Involvement of antibody-dependent apoptosis in graft rejection

Background. Both humoral factors and apoptosis have been recently suggested to play a role in chronic allograft rejection. However, a link between all oantibodies and grafted cell apoptosis has never been proposed. Using the a ortic allograft model in the rat, we have previously demonstrated the prese nce of IgG associated with the disappearance of donor endothelial and media l smooth muscle cells. In the present study, we tested the interaction betw een recipient allosera, enriched with antibodies by presensitization, and p rimary culture of cardiovascular cells of donor origin. Methods. For this purpose endothelial cells, smooth muscle cells, adventiti al fibroblasts, and cardiac myocytes of donor origin were cultured. Binding of alloantisera to these cells was analyzed by flow cytometry. Apoptosis o f donor cells was evaluated by Tdt-mediated d' UTP-FITC nick end labeling, 4',6-diamidino-2-phenylindole and DNA ladder techniques. The alloantisera w ere compared with anti-MHC class I monoclonal antibodies. Finally the coloc alization of antibodies and apoptosis was investigated in vivo. Results. In vitro, alloantisera bind to cardiovascular cells of donor origi n. These cells expressed MHC class I but not MHC class II. There was a part ial competition between anti-MHC I mouse monoclonal antibody and alloantise ra mainly of the IgG isotype. Alloantisera bound to, but did not induce lys is of, donor RBC. Alloantisera induced apoptosis of donor cardiovascular ce lls as assessed by the typical morphological aspect of the donor cells afte r 24 hr of incubation. These data were confirmed by the Tdt-mediated d' UTP -FITC nick end labeling positivity of the cells and the fragmentation of th e nucleus visualized by 4',6-diamidino-2-phenylindole and DNA ladder techni ques. Similar apoptosis was induced by specific monoclonal antibodies direc ted against the MHC class I of donor cells. Primary culture of similar vasc ular cells of recipient origin was insensitive to alloantisera directed aga inst donor alloantigens. Finally, in vivo, using allopresentization and aor tic allografts, an association of alloantibody binding and endothelial cell apoptosis was observed at day 5, and a similar association with smooth mus cle cell apoptosis on day 12 after grafting. Conclusion. These data demonstrate the role of humoral injury in chronic al lograft rejection and suggest new therapeutical approaches focused on the i nduction of resistance to antibody-dependent apoptosis.