Involvement of antibody-dependent apoptosis in graft rejection

Both humoral factors and apoptosis have been recently suggested to play a role in chronic allograft rejection. However, a link between alloantibodies and grafted cell apoptosis has never been proposed. Using the aortic allograft model in the rat, we have previously demonstrated the presence of IgG associated with the disappearance of donor endothelial and medial smooth muscle cells. In the present study, we tested the interaction between recipient allosera, enriched with antibodies by presensitization, and primary culture of cardiovascular cells of donor origin.

For this purpose endothelial cells, smooth muscle cells, adventitial fibroblasts, and cardiac myocytes of donor origin were cultured. Binding of alloantisera to these cells was analyzed by flow cytometry. Apoptosis of donor cells was evaluated by Tdt-mediated d' UTP-FITC nick end labeling, 4',6-diamidino-2-phenylindole and DNA ladder techniques. The alloantisera were compared with anti-MHC class I monoclonal antibodies. Finally the colocalization of antibodies and apoptosis was investigated in vivo.

In vitro, alloantisera bind to cardiovascular cells of donor origin. These cells expressed MHC class I but not MHC class II. There was a partial competition between anti-MHC I mouse monoclonal antibody and alloantisera mainly of the IgG isotype. Alloantisera bound to, but did not induce lysis of, donor RBC. Alloantisera induced apoptosis of donor cardiovascular cells as assessed by the typical morphological aspect of the donor cells after 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 the nucleus visualized by 4',6-diamidino-2-phenylindole and DNA ladder techniques. Similar apoptosis was induced by specific monoclonal antibodies directed against the MHC class I of donor cells. Primary culture of similar vascular cells of recipient origin was insensitive to alloantisera directed against donor alloantigens. Finally, in vivo, using allopresentization and aortic allografts, an association of alloantibody binding and endothelial cell apoptosis was observed at day 5, and a similar association with smooth muscle cell apoptosis on day 12 after grafting.

These data demonstrate the role of humoral injury in chronic allograft rejection and suggest new therapeutical approaches focused on the induction of resistance to antibody-dependent apoptosis.