Shikonin Suppresses Human T Lymphocyte Activation through Inhibition of IKK β Activity and JNK Phosphorylation

Abstract

The key role of T cells has been elaborated in mediating immune responses and pathogenesis of human inflammatory and autoimmune conditions. In the current study the effect of shikonin, a compound isolated from a medicinal plant, on inhibition of T-cell activation was firstly examined by using primary human T lymphocytes isolated from buffy coat. Results showed that shikonin dose dependently suppressed T-cell proliferation, IL-2 and IFN- γ secretion, CD69 and CD25 expression, as well as cell cycle arrest activated by costimulation of PMA/ionomycin or OKT-3/CD28 monoclonal antibodies. Moreover, these inhibitory responses mediated by shikonin were found to be associated with suppression of the NF- κ B signaling pathway via inhibition of the IKK α / β phosphorylation, I κ B- α phosphorylation and degradation, and NF- κ B nuclear translocation by directly decreasing IKK β activity. Moreover, shikonin suppressed JNK phosphorylation in the MAPKs pathway of T cells. In this connection, we conclude that shikonin could suppress T lymphocyte activation through suppressing IKK β activity and JNK signaling, which suggests that shikonin is valuable for further investigation as a potential immunosuppressive agent.

Figure 1

Effect of shikonin on suppression of cell proliferation and its cytotoxicity in human T lymphocytes. Chemical structure of shikonin (a). Effect of shikonin on T lymphocytes proliferation stimulated by PMA/ionomycin (b) or OKT-3/CD28 (c). Human T cells (10⁵/well) were pretreated with the indicated concentrations of shikonin for 2 h and then activated with PMA (20 ng/mL)/ionomycin (1 μM) (P/I) or with the coated OKT-3 (5 μg/mL)/CD28 (1 μg/mL) (OKT-3/CD28) for 72 h. BrdU was added to the cells for 14 h incubation before the end of cell culture, and then the amount of BrdU incorporation was measured by using plate reader at 450 nm. Data are expressed as relative folds of BrdU incorporation of the controlled cells and represent the mean ± SEM of three independent experiments. Cytotoxicity of shikonin on human T lymphocytes (d). The cells (10⁵/well) were treated with shikonin at the indicated concentrations for 3 days, and then MTT reagent was added to the cells for 4 h of incubation followed by addition of solubilization buffer. The absorbance was then read at 570 nm. Data are expressed as the percentage of absorbance of controlled cells and represent the mean ± SEM of three independent experiments.

Figure 2

Effect of shikonin on inhibition of IL-2 (a and b) and IFN-γ secretion (c and d) in human T lymphocytes stimulated by PMA/ionomycin or OKT-3/CD28. The cells (10⁵/well) were firstly treated with shikonin for 2 h and then stimulated with PMA (20 ng/mL)/ionomycin (1 μM) (P/I) or with the coated OKT-3 (5 μg/mL)/CD28 (1 μg/mL) (OKT-3/CD28) for 48 h. The level of IL-2 and IFN-γ in cell culture supernatants was determined by ELISA method. Data reported represent the mean ± SEM of three independent experiments. Significance of differences shows as *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Effect of shikonin on the cell cycle of human T lymphocytes stimulated by PMA/ionomycin. Human T cells (10⁶) were pretreated with shikonin for 2 h then cultured with or without PMA (20 ng/mL)/ionomycin (1 μM) (P/I) for 72 h. The cell populations were measured by flow cytometry, and total percentages of the cells entering the S and G2/M phases of the cell cycle were indicated. Data are a representative experiment out of three independent experiments with similar results.

Figure 4

Effect of shikonin on CD25, CD69, and CD71 expression on human T lymphocytes. Human T lymphocytes (10⁶) were pretreated with shikonin for 2 h and then stimulated by PMA (20 ng/mL)/ionomycin (1 μM) (P/I) for 48 h or 72 h, respectively. The cells were double stained with PE-CD3 and FITC-CD69 (a), PE-CD3 and FITC-CD25 (b), PE-CD3 or FITC-CD71 (c) antibodies and then analyzed by flow cytometry. The unstimulated cells were served as negative control. Values represent percentages of the double stained cells.

Figure 5

Effect of shikonin on inhibition of nuclear translocation of NF-κB subunit p65 (a), degradation and phosphorylation of IκB-α in human T lymphocytes stimulated by PMA/ionomycin (b and c). For analysis of the intercellular NF-κB expression, cells were incubated with shikonin for 60 min, and then fixed immediately by cytofix buffer after costimulation by PMA (20 ng/mL)/ionomycin (1 μM) for 120 min, stained with NF-κB antibody for 60 min avoiding light, and then analyzed by flow cytometry. The unstimulated cells were served as negative control (a). For detection of IκB-α, cells were incubated with or without shikonin for 60 min (b); for detection of pIκB-α, the human T lymphocytes were pretreated with or without shikonin and 100 μM ALLN for 60 min (c) and then stimulated with PMA (20 ng/mL)/ionomycin (1 μM) (P/I) at 37°C for 60 min. The whole-cell lysates were prepared, and the proteins were analyzed by Western blotting using antibodies against IκB-α and P-IκB-α. Data are representative of three independent experiments.

Figure 6

Effect of shikonin on inhibition of IKK-β activity (a) and IKK-α/β phosphorylation (b). Human HEK293 cells transfected with FLAG-IKK-β (wt) plasmid were immunoprecipitated (IP) with anti-Flag antibody, and the IP Flag-IKK-β was incubated with GST-IκBα substrate and ATP in the presence or absence of 100 μM shikonin. IKK-β kinase activity was determined by the level of phosphorylated GST-IκBα using antibody against p-IκBα (a). The human T lymphocytes were pretreated with shikonin at 37°C for 60 min and then stimulated with PMA (20 ng/mL)/ionomycin (1 μM) (P/I) at 37°C for different time points. The whole-cell lysates were prepared, and proteins were analyzed by Western blotting using antibodies against IKKα/β and the phosphorylated form of IKKα/β. Data are representative of three independent experiments.

Figure 7

Effect of shikonin on MAPK phosphorylation stimulated by PMA/ionomycin. The human T lymphocytes were pretreated with shikonin at 37°C for 60 min and then costimulated with PMA (20 ng/mL)/ionomycin (1 μM) (P/I) at 37°C for different time points. The whole-cell lysates were prepared, and proteins were analyzed by Western blotting using antibodies against ERK, JNK, and p38 and the phosphorylated forms of ERK, JNK, and p38 (a, b, and c). Data are representative of three independent experiments.