Increased multiple transmigration events of human monocytes with blocking antibody to JAM-C and solJAM-C in flow. (A) Monocytes captured from free flow became activated and firmly adhered to HUVEC luminal surfaces (step 1). Monocytes migrating on luminal surfaces could move into the ablumen by migrating between junctions of adjacent endothelial cells (transmigration; step 2), which could be followed by transmigration in the abluminal-to-luminal direction (reverse transmigration) back onto luminal surfaces (step 3). A further transendothelial migration event (repeat transmigration) led to a return to the ablumen (step 4). (B) Adherent monocytes in coculture with HUVECs were individually tracked and monitored for transmigration between different compartments over 60 minutes. Increased reverse transmigration was observed for cocultures treated with functional blocking mAbs to JAM-C (●) compared with nonfunctional blocking antibody D22 (○). Monocytes with a reverse-transmigratory phenotype treated with H33 repeat-transmigrate back into the ablumen at higher levels (■) compared with D22 (□). (C) This increases in reverse, and repeat transmigration was also seen with solJAM-C (■, ●) compared with flag peptide control (□, ○). Increased repeat transmigration was observed for mAb H33 and solJAM-C (*P < .05; **P < .01) at time points marked with gray boxes. (D) A summary of the number of monocytes that had repeat-transmigrated in 60 minutes demonstrates that mAb H33 and solJAM-C induced similar levels of repeat transmigration. Reverse and repeat transmigration are presented as a percentage of total monocytes captured from flow per unit field. Data are presented as the mean of 3 fields with SEM and represent a single representative experiment (N = 3). (E) Monocytes cocultured on HUVECs under flow transfected with siRNAs against JAM-C (▴) or a sham-transfected control (▵) exhibited similar transmigration profiles (steps 1-2), but increased reverse transmigration (steps 3-4) was associated with reduced JAM-C expression (●) compared with the control (○) at time points marked by the gray box (*P < .05; **P < .01). Error bars in all figures represent SEM.