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.

FIG. 1.

Relative level of IL-6 mRNA (mean ± standard error of the mean) in cultures of GO orbital preadipocytes (n = 3) treated with IL-6 (1 ng/mL), bovine thyrotropin (10 U/L), or M22 (10 ng/mL) during differentiation compared with parallel untreated cultures (3.1, 2.9, 2.7-fold; *p < 0.05). Data represent mean fold elevation relative to parallel untreated (control) GO cultures. GO, Graves' ophthalmopathy; IL-6, interleukin-6.

FIG. 2.

IL-6 protein secretion in cultures (n = 8) treated for 24 or 48 hours with bovine thyrotropin (10 U/L; 1.8-fold; p = 0.002 and 2.3-fold; p = 0.015, respectively) or M22 (10 ng/mL; 1.4-fold; p = 0.076 and 2.0-fold; *p = 0.005, respectively) after a 10-day period of adipocyte differentiation in the presence of rosiglitazone. Data represent mean fold elevation relative to parallel untreated (control) cultures.

FIG. 3.

Relative expression of IL-6 mRNA (normalized to 18S rRNA) in orbital adipose tissue specimens from patients with GO (n = 9) and normal individuals (n = 9). The mean IL-6 level in the GO specimens was 10-fold above the mean of the normal group (*p < 0.01) and was attributable to high levels in 3/9 GO patients.