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Increased expression of cystine/glutamate antiporter in multiple sclerosis.

Pampliega O, Domercq M, Soria FN, Villoslada P, Rodríguez-Antigüedad A, Matute C - J Neuroinflammation (2011)

Bottom Line: In addition, xCT expression is also increased in EAE and in the disease proper.In the later, high expression of xCT occurs both in the central nervous system (CNS) and in peripheral blood cells.Together, these results reveal that increased expression of the cystine/glutamate antiporter system x(c)⁻ in MS provides a link between inflammation and excitotoxicity in demyelinating diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurotek-UPV/EHU, Parque Tecnológico de Bizkaia, Zamudio, Bizkaia, Spain.

ABSTRACT

Background: Glutamate excitotoxicity contributes to oligodendrocyte and tissue damage in multiple sclerosis (MS). Intriguingly, glutamate level in plasma and cerebrospinal fluid of MS patients is elevated, a feature which may be related to the pathophysiology of this disease. In addition to glutamate transporters, levels of extracellular glutamate are controlled by cystine/glutamate antiporter x(c)⁻, an exchanger that provides intracellular cystine for production of glutathione, the major cellular antioxidant. The objective of this study was to analyze the role of the system x(c)⁻ in glutamate homeostasis alterations in MS pathology.

Methods: Primary cultures of human monocytes and the cell line U-937 were used to investigate the mechanism of glutamate release. Expression of cystine glutamate exchanger (xCT) was quantified by quantitative PCR, Western blot, flow cytometry and immunohistochemistry in monocytes in vitro, in animals with experimental autoimmune encephalomyelitis (EAE), the animal model of MS, and in samples of MS patients.

Results and discussion: We show here that human activated monocytes release glutamate through cystine/glutamate antiporter x(c)⁻ and that the expression of the catalytic subunit xCT is upregulated as a consequence of monocyte activation. In addition, xCT expression is also increased in EAE and in the disease proper. In the later, high expression of xCT occurs both in the central nervous system (CNS) and in peripheral blood cells. In particular, cells from monocyte-macrophage-microglia lineage have higher xCT expression in MS and in EAE, indicating that immune activation upregulates xCT levels, which may result in higher glutamate release and contribution to excitotoxic damage to oligodendrocytes.

Conclusions: Together, these results reveal that increased expression of the cystine/glutamate antiporter system x(c)⁻ in MS provides a link between inflammation and excitotoxicity in demyelinating diseases.

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Related in: MedlinePlus

Activated U-937 monocytes release glutamate through cystine/glutamate antiporter and show an increased expression of the xCT subunit. A. Glutamate release by U-937 cells after activation with LPS (1 μg/ml) for 48 h in the absence and in the presence of the inhibitor of cystine/glutamate antiporter, AAA (1 mM), the glutaminase inhibitor DON (1 mM) and the inhibitors of glutamate transporters, DHK (1 mM), and TBOA (100 μM). Ordinates indicate the difference between the amount of glutamate released by LPS-activated and resting monocytes. Data are mean ± SEM from 4 independent experiments performed in triplicate. B, Intracellular glutathione levels in control U-937 cells and after activation with LPS (1 μg/ml) for 48 h. C. Relative expression of xCT antiporter and glutamate transporters EAAT1, EAAT2 and EAAT3 in CD14+ monocytes. D. Histogram illustrates the increase of xCT mRNA expression, but not of glutaminase (GLS), in LPS-activated U-937 monocytes using qPCR. U-937 cells were treated with LPS (1 μg/ml) for 48 h and qPCR data were normalized using 4 housekeeping genes and GeNorm software. Data are mean ± SEM from 3 independent experiments performed in triplicate. E. Western blotting analysis shows an up-regulation of xCT protein in U-937 cells after LPS (1 μg/ml) treatment for 48 ch. Data were normalized to actin and expressed as mean ± SEM from 3 independent experiments performed in triplicate. F. xCT mRNA levels in U-937 monocyte cell line increase after LPS (1 μg/ml) treatment but not after incubation with glutamate (100 μM and 1 mM) for 48 h. Stimulation with the cytokine TNFα (10 ng/ml; 24 h) also induced a significant increase in xCT mRNA expression. qPCR data were normalized using 4 housekeeping genes and GeNorm software. Data are mean ± SEM from 3 independent experiments performed in triplicate. *, p < 0.05; **, p < 0.01.
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Figure 1: Activated U-937 monocytes release glutamate through cystine/glutamate antiporter and show an increased expression of the xCT subunit. A. Glutamate release by U-937 cells after activation with LPS (1 μg/ml) for 48 h in the absence and in the presence of the inhibitor of cystine/glutamate antiporter, AAA (1 mM), the glutaminase inhibitor DON (1 mM) and the inhibitors of glutamate transporters, DHK (1 mM), and TBOA (100 μM). Ordinates indicate the difference between the amount of glutamate released by LPS-activated and resting monocytes. Data are mean ± SEM from 4 independent experiments performed in triplicate. B, Intracellular glutathione levels in control U-937 cells and after activation with LPS (1 μg/ml) for 48 h. C. Relative expression of xCT antiporter and glutamate transporters EAAT1, EAAT2 and EAAT3 in CD14+ monocytes. D. Histogram illustrates the increase of xCT mRNA expression, but not of glutaminase (GLS), in LPS-activated U-937 monocytes using qPCR. U-937 cells were treated with LPS (1 μg/ml) for 48 h and qPCR data were normalized using 4 housekeeping genes and GeNorm software. Data are mean ± SEM from 3 independent experiments performed in triplicate. E. Western blotting analysis shows an up-regulation of xCT protein in U-937 cells after LPS (1 μg/ml) treatment for 48 ch. Data were normalized to actin and expressed as mean ± SEM from 3 independent experiments performed in triplicate. F. xCT mRNA levels in U-937 monocyte cell line increase after LPS (1 μg/ml) treatment but not after incubation with glutamate (100 μM and 1 mM) for 48 h. Stimulation with the cytokine TNFα (10 ng/ml; 24 h) also induced a significant increase in xCT mRNA expression. qPCR data were normalized using 4 housekeeping genes and GeNorm software. Data are mean ± SEM from 3 independent experiments performed in triplicate. *, p < 0.05; **, p < 0.01.

Mentions: We have previously demonstrated that activation of U-937 monocytes induces glutamate release [13]. So, we now analyzed the mechanism of glutamate release in U-937 cells as well as in peripheral blood monocytes in vitro. LPS-activated U-937 monocytes (LPS 1 μg/ml, 48 h) showed a significant increase in glutamate levels (n = 4; p < 0.001; Figure 1A), as previously described [13]. Incubating cells with the non competitive blocker of glutamate transporters DL-threo-beta-benzyloxyaspartate (TBOA; 100 μM) or with dihydrokainate (DHK; 1 mM), a selective inhibitor of EAAT2 glutamate transporter, did not block LPS-induced glutamate release by monocytes (n = 4; Figure 1A), excluding any role of glutamate transporters in such glutamate release. In contrast, incubating cells with aminoadipic acid (AAA, 1 mM), an inhibitor of cystine/glutamate antiporter [25,26], significantly reduced glutamate release by LPS-activated monocytes (n = 4; p = 0.02; Figure 1A), indicating that the xc- system is the main glutamate release mechanism in monocytes. Accordingly, intracellular glutathione levels were increased after LPS treatment (n = 3; p = 0.03; Figure 1B), as a result of an increased function of xCT antiporter.


Increased expression of cystine/glutamate antiporter in multiple sclerosis.

Pampliega O, Domercq M, Soria FN, Villoslada P, Rodríguez-Antigüedad A, Matute C - J Neuroinflammation (2011)

Activated U-937 monocytes release glutamate through cystine/glutamate antiporter and show an increased expression of the xCT subunit. A. Glutamate release by U-937 cells after activation with LPS (1 μg/ml) for 48 h in the absence and in the presence of the inhibitor of cystine/glutamate antiporter, AAA (1 mM), the glutaminase inhibitor DON (1 mM) and the inhibitors of glutamate transporters, DHK (1 mM), and TBOA (100 μM). Ordinates indicate the difference between the amount of glutamate released by LPS-activated and resting monocytes. Data are mean ± SEM from 4 independent experiments performed in triplicate. B, Intracellular glutathione levels in control U-937 cells and after activation with LPS (1 μg/ml) for 48 h. C. Relative expression of xCT antiporter and glutamate transporters EAAT1, EAAT2 and EAAT3 in CD14+ monocytes. D. Histogram illustrates the increase of xCT mRNA expression, but not of glutaminase (GLS), in LPS-activated U-937 monocytes using qPCR. U-937 cells were treated with LPS (1 μg/ml) for 48 h and qPCR data were normalized using 4 housekeeping genes and GeNorm software. Data are mean ± SEM from 3 independent experiments performed in triplicate. E. Western blotting analysis shows an up-regulation of xCT protein in U-937 cells after LPS (1 μg/ml) treatment for 48 ch. Data were normalized to actin and expressed as mean ± SEM from 3 independent experiments performed in triplicate. F. xCT mRNA levels in U-937 monocyte cell line increase after LPS (1 μg/ml) treatment but not after incubation with glutamate (100 μM and 1 mM) for 48 h. Stimulation with the cytokine TNFα (10 ng/ml; 24 h) also induced a significant increase in xCT mRNA expression. qPCR data were normalized using 4 housekeeping genes and GeNorm software. Data are mean ± SEM from 3 independent experiments performed in triplicate. *, p < 0.05; **, p < 0.01.
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Related In: Results  -  Collection

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Figure 1: Activated U-937 monocytes release glutamate through cystine/glutamate antiporter and show an increased expression of the xCT subunit. A. Glutamate release by U-937 cells after activation with LPS (1 μg/ml) for 48 h in the absence and in the presence of the inhibitor of cystine/glutamate antiporter, AAA (1 mM), the glutaminase inhibitor DON (1 mM) and the inhibitors of glutamate transporters, DHK (1 mM), and TBOA (100 μM). Ordinates indicate the difference between the amount of glutamate released by LPS-activated and resting monocytes. Data are mean ± SEM from 4 independent experiments performed in triplicate. B, Intracellular glutathione levels in control U-937 cells and after activation with LPS (1 μg/ml) for 48 h. C. Relative expression of xCT antiporter and glutamate transporters EAAT1, EAAT2 and EAAT3 in CD14+ monocytes. D. Histogram illustrates the increase of xCT mRNA expression, but not of glutaminase (GLS), in LPS-activated U-937 monocytes using qPCR. U-937 cells were treated with LPS (1 μg/ml) for 48 h and qPCR data were normalized using 4 housekeeping genes and GeNorm software. Data are mean ± SEM from 3 independent experiments performed in triplicate. E. Western blotting analysis shows an up-regulation of xCT protein in U-937 cells after LPS (1 μg/ml) treatment for 48 ch. Data were normalized to actin and expressed as mean ± SEM from 3 independent experiments performed in triplicate. F. xCT mRNA levels in U-937 monocyte cell line increase after LPS (1 μg/ml) treatment but not after incubation with glutamate (100 μM and 1 mM) for 48 h. Stimulation with the cytokine TNFα (10 ng/ml; 24 h) also induced a significant increase in xCT mRNA expression. qPCR data were normalized using 4 housekeeping genes and GeNorm software. Data are mean ± SEM from 3 independent experiments performed in triplicate. *, p < 0.05; **, p < 0.01.
Mentions: We have previously demonstrated that activation of U-937 monocytes induces glutamate release [13]. So, we now analyzed the mechanism of glutamate release in U-937 cells as well as in peripheral blood monocytes in vitro. LPS-activated U-937 monocytes (LPS 1 μg/ml, 48 h) showed a significant increase in glutamate levels (n = 4; p < 0.001; Figure 1A), as previously described [13]. Incubating cells with the non competitive blocker of glutamate transporters DL-threo-beta-benzyloxyaspartate (TBOA; 100 μM) or with dihydrokainate (DHK; 1 mM), a selective inhibitor of EAAT2 glutamate transporter, did not block LPS-induced glutamate release by monocytes (n = 4; Figure 1A), excluding any role of glutamate transporters in such glutamate release. In contrast, incubating cells with aminoadipic acid (AAA, 1 mM), an inhibitor of cystine/glutamate antiporter [25,26], significantly reduced glutamate release by LPS-activated monocytes (n = 4; p = 0.02; Figure 1A), indicating that the xc- system is the main glutamate release mechanism in monocytes. Accordingly, intracellular glutathione levels were increased after LPS treatment (n = 3; p = 0.03; Figure 1B), as a result of an increased function of xCT antiporter.

Bottom Line: In addition, xCT expression is also increased in EAE and in the disease proper.In the later, high expression of xCT occurs both in the central nervous system (CNS) and in peripheral blood cells.Together, these results reveal that increased expression of the cystine/glutamate antiporter system x(c)⁻ in MS provides a link between inflammation and excitotoxicity in demyelinating diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurotek-UPV/EHU, Parque Tecnológico de Bizkaia, Zamudio, Bizkaia, Spain.

ABSTRACT

Background: Glutamate excitotoxicity contributes to oligodendrocyte and tissue damage in multiple sclerosis (MS). Intriguingly, glutamate level in plasma and cerebrospinal fluid of MS patients is elevated, a feature which may be related to the pathophysiology of this disease. In addition to glutamate transporters, levels of extracellular glutamate are controlled by cystine/glutamate antiporter x(c)⁻, an exchanger that provides intracellular cystine for production of glutathione, the major cellular antioxidant. The objective of this study was to analyze the role of the system x(c)⁻ in glutamate homeostasis alterations in MS pathology.

Methods: Primary cultures of human monocytes and the cell line U-937 were used to investigate the mechanism of glutamate release. Expression of cystine glutamate exchanger (xCT) was quantified by quantitative PCR, Western blot, flow cytometry and immunohistochemistry in monocytes in vitro, in animals with experimental autoimmune encephalomyelitis (EAE), the animal model of MS, and in samples of MS patients.

Results and discussion: We show here that human activated monocytes release glutamate through cystine/glutamate antiporter x(c)⁻ and that the expression of the catalytic subunit xCT is upregulated as a consequence of monocyte activation. In addition, xCT expression is also increased in EAE and in the disease proper. In the later, high expression of xCT occurs both in the central nervous system (CNS) and in peripheral blood cells. In particular, cells from monocyte-macrophage-microglia lineage have higher xCT expression in MS and in EAE, indicating that immune activation upregulates xCT levels, which may result in higher glutamate release and contribution to excitotoxic damage to oligodendrocytes.

Conclusions: Together, these results reveal that increased expression of the cystine/glutamate antiporter system x(c)⁻ in MS provides a link between inflammation and excitotoxicity in demyelinating diseases.

Show MeSH
Related in: MedlinePlus