Figure 2.

MIF and SDF-1 trigger chemotactic migration of OCPs and promote osteoclastogenesis via ligation to CXCR4 receptor in vitro. A) Transmigration of FACS-sorted eGFP⁺CD11b⁺OCPs across the Transwell culture insert in response to various concentrations of MIF and SDF-1 chemokines. B) Number of differentiated TRAP+ osteoclasts differentiated from the transmigrated eGFP⁺CD11b⁺ OCPs in response to MIF and SDF-1. C) Effects of anti-MIF-mAb and control-mAb on RANKL-induced osteoclastogenesis in the absence of MIF or SDF-1 chemokines. D) Inhibitory effects of anti-MIF-mAb, but not control-mAb, on chemotactic migration of eGFP⁺CD11b⁺ OCPs to MIF and SDF-1. E) Number of TRAP+ osteoclasts per well formed from the RANKL-stimulated, transmigrated eGFP⁺CD11b⁺ OCPs in the presence of MIF and SDF-1. Freshly FACS-sorted OCPs (5×10⁵) were loaded in the upper chamber of a Transwell culture insert and allowed to migrate for 12 h toward the lower chamber of the transwell device that contained MIF or SDF-1 (both 100 ng/ml). The number of transmigrated cells in the lower chamber was counted microscopically. Data are expressed as chemotactic index (mean±SD) and representative of three independent experiments is shown (A). To induce osteoclastogenesis from the transmigrated OCPs in response to MIF or SDF-1, the transmigrated respective OCPs were incubated in 96 well plate (1×10⁴ cells/well) in the presence of RANKL and M-CSF (50 and 30 ng/ml, respectively) with or without MIF or SDF-1 (100 ng/ml, respectively) for 5 days. The number of TRAP+ osteoclasts per well is presented as mean±SD (B). To evaluate the possible effects of anti-MIF-mAb or control-mAb on osteoclastogenesis process, FACS-sorted eGFP⁺CD11b⁺ OCPs were incubated with RANKL and M-CSF (50 and 30 ng/ml, respectively) in the presence or absence of anti-MIF-mAb or control-mAb (100 μg/ml, respectively) for 4 days (C). Anti-MIF-mAb or control-mAb (100 μg/ml, respectively) was also applied to the OCPs transmigration toward MIF or SDF-1 (D) and RANKL-induced osteoclastogenesis assay from the transmigrated respective OCPs in the presence or absence of MIF or SDF-1 (E) following the protocols described for (A) & (B). *p<0.05, **p<0.01, ***p<0.001.