Muscarinic type 3 receptor autoantibodies are associated with anti-SSA/Ro autoantibodies in Sjögren's syndrome

• DOI: 10.1016/j.jim.2016.07.003
• Publication Date: 2016-07-26T09:13:18Z
• Authors: Jian Zuo; Adrienne E.G. Wil...; Yun-Jong Park; Kevin Choi; Annie L. Chan; Westley H. Reeves; Michael R. Bubb; Yun Jong Lee; Kyungpyo Park; Carol M. Stewart; Seunghee Cha
• Keywords: Adult; Male; Receptor, Muscarinic M3; Recombinant Fusion Proteins; Green Fluorescent Proteins; Middle Aged; Autoantigens; 3. Good health; Arthritis, Rheumatoid; 03 medical and health sciences; HEK293 Cells; Sjogren's Syndrome; 0302 clinical medicine; ROC Curve; Ribonucleoproteins; Predictive Value of Tests; RNA, Small Cytoplasmic; Humans; Lupus Erythematosus, Systemic; Female; Serologic Tests; Aged; Autoantibodies

Anti-muscarinic type 3 receptor autoantibodies (anti-M3R) are reported as potential inhibitors of saliva secretion in Sjögren's syndrome (SjS). However, despite extensive efforts to establish an anti-M3R detection method, there is no clinical test available for these autoantibodies. The purpose of this study was to propose inclusion of anti-M3R testing for SjS diagnosis through investigation of their prevalence using a modified In-Cell Western (ICW) assay. A stable cell line expressing human M3R tagged with GFP (M3R-GFP) was established to screen unadsorbed and adsorbed plasma from primary SjS (n=24), rheumatoid arthritis (RA, n=18), systemic lupus erythematosus (SLE, n=18), and healthy controls (HC, n=23). Anti-M3R abundance was determined by screening for the intensity of human IgG interacting with M3R-GFP cells by ICW assay, as detected by an anti-human IgG IRDye800-conjugated secondary antibody and normalized to GFP. Method comparisons and receiver-operating-characteristic (ROC)-curve analyses were performed to evaluate the diagnostic value of our current approaches. Furthermore, clinical parameters of SjS were also analyzed in association with anti-M3R. Anti-M3R was significantly elevated in SjS plasma in comparison with HC, SLE, or RA (P<0.01). SjS anti-M3R intensities were greater than two-standard deviations above the HC mean for both unadsorbed (16/24, 66.67%) and adsorbed (18/24, 75%) plasma samples. Furthermore, anti-M3R was associated with anti-SjS-related-antigen A/Ro positivity (P=0.0353). Linear associations for anti-M3R intensity indicated positive associations with focus score (R(2)=0.7186, P<0.01) and negative associations with saliva flow rate (R(2)=0.3052, P<0.05). Our study strongly supports our rationale to propose inclusion of anti-M3R for further testing as a non-invasive serological marker for SjS diagnosis.