Background/Purpose: In Sjörgen’s Syndrome, an autoimmune disease, the lacrimal and salivary glands are the primarily affected glands. Previous studies have proposed autoantibodies that target type-3 muscarinic acetylcholine receptors (M3R) found in salivary and lacrimal glands as a clinical marker for SS. In this study, we hypothesize that rabbits and mice immunized with 4-hydroxy-2-noneal (HNE)-modified or unmodified 60-kDa Ro will have increased levels of circulating anti-M3R and anti-Ro antibodies mirroring some of the symptoms of Sjörgen’s Syndrome.

Methods: In the first experiment, two rabbits were immunized multiple times with 500μg of 10mM of HNE-modified Ro60 and two rabbits were immunized with 500μg unmodified Ro60. In the second experiment, 50 mice, ten mice from five different strains (SJL/J, DBA, BALB/c, C57BL/6, and PL/J) were immunized subsequent times with 50μg of Ro60 peptide. In the third experiment, fifty mice were in immunized with unmodified Ro60, or low (.4mM), moderate (2mM), or high (10mM)-HNE modified Ro60. ELISA analysis was used to detect the presence of antibodies specific to Ro60, HNE-modified Ro60, and the second extracellular loop (amino acids 213-228) or the third extracellular loop (amino acids 514-527) of the type-3 muscarinic acetylcholine receptor using the serial bleeds from the immunized rabbits and mice.

Results: Immunization with HNE-modified Ro60 or unmodified Ro60 abrogates tolerance to autoimmunity. Rabbits immunized with unmodified Ro60 showed in an induction of antibodies to Ro60 and the second loop (213-228) of M3R, but not 514-527. Conversely, rabbits immunized with HNE-modified Ro60 had suppressed levels of antibodies to either variant of the M3R peptide, but showed a rapid response after immunization with increased levels of anti-Ro60 and anti-HNE Ro60 antibodies. The five strains of mice immunized with HNE-modified Ro60 or unmodified Ro60 , as well as, the mice immunized with various concentrations  of HNE-modified Ro60 had negligible levels of antibodies directed toward M3R, but both experimental groups showed an induction in anti-Ro60 antibodies.

Conclusion: We previously published data showing that the rabbits and mice immunized with unmodified or HNE-modified Ro60 developed anti-Ro antibodies, as commonly seen in circulation of SS patients. Add to that, this data which shows increasing levels of anti-M3R antibodies in rabbits immunized with Ro60, but not mice. Taken together this data suggests that second extracellular loop of the type-3 muscarinic receptor is a target in the lacrimal and salivary glands during SS autoimmunity progression in rabbits.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/antibodies-to-anti-type-3-muscarinic-acetylcholine-receptor-are-highly-prevalent-to-rabbits-immunized-with-4-hydroxy-2-noneal-modified-and-60-kda-ro/