rhMIF induces the adhesion of HL-60 cells to HMVECs/HMEC-1 cells. (A) To evaluate the functional significance of rhMIF-induced ICAM-1 and VCAM-1 expression, we performed cell adhesion assays using a human myelomonocytic cell line, HL-60, and HMVECs. HMVECs (12.5 × 10³ cells/well) were plated on fibronectin-coated 96-well plates in EBM with 10% FBS. Medium was switched to 0.1 BSA in EBM when HMVECs were 70% confluent. HMVECs were stimulated with rhMIF (50 nM) for 12 hours, and cell adhesion assays were performed. HL-60 cells (8 × 10⁶ cells/well) labeled with calcAM were added to each well and incubated for 1 hour with HMVECs. Plates were carefully washed 4 times with PBS, and fluorescence was determined by a fluorescent plate reader set to 495 nm for excitation and 517 nm for emission. Adhesion was expressed in relative fluorescence units. We found an approximate 2.5-fold significant increase in the adhesion index in rhMIF-stimulated HMVECs compared with nonstimulated cells (A). For better comparisons of the differentially treated groups, the adhesion of HL-60 cells to nonstimulated HMEC-1 cells was chosen as a reference. The adhesion index was, therefore, defined as the ratio of adhesion of HL-60 cells to stimulated HMEC-1 cells (in relative fluorescence units) to adhesion of HL-60 cells to unstimulated HMEC-1 cells (in relative fluorescence units). (B) To elucidate the signaling mechanisms involved rhMIF-induced VCAM-1 and ICAM-1 expression in the functional assays, we performed cell adhesion assays in the presence and absence of signaling inhibitors. In this assay we used an endothelial cell line, HMEC-1. HMEC-1 cells were incubated with signaling inhibitors, anti–ICAM-1, and anti–VCAM-1 or isotype mouse-matched control (2.5 μg/mL) for 1 hour before they were stimulated with rhMIF (50 nM) or 1.15 nM TNF-α (positive control) for 8 hours at 37°C, 5% CO₂. HL-60 cells (2.5 × 10⁶ cells/mL, 100 μL) labeled with calcAM were added and incubated for 1 hour with HMEC-1 cells, and adhesion assays were performed. The Src inhibitor (PP2), the PI3K inhibitor (LY), the NFκB inhibitor, anti–ICAM-1, and anti–VCAM inhibited HL-60 cell adhesion to HMEC-1 cells (Figure 5B), but the Erk1/2 inhibitor (PD) did not inhibit rhMIF-induced adhesion. Data from 3 separate experiments are presented as the mean (n) ± SE. *P < .05 was considered significant. (C) To confirm the role of rhMIF in up-regulating these adhesion molecules, we used siRNA directed against MIF. HMVECs were plated on fibronectin-coated 96-well plates and transfected with siRNA directed against MIF or control-scrambled MIF siRNA for 24 hours using TransIT-siQUEST transfection reagent. HMVECs were stimulated with rhMIF (50 nM) for 12 hours, and cell adhesion assays were performed (C). Data represent the mean of 3 individual experiments (n) ± SEM. *P < .05 was considered significant.