Endothelial NF-kB blockade abrogates ANCA-induced GN

Abstract

ANCA-associated vasculitis (AAV) is a highly inflammatory condition in which ANCA-activated neutrophils interactwith the endothelium, resulting in necrotizing vasculitis. We tested the hypothesis that endothelial NF-kBmediates necrotizing crescenticGN(NCGN) and provides a specific treatment target. Reanalysis of kidneys from previously examined murine NCGN disease models revealed NF-kB activation in affected kidneys, mostly as a p50/p65 heterodimer, and increased renal expression of NF-kB-dependent tumor necrosis factor a (TNF-a). NF-kB activation positively correlated with crescent formation, and nuclear phospho-p65 staining showedNF-kB activation within CD31-expressing endothelial cells (ECs) in affected glomeruli. Therefore, we studied the effect of ANCA on NF-kB activation in neutrophil/EC cocultures in vitro. ANCA did not activate NF-kB in primed human neutrophils, but ANCA-stimulated primed neutrophils activated NF-kB in ECs, at least in part via TNF-a release. This effect increased endothelial gene transcription and protein production of NF-kB-regulated interleukin-8. Moreover, upregulation of endothelial NF-kB promoted neutrophil adhesion to EC monolayers, an effect that was inhibited by a specific IKKb inhibitor. In a murine NCGN model, prophylactic application of E-selectin-targeted immunoliposomes packed with p65 siRNA to downregulate endothelial NF-kB significantly reduced urine abnormalities, renal myeloid cell influx, and NCGN. Increased glomerular endothelial phospho-p65 staining in patients with AAV indicated that NF-kB is activated in human NCGN also. We suggest that ANCAstimulated neutrophils activate endothelial NF-kB, which contributes to NCGN and provides a potential therapeutic target in AAV.