Endothelial oxidative stress activates the lectin complement pathway - Role of cytokeratin 1

Oxidative stress increases endothelial mannose-binding lectin (MBL) binding and activates the lectin complement pathway (LCP). However, the molecular mechanism of MBL binding to the endothelium after oxidative stress is unkno wn. Intermediate filaments have been previously reported to activate the cl assical complement pathway in an antibody-independent manner. We investigat ed whether oxidative stress increases human umbilical vein endothelial. cel l (HUVEC) cytokeratin 1 (CK1) expression and activates the LCP via MBL bind ing to CK1. Reoxygenation (3 hours, 21% O-2) of hypoxic HUVECs (24 hours, 1 % O-2) significantly increased CK1 mRNA (in situ hybridization) and membran e protein expression [enzyme-linked immunosorbent assay (ELISA)/confocal mi croscopy]. incubating human serum (HS) with N-acetyl-D-glucosamine or anti- human MBL monoclonal antibody attenuated MBL and C3 deposition on purified CK1 (ELISA). CK1 and MBL were co-immunoprecipitated from hypoxic HUVECs reo xygenated in HS. Treatment with anti-human cytokeratin Fab fragments attenu ated endothelial MBL and C3 deposition after oxidative stress (ELISA/confoc al microscopy). We conclude that. 1) endothelial oxidative stress increases CK1 expression, MBL binding, and C3 deposition; 2) inhibition of MBL atten uates purified CK1-induced complement activation; and 3) anti-human cytoker atin Fab fragments attenuate endothelial MBL and C3 deposition after oxidat ive stress. These results suggest that MBL binding to endothelial cytokerat ins may mediate LCP activation after oxidative stress.