CD157 is involved in neutrophil transendothelial migration. PMNs (10⁶/mL) pretreated with purified murine IgG Fc fragments (150 μg/mL) to block Fc receptor were seeded on HUVEC monolayers grown on fibronectin-coated transwell filters. Migration is shown in the presence of a 10-nM fMLP transwell gradient. (A) Anti-CD157 mAb (20 μg/mL) significantly blocked transendothelial migration of PMNs. After 1 hour of incubation at 37°C, the transmigrated cells were counted. Results are expressed as the percentage of transmigrated cells. There was no significant difference in the ability of CD157 or CD31 ligation to block transmigration across resting(▪) or TNF-α activated (▦) HUVEC monolayers. *P < .001 anti-CD157 and anti-CD31 versus no antibody or irrelevant IgG. Data are the mean ± SD of 5 independent experiments. (B) Transmigration assays across TNF-α–activated HUVEC monolayers were run for 1 hour in the presence of decreasing concentrations of anti-CD157 mAb, 20 μg/mL anti-CD31 mAb (positive control), or anti-HLA class I mAb (negative control). Data are the mean ± SD of 3 independent experiments. **P < .001 anti-CD157 or anti-CD31 mAb (20 μg/mL) versus no mAb or irrelevant IgG (20 μg/mL). **P < .001 anti-CD157 mAb (5 μg/mL). *P < .05 anti-CD157 mAb (1 μg/mL). (C-D) Anti-CD157 mAb blocks transmigration reversibly. (C) PMNs and HUVECs were treated with 20 μg/mL of the mAb indicated (15 minutes at 20°C), then the mAb was washed away. Transmigration assays were run and the percentage of transmigrated cells was evaluated every 30 minutes for 3 hours. (D) Experiments were run as in panel C, with mAb refilled during the time course, as indicated (arrow). Data are the mean ± SD (n = 3) of a representative experiment; similar results were observed in 3 separate experiments. *P < .05 anti-CD157 mAb along with anti-CD31 mAb versus anti-CD157 or anti-CD31 alone. (E) PMNs and HUVECs use CD157 for transmigration. PMNs and HUVECs were incubated separately with 20 μg/mL anti-CD157 or anti-CD31 mAb, then unbound mAb was washed away. The percentage of transmigrated PMNs was evaluated after 1 hour. Data are the mean ± SD of 4 independent experiments. Ligation of CD157 expressed either by PMNs or by HUVECs strongly inhibits transmigration; however, ligation of CD157 on neutrophils is more efficient. *P < .05 versus ligation of CD157 on HUVECs. Reduction of transmigration using any methods of blocking of CD157 or CD31 was always significant at P < .001 versus irrelevant IgG.