Detection of anti-type 3 muscarinic acetylcholine receptor Autoantibodies in the sera of Sjogren's Syndrome patients by use of a transfected cell line assay

Objective. Sjogren's syndrome (SS) is an autoimmune disease affecting primarily the salivary and lacrimal glands, leading to dry mouth and dry eyes. Recent studies have suggested that autoantibodies reactive with the type 3 muscarinic acetylcholine receptors (M3Rs) expressed on salivary and lacrimal gland cells may be highly specific for SS. To test this hypothesis, we constructed a cell line expressing the human M3R gene in order to screen for anti-M3R autoantibodies in sera from SS patients. Methods. Complementary DNA encoding the open-reading frame (ORF) of the human M3R gene was amplified, ligated into the pcDNA5/FRT/V5-His-TOPO TA vector, and then used to transform Escherichia coli bacteria. Plasmid DNA containing the M3R ORF with the correct orientation was transfected into Flp-In Chinese hamster ovary (CHO) cells using Flp recombinase-mediated site-specific recombination. An M3R-transfected CHO cell line, selected and propagated in hygromycin, was used to detect anti-M3R autoantibodies in SS patient and healthy control sera by flow cytometry. Results. Testing of sera for the presence of anti-M3R antoantibodies bound to CHO-transfected cells revealed the presence of anti-M3R autoantibodies in SS patients (9 of 11) but not in healthy controls (0 of 11). Although the anti-M3R autoantibodies detected in patient sera were of multiple isotypes, the most consistently detected were IgG1, IgG3, and IgA. Conclusion. Using a newly constructed cell line expressing human M3R, anti-M3R autoantibodies were easily detected in sera from SS patients. These autoantibodies were skewed toward the IgG1, IgG3, and IgA isotypes, probably recognizing a tertiary epitope created by extracellular domains of the receptor protein. Anti-M3R autoantibodies represent a highly promising clinical marker for the identification of SS.