OR22: LIGATION OF HLA CLASS II MOLECULES BY HLA ANTIBODIES INDUCES ENDOTHELIAL CELL PERMEABILITY AND MONOCYTE TRANSENDOTHELIAL MIGRATION

Aim Leukocyte extravasation plays a major rolein the development of allograft rejection and transplant vasculopathy and consists of multiple processes, including leukocyte capture, firm adhesion, transmigration, and loss of endothelial cell integrity causing barrier dysfunction. Ligation of HLA class I molecules by antibodies (Ab) elicits signaling events in endothelial cells (EC) leading to EC cell migration, proliferation and leukocyte recruitment. We hypothesize that ligation of HLA class II molecules on EC by Ab contributes to chronic rejection by transducing intracellular signaling cascades leading to cytoskeletal remodeling, increase in EC permeability and monocyte transmigration. Methods Class II expression on primary EC was achieved by infection with adenoviral recombinant Class II Transactivator (CIITA) subcloned in pAd/PL-DEST. EC were treated with murine mAb against class II (F26C6G1). Stress fiber formation was assessed using Texas Red-phalloidin staining. Protein phosphorylation was assessed by Western blot. Changes in EC permeability were measured by VE-cadherin staining and FITC-dextran permeability. Monocyte MM6 recruitment to EC was measured by immunofluorescence microscopy and monocyte transendothelial migration was determined using a transwell assay. Results Anti-class II Ab significantly increased ERK phosphorylation, stimulated stress fiber formation, reduced VE-cadherin localization in EC junctions and enhanced FITC-dextran permeability. ERK inhibitor UO126 pretreatment abrogated class II-induced stress fiber formation, reversed VE-cadherin expression in EC junctions, and attenuated class II-stimulated FITC-dextran permeability. In addition, treatment of EC with anti-class II antibodies induced a 2-4-fold increase in the recruitment and transmigration of monocytes, which was attenuated by treatment with the ERK inhibitor U0126 or recombinant PSGL1 that blocks monocyte capture. Conclusions Ab ligation of HLA class II molecules on EC stimulates activation of the ERK signaling pathway and actin cytoskeleton remodeling leading to barrier dysfunction and monocyte transendothelial migration. Elucidation of the class II signaling pathways has the potential to identify novel therapeutic targets for treatment of chronic rejection.