Anti-PR-3 antibodies induce endothelial IL-8 release

Background It has been shown that interaction of anti-PR-3 antibodies with human endothelial cells (EC) leads to an activation of EC in vitro, i.e. in duction of adhesion molecules like E-selectin, VCAM-1 and tissue factor. Th e aim of this study was to investigate the effect of anti-PR-3 antibodies o n endothelial IL-8 expression. Materials and methods EC were cultured in 96-well plates and stimulated wit h TNF-alpha and IL-1 beta for 1 h to induce membrane expression of endothel ial PR-3. Anti-PR-3 antibodies were purified from sera from patients with c linically active Wegener's granulomatosis. Purified anti-Ro, anti-centromer e, anti-dsDNA antibodies and a monoclonal anti-PR-3 antibody (WGM2) served as controls. Induction of IL-8 mRNA was detected by RT-PCR. IL-8 was measur ed in the supernatant of EC by ELISA. In addition, induction of NF kappa B was investigated by PAGE of nuclear extracts of EC and Western blot with ab against p65. Results In contrast to controls, interaction of anti-PR-3 antibodies (patie nts and WGM2) with cytokine activated EC led to the highest amount of IL-8 synthesis. Priming of EC with cytokines alone induced a markedly lower IL-8 level. The lowest levels of IL-8 could be measured after incubation of unp rimed EC with anti-PR-3 antibodies. Anti-PR-3 antibody induced endothelial IL-8 expression could be inhibited by cycloheximide. In addition, we establ ished that the activation of NF-kappa B is critically involved in anti-PR-3 antibody induced endothelial IL-8 production. Conclusion In summary, we were able to show that anti-PR-3 antibodies induc e endothelial IL-8 synthesis by activating NF-kappa B. As IL-8 represents a powerful neutrophil chemoattractant, our data provide further evidence for a direct pathogenic effect of anti-PR-3 antibodies in ANCA related disease s.