A Stimulatory Thyrotropin Receptor Antibody (M22) and Thyrotropin Increase Interleukin-6 Expression and Secretion in Graves' Orbital Preadipocyte Fibroblasts

Abstract

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. 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). 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. 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.