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 unknown. Intermediate filaments have been previously reported to activate the classical complement pathway in an antibody-independent manner. We investigated whether oxidative stress increases human umbilical vein endothelial cell (HUVEC) cytokeratin 1 (CK1) expression and activates the LCP via MBL binding to CK1. Reoxygenation (3 hours, 21% O(2)) of hypoxic HUVECs (24 hours, 1% O(2)) significantly increased CK1 mRNA (in situ hybridization) and membrane protein expression [enzyme-linked immunosorbent assay (ELISA)/confocal microscopy]. 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 reoxygenated in HS. Treatment with anti-human cytokeratin Fab fragments attenuated endothelial MBL and C3 deposition after oxidative stress (ELISA/confocal microscopy). We conclude that: 1) endothelial oxidative stress increases CK1 expression, MBL binding, and C3 deposition; 2) inhibition of MBL attenuates purified CK1-induced complement activation; and 3) anti-human cytokeratin Fab fragments attenuate endothelial MBL and C3 deposition after oxidative stress. These results suggest that MBL binding to endothelial cytokeratins may mediate LCP activation after oxidative stress.
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Department of Anesthesiology, Perioperative, and Pain Medicine, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Montalto
M C
MC
Reenstra
W R
WR
Buras
J A
JA
Stahl
G L
GL
D000906:Antibodies / Q000494:pharmacology; D015153:Blotting, Western; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002478:Cells, Cultured; D037222:Collectins; D003176:Complement C3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D003165:Complement System Proteins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D007633:Keratins / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D037102:Lectins / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000378:metabolism
D000906:Antibodies; D002352:Carrier Proteins; D037222:Collectins; D003176:Complement C3; D037102:Lectins; D012333:RNA, Messenger; D007633:Keratins; D003165:Complement System Proteins