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

Pathogenic autoantibodies from two dogs with autoimmune haemolytic anaemia (AIHA) were shown to react with glycophorin from the canine red blood cell (RBC) membrane. Autoantibodies in both cases bound to purified glycophorin in enzyme-linked immunosorbent assays (ELISAs), and the major autoantigen immunoprecipitated by the antibodies corresponded in apparent molecular mass with glycophorin. Furthermore, neuraminidase treatment of the precipitated antigen, or of canine glycophorin, resulted 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 SDS-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 present 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-cells from a case of AIHA proliferated in vitro in response to autologous RBC, or to multiple RBC membrane components fractionated by SDS-PAGE. Three fractions, corresponding to major glycophorins, to the RBC anion channel band 3, and to spectrin from the membrane skeleton, were stimulatory. In contrast, T-cells from healthy dogs failed to respond to RBC, or to any blot fractions with the exception, in one animal, of the fraction bearing spectrin. It is suggested that activation of autoreactive T-cells with multiple specificities may be necessary to provide sufficient help for pathogenic autoantibody production.