IgG binding to ECs augments TNFα-mediated leukocyte recruitment. (A) Mouse antiendoglin mAb RMAC8 (10 μg/mL) was perfused over HMEC-1 cells at 0.83 mL/minute for 5 minutes, after which antibody binding was detected by flow cytometry using FITC rabbit anti–mouse Ig. The figure shows similar mAb RMAC8 binding to unstimulated (black line) and TNFα (10 ng/mL, 5 hours)–activated HMEC-1 cells (gray line). The filled histogram shows lack of binding of control mAb MOPC21. (B) Confirmation by confocal imaging of the deposition of RMAC8 on the surface of an EC, with the main panel showing composite images of Z-stack views and the lower insert showing cell cross-section. Arrows point to deposition of antibody on the apical surface. (C) PMNs were perfused at 1.5 dynes/cm2 over ECs precoated with ± RMAC8, with or without prestimulation with TNFα. The figure shows that mAb RMAC8 enhanced PMN adhesion, but only if ECs were TNFα activated. Values are mean + SEM of 3 experiments (*P < .01). HL60 cell rolling (D) and arrest (E) on TNFα-stimulated ECs, showing increased arrest but not rolling on mAb RMAC8-coated ECs over a range of 0.5 to 1.5 dynes/cm2; (F) shows that increasing RMAC8 deposition resulted in increasing HL60 cell arrest on TNFα-stimulated ECs. Relative fluorescent intensities of RMAC8 binding by flow cytometry are depicted by the line (right axis), with HL60 arrest shown in bars (left axis).
