Antagonistic effects of sJAM-A on leukocyte recruitment. (A) HUVECs and neutrophils were pretreated for 1 hour with GW280264X (GW) or left untreated. After removal of the inhibitor by washing, both cell types were either coincubated or incubated separately for 2 hours, and subsequently conditioned media were investigated for the release of sJAM-A by Western blotting. Western blot signals were quantified and shown as mean plus or minus SD for 3 independent experiments. *Significant inhibition of JAM-A from cocultured cells after pretreatment of neutrophils with GW280264X. (B) HUVECs or isolated neutrophils were pretreated with the indicated concentrations of sJAM-A.Fc, monovalent sJAM, or irrelevant hIgG in the presence and absence of Fc block (50 μg/mL) for 30 minutes and subsequently washed. Neutrophils were then assayed for transendothelial migration toward IL-8. *Statistically significant changes in IL-8–induced neutrophil transmigration compared with cells receiving IgG control. (C) Isolated neutrophils were pretreated with sJAM-A.Fc, sJAM-A.D1.Fc, or sJAM-A.D2.Fc (all 10 μg/mL) in the presence of Fc block (50 μg/mL) for 30 minutes and subsequently assayed for transendothelial migration as described in panel B. *Statistically significant changes in IL-8–induced neutrophil transmigration compared with the control treated with Fc block only. (D) JAM-A.Fc (60 μg), monovalent sJAM (40 μg), or human IgG (60 μg) were intravenously administered to wild-type and jam-a^−/− mice (n = 4 per group). One hour later, the neutrophil attracting chemokine KC or vehicle control was injected into experimentally induced air pouches. Four hours after KC injection, neutrophil recruitment into the air pouches was determined by flow cytometric analysis of the differentially labeled leukocyte populations. *Statistically significant changes in neutrophil infiltration compared with the IgG-treated control (P < .05).