Identification of murine erythrocyte autoantigens and cross-reactive rat antigens

Autoimmune haemolytic anaemia (AIHA) can be induced in mice by repeated injections with rat red blood cells (RBC). Here we describe the identification of murine RBC autoantigens and cross-reactive rat antigens. Sera and RBC eluates from the AIHA-positive mice immunoprecipitated a murine RBC autoantigen of 42,000 MW that comigrates with a zone of glycophorin in PAS-stained polyacrylamide gels. In addition, the eluates immunoprecipitated a 105,000 MW protein corresponding to band 3, the erythrocyte anion channel, and two further components of 34,000 and 29,000 MW within the glycophorin zone. The 42,000 MW band was not detected by immunoblotting, indicating that it bears autoantigenic epitope(s) that are denatured during electrophoresis. Cross-reactive autoantibody in the eluates immunoprecipitated a rat RBC protein that comigrated with band 3, together with two bands of 36,000 MW and 34,000 MW that may represent minor rat glycophorins. In contrast, rat-specific serum IgG from mice with AIHA reacted predominantly with major rat glycophorins of 75,000 MW and 38,000 MW. Immunoblotting revealed that normal murine sera contain IgG that binds autologous spectrin from the RBC membrane skeleton, and that this activity is increased in mice with AIHA. Sera from AIHA-positive mice also reacted with other internal murine RBC components that are not exposed on the surface of intact cells. It is evident from the data that immunization of mice with rat RBC results in the generation of multiple autoantibodies with a complex range of specificities.