5). RT–PCR analysis showed significantly elevated MHC-II expression levels in the spinal cords at 16 dpi Alectinib purchase EAE mice compared to CFA mice (P < 0·05). In the spinal cords of EAE mice, MHC-II expression peaked at 16 dpi compared to levels observed at 7 dpi (P < 0·01) and 28 dpi (P < 0·05) (Fig. 4a,b). In order to strengthen the observations in RT–PCR, real-time PCR was employed to determine MHC-II mRNA levels in the spinal cord. The data shown were normalized to GAPDH expression, and the expression levels in the CFA group were set to 1. As shown in Fig. 4c, MHC-II mRNA level

was promoted significantly in the spinal cords at 16 dpi EAE mice compared to either 7 dpi (P < 0·001) or 28 dpi (P < 0·01). MHC-II expression levels were correlated positively with disease progression and IFN-γ production levels in the spinal cord. Double-labelled immunofluorescence staining was employed to localize MHC-II expression on astrocytes. Spinal cords harvested from EAE mice presented with undetectable MHC-II expression levels on astrocytes at 7 dpi, peaked at 16 dpi and then expression was learn more down-regulated at 28 dpi (Fig. 5). MHC-II expression could not be detected on astrocytes

harvested from mice in the CFA group (data not shown). For proliferation assay, astrocytes were treated with different concentrations of IFN-γ ranged from 0 to 200 U/ml for 24 h. They were then co-cultured with lymph node lymphocytes at a lymphocyte : astrocyte ratio of 10:1. Proliferation of lymphocyte was promoted when co-cultured with IFN-γ-treated

astrocytes (P < 0·001). These data indicate that IFN-γ-treated astrocytes could promote the proliferation of MOG35–55-specific lymphocytes (Fig. 6a). Bay 11-7085 For cytokine production assay, astrocytes were treated with 100 U/ml IFN-γ for 24 h. They were then co-cultured with lymph node lymphocytes at a lymphocyte : astrocyte ratio of 10:1. Supernatants were harvested 72 h later and cytokine levels were determined by ELISA. IFN-γ levels in the supernatants of EAE lymphocytes and IFN-γ-treated astrocytes in the co-culture group were elevated significantly (P < 0·001). Levels of IL-4, IL-17 and TGF-β were also elevated compared to levels observed in supernatants from EAE lymphocytes cultured alone. There were no significant differences in cytokine production levels by cells harvested from mice in the CFA group. Levels of the cytokines described above were low in the supernatants of astrocytes cultured (without lymphocytes) in the presence of IFN-γ (Fig. 6b). Astrocytes were treated in the presence or absence of 100 U/ml IFN-γ for 24 h and then co-cultured with lymphocytes at a lymphocyte : astrocyte ratio of 10:1 for 72 h. Total astrocyte RNA was extracted and MHC-II mRNA levels were detected by real-time RT–PCR.