Figure 5. The expression levels of two representative neutrophil chemoattractants are diminished by blocking TIM-3 in vitro and in vivo.

(a) Mouse primary mixed glial cells (2 × 10⁵) were plated into the lower chamber of the Transwell system, and pretreated with the indicated concentration of TIM-3-blocking antibody or control IgG, after which 5 × 10⁵ splenocytes were plated into the upper chamber. After incubation for 24 h under hypoxia, the splenocytes that had migrated to the lower chamber were analysed by flow cytometry. The percentage of Gr-1^highCD11b^high cells is expressed as the mean±s.d. from three independent experiments. (b) Gr-1^highCD11b^high neutrophils were sorted from the bone marrow of C57BL/6 mice, and incubated with control IgG- or anti-TIM-3-treated primary glial cells under 1% O₂. The results from three independent experiments are expressed relative to the results from control IgG-treated primary glial cells, which were arbitrarily set to 1. (c) RT–PCR analysis was performed on tissue-specific samples from control IgG- or TIM-3-blocking antibody-treated H/I mice. (d) Graphs show quantified values normalized to those of actin (n=3). (e) Primary cultured glial cells were treated with control IgG or TIM-3-blocking antibody, and then incubated under 1 or 20% O₂ for 24 h. The transcript levels of IL-1β and CXCL1 were determined by RT–PCR analysis. (f) The graphs show the results from at least three independent experiments. NS, not significant.