Thyroid-stimulating hormone receptor (TSHR) plays a central role in regulating thyroid function and is targeted by IgGs in Graves’ disease (GD-IgG). Whether TSHR is involved in the pathogenesis of thyroid-associated ophthalmopathy (TAO), the orbital manifestation of GD, remains uncertain. TSHR signaling overlaps with that of insulin-like grow factor 1 receptor (IGF-1R). GD-IgG can activate fibroblasts derived from donors with GD to synthesize T cell chemoattractants and hyaluronan, actions mediated through IGF-1R. In this study, we compare levels of IGF-1R and TSHR on the surfaces of TAO and control orbital fibroblasts and thyrocytes and explore the physical and functional relationship between the two receptors. TSHR levels are 11-fold higher on thyrocytes than on TAO or control fibroblasts. In contrast, IGF-1R levels are 3-fold higher on TAO vs control fibroblasts. In pull-down studies using fibroblasts, thyrocytes, and thyroid tissue, Abs directed specifically against either IGF-1Rβ or TSHR bring both proteins out of solution. Moreover, IGF-1Rβ and TSHR colocalize to the perinuclear and cytoplasmic compartments in fibroblasts and thyrocytes by confocal microscopy. Examination of orbital tissue from patients with TAO reveals similar colocalization to cell membranes. Treatment of primary thyrocytes with recombinant human TSH results in rapid ERK phosphorylation which can be blocked by an IGF-1R-blocking mAb. Our findings suggest that IGF-1R might mediate some TSH-provoked signaling. Furthermore, they indicate that TSHR levels on orbital fibroblasts are considerably lower than those on thyrocytes and that this receptor associates with IGF-1R in situ and together may comprise a functional complex in thyroid and orbital tissue.

Purpose Fibroblast diversity represents an emerging concept critical to our understanding of tissue inflammation, repair and remodeling. Orbital fibroblasts heterogeneously display Thy-1 and exhibit unique phenotypic attributes that may explain the susceptibility of the human orbit to thyroid-associated ophthalmopathy (TAO). In the current study we investigate the role of CD40 ligation upon macrophage chemoattractant protein -1 (MCP-1), IL-6 and IL-8 expression in fibroblasts from patients with TAO. Methods Human orbital fibroblasts were cultured from tissues obtained with informed consent from patients with TAO and from patients undergoing surgery for other, non-inflammatory conditions. The fibroblasts were then examined by flow cytometry, microscopy, and cytokine assays. Results We report that orbital fibroblasts from patients with TAO express elevated levels of CD40. Surface CD40 can be further up-regulated by interferon.γ (IFN-γ) in both TAO and control fibroblasts. This up-regulation is mediated through Jak2 and can be blocked by dexamethasone and AG490, a powerful and specific inhibitor of the tyrosine kinase. Treatment with CD154, the ligand for CD40, up-regulates the expression of IL-6, IL-8 and MCP-1 in TAO fibroblasts, but fails to do so in control cultures. Thy-1+ fibroblasts displayed higher CD40 levels than do their Thy-1− counterparts and are largely responsible for this cytokine production. IL-1β also induces MCP-1, IL-6 and IL-8 more vigorously in TAO-derived fibroblasts. Conclusion Characterization of orbital fibroblasts and their differential expression of cytokines and receptors should prove invaluable in understanding the site-specific nature of TAO and the development of specific therapies.

Graves disease represents a systemic autoimmune process targeting the thyroid, orbit, and pretibial skin. The thyroid dysfunction is treatable, but no consistently effective medical therapy has yet been described for the orbital manifestations of Graves disease, also known as thyroid-associated ophthalmopathy or thyroid eye disease. Several autoantigens are potentially relevant to the pathogenesis of thyroid eye disease. Activating antibodies generated against the thyrotropin receptor can be detected in a majority of patients, and these drive hyperthyroidism. However, stimulating antibodies against the insulin-like growth factor-1 receptor (IGF-1R) may also play a role in the extra-thyroid manifestations of GD. IGF-1R is over-expressed by orbital fibroblasts derived from patients with TED, while IGF-1R + T and IGF-1R + B cells are considerably more frequent in GD. Actions of several cytokines and the molecular interplay peculiar to the orbit appear to provoke the inflammation, fat expansion, and deposition of excessive extracellular matrix molecules in thyroid eye disease. Based upon these new insights, several therapeutic strategies can now be proposed that, for the first time, might specifically interrupt its pathogenesis.

Graves’ disease (GD) is an autoimmune process involving the thyroid and connective tissues in the orbit and pretibial skin. Activating anti-thyrotropin receptor Abs are responsible for hyperthyroidism in GD. However, neither these autoAbs nor the receptor they are directed against have been convincingly implicated in the connective tissue manifestations. Insulin-like growth factor-1 receptor (IGF-1R)-bearing fibroblasts overpopulate connective tissues in GD and when ligated with IgGs from these patients, express the T cell chemoattractants, IL-16, and RANTES. Disproportionately large fractions of peripheral blood T cells also express IGF-1R in patients with GD and may account, at least in part, for expansion of IGF-1R+ memory T cells. We now report a similarly skewed B cell population exhibiting the IGF-1R+ phenotype from the blood, orbit, and bone marrow of patients with GD. This expression profile exhibits durability in culture and is maintained or increased with CpG activation. Moreover, IGF-1R+ B cells produce pathogenic Abs against the thyrotropin receptor. In lymphocytes from patients with GD, IGF-1 enhanced IgG production (p < 0.05) and increased B cell expansion (p < 0.02) in vitro while those from control donors failed to respond. These findings suggest a potentially important role for IGF-1R display by B lymphocytes in patients with GD in supporting their expansion and abnormal Ig production.