Characteristics of CD59 up-regulation induced in porcine endothelial cells by alphaGal ligation and its association with protection from complement.
BACKGROUND
Activation of endothelial cells may result in proinflammatory and procoagulant changes, or in changes that protect the endothelial cells (EC) from injurious insults. Stimulation of porcine EC with human anti-porcine antibodies, or lectins from Bandeiraea simplicifolia that bind terminal Galalpha(1-3)Gal (abbreviated alphaGal), can induce EC protection from cytotoxicity by human complement. These EC also exhibit up-regulation of CD59 protein and mRNA expression. Porcine CD59 has been reported to protect porcine cells from human complement. Therefore we investigated the specificity requirements and other characteristics of the induced CD59 up-regulation, as well as the role of up-regulated CD59 in lectin-induced protection of EC from human complement.


METHODS
Aortic EC were incubated in vitro with alphaGal-binding lectins B. simplicifolia lectin I isolectin B4 (IB4) and B. simplicifolia lectin I (BS-I) and CD59 expression was assessed by flow cytometry and enzyme linked immunosorbent assay (ELISA). Binding requirement was studied using disaccharides containing either alphagalactosyl or betagalactosyl moieties to inhibit CD59 up-regulation. Protection from complement killing was assessed after incubation of EC with human serum as a source of anti-porcine antibodies and complement. The role of CD59 in lectin-induced protection was studied in the presence of an anti-pig CD59 antibody and after removal of CD59 using phosphatidylinositol (PI)-specific phospholipase C (PI-PLC).


RESULTS
We found that induction of CD59 up-regulation required specific binding of the lectin to terminal alphaGal and was not induced either by soluble factors that may be released from EC by stimulation with the lectin or by TNF-alpha, IFN-gamma, or IL-1alpha. Unstimulated or BS-I-treated EC showed little or no expression of decay accelerator factor (DAF). Removal of membrane-associated CD59 (and other proteins that are associated with the membrane through PI linkage) with PI-PLC from EC that had been exposed to lectin restored their complement sensitivity to various degrees, depending on the extent of lectin-induced protection. Cytotoxicity was completely restored in cells that exhibited partial protection induced with lectin at low doses or for a short period of time. However, EC that were fully resistant to complement did not regain sensitivity to complement after removal of CD59. Changes in CD59 expression did not modify the degree of C9 binding.


CONCLUSIONS
Induction of CD59 expression required specific binding of the lectin to terminal alphaGal and was not induced by soluble factors that may be released from EC by lectin stimulation. Increased CD59 expression may contribute to this form of protection from complement; however, mechanisms other than CD59 up-regulation appear to be essential for the development of full protection.
Grubbs
Brian C
BC
Department of Surgery, School of Medicine, University of Minnesota, Minneapolis, MN 55455, USA.
Benson
Barbara A
BA
Dalmasso
Agustin P
AP
D000818:Animals; D000910:Antibodies, Heterophile / Q000276:immunology*; D001011:Aorta; D018958:CD55 Antigens / Q000235:genetics; D018957:CD59 Antigens / Q000235:genetics / Q000276:immunology*; D002241:Carbohydrates / Q000494:pharmacology; D003165:Complement System Proteins / Q000276:immunology; D017077:Culture Media, Conditioned; D003602:Cytotoxicity, Immunologic; D004187:Disaccharides / Q000276:immunology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000276:immunology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D006801:Humans; D037102:Lectins; D043265:Phosphatidylinositol Diacylglycerol-Lyase / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D013552:Swine
D000910:Antibodies, Heterophile; D018958:CD55 Antigens; D018957:CD59 Antigens; D002241:Carbohydrates; D017077:Culture Media, Conditioned; D004187:Disaccharides; D037102:Lectins; C055075:galactosyl-(1-3)galactose; D003165:Complement System Proteins; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase