Figure 6.

Lack of endothelial PECAM-1 favors transcellular over paracellular diapedesis of encephalitogenic CD4⁺ T cells across pMBMEC monolayers under physiological flow. Confluent WT and PECAM-1^−/− VE-CadGFP pMBMEC monolayers were stimulated with IL-1β (20 ng/ml) for 16–24 h prior to live cell imaging. Activated encephalitogenic CD4+ T cells were allowed to arrest on pMBMECs at low flow (0. 1 dyn/cm²). After 5 min, the flow was increased to 1.5 dyn/cm². For time-lapse videos, pictures were taken every 30 s for a total of 30 min. For each diapedesis event, the transmigration route was determined as follows: An opening of GFP-expressing endothelial junctions was judged as paracellular diapedesis, while a sustained junctional integrity was indicative of transcellular diapedesis. (A,B) Representative pictures of paracellular (A) and transcellular (B) diapedesis taken before and during T-cell diapedesis. Dashed line, site of transmigration; scale bar, 10 μm; DIC, differential interference contrast. (C) In total, 144 and 140 T cells, which completely transmigrated through WT and PECAM-1^−/− endothelial monolayers, respectively, recorded in 68 videos (WT, 34 videos; PECAM-1^−/−, 34 videos) were analyzed. Reported statistics of categorical data result from Pearson's qui-square test. Test statistics: p = 0.0016, χ²(1) = 9.991, odds ratio = 2.160; ^**p < 0.01. In order to provide an estimate for data variability, a video-wise representation is provided in Supplementary Figure 2.