Expression and regulation of erythrocyte autoantibodies in mice following immunization with rat erythrocytes*(1)
Mice immunized with rat RBC produce antibodies to their own erythrocytes as well as anti-rat RBC specific anti- bodies. If naive recipient mice are given lymphoid cells from such donors and challenged with rat RBC, then their autoantibody response is suppressed while their anti-rat RBC response remains unimpaired. Monoclonal antibodies have been generated from fusions of rat RBC-primed spleen cells and used in immunoprecipitation and Western blotting studies. It has been shown that the anti-rat response is largely directed towards glycophorin components, particularly at 81 and 38 kDa, and a cross-reactive autoantigen of 52 kDa has been identified.
Each antigen preparation was analyzed for content by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. The banding pattern observed with each antigen preparation is shown.
The anti-rat and Coombs’ autoantibody responses for mice challenged with intact rat RBC can be seen that mice became positive in both assays, the antibody titers reaching a plateau by day 28.
Serum (day 28) from each mouse was tested by Western blotting against rat and mouse RBC ghosts. Against rat RBC ghosts a range of specificities were detected with major reactivity to one or more of spectrin or to antigens at 100 and 81 kDa, with a more consistent response to the 38-kDa glycophorin and to bands of 50-55 kDa. By contrast, against mouse ghosts the same sera recognized only spectrin or the 100-kDa or 81-kDa bands corresponding to those antigens defined on the rat ghost blot.
Autoantibodies are more commonly generated to spectrin (60%) than to either the 100-kDa (30%) or the 81-kDa (20%) glycophorins. Mice immunized with intact mouse RBC failed to develop any anti-RBC antibodies in ELISA. Mice receiving SC primed with intact rat RBC had selective suppression of the Coombs’ response with no effect on the anti-rat responses which were in fact higher in suppressed sera relative to non suppressed. Sera from recipients of intact rat RBC-primed SC failed to react against mouse RBC ghosts on Western blots. The same sera tested against rat RBC ghosts did not react with the spectrin and 81-kDa bands although with one serum the spectrin band was defined. There was no loss of definition of the rat 100-kDa band. With another serum failure to react with the rat-specific component at 38 kDa was also observed,
Anti-spectrin autoantibodies were detected in suppressed sera by ELISA with diminished titers up to day 35. The antibody titers of suppressed mice were not significantly greater than those of the control group (no SC) except on day 21.
RBC ghost was prepared. Red cells were incubated in Tris lysis buffer (pH 7.6, 20 mOsm Tris) at 4°C followed by centrifugation at 20,000 x g for 30 min at 4C. This procedure was repeated with the pelleted membranes until no more hemoglo- bin was released into the supernatant. The final pellet was reconstituted in lysis buffer and aliquots stored at -70°C.