Fig. 4.

Purified anti-ICAM-1 antibodies induce VCAM-c1 expression on human endothelial cells. Purified anti-ICAM-1 antibodies, in the presence or absence of crosslinking RAH antibody, were incubated with HUVEC for 18 h at 37 °C followed by indirect immunofluorescence staining for VCAM-1 and analysis by flow cytometry. A. Representative (n = 6) histograms for staining with CRL1724 isotype control, RAH (negative control) treated cells, and cells treated with purified anti-ICAM-1 (diluted 1:100) with or without crosslinking. B. Percentage of VCAM-1 positive HUVEC from n = 6 experiments after treatment with TNF (positive control), negative serum (diluted 1:100), wash buffer (diluted 1:100) with or without RAH cross-linking (negative controls), or purified patient anti-ICAM-1 antibodies (diluted 1:100) with or without crosslinking. **p < 0.01, ***p < 0.001 compared to untreated cells. 1 way ANOVA with Bonferroni post-test. Antibodies purified from n = 6 patients. C. Time course of VCAM-1 expression after treatment with purified patient anti-ICAM-1 antibodies (diluted 1:100) with or without RAH cross-linking for 6, 12, 18, 24 h (n = 4), or VCAM-1 staining after TNF treatment 18 h (n = 2 HUVEC isolates) (Friedman ANOVA for non-parametric data followed by Dunns post-test comparison *p < 0.05 crosslinked purified Ab 6 h compared to crosslinked purified Ab 12 h, 18 h, or 24 h). D. Concentration effect of purified patient anti-ICAM-1 antibodies (diluted 1:100; 1:250; 1:500) with or without RAH cross-linking on VCAM-1 expression in HUVEC or VCAM-1 staining after TNF treatment 18 h (n = 2 HUVEC isolates) (Friedman ANOVA for non-parametric data followed by Dunns post-test comparison *p < 0.05 crosslinked purified Ab diluted 1:100 compared to crosslinked purified Ab diluted 1:500).