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.