Vascular endothelial cell apoptosis induced by non-MHC alloantibodies: an important injury mechanism in the development of chronic graft rejection

Abstract

Non-MHC alloantibodies may play a pathogenic role in chronic rejection but remain poorly characterized. This study investigated the kinetics of antidonor antibody production and the injury mechanism in a LEW-to-F344 rat model of cardiac transplantation, in which donor and recipient strains differ for non-MHC alloantigens. All the F344 recipients of LEW allografts produced antidonor IgG antibodies reactive with LEW endothelial cells (EC). A subgroup of recipients that rejected their grafts in 30-60 days exhibited markedly higher levels of anti-donor IgG antibodies (n=6, MFI:23.85±2.7) prior to graft rejection when compared to those with long-surviving allografts (n=4, MFI:11.23±0.81; P=0.00058). F344-anti-LEW sera were capable of inducing lesions of chronic rejection when passively transferred in an antibody-free heart graft model in which immune non-responsiveness of F344 rats to LEW alloantigens was induced by intrathymic inoculation of LEW lymphocytes. Five of six cardiac allografts exposed to the antisera were rejected in 2-5 weeks after passive transfer and developed severe chronic lesions while control allografts (N=3) treated with normal rat sera survived for > 90 days (p=0.015). IgG antibodies purified from anti-LEW sera exhibited complement-dependent cytotoxicity against LEW EC in culture. The EC displayed early (Annexin V+) and late (TUNEL+) evidence for programmed cell death examined with flowcytometry. Western blot analysis of poly (ADP-ribose) polymerase (PARP) cleavage showed that an 116 kDa intact form of PARP was degraded into 85 kDa and 25 kDa fragments in lysates of the ECs treated with the IgG, indicating an apoptosis-associated caspase activity. In addition, in situ TUNEL experiment demonstrated that vascular EC apoptosis was consistently present inall the heart allografts examined. We conclude that non-MHC alloantibodies are pathogenic and capable of causing chronic graft injury via an antibody-induced cell apoptosis mechanism. The results emphasize the importance of non-MHC antibodies as a common predisposing factor in the development of chronic rejection.