CX3CL1 shedding by PMA down-regulates adhesion of THP-1 cells to ECV-304 cells, and adhesion is restored by MP inhibitors. (A) CX3CL1 mediates adhesion of THP-1 cells: fluorescently labeled THP-1 cells were seeded onto confluent wild-type (WT) or CX3CL1-expressing ECV-304 cells. After 20 minutes of incubation and 2-fold washing, adherent cells were detected by fluorescence microscopy (right panel). The confluence of the ECV-304 cell layer was controlled by phase contrast microscopy (left panel). (B) PMA down-regulates THP-1 cell adhesion: WT and CX3CL1–ECV-304 cells were treated with 200 ng/mL PMA for 1 hour or left untreated. Subsequently, fluorescently labeled cells were added and, after 2-fold washing, adhesion was quantified as relative light units (RLU) of the fluorescence signal emitted from the adherent cells (mean and SD, n = 4). (C) MP inhibitors differentially restore THP-1 cell adhesion: CX3CL1–ECV-304 cells were treated with 3 μM of the mixed ADAM10/TACE inhibitor GW280264X or the selective ADAM10 inhibitor GI254023X and stimulated with 200 ng/mL PMA for 1 hour or left untreated. Fluorescently labeled cells were added and, after 2-fold washing, adhesion was quantified by reading the fluorescence (mean and SD, n = 4). While both inhibitors significantly increased THP-1 cell adhesion to unstimulated CX3CL1–ECV-304 cells, only GW280264X could significantly increase adhesion to the PMA-stimulated cells (P < .05, indicated by asterisk). In panels A-C, 1 representative of 3 independent experiments is shown.