Abstract
Epstein-Barr virus (EBV) is a human herpes virus that latently infects B lymphocytes. When EBV is reactivated, host B cells differentiate into plasma cells and produce IgM-dominant antibodies as well as many progeny virions. The aims of the present study were to confirm the IgM dominance of thyrotropin-receptor antibodies (TRAbs) produced by EBV reactivation and investigate the roles of TRAb-IgM in Graves’ disease. Peripheral blood mononuclear cells (PBMCs) containing TRAb-producing cells were stimulated for EBV reactivation, and TRAb-IgM and TRAb-IgG were measured by ELISA. TRAb-IgM were purified and TSH-binding inhibitory activities were assessed using a radio-receptor assay. Porcine thyroid follicular epithelial cells were cultured with TRAb-IgM and/or complements to measure the intracellular levels of cAMP and the amount of LDH released. TRAb-IgM/TRAb-IgG (the MG ratio) was examined in sequential serum samples of Graves’ disease and compared among groups of thyroid function. The results obtained showed that IgM-dominant TRAb production was induced by EBV reactivation. TRAb-IgM did not inhibit TSH binding to TSH receptors and did not transduce hormone-producing signals. However, it destroyed thyroid follicular epithelial cells with complements. The MG ratio was significantly higher in samples of hyperthyroidism or hypothyroidism than in those with normal function or in healthy controls. A close relationship was observed between TRAb-IgM produced by EBV reactivation and the development and exacerbation of Graves’ disease. The present results provide novel insights for the development of prophylaxis and therapeutics for Graves’ disease.
GRAVES’ DISEASE is an autoimmune disorder that causes hyperthyroidism. The causative autoantibodies, thyrotropin (TSH) receptor antibodies (TRAbs), competitively bind to the N terminus of the TSH receptor (TSHR) and induce the chronic and excessive production of thyroid hormones [1]. Known risk factors for Graves’ disease are genetic susceptibility, sex, and environmental factors. We have been investigating Epstein-Barr virus (EBV) reactivation-induced antibody production as an environmental factor [2-6]. Previous studies reported relationships between EBV and autoimmune diseases [7-12].
EBV is a human herpes virus that latently infects B lymphocytes which differentiate into antibody-producing plasma cells [7]. Herpes viruses occasionally reactivate, shift their gene expression from latent to lytic, and then generate a large number of infectious virions. During the reactivation of persistent herpes viruses, host cells become lytic and their function is modified. We sometimes encounter the reactivation of varicella zoster virus or herpes simplex virus as shingles or oral herpes, respectively. In EBV reactivation, host B cells differentiate into plasma cells and produce immunoglobulin (Ig) in vitro [4, 6]. Previous studies reported the presence of EBV reactivation markers in plasma cells [13, 14]. Ig production via EBV reactivation does not need to pass through bone marrow or germinal centers, which is necessary for regular antibody production. Therefore, it may represent an alternative Ig production system. Previous findings on Ig isotypes in EBV-reactivated culture media indicated that Igs produced by this system were dominant for IgM [6]. Rosén [15], Steinitz [16], and Nakamura [17] reported IgM-dominant Ig production in EBV-infected lymphocytes.
In the present study, we confirmed the IgM dominance of TRAbs produced by EBV reactivation. However, thyroid-stimulating TRAbs are TRAb IgG [1, 18, 19]. Therefore, we herein investigated the roles of TRAb-IgM in Graves’ disease and the results obtained provide novel insights into the pathophysiology of Graves’ disease.
Materials and Methods
Subjects
Seventy-seven patients with Graves’ disease (all females) and 21 healthy controls (9 males and 12 females) participated in the present study. All patients were receiving treatment in Tottori University Hospital and clinics in Yonago, Japan. Since the majority of patients with Graves’ disease are females, our patients were all females. In some experiments, females were selected as the healthy controls, while males and females were both examined in other experiments as a representative of the general population. Several subjects participated in some protocols at different time periods. The mean (SD) ages of patients and healthy controls were 40.7 (8.6) and 29.3 (11.5) years, respectively. All subjects provided written informed consent for participation. The present study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Tottori University. The study protocols were approved by the Medical Ethics Committee for Human Subject Research (No. 707) at the Faculty of Medicine, Tottori University, Yonago, Japan. At the time of their diagnosis, patients had symptoms that included at least one of the following: (1) signs of thyrotoxicosis such as tachycardia, weight loss, finger tremors, and sweating; (2) diffuse enlargement of the thyroid gland; and (3) exophthalmos and/or specific ophthalmopathy. Moreover, all patients met the following criteria: (1) elevated serum levels of free T4 and/or free T3; (2) the suppression of serum TSH (<0.1 μU/mL); and (3) a positive test result for TRAbs or thyroid-stimulating antibody (TSAb). Controls were healthy laboratory staff and students with no remarkable symptoms. We confirmed that all subjects had persistent EBV infection by serum anti-EBV antibodies.
Preparation of PBMCs and induction of EBV reactivation
PBMCs were purified from blood samples by a Ficoll-Conray density gradient and stored at –80°C until analyzed. In total, 1 × 106 of PBMCs were pre-cultured in 48-well culture plates for several days in RPMI1640 with 10% fetal bovine serum and 0.1 μg/mL cyclosporine A at 37°C to inhibit T cell function [20]. We previously confirmed that all pre-cultured 1 × 106 PBMCs contained EBV-infected TRAb-producing cells (TRAb(+) EBV(+) cells) [3, 4, 6]. Therefore, we used 1 × 106 cells to detect TRAb(+) EBV(+) cells. As previously reported, culture plates were incubated at 33°C to induce the reactivation of EBV persisting in B cells [4, 6], because it is a physiological approach for EBV reactivation [21-23] without the use of chemical agents; phorbol ester, calcium ionophores or histone deacetylase [7]. On days 0, 5, 10, and 12 of the culture at 33°C (reactivation), half of the culture medium was sampled and replaced with the same amount of fresh medium. Therefore, for each sample PBMC, TRAb production at each sampling point was obtained by subtracting half of the values of the previous point from those at the sampling point.
Enzyme-linked immunosorbent assay (ELISA) for TRAb isotypes
TRAb-IgG and TRAb-IgM were measured in sera diluted two-fold by assay buffer (50 mM Tris, 0.14 M NaCl, 1% BSA, and 0.05% Tween 20) and in culture media (on days 0, 5, 10, and 12) using a previously described method [5]. In brief, 96-well ELISA plates were coated with goat anti-TSHR IgG against the human TSHR C terminus (Santa-Cruz Biotechnology, Santa Cruz, CA, Catalog #sc-7817, RRID: AB_2208382) (0.1 mg/L 0.05 M carbonate-bicarbonate buffer), followed by full-length recombinant human TSHR (Abnova, Taipei, Taiwan, Catalog # H00007253-G01) (4 ng/mL assay buffer). Samples were then applied to each well, incubated at 37°C for 60 min, and Igs were detected by a HRP-conjugated goat anti-human IgM antibody (Bethyl Laboratories, Montgomery, TX, Catalog #A80-100P, RRID: AB_67082) (1:75,000) or HRP-conjugated goat anti-human IgG-Fc (Bethyl Laboratories, Montgomery, TX, Catalog #A80-104P, RRID: AB_67064) (1:100,000).
Purification of TRAb-IgM
TRAb-IgM were purified from 6 samples of culture medium mixtures (days 5, 10, and 15) of EBV-reactivated PBMCs and 2 serum samples from patients with Graves’ disease (Fig. 1). Fifty microliters of protein G-bound magnetic beads (Dynabeads Protein G; Invitrogen/Thermo Fisher Scientific, Waltham, MA, Catalog # DB10003) was placed into each tube and washed by phosphate-buffered saline (PBS). This was followed by the addition of 250 μL of the 500 × diluted anti-TSHR (C terminus) antibody (Abcam, Cambridge, UK, Catalog # ab218329, RRID:AB_2904538), rotation at room temperature for 20 min, and washing with PBS. A total of 250 μL of 25,000 × diluted (2 ng/mL) full-length recombinant human TSHR was then added to each tube, followed by rotation at room temperature for 20 min and washing with PBS. To remove non-specific IgG fractions, each culture medium or 10 × diluted serum sample was passed through an equilibrated protein G affinity column (HiTrap protein G HP column: Cytiva, Uppsala, Sweden, Catalog # 17040401) at 0.2 mL/min. Eluted samples were added to TSHR-bound magnetic beads, rotated at room temperature for 20 min, washed with PBS, and then suspended in 100 μL of PBS. We obtained magnetic beads combined to IgM (non-IgG antibodies), which bound to TSHR, and utilized them as TRAb-IgM in subsequent experiments.
Porcine thyroid follicular epithelial cells have TSHRs that react to both human TSH and human TRAbs [24-26]. cAMP produced from porcine thyroid cells was measured using a TSAb bioassay kit (TSAb kit YAMASA EIA; Yamasa Corporation, Chiba, Japan, Catalog #80104, RRID: AB_2784529) [27, 28] to evaluate the thyroid-stimulating effects of TRAb-IgM samples (5 μL/well) after a culture for 4 hours. A total of 2.5 ng/mL of human TRAb-IgG (M22) (RSR Limited, Cardiff, UK, Catalog #M22/FD/0.004, RRID: AB_2892140) was used as the positive control [29]. All samples were pre-absorbed by dextran coated charcoal to avoid the interference of endogenous cAMP. We utilized porcine thyroid follicular epithelial cells, which were included in the TSAb bioassay kit, in experiments involving thyroid cells. They were cultured in serum-free medium provided in the kit as per the instructions.
TSH-binding inhibitory activity
The TSH-binding inhibitory activity of TRAb-IgM was measured using a commercial radio-receptor assay kit (DYNOtest TRAb Human kit YAMASA: Yamasa Corporation, Chiba, Japan, Catalog # 7773) (Fig. 2a, b) with the following minor modifications. Following the addition of 100 μL/tube of TRAb-IgM samples and standards to TSHR-precoated test tubes, sufficient, gentle, and continuous shaking of the whole tube-rack was performed by hand at room temperature for 45 min. Two hundred microliters of 125I-labeled TSH was then added without washing because of concerns regarding the cessation of binding by washing. Tubes were incubated at room temperature for 30 min with moving by a shaker. The sufficient washing of tubes with washing solution was performed 3 times, and gamma rays were then counted for 1 min.
Human complements
To remove a non-specific IgG fraction, 1 mL of TRAb-negative serum was 2-fold diluted by PBS, and passed through three equilibrated protein G affinity columns at 0.2 mL/min twice per column [30]. The elution was 100K-ultra filtrated at 800 × g for 45 min three times using a spin column (Microsep 100K: Pall Corporation, Port Washington, NY, Catalog # MCP100C41). The retentate of the first 100K-ultra filtration was 300K-ultra filtrated at 800 × g for 45 min (Nanosep 300K: Pall Corporation, Port Washington, NY, Catalog # OD300C34). The 300K retentate contained a complement fraction with a relatively large molecular weight. Therefore, we added 1/4 of the 300K retentate to the 100K filtrate and utilized it as human complements.
Evaluation of cytotoxicity
Porcine thyroid follicular epithelial cells (4-fold dilution of the cell suspension for the cAMP assay) were cultured with TRAb-IgM (10 μL/well) and/or human complements (50 μL/well) for 4 hours. The release of leucine dehydrogenase (LDH) into culture media was then measured based on the formation of formazan using a commercial kit (cytotoxicity LDH assay kit-WST: Dojindo Molecular Technologies Inc., MD, USA, Catalog # CK-12) according to the manufacturer’s instructions to assess cytotoxicity (%) as follows.
[(experimental value of LDH) – (spontaneously released LDH)] × 100 / [(maximum release of LDH) – (spontaneously released LDH)]. The cytotoxicity of 2.5 ng/mL of human TRAb-IgG (M22) (RSR Limited, Cardiff, UK, Catalog #M22/FD/0.004, RRID: AB_2892140) [29] was measured for comparison.
Statistical analysis
Statistical analyses were performed with IBM SPSS Statistics 27 (IBM, Armonk, NY). The Wilcoxon test was used for comparisons between the quantities of TRAb-IgG and TRAb-IgM as well as LDH levels between two culture conditions. The Mann-Whitney test was employed for comparisons of the serum MG ratio among the groups with different clinical conditions.
Results
EBV reactivation induced IgM-dominant TRAb production
Total TRAb production throughout the EBV-reactivation period was assessed by the sum of TRAb production after the beginning of EBV reactivation (days 5, 10, and 12) in each subject. The sums of TRAb-IgM production were significantly higher than those of TRAb-IgG in patients (p < 0.001) and healthy controls (p = 0.010) (Fig. 3).
The signal for thyroid hormone production was not transduced by TRAb-IgM
We purified TRAb-IgM from the culture media of EBV-reactivated PBMCs and the sera of patients as fractions other than IgG, which bound to TSHR. Then we measured cAMP as one of thyroid hormone-producing signals, by ELISA. TRAb-IgM did not increase intracellular cAMP levels in porcine thyroid follicular epithelial cells, whereas TRAb-IgG (M22), which was used as the control, markedly increased its levels (Fig. 4).
TRAb-IgM did not occupy the TSH-binding site of TSHR
The TSH-binding inhibitory activities of TRAb-IgM were assessed using a radio-receptor assay kit, and all TRAb-IgM samples showed gamma ray counts that were approximately two-fold higher than that of the 0 standard (Fig. 5). The radio-receptor assay detects gamma rays emitted by 125I-TSHs bound to the remainder of tube-coated TSHRs partially occupied by TRAbs in samples (Fig. 2a, b). Therefore, the 0 standard, which has no TRAb, shows the highest gamma ray count. However, in the present study, the gamma ray count of TRAb-IgM was approximately two-fold higher than that of the 0 standard. Therefore, we were unable to assess the inhibitory effects of TRAb-IgMs on TSH binding. However, these results indicate that two molecules of 125I-TSH bound to one TRAb-IgM binding complex because we purified TRAb-IgM together with TSHR (Fig. 2c). That is to say, TRAb-IgM bound to all of the pre-coated TSHRs and did not inhibit TSH binding to TSHR.
Cytotoxicity caused by TRAb-IgM
Porcine thyroid follicular epithelial cells were cultured with TRAb-IgM and/or human complements to investigate the cytotoxicity of TRAb-IgM through complement activation. The LDH cytotoxicity assay showed that TRAb-IgM or human complements themselves did not induce the release of LDH, whereas TRAb-IgM with human complements caused the release of a significant amount of LDH in all samples (all TRAb-IgM: p = 0.012, TRAb-IgM from medium: p = 0.028). In contrast, human TRAb-IgG (M22) did not induce the release of LDH even with human complements (Fig. 6).
Serum TRAb-IgM/TRAb-IgG (the MG ratio) increased in hyperthyroidism and hypothyroidism.
Serum levels of TRAb-IgM/TRAb-IgG (the MG ratio) were calculated in 50 serum samples from 14 patients with Graves’ disease and 10 samples from 10 healthy subjects. Each patient provided several sequential samples under the following conditions; 7 samples were from patients with hyperthyroidism, 8 from hypothyroidism, and 35 from euthyroidism.
No significant differences were observed in the MG ratio between the euthyroidism group and healthy controls. The MG ratio was significantly higher in the hyperthyroidism group than in the euthyroidism group (p < 0.001) and healthy controls (p < 0.001), and was significantly higher in the hypothyroidism group than in the euthyroidism group (p < 0.001) and healthy controls (p = 0.001) (Fig. 7).
Discussion
EBV latently infects peripheral B cells, 75% of which are naive B cells with IgM on their surface [31]. EBV reactivation induces IgM-dominant antibody production; however, activation-induced cytidine deaminase (AID), which catalyzes class-switch recombination, has been detected in EBV-infected cells [6]. Heath et al. previously analyzed the Ig genes of EBV-transformed lymphoblastoid cells and detected the expression of AID, but not class-switch recombination [32].
We herein measured TRAb-IgM and TRAb-IgG released into the culture media of EBV-reactivated PBMCs, and confirmed that TRAb induced by EBV reactivation was also dominant for IgM (Fig. 3). The results obtained are the first to demonstrate the possible roles of TRAb-IgM in Graves’ disease, which was cytotoxic to thyroid follicular cells in vitro. In the present study, TRAb-IgM did not increase intracellular cAMP levels, representing a thyroid hormone-producing signal from TSHR (Fig. 4). Furthermore, we expected TRAb-IgM to occupy the TSH-binding sites of the TSHR N terminus; however, it did not inhibit TSH binding. TRAb that binds to the cleavage region of TSHR is known as a neutral antibody [1, 33]. TRAb-IgM may also bind to sites other than the N terminus that are similar to cleavage regions.
Prior to further investigations on the role of TRAb-IgM, we hypothesized that TRAb-IgM destroys thyroid follicular epithelial cells through the activation of complements, and the thyroid antigen released may contribute to thyroid autoimmunity. The system of immune tolerance does not eliminate autoreactive immature B cells, the specific antigens of which are not in the circulation [34]. Therefore, TRAb-producing B cells are also found in healthy controls [3] (Fig. 3). Autoreactive mature naive B cells, including TRAb-producing B cells cannot enter lymphoid tissue and die because their antigens are not in the circulation (Fig. 8). Once TSHR is released, TRAb-producing B cells are exposed to their specific antigen and remain in lymphoid tissue. On the other hand, dendritic cells (DCs) that have digested the released TSHR activate naive T cells into cognate follicular helper T cells (TFH) by presenting the digested TSHR and co-stimulator (B7) [34] (Fig. 8). TRAb-producing B cells then interact with cognate TFH cells and differentiate towards antibody production in a regular system through germinal centers and bone marrow to produce high-affinity TRAb-IgG (Fig. 8).
We purified TRAb-IgM from the culture media of the EBV-reactivated lymphocytes of patients with Graves’ disease as well as their sera. When TRAb-IgMs were added to the culture of porcine thyroid follicular epithelial cells with human complements, LDH release from thyroid cells was observed (Fig. 6), which indicated the destruction of thyroid cells and release of cell components. Necrotic cell components stimulate TLR and induce a co-stimulator on DCs [34, 35]; therefore, the destruction of thyroid cells may induce co-stimulators. This appears to be supported by increased serum levels of TRAb after radioiodine therapy [1, 36]. We intend to identify the thyroid antigens, including TSHR, released into the culture media of thyroid follicular epithelial cells by TRAb-IgM in future studies.
To investigate the potential of the present results to provide insights into the clinical conditions of patients, we calculated the MG ratio in serum samples and found that it was significantly higher in the hyperthyroidism and hypothyroidism groups than in the euthyroidism group and healthy controls (Fig. 7). These results suggest that TRAb-IgM produced through EBV reactivation increases with the exacerbation of Graves’ disease.
Conclusions
In the present study, EBV reactivation induced IgM-dominant TRAb production. TRAb-IgM neither inhibited TSH binding to TSHR nor transduced the receptor signal for hormone production. TRAb-IgM destroyed thyroid follicular epithelial cells through complement activation, which may release thyroid antigens, including TSHR, and contribute to the production of pathogenic high-affinity TRAb-IgG. The MG ratio of serum TRAb increased with the exacerbation of Graves’ disease, and thus, it may be helpful for predicting the course of Graves’ disease.
EBV reactivation is closely associated with the development and exacerbation of Graves’ disease, which may provide insights into the pathophysiology of Graves’ disease and contribute to the development prophylaxis and therapeutics based on the inhibition of EBV reactivation.
Acknowledgments
We are grateful to Takeshi Sairenji, Emeritus Professor of Tottori University for his kind help. We thank Dr. Takehito Kumata and Dr. Hiroki Nagata for technical assistances. We also thank Medical English Service and Mr. Glenn Squires for proofreading the manuscript.
Disclosures
Authors’ contributions
Keiko Nagata: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Visualization; Writing-original draft; Writing-Review and Editing. Kazuhiko Hayashi: Conceptualization; Funding acquisition; Methodology; Project administration; Supervision; Writing-Review and Editing. Keisuke Kumata: Data curation; Formal analysis; Investigation; Methodology; Validation. Yukio Satoh: Methodology; Resources; Validation. Mitsuhiko Osaki: Investigation, Methodology; Resources; Validation. Yuji Nakayama: Resources; Software; Validation. Satoshi Kuwamoto: Conceptualization; Resources; Validation. Yoshinori Ichihara: Resources; Validation. Tsuyoshi Okura: Conceptualization; Resources; Validation. Kazuhiko Matsuzawa: Investigation; Resources; Validation. Junichiro Miake: Resources; Validation. Shuji Fukata: Conceptualization; Investigation; Resources. Takeshi Imamura: Conceptualization; Funding acquisition; Project administration; Supervision; Writing-Review and Editing.
Conflict of interest disclosure
Keiko Nagata, Kazuhiko Hayashi, Keisuke Kumata, Yukio Satoh, Mitsuhiko Osaki, Yuji Nakayama, Satoshi Kuwamoto, Yoshinori Ichihara, Tsuyoshi Okura, Kazuhiko Matsuzawa, Junichiro Miake, Shuji Fukata and Takeshi Imamura declare no conflicts of interest associated with this manuscript.
Funding statement
Keiko Nagata was supported by JSPS KAKENHI Grant Numbers 17K08694 and 20K07374.
Kazuhiko Hayashi, Keisuke Kumata, Yukio Satoh, Mitsuhiko Osaki, Yuji Nakayama, Satoshi Kuwamoto, Yoshinori Ichihara, Tsuyoshi Okura, Kazuhiko Matsuzawa, Junichiro Miake, Shuji Fukata and Takeshi Imamura declare no funding information to declare on the present study.
Data availability statement
Some or all of the datasets generated and/or analyzed during the present study are not publicly available, but are available from the corresponding author upon reasonable request.
Ethics approval statement
This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Tottori University. The study protocols were approved by the Medical Ethics Committee for Human Subject Research (No. 707) at the Faculty of Medicine, Tottori University, Yonago, Japan.
Subjects’ consent statement
All subjects provided written informed consent for participation.
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