Putative anti-muscarinic antibodies cannot be detected in patients with primary Sjogren's syndrome using conventional immunological approaches

Abstract

Objective: To determine whether autoantibodies directed against muscarinic M3 receptors are present in the serum of patients with primary Sjogren's syndrome (pSS), and if so whether these autoantibodies inhibit secretion by intact salivary acinar cells.

Methods: IgG was purified by affinity chromatography using protein G from sera collected from 15 patients with pSS. Antibody detection was by Western blotting, whole-cell enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The antisecretory activity of the IgG faction was determined using fura-2 microfluorimetry to measure changes in intracellular Ca(2+) activity ([Ca(2+)](i)) in human and mouse salivary gland acinar cells and in Chinese hamster ovary (CHO) cells transfected with human M3 receptors (CHO-M3).

Results: We found no specific M3 receptor recognition by the IgG fraction obtained from pSS patients using either Western blotting or ELISA or immunoblot techniques in which epitope conformation were preserved. Chronic exposure to pSS IgG had no effect on agonist-evoked Ca(2+) signals measured in human or mouse submandibular acinar cells or in CHO-M3 cells. However, acute application of IgG from Sjogren's syndrome patients produced a rapidly reversible reduction in the agonist-stimulated elevation in [Ca(2+)](i).

Conclusion: These data represent the first demonstration of salivary acinar cell inhibition by pSS IgG; however, this inhibition was found to be reversible. Our data also show that pSS IgG binding to M3R cannot be visualized by conventional immunological approaches.