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.
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.
Chemokines were significantly downregulated in GO orbital tissues compared to non-GO orbital tissues (
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.
Graves' orbitopathy (GO) is an autoreactive inflammatory disease that causes expansion of orbital adipose tissue and edema of extraocular muscles within the orbit [
Chemokines are small proteins that induce chemotactic migration of leukocytes during immune and inflammatory reactions [
In this study, we compared the basal messenger RNA (mRNA) expression levels of chemokines in GO and non-GO orbital tissues. We also investigated changes in the mRNA expression levels of chemokines at different time points during adipogenesis and inflammatory reactions to understand the pathogenesis of GO and suggest therapeutic strategies.
Recombinant human interleukin (IL)-1β and TNF-α were purchased from R&D Systems (Minneapolis, MN, USA). Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F-12), DMEM, and penicillin–streptomycin were purchased from Welgene (Daegu, Korea). Fetal bovine serum (FBS) was purchased from Invitrogen (Carlsbad, CA, USA).
Orbital tissue specimens were collected from patients with severe, inactive GO during orbital decompression surgery (n = 6). At the time of surgery, patients with GO were in the euthyroid state and had not received steroids or radiation therapy for at least three months. Patient characteristics are summarized in
Orbital tissues were homogenized using a tissue homogenizer (Precellys 24; Bertin Instruments, Montigny-le-Bretonneux, France) and a Precellys Lysing Kit (Bertin Instruments) with TRIzol (Invitrogen). Total RNA contents were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
For orbital fibroblast analysis, specimens were primarily cultured as previously described [
To study the time course of adipogenesis, cells were cultured and differentiated into adipocytes using the following protocol. The culture medium was changed to serum-free DMEM supplemented wit h 100 µM of isobutylmethylxanthine (Sigma-Aldrich, St. Louis, MO, USA), 10 µM of dexamethasone (Sigma-Aldrich), 1 µM of insulin (Roche Holding, Basel, Switzerland), 33 µM of biotin (Sigma-Aldrich), 17 µM of pantothenic acid (Sigma-Aldrich), 10 µg/mL of transferrin (Sigma-Aldrich), 0.2 µM of T3 (Sigma-Aldrich), 0.2 µM of carba-prostaglandin (Sigma-Aldrich), and 10 µM of rosiglitazone (Santa Cruz Biotechnology, Dallas, TX, USA). Differentiation was continued for 10 days, during which the medium was replaced every two or three days. Chemokine mRNA levels were analyzed at one, three, five, seven, and 10 days of adipogenesis, and differentiating medium was added or replaced at the same time. To verify adipogenesis, cells were stained with oil red O on days 0 and 10. Six milliliters of a stock solution prepared with 0.5% oil red O in isopropanol was mixed with 4 mL of distilled water and left at room temperature for one hour. The solution was then filtered and added to cells that had been washed with phosphate-buffered saline and fixed with 3.7% formalin for one hour at 4℃. The cell-oil red O solution mixture was left for one hour at room temperature, inspected using a light microscope (Axiovert; Carl Zeiss AG, Oberkochen, Germany), and photographed (Olympus BX60; Olympus Corp., Melville, NY, USA) (×40).
Total RNA was isolated using TRIzol. One microgram of RNA was used for first-strand complementary DNA synthesis according to the manufacturer's instructions (SensiFAST cDNA Synthesis Kit; Meridian Life Science, Memphis, TN, USA). mRNA levels were measured by real-time polymerase chain reaction using the LightCycler 480 System (Roche Holding) with SYBR Green PCR Master Mix (Takara Bio, Shiga, Japan) and Realtime PCR Master Mix (TOYOBO, Osaka, Japan). The TaqMan Gene Expression Assay (Applied Biosystems, Foster City, CA, USA) was used for CXCL16 (assay identification no. Hs00222859_m1).
All PCRs were performed in triplicate, and the housekeeping gene
IBM SPSS Statistics ver. 20.0 (IBM Corp., Armonk, NY, USA) was used for statistical analyses. All experiments were performed at least three times using samples from different patients, with results expressed as mean ± standard deviation (SD). Comparisons of data between groups or within cell groups were analyzed by
We compared the basal mRNA expression levels of chemokines and their receptors in GO (n = 6) and non-GO (n = 5) orbital tissues.
We induced adipogenesis in confluent orbital fibroblasts from patients with GO (n = 5) for 10 days and evaluated mRNA expression levels of chemokines and their receptors over time. Of the six GO samples, five orbital cell cultures were further analyzed due to contamination of one cell culture. Increased intracytoplasmic lipid droplets were observed over time during adipogenesis. Differentiated cells at day 10 showed a significant level of staining of oil red O in all five GO samples (
We treated primary cultured orbital fibroblasts from patients with GO (n = 5) with the proinflammatory cytokines IL-1β and TNF-α. The mRNA expression levels of
Responses of chemokine receptors during inflammatory reactions were similar to those of chemokines. Further, the expression levels of
The important roles of chemokines in immune and inflammatory reactions via induction of chemotactic migration of leukocytes suggest that they are involved in the pathogenesis of autoimmune diseases [
To our surprise, we found that mRNA levels of chemokines including
Previous studies have reported that, unlike IL-4, IL-5, IL-10, and interferon-γ, the proinflammatory cytokines TNF-α, IL-1β, and IL-6 are mainly detected in early active GO, indicating the predominance of a Th1-like immune response [
However, there is a limitation because this study was performed only with fibroblasts and did not include lymphocytes attracted by chemokines. Also, the adipogenesis condition in vitro could be quite different from that in vivo; therefore, it remains difficult to determine the exact point of chemokine upregulation during adipogenesis in a clinical environment. The use of orbital tissues harvested during orbital fracture repair or evisceration might not be a perfect control to use in comparison with GO tissues. In vivo animal studies might be required to investigate a causal relationship between adipogenesis and chemokines and to support the development of a new therapeutic drug targeting chemokines.
In conclusion, we investigated the strong upregulation of chemokines at the initial phase of adipogenesis and subsequent downregulation until the end of differentiation. Although the in vitro pathophysiology of adipogenesis cannot reflect precise orbital fat volume increase in GO patients, we carefully speculate that anti-inflammatory therapy is expected to be more effective in the early active stage of GO, when chemokines are upregulated. We also demonstrated that chemokines and their receptors are upregulated after stimulation by IL-1β and TNF–α, suggesting a crucial role of proinflammatory cytokines in the pathogenesis of GO and further indicating that chemokines could be used as biomarkers of GO activity.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2017R1A2B4009565).
Supplemental Fig. 1. is available from:
Oil red O staining in cells with adipogenesis. Adipogenesis was induced in Graves' orbitopathy (GO) orbital fibroblasts (n = 5) for 10 days. At days 0 and 10 of differentiation, cells were stained with oil red O solution mixture and photographed using a light microscope (Axiovert; Carl Zeiss AG, Oberkochen, Germany) (×40).
CAS = clinical activity score; GO = Graves' orbitopathy; R = right eye; L = left eye; M = male; F = female; NA = not applicable.