RED-BLOOD-CELL GLYCOPHORINS AS B-CELL AND T-CELL ANTIGENS IN CANINE AUTOIMMUNE HEMOLYTIC-ANEMIA

Pathogenic autoantibodies from two dogs with autoimmune haemolytic ana emia (AIHA) were shown to react with glycophorin from the canine red b lood cell (RBC) membrane. Autoantibodies in both cases bound to purifi ed glycophorin in enzyme-linked immunosorbent assays (ELISAs), and the major autoantigen immunoprecipitated by the antibodies corresponded i n apparent molecular mass with glycophorin. Furthermore, neuraminidase treatment of the precipitated antigen, or of canine glycophorin, resu lted in identical changes in apparent molecular mass in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Such removal of sialic acid from glycophorins was demonstrated to cause shifts in S DS-PAGE migration that are unique among RBC membrane proteins. In two further cases of AIHA, where autoantibodies did not immunoprecipitate the glycophorin pattern, ELISAs revealed that RBC-reactive IgG was pre sent in serum and RBC elutes, but that these antibodies failed to bind to canine,glycophorin. Thus, we consider that autoantibodies specific for glycophorin are present in some, but not all, dogs with AIHA. T-c ells from a case of AIHA proliferated in vitro in response to autologo us RBC, or to multiple RBC membrane components fractionated by SDS-PAG E. Three fractions, corresponding to major glycophorins, to the RBC an ion channel band 3, and to spectrin from the membrane skeleton, were s timulatory. In contrast, T-cells from healthy dogs failed to respond t o RBC, or to any blot fractions with the exception, in one animal, of the fraction bearing spectrin. It is suggested that activation of auto reactive T-cells with multiple specificities may be necessary to provi de sufficient help for pathogenic autoantibody production.