Figure 1.

High molecular weight neuronal soluble factors induce downregulation of CD45 and MHC class II and upregulation of miR‐124 in macrophages. (a–c) Bone marrow‐derived macrophages (BMDMs) were obtained from DsRed transgenic mice as described in Materials and Methods and cultured alone (BMDMs alone), directly co‐cultured (co‐culture), or co‐cultured separately using a Transwell system (Transwell) with neuronal cell line (top contour plots and b) or primary cortical neurons (bottom contour plots and c) for 5 days and analyzed for the expression of DsRed, CD11b, CD45, and MHC class II using 4‐color cytometry. DsRed⁺CD11b⁺ gated macrophages were analyzed for the expression of CD45 (x‐axis) and MHC class II (y‐axis) (a), or FACS‐sorted DsRed⁺CD11b⁺ macrophages were used for isolation of RNA and analysis of miR‐124 expression by real‐time RT PCR as described in Materials and Methods (b, c). (d, e) Fractionation of neuronal conditioned medium (NCM) according to molecular weight and analysis of each fraction for the ability to induce expression of miR‐124 in macrophages. NCM (d) or brain slice conditioned medium (e; BSCM) were prepared and fractionated by size filtration as described in Materials and Methods. BMDMs were cultured alone in 50% neurobasal medium (untreated) or cultured in the presence of 50% non‐fractionated NCM (NCM‐all) or BSCM (BSCM‐all) or in fractionated NCM (<3; 3–30; 30–50; 50–100; >100 kDa) or BSCM (10–100; >100 kDa) for 4 hr and analyzed for miR‐124 expression by real‐time RT PCR as described in Materials and Methods. In (b–e), the median ± SD is shown on box and whisker plots (n = 4) with the mean value indicated by “+” symbol. The indicated differences were statistically significant as determined by one‐way ANOVA followed by Bonferroni post hoc test (**p < 0.01 for comparisons between two groups; b: F(2, 9) = 86.42, p < 0.0001; c: F(2, 9) = 190.3, p < 0.0001; d: F(6, 21) = 216.7, p < 0.0001; e: F(3, 12) = 775.6, p < 0.0001) [Colour figure can be viewed at wileyonlinelibrary.com]