Figure 4.

Role of CAR in transepithelial migration. A) Transepithelial migrations assays were performed as described. Preincubation of T cells with a blocking mAb against LFA-1, or of the epithelial monolayer with a blocking mAb against CAR, caused a significant reduction in transepithelial migration. B–E) LSCM images of basal (B, D) and apical (C, E) epithelium 1 h into a transepithelial migration assay, following preincubation of the epithelial monolayer with an isotype-matched control mAb (B, C) or a blocking mAb against CAR (D, E). Images were taken at 0.2-μm intervals extending 1 μm basal to the ZO-1 staining (B, D) and 1 μm apical to the ZO-1 staining (C, E) and then projected as a composite. In addition, the ZO-1 staining from the same series was analyzed frame by frame, and the composite was imported into Adobe Illlustrator and recreated as an overlay in orange to show the tight junctions. Scale bar = 15 μm. F) Epithelial cell monolayers were untreated (white bars) or pretreated with 5 μM cytochalasin D (black bars) and/or a control mAb or a blocking mAb to CAR. Monolayers were washed and transepithelial migration assays were performed as described above. T cells were untreated or treated with a blocking mAb to LFA-1. Data are means ± sd of triplicates; 1 representative experiment of 3 is shown. NS, not significant (P≥0.05). **P < 0.005, ***P < 0.0005; 2-tailed Student’s t test.