Hinokitiol Negatively Regulates Immune Responses through Cell Cycle Arrest in Concanavalin A-Activated Lymphocytes

Abstract

Autoimmune diseases are a group of chronic inflammatory diseases that arise from inappropriate inflammatory responses. Hinokitiol, isolated from the wood of Chamaecyparis taiwanensis, engages in multiple biological activities. Although hinokitiol has been reported to inhibit inflammation, its immunological regulation in lymphocytes remains incomplete. Thus, we determined the effects of hinokitiol on concanavalin A- (ConA-) stimulated T lymphocytes from the spleens of mice. In the present study, the MTT assay revealed that hinokitiol (1-5 μM) alone did not affect cell viability of lymphocytes, but at the concentration of 5 μM it could reduce ConA-stimulated T lymphocyte proliferation. Moreover, propidium iodide (PI) staining revealed that hinokitiol arrested cell cycle of T lymphocytes at the G0/G1 phase. Hinokitiol also reduced interferon gamma (IFN-γ) secretion from ConA-activated T lymphocytes, as detected by an ELISA assay. In addition, hinokitiol also downregulated cyclin D3, E2F1, and Cdk4 expression and upregulated p21 expression. These results revealed that hinokitiol may regulate immune responses. In conclusion, we for the first time demonstrated that hinokitiol upregulates p21 expression and attenuates IFN-γ secretion in ConA-stimulated T lymphocytes, thereby arresting cell cycle at the G0/G1 phase. In addition, our findings also indicated that hinokitiol may provide benefits to treating patients with autoimmune diseases.

Figure 1

Effects of hinokitiol on cell viability and interferon gamma (IFN-γ) secretion in ConA-activated T lymphocytes. Cells were treated with hinokitiol (1–5 μM) in the absence or presence of ConA (10 μg/mL) for 24 or 48 h. (a, b) Cell viability was determined using a MTT assay (n = 4). (c) The level of IFN-γ was measured by an ELISA assay (n = 3). Data (b, c) are presented as the mean ± SEM (^* P < 0.05 and ^** P < 0.01 compared with solvent control (DMSO); ^# P < 0.05 and ^## P < 0.01 compared with the ConA-treated group).

Figure 2

Effects of hinokitiol on the cell cycle in ConA-activated T lymphocytes. Cells were treated with hinokitiol (1–5 μM) in the absence or presence of ConA (10 μg/mL) for 48 h. (a) Cell cycle was determined by PI staining under a flow cytometry. (b) The panel shows the population of the G0/G1 and S-G2/M phases. Data (b) are presented as the mean ± SEM (n = 3; ^** P < 0.01 compared with solvent control (DMSO); ^## P < 0.01 compared with the ConA-treated group).

Figure 3

Effects of hinokitiol on positive regulators of the cell cycle. Cells were treated with hinokitiol (1–5 μM) in the absence or presence of ConA (10 μg/mL) for 24 h. The specific antibodies were used to detect (a) cyclin D3, (b) Cdk4, and (c) E2F1. Data (a–c) are presented as the mean ± SEM (n = 3; ^** P < 0.01 and ^*** P < 0.001 compared with solvent control (DMSO); ^# P < 0.05, ^## P < 0.01, and ^### P < 0.001 compared with the ConA-treated group).

Figure 4

Effects of hinokitiol on negative regulators of the cell cycle. (a) Cells were treated with hinokitiol (1–5 μM) in the presence of ConA (10 μg/mL) for 24 h. The specific antibody was used to detect p21. Data are presented as the mean ± SEM (n = 3; ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001 compared with the ConA (alone)-treated group). (b) Schematic illustration of hinokitiol-mediated inhibition of immune responses in ConA-activated T lymphocytes. Hinokitiol downregulates cyclin D3, Cdk4, and E2F1 expression and upregulates p21 expression and subsequently arrests the cell cycle at the G0/G1 phase. Hinokitiol also attenuates IFN-γ secretion. Finally, hinokitiol negatively regulates immune responses.