The role of RAGE in high mobility group box-1 protein1-mediated CD4+ T cells differentiation to Th1/Th2

Abstract

Objective To explore the role of receptor for advanced glycation end products (RAGE) in HMGB1-mediated CD4+ T cells differentiation to Th1/Th2. Methods CD4+ T lymphocytes isolated from the spleens of male BALB/C mice by magnetic beads were suspended in RPMI-1640 with 10% FCS in 2×106cell/well on 96-well cell culture plates in vitro. The cells were randomly divided 4 groups according to concentration of HMGB1 treatment: control group, 10 ng/mL group, 100 ng/mL group, 1 000 ng/mL group after stimulation with ConA in 3 μg/mL for 12 hours. IL-4 and IFN-γ levels in culture supernatants were quantitated by ELISA kits after HMGB1 stimulation for 12, 24, and 48 h. According to the results, cultured cells were exposed to HMGB1 in 100 ng/mL for 24 h in the following experiments. The cells were randomly divided into 4 groups: control group, A group, B group, C group, and each group were cultured with ConA in 3 μg/mL for 24 h. The cells of control group and other three groups were stimulated with PBS or 100 μg/L HMGB1 for 24 h. The cells of B, C groups were incubated with 1/200 diluted 5 μg/L anti-RAGE Abs (anti-bodies) or PBS for 2 h before HMGB1 stimulation. The cell suspension was obtained to detect the levels of IL-4 and IFN-γ by EILSA and the protein levels and mRNA expressions of RAGE, CATA-3 were detected by western-blot and real-time fluorescent quantitative PCR, respectively. Results Compared with control group, CD4+ T cells incubated with increasing concentrations of HMGB1 (100, 1 000 ng/mL) for 24 h resulted in a decrease in IFN-γ/IL-4 ratio (P<0.05). When CD4+ T cells were exposed to 100 ng/mL HMGB1 for 12 h, IFN-γ/IL-4 ratio was markedly increased. However, CD4+ T cells treated with 100 ng/mL HMGB1 for 24, 48 h, IFN-γ/IL-4 ratio was markedly inhibited (P<0.05). Compared with control group, protein levels and mRNA expressions of RAGE and GATA3 of cells in A group were significantly increased (P<0.05), and IFN-γ/IL-4 ratio of cell suspension in A group and B group was significantly down-regulated (P<0.05). Compared with A group, IFN-γ/IL-4 ratio of cell suspension in C group was increased (P<0.05), and expression of GATA3 mRNA was down-regulated (P<0.05). Compared with A group, protein level of RAGE of cells in C group was significantly down-regulated (P<0.05), but protein level of RAGE of cells in C group was still increased compared with control group (P<0.05). Conclusion Th1/Th2 differentiation induced by HMGB1 on CD4+ T lymphocytes in vitro was at least partly mediated by over-activating RAGE/GATA3 pathway. Key words: High mobility group box-1 protein; Receptor for advanced glycation endproducts; CD4+ T cells; Th1/Th2