Human monoclonal anti-endothelial cell IgG-derived from a systemic lupus erythematosus patient binds and activates human endothelium in vitro

Our objectives were to obtain monoclonal anti-endothelial cell antibodies ( AECA) from systemic lupus erythematosus (SLE) patients, to characterize the ir antigen specificity, and their capability to induce a pro-inflammatory a nd pro-adhesive endothelial phenotype, and to investigate the mechanism of endothelial cell (EC) activation in vitro. Monoclonal IgG AECA were generat ed by hybridoma formation with human SLE B cells. Antigen specificity was c haracterized by immunoblotting with enriched cell membrane fractions, by cy tofluorimetry and by cell solid-phase ELISA, Endothelial activation was eva luated by measuring increases in U937 cell adhesiveness, adhesion molecule (E-selectin and ICAM-1) expression and IL-6 production. In addition, mechan isms 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 throu gh NF-kappaB activation.