Background/Purpose: Primary Sjögren’s syndrome (SS) is a systemic autoimmune disorder characterized by exocrine failure and widespread autonomic dysfunction. Functional autoantibodies (Abs) directed against the muscarinic type 3 receptor (M3R) have been postulated to underpin gastrointestinal, bladder and cardiac dysfunction in SS, and we have recently demonstrated that SS IgG acts specifically at the M3R to disrupt cholinergic neurotransmission and motility in murine gastrointestinal tissues.To date, the mechanism by which anti-M3R Abs exert an effect on receptor signalling has remained difficult to determine, due to a lack of suitable functional assays. In the current study, we use a novel, real-time cell bioassay of M3R signalling to explore the physiological mechanism by which anti-M3R Abs inhibit M3R activity.  

Methods: HEK293 cells (2 X 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 300 µM) 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 in vitro bladder strip assays. IgG from healthy donors (n = 6) was used as controls. Luciferase gene activity was determined on a DTX 880 MultiMode Detector (Beckman Coulter). 

Results: All M3R+ IgGs, but not control or M3R- IgG, significantly inhibited carbachol-induced luciferase activity at carbachol concentrations ranging from 0.3 to 30 µM, with a maximum inhibition of approximately 40%.  In contrast, inhibition of luciferase activity by anti-M3R Abs was lost at carbachol concentrations of 100 and 300 µM, consistent with competitive antagonist activity by the Abs at the carbachol-binding site.  Anti-M3R Abs had no effect on luciferase activity in M3R-expressing cells in the absence of carbachol.

Conclusion: We have used a real-time cell-based bioassay incorporating a luciferase reporter to characterise inhibitory anti-M3R antibodies in IgG from patients with SS.  The bioassay allows a functional measure of receptor signalling activity, thereby facilitating investigation of the mechanism by which patient Abs alter M3R activity. We found that Ab inhibition of carbachol-mediated M3R activity was reversible at high agonist concentrations, consistent with competitive displacement of agonist at the carbachol-binding site. These findings contrast previous studies suggesting non-competitive interactions between anti-M3R Abs and receptor agonists, and confirm the carbachol binding site as the target of the functional anti-M3R Abs in SS.  The ability of the assay to detect anti-M3R Abs compare favourably with whole-tissue in vitro assays, 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 studies required to establish the role of anti-M3R antibodies in the autonomic dysfunction associated with SS.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/pathogenic-autoantibodies-to-the-anti-muscarinic-type-3-receptor-act-by-competitive-inhibition-of-acetylcholine-mediated-receptor-signalling-in-sjogrens-syndrome/