Glycogen Synthase Kinase-3β Facilitates Cytokine Production in 12-O-Tetradecanoylphorbol-13-Acetate/Ionomycin-Activated Human CD4+ T Lymphocytes

Abstract

Cytokines are the major immune regulators secreted from activated CD4⁺ T lymphocytes that activate adaptive immunity to eradicate nonself cells, including pathogens, tumors, and allografts. The regulation of glycogen synthase kinase (GSK)-3β, a serine/threonine kinase, controls cytokine production by regulating transcription factors. The artificial in vitro activation of CD4⁺ T lymphocytes by a combination of 12-O-tetradecanoylphorbol-13-acetate and ionomycin, the so-called T/I model, led to an inducible production of cytokines, such as interferon-γ, tumor necrosis factor-α, and interleukin-2. As demonstrated by the approaches of pharmacological targeting and genetic knockdown of GSK-3β, T/I treatment effectively caused GSK-3β activation followed by GSK-3β-regulated cytokine production. In contrast, pharmacological inhibition of the proline-rich tyrosine kinase 2 and calcineurin signaling pathways blocked cytokine production, probably by deactivating GSK-3β. The blockade of GSK-3β led to the inhibition of the nuclear translocation of T-bet, a vital transcription factor of T lymphocyte cytokines. In a mouse model, treatment with the GSK-3β inhibitor 6-bromoindirubin-3'-oxime significantly inhibited T/I-induced mortality and serum cytokine levels. In summary, targeting GSK-3β effectively inhibits CD4⁺ T lymphocyte activation and cytokine production.

Keywords: 12-O-tetradecanoylphorbol-13-acetate/ionomycin; CD4+ T lymphocyte; Glycogen synthase kinase-3; cytokine.

Figure 1

A combination of 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin causes in vitro activation of CD4⁺ T lymphocytes. (A) Representative dot plots of immunostaining followed by flow cytometric analysis detected the IFN-γ expression in CD4⁺ and CD8⁺ T lymphocytes 6 h posttreatment with TPA plus ionomycin (T/I). In addition, ELISA determined the levels of IFN-γ in the cell supernatants of TPA-, ionomycin (Iono)-, and T/I-treated CD4⁺ and CD8⁺ T lymphocytes (B) and Jurkat T cells (C). (D) Representative dot plots and histograms of immunostaining followed by flow cytometric analysis detected the IFN-γ expression in T/I-stimulated murine CD4⁺CD8⁻ (a), CD4⁺CD8⁺ (b), and CD4⁻CD8⁺ (c) thymocytes. Primary CD4⁺ T cells were obtained from three individual volunteers of number (No.) 1 to 3. ELISA determined the levels of cytokines in the cell supernatants of TPA-, ionomycin-, or T/I-treated CD4⁺ T cells 6 h posttreatment (E) without or (F) with Bis (5 μM) and BAPTA (10 μM) pretreatment for 0.5 h. An LDH assay was used to detect cell cytotoxicity, and the results are normalized to the untreated group. For flow cytometric analysis, the percentages of IFN-γ-expressing cells are shown. For ELISA, the data are shown as the mean ± SD from three individual experiments. * p < 0.05 and *** p < 0.001 compared to untreated cells. # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to the T/I-treated group. ns, not significant.

Figure 2

Treatment with T/I activates GSK-3β and then induces GSK-3β-regulated cytokine production. (A) Western blotting detected the phosphorylation of GSK-3β (47 kDa) in CD4⁺ lymphocytes treated with the T/I combination. (B) Primary CD4⁺ T cells were obtained from three individual volunteers of number (No.) 1 to 3. ELISA determined the levels of cytokines in the cell supernatants of T/I-treated CD4⁺ T lymphocytes and Jurkat T cells 6 h posttreatment after BIO (10 μM) pretreatment for 0.5 h. DMSO was used as a control. (C) Western blot analysis showed GSK-3β (47 kDa) knockdown in Jurkat T cells (shGSK-3β). Luciferase shRNA (shLuc) was used as a control. (D) ELISA determined the levels of cytokines in the shGSK-3β-transfected cells pretreated with BIO (10 μM) for 0.5 h and then stimulated with or without T/I for an additional 6 h. For Western blotting, β-actin (42 kDa) was used as an internal control. The ratio of phosphorylated protein/total protein/β-actin is shown. For ELISA, an LDH assay was used to detect cell cytotoxicity, and the results are normalized to the untreated group. The data are shown as the mean ± SD from three individual experiments. *** p < 0.001 compared to the untreated cells. # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to the T/I-treated group. ns, not significant.

Figure 3

Treatment with T/I induces Pyk-2-regulated GSK-3β activation and cytokine production. (A) Western blotting detected the phosphorylation of GSK-3β (47 kDa) in CD4⁺ lymphocytes treated with the T/I combination in the presence of A9 (10 μM). (B) ELISA determined the levels of cytokines in the cell supernatants of T/I-treated CD4⁺ T lymphocytes and Jurkat T cells 6 h posttreatment after A9 pretreatment for 0.5 h. For Western blotting, β-actin (42 kDa) was used as an internal control. The ratio of phosphorylated protein/total protein/β-actin is shown. For ELISA, an LDH assay was used to detect cell cytotoxicity, and the results are normalized to the untreated group. The data are shown as the mean ± SD from three individual experiments. *** p < 0.001 compared to the untreated cells. ## p < 0.01 and ### p < 0.001 compared to the T/I-treated group. ns, not significant.

Figure 4

Treatment with T/I induces GSK-3β activation and cytokine production through calcineurin (PP2B) signaling. (A) Western blotting detected the phosphorylation of GSK-3β (47 kDa) in CD4⁺ lymphocytes treated with the T/I combination in the presence of cyclosporine A (CsA, 10 μM). CD4⁺ lymphocytes were pretreated with Bis, BAPTA, CsA, VIVIT, CAPE, PD98059, or a combination of VIVIT, CAPE, and PD98059 for 0.5 h and then stimulated with T/I for 6 h. (B) The PP2B activity assay detected the phosphatase activity. (C, D, and E) ELISA determined the levels of cytokines in the cell supernatants of the T/I-treated cells. For Western blotting, β-actin (42 kDa) was used as an internal control. The ratio of phosphorylated protein/total protein/β-actin is shown. For ELISA, an LDH assay was used to detect cell cytotoxicity, and the results are normalized to the untreated group. The data are shown as the mean ± SD from three individual experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the untreated cells. ### p < 0.001 compared to the T/I-treated group. ns, not significant.

Figure 5

Pharmacological inhibition of GSK-3β decreases the nuclear translocation of T-bet, which is required for controlling T lymphocyte cytokines. (A) Representative selected images of T-bet (green) immunostaining, which are obtained from the field of view (100× total magnification), in Jurkat T cells pretreated with BIO for 0.5 h and then stimulated with T/I for an additional 6 h. DAPI (blue) was used for nuclear staining. The percentages of positive cells, which were calculated from three fields of view (100× total magnification), are shown. (B) ELISA determined the levels of cytokines in the cell supernatants of T/I-treated Jurkat T cells transfected with siRNA targeting T-bet (siT-bet). siRNA targeting luciferase (siLuc) was used as a nonspecific control. For ELISA, an LDH assay was used to detect cell cytotoxicity, and the results are normalized to the untreated group. The data are shown as the mean ± SD from three individual experiments. *** p < 0.001 compared to the untreated cells. ### p < 0.001 compared to the T/I-treated group. ns, not significant.

Figure 6

Treatment with a GSK-3β inhibitor reduces T/I-induced mortality and cytokine production in vivo. (A) Survival rate of mice following T/I treatment with or without BIO therapy. Six groups were included: PBS (n = 3 for control), T200 (n = 3; TPA, 200 μg/kg), T50/I (n = 3; TPA, 50 μg/kg plus ionomycin, 250 μg/kg), T100/I (n = 5; TPA, 100 μg/kg plus ionomycin, 250 μg/kg), T200/I (n = 6; TPA, 200 μg/kg plus ionomycin, 250 μg/kg), and T200/I/BIO (n = 12; TPA, 200 μg/kg plus ionomycin, 250 μg/kg plus BIO 2 mg/kg). (B and C) Mice were pretreated with BIO (2 mg/kg) for 0.5 h and were then stimulated with T/I (n = 3; TPA, 200 μg/kg plus ionomycin, 250 μg/kg) for the indicated time. ELISA determined the levels of IFN-γ and TNF-α in the mouse serum. The data are shown as the mean ± SD obtained from three mice. * p < 0.05 and *** p < 0.001 compared with the PBS-treated group. ## p < 0.01 and ### p < 0.001 compared with the T/I-treated group. (D) A model of GSK-3β-regulated cytokine production in T/I-activated human CD4⁺ T lymphocytes.