title: A stimulatory thyrotropin receptor antibody (M22) and thyrotropin increase interleukin-6 expression and secretion in Graves' orbital preadipocyte fibroblasts.
abstract: BACKGROUND
  Patients with Graves' ophthalmopathy (GO) have circulating autoantibodies directed against the thyrotropin receptor (TSHR) and elevated levels of the proinflammatory cytokine interleukin-6 (IL-6) in both serum and orbital tissues. We hypothesized that these autoantibodies might increase IL-6 expression and secretion in preadipocyte fibroblasts and adipocytes from patients with GO, and thus directly impact the clinical activity of the disease.
  
  
  METHODS
  IL-6 mRNA levels were measured in cultures of GO orbital preadipocytes (n = 3) treated during adipocyte differentiation with a monoclonal stimulatory TSHR antibody (M22; 10 ng/mL), IL-6 (1 ng/mL), or TSH (10 U/L). Additionally, levels of IL-6 protein secretion were assessed after adipocyte differentiation in orbital cultures exposed to TSH or M22 for 24 or 48 hours (n = 8). IL-6 mRNA levels were also measured in orbital adipose tissue specimens from well-characterized GO patients (n = 9) and normal individuals (n = 9).
  
  
  RESULTS
  Treatment of GO orbital preadipocyte cultures with IL-6, TSH, or M22 during adipocyte differentiation resulted in increased IL-6 mRNA levels (3.1-fold, 2.9-fold, and 2.7-fold, respectively; p < 0.05). Treatment of orbital cultures with M22 or TSH after adipocyte differentiation enhanced the release of IL-6 protein into the medium at both 24 and 48 hours for TSH (mean 1.9- and 2.3-fold; p = 0.002 and 0.015, respectively) and at 48 hours for M22 (mean 2.0-fold; p = 0.005). In addition, we found mean IL-6 mRNA levels to be significantly increased in GO orbital adipose tissue specimens (10-fold; p < 0.01), primarily attributable to high levels in three of the four patients with clinical activity scores >or=5.
  
  
  CONCLUSIONS
  Both TSH and M22 increase IL-6 expression in orbital preadipocyte fibroblasts and IL-6 secretion by mature adipocytes. These results suggest that circulating TSHR autoantibodies in GO might play a direct role in the clinical activity of the disease.
authors.affiliation: Division of Pediatric Endocrinology, Mayo Clinic, Rochester, Minnesota 55905, USA.
authors.forename: Seema
authors.initials: S
authors.lastname: Kumar
authors.forename: Reagan
authors.initials: R
authors.lastname: Schiefer
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Coenen
authors.forename: Rebecca S
authors.initials: RS
authors.lastname: Bahn
mesh_terms: D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D052436:Adipose Tissue, White / Q000187:drug effects / Q000378:metabolism; D000328:Adult; D000368:Aged; D000911:Antibodies, Monoclonal / Q000276:immunology; D001323:Autoantibodies / Q000276:immunology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D049970:Graves Ophthalmopathy / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D009915:Orbit; D012333:RNA, Messenger / Q000378:metabolism; D011989:Receptors, Thyrotropin / Q000276:immunology*; D013972:Thyrotropin / Q000494:pharmacology* / Q000502:physiology; D013997:Time Factors; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D015850:Interleukin-6; D012333:RNA, Messenger; D011989:Receptors, Thyrotropin; D013972:Thyrotropin