title: The 52-kd protein as a target of intermolecular spreading of the immune response to components of the SS-A/Ro-SS-B/La complex.
abstract: OBJECTIVE
  To determine whether immunization of healthy non-autoimmune mice with 52-kd SS-A/Ro induces a secondary antibody response to other components of the 48-kd SS-B/La-60-kd SS-A/Ro RNP complex and vice versa, since anti-52-kd antibodies have been invariably linked to these antigens in patients with Sjogren's syndrome and in mothers whose children have neonatal lupus.
  
  
  METHODS
  Female BALB/c mice were immunized with 100 microg of 6xHis recombinant human 48-kd SS-B/La, 52-kd SS-A/Ro, or 60-kd SS-A/Ro proteins, or the 6xHis polypeptide control, each purified by Ni2+ affinity chromatography. Mice subsequently received booster injections with 50 microg of the same antigen every 10-21 days. Immune responses were measured by enzyme-linked immunosorbent assay (ELISA), immunoblotting of recombinant antigens, and immunoprecipitation of 35S-methionine-labeled in vitro translation products.
  
  
  RESULTS
  Immunization with 48-kd SS-B/La resulted in anti-48-kd SS-B/La antibodies within 45 days, followed 10 days later by a secondary response to 52-kd SS-A/Ro, as measured by ELISA. Antibody spreading to 60-kd SS-A/Ro was not detected. Immunization with 52-kd SS-A/Ro resulted in rapid high-titer anti-52-kd SS-A/Ro responses within 27 days. Spreading to 48-kd SS-B/La occurred in only 1 mouse and 60-kd SS-A/Ro was detected in a minority of the mice after prolonged antigen exposure. Immunization with 60-kd SS-A/Ro led to anti-60-kd SS-A/Ro responses within 37 days, followed 3 months later by low-titer anti-48-kd SS-B/La and anti-52-kd SS-A/Ro antibodies. All primary immune responses were confirmed by immunoblotting and immunoprecipitation. While immunoblotting of the recombinant proteins revealed reciprocal intermolecular spreading in the majority of mice, immunoprecipitation clearly demonstrated that predominant spreading was generated after immunization with 48-kd SS-B/La, which consistently resulted in antibodies to 52-kd SS-A/Ro.
  
  
  CONCLUSIONS
  The murine responses observed in the present study, demonstrating reciprocal intermolecular spreading to 48-kd SS-B/La, 52-kd SS-A/Ro, and 60-kd SS-A/Ro, support the linkage of 52-kd SS-A/Ro with the other proteins, despite their as-yet-undetected association in vivo. The marked recruitment of anti-52-kd SS-A/Ro responses elicited by 48-kd SS-B/La may provide a lead to exploring the physical interaction, direct or indirect, of 52-kd SS-A/Ro with the SS-A/Ro-SS-B/La RNP particle and its presentation to the immune system. These data should facilitate the establishment of a murine model of neonatal lupus.
authors.affiliation: New York University School of Medicine, and the Hospital for Joint Diseases, New York 10003, USA.
authors.forename: C E
authors.initials: CE
authors.lastname: Tseng
authors.forename: E K
authors.initials: EK
authors.lastname: Chan
authors.forename: E
authors.initials: E
authors.lastname: Miranda
authors.forename: M
authors.initials: M
authors.lastname: Gross
authors.forename: F
authors.initials: F
authors.lastname: Di Donato
authors.forename: J P
authors.initials: JP
authors.lastname: Buyon
mesh_terms: D000818:Animals; D000917:Antibody Formation; D000936:Antigen-Antibody Complex / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007114:Immunization; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D011233:Precipitin Tests; D020733:RNA, Small Cytoplasmic; D011994:Recombinant Proteins / Q000276:immunology; D012261:Ribonucleoproteins / Q000276:immunology*; D000097585:SS-B Antigen; D000099207:SS-A Antigen
chemical_list: D000936:Antigen-Antibody Complex; D001324:Autoantigens; D020733:RNA, Small Cytoplasmic; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D000097585:SS-B Antigen; D000099207:SS-A Antigen; C116006:RO60 protein, human