Background/Purpose: Sjogren’s syndrome (SS) is a chronic autoimmune inflammatory disease characterized by impaired function of salivary and lacrimal glands leading to dry mouth and dry eyes. The hallmark of SS is the presence of autoantibodies against Ro and La antigens. High titers of anti-Ro60 autoantibodies have shown to correlate with severity of the disease. It is yet unclear how these antiRo60 antibodies correlate with severity. Autoantibodies against M3R are also prevalent in SS. Muscarinic receptor 3 abbr. as M3R is an end organ parasympathetic GPCR mainly present in lacrimal and salivary glands. The stimulation of M3R is well studied and is known to induce secretion in lacrimal and salivary glands and so an effect to M3R could possibly lead to a reduction in secretion.We immunized rabbits with Ro60 and found that immunization not only resulted in the formation of antibodies against Ro60 but also to 2^nd and 3^rd extracellular domains of the M3 receptor. The reactivity to Ro60 and M3R 2^nd and 3^rd extracellular domains has also shown a high correlation.

Methods:

Immunization of rabbits: NZW rabbits were immunized with 500µg of either unmodified Ro60, Smith or RNP antigen) emulsified in 0.5 ml of complete Freund’s adjuvant given I/P with subsequent boosters at days 26, 53 and 99 with a final I/V boost on day 152. The sera were collected weekly in pre and post-immunized rabbits. Ro60 and M3R experiments: Reactivity of sera towards Ro60, MAPS of M3R 2^nd or 3^rd extracellular domains were tested using ELISA. Seral dilution ELISA was done using dilution 1:100 to 1: 100,000 and competition ELISA by preincubating sera with either Ro60 and MAPS of 2nd ECL was done to see the specificity of antibody binding.

Results: Rabbits immunized with Ro60 developed antibodies against Ro60, M3R 2^nd and 3^rd ECLs in a progressive way. A positive correlation was found between both Ro60 and M3R 2^nd ECL (R² =0.66, p = <0.0001) and between Ro60 and M3R 3^rd ECL(R² =0.789, p = <0.0001) . A positive correlation was also found between M3R 2^nd and 3rd ECL ELISA (R² =0.56, p = 0.0001). Rabbits immunized with Smith and RNP antigen did not develop antibodies to any of the extracellular domains of M3R to significant levels. Seral dilution ELISA showed a sequential decline in reactivity for both the 2nd and the 3rd ECL of M3R. Competition ELISA for 2nd ECL of M3R using sera preincubated with M3R 2^nd ECL showed an inhibition of 48.2% to 58.99%, sera preincubated with Ro60 showed an inhibition of 54.5 to 89.03% verifying the specific binding.

Conclusion: Previously our studies found high reactivity for M3R and Ro60 antigen in fully human recombinant monoclonal antibodies produced from plasmablasts isolated from salivary glands of SS patients and was found to be inhibitory. The subsequent step, immunization of rabbits with Ro60 also supported the same hypothesis. The rabbits immunized with Ro60 developed antibodies to M3R extracellular domains supporting the concept of cross-reactivity. Our next step is to see whether the immunization of rabbits with M3R develops anti-Ro60 antibodies. Our future studies are also aimed at passive transfer of IgGs (from Ro60 immunized rabbit sera as well as human recombinant IgGs positive for M3R and Ro) to mice to see the invivo functional consequences of this cross-reactivity.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/anti-muscarinic-receptor-3-antibodies-a-cross-reactive-result-of-ro60-immunization/