Ligation of HLA class ii molecules on endothelial cells induces p-selectin cell surface expression and myeloid cell recruitment

Abstract

Aim A hallmark of chronic antibody-mediated rejection is the presence of leukocytes infiltrates within the allograft. We tested the hypothesis that ligation of HLA class II on EC mediates myeloid cell recruitment by inducing p-selectin presentation through Weibel Palade body exocytosis. Methods Class II expression was induced by adenoviral transfection of EC with CIITA. Western blots were used to quantitate protein phosphorylation. Monocyte adherence was measured by counting CFSE-labeled monocytes (MonoMac 6) recruited to HLA-class II Ab stimulated EC in the presence/absence of Fc receptor blockade with human IgG. Transendothelial cell migration was determined using a transwell assay. P-selectin expression was detected by Cell ElISA. Results Class II ligation by antibody (Ab) against a monomorphic epitope determinant on class II antigens significantly increased monocyte transendothelial migration in a Fc-independent manner. Class II Ab-mediated myeloid cell transendothelial migration was attenuated by blockading with recombinant PSGL-1 (rPSGL-1). Class II Ab caused a 1.54 ±± 0.11 fold increases in monocyte adherence that was inhibited by pretreatment with rPSGL-1. In addition, pretreatment of EC with Src inhibitor PP2 and ERK inhibitor U0126 resulted in decreased myeloid cell recruitment (Fig. 1). Furthermore, PP2 and U0126 diminished class II induced p-selectin cell surface expression (Fig. 2). Conclusions We provide evidence that class II DSA promotes a MAPK-mediated monocyte adherence by upregulating EC p-selectin expression, stimulating monocyte adherence and infiltration into allografts, leading to transplant vasculopathy. Figure options Download full-size image Download as PowerPoint slide Figure options Download full-size image Download as PowerPoint slide