Figure 4

In vitro assay for IL-10 detection in MG/MΦ culture and in vivo experiment using il-10-knockout mice. Neonatal mouse brain-derived MG or mouse femoral bone marrow-derived MΦs were cultured on 96-well plate. The MG or the MΦs were treated with the T-cell conditioned media (CM) or recombinant IFN-γ and, thereafter, IL-10 levels at different time points were measured by using ELISA (schematic drawn in an upper panel). IL-10 surge from MG/MΦs was not observed in the in vitro assay unlike in the injured spinal cord after Th1-cell transfer. However, Th1 cells secreted robust IL-10 in an ifn-γ-dependent manner, and this IL-10 secretion from Th1 cells had a pivotal role in improved recovery after SCI. (a) Time course of IL-10 level detected by ELISA in MG culture media after treatment with T-cell CM or IFN-γ (medium, n=4; Th1, n=4; Th1 w/o MG: Th1 cells without MG, n=4; Th1 ifn-γ^−/−, n=3; IFN-γ (recombinant IFN-γ at 20 ng/ml), n=3; Th17, n=2). (b) Time course of IL-10 level detected by ELISA in macrophage (MΦ) culture media after treatment with T-cell CM or IFN-γ (medium, n=4; Th1, n=4; Th1 w/o microglia (MG): Th1 cells without MΦs, n=4; Th1 ifn-γ^−/−, n=3; IFN-γ: recombinant IFN-γ at 20 ng/ml, n=3; Th17, n=2). (c) IL-10 level in MG or MΦ culture media at 3 h after treatment with T-cell CM or IFN-γ (MG: medium, n=4; Th1, n=4; Th1 cells without MG, n=4; Th1 ifn-γ^−/−, n=3; IFN-γ: recombinant IFN-γ at 20 ng/ml, n=3; MΦs: medium, n=4; Th1, n=4; Th1 w/o MΦ: Th1 cells without MG, n=4; Th1 ifn-γ^−/−, n=3; IFN-γ: recombinant IFN-γ at 20 ng/ml, n=3). **P<0.01, ***P<0.001 versus the medium group, ^##P<0.01, ^###P<0.001 versus Th1 group (one-way ANOVA, Bonferroni post-test). (d) BMS scores in Th1 or Th1 il-10^−/−-transferred SCI mice (PBS, n=5; Th1, n=5; Th1 il-10^−/−, n=6). *P<0.05, **P<0.01, ***P<0.001 versus the PBS group (two-way ANOVA with repeated measures, Bonferroni post-test)