Purpose To investigate the characteristics of human orbital fibroblasts (OFs) cultivated from intraconal, nasal and central adipose tissues.
Methods Intraconal adipose tissues were obtained during orbital decompression surgery for severe proptosis in nine patients with Graves’ orbitopathy (GO). Nasal and central adipose tissues were obtained during upper eyelid blepharoplasty in nine patients with no history of GO. Human OFs were separately cultured from GO intraconal, non-GO nasal, non-GO central orbital adipose deposits. Human dermal fibroblasts were also cultured from redundant resected skin tissue obtained during upper eyelid blepharoplasty in normal controls. Expression of insulin-like growth factor 1 (IGF-1) and thyroid-stimulating hormone (TSH) receptors were investigated using real-time quantitative reverse transcription PCR. Protein levels of interleukin-1β (IL-1β)-induced inflammatory cytokines and generated intracellular reactive oxygen species (ROS) were determined.
Results IGF-1 and TSH receptor RNA expressions of GO intraconal OFs and non-GO nasal OFs were higher than non-GO central OFs and dermal fibroblasts. The expression of IL-1β induced the IL-6, IL-8, intercellular adhesion molecule-1 and cyclooxygenase-2 of GO intraconal OFs, and non-GO nasal OFs were higher than non-GO central OFs and dermal fibroblasts. Intracellular ROS generation in GO intraconal OFs and non-GO nasal OFs were higher than in non-GO central OFs and dermal fibroblasts, although the differences were not statistically significant.
Conclusions Non-GO nasal OFs had similar characteristics to GO intraconal OFs. We recommend the use of nasal adipose tissue in order to culture OFs as a normal control involving in vitro experiments.
- Graves’ orbitopathy
- orbital adipose tissue
- orbital fibroblast
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Graves’ disease (GD) is an autoimmune inflammatory disorder characterised by hyperthyroidism, diffuse goitre, dermopathy and Graves’ orbitopathy (GO).1 GO involves localised inflammation in orbital connective tissue, and its incidence among patients with GD is approximately 25%–50%.2 The role of the thyroid-stimulating hormone (TSH) receptor and activating antibodies directed against it in the hyperthyroidism of GD is well-established,3 whereas the role of the TSH receptor pathway in GO is less well-established. Discovery of the TSH receptor expression in retro-orbital tissues was consistent with the possibility that the TSH receptor pathway contributes to the ocular involvement of GD.4 5 TSH receptor activation in orbital fibroblasts (OFs) enhances hyaluronic acid synthesis and adipogenesis.6 Furthermore, the clinical activity and severity of GO has been correlated with serum levels of anti-TSH receptor antibodies.7–9 Besides the TSH receptor, immunoglobulins that activate insulin-like growth factor 1 (IGF-1) receptor signalling have been found in patients with GD,10 and IGF-I synergistically enhances the actions of thyrotropin.11 12 Furthermore, an inhibitor of the IGF-I receptor, teprotumumab, has recently been used as a new therapeutic strategy to attenuate the underlying autoimmune pathogenesis of ophthalmopathy.13
An animal model is usually used to elucidate the pathogenesis and establish a new therapeutic option for human diseases, but no ideal GO animal models are currently available. Although efforts to establish an animal model mimicking GO have been made,14 15 GO studies are still usually being conducted using an in vitro model of OFs. In experimental in vitro GO settings, human GO OFs are primarily cultured from intraconal orbital adipose tissue obtained during orbital decompression surgery to treat the severe proptosis of patients with GO.16–19 However, as orbital decompression surgery is not required by normal controls, nasal and/or central (pre-aponeurotic) fat pads from patients with no history of GO were obtained (as surgical waste) during upper eyelid blepharoplasty; these yielded normal control OFs.18 20 21 Investigators have already recognised that studies using GO OFs and OFs from normal controls have limitations because the tissues were obtained from different anatomical sites. Prior reports on OFs used specific selection criteria for ‘control group’ subjects in efforts to obtain fat very similar to that from patients with GO. Normal orbital adipose tissue was harvested from patients undergoing orbital or eyelid reconstruction.22 To render the anatomical collection sites similar, other groups collected normal control tissues during surgery to treat eye enucleation,23 24 restrictive strabismus23 or blowout fractures. However, it remains ethically difficult to obtain intraconal orbital fat/connective tissue from normal controls.
Therefore, we investigated the characteristics of human OFs cultivated from nasal and central adipose tissues. We explored differences between OFs obtained from patients with GO and normal controls in an in vitro experimental (bench) setting. As usual, intraconal fat was obtained during orbital decompression surgery to treat proptosis in patients with GO. Nasal and central adipose tissues were obtained during upper eyelid blepharoplasty for patients with no history of GO. OFs were separately cultured from GO intraconal, non-GO nasal and non-GO central orbital adipose deposits. We investigated the characteristics of OFs cultivated from these three tissues using analyses of TSH and IGF-1 receptor expression levels.
As previously mentioned, intraconal adipose tissue was obtained during orbital decompression surgery treating severe proptosis in nine patients with GO. All patients with GO were euthyroid status at the time of surgery and had not been treated with steroids or radiation therapy for at least 3 months. To obtain OFs from normal controls, nasal and central fat pads from nine age-matched and sex-matched patients with no history of GO were obtained during upper eyelid blepharoplasty. Upper eyelid skin was obtained during blepharoplasty as surgical waste to culture dermal fibroblasts.
All experiments were independently performed at least three times using at least three cell cultures harvested from each individual. The results are presented as the mean±SD. Between-group differences were assessed using one-way analysis of variance (ANOVA) and the independent-samples t-test. In all analyses, p<0.05 was assumed to indicate statistical significance.
Orbital and dermal fibroblast culture
OF cell culture was performed as previously described.18 20 21 Briefly, adipose tissue explants were minced and placed in plastic culture dishes; we were careful to avoid contamination. Dulbecco’s Modified Eagle Medium (DMEM) (GenDEPOT, Katy, Texas, USA), 20% fetal bovine serum (FBS; GenDEPOT) and penicillin-streptomycin (GenDEPOT) constituted the culture medium. After OFs grew out from the explants, monolayers were subjected to gentle passage via trypsin/EDTA (Lonza) resuspension. The cells were next incubated in DMEM with 10% FBS and 1% penicillin-streptomycin in a humidified 5% CO2 incubator at 37°C. Cells were stored in liquid N2 until needed and used between passage 3 and 6.
Dermal fibroblasts were cultured using a standard protocol.25 Upper eyelid skin was obtained during blepharoplasty. The dermis was separated from the epidermis using sterile forceps and cut into small pieces. Dermal explants were minced and placed in plastic culture dishes containing DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Monolayers were grown in a humidified incubator at 37°C under 5% CO2. The cells were stored in liquid N2 until needed and used between passage 3 and 6.
Quantitative real-time PCR
To compare the mRNA levels of IGF-1 and TSH receptors between GO intraconal OFs, non-GO nasal OFs, non-GO central OFs and dermal fibroblasts, we performed real-time quantitative reverse transcription PCR (qRT-PCR).
Total mRNA was isolated from the OFs of each group using the miScript II RT Kit (Qiagen GmbH, Hilden, Germany). One µg of RNA was reverse-transcribed for 1 hour at 37°C in a reaction volume using the miScript SYBR Green PCR Kit (Qiagen). Next, 500 ng reverse-transcribed complementary DNA was amplified using the miScript Primer/Precursor Assay (Qiagen). PCR amplifications were processed in reaction mixtures containing specific primers and MgCl2 in the presence of DNA polymerase. Second-round PCR amplifications were carried out following the protocol described above. To decrease variations in the amount of starting RNA, amplification of β-actin mRNA was performed as an internal reference for normalisation of other RNA values. The PCR amplification was performed using specific primer pairs as follows:
OFs were prepared with polyethylene for 1 hour at room temperature on coverslips. After drying completely, coverslips were sterilised using ultraviolet light for 4 hours. After the coverslips were well-rinsed with phosphate-buffered saline (PBS), the cells were incubated in 100% methanol (chilled to −20°C) at room temperature for 5 min. The cells were then incubated with PBS containing 0.1% Triton X-100 (PBST) for 10 min to improve penetration of the antibody. After blocking, the samples were incubated with primary antibodies, including anti-TSH receptor antibody (1:100, #ab27974; Abcam, Cambridge, Massachusetts, USA) and anti-GF-1 receptor antibody (1:100, #ab182408; Abcam) in 1% bovine serum albumin (BSA) in PBS containing 0.1% Triton X-100 (PBST) for 1 hour at room temperature, or overnight at 4°C. The solution was then decanted, and the cells were washed three times in PBS, with 5 min for each wash. The cells were then incubated with the secondary antibody in 1% BSA for 1 hour at room temperature in the dark. The secondary antibody solution was decanted, followed by washing with PBS three times, for 5 min each. Nuclear counterstaining was performed using Hoechst 33 258 (2.5 μg/mL, H1399; Thermo Fisher Scientific, Waltham, Massachusetts, USA) in the dark for 15 min at room temperature.
Images were obtained using confocal fluorescence microscopy (LSM710; Carl Zeiss, Oberkochen, Germany). Alexa Fluor-488 antimouse (1:1000, A-11001; Thermo Fisher Scientific) and Alexa Fluor-594 antirabbit (1:1000, A-11012; Thermo Fisher Scientific) antibodies were used as the secondary antibodies.
Western blot analysis
Protein levels of inflammatory cytokines such as interleukin (IL)-6, IL-8, intercellular adhesion molecule (ICAM)-1 and cyclooxygenase-2 (COX-2) without any stimulation and with stimulation with IL-1β were analysed by western blot analysis. OFs were plated onto six-well plates to determine cytokine protein levels. GO intraconal OFs, non-GO nasal OFs, non-GO central OFs and dermal fibroblasts were washed with ice-cold PBS, and whole cell lysates were obtained by incubating on ice for 30 min in cell lysis buffer (1× RIPA Cell Lysis Buffer; Invitrogen, Carlsbad, California, USA) with EDTA; 100 mL contained 150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris-HCl, pH 7.5 and 2 mM EDTA in a sterile solution. Reagents were purchased from GenDEPOT. Lysates were centrifuged at 12 000× g for 10 min and the cell homogenate fractions were stored at −70°C until use.
Protein concentrations were determined by the Bradford assay. Equal amounts of protein (50 µg) were boiled in sample buffer and resolved by 8% (w/v) SDS-polyacrylamide gel electrophoresis. Proteins were transferred onto polyvinylidene difluoride membranes (Immobilon; Millipore, Billerica, Massachusetts, USA). The samples were probed overnight with primary antibodies against IL-6, IL-8, ICAM-1 or COX-2 in Tris-buffered saline containing Tween 20 (TBST), and washed three times with TBST. Immunoreactive bands were detected with horseradish peroxidase-conjugated secondary antibody and developed using an enhanced chemiluminescence kit (GenDEPOT) and exposed to X-ray film (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA). The immunoreactive bands were quantified by densitometry.
IL-1β-induced inflammatory cytokine (IL-6, IL-8, ICAM-1 and COX-2) release was measured in GO intraconal OFs, non-GO nasal OFs, non-GO central OFs and dermal fibroblasts. OFs and dermal fibroblasts were stimulated in DMEM with IL-1β (5 ng/mL for 16 hours).
Enzyme-linked immunosorbent assay
IL-1β-induced inflammatory cytokine (IL-6, IL-8) release was measured using ELISA kit (Abcam) according to the manufacturer’s protocol. We assayed cytokine production by GO intraconal, non-GO nasal and non-GO central OFs, as well as dermal fibroblasts. OFs were plated into six-well plates and allowed to adhere overnight. Six-well plates were replated in poly-L-lysin coated for all experiments. The cells were then starved for 6 hours prior to stimulation with 5 ng/mL IL-1β. Supernatants were removed 16 hours later and IL-6 and IL-8 levels were determined via ELISA.
Intracellular reactive oxygen species measurements
Reactive oxygen species (ROS) production was determined using 5-(6)-carboxy-2’,7’-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Invitrogen, Eugene, Oregon, USA), as an oxidant-sensitive fluorescent probe as previously described.18 26 CM-H2DCFDA is deacetylated intracellularly by esterase, forming H2DCF, which is oxidised by ROS to 2’,7’-dichlorodihydrofluorescein diacetate, a highly fluorescent compound.
GO intraconal OFs, non-GO nasal OFs, non-GO central OFs and dermal fibroblasts were seeded in six-well plates to a total final volume of 2 mL. To determine the ROS production stimulated by H2O2, the cells were incubated with 10 µM H2DCFDA at 37°C for 30 min, and then stimulated with 10 µM H2O2 for 30 min. The cells were then trypsinised, washed and resuspended in PBS, and flow cytometric analysis was performed (Beckman Coulter Epics XL Flow Cytometer, Beckman Coulter, Pasadena, California, USA).
Demographic data are shown in table 1. The mean age of all patients with GO was 53.8±13.8 years, and seven (77.8%) were female. The mean age of control group was 59.8±16.3 years, and six (66.7%) were female. Smoker and ex-smokers numbered three in both group.
IGF-1 and TSH receptor expression
To compare the mRNA expression levels of TSH and IGF-1 receptors between GO intraconal OFs, non-GO OFs from the nasal fat pad, non-GO OFs from the central fat pad and dermal fibroblasts, we performed qRT-PCR. The IGF-1 and TSH receptor RNA expressions of GO intraconal OFs were higher than those from the central adipose tissue and dermal fibroblasts (p=0.04 and p=0.02, respectively). IGF-1 and TSH receptor RNA expressions of GO intraconal OFs were similar to those of non-GO OFs from nasal fat tissue (p=0.34 and p=0.96, respectively) (figures 1 and 2).
Confocal microscopy images showed the expressions of IGF-1 receptor (red), TSH receptor (green) and live cell nuclei (Hoechst; blue) in OFs and dermal fibroblasts (figure 3). Figure 3A,C show the presence of IGF-1 and TSH receptors in primary cultured OFs from intraconal fat from patients with GO and OFs from nasal fat pads from normal controls. However, immunostaining of IGF-1 and TSH receptors was barely found in primary cultured OFs from the central fat pad and dermal fibroblasts from normal controls (figure 3B,D).
Western blot analysis
Pro-inflammatory cytokines including IL-6, IL-8, ICAM-1 and COX-2 were measured and compared with each of the following four groups: GO intraconal OFs, non-GO nasal OFs, non-GO central OFs and dermal fibroblasts. Protein levels were evaluated in both conditions: with and without stimulation by IL-1β.
Figure 4 shows pro-inflammatory cytokine protein levels without IL-1β stimulation. The protein expressions of IL-6 and IL-8 in GO intraconal OFs and non-GO OFs from nasal fat pads were higher than non-GO OFs from the central fat pad and non-GO dermal fibroblasts. The levels of IL-6, IL-8 and ICAM-1 were barely detectable in dermal fibroblasts. COX-2 was found in all OF and dermal fibroblasts.
Figure 5 shows pro-inflammatory cytokine protein levels after IL-1β stimulation. After IL-1β stimulation, the IL-1β-induced increase of protein expressions of IL-6, IL-8, ICAM-1 and COX-2 in GO intraconal OFs and non-GO OFs from nasal fat tissue were higher than those from central fat and dermal fibroblasts. After stimulation, non-GO nasal OFs had more similar characteristics to GO intraconal OFs.
Enzyme-linked immunosorbent assay
IL-1β-induced inflammatory cytokine (IL-6, IL-8) release from GO intraconal, non-GO nasal and non-GO central OFs, as well as dermal fibroblasts, were measured via ELISAs. The levels of IL-6 and IL-8 induced by IL-1β in GO intraconal and non-GO nasal OFs were greater than those in non-GO central or dermal fibroblasts (figure 6). Compared with GO intraconal OFs, cells derived from non-GO nasal adipose tissue exhibited somewhat lower levels of IL-6 and IL-8 induction by IL-1β, but the differences were not statistically significant (p=0.06 and 0.35, respectively). Compared with GO intraconal OFs, cells derived from non-GO central adipose tissue exhibited significantly lower levels of IL-6 and IL-8 induction by IL-1β (p<0.001 and p<0.001, respectively). Compared with GO intraconal OFs, cells derived from skin exhibited significantly lower levels of IL-6 and IL-8 induction by IL-1β (p<0.001 and p<0.001, respectively).
Intracellular ROS measurements
Intracellular ROS generation in GO intraconal OFs and non-GO OFs from nasal fat pads were higher than those from central fat pads and dermal fibroblasts, although the differences were not statistically significant (p=0.516, one-way ANOVA) (figure 7).
In the present study, we investigated the characteristics of OFs cultivated from intraconal, nasal and central adipose tissues. We obtained nasal and central fat pads from tissue discarded during upper blepharoplasty; these served as primary cultures of OFs from normal controls. However, it was difficult to obtain intraconal fat from controls; thus, we obtained intraconal adipose tissue during decompression surgery in patients with GO. Accordingly, we grouped orbital fat as GO intraconal, non-GO nasal and non-GO central orbital fat and found that non-GO nasal OFs were similar to GO intraconal OFs.
The upper eyelid fat, separated by the superior oblique muscle tendon, is divided in the nasal and central (preaponeurotic) fat pads. During fetal development, the nasal and central orbital fat tissues develop from different tissue layers. The nasal fat pad is continuous with the intraconal fat and has a similar gross morphological appearance.27 Orbital connective tissue has been shown to originate from neural crest cells,28 whereas central orbital fat grossly resembles adipose tissue from the rest of the body, and is presumed to be derived from the mesoderm.27 We therefore assumed that the reason why OFs from nasal adipose tissue had more similar characteristics to GO intraconal OFs when compared with OFs from the central adipose tissue might be explained by the differences of embryological origin.
In addition to IGF-1 and TSH receptor expressions, with regard to pro-inflammatory cytokine levels and the response of IL-1β stimulation, non-GO nasal OFs had more similar characteristics to GO intraconal OFs. Pro-inflammatory cytokine levels including IL-6, IL-8, ICAM-1 and COX-2 were higher in GO intraconal OFs and non-GO nasal OFs, compared with non-GO central OFs and dermal fibroblasts. These results were consistent with previous experimental results, which showed that IL-6, IL-8, IL-1α and IL-1β protein levels were higher in GO intraconal orbital tissue than in normal control orbital tissue.21 Moreover, we found that the increases in cytokine levels after IL-1β stimulation were higher in GO intraconal and non-GO nasal OFs than non-GO central OFs and dermal fibroblasts. These findings were confirmed by ELISAs of culture cell supernatants. We found that nasal OFs were similar to intraconal OFs even though the first cells were obtained from patients without GO and the second cells from patients with GO. Previous reports exploring the response to IL-1β stimulation of GO and non-GO OFs reported very similar trends.18 20 29 30 We found that dermal fibroblasts differed from GO intraconal OFs, consistent with the findings of previous reports.29 However, we believe that analysis of fibroblasts characteristic of both the intraconal and nasal fat pads of patients might reveal differences associated with fat location.
The colour difference in nasal and central orbital fat is well-known and is used as an important surgical landmark during the upper blepharoplasty procedure. Preaponeurotic orbital fat is more yellow than the nasal fat pad. Sires et al 31 used high-performance liquid chromatography and reported that yellow central fat contains higher quantities of β-carotene and lutein than nasal fat. However, their report did not address the biological and physiological significance of the colour difference between the two fat pads. Carotenoids are antioxidants that play a role in terminating ROS and radical-mediated chain reactions.32 33 In this study, we found that the intracellular ROS generation after H2O2 stimulation in GO intraconal OFs and non-GO nasal OFs was higher than those from central aponeurotic fat tissue and dermal fibroblasts. Although the differences were not statistically significant, we assumed that the anatomical differences between nasal and central fat pads, such as β-carotene and lutein, might lead to the differences in ROS production. Moreover, it has been suggested that β-carotene may enhance immune cell functioning.34 In the present study, the increases of cytokine levels responding to IL-1β stimulation were also higher in non-GO nasal OFs, compared with non-GO central OFs, which may be explained by the higher carotenoid content of the central fat pad.
Hwang et al 35 reported that the levels of IGF-1 and TSH receptors were higher in the eyebrow fat of patients with GO compared with normal controls, supporting the findings of a clinical study that suggested that brow enlargement might be a clinical sign of thyroid-associated ophthalmopathy.36 As no prior clinical study has investigated the lid fat appearance of patients with GO, we believe that clinical or radiological studies on this topic may be of interest to physicians. Indeed, given the radiographic evidence of prominent retro-orbicularis and suborbicularis oculi fat in patients with GO,37 we suggest that the retro-orbital and the peri-orbital fat pads are involved in thyroid-associated orbitopathy.
In conclusion, non-GO nasal OFs had similar characteristics to GO intraconal OFs. These findings may be explained by observations that the nasal adipose tissues embryologically originated from the neural crest and by the presence of a different level of antioxidants. We therefore recommend the selection of nasal adipose tissue to culture OFs as normal controls during in vitro experiments.
JAK and DA contributed equally.
Contributors JAK, DA and SYJ were responsible for the study conception and design, as well as the intellectual content of the paper. YJC and HJS performed experiments. BYK, DA and SYJ revised the article critically for intellectual content. All authors read and approved the final manuscript.
Funding This study was supported by a grant from the National Research Foundation of Korea (NRF-2017R1A1A1A05001051) and the Soonchunhyang University Research Fund.
Competing interests None declared.
Patient consent for publication Obtained.
Ethics approval This study was approved by the Institutional Review Board of the Soonchunhyang University Bucheon Hospital, Soonchunhyang University College of Medicine.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information.