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Abstracts / Human Immunology 74 (2013) 1–49
Special Abstract Session: Scholar Awards
Wednesday, November 20, 2013 2:00 PM - 3:30 PM
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ACTIVATION AND CYTOSKELETAL REMODELING OF HUMAN ENDOTHELIAL CELLS BY HLA CLASS II ANTIBODIES. Fang Li, Sahar Salehi, Xiaohai Zhang, Elaine F. Reed. Immunogenetics Center, Department of Pathology and Laboratory Medicine, University of California, Los Angeles, CA, USA. Aim: The development of anti-donor HLA class II antibodies (Ab) is associated with chronic allograft rejection. The endothelial cell (EC) barrier is controlled by adhesive structures called cadherins which regulate permeability, migration and proliferation. We hypothesize that Ab ligation of HLA class II molecules on EC contributes to chronic rejection by transducing intracellular signaling cascades leading to remodeling of the actin cytoskeleton, redistribution of VE-cadherin and changes in EC barrier function. Methods: Class II expression on primary cultures of EC was achieved by adenoviral infection with recombinant Class II Transactivator (CIITA) sub-cloned with pAd/PL-DEST. Mouse monoclonal Ab that reacts with a monomorphic determinant of class II antigens (F26C6G1) was used to treat EC. Stress fiber formation was assessed using Texas Red-phalloidin staining. Protein phosphorylation was assessed by Western blot. VE-cadherin localization was measured by immunofluoresence. Results: Treatment of class II-positive EC with class II Ab significantly increased Akt, ERK and p70 S6 Kinase phosphorylation. Class II-augmented ERK phosphorylation was accompanied by a substantial increase in stress fiber formation that was abrogated by pretreatment with the ERK inhibitor UO126 (Fig. 1A). Treatment of EC with class II Ab markedly reduced VE-cadherin localization in EC tight junctions which could be reversed by pretreatment with U0126 (Fig. 1B). Conclusions: Ligation of HLA class II molecules with Ab stimulates intracellular signaling cascades leading to activation of the ERK signaling pathway, actin cytoskeleton remodeling, relocalization of VE cadherin and changes in EC barrier function.
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Special Abstract Session: Scholar Awards
Wednesday, November 20, 2013 2:00 PM - 3:30 PM
56-OR
ACTIVATION AND CYTOSKELETAL REMODELING OF HUMAN ENDOTHELIAL CELLS BY HLA CLASS II ANTIBODIES. Fang Li, Sahar Salehi, Xiaohai Zhang, Elaine F. Reed. Immunogenetics Center, Department of Pathology and Laboratory Medicine, University of California, Los Angeles, CA, USA. Aim: The development of anti-donor HLA class II antibodies (Ab) is associated with chronic allograft rejection. The endothelial cell (EC) barrier is controlled by adhesive structures called cadherins which regulate permeability, migration and proliferation. We hypothesize that Ab ligation of HLA class II molecules on EC contributes to chronic rejection by transducing intracellular signaling cascades leading to remodeling of the actin cytoskeleton, redistribution of VE-cadherin and changes in EC barrier function. Methods: Class II expression on primary cultures of EC was achieved by adenoviral infection with recombinant Class II Transactivator (CIITA) sub-cloned with pAd/PL-DEST. Mouse monoclonal Ab that reacts with a monomorphic determinant of class II antigens (F26C6G1) was used to treat EC. Stress fiber formation was assessed using Texas Red-phalloidin staining. Protein phosphorylation was assessed by Western blot. VE-cadherin localization was measured by immunofluoresence. Results: Treatment of class II-positive EC with class II Ab significantly increased Akt, ERK and p70 S6 Kinase phosphorylation. Class II-augmented ERK phosphorylation was accompanied by a substantial increase in stress fiber formation that was abrogated by pretreatment with the ERK inhibitor UO126 (Fig. 1A). Treatment of EC with class II Ab markedly reduced VE-cadherin localization in EC tight junctions which could be reversed by pretreatment with U0126 (Fig. 1B). Conclusions: Ligation of HLA class II molecules with Ab stimulates intracellular signaling cascades leading to activation of the ERK signaling pathway, actin cytoskeleton remodeling, relocalization of VE cadherin and changes in EC barrier function.
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