Purpose: Orbital fibroblasts are now recognized as the key effectors in the development of thyroid associated ophthalmopathy (TAO). TAO is clinically apparent in approximately 50% of patients with Graves' hyperthyroidism. High levels of plasma free fatty acids (FFAs) are frequently seen in patients with hyperthyroidism. Palmitate is one of the most abundant FFAs in plasma and aggravates inflammation by promoting secretion of pro-inflammatory cytokines in various cells. In the present study, we characterized orbital fibroblasts from patients with TAO and then examined the effect of palmitate on the production of pro-inflammatory cytokines and hyaluronic acid (HA) in orbital fibroblasts.
Methods: Orbital fat explants were obtained from patients with TAO undergoing orbital decompression surgery (n=5). The fibroblasts were characterized by antibodies specific for fibroblast markers and Thy-1 (cluster differentiation 90, CD90) by immunostaining and flow cytometry. We then investigated the capability of orbital fibroblasts to secrete cytokines and HA in response to interleukin (IL)-1β using an enzyme-linked immunosorbent assay (ELISA). The effect of palmitate on cytokine and HA production in orbital fibroblasts was examined at the protein level by ELISA and at the mRNA level by quantitative real time RT-PCR. The level of phosphorylation of mitogen-activated protein kinase (MAPK)s, including p38 MAPK (p38), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), was measured by immunoblot analysis. We then examined the role of MAPKs on palmitate-induced cytokine production using specific inhibitors to p38, ERK, and JNK, respectively.
Results: The orbital fibroblasts from patients with TAO were Thy-1- positive fibroblasts (>90%) with the ability to secrete IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), and HA in response to IL-1β. Treatment with palmitate induced significant production of IL-6 and MCP-1, but not IL-8 and HA, in orbital fibroblasts. IL-6 and MCP-1 expression by palmitate were differentially regulated by MAPKs. IL-6 expression was mediated by the p38, ERK, JNK pathways, whereas MCP-1 expression was mediated by ERK and JNK, but not by p38, in palmitate-treated orbital fibroblasts.
Conclusions: We show the possible involvement of palmitate in the promotion of inflammation within orbital tissues. This finding may be helpful for understanding the development of TAO in patients with hyperthyroidism.