Our objectives were to obtain monoclonal anti-endothelial cell antibodies (AECA) from systemic lupus erythematosus (SLE) patients, to characterize their antigen specificity, and their capability to induce a pro-inflammatory and pro-adhesive endothelial phenotype, and to investigate the mechanism of endothelial cell (EC) activation in vitro. Monoclonal IgG AECA were generated by hybridoma formation with human SLE B cells. Antigen specificity was characterized by immunoblotting with enriched cell membrane fractions, by cytofluorimetry and by cell solid-phase ELISA. Endothelial activation was evaluated by measuring increases in U937 cell adhesiveness, adhesion molecule (E-selectin and ICAM-1) expression and IL-6 production. In addition, mechanisms of endothelial activation were investigated by assessment of NF-kappaB by measuring the loss of its inhibitor I-kappaB. mAb E-3 bound live EC and recognized a 42 kDa EC membrane protein, it enhanced U937 adhesiveness, E-selectin and ICAM-1 expression and IL-6 production, and caused the loss of I-kappaB. We conclude this is the first in vitro demonstration that a human monoclonal AECA from a SLE patient reacts with a constitutive endothelial membrane antigen and induces a pro-inflammatory endothelial phenotype through NF-kappaB activation.
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