Increase of Intracellular Zinc Levels Rather Than Zinc Influx Inhibits Interleukin‐2 Production in Zinc Supplemented Jurkat Cells

Figures

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  Figure 1. Zinc inhibits the PMA/Cal activated NFAT signaling pathway. Jurkat cells were stimulated as described in the experimental section. (A) qPCR was performed to quantify IL‐2 mRNA after 3 h (n = 6) and (B) 6 h (n = 11). (C) IL‐2 secretion after 24 h, measured by ELISA (n = 11). (D) One representative western blot after 30 min is shown. The control band belongs to the same membrane but is excised and shown individually, as the samples stimulated with PHA are not shown. (E) pNFAT and NFAT (n = 9) were normalized to β‐actin on their respective membrane and their ratio was calculated. The controls shown in A, B, C, and E are also used in Figure 3A, B, C, and E, respectively. Results show mean values + SEM. Data were analyzed by repeated‐measures ANOVA and Tukey post hoc test (A–C, E). Significantly different means do not share the same letters.

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  Figure 2. Zinc has no inhibiting effect on PHA stimulated IL‐2 expression. Jurkat cells were stimulated as described in the experimental section. (A) qPCR was performed to quantify IL‐2 mRNA after 3 h (n = 7) and (B) 6 h (n = 15). (C) IL‐2 secretion after 24 h measured by ELISA (n = 10). Results show mean values + SEM. Data were analyzed by repeated‐measures ANOVA Friedman test and Dunn's post hoc test (A) and Tukey post hoc test (B and C). Significantly different means do not share the same letters.

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  Figure 3. Zinc inhibits PHA/Cal stimulated NFAT signaling pathway. Jurkat cells were stimulated as described in the experimental section. (A) qPCR was performed to quantify IL‐2 mRNA after 3 h (n = 8) and (B) 6 h (n = 9). (C) IL‐2 secretion after 24 h, measured by ELISA (n = 8). (D) One representative experiment after 30 min is shown. (E) pNFAT and NFAT (n = 10) were normalized to β‐actin on their respective membrane and their ratio was calculated. The controls shown in A–C, E are also used in Figure 1A–C, E, respectively. Results show mean values + SEM. Data were analyzed by repeated‐measures ANOVA Friedman test and Dunn's post hoc test (A) and Tukey post hoc test (B–C, E). Significantly different means do not share the same letters.

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  Figure 4. Pyrithione leads to a strong and persistent increased intracellular zinc amount. Jurkat cells were stimulated as described in the experimental section. After 3 h, labile zinc was measured with FluoZin‐3AM. (A) Cells after preincubation without stimulation (n = 5) or with (B) PHA (n = 10), (C) PMA/Cal (n = 7), or (D) PHA/Cal (n = 7) stimulation. Results show mean values + SEM. Data were analyzed by repeated‐measures ANOVA and Tukey post hoc test (A), (C) and (D) and Kruskal–Wallis test and Dunn's post hoc test (B). Significantly different means do not share the same letters.

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  Figure 5. PMA/Cal‐induced IL‐2 production. Jurkat cells were stimulated as described in the experimental section. (A) qPCR was performed to quantify IL‐2 mRNA after 3 h (n = 4) and (B) 6 h (n = 14). (C) IL‐2 (n = 9) secretion after 24 h, measured by ELISA. The controls shown in A–C are also used in Figure 6A–C, respectively. Results show mean values + SEM of densitometric quantifications and measured results. Data were analyzed by repeated‐measures ANOVA Friedman test and Dunn's post hoc test (A) and Tukey post hoc test (B–C). Significantly different means do not share the same letters.

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  Figure 6. PHA/Cal‐induced IL‐2 production. Jurkat cells were stimulated as described in the experimental section. (A) qPCR was performed to quantify IL‐2 mRNA after 3 h (n = 8) and (B) 6 h (n = 11). (C) IL‐2 secretion after 24 h, measured by ELISA (n = 10). The controls shown in A–C are also used in Figure 5A–C, respectively. Results show mean values + SEM. Data were analyzed by repeated‐measures ANOVA Friedman test and Dunn's post hoc test (A) and Tukey post hoc test (B–C). Significantly different means do not share the same letters.

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  Figure 7. Zinc has no impact on calcimycin‐stimulated calcium influx. Jurkat cells were stimulated as described in the experimental section. Calcium was measured with Fluo‐4. (A) PHA and/or Cal (n = 5), or (B) PMA/Cal (n = 5) was used for stimulation. Same control was used in (A) and (B). Results show mean values + SEM. Data were analyzed by repeated‐measures ANOVA and Tukey post hoc test. Significantly different means do not share the same letters.

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  Figure 8. The impact of increased intracellular zinc on signaling pathways in T cells. Schematic view of the effects of zinc on IL‐2 related pathways. The inhibitory effect of zinc on pNFAT is represented by the red bar. Solid‐lined arrows indicate an activating impact. Dotted arrows indicate translocation of calcium or signaling molecules within the cell. The curved arrow indicates dephosphorylation of pNFAT. Upward arrow indicates an increased intracellular concentration of Zn2+, with a concurrent decrease in IL‐2 production, indicated by the downward arrow. The question mark indicates a possible additional signaling molecule which would be affected by zinc.