Manganese Potentiates In Vitro Production of Proinflammatory Cytokines and Nitric Oxide by Microglia Through a Nuclear Factor kappa B–Depend
http://www.100md.com
《毒物学科学杂志》
Wadsworth Center, New York State Department of Health
Albany, New York 11201
Center for Environmental Health Sciences, Department of Basic Sciences, College of Veterinary Medicine, Mississippi State, Mississippi 39762
ABSTRACT
Recent evidence suggests that the mechanism of manganese (Mn) neurotoxicity involves activation of microglia and/or astrocytes; as a consequence, neurons adjacent to the activated microglia may be injured. Mn modulation of proinflammatory cytokine expression by microglia has not been investigated. Therefore, the objectives of this research were to (1) assess whether Mn induces proinflammatory cytokine expression and/or modulates lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines and (2) investigate possible mechanisms for such an induction. N9 microglia were exposed in vitro to increasing concentrations (50–1000 μM) of Mn in the presence or absence of LPS (10, 100, or 500 ng/ml). After various incubation times (up to 48 h), media levels of several cytokines and nitric oxide (NO) were determined, as was the expression of the inducible form of NO synthase (iNOS). Lactate dehydrogenase (LDH) release into the medium and the cellular uptake of Neutral Red were used as general measures for cytotoxicity. In the absence of LPS, Mn moderately increased interleukin-6 and tumor necrosis factor alpha (TNF-a) production only at higher Mn concentrations, which were cytotoxic. At all LPS doses, however, proinflammatory cytokine production was dose-dependently increased by Mn. Similarly, LPS-induced NO production and iNOS expression were substantially enhanced by Mn. Pharmacological manipulations indicated that nuclear factor kappa B (NFB) activation is critical for the observed enhancement of cytokine and NO production. Within the context of inflammation, increased production of proinflammatory cytokines and NO by Mn could be an important part of the mechanism by which Mn exerts its neurotoxicity.
Key Words: manganese; microglia; neurotoxicity; inflammation; cytokines; nitric oxide.
INTRODUCTION
Manganese (Mn) is ubiquitously distributed in the environment and, although an essential metal, is a common environmental contaminant. Major sources of environmental contamination by Mn include the manufacturing of alloys, steel and iron, fertilizers, fungicides, and dry-cell batteries, as well as mining operations (Aschner, 2000). Re-introduction of the fuel additive methylcyclopentadienyl manganese tricarbonyl (MMT) represents another potential source of Mn pollution in the U.S. (Frumkin and Solomon, 1997) and adds a sense of urgency for understanding the mechanisms of Mn neurotoxicity. Manganism, the disorder associated with Mn exposure, is characterized by clinical signs and morphological lesions similar to those seen in Parkinson's disease (PD), and the occurrence of parkinsonism in humans exposed to the Mn-containing fungicide maneb has been reported (Meco et al., 1994). As in PD, Mn in manganism affects dopaminergic neuronal circuitry, primarily damaging the basal ganglia, the globus pallidus, and the substantia nigra; importantly, patients with manganism continue to deteriorate years after exposure to Mn has ceased (Huang et al., 1998). At the cellular level, Mn preferentially accumulates in mitochondria, where it causes mitochondrial dysfunction (Aschner and Aschner, 1991; Gavin et al., 1999) that is thought to be at least partially responsible for the toxic effects of this metal in the brain.
An uncontrolled or chronic inflammatory response, while it is an essential defense against infection, may cause irreversible tissue damage. In the brain, inflammatory mediators released mainly by microglia are implicated in the etiology of human PD (Olanow and Tatton, 1999) as well as in animal models of PD (Castano et al., 1998).
Recent evidence suggests that Mn neurotoxicity involves activation of microglia (Chang and Liu, 1999) and/or astrocytes (Spranger et al., 1998) manifested with an increase in nitric oxide (NO) production; as a consequence, neurons adjacent to the activated microglia could be injured. Importantly, mice pretreated with maneb and challenged subsequently with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), a model dopaminergic toxicant that causes microglial activation, exhibited greater PD-like pathology than mice treated with maneb or MPTP alone (Takahashi et al., 1989). This finding suggests that Mn exposure, when combined with exposure to another microglial activator, further enhances the microglial activation that ultimately results in basal ganglia degeneration. Once activated, microglial cells produce several proinflammatory cytokines (e.g., tumor necrosis factor alpha [TNF-]; interleukins 1 and 6 [IL-1 and IL-6], Chao et al., 1992). When chronically elevated, proinflammatory cytokines may induce NO-dependent or -independent cytotoxicity of neighboring neurons (Bronstein et al., 1995; Heales et al., 1999). Moreover, NO-induced neurotoxicity is a result of mitochondrial impairment (Heales et al., 1999) and glutathione depletion (Garcia-Nogales et al., 1999). Because Mn modulation of proinflammatory cytokine expression by microglia has not been investigated, the objectives of this study were to (1) evaluate the ability of Mn to stimulate microglial proinflammatory cytokine production, either alone or in combination with an inflammogen, lipopolysaccharide (LPS), and (2) determine possible mechanism(s) for such stimulation.
MATERIALS AND METHODS
Chemicals. Unless specified, all chemicals and reagents were purchased from Sigma (St. Louis, MO). In the case of MnCl2 and MnSO4, products with purity >99% were used.
Cell culture. The N9 murine microglial cell line used in the experiments was a gift kindly provided by Dr. P. Ricciardi-Castagnoli (University of Milan, Italy). These cells, derived by a retroviral immortalization of day-13 embryonic mouse brain cultures, are similar to primary microglia in that, upon activation, they produce proinflammatory cytokines, such as IL-1, IL-6, and TNF-; Righi et al., 1989). Also, when activated with lipopolysaccharide (LPS)/interferon gamma (IFN-), the N9 microglia secrete copious amounts of nitric oxide with the inducible form of nitric oxide synthase (iNOS/NOS2) being responsible for the NO production (Corradin et al., 1993).
The cultures were maintained (5% CO2, 95 % air, at 37°C) in Dulbecco's Modified Eagle's Medium (DMEM; Gibco BRL, Grand Island, NY) supplemented with 5% heat-inactivated fetal bovine serum, 0.075 % sodium bicarbonate (Gibco), 1 mM sodium pyruvate, 1 mM non-essential amino acids, 2 mM L-glutamine (Gibco), 50 μM 2-mercaptoethanol, 100 U/ml penicillin G, and 100 μg/ml streptomycin. For the cytotoxicity, cytokine, and NO analyses, cells were seeded at 0.25x106 cells per well density (0.5 ml volume) in 48-well plates and incubated for up to 48 h in the presence of Mn (50 to1000 μM) and/or LPS (Escherichia coli 055: B5; Calbiochem, La Jolla, CA; 10, 100, or 500 ng/ml). Concentrations of Mn were selected to include brain levels found after in vivo exposures. For example, nonhuman primates exposed to Mn (as manganese dioxide) for 3 months had brain Mn concentrations ranging from approximately 35 μM to approximately 350 μM (Suzuki et al., 1975). Concentrations of LPS were selected based on a lack of substantial cytotoxicity and on sufficient amounts present to induce proinflammatory cytokine and NO production in this cell line (Corradin et al., 1993). For each condition, two to three identical experiments were performed, with three replicates in each experiment.
Cytokine analysis. After incubation, plates were centrifuged, supernatants collected, and the concentrations of IL-1, IL-6, and TNF- in the supernatants were determined by mouse-specific Duo-Set ELISA sets (R&D Systems, Minneapolis, MN). The overall sensitivity of the assays was routinely <10 pg/ml, with the ELISA for IL-1, being the most sensitive. Initial time-course experiments were performed to determine optimal exposure duration. Based on these pilot studies, cytokine analyses subsequently were performed on supernatants collected after 24 or 48 h incubation. All samples for each analyte were assayed in duplicate, and the mean was used in the subsequent statistical analyses. A standard curve was generated with each run.
Cell viability. Two independent measures of cell viability were used. First, at the end of each sample's respective incubation period, the activity of lactate dehydrogenase (LDH) released in the supernatant was assessed by a modified Sigma kit that was suited to a microplate format. Formation of reduced nicotinamide adenine dinucleotide (NADH), during the LDH-mediated catalysis of the oxidation of lactate to pyruvate, results in an increased absorbance at 340 nm that is proportional to the LDH activity in the sample (50 μl). Assessment of LDH release after exposure to a toxicant correlates well with other measures of cell viability, such as the Trypan blue exclusion test (Legrand et al., 1992). Maximal LDH release was determined by incubating cells in medium containing 0.5% of the potent nonionic detergent Triton X-100 for 3 h at 37°C (Arechabala et al., 1999). All LDH data are expressed as units (U) per liter (l).
Second, in a subset of samples, the Neutral Red (NR) assay was used as previously described (Spranger et al., 1998). For this analysis, cells were incubated (after their LPS/Mn exposures) with fresh medium containing 40 μg/ml of NR dye that had been filtered through a 0.22 μm filter for 3 h at 37°C. The cells were then washed with phosphate-buffered saline (PBS), and the NR incorporated in viable cells was released into the supernatant with 1% acetic acid, 50% ethanol solution. Absorbance was read at 540 nm.
Nitric oxide (NO) analysis. The Sievers Nitric Oxide Analyzer (NOA 280i; Ionics Instruments, Boulder, CO) was used for analyses of NO in the cell supernatants. Because some of the culture-medium ingredients, including the fetal bovine serum (FBS), contain nitrites, all media that were used for the NO experiments were dialyzed for 48 h at 4°C against PBS. In the NO analysis, standard curves, ranging from 50 nM to 50 μM, were prepared in nitrite-free deionized H2O from 100 mM stocks of NaNO3 and NaNO2 (for the measurement of nitrate- and nitrite-derived NO, respectively). In preliminary experiments, we determined that, under the current experimental conditions and in agreement with manufacturer's suggestions, only negligible amounts of NO3 were generated (data not shown). Therefore, in the actual experiments, only NO2-derived NO was analyzed and quantified against a NaNO2-constructed standard curve. Briefly, at the end of incubation (48 h), supernatants were collected and cleared by centrifugation (10,000 x g for 10 min at 4°C), and duplicate 20 μl aliquots were injected into the NO analyzer. All other procedures were performed according to manufacturers' instructions.
Inducible nitric oxide synthase (iNOS) analysis. The expression of iNOS was assessed by Western blotting using mouse-specific polyclonal primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Cells (2.0 x 106 cells per well) were grown in 6-well culture plates for 18 h. The other culture conditions and treatments represented a subset (using only certain LPS/Mn combinations) of the experiments that were performed for cytokine analysis. At the end of the incubation, cells were washed with PBS and lysed by incubation for 1 h on ice with modified radioimmunoprecipitation buffer (RIPA: Santa Cruz), to which phenylmethylsulphonyl fluoride (PMSF; 10 μg/ml), freshly thawed protease inhibitor cocktail (10 μl/ml), and sodium orthovanadate (10 μl/ml of 100 mM stock) had been added. Total cell lysate was then collected after centrifugation at 10,000 x g for 10 min at 4°C, and its protein content was analyzed by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Twenty micrograms of protein per lane were loaded and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels. After the electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Corp., Billerica, MA), blocked with 5% milk blocking solution (1 h at RT), and incubated overnight at 4°C with the anti-iNOS primary antibody (1:1,000). The blot was then washed (3x), incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H + L) (1:3,000; Bio-Rad, Richmond, CA; 1 h at room temperature), washed again, and developed with the ECL Western Blotting Chemiluminescence Detection Reagents (Amersham Biosciences Corp., Piscataway, NJ).
Pharmacological manipulations. In cases where pharmacological manipulations were employed, the pharmacological agent was added 1 h prior to addition of LPS/Mn to the cell cultures. The agents used were as follows: N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase (Singhal et al., 1998); pentoxifylline (PTX), a phosphodiesterase inhibitor that potently inhibits TNF- synthesis in vivo and in vitro, including in microglia (Chao et al., 1992); tosyl-Phe-chloromethylketone (TPCK), a chymotrypsin-like serine protease inhibitor that potently inhibits nuclear factor kappa B (NFB) activation, most likely by preventing the degradation of the NFB–IB complex in various cell types, including microglia (Heyen et al., 2000); the water-soluble vitamin-E analog Trolox, a potent antioxidant (Radesater et al., 2003); N-acetyl-L-cysteine (NAC), a reactive oxygen species scavenger and a glutathione (GSH) precursor (Song et al., 2004); and buthionine sulfoximine (BSO), an inhibitor of intracellular GSH synthesis (Garaci et al., 1997).
Statistical analysis. Data were analyzed by analysis of variance (ANOVA). When statistical differences were detected (p < 0.05) treatment means were separated by the Student-Newman-Keuls (SNK) post hoc test. All data are presented as mean ± S.E.M.
RESULTS
Cytotoxicity
In the absence of LPS, concentrations of Mn up to 200 μM were not cytotoxic to the N9 microglial cells, as assessed by the measurement of LDH release into the culture medium (Fig. 1). Exposure to Mn at a concentration of either 500 μM or 1 mM elicited a dose-dependent increase in the LDH release (p 0.05). Although significant, this increase was only 23% and 35.3% above control LDH values (500 μM and 1 mM, respectively), whereas the Triton X-100–elicited maximal release of LDH was more than 630% above control values. Lipopolysaccharide, at concentrations of 100 ng/ml or greater, also caused a significant increase of LDH release (p 0.05). Combined exposure to LPS and Mn resulted in a biphasic interaction: on the one hand, higher doses of Mn combined with LPS were more toxic than was a dose of either LPS or Mn alone (p 0.05); on the other hand, lower (50 and 100 μM) amounts of Mn prevented the LPS-induced increase in LDH release and, in the case of 100 ng/ml LPS, this effect was significant (p 0.05). The greatest release of LDH into the culture medium was determined for the 1 mM Mn plus 500 ng/ml LPS combination (118.2% increase over control), but even this increase was more that 500% less than the Triton X-100–elicited maximal release of LDH. The effects of MnSO4, either alone or in combination with LPS, were virtually identical to the effects of MnCl2.
The cytotoxicity assay with NR uptake yielded similar results at the highest dose of Mn and for the combination of the 500 μM and 1 mM doses of Mn with LPS (p 0.05; Fig. 1 insert). At media concentrations of Mn lower than 500 μM, NR uptake was not affected (data not shown). Overall, at least under the present experimental conditions, this assay was less sensitive than the LDH release method in that it did not detect the effects of LPS in the absence of Mn. Based on the cytotoxic analyses, the highest (1 mM) dose of Mn was omitted from the remainder of the experiments.
Cytokines
In the absence of LPS, only the highest (500 μM) concentration of Mn increased the amount of TNF- and IL-6 (p 0.05) produced by the N9 microglia, and the increase was only moderate (Fig. 2A and 2B). Exposure to LPS alone dose-dependently (p 0.05) increased media concentrations of both TNF- and IL-6. Of the two cytokines, the magnitude of the TNF- increase was greater. Combined exposure to LPS and Mn substantially enhanced the proinflammatory cytokine production by the N9 cells. Moreover, this enhancement was both Mn dose-dependent and LPS dose-dependent (p 0.05). Thus, at the highest (500 ng/ml) dose of LPS, even the lowest (50 μM) concentration of Mn enhanced the media levels of TNF- and IL-6 above the increases observed by LPS alone; on the other hand, 200 μM Mn was necessary to produce this enhancement when the amount of LPS in the culture medium was 10 ng/ml. As with the cytotoxic analyses, the effects of MnSO4, either alone or in selected combinations with LPS, were similar to the effects of MnCl2. Based on the similar responses elicited by MnSO4 and MnCl2, subsequent experiments were conducted only with MnCl2.
In addition to TNF- and IL-6, IL-1 levels were also assayed (data not shown). The effects of LPS, either alone or combined with Mn, on IL-1 expression were similar to the effects on TNF- and IL-6, but the magnitude was smaller. In the absence of LPS, IL-1 levels in the culture medium were below the limit of detection.
NO and iNOS
Forty-eight hour exposure to Mn alone failed to increase microglial NO production (data not shown), whereas only the highest (500 μM) dose of Mn mildly increased iNOS expression (Fig. 3B). In the presence of LPS, however, Mn dose-dependently (p 0.05) increased the media NO concentrations, and this increase was prevented by the NOS inhibitor L-NAME (2 mM; Fig. 3A). The increased media NO levels were associated with marked enhancement of microglial iNOS expression; i.e., the expression of iNOS in cells exposed to 500 μM Mn + 100 ng/ml LPS was well above the increase resulting from exposure to a 10-fold (1 μg/ml) higher amount of LPS (Fig. 3B; lanes 3 and 5, respectively).
Pharmacological Manipulations
L-NAME. While incubation of the N9 cells with the NOS inhibitor L-NAME blocked the NO increases caused by Mn and LPS (Fig. 3A), L-NAME was largely ineffective in modulating the proinflammatory cytokine production induced by the LPS + Mn combination. The only significant (p 0.05) effect that was observed was a moderate further increase of the TNF- levels in the presence of LPS alone or LPS + 100 μM Mn (Fig. 4A).
Pentoxifylline. As expected, the phosphodiesterase inhibitor pentoxifylline (PTX) dose-dependently inhibited the LPS-induced TNF- production (Fig. 4B; p 0.05). The low (100 μM) dose of PTX did not affect the Mn-induced enhancement of TNF-, whereas the high (1000 μM) PTX dose significantly (p 0.05), although moderately, decreased this enhancement. Pentoxifylline did not affect the media levels of IL-6 under any experimental conditions (data not shown).
Buthionine sulfoximine, N-acetyl cysteine, and Trolox. Treat ment of the N9 microglia with the reactive oxygen species scavenger/GSH precursor N-acetyl cysteine (NAC) significantly (p 0.05), but not completely, decreased the amounts of TNF- (Fig. 5A), but not of IL-6 (data not shown) in the media from cells exposed to LPS or LPS + Mn. Treatment of the microglia with the GSH synthesis inhibitor buthionine sulfoximine (BSO) significantly (p 0.05) decreased TNF- (500 μM Mn; Fig. 5A) and IL-6 (100–500 μM Mn; data not shown) cytokine production. However, the amount of LDH released into the culture medium under these conditions was increased above the increase induced by the 100 ng/ml LPS + 500 μM Mn combination, thus suggesting this decrease was due to general cytotoxicity (data not shown).
Treatment with the antioxidant Trolox, on the other hand, had no effect on the LPS or LPS + Mn-induced TNF- production (data not shown), but it did significantly (p 0.05), although not completely, decrease the media levels of IL-6 (Fig. 5B).
N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). The NFB inhibitor TPCK dose-dependently inhibited the LPS and LPS + Mn–induced TNF- and IL-6 production by the N9 microglia (p 0.05; Fig. 6A and 6B, respectively). In the case of IL-6, 10 μM TPCK completely blocked the Mn-induced enhancement of the production of this cytokine, whereas the blockage of TNF- was almost complete. Similarly, TPCK also blocked the Mn + LPS-induced enhancement of iNOS expression (Fig. 7).
DISCUSSION
Although the exact mechanisms of Mn neurotoxicity are still being unraveled, the neurotoxic effects of Mn have traditionally been associated with direct effects of this metal on neuronal cells, with selective accumulation within the basal ganglia, induction of mitochondrial dysfunction, and induction of neurotransmitter imbalance contributing to the neurotoxicity of Mn (e.g., Aschner, 2000; Aschner and Aschner, 1991; Verity, 1999). However, the present studies suggest an additional mechanism, which might be particularly important within the context of ongoing inflammation—namely, a possible contribution to the neurotoxicity of Mn by inflammatory products from microglia.
The current results clearly demonstrate that Mn enhances TNF-, IL-6, and NO production by LPS-activated microglia, and that this enhancement is NFB-dependent. The increased iNOS expression and subsequent NO production confirm the Mn effects reported elsewhere (Chang and Liu, 1999). However, the proinflammatory cytokine increases and the overall dependency of the cytokine and NO increases caused by Mn on NFB activation have not been reported previously. In the absence of LPS, Mn was not capable of stimulating microglial NO production (our results, as well as those of Chang and Liu [1999]); Mn also induced minimal cytokine production. However, in the presence of LPS, cytokine production was markedly enhanced by Mn (either as MnCl2 or as MnSO4). This indicates that, for Mn to exert its potentiating effects, the presence of an inflammogen, such as LPS, is necessary.
Elevated proinflammatory cytokine expression in the brain has been found in post mortem examination of brains of PD patients, as well as in animal models of PD (Nagatsu et al., 2000; Sriram et al., 2002). Moreover, pharmacological inhibition of TNF- synthesis, or deletion of TNF- (Ferger et al., 2004) or its receptors (Sriram et al., 2002), attenuates the basal ganglia toxicity of MPTP in mice.
In addition to proinflammatory cytokines, an increase in NO, via increased expression of iNOS, has been associated with PD-like pathology (Tieu et al., 2003). For example, mice deficient in iNOS were protected from MPTP toxicity, which suggests that reactive nitrogen intermediates, such as NO and its toxic metabolite peroxynitrite (ONOO–), contribute to dopaminergic neuronal cell death, possibly by inhibiting components of the mitochondrial respiratory chain and, as a result, they compromise the energy state of the cells (Heales et al., 1999). Even more interesting is the fact that neurons, in contrast to other resident cells, such as astrocytes and microglia, are susceptible to NO and ONOO– exposure, whereas the principal producers of NO/ONOO– in the brain, the microglia and the astrocytes, are relatively resistant (Heales et al., 1999). Moreover, dopaminergic neurons are twice as sensitive to LPS toxicity as neurons that are negative for tyrosine hydroxylase (TH, the rate limiting-enzyme for dopamine synthesis, Bronstein et al., 1995). Considering the fact that cytokines, like TNF-, can be directly cytotoxic to neurons (Sipe et al., 1996) and can (either by themselves or in combination with LPS) enhance NO-dependent cytotoxicity, e.g., in PC12 cells (Heneka et al., 1998), the importance of increased proinflammatory cytokine generation in PD patients (Nagatsu et al., 2000) and in the MPTP model of PD (Sriram et al., 2002) becomes apparent. Of particular interest is the finding that maneb enhanced the neuronal damage caused by MPTP, the basal ganglia effects of which are dependent on microglial activation (Takahashi et al., 1989).
The present results suggest that microglia-derived proinflammatory cytokines and NO might play a major role in Mn neurotoxicity when microglia are also exposed to an inflammagen. This finding does not appear to be unique to Mn as, for example, the basal ganglia toxicity of MPTP was substantially enhanced by LPS (Gao et al., 2003). However, MPTP is a model toxicant, and the likelihood for exposure to it, other than accidentally, is minimal. Mn, on the other hand, is both occupationally and environmentally relevant (Aschner, 2000). Similarly, LPS, a component of the Gram-negative bacterial cell, is widely distributed in the environment, including in house dust (Michel et al., 1996) and various agricultural settings (Kullman et al., 1998). Importantly, systemic inflammation activates brain microglia and has been associated with neurodegeneration (Perry et al. 2003). Moreover, proinflammatory cytokines and bacterial products, such as LPS, readily cross the blood–brain barrier (Banks et al. 2002). Thus, the possibility of Mn and LPS interacting within the brain and stimulating microglial production of inflammatory mediators is distinct in cases where the opportunity for co-exposure exists. For example, many agricultural workers are exposed to other hazardous chemicals, such as pesticides, in conjunction with LPS, and some of these pesticides, such as maneb, contain Mn, which has already been reported to synergistically increase the dopaminergic toxicity of paraquat (Thiruchelvam et al., 2003; Takahashi et al., 1989). Even more intriguing, however, is recent evidence suggesting that pretreatment with Mn increased the susceptibility of mice to a subsequent viral infection (viruses that infect the brain), and that the increased susceptibility was manifested with brain inflammation of earlier onset and greater magnitude than that observed in control, non-Mn-treated mice (Seth et al., 2003).
Although the results of the present study and those of Chang and Liu (1999) have implicated microglia as important players in Mn-induced neurotoxicity, especially in the presence of an inflammogen (Spranger et al., 1998), and, very recently (Barhoumi et al., 2004) have demonstrated that astrocytes exposed to Mn combined with the cytokines IL-1 and IFN- (Spranger et al., 1998), or with LPS (Barhoumi et al., 2004) have iNOS-derived enhanced production of NO. These reports demonstrate that the effects of combined Mn plus inflammagen exposure on iNOS/NO do not appear to be specific only to microglia. More importantly, however, these reports raise the question of the importance of astrocytes relative to microglia in Mn neurotoxicity, within the context of an inflammatory stimulus. Even though astrocytes outnumber microglia in the brain, it has been demonstrated that microglia are non-uniformly distributed in the brain (the substantia nigra, basal ganglia, hippocampus have the greatest numbers; Lawson et al., 1990), and that addition of microglia to mixed glial–neuronal cultures from brain regions with low microglial numbers such as the cortex, renders these cultures as sensitive to the effects of LPS, as are mixed striatal cultures (Kim et al., 2000). Moreover, in vivo, as well as in mixed microglia-astrocyte cultures, increased NO and iNOS expression after LPS + IFN- stimulation is exclusively microglia-derived (Possel et al., 2000). Also, microglia, but not astrocytes, possess NADPH oxidase, which allows them to generate superoxide free radical, and, ultimately peroxynitrite (Gao et al., 2003). In addition, LPS alone was capable of inducing proinflammatory cytokine expression, including that of cell-associated IL-1, in highly purified cultures of microglia, whereas LPS failed to stimulate cytokine expression by astrocytes. On the other hand, IL-1 was a strong stimulus for astrocytes, suggesting that microglia are key regulators of astrocyte response, perhaps working primarily through the expression of cell-associated IL-1 (Lee et al., 1993). Interestingly, the studies of Spranger et al. (1998), which suggested that astrocytes are more sensitive to Mn than are microglia, used the IFN- + IL-1 combination as the additional stimulus for iNOS expression. Overall, these findings, combined with the facts that activated microglia are readily detected in striatum and substantia nigra of PD patients and in MPTP-treated mice (Olanow and Tatton, 1999; Czlonkowska et al., 1996) and more importantly, that microglial activation preceded astrocytosis in the MPTP model of PD (Kohutnicka et al., 1998), suggest that microglia are important contributors to dopaminergic neurotoxicity. Their importance arises from their differential distribution, reactivity to stimuli, ability to stimulate astrocytes, and relative insensitivity to potential toxic products that they produce.
So far, all of the studies evaluating the potential of Mn as a modulator of inflammatory mediators, including our study, have been conducted in vitro. Although they all demonstrate the ability of Mn to enhance proinflammatory cytokine and/or NO production by either microglia or astrocytes, only future in vitro studies with mixed cultures and, ultimately, in vivo experiments will (1) confirm these findings and (2) decipher the relative importance of microglia and astrocytes in Mn-induced neurodegeneration with and without ongoing inflammation. It must not be overlooked in such studies that neuronal cells themselves are capable of producing NO (derived from neuronal NOS) when stimulated with cytokines and/or LPS (e.g., Heneka et al., 1998). Moreover, the role of reactive substances that are released from damaged neurons and are capable of activating microglia should be taken into account. In light of the known neuronal mitochondrial dysfunction caused by Mn exposure (Aschner, 2000), the ability of signals from Mn-damaged neurons to enhance the direct effect of this metal on microglial activation, even in the absence of an inflammagen, needs to be considered.
Our pharmacological experiments suggest that both increased cytokine production and iNOS expression are NFB-dependent. The exact mechanism of the NFB activation by the Mn-LPS combination is unknown at present. One possibility, however, is an alteration of iron homeostasis by Mn. In this regard, Zheng and Zhao (2001) reported that Mn exposure increased cellular iron uptake in PC12 cells, but not in astrocytes. Moreover, iron plays a major role in NFB activation and subsequent proinflammatory cytokine production by inflammation activated peripheral macrophages (Xiong et al., 2004). The transcriptional regulatory factor NFB binds to elements present in the promoter regions of many of the proinflammatory cytokines, TNF- and IL-6 included, as well as to the promoter of iNOS (Abraham, 2000; Aktan, 2004). As a result of increased activation of NFB, the expression of these proinflammatory cytokines and the iNOS-dependent production of NO are enhanced. It has been suggested that NFB activation is a central event in the development of acute inflammatory injury associated with critical illness (Abraham, 2000). Additionally, a central role of NFB activation and ensuing transactivation of promoters of inflammatory cytokines in neurodegenerative processes, such as PD, has been suggested (Youdim et al., 1999).
Although inhibition of NFB was highly effective in preventing the enhancing effects of Mn on TNF-, IL-6 and iNOS, addition of either of the antioxidants Trolox or NAC was only marginally effective. Somewhat in contrast, Barhoumi et al. (2004) reported that in C6 gliomas, addition of the mitochondrion-specific antioxidant MitoQ diminished the enhancement of iNOS caused by the Mn plus LPS combination. It should be noted however, that (1) the cell line used by these investigators is of astrocytic origin and, thus, possibly accumulated more Mn; (2) Trolox and NAC are not mitochondrion-specific; and (3) iNOS enhancement was diminished, but not eliminated, by MitoQ. In this regard, the central role of increased oxidative stress within the cell on subsequent NFB activation, even though demonstrated in various experimental paradigms, has been recently questioned. At best, the evidence for the involvement of oxidative stress is inconclusive; such stress may be only facilitative (Bowie and O'Neill, 2000) or nonexistent, as it was for TNF- mRNA induction by LPS (White and Tsan, 2001). The differential effects of NAC and Trolox on TNF- and IL-6 production by microglia seen in the present experiments have been observed in other experimental paradigms. For example, in vivo pretreatment of mice challenged with LPS with NAC, diminished the subsequent induction of serum TNF-a, but not that of IL-6 (Peristeris et al., 1992). Moreover, the activation of the transcription factor AP-1, but not the activation of NFB, was inhibited by Trolox pretreatment (Rensing et al., 2001), raising the possibility that AP-1 plays a greater role in IL-6 induction in microglia than it does in TNF- induction in the absence of Mn.
Lipopolysaccharides and other inflammatory stimuli activate numerous intracellular signaling pathways, many of which converge on NFB and, ultimately, lead to increased cytokine production. Thus, LPS activates several mitogen-activated protein (MAP) kinases, including the extracellular signal-regulated kinase (ERK), the stress-activated protein kinases (SAPK), and the p38 kinase. Often these kinases function in a cooperative manner (Zhu et al., 2000), but addition of a p38 inhibitor to LPS-stimulated macrophages blocks LPS-stimulated TNF- production, thus suggesting a critical role for the p38 kinase (Kraatz et al., 1999). Although the exact mechanism of the Mn-induced enhancement of NFB activation is unknown at present, preliminary evidence indicates that p38 kinase is involved (Crittenden and Filipov, 2004).
In summary, microglia exposed to Mn, in combination with an inflammagen, respond with enhanced production of proinflammatory cytokines and NO. Thus, the role of brain microglia in the mechanisms of Mn neurotoxicity, especially within the context of inflammation, should not be overlooked.
ACKNOWLEDGMENTS
This research was supported in part by grant ES11654 (K22) and ES05879 (F32) to N.M.F. from the National Institute of Environmental Health Sciences (NIEHS, NIH). Part of this research was presented at the 40th Annual Meeting of the Society of Toxicology in San Francisco, CA; March 25–29, 2001.
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Albany, New York 11201
Center for Environmental Health Sciences, Department of Basic Sciences, College of Veterinary Medicine, Mississippi State, Mississippi 39762
ABSTRACT
Recent evidence suggests that the mechanism of manganese (Mn) neurotoxicity involves activation of microglia and/or astrocytes; as a consequence, neurons adjacent to the activated microglia may be injured. Mn modulation of proinflammatory cytokine expression by microglia has not been investigated. Therefore, the objectives of this research were to (1) assess whether Mn induces proinflammatory cytokine expression and/or modulates lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines and (2) investigate possible mechanisms for such an induction. N9 microglia were exposed in vitro to increasing concentrations (50–1000 μM) of Mn in the presence or absence of LPS (10, 100, or 500 ng/ml). After various incubation times (up to 48 h), media levels of several cytokines and nitric oxide (NO) were determined, as was the expression of the inducible form of NO synthase (iNOS). Lactate dehydrogenase (LDH) release into the medium and the cellular uptake of Neutral Red were used as general measures for cytotoxicity. In the absence of LPS, Mn moderately increased interleukin-6 and tumor necrosis factor alpha (TNF-a) production only at higher Mn concentrations, which were cytotoxic. At all LPS doses, however, proinflammatory cytokine production was dose-dependently increased by Mn. Similarly, LPS-induced NO production and iNOS expression were substantially enhanced by Mn. Pharmacological manipulations indicated that nuclear factor kappa B (NFB) activation is critical for the observed enhancement of cytokine and NO production. Within the context of inflammation, increased production of proinflammatory cytokines and NO by Mn could be an important part of the mechanism by which Mn exerts its neurotoxicity.
Key Words: manganese; microglia; neurotoxicity; inflammation; cytokines; nitric oxide.
INTRODUCTION
Manganese (Mn) is ubiquitously distributed in the environment and, although an essential metal, is a common environmental contaminant. Major sources of environmental contamination by Mn include the manufacturing of alloys, steel and iron, fertilizers, fungicides, and dry-cell batteries, as well as mining operations (Aschner, 2000). Re-introduction of the fuel additive methylcyclopentadienyl manganese tricarbonyl (MMT) represents another potential source of Mn pollution in the U.S. (Frumkin and Solomon, 1997) and adds a sense of urgency for understanding the mechanisms of Mn neurotoxicity. Manganism, the disorder associated with Mn exposure, is characterized by clinical signs and morphological lesions similar to those seen in Parkinson's disease (PD), and the occurrence of parkinsonism in humans exposed to the Mn-containing fungicide maneb has been reported (Meco et al., 1994). As in PD, Mn in manganism affects dopaminergic neuronal circuitry, primarily damaging the basal ganglia, the globus pallidus, and the substantia nigra; importantly, patients with manganism continue to deteriorate years after exposure to Mn has ceased (Huang et al., 1998). At the cellular level, Mn preferentially accumulates in mitochondria, where it causes mitochondrial dysfunction (Aschner and Aschner, 1991; Gavin et al., 1999) that is thought to be at least partially responsible for the toxic effects of this metal in the brain.
An uncontrolled or chronic inflammatory response, while it is an essential defense against infection, may cause irreversible tissue damage. In the brain, inflammatory mediators released mainly by microglia are implicated in the etiology of human PD (Olanow and Tatton, 1999) as well as in animal models of PD (Castano et al., 1998).
Recent evidence suggests that Mn neurotoxicity involves activation of microglia (Chang and Liu, 1999) and/or astrocytes (Spranger et al., 1998) manifested with an increase in nitric oxide (NO) production; as a consequence, neurons adjacent to the activated microglia could be injured. Importantly, mice pretreated with maneb and challenged subsequently with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), a model dopaminergic toxicant that causes microglial activation, exhibited greater PD-like pathology than mice treated with maneb or MPTP alone (Takahashi et al., 1989). This finding suggests that Mn exposure, when combined with exposure to another microglial activator, further enhances the microglial activation that ultimately results in basal ganglia degeneration. Once activated, microglial cells produce several proinflammatory cytokines (e.g., tumor necrosis factor alpha [TNF-]; interleukins 1 and 6 [IL-1 and IL-6], Chao et al., 1992). When chronically elevated, proinflammatory cytokines may induce NO-dependent or -independent cytotoxicity of neighboring neurons (Bronstein et al., 1995; Heales et al., 1999). Moreover, NO-induced neurotoxicity is a result of mitochondrial impairment (Heales et al., 1999) and glutathione depletion (Garcia-Nogales et al., 1999). Because Mn modulation of proinflammatory cytokine expression by microglia has not been investigated, the objectives of this study were to (1) evaluate the ability of Mn to stimulate microglial proinflammatory cytokine production, either alone or in combination with an inflammogen, lipopolysaccharide (LPS), and (2) determine possible mechanism(s) for such stimulation.
MATERIALS AND METHODS
Chemicals. Unless specified, all chemicals and reagents were purchased from Sigma (St. Louis, MO). In the case of MnCl2 and MnSO4, products with purity >99% were used.
Cell culture. The N9 murine microglial cell line used in the experiments was a gift kindly provided by Dr. P. Ricciardi-Castagnoli (University of Milan, Italy). These cells, derived by a retroviral immortalization of day-13 embryonic mouse brain cultures, are similar to primary microglia in that, upon activation, they produce proinflammatory cytokines, such as IL-1, IL-6, and TNF-; Righi et al., 1989). Also, when activated with lipopolysaccharide (LPS)/interferon gamma (IFN-), the N9 microglia secrete copious amounts of nitric oxide with the inducible form of nitric oxide synthase (iNOS/NOS2) being responsible for the NO production (Corradin et al., 1993).
The cultures were maintained (5% CO2, 95 % air, at 37°C) in Dulbecco's Modified Eagle's Medium (DMEM; Gibco BRL, Grand Island, NY) supplemented with 5% heat-inactivated fetal bovine serum, 0.075 % sodium bicarbonate (Gibco), 1 mM sodium pyruvate, 1 mM non-essential amino acids, 2 mM L-glutamine (Gibco), 50 μM 2-mercaptoethanol, 100 U/ml penicillin G, and 100 μg/ml streptomycin. For the cytotoxicity, cytokine, and NO analyses, cells were seeded at 0.25x106 cells per well density (0.5 ml volume) in 48-well plates and incubated for up to 48 h in the presence of Mn (50 to1000 μM) and/or LPS (Escherichia coli 055: B5; Calbiochem, La Jolla, CA; 10, 100, or 500 ng/ml). Concentrations of Mn were selected to include brain levels found after in vivo exposures. For example, nonhuman primates exposed to Mn (as manganese dioxide) for 3 months had brain Mn concentrations ranging from approximately 35 μM to approximately 350 μM (Suzuki et al., 1975). Concentrations of LPS were selected based on a lack of substantial cytotoxicity and on sufficient amounts present to induce proinflammatory cytokine and NO production in this cell line (Corradin et al., 1993). For each condition, two to three identical experiments were performed, with three replicates in each experiment.
Cytokine analysis. After incubation, plates were centrifuged, supernatants collected, and the concentrations of IL-1, IL-6, and TNF- in the supernatants were determined by mouse-specific Duo-Set ELISA sets (R&D Systems, Minneapolis, MN). The overall sensitivity of the assays was routinely <10 pg/ml, with the ELISA for IL-1, being the most sensitive. Initial time-course experiments were performed to determine optimal exposure duration. Based on these pilot studies, cytokine analyses subsequently were performed on supernatants collected after 24 or 48 h incubation. All samples for each analyte were assayed in duplicate, and the mean was used in the subsequent statistical analyses. A standard curve was generated with each run.
Cell viability. Two independent measures of cell viability were used. First, at the end of each sample's respective incubation period, the activity of lactate dehydrogenase (LDH) released in the supernatant was assessed by a modified Sigma kit that was suited to a microplate format. Formation of reduced nicotinamide adenine dinucleotide (NADH), during the LDH-mediated catalysis of the oxidation of lactate to pyruvate, results in an increased absorbance at 340 nm that is proportional to the LDH activity in the sample (50 μl). Assessment of LDH release after exposure to a toxicant correlates well with other measures of cell viability, such as the Trypan blue exclusion test (Legrand et al., 1992). Maximal LDH release was determined by incubating cells in medium containing 0.5% of the potent nonionic detergent Triton X-100 for 3 h at 37°C (Arechabala et al., 1999). All LDH data are expressed as units (U) per liter (l).
Second, in a subset of samples, the Neutral Red (NR) assay was used as previously described (Spranger et al., 1998). For this analysis, cells were incubated (after their LPS/Mn exposures) with fresh medium containing 40 μg/ml of NR dye that had been filtered through a 0.22 μm filter for 3 h at 37°C. The cells were then washed with phosphate-buffered saline (PBS), and the NR incorporated in viable cells was released into the supernatant with 1% acetic acid, 50% ethanol solution. Absorbance was read at 540 nm.
Nitric oxide (NO) analysis. The Sievers Nitric Oxide Analyzer (NOA 280i; Ionics Instruments, Boulder, CO) was used for analyses of NO in the cell supernatants. Because some of the culture-medium ingredients, including the fetal bovine serum (FBS), contain nitrites, all media that were used for the NO experiments were dialyzed for 48 h at 4°C against PBS. In the NO analysis, standard curves, ranging from 50 nM to 50 μM, were prepared in nitrite-free deionized H2O from 100 mM stocks of NaNO3 and NaNO2 (for the measurement of nitrate- and nitrite-derived NO, respectively). In preliminary experiments, we determined that, under the current experimental conditions and in agreement with manufacturer's suggestions, only negligible amounts of NO3 were generated (data not shown). Therefore, in the actual experiments, only NO2-derived NO was analyzed and quantified against a NaNO2-constructed standard curve. Briefly, at the end of incubation (48 h), supernatants were collected and cleared by centrifugation (10,000 x g for 10 min at 4°C), and duplicate 20 μl aliquots were injected into the NO analyzer. All other procedures were performed according to manufacturers' instructions.
Inducible nitric oxide synthase (iNOS) analysis. The expression of iNOS was assessed by Western blotting using mouse-specific polyclonal primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Cells (2.0 x 106 cells per well) were grown in 6-well culture plates for 18 h. The other culture conditions and treatments represented a subset (using only certain LPS/Mn combinations) of the experiments that were performed for cytokine analysis. At the end of the incubation, cells were washed with PBS and lysed by incubation for 1 h on ice with modified radioimmunoprecipitation buffer (RIPA: Santa Cruz), to which phenylmethylsulphonyl fluoride (PMSF; 10 μg/ml), freshly thawed protease inhibitor cocktail (10 μl/ml), and sodium orthovanadate (10 μl/ml of 100 mM stock) had been added. Total cell lysate was then collected after centrifugation at 10,000 x g for 10 min at 4°C, and its protein content was analyzed by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Twenty micrograms of protein per lane were loaded and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels. After the electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Corp., Billerica, MA), blocked with 5% milk blocking solution (1 h at RT), and incubated overnight at 4°C with the anti-iNOS primary antibody (1:1,000). The blot was then washed (3x), incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H + L) (1:3,000; Bio-Rad, Richmond, CA; 1 h at room temperature), washed again, and developed with the ECL Western Blotting Chemiluminescence Detection Reagents (Amersham Biosciences Corp., Piscataway, NJ).
Pharmacological manipulations. In cases where pharmacological manipulations were employed, the pharmacological agent was added 1 h prior to addition of LPS/Mn to the cell cultures. The agents used were as follows: N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase (Singhal et al., 1998); pentoxifylline (PTX), a phosphodiesterase inhibitor that potently inhibits TNF- synthesis in vivo and in vitro, including in microglia (Chao et al., 1992); tosyl-Phe-chloromethylketone (TPCK), a chymotrypsin-like serine protease inhibitor that potently inhibits nuclear factor kappa B (NFB) activation, most likely by preventing the degradation of the NFB–IB complex in various cell types, including microglia (Heyen et al., 2000); the water-soluble vitamin-E analog Trolox, a potent antioxidant (Radesater et al., 2003); N-acetyl-L-cysteine (NAC), a reactive oxygen species scavenger and a glutathione (GSH) precursor (Song et al., 2004); and buthionine sulfoximine (BSO), an inhibitor of intracellular GSH synthesis (Garaci et al., 1997).
Statistical analysis. Data were analyzed by analysis of variance (ANOVA). When statistical differences were detected (p < 0.05) treatment means were separated by the Student-Newman-Keuls (SNK) post hoc test. All data are presented as mean ± S.E.M.
RESULTS
Cytotoxicity
In the absence of LPS, concentrations of Mn up to 200 μM were not cytotoxic to the N9 microglial cells, as assessed by the measurement of LDH release into the culture medium (Fig. 1). Exposure to Mn at a concentration of either 500 μM or 1 mM elicited a dose-dependent increase in the LDH release (p 0.05). Although significant, this increase was only 23% and 35.3% above control LDH values (500 μM and 1 mM, respectively), whereas the Triton X-100–elicited maximal release of LDH was more than 630% above control values. Lipopolysaccharide, at concentrations of 100 ng/ml or greater, also caused a significant increase of LDH release (p 0.05). Combined exposure to LPS and Mn resulted in a biphasic interaction: on the one hand, higher doses of Mn combined with LPS were more toxic than was a dose of either LPS or Mn alone (p 0.05); on the other hand, lower (50 and 100 μM) amounts of Mn prevented the LPS-induced increase in LDH release and, in the case of 100 ng/ml LPS, this effect was significant (p 0.05). The greatest release of LDH into the culture medium was determined for the 1 mM Mn plus 500 ng/ml LPS combination (118.2% increase over control), but even this increase was more that 500% less than the Triton X-100–elicited maximal release of LDH. The effects of MnSO4, either alone or in combination with LPS, were virtually identical to the effects of MnCl2.
The cytotoxicity assay with NR uptake yielded similar results at the highest dose of Mn and for the combination of the 500 μM and 1 mM doses of Mn with LPS (p 0.05; Fig. 1 insert). At media concentrations of Mn lower than 500 μM, NR uptake was not affected (data not shown). Overall, at least under the present experimental conditions, this assay was less sensitive than the LDH release method in that it did not detect the effects of LPS in the absence of Mn. Based on the cytotoxic analyses, the highest (1 mM) dose of Mn was omitted from the remainder of the experiments.
Cytokines
In the absence of LPS, only the highest (500 μM) concentration of Mn increased the amount of TNF- and IL-6 (p 0.05) produced by the N9 microglia, and the increase was only moderate (Fig. 2A and 2B). Exposure to LPS alone dose-dependently (p 0.05) increased media concentrations of both TNF- and IL-6. Of the two cytokines, the magnitude of the TNF- increase was greater. Combined exposure to LPS and Mn substantially enhanced the proinflammatory cytokine production by the N9 cells. Moreover, this enhancement was both Mn dose-dependent and LPS dose-dependent (p 0.05). Thus, at the highest (500 ng/ml) dose of LPS, even the lowest (50 μM) concentration of Mn enhanced the media levels of TNF- and IL-6 above the increases observed by LPS alone; on the other hand, 200 μM Mn was necessary to produce this enhancement when the amount of LPS in the culture medium was 10 ng/ml. As with the cytotoxic analyses, the effects of MnSO4, either alone or in selected combinations with LPS, were similar to the effects of MnCl2. Based on the similar responses elicited by MnSO4 and MnCl2, subsequent experiments were conducted only with MnCl2.
In addition to TNF- and IL-6, IL-1 levels were also assayed (data not shown). The effects of LPS, either alone or combined with Mn, on IL-1 expression were similar to the effects on TNF- and IL-6, but the magnitude was smaller. In the absence of LPS, IL-1 levels in the culture medium were below the limit of detection.
NO and iNOS
Forty-eight hour exposure to Mn alone failed to increase microglial NO production (data not shown), whereas only the highest (500 μM) dose of Mn mildly increased iNOS expression (Fig. 3B). In the presence of LPS, however, Mn dose-dependently (p 0.05) increased the media NO concentrations, and this increase was prevented by the NOS inhibitor L-NAME (2 mM; Fig. 3A). The increased media NO levels were associated with marked enhancement of microglial iNOS expression; i.e., the expression of iNOS in cells exposed to 500 μM Mn + 100 ng/ml LPS was well above the increase resulting from exposure to a 10-fold (1 μg/ml) higher amount of LPS (Fig. 3B; lanes 3 and 5, respectively).
Pharmacological Manipulations
L-NAME. While incubation of the N9 cells with the NOS inhibitor L-NAME blocked the NO increases caused by Mn and LPS (Fig. 3A), L-NAME was largely ineffective in modulating the proinflammatory cytokine production induced by the LPS + Mn combination. The only significant (p 0.05) effect that was observed was a moderate further increase of the TNF- levels in the presence of LPS alone or LPS + 100 μM Mn (Fig. 4A).
Pentoxifylline. As expected, the phosphodiesterase inhibitor pentoxifylline (PTX) dose-dependently inhibited the LPS-induced TNF- production (Fig. 4B; p 0.05). The low (100 μM) dose of PTX did not affect the Mn-induced enhancement of TNF-, whereas the high (1000 μM) PTX dose significantly (p 0.05), although moderately, decreased this enhancement. Pentoxifylline did not affect the media levels of IL-6 under any experimental conditions (data not shown).
Buthionine sulfoximine, N-acetyl cysteine, and Trolox. Treat ment of the N9 microglia with the reactive oxygen species scavenger/GSH precursor N-acetyl cysteine (NAC) significantly (p 0.05), but not completely, decreased the amounts of TNF- (Fig. 5A), but not of IL-6 (data not shown) in the media from cells exposed to LPS or LPS + Mn. Treatment of the microglia with the GSH synthesis inhibitor buthionine sulfoximine (BSO) significantly (p 0.05) decreased TNF- (500 μM Mn; Fig. 5A) and IL-6 (100–500 μM Mn; data not shown) cytokine production. However, the amount of LDH released into the culture medium under these conditions was increased above the increase induced by the 100 ng/ml LPS + 500 μM Mn combination, thus suggesting this decrease was due to general cytotoxicity (data not shown).
Treatment with the antioxidant Trolox, on the other hand, had no effect on the LPS or LPS + Mn-induced TNF- production (data not shown), but it did significantly (p 0.05), although not completely, decrease the media levels of IL-6 (Fig. 5B).
N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). The NFB inhibitor TPCK dose-dependently inhibited the LPS and LPS + Mn–induced TNF- and IL-6 production by the N9 microglia (p 0.05; Fig. 6A and 6B, respectively). In the case of IL-6, 10 μM TPCK completely blocked the Mn-induced enhancement of the production of this cytokine, whereas the blockage of TNF- was almost complete. Similarly, TPCK also blocked the Mn + LPS-induced enhancement of iNOS expression (Fig. 7).
DISCUSSION
Although the exact mechanisms of Mn neurotoxicity are still being unraveled, the neurotoxic effects of Mn have traditionally been associated with direct effects of this metal on neuronal cells, with selective accumulation within the basal ganglia, induction of mitochondrial dysfunction, and induction of neurotransmitter imbalance contributing to the neurotoxicity of Mn (e.g., Aschner, 2000; Aschner and Aschner, 1991; Verity, 1999). However, the present studies suggest an additional mechanism, which might be particularly important within the context of ongoing inflammation—namely, a possible contribution to the neurotoxicity of Mn by inflammatory products from microglia.
The current results clearly demonstrate that Mn enhances TNF-, IL-6, and NO production by LPS-activated microglia, and that this enhancement is NFB-dependent. The increased iNOS expression and subsequent NO production confirm the Mn effects reported elsewhere (Chang and Liu, 1999). However, the proinflammatory cytokine increases and the overall dependency of the cytokine and NO increases caused by Mn on NFB activation have not been reported previously. In the absence of LPS, Mn was not capable of stimulating microglial NO production (our results, as well as those of Chang and Liu [1999]); Mn also induced minimal cytokine production. However, in the presence of LPS, cytokine production was markedly enhanced by Mn (either as MnCl2 or as MnSO4). This indicates that, for Mn to exert its potentiating effects, the presence of an inflammogen, such as LPS, is necessary.
Elevated proinflammatory cytokine expression in the brain has been found in post mortem examination of brains of PD patients, as well as in animal models of PD (Nagatsu et al., 2000; Sriram et al., 2002). Moreover, pharmacological inhibition of TNF- synthesis, or deletion of TNF- (Ferger et al., 2004) or its receptors (Sriram et al., 2002), attenuates the basal ganglia toxicity of MPTP in mice.
In addition to proinflammatory cytokines, an increase in NO, via increased expression of iNOS, has been associated with PD-like pathology (Tieu et al., 2003). For example, mice deficient in iNOS were protected from MPTP toxicity, which suggests that reactive nitrogen intermediates, such as NO and its toxic metabolite peroxynitrite (ONOO–), contribute to dopaminergic neuronal cell death, possibly by inhibiting components of the mitochondrial respiratory chain and, as a result, they compromise the energy state of the cells (Heales et al., 1999). Even more interesting is the fact that neurons, in contrast to other resident cells, such as astrocytes and microglia, are susceptible to NO and ONOO– exposure, whereas the principal producers of NO/ONOO– in the brain, the microglia and the astrocytes, are relatively resistant (Heales et al., 1999). Moreover, dopaminergic neurons are twice as sensitive to LPS toxicity as neurons that are negative for tyrosine hydroxylase (TH, the rate limiting-enzyme for dopamine synthesis, Bronstein et al., 1995). Considering the fact that cytokines, like TNF-, can be directly cytotoxic to neurons (Sipe et al., 1996) and can (either by themselves or in combination with LPS) enhance NO-dependent cytotoxicity, e.g., in PC12 cells (Heneka et al., 1998), the importance of increased proinflammatory cytokine generation in PD patients (Nagatsu et al., 2000) and in the MPTP model of PD (Sriram et al., 2002) becomes apparent. Of particular interest is the finding that maneb enhanced the neuronal damage caused by MPTP, the basal ganglia effects of which are dependent on microglial activation (Takahashi et al., 1989).
The present results suggest that microglia-derived proinflammatory cytokines and NO might play a major role in Mn neurotoxicity when microglia are also exposed to an inflammagen. This finding does not appear to be unique to Mn as, for example, the basal ganglia toxicity of MPTP was substantially enhanced by LPS (Gao et al., 2003). However, MPTP is a model toxicant, and the likelihood for exposure to it, other than accidentally, is minimal. Mn, on the other hand, is both occupationally and environmentally relevant (Aschner, 2000). Similarly, LPS, a component of the Gram-negative bacterial cell, is widely distributed in the environment, including in house dust (Michel et al., 1996) and various agricultural settings (Kullman et al., 1998). Importantly, systemic inflammation activates brain microglia and has been associated with neurodegeneration (Perry et al. 2003). Moreover, proinflammatory cytokines and bacterial products, such as LPS, readily cross the blood–brain barrier (Banks et al. 2002). Thus, the possibility of Mn and LPS interacting within the brain and stimulating microglial production of inflammatory mediators is distinct in cases where the opportunity for co-exposure exists. For example, many agricultural workers are exposed to other hazardous chemicals, such as pesticides, in conjunction with LPS, and some of these pesticides, such as maneb, contain Mn, which has already been reported to synergistically increase the dopaminergic toxicity of paraquat (Thiruchelvam et al., 2003; Takahashi et al., 1989). Even more intriguing, however, is recent evidence suggesting that pretreatment with Mn increased the susceptibility of mice to a subsequent viral infection (viruses that infect the brain), and that the increased susceptibility was manifested with brain inflammation of earlier onset and greater magnitude than that observed in control, non-Mn-treated mice (Seth et al., 2003).
Although the results of the present study and those of Chang and Liu (1999) have implicated microglia as important players in Mn-induced neurotoxicity, especially in the presence of an inflammogen (Spranger et al., 1998), and, very recently (Barhoumi et al., 2004) have demonstrated that astrocytes exposed to Mn combined with the cytokines IL-1 and IFN- (Spranger et al., 1998), or with LPS (Barhoumi et al., 2004) have iNOS-derived enhanced production of NO. These reports demonstrate that the effects of combined Mn plus inflammagen exposure on iNOS/NO do not appear to be specific only to microglia. More importantly, however, these reports raise the question of the importance of astrocytes relative to microglia in Mn neurotoxicity, within the context of an inflammatory stimulus. Even though astrocytes outnumber microglia in the brain, it has been demonstrated that microglia are non-uniformly distributed in the brain (the substantia nigra, basal ganglia, hippocampus have the greatest numbers; Lawson et al., 1990), and that addition of microglia to mixed glial–neuronal cultures from brain regions with low microglial numbers such as the cortex, renders these cultures as sensitive to the effects of LPS, as are mixed striatal cultures (Kim et al., 2000). Moreover, in vivo, as well as in mixed microglia-astrocyte cultures, increased NO and iNOS expression after LPS + IFN- stimulation is exclusively microglia-derived (Possel et al., 2000). Also, microglia, but not astrocytes, possess NADPH oxidase, which allows them to generate superoxide free radical, and, ultimately peroxynitrite (Gao et al., 2003). In addition, LPS alone was capable of inducing proinflammatory cytokine expression, including that of cell-associated IL-1, in highly purified cultures of microglia, whereas LPS failed to stimulate cytokine expression by astrocytes. On the other hand, IL-1 was a strong stimulus for astrocytes, suggesting that microglia are key regulators of astrocyte response, perhaps working primarily through the expression of cell-associated IL-1 (Lee et al., 1993). Interestingly, the studies of Spranger et al. (1998), which suggested that astrocytes are more sensitive to Mn than are microglia, used the IFN- + IL-1 combination as the additional stimulus for iNOS expression. Overall, these findings, combined with the facts that activated microglia are readily detected in striatum and substantia nigra of PD patients and in MPTP-treated mice (Olanow and Tatton, 1999; Czlonkowska et al., 1996) and more importantly, that microglial activation preceded astrocytosis in the MPTP model of PD (Kohutnicka et al., 1998), suggest that microglia are important contributors to dopaminergic neurotoxicity. Their importance arises from their differential distribution, reactivity to stimuli, ability to stimulate astrocytes, and relative insensitivity to potential toxic products that they produce.
So far, all of the studies evaluating the potential of Mn as a modulator of inflammatory mediators, including our study, have been conducted in vitro. Although they all demonstrate the ability of Mn to enhance proinflammatory cytokine and/or NO production by either microglia or astrocytes, only future in vitro studies with mixed cultures and, ultimately, in vivo experiments will (1) confirm these findings and (2) decipher the relative importance of microglia and astrocytes in Mn-induced neurodegeneration with and without ongoing inflammation. It must not be overlooked in such studies that neuronal cells themselves are capable of producing NO (derived from neuronal NOS) when stimulated with cytokines and/or LPS (e.g., Heneka et al., 1998). Moreover, the role of reactive substances that are released from damaged neurons and are capable of activating microglia should be taken into account. In light of the known neuronal mitochondrial dysfunction caused by Mn exposure (Aschner, 2000), the ability of signals from Mn-damaged neurons to enhance the direct effect of this metal on microglial activation, even in the absence of an inflammagen, needs to be considered.
Our pharmacological experiments suggest that both increased cytokine production and iNOS expression are NFB-dependent. The exact mechanism of the NFB activation by the Mn-LPS combination is unknown at present. One possibility, however, is an alteration of iron homeostasis by Mn. In this regard, Zheng and Zhao (2001) reported that Mn exposure increased cellular iron uptake in PC12 cells, but not in astrocytes. Moreover, iron plays a major role in NFB activation and subsequent proinflammatory cytokine production by inflammation activated peripheral macrophages (Xiong et al., 2004). The transcriptional regulatory factor NFB binds to elements present in the promoter regions of many of the proinflammatory cytokines, TNF- and IL-6 included, as well as to the promoter of iNOS (Abraham, 2000; Aktan, 2004). As a result of increased activation of NFB, the expression of these proinflammatory cytokines and the iNOS-dependent production of NO are enhanced. It has been suggested that NFB activation is a central event in the development of acute inflammatory injury associated with critical illness (Abraham, 2000). Additionally, a central role of NFB activation and ensuing transactivation of promoters of inflammatory cytokines in neurodegenerative processes, such as PD, has been suggested (Youdim et al., 1999).
Although inhibition of NFB was highly effective in preventing the enhancing effects of Mn on TNF-, IL-6 and iNOS, addition of either of the antioxidants Trolox or NAC was only marginally effective. Somewhat in contrast, Barhoumi et al. (2004) reported that in C6 gliomas, addition of the mitochondrion-specific antioxidant MitoQ diminished the enhancement of iNOS caused by the Mn plus LPS combination. It should be noted however, that (1) the cell line used by these investigators is of astrocytic origin and, thus, possibly accumulated more Mn; (2) Trolox and NAC are not mitochondrion-specific; and (3) iNOS enhancement was diminished, but not eliminated, by MitoQ. In this regard, the central role of increased oxidative stress within the cell on subsequent NFB activation, even though demonstrated in various experimental paradigms, has been recently questioned. At best, the evidence for the involvement of oxidative stress is inconclusive; such stress may be only facilitative (Bowie and O'Neill, 2000) or nonexistent, as it was for TNF- mRNA induction by LPS (White and Tsan, 2001). The differential effects of NAC and Trolox on TNF- and IL-6 production by microglia seen in the present experiments have been observed in other experimental paradigms. For example, in vivo pretreatment of mice challenged with LPS with NAC, diminished the subsequent induction of serum TNF-a, but not that of IL-6 (Peristeris et al., 1992). Moreover, the activation of the transcription factor AP-1, but not the activation of NFB, was inhibited by Trolox pretreatment (Rensing et al., 2001), raising the possibility that AP-1 plays a greater role in IL-6 induction in microglia than it does in TNF- induction in the absence of Mn.
Lipopolysaccharides and other inflammatory stimuli activate numerous intracellular signaling pathways, many of which converge on NFB and, ultimately, lead to increased cytokine production. Thus, LPS activates several mitogen-activated protein (MAP) kinases, including the extracellular signal-regulated kinase (ERK), the stress-activated protein kinases (SAPK), and the p38 kinase. Often these kinases function in a cooperative manner (Zhu et al., 2000), but addition of a p38 inhibitor to LPS-stimulated macrophages blocks LPS-stimulated TNF- production, thus suggesting a critical role for the p38 kinase (Kraatz et al., 1999). Although the exact mechanism of the Mn-induced enhancement of NFB activation is unknown at present, preliminary evidence indicates that p38 kinase is involved (Crittenden and Filipov, 2004).
In summary, microglia exposed to Mn, in combination with an inflammagen, respond with enhanced production of proinflammatory cytokines and NO. Thus, the role of brain microglia in the mechanisms of Mn neurotoxicity, especially within the context of inflammation, should not be overlooked.
ACKNOWLEDGMENTS
This research was supported in part by grant ES11654 (K22) and ES05879 (F32) to N.M.F. from the National Institute of Environmental Health Sciences (NIEHS, NIH). Part of this research was presented at the 40th Annual Meeting of the Society of Toxicology in San Francisco, CA; March 25–29, 2001.
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