Background/Purpose: Circulating autoantibodies reactive against Ro52 are detected in the sera of 70% of patients with Sjögren’s syndrome (SS), and their presence has been associated with higher severity of the disease. Previous work from our laboratory has demonstrated that interaction between anti-Ro52 and activated innate immunity induces salivary gland (SG) dysfunction in the New Zealand Mixed (NZM) 2758 strain of mice. This study was undertaken to investigate the mechanisms involved in anti-Ro52 mediated salivary gland dysfunction.

Methods: To determine the role of B cell epitope specificity in glandular dysfunction, NZM 2758 mice (5-10 per group) were immunized with recombinant fusion proteins representing the 3 structural domains of Ro52: Ring-B-Box (amino acids 16-123), Coiled-coil (amino acids 128-238) and SPRY (amino acids 268-465). Control mice were immunized with Maltose-binding protein (MBP). All immunization used Alum as the adjuvant. Antibody reactivity was analyzed by immunoprecipitation assay employing 35S-Met labeled Ro52, and by flow cytometry, using mouse ductal cell line SCA9-15. The ability of antibodies to penetrate live SCA9-15 cells was investigated by immunofluorescence. To determine end-organ susceptibility, anti-Ro52 serum generated in rabbits was passively transferred into either untreated or alum treated NZM2758 and lupus-prone NZM2328 mouse strains. SG function was assessed by measuring pilocarpine stimulated saliva.

Results: All groups of NZM2758 mice, except the MBP immunized control mice, generated IgG antibodies capable of immunoprecipitating whole Ro52. While antibodies in the sera from most of the coiled-coil immunized mice (7/8) penetrated live SCA9-15 cells, only 1/5 SPRY and 0/10 Ring-B-box immunized mice had cell penetrating antibodies. This antibody uptake was not dependent on the Fc gamma receptor expression. Only mice immunized with the coiled-coil domain showed a significant drop (p=0.0027) in saliva production. Passive transfer of rabbit anti-Ro52 readily induced SG dysfunction in NZM2758 mice, which was further amplified, if the mice were pretreated with alum. No such functional loss was observed in the lupus-prone NZM2328 mice.

Conclusion:  Our study demonstrates that anti-Ro52 mediated salivary gland dysfunction is regulated by antibody epitope specificity and the genetic susceptibility of the end-organ. Our data provides an explanation for the lack of concordance between the presence of anti-Ro52 and the extent of dry mouth observed in some patients. Thus, in SS patients, measuring the relative levels of anti-Ro52 domain reactive antibodies might be more informative than just determining the presence or absence of anti-Ro52.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/epitope-specificity-and-end-organ-susceptibility-dictates-anti-ro52-mediated-salivary-gland-dysfunction/