Figure 6.
PVR and DNAM-1 contribute to monocyte transmigration. Monocyte transmigration through the HUVEC monolayer was performed in the presence of 20 μg/ml of the indicated anti-PVR or anti–DNAM-1 (sodium azide free) mAbs, and 250 ng/ml MCP-1 was added to the lower chamber. Each measurement was done in quadruplicate. The results are representative of three independent experiments. (a) The N4.40 mAb that does not bind monocytes and HUVECs and Immu-133 mAb that strongly binds to HUVECs were used as isotype-matched irrelevant antibodies. Anti-CD99 mAb 12E7 was used as an inhibitory control. The value 100% corresponds to the number of monocytes that migrate in the presence of the Immu-133 mAb; FACS® analysis confirmed that almost every monocyte of the PBMC suspension transmigrates under this condition. Fab fragments of PV.404 or FS123 mAbs were used at 20 μg/ml. (b) Anti-CD11a mAb was used as a positive control. In this experiment, anti-PVR and anti–DNAM-1 mAbs were incubated on both monocytes and HUVECs (M+E) or only on endothelial cells (E). In the latter condition (E), mAbs were incubated for 30 min with HUVECs and washed with fresh medium. Monocytes were incubated, and TEM was processed as in a. In this experiment, the different antibodies used were treated with 10 μM polymyxin B. (c) Chemotaxis assay. Chemotaxis, in response to 250 ng/ml MCP-1, was assessed for 1 h on costar transwells (5-μm pore size and 6.5-mm diameter). Experimental conditions were similar to the TEM assay, except that filters were not layered with endothelial cells. Fabs were incubated through the process of migration.