title: Autoantibodies in primary Sjögren's syndrome patients induce internalization of muscarinic type 3 receptors.
abstract: OBJECTIVE
  Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized by lymphocyte infiltration into the salivary and lachrymal glands, leading to dry mouth and eyes. The presence of functional autoantibodies against muscarinic type 3 receptor (M3R) has been reported in pSS patients. However, the pathological role of anti-M3R autoantibodies in pSS salivary dysfunction remains controversial.
  
  
  METHODS
  Purified IgGs were obtained from normal (control) and primary SS patients' sera (pSS IgG). Internalization of M3R and clathrin was analyzed by biochemical assay and immunofluorescence confocal microscopy using human submandibular gland (hSMG) cells. Cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) was measured by microspectrofluorimetry.
  
  
  RESULTS
  Incubation of hSMG cells with pSS IgG (1mg/ml) significantly decreased M3R expression levels at the membrane. Carbachol-induced [Ca(2+)](i) transients (CICTs) in these cells were also inhibited by pSS IgG. In contrast to pSS IgG, control IgG had no effect on both the M3R expression level and CICTs. We found that binding of pSS IgG to M3R induces phosphorylation of the receptor, and that the pSS IgG-induced M3R internalization is prevented by the lysosomal inhibitor, chloroquine. In addition, pSS IgG decreased membrane clathrin expression, which was inhibited by atropine. Our immunofluorescence study further confirmed that pSS IgG induces a co-localization of M3R with clathrin and subsequent internalization of M3R.
  
  
  CONCLUSIONS
  pSS IgG induces internalization of M3R partly through a clathrin-mediated pathway. The results suggest M3R internalization as a potential mechanism to explain the exocrinopathy seen in pSS patients.
authors.affiliation: Department of Physiology, College of Medicine, Seoul National University and Dental Research Institute, Seoul 110-749, Republic of Korea.
authors.forename: Meihong
authors.initials: M
authors.lastname: Jin
authors.forename: Sung-Min
authors.initials: SM
authors.lastname: Hwang
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Davies
authors.forename: Yonghwan
authors.initials: Y
authors.lastname: Shin
authors.forename: Jun-Seok
authors.initials: JS
authors.lastname: Bae
authors.forename: Jong-Ho
authors.initials: JH
authors.lastname: Lee
authors.forename: Eun Bong
authors.initials: EB
authors.lastname: Lee
authors.forename: Yeong Wook
authors.initials: YW
authors.lastname: Song
authors.forename: Kyungpyo
authors.initials: K
authors.lastname: Park
mesh_terms: D000328:Adult; D000368:Aged; D001323:Autoantibodies / Q000097:blood / Q000276:immunology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002738:Chloroquine / Q000494:pharmacology; D002966:Clathrin / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D008875:Middle Aged; D010766:Phosphorylation / Q000187:drug effects; D043587:Receptor, Muscarinic M3 / Q000235:genetics / Q000378:metabolism*; D012859:Sjogren's Syndrome / Q000097:blood / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D013363:Submandibular Gland / Q000187:drug effects / Q000276:immunology / Q000378:metabolism
chemical_list: D001323:Autoantibodies; D002966:Clathrin; D007074:Immunoglobulin G; D043587:Receptor, Muscarinic M3; D002738:Chloroquine; D002217:Carbachol; D002118:Calcium