Figure 5.

Interference with MIF signaling pathways reveals mutual regulation of CD74, CXCR4, and MIF expression in in vitro‐activated B cells. B cells sorted from CIS, RRMS, and HC blood (n = 9–12 per group) were activated in vitro for 24 h using anti‐IgM and analyzed for MIF expression (A; qPCR) and secretion into culture media (B; ELISA). Stimulations have been done in three individual experiments with 3–4 patients per group per experiment. Each dot represents the mean value of one individual, measured in duplicates. (C) In vitro‐activated B cells from healthy blood were treated with and without MIF inhibitor ISO‐1 for 24 h and assessed for CXCR4 and CD74 surface expression using FACS (n = 6). Stimulations have been done in three individual experiments with two controls per group per experiment. (D, E) The human B‐cell line Raji (CD74^hi) was transfected with three distinct MIF shRNA constructs and compared with scrambled controls for MIF mRNA levels (D) and the percentage of CD74^hi cells (E) at day 3 (n = 5). Data were measured in five individual experiments with one set of scrambled and three different shRNA's per experiment. In vitro‐activated B cells were also evaluated for CXCR4 (F, n = 7), CD74 (G, n = 7) and MIF mRNA (H, n = 5–6, measured in duplicates) expression after 24 h treatment with anti‐CD74 (LN2) or isotype antibody (F, H) and CXCR4 antagonist AMD3100 (G, H). Stimulations have been done in three individual experiments with 2–3 controls per group per experiment. All used controls were set at 1 (dotted line). Data are shown as mean ± SEM. Paired t‐tests were performed to compare groups. *p < 0.05, **p < 0.01, ***p < 0.001.