Chemokine Expression during Adipogenesis and Inflammation in Orbital Fibroblasts from Patients with Graves' Orbitopathy

Abstract

Graves' orbitopathy (GO) is an autoreactive inflammatory disease that causes expansion of orbital adipose tissue and edema of extraocular muscles within the orbit [1-3]. Generation of antibodies against self-antigens such as thyroid-stimulating hormone receptor and insulin-like growth factor-1 receptor, inflammatory infiltration, and accumula-Purpose: Chemokines are involved in the pathogenesis of various autoimmune diseases, including Graves' orbitopathy (GO), but comprehensive analyses of the dynamics of these cytokines and their receptors in such diseases remain lacking. In this study, we investigated the expressions of chemokines and their receptors during adipogenesis and inflammation in primary cultured orbital fibroblasts from patients with GO. Methods: The messenger RNA (mRNA) expression levels of chemokines were compared between GO (n = 6) and non-GO (n = 5) orbital tissues by real-time polymerase chain reaction. After adipogenesis was induced in primary cultured orbital fibroblasts from patients with GO (n =5) and following stimulation with interleukin (IL)-1β and tumor necrosis factor (TNF)-α, the mRNA expression levels of chemokines and their receptors were analyzed. Results: Chemokines were significantly downregulated in GO orbital tissues compared to non-GO orbital tissues (p < 0.05). Adipogenesis resulted in a strong increase in mRNA expression levels of chemokines and their receptors at an early stage (day 1); however, expression levels started to decrease thereafter and, eventually, decreased to below basal levels at the end of adipogenesis (day 10). Following stimulation with IL-1β and TNF-α, the mRNA expression levels of chemokines and their receptors increased, showing different responses to various proinflammatory cytokines. Conclusions: Chemokines were strongly upregulated in the early phase of adipogenesis before decreasing continuously until the end of adipogenesis. Also, overt mature GO tissues showed reduced mRNA expression of chemokines compared to controls, which might indicate the existence of a shorter window for effective medical inflammatory treatment. The heightened levels of chemokines and their receptors observed after stimulation with IL-1β and TNF-α suggest a crucial role of proinflammatory cytokines in the pathogenesis of GO and, further, support the idea that chemokines could be used as biomarkers of GO activity.

Serum Levels of CXCL-13, RBP-4, and IL-6, and Correlation Analysis of Patients with Graves’ Disease

“…Interleukin-6 (IL-6), a multifunctional cytokine produced by macrophages and B lymphocytes, is involved in the differentiation, expression, growth, and activity of cells in the immune response of the body. It is able to regulate the immune system and participate in the inflammatory response, and is related to a variety of autoimmune diseases [12]. e basal metabolic rate (BMR) even refers to the unit basal metabolism of the human body in an awake and very quiet state, not affected by muscle activity, environmental temperature, food, mental stress, and other factors.…”

Identification of immune associated potential molecular targets in proliferative diabetic retinopathy

“…IFN-γ-expressing Th1 cells and IL-17-expressing Th17 cells could be selectively enriched based on the expression of surface CCR3 + , CCR4 + , CCR6 + [39]. CXCR6 was upregulated in the primary culture of orbital fibroblasts from patients with Graves' orbitopathy, following treatment with pro-inflammatory cytokines IL-1β and TNF-α [40]. Furthermore, CXCR6 expression was highly confined to memory Th1 cells, which can be categorized into activated memory Th1 and Tc1 cells secreting IFN-γ [41].…”

The Role of Adipsin, Complement Factor D, in the Pathogenesis of Graves’ Orbitopathy

“…Tissue explants were minced and placed in DMEM/F12 containing 20% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) according to the previous study methods. 17 As orbital fibroblasts proliferated, the cells were passaged serially with trypsin/ethylenediaminetetraacetic acid (EDTA) and antibiotics and were incubated. Strains between the third and sixth passages were stored in liquid nitrogen and were used for subsequent experiments.…”

“…Orbital pre-adipocyte fibroblasts were differentiated into adipocytes for 14 days using previously described methods. 17 , 18 To assess the effect of silencing adipsin on adipogenesis, cells were transfected with si-adipsin or si-con for 48 hours before 14-day differentiation period. Briefly, cells grown in 6-well plates were stimulated with adipogenic solutions; 10% FBS DMEM, 33 µM biotin, 17 µM pantothenic acid, 10 µg/mL transferrin, 0.2 nM T3, 1 µM insulin (Boehringer-Mannheim, Mannheim, Germany), 0.2 µM carbaprostaglandin (cPGI2; Calbiochem, La Jolla, CA, USA), and 10 µM rosiglitazone (Cayman, Ann Arbor, MI, USA).…”