In vitro DC migration on inflamed lymphatic endothelium is ICAM-1 and ROCK-dependent. (A) FACS analysis of CD11a and CD11b expression in LPS-matured BM-DCs. (B-F) Flow chamber experiments were performed on monolayers of TNFα/IFNγ-stimulated murine imLECs, in presence of low shear-stress conditions (0.15 dyne/cm2). (B) Overnight stimulation with TNFα/IFNγ lead to a strong up-regulation of ICAM-1 expression in LECs, as demonstrated by FACS. One of at least 5 independent experiments is shown. (C) Pretreatment of LECs with ICAM-1 blocking antibodies (clone YN1/1.7.4 and 29G1), significantly reduced the speed of DC migration on LECs in comparison to pretreatment with an unspecific control antibody (clone 9B5). (D) Track plots of DCs migrating on anti-ICAM1 and ctrl-treated LECs. The starting point of each track was set to the center point of the respective diagram. End points of tracks are indicated by dots. The red line indicates the overall migration directionality and represents the addition of the displacement vectors of all tracked cells. (E) Pretreatment of DCs with LFA-1 or Mac-1 blocking antibodies (clone FD441.8 and M1/70, respectively) reduced the speed of DC migration on LECs in comparison to pretreatment with an unspecific control antibody (clone A95-1). (F) Track plots of anti–LFA-1, anti–Mac-1, or control-treated DCs. (G) Pretreatment of DCs with Y27632 (Y27) significantly reduced the speed of DC migration on LECs. (H) Track plots of Y27632 and control-treated DCs migrating on LECs. Pooled data from 3 (Y27/ctrl/29G1/), 4 (9B5), 5 (YNI) and 6 (FD441.8/M1/70/A95-1) different experiments are shown in panels C through F (*P < .05; **P < .01; ***P < .001).
