rhMIF induces MN VCAM-1 expression via Src kinase, PI3K, and NFκB. (A) To define the signaling mechanisms involved in VCAM-1 up-regulation by rhMIF, MNs were incubated in 96-well plates. Signaling inhibitors (10 μM) were added to the cells an hour before stimulating with rhMIF and remained in the medium during the experiments. MNs were stimulated with rhMIF for 8 to 12 hours in the presence and absence of different signaling inhibitors. rhMIF-induced VCAM-1 expression was significantly inhibited by an Src inhibitor, PP2, a PI3K inhibitor, LY, and a NFκB inhibitor, PDTC, (*P < .05). PD, an Erk1/2 inhibitor, did not inhibit VCAM-1 expression, suggesting that rhMIF induces VCAM-1 expression in MNs via Src, PI3K, and NFκB, whereas Erk1/2 is not involved in VCAM-1 expression. VCAM-1 up-regulation by rhMIF was more than 2-fold compared with nonstimulated MNs. (B) To confirm our results, we transfected MNs with sense and antisense ODNs of Src, PI3K, NFκB, and Erk1/2 before stimulating cells with rhMIF using lipofectAmine Plus reagent in cell-surface ELISAs. MNs (1 × 10⁶ cells/well) were incubated in 96-well plates in RPMI with 5% FBS for 2 hours at 37°C, and medium was switched to serum free. MNs were transfected with ODNs for 24 hours before stimulation with rhMIF (50 nM). MNs were stimulated with rhMIF for 8 to 12 hours. rhMIF-induced VCAM-1 expression was significantly decreased by antisense ODNs of Src, PI3K, and NFκB compared with MNs transfected with corresponding sense ODNs (*P < .05) using an ELISA. We did not find a decrease in rhMIF-induced VCAM-1 expression by antisense ODNs of Erk1/2. Data represent the mean of 3 individual experiments (n) ± SEM. *P < .05 was considered significant.