Migration inhibitory factor up-regulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 via Src, PI3 kinase, and NFκB by M. Asif Amin, Christian S. Haas, Kui Zhu, Pamela J. Mansfield, Michael J. Kim, Nicholas P. Lackowski, and Alisa E. Koch Blood Volume 107(6):2252-2261 March 15, 2006 ©2006 by American Society of Hematology

rhMIF up-regulates VCAM-1 and ICAM-1 expression on human PB MNs by cell-surface ELISA. (A) MNs (1 × 106 cells/well) were incubated in 96-well plates in RPMI with 5% FBS for 2 hours at 37°C. rhMIF up-regulates VCAM-1 and ICAM-1 expression on human PB MNs by cell-surface ELISA. (A) MNs (1 × 106 cells/well) were incubated in 96-well plates in RPMI with 5% FBS for 2 hours at 37°C. Medium was switched to serum free for 6 to 8 hours to achieve quiescence. MNs were stimulated with rhMIF (50 nM), and cell-surface ELISAs were performed. rhMIF induced a time-dependent increase in MN VCAM-1 expression. rhMIF-induced VCAM-1 expression became significantly higher at 4 hours, and it decreased to the basal level after 24 hours (*P < .05). TNF-α served as a positive control. Fold change in VCAM-1 expression compared with PBS is shown. (B) rhMIF induced expression of ICAM-1 in a time-dependent manner. rhMIF increased ICAM-1 expression at 4 hours, and the maximum response was between 8 and 12 hours. ICAM-1 expression in MNs remained significantly higher even after 24 hours compared with PBS (*P < .05). Panel B shows fold increase in ICAM-1 up-regulation compared with PBS. Data represent the mean of 4 individual experiments (n) ± SEM. *P < .05 was considered significant. M. Asif Amin et al. Blood 2006;107:2252-2261 ©2006 by American Society of Hematology

rhMIF increases VCAM-1 and ICAM-1 expression on human PB MNs in a concentration-dependent manner. rhMIF increases VCAM-1 and ICAM-1 expression on human PB MNs in a concentration-dependent manner. (A) MNs were stimulated with different concentrations of rhMIF for 12 hours. rhMIF-induced VCAM-1 expression was significantly higher between 10 pM and 1 μM (A). We did not observe VCAM-1 expression at 10 μM. (B) Similarly, rhMIF-induced ICAM-1 expression was significantly higher compared with nonstimulated MNs between 1 pM and 1 μM. We found the maximum up-regulation of VCAM-1 and ICAM-1 by rhMIF between 1 and 100 nM (A-B). NS indicates nonstimulated. Data represent the mean of 3 individual experiments (n) ± SEM. *P < .05 was considered significant. M. Asif Amin et al. Blood 2006;107:2252-2261 ©2006 by American Society of Hematology

rhMIF induces MN VCAM-1 expression via Src kinase, PI3K, and NFκB. 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. M. Asif Amin et al. Blood 2006;107:2252-2261 ©2006 by American Society of Hematology

rhMIF induces MN ICAM-1 up-regulation via Src kinase, PI3K, and NFκB. rhMIF induces MN ICAM-1 up-regulation via Src kinase, PI3K, and NFκB. (A) To investigate the signaling cascades involved in ICAM-1 up-regulation by rhMIF, we performed cell-surface ELISAs using chemical signaling inhibitors. rhMIF-increased ICAM-1 expression was significantly inhibited by an Src inhibitor, PP2, a PI3K inhibitor, LY, and a NFκB inhibitor, PDTC, (*P < .05) but not by a Jak2 inhibitor, AG-490, suggesting that rhMIF induces ICAM-1 expression in MNs via Src, PI3K, and NFκB. rhMIF induced a 3-fold increase in MN ICAM-1 expression compared with NS. (B) rhMIF-induced ICAM-1 expression was significantly decreased by antisense ODNs of Src, PI3K, and NFκB compared with MNs transfected with sense ODNs of Src, PI3K, and NFκB in 8 to 12 hours (*P < .05). Panel B shows the percentage of inhibition in ICAM-1 expression by antisense ODNs of Src, PI3K, and NFκB compared with corresponding sense ODNs. Data represent the mean of 3 individual experiments (n) ± SEM. *P < .05 was considered significant. M. Asif Amin et al. Blood 2006;107:2252-2261 ©2006 by American Society of Hematology

rhMIF induces the adhesion of HL-60 cells to HMVECs/HMEC-1 cells. 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. M. Asif Amin et al. Blood 2006;107:2252-2261 ©2006 by American Society of Hematology

Immunoblotting of MNs stimulated with rhMIF for various time points. Immunoblotting of MNs stimulated with rhMIF for various time points. (A) MNs were stimulated with rhMIF (50 nM) for time periods of 1 minute to 45 minutes. rhMIF induced a marked increase in Src, Akt, and NFκB phosphorylation in a time-dependent manner compared with nonstimulated cells. This phosphorylation was inhibited by relatively specific chemical inhibitors (A-E). (B) Western blotting was performed with MNs stimulated with rhMIF (50 nM) for 12 hours to examine the up-regulation of VCAM-1 and ICAM-1. For inhibitor studies, MNs were pretreated with PP2, LY, PD, and PDTC for 1 hour prior to stimulating with rhMIF. All inhibitors were used at 10 μM concentration except PDTC (100 μM). rhMIF induced a marked increase in VCAM-1 and ICAM-1 in MNs at 12 hours. rhMIF-induced MN VCAM-1 and ICAM-1 up-regulations were abrogated by the inhibitors of Src, PI3K, and NFκB, but an inhibitor of Erk1/2 did not affect the expression of the adhesion molecules (F-G). Each blot represents 1 of 3 experiments. M. Asif Amin et al. Blood 2006;107:2252-2261 ©2006 by American Society of Hematology

rhMIF induces VCAM-1 and ICAM-1 on MNs via Src and NFκB as determined by immunofluorescence. rhMIF induces VCAM-1 and ICAM-1 on MNs via Src and NFκB as determined by immunofluorescence. MNs were plated in 8-well chamber slides overnight. MNs were stimulated with rhMIF (50 nM) for 10 hours and studied for VCAM-1 and ICAM-1 expression using FITC-conjugated and PE-conjugated secondary antibodies. VCAM-1 and ICAM-1 expression was visible on the cell surface (A). To investigate Src activation by rhMIF, MNs were stimulated for 20 minutes. Panels B and C show immunopositivity for phospho-Src and phospho-NFκBp65 compared with NS cells. DAPI was added to stain nuclei. We have merged the DAPI and phosphorylation images in panels B and C to show the nuclear and cytoplasmic localization of phospho-NFκBp65 and phospho-Src. NS indicates nonstimulated. Images were captured using an Olympus BX fluorescence microscope with attached Olympus camera (Olympus, Melville, NY) and a 100 ×/1.3 numeric aperture objective, with oil as an imaging medium. Images were assembled using Adobe Photoshop software, version 7.01 (Adobe Systems, San Jose, CA). M. Asif Amin et al. Blood 2006;107:2252-2261 ©2006 by American Society of Hematology