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Estradiol Affects Epstein–Barr Virus Reactivation-Induced Thyrotropin Receptor Antibody and Immunoglobulin Production in Graves’ Disease Patients and Healthy Controls

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Estradiol Affects Epstein–Barr Virus Reactivation-Induced Thyrotropin Receptor Antibody and Immunoglobulin Production in Graves’ Disease Patients and Healthy Controls

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Epstein-Barr virus (EBV) is a gamma-herpesvirus persisting mainly in human B lymphocytes. EBV reactivation induces host cells to differentiate into plasma cells and is related to autoimmune diseases. Graves' disease, an autoimmune hyperthyroidism, is caused by the thyrotropin receptor antibody (TRAb), which overstimulates thyroid stimulating hormone receptor. The disease occurs predominantly in women, which suggests involvement with estrogen. Graves' disease patients and healthy controls have EBV-infected lymphocytes with TRAb on the surface (TRAb(+)EBV(+) cells) in peripheral blood mononuclear cells (PBMCs). TRAb can be produced by reactivation of EBV in vitro, which is an alternative system of antibody production. In this study, we cultured PBMCs from Graves' disease patients and healthy controls with 0, 1, and 100 nM estradiol, corresponding to control, midluteal, and pregnancy levels, respectively, and analyzed the levels of TRAb, total-IgG, and total-IgM during EBV reactivation. We found that 1 nM estradiol increased TRAb levels and 100 nM estradiol slightly lowered them in both patients and controls. In patients, IgM production at 100 nM estradiol was significantly lower than that at 0 nM estradiol (p = 0.028). Estradiol increased the ratio of IgG production to immunoglobulin G (IgG) and immunoglobulin M (IgM) production (IgG/IgG + IgM), which suggested an increase in class switch recombination in the process of EBV reactivation-induced Ig production. Moreover, TRAb production was stimulated by a midluteal level of estradiol and was suppressed by a pregnancy level of estradiol in controls and patients. These results were consistent with premenstrual worsening and maternity improving of autoimmune diseases, including Graves' disease.
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Estradiol Affects Epstein–Barr Virus
Reactivation-Induced Thyrotropin Receptor Antibody
and Immunoglobulin Production in Graves’
Disease Patients and Healthy Controls
Sayuri Hara,
1
Keiko Nagata,
1
Keisuke Kumata,
1
Michiko Matsushita,
2
Satoshi Kuwamoto,
1
Masako Kato,
1
and Kazuhiko Hayashi
1
Abstract
Epstein–Barr virus (EBV) is a gamma-herpesvirus persisting mainly in human B lymphocytes. EBV re-
activation induces host cells to differentiate into plasma cells and is related to autoimmune diseases. Graves’
disease, an autoimmune hyperthyroidism, is caused by the thyrotropin receptor antibody (TRAb), which
overstimulates thyroid stimulating hormone receptor. The disease occurs predominantly in women, which
suggests involvement with estrogen. Graves’ disease patients and healthy controls have EBV-infected lym-
phocytes with TRAb on the surface (TRAb(+)EBV(+) cells) in peripheral blood mononuclear cells (PBMCs).
TRAb can be produced by reactivation of EBV in vitro, which is an alternative system of antibody production.
In this study, we cultured PBMCs from Graves’ disease patients and healthy controls with 0, 1, and 100 nM
estradiol, corresponding to control, midluteal, and pregnancy levels, respectively, and analyzed the levels of
TRAb, total-IgG, and total-IgM during EBV reactivation. We found that 1 nM estradiol increased TRAb levels
and 100 nM estradiol slightly lowered them in both patients and controls. In patients, IgM production at 100 nM
estradiol was significantly lower than that at 0 nM estradiol ( p=0.028). Estradiol increased the ratio of IgG
production to immunoglobulin G (IgG) and immunoglobulin M (IgM) production (IgG/IgG +IgM), which
suggested an increase in class switch recombination in the process of EBV reactivation-induced Ig production.
Moreover, TRAb production was stimulated by a midluteal level of estradiol and was suppressed by a preg-
nancy level of estradiol in controls and patients. These results were consistent with premenstrual worsening and
maternity improving of autoimmune diseases, including Graves’ disease.
Keywords: estradiol, EBV reactivation, Ig production, Graves’ disease
Introduction
Most autoimmune diseases predominantly occur in
women and are influenced by various factors, includ-
ing reproductive function, genetics (14,16), and sex hor-
mones (5,6,19,20). Estrogen binds to estrogen receptors (ERs),
ERaand ERb, and regulates the menstrual cycle and maternity.
Estrogen triggers distinct signaling pathways (6) and can
stimulate antibody production from B cells (20). Estradiol is the
most potent estrogen. The levels of immunoglobulin G (IgG)
and immunoglobulin M (IgM) in human peripheral blood
mononuclear cells (PBMCs) are increased by 0.1–10 nM es-
tradiol and are sustained at concentrations of estradiol higher
than 10 nM after 7 days in culture (5).
Nagata et al. reported that Graves’ disease patients and
healthy controls have Epstein–Barr virus (EBV)-infected
lymphocytes with thyrotropin receptor antibody (TRAb) on
their surface (TRAb(+)EBV(+) cells) in PBMCs, and these
PBMCs produce TRAb in response to reactivation of EBV
in vitro (11,13). Nagata et al. also reported that there is a
1
Division of Molecular Pathology, Department of Pathology, Faculty of Medicine, Tottori University, Yonago, Japan.
2
Department of Pathobiological Science and Technology, School of Health Science, Tottori University, Yonago, Japan.
ªHara et al., 2018; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative
Commons Attribution Noncommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use,
distribution, and reproduction in any medium, provided the original author(s) and the source are cited.
VIRAL IMMUNOLOGY
Volume 31, Number 7, 2018
Mary Ann Liebert, Inc.
Pp. 1–6
DOI: 10.1089/vim.2018.0032
1
positive relationship between serum levels of TRAb and the
EBV-early antigen antibody, a marker of EBV reactivation
(10). Therefore, EBV reactivation may contribute to auto-
antibody production. Graves’ disease, an autoimmune hy-
perthyroidism, is caused by TRAb, which overstimulates
thyroid stimulating hormone receptor (8).
We examined the effects of estradiol on antibody pro-
duction induced by EBV reactivation in PBMCs of healthy
controls and Graves’ disease patients. We used 0, 1, and
100 nM concentrations of estradiol, corresponding to con-
trol, midluteal phase, and pregnancy levels, respectively.
We discuss the effects of estradiol levels in the menstrual
cycle and pregnancy on EBV reactivation-induced antibody
production.
Materials and Methods
Subjects
PBMC samples were obtained from seven Graves’ dis-
ease patients and eight healthy controls (Table 1). All sub-
jects provided written informed consent, and the study
protocol was approved by the Medical Ethics Committee for
Human Subject Research (No. 707, 707-1-13) at the Faculty
of Medicine, Tottori University, Yonago, Japan. The mean
ages (SD) of healthy controls and Graves’ disease patients
were 29.75 (10.89) years and 40.00 (6.22) years, re-
spectively. All subjects were female. At the time of diag-
nosis, patients exhibited symptoms and had laboratory data
that included at least one of the following: (1) signs of thy-
rotoxicosis such as tachycardia, weight loss, finger tremors,
and sweating; (2) diffuse enlargement of the thyroid gland;
and (3) exophthalmos and/or specific ophthalmopathy. All
patients met the following criteria: (1) elevated serum levels
of free T4 and/or free T3; (2) suppression of serum thyrotropin
(thyroid stimulating hormone: TSH) (<0.1 lU/mL); and (3)
positivity for TRAb or thyroid-stimulating antibody. Five of
the seven patients were receiving treatments with antithyroid
drugs (methylmercaptoimidazole or propylthiouracil). Three
patients were receiving treatments with levothyroxine and No.
11 patient was treated with only levothyroxine. Control sub-
jects were enrolled voluntarily, their thyroid functions were
normal, and they had no family history of thyroid disease.
PBMC preparation
Peripheral blood samples were obtained from Graves’
disease patients and healthy controls. PBMCs were sepa-
rated using a Ficoll-Conray density gradient and stored at
-80C until use.
Estradiol
17b-estradiol (Sigma-Aldrich, Saint Louis, MO) was
dissolved in ethanol to prepare a 1 mg/mL stock solution and
stored at 4C until use. Estradiol was added to medium and
maintained at 0, 1, or 100 nM, corresponding to control,
midluteal, or pregnancy levels, respectively. Except for
doses of estradiol, every culture was performed with the
same medium, and we confirmed that the estradiol con-
centration of the medium was neglectable.
Sampling protocol
PBMCs were cultured at 37C for 2 days in RPMI 1640/
10% FBS with cyclosporin A (0.1 lg/mL) to inhibit T cell
effects. PBMCs were transferred to an incubator at 33Cto
induce EBV reactivation and were regarded as day 0 sam-
ples. On days 0, 5, 10, and 12, half of the culture supernatant
was sampled and replaced by fresh medium with estradiol,
and the estradiol concentration was maintained at 0, 1, or
100 nM.
Measurement of total immunoglobulin
Immunoglobulin (Ig) concentrations in culture supernatant
were measured by ELISA. The total IgG and IgM concen-
trations were measured using a Human IgG/IgM ELISA
Quantitation Set (Bethyl Laboratories, Montgomery, TX).
Table 1. Subject Profiles
No. Sex Age (years) Allergy Treatment
a
Controls 1 F 24
2F 26
3 F 22 Allergic conjunctivitis, allergic rhinitis
4F 27
5 F 24 Allergic rhinitis
6F 22
7 F 52 Bronchial asthma
8 F 41 Atopic dermatitis
Mean 29.75
Patients (Graves’ disease) 9 F 48 Atopic dermatitis MMI 1T, LT4 50 mg
10 F 39
11 F 41 LT4 50 mg
12 F 30 PTU 2T
13 F 36 PTU 1T
14 F 47 MMI 1T, LT4 50 mg
15 F 39 Bronchial asthma, atopic dermatitis PTU 3T
Mean 40
a
Treatment for patients with Graves’ disease.
LT4, levothyroxine; MMI, methylmercaptoimidazole; PTU, propylthiouracil.
2 HARA ET AL.
Determination of TRAb concentration
TRAb concentration in culture fluids was quantified by a
radioreceptor assay according to the manufacturer’s in-
structions (DYNOtest TRAb Human; Yamasa Corporation,
Choshi, Japan).
Statistical analyses
SPSS Statistics 21 (IBM, Armonk, NY) was used for sta-
tistical analyses. A Mann–Whitney Utest was adopted for
comparisons between Graves’ disease patients and healthy
controls. The Wilcoxon rank sum test was used for the ana-
lyses of the effects of estradiol compared with that of baseline
(0 nM estradiol).
Results
Effect of estradiol on EBV reactivation-induced
Ig production
TRAb production. Compared with TRAb production in
the culture at 0 nM estradiol, means of TRAb production
were increased with 1 nM estradiol and decreased with
100 nM in healthy controls and patients, although the dif-
ferences were not statistically significant. There were no
significant differences between healthy controls and patients
in TRAb production at each estradiol concentration (Fig. 1).
IgG production. The concentration of estradiol did not
have a significant effect on IgG production in healthy con-
trols or patients. There were no significant differences in
IgG production at each estradiol concentration between
healthy controls and patients (Fig. 2).
IgM production. In patients, IgM production at 100 nM
estradiol was significantly lower than that at 0nM estradiol
(p=0.028). There were no significant differences in IgM
production at each concentration of estradiol between heal-
thy controls and patients (Fig. 3).
Effect of estradiol on the ratio of IgG to IgG and IgM
Figure 4a and b is a pileup of mean IgG and IgM production
to overview the ratio of IgG production. Estradiol increased
the mean production of IgG in controls (Fig. 4a) and de-
creased the mean production of IgM in patients (Fig. 4b).
To perform statistical analyses, we calculated the ratio of
IgG production to the sum of IgG and IgM production (IgG/
IgG +IgM) as for every sample of controls and patients
(Fig. 4c, d). In patients, we detected the trend of an increase
in the ratio of IgG/IgG +IgM between 0 and 1 nM estradiol
(p=0.063) and between 0 and 100 nM estradiol ( p=0.128)
(Fig. 4d), but there were no significant differences (Fig. 4d).
There were no significant differences in the ratio of IgG/IgG
+IgM between healthy controls and patients at each con-
centration of estradiol (Fig. 5).
Discussion
Similar to previous reports (11–13), we found that EBV
reactivation induced production of IgG, IgM,and TRAb in the
supernatants of PBMCs from Graves’ disease patients and
healthy controls (Figs. 1–3). We observed that 1 nM estradiol
considerably increased TRAb levels and 100 nM estradiol
slightly lowered them in both patients and controls (Fig. 1),
although the differences were not statistically significant.
TRAb production was stimulated by a midluteal level of es-
tradiol and suppressed by a pregnant level of estradiol, but the
difference was not significant probably because there were
only a few subjects and large differences in TRAb production
between individuals. In a study of menstrual cycle, we often
realize the large individual difference probably derived from
the number or function of ERs in each subject, as well as the
cyclicity of estrogen and progesterone secretion (1–3). The
increase in TRAb at midluteal level of estradiol matches
FIG. 1. TRAb production was increased by 1 nM estradiol and decreased by 100 nM estradiol in healthy controls and
patients, but differences were not significant. (a) Mean TRAb production in healthy controls. (b) Mean TRAb production in
patients. TRAb, thyrotropin receptor antibody.
ESTRADIOL AFFECTS IMMUNOGLOBULIN PRODUCTION 3
premenstrual worsening of autoimmune diseases, including
Graves’ disease (7,15,18). On the contrary, TRAb suppression
by a pregnant level of estradiol is consistent with the im-
provement of autoimmune diseases, including Graves’ dis-
ease in maternity (8).
In patients, IgM production at 100 nM estradiol was sig-
nificantly lower than that at 0 nM estradiol ( p=0.028),
which suggested a relative increase in IgG. In particular,
estradiol increased the ratio of IgG/IgG +IgM in both pa-
tients and healthy controls. In patients, the IgG/IgG +IgM
ratio increased between 0 and 1 nM estradiol (p=0.063) and
between 0 and 100 nM estradiol ( p=0.128), but there were
no differences in control patients (Fig. 4).
When persistent EBVs are reactivated, host B cells dif-
ferentiate into plasma cells and produce various isotypes of
Ig, which is the result of class switch recombination (CSR)
during Ig production because naive B cells have IgM on
their surface. CSR replaces the Ig heavy chain and switches
the Ig isotype from IgM to high affinity Ig (9). In EBV
reactivation-induced Ig production, expression of activation-
induced cytidine deaminase (AID) is induced by EBV-LMP1
(12). Therefore, Ig production involves AID expression and
subsequent CSR. An increase in the ratio of IgG/IgG +IgM
may result from increased IgG production due to AID in-
duction and CSR. Estrogen binds to the ER and the estrogen-
ER complex binds to the Ig gene heavy chain locus in
B cells, stimulating antibody expression and CSR (4).
Moreover, the estrogen-ER complex binds to the AID
promoter and increases AID protein production (17). We
observed that estradiol increased the ratio of IgG/IgG +IgM,
FIG. 3. IgM production was decreased by 100 nM estradiol. (a) Mean IgM production in healthy controls. (b) Mean IgM
production in patients. IgM production at 100 nM estradiol was significantly lower than that at 0 nM estradiol ( p=0.028).
IgM, immunoglobulin M.
FIG. 2. The concentration of estradiol did not significantly affect IgG production in healthy controls and patients. (a)
Mean IgG production in healthy controls. (b) Mean IgG production in patients. IgG, immunoglobulin G.
4 HARA ET AL.
which suggests that estradiol increased CSR in the process of
EBV reactivation-induced Ig production.
The ratio of IgG/IgG +IgM in controls was higher than that
in patients at all three concentrations of estradiol. However,
there were no significant differences between patients and
healthy controls at each concentration probably because of a
small sample size and large individual differences in controls.
EBV reactivation-induced Ig production is influenced by how
easily persistent EBV is reactivated. EBV serum antibodies
are high in patients with allergies and autoimmune diseases
(7,15). Therefore, allergen history is important for selecting
control subjects, and examination of how estradiol directly
affects EBV reactivation is needed.
In this study, we examined the tonic effect of estradiol on B
cell in vitro although estradiol may have a tonic and cyclic
effect on B cell in vivo, for instance, estradiol levels fluctuate
during menstrual cycle (1). Therefore, we should investigate
further possibility between estradiol and B cell, including the
estradiol withdrawal test. Estrogen affects various cells
through multiple pathways (2,6). B cells are known to be ER-
positive cells, but the receptor number and function may be
different individually, similar to mammary glandular cells
(1,3). This might be one of the causes of individual difference.
Two of our seven patients (No. 10, 11) were not treated
with antithyroid drugs. The levels of TRAb released in
culture mediums were lower in No. 11 treated with only
FIG. 4. Estradiol increased the ratio of IgG to IgG and IgM (IgG/IgG +IgM) in patients. Pileup production of Ig in
healthy controls (a) and patients (b). The ratio of IgG to IgG and IgM (IgG/IgG +IgM) in healthy controls (c) and patients
(d). In patients, the IgG/IgG +IgM ratio increased between 0 and 1 nM estradiol ( p=0.063) and between 0 and 100 nM
estradiol ( p=0.128).
FIG. 5. The effect of estradiol on the ratio of IgG to IgG and IgM (IgG/IgG +IgM) in healthy controls and patients. There
were no significant differences between healthy controls and patients for each concentration of estradiol.
ESTRADIOL AFFECTS IMMUNOGLOBULIN PRODUCTION 5
levothyroxine than the means of patients in every estradiol
concentration, but in No. 10 without treatment, they were
higher.
Antithyroid drugs are reported to have immunomodula-
tory effects and that is because the patients receiving anti-
thyroid drug show lowered serum TRAb levels (clinical
observation). These are also future tasks.
In conclusion, we examined the effect of estradiol on
EBV reactivation-induced Ig production, an alternative
system of antibody production. Because of a small sample
size and individual difference, including the numbers and
function of ER, we did not observe sufficiently significant
differences. However, we detected a significant decrease in
IgM production at 100 nM estradiol in patients and showed
the trend that estradiol increased the ratio of IgG/IgG +IgM,
which suggests that estradiol increased CSR. Furthermore,
1 nM estradiol (the midluteal level) may have the effect to
increase TRAb production and 100 nM estradiol (the preg-
nancy level) may suppress TRAb production. These results
are consistent with clinical observations of premenstrual
worsening and maternity improving of autoimmune disease,
including Graves’ disease (7,15,18).
Acknowledgments
We are grateful to the laboratory staff of Molecular
Pathology Division, Tottori University. This work was sup-
ported by JSPS KAKENHI Grant No. 17K08694 (K.N.).
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Prof. Kazuhiko Hayashi
Division of Molecular Pathology
Department of Pathology
Faculty of Medicine
Tottori University
86 Nishi-cho
Yonago 683-8503
Japan
E-mail: hayashik@med.tottori-u.ac.jp
6 HARA ET AL.
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... The present results and our previous results showing that high estradiol concentrations decrease TRAb and IgM production are consistent with the clinical observations of improvement of Graves' disease during maternity and exacerbation postpartum. 6,12,13 ...
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Graves’ disease occurs predominantly in women. Epstein-Barr virus (EBV) mainly persists in human B lymphocytes, and its reactivation stimulates antibody production. We previously suggested that the EBV reactivation-induced production of TRAb and IgM at 100 nM estradiol (pregnant level) was lower than that at 0 nM estradiol and that class switch recombination may be increased by estradiol. In this study, we examined the effect of estradiol on EBV reactivation. We identified the expression of EBV-glycoprotein 350/220 (gp350/220) in the late phase of reactivation and plasma cell differentiation of EBV-infected cells using 72A1 antibody and CD138 antibody, respectively. We found the mean ratio of gp 350/220(+) CD138(+) cells at 100 nM estradiol was higher than that at 0 nM estradiol. These results suggested that EBV-infected cells could survive with keeping the ability of antibody production in 100 nM estradiol, which is consistent with the improvement of Graves’ disease during maternity and exacerbation postpartum.
... Thyroid disease was also attributed to the reactivation of Epstein-Barr virus (EBV) in a recent study from Japan. This study had noted that during EBV reactivation, thyrotropin receptor antibodies (TRAbs) are formed, which in turn stimulates the TSH receptors and causes thyroid dysfunction [30]. ...
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Abstract Graves' disease is an autoimmune hyperthyroidism caused by thyrotropin receptor antibodies (TRAbs). Because Epstein-Barr virus (EBV) persists in B cells and is occasionally reactivated, we hypothesized that EBV contributes to TRAbs production in Graves' disease patients by stimulating the TRAbs-producing B cells. In order for EBV to stimulate antibody-producing cells, EBV must be present in those cells but that have not yet been observed. We examined whether EBV-infected (EBV(+)) B cells with TRAbs on their surface (TRAbs(+)) as membrane immunoglobulin were present in peripheral blood of Graves' disease patients. We analyzed cultured or non-cultured peripheral blood mononuclear cells (PBMCs) from 13 patients and 11 healthy controls by flow-cytometry and confocal laser microscopy, and confirmed all cultured PBMCs from 8 patients really had TRAbs(+) EBV(+) double positive cells. We unexpectedly detected TRAbs(+) cells in all healthy controls, and TRAbs(+) EBV(+) double positive cells in all cultured PBMC from eight healthy controls. The frequency of TRAbs(+) cells in cultured PBMCs was significantly higher in patients than in controls (p = 0.021). In this study, we indicated the presence of EBV-infected B lymphocytes with TRAbs on their surface, a possible player of the production of excessive TRAbs, the causative autoantibody for Graves' disease. This is a basic evidence for our hypothesis that EBV contributes to TRAbs production in Graves' disease patients. Our results further suggest that healthy controls have the potential for TRAbs production. This gives us an important insight into the pathogenesis of Graves' disease.
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The immunological targets of estrogen at the molecular, humoral, and cellular level have been well documented, as has estrogen's role in establishing a gender bias in autoimmunity and cancer. During a healthy immune response, activation-induced deaminase (AID) deaminates cytosines at immunoglobulin (Ig) loci, initiating somatic hypermutation (SHM) and class switch recombination (CSR). Protein levels of nuclear AID are tightly controlled, as unregulated expression can lead to alterations in the immune response. Furthermore, hyperactivation of AID outside the immune system leads to oncogenesis. Here, we demonstrate that the estrogen–estrogen receptor complex binds to the AID promoter, enhancing AID messenger RNA expression, leading to a direct increase in AID protein production and alterations in SHM and CSR at the Ig locus. Enhanced translocations of the c-myc oncogene showed that the genotoxicity of estrogen via AID production was not limited to the Ig locus. Outside of the immune system (e.g., breast and ovaries), estrogen induced AID expression by >20-fold. The estrogen response was also partially conserved within the DNA deaminase family (APOBEC3B, -3F, and -3G), and could be inhibited by tamoxifen, an estrogen antagonist. We therefore suggest that estrogen-induced autoimmunity and oncogenesis may be derived through AID-dependent DNA instability.
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Autoimmune diseases are characterized by an exaggerated immune response leading to damage and dysfunction of specific or multiple organs and tissues. Most autoimmune diseases are more prevalent in women than in men. Symptom severity, disease course, response to therapy and overall survival may also differ between males and females with autoimmune diseases. Sex hormones have a crucial role in this sex bias, with estrogens being potent stimulators of autoimmunity and androgens playing a protective role. Accumulating evidence indicates that genetic, epigenetic and environmental factors may also contribute to sex-related differences in risk and clinical course of autoimmune diseases. In this review, we discuss possible mechanisms for sex specific differences in autoimmunity with a special focus on three paradigmatic diseases: systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis.
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Epstein-Barr virus (EBV) is a ubiquitous virus that infects most adults latently. It persists in B lymphocytes and reactivates occasionally. Graves' disease is an autoimmune hyperthyroidism caused by thyrotropin receptor antibodies (TRAbs). We have reported that Graves' disease patients and healthy controls have EBV-infected lymphocytes that have TRAbs on their surface (TRAb(+)EBV(+) cells) in peripheral blood mononuclear cells (PBMCs). EBV reactivation is known to be associated with plasma cell differentiation and antibody production of B cells. In this study, we investigated whether TRAb(+)EBV(+) cells really produce TRAbs or not when persistent EBV is reactivated. We cultured PBMCs from 12 Graves' disease patients and 12 healthy controls for several days with cyclosporine A to expand the EBV-infected cell population, and then compared TRAb levels between EBV reactivation by 33 °C culture and EBV nonreactivation by 37 °C culture of PBMCs. Flow cytometry confirmed that all samples at day 0 (reactivation starting point) contained TRAb(+)EBV(+) cells. During 33 °C culture, EBV-reactivated cells with EBV-gp350/220 expression increased from about 1 to 4%. We quantified TRAb levels in culture fluids by radio-receptor assay, and detected an increased concentration for at least one sampling point at 33 °C (from days 0 to 12) for all patients and healthy controls. TRAb levels were significantly higher in supernatants of 33 °C culture than of 37 °C culture, and also significantly higher in supernatants from patients than those from controls. This study revealed TRAb production from TRAb(+)EBV(+) cells in response to reactivation induction of persistent EBV in different efficiencies between patients and controls.