A stimulatory thyrotropin receptor antibody (M22) and thyrotropin increase interleukin-6 expression and secretion in graves' orbital preadipocyte fibroblasts

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; 10ng/mL), IL-6 (1ng/mL), or TSH (10U/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≥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.