A novel luminescence-based method for the detection of functionally active antibodies to muscarinic acetylcholine receptors of the M3 type (mAchR3) in patients' sera

Abstract

In different bioassays, functional antibodies reacting with the human muscarinic acetylcholine receptor M3(mAchR3) have been detected in sera from patients with Sjögren's syndrome (SS), and there is strong evidence that those antibodies may have pathogenetic relevance. However, depending on the method of detection, their prevalence varied. Furthermore, those bioassays are difficult to standardize. We report on the development and optimization of a novel test system based on a luminometric method to determine downstream signalling of mAchR3 which produces specific and reproducible results. Chinese hamster ovarian (CHO) cells were transfected with plasmids encoding mAchR3 and a green fluorescence protein (GFP)/aequorin fusion protein. Incubation of cells with carbachol resulted in an increase in intracellular [Ca(2+)], which was detected by measuring light emission with a luminometer, and the effect of incubation with patients' immunoglobulins (Ig) was evaluated. Optimal cell density, Ig preparation and time of incubation with patients' sera were determined. Sera from patients with primary Sjögren's syndrome (pSS; n = 40), systemic sclerosis (SSc; n = 47), myasthenia gravis (MG; n = 133) and 50 blood donors were analysed. Optimal assay conditions were obtained with a cell density of 100 000 cells/ml, isolation of Ig by ammonium sulphate precipitation and short-term incubation. Based on this highly reliable assay, 50% of the pSS patients had antibodies which inhibited carbachol-induced activation of mAchR3; none of the SSc patients, 6% of the patients with MG and 12% of the blood donors had antibodies which reacted with the mAchR3. This method facilitates the determination of functional anti-mAchR3 antibodies in patients' sera, confirmed their high prevalence in pSS patients and may, therefore, help to analyse their pathogenetic and clinical relevance in more detail.

Evaluation of muscarinic acetylcholine receptor type 3 gene polymorphisms in patients with primary biliary cholangitis and primary sclerosing cholangitis

“…Second, two independent studies identified specific autoantibodies against mAChR3 in PBC patients . It might be of interest to add that inhibitory mAChR3 autoantibodies have also been described in patients with Sjögren's syndrome and systemic sclerosis leading to impaired mAChR3‐mediated signaling in vitro . Most important, genetic loss of mAChR3 aggravated bile duct destruction in a well‐established mouse model of chronic biliary inflammation .…”

“…18,19 It might be of interest to add that inhibitory mAChR3 autoantibodies have also CHRM3 polymorphisms in PBC and PSC 327 Hepatology Research 2020; 50: 321-329 been described in patients with Sjögren's syndrome and systemic sclerosis leading to impaired mAChR3mediated signaling in vitro. 20,21 Most important, genetic loss of mAChR3 aggravated bile duct destruction in a well-established mouse model of chronic biliary inflammation. 22 From that, we hypothesize that impaired mAChR3-mediated signaling might affect the so-called biliary bicarbonate umbrella, which has been postulated to protect cholangiocytes from aggressive bile acids in the bile ducts.…”

Antibodies to the Muscarinic Acetylcholine Receptor M3 in Primary Biliary Cholangitis Inhibit Receptor Function on Cholangiocytes

“…CHO cells (CHO-K1) stably transfected with a calcium-sensitive bioluminescent fusion protein consisting of aequorin and green fluorescent protein (GFP) were kindly provided by Dr. Stefan Offermanns (Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany) and were cultured as described ( 30 ). These cells are indicated as CHO/G5A.…”

“…We have, therefore, recently established a novel test system for the demonstration of functional anti-mAChR3-antibodies in patients' sera. It is based on the determination of downstream signaling of mAChR3 using Chinese hamster ovarian (CHO) cells transfected with plasmids encoding mAChR3 and a green fluorescence protein (GFP)/aequorin fusion protein ( 30 ). Thus, activation of G protein-coupled receptors (GPCR) leads to an opening of Ca 2+ channels in the endoplasmic reticulum; the resulting efflux of Ca 2+ can be visualized with a luminometric assay.…”

Fluorescent Protein–photoprotein Fusions and Their Applications in Calcium Imaging

“…A bioassay was developed to screen for functional antibodies against muscarinic acetylcholine receptor M3 in sera from patients with Sjogren′s syndrome . Rises in Ca 2+ were detected in response to carbachol by a GFP‐Aeq fusion expressed in CHO cells, and antibodies in patients’ sera inhibited the activation of the receptor.…”