Increased expression of cystine/glutamate antiporter in multiple sclerosis - PubMed (original) (raw)
Increased expression of cystine/glutamate antiporter in multiple sclerosis
Olatz Pampliega et al. J Neuroinflammation. 2011.
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.
Figures
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.
Figure 2
Activation of human peripheral blood monocytes by LPS induces glutamate release through the cystine/glutamate antiporter and xCT upregulation. A. Monocytes, isolated by MACS, showed an increase in glutamate release after their activation with LPS (1 μg/ml, 48 h). This effect is prevented in the presence of AAA (1 mM), an inhibitor of the cystine/glutamate antiporter. B. Flow cytometry analysis of peripheral blood monocytes (CD14-PE) reveals a change in the morphology of monocyte population after their activation with LPS (left-up). Staining with xCT-FITC demonstrates higher expression of this exchanger in LPS-activated CD14+ monocytes (left-down and right). *, p < 0.05; **, p < 0.01; #, p < 0.05 vs. LPS treatment.
Figure 3
xCT expression is increased in the CNS of rats with EAE. A. Histogram showing the neurological score during the course of acute EAE induced in Lewis rats by immunization with myelin basic protein. The peak of neurological disability was at day 14 post-immunization, which was selected for obtaining tissue samples. B. xCT mRNA (left) and protein (right) expression in spinal cord from control and acute EAE rats, as assessed by qPCR and Western blot analysis. Data are referred to mean expression level of controls (n = 5-6). C. Double immunofluorescence for xCT (green) and OX-42 (red), a marker of microglia and infiltrating macrophages. OX42+ cells express high xCT levels in acute EAE. Both meninges (asterisk in top) and infiltrating cells (bottom) in inflammatory foci show high levels of xCT in rat spinal cord with EAE as compared to controls. D. Microglial cells (OX42+ cells) of EAE rats have higher xCT levels in spinal cord than controls. Notice the difference between resting microglia in control rats, with ramified morphology (arrows in control) and microglia in EAE showing round shaped morphology, characteristic of its activated state (arrows in EAE). Scale bar = 20 μm.E. xCT mRNA (left) and protein (right) expression in spinal cord from control and chronic EAE mice, assessed by qPCR and Western blot analysis. Data are referred to mean expression level of controls (n = 5).
Figure 4
xCT mRNA expression is increased in MS patients. A. Data show a significant increase in xCT mRNA expression in leukocytes in relapsing-MS, which is more prominent during relapses. Controls, n = 39; Total R-MS, n = 42; R-MS in remission, n = 24; R-MS in relapse, n = 18. B. Data show a significant increase in xCT mRNA in human optic nerve from MS patients as compared to matched controls. Damage optic nerves (DON), showing macroscopic plaques, atrophy and/or optic neuritis have a significantly higher increase in xCT mRNA. *, p < 0.05; **, p < 0.01; #, p < 0.05. C, D. xCT mRNA expression correlates with CD8 and EAAT2 mRNA expression in MS optic nerve. Pearson r = 0.83; n = 10; p = 0.0013 for CD8 vs. xCT. Pearson r = 0.81; n = 10; p = 0.005 for EAAT2 vs. xCT.
Figure 5
xCT expression is enhanced in CD68+ cells from MS spinal cord. A. Triple immunofluorescence staining for xCT (green), CD68 (red) and Hoechst 33258 (blue) in spinal cord of control (left) and MS patients (right). A high expression of xCT was detected in CD68+ infiltrating macrophages (arrows) associated with blood vessels, which are virtually absent in controls. Note that overall xCT expression is enhanced in MS tissue. B. CD68+ cells (arrows) show enhanced xCT expression in MS patients as compared to controls. CD68+ macrophages are round shaped and form clusters in MS patients, whereas in controls, CD68+ cells appear isolated and long shaped. Scale bar = 50 μm.
References
- Hemmer B, Archelos JJ, Hartung HP. New concepts in the immunopathogenesis of multiple sclerosis. Nat Rev Neurosci. 2002;3:291–301. -PubMed
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