Department of Kidney desease and Transplant Immunology
Aichi Medical University School of Medicine
Human Endothelial Cell Activation through ERK by Non-Gal Antibody
Kenta Iwasaki1, Yukiko Inanaga2, Yuko Miwa2, Masataka Haneda2, Akira Onishi3, Haruko Ogawa4, Takaaki Kobayashi5.
1Department of Kidney desease and Transplant Immunology, Aichi Medical University School of Medicine, Nagakute, Japan; 2Nagoya University School of Medicine, Nagoya, Japan; 3Nihon University, Fujisawa, Japan; 4Obihiro University, Obihiro, Japan; 5Aichi Medical University School of Medicine, Nagakute, Japan
Purpose: The deleterious effect of non-Gal antibody has been debated. We previously demonstrated that anti-HLA or anti-ABO antibody induced intracellular signaling cascade differently on endothelial cells, and protected cells against complement-dependent cell toxicity (CDC) under certain conditions, i.e., through AKT activation or ERK inactivation. In current study, we analyzed the character of non-Gal and Gal antibody-mediated signaling pathway on endothelial cell, and to develop the next strategy for xenotransplantation.
Method: Fibroblast were harvested by a combined collagenase and scraping technique from newborn GTKO (-/-) transgenic pigs and were cultured in DMEM cell medium containing 10% FBS. Human serum were obtained from 4 healthy volunteers. IgG antibody were purified from serum by using IgG purification column. Western blots were performed using whole-cell lysates with the antibodies against ERK, phospho-ERK. CDC and cell proliferation were estimated by MTT assay.
Result: Non-Gal antibody dramatically reduced CDC compared with Gal antibody, but still had about 15 to 20% toxicity. To the contrary, non-Gal antibody induced cell proliferation strongly than Gal-antibody in the absence of complement, and did not influence AKT cell protection signaling. Anti-ABO antibody inactivated ERK cell activation signaling, whereas non-Gal antibody upregulated ERK.
Discussion: Endothelial cell activation through ERK has been reported to be correlated with antibody-mediated rejection via complement dependent or independent pathway. Although GTKO strongly enhanced successful rate of xenotransplantation, there seems to exist the risk of non-Gal antibody-mediated rejection. We have demonstrated that non-Gal antibody as well as HLA antibody enhanced ERK signaling pathway, which could be the inhibitory factor for accommodation in kidney transplantation. These results suggest that we may need to conceive a common strategy to induce accommodation in HLA-incompatible transplantation and xenotransplantation.
11:00 - 12:30
|Inflammation & Coagulation||Human Endothelial Cell Activation through ERK by Non-Gal Antibody||Room 109|