Figure 1. bTSH induces IL-6 in orbital fibroblasts and fibrocytes.

(A) Confluent cultures were shifted to medium containing 1% FBS for 20 h and then treated without or with bTSH (5 mIU/mL) for 16 h. Media were collected and subjected to IL-6-specific ELISA. Cell layers were analyzed for protein content. Data are expressed as mean ± SD of three independent determinations (***, p<0.001). In a total of 3 experiments, IL-6 induction by bTSH was 18.1±5.1-fold in fibroblasts and 24.1±4.4-fold in fibrocytes. (B) Cultures were treated without or with bTSH for 6 h. Cellular RNA from three strains each of TAO orbital fibroblasts, healthy orbital fibroblasts, fibrocytes from healthy donors, and those with GD. Real-time RT-PCR was performed using the comparative critical threshold (C_T) method. Ct values were normalized to respective GAPDH levels. Data are expressed as the mean±SD of three independent determinations. (C) IL-6 levels were determined as in panel A following treatment without or with M22 (2 µg/ml) for 16 h. (***, p<0.001). The induction of IL-6 by M22 in 3 experiments was 22.4±9.2-fold in fibroblasts and 47.1±10.1-fold in fibrocytes. (D) IL-6 levels were determined in fibroblasts from two individuals harboring a loss of function TSHR mutation (TSH-resistant) and two unaffected family members (control), treated without or with bTSH for 16 h as in panel A. Data are expressed as mean ± SD fold-change of three independent determinations (**, p<0.01; ***, p<0.001 vs untreated controls). In 3 separate experiments, TSH induced IL-6 by 12.7±1.6-fold and 14.5±1.6-fold in cultures from the healthy donors and 6.4±0.5 and 8.2±0.6-fold respectively in those from the two affected individuals.