Figure 1.

Neutrophil adhesion to complement-challenged BOECs under static and dynamic conditions. (a) BOECs cultured as monolayers in 96-well plates were treated with 50% NHS with or without prior incubation with anti-CD46, -CD55, and -CD59 antibodies. Cells incubated for 4 hours with TNF-α 20 ng/ml served as positive control. Calcein-labeled neutrophils were introduced for 30 minutes and then removed before wells were read for green fluorescence emission. Total fluorescence was normalized to values obtained for BOECs incubated with media only; n = 5, **P < 0.01 (1-way ANOVA with Dunnett’s multiple comparison test). (b–d) Calcein-labeled neutrophils were perfused over BOECs in a Bioflux chamber. (b) Markedly enhanced neutrophil adhesion was observed when neutrophils were co-perfused with 50% NHS for 5 minutes compared with preincubation with 50% NHS for 1 hour and then subsequent exposure to neutrophils (2-tailed t-test). (c) Calcein-labeled neutrophils perfused in a Bioflux system at 1 dyne/cm² with 50% NHS over untreated BOECs (top channel) or cells incubated with anti-CD46, -CD55, and -CD59 antibodies (bottom channel) showed marked adhesion after 5 minutes to cells with combined complement regulator blockade. (d) Quantification of neutrophil adhesion to BOECs within the measurement channel via total green fluorescence intensity shows the effect of various complement activation conditions, with combined incubation with anti-CD46, -CD55, and -CD59 antibodies showing significant neutrophil adhesion (n = 3–8, *P < 0.05, ***P < 0.001; 1-way ANOVA with Dunnett’s multiple comparison test). ANOVA, analysis of variance; BOECs, blood outgrowth endothelial cells; NHS, normal human serum; TNF-α, tumor necrosis factor-α.