Migration pattern of human monocytes in HUVEC cocultures under flow. (A) Monocytes were flowed over activated HUVECs for 5 minutes (area marked in gray). Monocytes were individually tracked, and their positions were marked at 1-minute intervals. Monocytes captured from free flow become rapidly activated by rolling on the luminal surface, changing phase from white (■) to gray (▴) before transmigrating into the ablumen (phase-black; ▾). (B) A functional blocking antibody to JAM-C and solJAM-C induced reverse transmigration. The abluminal (B) and luminal positions (D) of monocytes in HUVEC cocultures are shown untreated with antibody (●) or treated with D22 (▴) or H33 (▾). The abluminal (C) and luminal positions (E) of monocytes in HUVEC cocultures are shown untreated (●) or treated with flag peptide control (▴) or solJAM-C (▾). A decrease in abluminal monocytes was paralleled by an increase in abluminal monocytes for H33 (B,D) and solJAM-C (C,E), indicating an increased rate of reverse transmigration. Data are presented as the means of 3 fields (± SEM).