ICAM-2 supports homophilic interaction. (A) ICAM-2 surface expression on CHO cells stably transfected with human ICAM-2 (CHO-IC2), detected by immunofluorescence using an anti–ICAM-2 mAb (clone B-T1). ICAM-2 expression is concentrated at the cell junctions, similarly to what is observed in endothelial cells. Staining of mock-transfected CHO cells with the same antibody is also shown. (B) ICAM-2 homophilic interaction. Polystyrene beads conjugated with anti–human Fc Ab, preincubated with soluble ICAM-2–Fc, were added to CHO-IC2 (top) or control cells (CHO-Esel, bottom) in 96-well plates. ICAM-1–Fc (middle) or VCAM-1–Fc (image not shown) were used as control proteins. Images were acquired as in Figure 1A. (C) The number of beads retained in the wells after washings was counted per 400 × field of each replicate. Experiments were performed in triplicate. ICAM-2–Fc significantly bound to CHO-IC2 cells, compared with ICAM-1–Fc and VCAM-1–Fc. None of the protein-Fc constructs bound to CHO-Esel. ***P < .001, compared with control protein constructs and binding to CHO-Esel, ANOVA. n = 4. (D) Two anti–ICAM-2 Abs were tested for their ability to block ICAM-2 homophilic interaction, by preincubation with the protein-Fc-beads complex before adding to CHO-IC2. The polyclonal anti–ICAM-2 Ab (pAb), but not the monoclonal anti–ICAM-2 Ab (CBR-IC2/2) or goat IgG isotype control (Ctrl), significantly inhibited binding of ICAM-2 Fc to CHO-IC2 by 50%. ***P < .001, ANOVA. n = 3. (E) Matrigel tube formation is inhibited by anti–ICAM-2 mAb. HUVECs were preincubated with pAb anti–ICAM-2 or goat IgG control for 15 minutes before plating onto Matrigel. Photos were taken hourly after seeding, and branches counted as described (see “Materials and methods”). In the presence of anti–ICAM-2 pAb, HUVEC tube formation was inhibited by approximately 30% compared with control Ab (Ctrl Ab) or untreated samples (Ctrl) at 3, 4, and 5 hours. ***P < .001, ANOVA. n = 3. (C-E) Error bars indicate mean ± SEM.