Experimental induction of anti-muscarinic type-3-receptor extracellular loop antibodies by immunization with 4-hydroxy-2-nonenal modified Ro60 and unmodified Ro60.

OBJECTIVE

Sjögren's Disease (SjD) subjects have decreased lacrimal/salivary gland function. Studies have proposed that autoantibodies targeting G-protein-coupled muscarinic acetylcholine-type-3-receptor (M3R) are potential clinical markers for SjD. We hypothesized that rabbits/mice immunized with 4-hydroxy-2-nonenal (HNE)-modified/unmodified Ro60 will develop an autoimmunity, specifically a SjD phenotype, thus expressing increased levels of anti-M3R antibodies.

METHODS

We immunized two rabbits each with 10 mM HNE-modified Ro60/unmodified Ro60 antigen or Ro274-290/Ro413-428/Ro500-517 Ro60 peptides. Two rabbits each were immunized with either M3R second extracellular loop (ECL2) or M3R ECL3 peptide. Finally, five groups of BALB/c mice were immunized as follows-Group-I immunized with Ro60, Groups-II-IV immunized with Ro60 modified with 0.4 mM (low), 2 mM (medium), and 10 mM (high) HNE, respectively and Group-V-Freund's adjuvant. Serum antibodies to M3R ECL2/ECL3/Ro60/La or Sm were detected by ELISA. Functional assays were also performed.

RESULTS

Immunization with HNE-modified Ro60/unmodified Ro60 antigen or Ro274/Ro 413/Ro500 peptides induced a rapid intermolecular epitope spreading to M3R ECL2/ECL3, especially to M3R ECL3 in HNE-Ro immunized rabbits. These animals did not bind to scrambled M3R peptides. Ro60-immunized rabbit IgG inhibited M3R activity in a functional assay. Rabbits immunized with ECL2/ECL3 developed high reactivity to Ro60 but not against Sm/RNP. We found a differential antibody-induction against M3R ECL2 with Group-3 mice developing significant reactivity.

CONCLUSION

Our data show induction of increasing anti-M3R antibodies in rabbits immunized with Ro60/HNE-Ro60 or Ro60 peptides and differential induction of these antibodies in mice immunized with Ro60 modified with increasing HNE. These findings suggest that M3R ECL2/ECL3 are involved in SjD autoimmunity progression.