SPREADING OF THE IMMUNE-RESPONSE FROM 52 KDARO AND 60 KDARO TO CALRETICULIN IN EXPERIMENTAL AUTOIMMUNITY

Calreticulin (CR) is widely recognized as a new human autoantigen but there are conflicting data concerning its relationship with the Ro(SS- A) ribonucleoprotein (RNP). Recent evidence suggests that CR binds to 52kDaRo (Ro52) by a protein/protein interaction and binds to hY RNA an d rubella virus RNA. Other studies have shown that initiation of immun ity to either Ro52 or 60 kDaRo (Ro60) can lead to reciprocal spreading of autoimmunity to Ro60 or Ro52, respectively, and induce anti-La aut oantibodies in some strains of mice. These findings support a physical association of these polypeptides in Ro/La complexes. To test the hyp othesis that CR is physically associated with Ro52 and/or Ro60 we exam ined the sera of Ro52-, Ro60- and La-immunized mice for intermolecular spreading to CR. Immune sera from BALB/c and C3H/HeJ mice immunized w ith recombinant 6xHis-mouse Ro52, 6xHis-human Ro60 or 6xHis-human La w ere tested for reactivity by ELISA and immunoblotting with a full-leng th human CR protein expressed as a soluble maltose binding protein fus ion protein (CR-MBP). Five of the six Ro52-immunized C3H/HeJ mice sera and all six Ro60-immunized C3H/HeJ mice sera reacted with the CR-MBP (but not a MBP control) on ELISA. In the BALB/c group, the responder r ate was lower with one in six of the Ro52-immunized and one in five of the Ro60-immunized mice spreading to CR. In contrast, none of the BAL B/c or C3H/HeJ mice which was immunized with La showed evidence of a r ecruited anti-CR antibody response. Immunoblotting of the different re combinant proteins with immune sera from the C3H/HeJ mice confirmed th e specificity of the initial and recruited antibody responses. The spr eading of immunity from Ro52 and Ro60 to CR in Re-immunized mice sugge sts that a subpopulation of CR or CR-like molecules must associate und er certain circumstances with Ro52 and Ro60 polypeptides in vivo, poss ibly as Ro/CR complexes concentrated in surface membrane blebs of apop totic cells. The lack of spreading to CR in La-immunized mice suggests that CR may be associated with a subpopulation of Ro particles from w hich La has already dissociated.