CLEVER-1 mediates transmigration through vascular endothelium under shear flow. (A) In the in vitro frozen section assay, anti-CLEVER-1 mAbs significantly inhibit PBMC binding to blood vessel endothelium in inflamed skin samples. Results are shown as mean ± SEM (compared with binding in the presence of a nonblocking negative control mAb). (B) PBMCs were perfused over a confluent monolayer of HUVECs at a laminar shear stress of 1.0 dyne/cm². In 2 frames taken 8 seconds apart, the 2 rolling cells (white arrows) can be seen to travel downward (the direction of flow is from top to bottom). The 2 other PBMCs remain stably adherent. In the third panel, 3 PBMCs, which have transmigrated (and have become completely phase dark) through the HUVEC monolayer, are pointed out by white solid arrows. All other PBMCs (3 pointed out by open arrows) remain surface adherent (phase bright; the brightness of these cells varies depending on the extent of flattening during the firm adhesion). Bar, 10 μm. These 3 different forms of interactions between PBMCs and HUVECs can be much more readily seen in the video (Supplementary video 1 on the Blood website; see the Supplemental Video link at the top of the online article). (C) The numbers of rolling, adherent, and transmigrated cells were enumerated from video playbacks after different pretreatments. The results are expressed as percentage of binding when compared with control mAb (a mAb against HLA class I, which stains the HUVECs but does not block PBMC adhesion). The results are mean ± SEM from 5 to 6 independent experiments using different HUVEC and PBMC donors.