenuation of formation of H2O2, clearly underscores the involvement of H2O2 in the activation of gene expression. The more limited efficacy of the pharmacological inhibitors of NF-kB and p38 MAPK probably reflects multiple redox-signaling mechanisms activated by H2O2, rendering selective inhibitors of a single pathway relatively
The results of the current study are compatible with a prooxidative mechanism whereby MnCl2, as a consequence of its superoxide dismutase mimetic activity, potentiates the generation of H2O2 by monocyte-derived macrophages. In turn, H2O2, via its redox intracellular signaling activities, promotes the activation of genes encoding a range of pro-inflammatory cytokines. A summary of the potential mechanisms by which Mn2+ increases production of pro-inflammatory cytokines by macrophages is shown in Figure 6. Although remaining to be conclusively proven, this pro-oxidative pro-inflammatory mechanism is likely to be implicated in the (immuno)- pathogenesis of Mn-associated neurologic and respiratory dysfunction.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References Barnes, P. J., Adcock, I. M., and Ito, K. 2005. Histone acetylation and deacetylation: Importance in inflammatory lung diseases. Eur. Respir. J. 25:552–563. Basic Kes, V., Simundic, A. M., Nicolac, N., et al. 2008. Pro-inflammatory and anti-inflammatory cytokines in acute ischemic stroke and their relation to early neurological deficit and stroke outcome. Clin. Biochem. 41:1330–1334. Bird, E. D., Anton, A. H., and Bullock, B. 1984. The effect of manganese inhalation on basal ganglia dopamine concentrations in rhesus monkey. Neurotoxicology 5:59–65. Boojar, M. M., and Goodarzi, F. 2002. A longitudinal follow-up of pulmonary function and respiratory symptoms in workers exposed to manganese. J. Occup. Environ. Med. 44:282–290. Brodacki, B., Staszewski, J., Toczylowska, B., et al. 2008. Serum interleukin (IL-2, IL-10, IL-6, IL-4), TNFa, and INFg concentrations are elevated in patients with atypical and idiopathic Parkinsonism. Neurosci. Lett. 441:158–162. Dobson, A. W., Erikson, K. M., and Aschner, M. 2004. Manganese neurotoxicity. Ann. N.Y. Acad. Sci. 1012:115–128. Dodd, C. A., and Filipov, N. M. 2011. Manganese potentiates LPSinduced heme oxygenase-1 in microglia but not dopaminergic cells: Role in controlling microglial hydrogen peroxide and inflammatory cytokine output. Neurotoxicology 32:683–692. Environmental Protection Agency (EPA). 2007. Manganese TEACH Chemical Summary Revised 2007. Available online at: http:// www.epa.gov/teach/chem_summ/manganese summary.pdf [last accessed 11 Jul 2013]. Fisher, A. B. 2009. Redox signaling across cell membranes. Antiox. Redox Signal. 11:1349–1356. Frumkin, H., and Solomon, G. 1997. Manganese in the U.S. Gasoline Supply. Am. J. Ind. Med. 31:107–115. Glass, C. K., Saijo, K., Winner, B., et al. 2010. Mechanisms underlying inflammation in neuro-degeneration. Cell 140:918–934. Goodman, J. C., Van, M., Gopinath, S. P., and Robertson, C. S. 2008. Proinflammatory and pro-apoptotic elements of the neuro-inflammatory response are activated in traumatic brain injury. Acta. Neurochir. S102: 437–439.
Journal of Immunotoxicology Downloaded from informahealthcare.com by Astellas Pharma Japan on 02/11/15 For personal use only.
DOI: 10.3109/1547691X.2014.902877
Hochberg, F., Miller, G., Valenzuela, R., et al. 1996. Late motor deficits of Chilean manganese miners: A blinded control study. Neurology 47: 788–795. Laskin, D. L., Sunil, V. R., Gardner, C. R., and Laskin, J. D. 2011. Macrophages and tissue injury: Agents of defense or destruction? Annu. Rev. Pharmacol. Toxicol. 81:267–288. Littlejohn, D., Mangano, E., Clarke, E., et al. 2011. Inflammatory mechanisms of neurodegeneration in toxin-based models of Parkinson’s Disease. Parkinson’s Dis. 2011:713517. Liu, X., Sullivan, K. A., Madl, J. E., et al. 2006. Manganese-induced neurotoxicity: The role of astroglial-derived nitric oxide in striatal interneuron degeneration. Toxicol. Sci. 91:521–531. Liu, Y., Barber, D. S., Zhang, P., and Liu, B. 2013. Complex II of the mitochondrial respiratory chain is the key mediator of divalent manganese-induced hydrogen peroxide production in microglia. Toxicol. Sci. 132:298–306. Meco, G., Bonifati, V., Vanacore, N., and Fabrizio, E. 1994. Parkinsonism after chronic exposure to the fungicide Maneb (manganese ethylenebis-dithiocarbamate). Scand. J. Work. Environ. Health 20:301–305. Mokgobu, M. I., Anderson, R., Steel, H. C., et al. 2012. Manganese promotes increased formation of hydrogen peroxide by activated human macrophages and neutrophils in vitro. Inhal. Toxicol. 24: 634–644. O’Donnell, B. V., Tew, D. G., Jones, O. T., and England, P. J. 1993. Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase. Biochem. J. 290: 41–49. Reale, M., Iarlori, C., Thomas, A., Gambi, D., et al. 2009. Peripheral cytokines profile in Parkinson’s disease. Brain Behav. Immun. 23: 55–63.
Pro-inflammatory effects of MnCl2 on human macrophages
103
Rhee, S. G., Chang, T. S., Bae, Y. S., et al. 2003. Cellular regulation by hydrogen peroxide. J. Am. Soc. Nephrol. 14:S211–215. Roels, H. A., Ghyselen, P., Buchet, J. P., et al. 1992. Assessment of permissible exposure level to manganese in workers exposed to manganese dioxide dust. Br. J. Ind. Med. 49:25–34. Santamaria, A. B. 2008. Manganese: Exposure, essentiality, and toxicity. Indian J. Med. Res. 128:484–500. Shi, C., and Pamer, E. G. 2011. Monocyte recruitment during infection and inflammation. Nat. Rev. Immunol. 11:762–774. Sistrunk, S. C., Ross, M. K., and Filipov, N. M. 2007. Direct effects of manganese compounds on dopamine and its metabolite Dopac: An in vitro study. Environ. Toxicol. Pharmacol. 23:286–296. Spranger, M., Schwab, S., Desiderato, S., et al. 1998. Manganese augments nitric oxide synthesis in murine astrocytes: A new pathogenetic mechanism in manganism? Exp. Neurol. 149:277–283. Suzuki, Y., Mouri, T., Nishiyama, K., and Fujii, N. 1975. Study of subacute toxicity of manganese dioxide in monkeys. Tokashima J. Exp. Med. 22:5–10. Wang, J. D., Huang, C. C., Hwang, Y. H., et al. 1989. Manganese induced Parkinsonism: An outbreak due to an unrepaired ventilation control system in a ferromanganese smelter. Br. J. Ind. Med. 46:856–859. Wennberg, A., Iregren, A., Struwe, G., et al. 1991. Manganese exposure in steel smelters a health hazard to the nervous system. Scand. J. Work Environ. Health 17:255–262. Wittmann, C., Chockley, P., Singh, S. K., et al. 2012. Hydrogen peroxide in inflammation: Messenger, guide, and assassin. Adv. Hematol. 2012:541471. Zhang, P., Lokuta, K. M., Turner, D. E., and Liu, B. 2010. Synergistic dopaminergic neuro-toxicity of manganese and lipopolysaccharide: Differential involvement of microglia and astroglia. J. Neurochem. 112:434–443.
