Arthritis & Rheumatism, Volume 60,
October 2009 Abstract Supplement
The 2009 ACR/ARHP Annual Scientific Meeting
Philadelphia October 16-21, 2009.
New Epitopes and Functions of Anti-M3 Muscarinic Acetylcholine Receptor Antibodies in Patients with Sjgrens Syndrome
Tsuboi, Hiroto, Matsumoto, Isao, Wakamatsu, Ei, Nakamura, Yumi, Iizuka, Mana, Hayashi, Taichi, Goto, Daisuke
M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. Data from a recent study have suggested some patients with Sjögren's syndrome (SS) carried inhibitory auto-antibodies (Abs) against M3R. We reported a subgroup of SS patients had anti-M3R Abs recognizing the 2nd extracellular loop of M3R. In functional assays using salivary gland cells, IgG fractions from SS patients (SS-IgG) inhibited the rise in intracellular Ca2+ concentrations ([Ca2+]i) induced by carbachol. The precise epitopes of anti-M3R Abs and the relationship between the functions and epitopes are currently unknown. To clarify the precise B cell epitopes and the functions of anti-M3R Abs in patients with SS, we carried out this study.
1) We synthesized four different peptides encoding the extracellular domains of human M3R such as N terminal region, 1st, 2nd, and 3rd extracellular loops. Abs against those regions of M3R were examined by ELISA in the sera from 42 SS patients and 42 healthy controls (HC). We assessed the correlation between anti-M3R Abs and various clinical features.
2) Human salivary gland (HSG) cells were pre-cultured with IgG fractions (1.0 mg/ml) separated from sera of anti-M3R Abs positive SS, negative SS, and HC for 12 h. After loading with Fluo-3, HSG cells were stimulated with cevimeline hydrochloride (20 mM), and then [Ca2+]i was measured by the fluorescence plate reader.
1) Abs to N terminal region were detected in 42.9% (18/42) of SS patients and 4.8% (2/42) of HC. Abs to 1st extracellular loop were detected in 47.6% (20/42) of SS and 7.1% (3/42) of HC. Abs to 2nd were found in 54.8% (23/42) of SS and 2.4% (1/42) of HC. Abs to 3rd were detected in 45.2% (19/42) of SS and 2.4% (1/42) of HC. The titers of anti-M3R Abs against all extracellular domains were significantly higher in SS patients than HC. Some SS patients carried anti-M3R Abs that recognized several extracellular domains of M3R. The positivity for anti SS-A antibody was significantly higher among anti-M3R Abs positive SS than negative SS (P<0.05). In contrast, there was no difference in other clinical features between anti-M3R Abs positive and negative SS.
2) Anti-M3R Abs to the 2nd extracellular loop positive SS-IgG inhibited the increase of [Ca2+]i induced by cevimeline hydrochloride. Abs to the N terminal positive SS-IgG and Abs to the 1st extracellular loop positive SS-IgG enhanced it, while Abs to the 3rd extracellular loop positive SS-IgG showed no effect on [Ca2+]i as well as anti-M3R Abs negative SS-IgG.
These findings indicated the presence of several B cell epitopes on M3R in SS patients and some SS patients were reactive to several extracellular domains of the M3R. Influence of anti-M3R Abs on the secretion of saliva might differ with these B cell epitopes on M3R.
To cite this abstract, please use the following information:
Tsuboi, Hiroto, Matsumoto, Isao, Wakamatsu, Ei, Nakamura, Yumi, Iizuka, Mana, Hayashi, Taichi, et al; New Epitopes and Functions of Anti-M3 Muscarinic Acetylcholine Receptor Antibodies in Patients with Sjgrens Syndrome [abstract]. Arthritis Rheum 2009;60 Suppl 10 :493