The Jurkat T-cell line was used as a positive control. All immunostaining was performed on archival formalin-fixed, paraffin-embedded tissues using a Dako automated immunostainer and epitope unmasking carried out using the Dako PT link. After hydrogen peroxide and protein blocks, mouse monoclonal anti-CD20 (M0755; Dako) was applied for 1 hour at a 1:1,000 GDC-0068 price dilution and visualized with ImmPact Nova Red (Vector Laboratories, Burlingame, CA) or the EnVision HRP kit (Dako). Double immunolabeling was carried out after low-temperature epitope unmasking (ALTER).17 CD20 was

visualized with ImmPACT DAB nickel (Vector Laboratories), and, after an avidin/biotin block (Vector Laboratories) and a second protein block, rabbit polyclonal pan-cytokeratin CDK inhibitor (Z0622; Dako) was applied at a 1:100 dilution for 1 hour and detected with alkaline phosphatase avidin biotin and Vector blue (Vector Laboratories). Paired two-tailed t tests were used to assess significance in single treatments; for multiple treatments, variation was assessed using analysis of variance, followed by Dunnett’s test for comparison of control. Statistical calculations were performed using Prism 5 software (GraphPad Software, Inc., La Jolla, CA). A P value <0.05 was considered

statistically significant. When B cells were perfused over monolayers of TNF-α- and IFN-γ-treated HSECs, they were captured from flow and underwent firm adhesion. Unlike T cells, they did not undergo a tethering step before adhesion, but appeared to arrest and bind directly from flow. In addition, after arresting, they displayed minimal crawling across the endothelial monolayer, which was in marked contrast to adherent T cells that demonstrate clear crawling behavior (Fig. 1A). We analyzed the molecules involved in B-cell recruitment by HSECs. We studied classical adhesion receptors ICAM-1 and VCAM-1 as well as VAP-1 and CLEVER-1, which our group has shown mediate medchemexpress the transendothelial migration of T cells across HSECs.3, 4 VCAM-1 was the predominant capture

receptor for primary B cells (Fig. 1B). Furthermore, B-cell capture and adhesion was chemokine independent, because pertussis blockade had no effect on the numbers of B cells undergoing firm adhesion (Fig. 1B), although it did inhibit transendothelial migration (Fig. 1C). We have reported previously that the proportion of adherent CD4+ and CD8+ T cells undergoing transendothelial migration across HSECs ranges from 12% to 23%.4 In this study, the proportion of adherent B cells undergoing transmigration ranged from 6.5% to 8.6%. The transmigration of B cells was reduced significantly by the blockade of ICAM-1, VAP-1, and CLEVER-1, and blocking all three receptors had an additive effect and abolished 75% of the transmigration (Fig. 1C).