OR20 HLA Class II ligation by antibody induces endothelial cell permeability by promoting endocytosis of Ve-cadherin

Abstract

Aim Increased microvascular dilatation and permeability occurs during allograft rejection and contributes to fibrotic remodeling and organ dysfunction in transplant recipients. Endothelial cells (EC) control vascular permeability by regulating cell-to-cell junctions. We hypothesize that HLA class II ligation on EC transduces signals that disrupt cell–cell tight junctions resulting in increased cell permeability. Methods Class II expression was achieved by transducing EC with adenoviral recombinant CIITA. EC were stimulated with mAb against monomorphic determinant present on all HLA class II antigens (F26C6G1). Western blots were used to characterize protein phosphorylation. Cell permeability was measured by FITC-dextran transwell permeability assay and Ve-Cadherin (Ve-Cad) surface expression was determined by flow cytometry. Cell junctions and Ve-cad internalization were quantified by immunofluorescence microscopy. Results Treatment of EC with anti-class II Ab activated Src, stimulated Ve-Cad phosphorylation at tyr685 and increased Ve-Cad internalization (Fig). Class II ligation decreased Ve-cad surface expression by 42 ± 5%, which was blocked by PP2. In the presence of chloroquine, class II ligation resulted in a 27 ± 4% increase in Ve-cad internalization. Immunofluorescent studies showed that Ve-Cad in the vesicles was phosphorylated and class II ligation increased pY685-Ve-Cad expression. Preincubation of EC with PP2 decreased Ve-cad phosphorylation and internalization. Class II Ab increased FITC-dextran permeability confirming that class II signaling resulting in increased vascular permeability. Our data show that class II ligation on EC mediates the disassembly of intercellular junctions by stimulating Ve-Cad phosphorylation and internalization in a Src dependent manner. Conclusions We provide evidence that HLA class II DSA promote endocytosis of Ve-Cad via Src activation, disrupting endothelial barrier function and contributing to fibrotic remodeling and organ dysfunction. Download : Download full-size image