Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.
A Novel Cell-Based Assay for Inhibitory Anti-Muscarinic Type 3 Receptor Antibodies in Patients with Sjgren's Syndrome.
Jackson1, Michael W., Bastian1, Isabell, Gordon2, Thomas P.
Primary Sjögren's syndrome (SS) is a systemic autoimmune disorder characterized by exocrine failure and widespread autonomic dysfunction. Functional autoantibodies directed against the muscarinic type 3 receptor (M3R) have been postulated to underpin gastrointestinal, bladder and cardiac dysfunction in SS (Cai et al, Arthritis Res Ther 2008;10:R31), and we have recently demonstrated that SS IgG acts specifically at the M3R to disrupt cholinergic neurotransmission and motility in murine gastrointestinal tissues (Park et al, Arthritis Rheum, 2011, 63: 142634). To date, detailed studies correlating the presence in patients of anti-M3R antibodies and symptoms of autonomic dysfunction have been hampered by a lack of suitable screening assays (Dawson et al, Arthritis Rheum 2004;52:298495). Hence, the aim of the current study was to develop a cell-based assay to screen patient IgG samples for anti-M3R activity.
HEK293 cells (2 × 105) were transiently transfected in 96 well culture plates for 24 hours with DNA encoding the human M3R, and with the pGL4.33 vector (Promega) containing a luciferase gene driven by the serum response element promoter. Cells were then incubated for 4 hours in the presence of the cholinergic agonist, carbachol, (0.3 to 100 mM) and patient IgG (4 mg/ml) characterised as positive (M3R+; n = 4) or negative (M3R-; n = 2) for inhibitory anti-M3R activity, as determined by the in vitro bladder strip assay (Waterman et al, Arthritis Rheum 2000;43:164754). IgG from healthy donors (n = 6) was used as controls. Luciferase gene activity was determined on a DTX 880 MultiMode Detector (Beckman Coulter).
M3R+ IgG, but not control or M3R- IgG, significantly inhibited carbachol-induced luciferase activity across a range of carbachol concentrations, with a maximum inhibition of approximately 40%. M3R+ IgG had no effect on luciferase activity in the absence of carbachol, or in cells transfected with the pGL4.33 vector alone. The inhibitory action of the autoantibody on carbachol-induced receptor activity was non-competitive, consistent with allosteric modification of receptor signalling.
We have developed a real-time cell-based bioassay incorporating a luciferase reporter to detect inhibitory anti-M3R antibodies in IgG from patients with SS. The assay does not rely on direct detection of immobilised antibody at the receptor, thereby overcoming the limitations of conventional immunological techniques such as ELISA or Western blotting. The results from the cell assay compare favourably with those from whole-tissue in vitro assays (Waterman et al, Arthritis Rheum 2000;43:164754; Cavill et al, Arthritis Rheum 2003;48:359702), thereby combining the sensitivity of ex-vivo tissues with the convenience of a 96-well assay format. The bioassay should facilitate both detailed pharmacological characterization of the mechanism of antibody action at the M3R, and the screening studies required to establish the role of anti-M3R antibodies in mediating the autonomic dysfunction associated with SS.
To cite this abstract, please use the following information:
Jackson, Michael W., Bastian, Isabell, Gordon, Thomas P.; A Novel Cell-Based Assay for Inhibitory Anti-Muscarinic Type 3 Receptor Antibodies in Patients with Sjgren's Syndrome. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :469