The Effects of Royal Jelly on Autoimmunity in Graves' Disease

Abstract

Graves' disease is an organ-specific autoimmune disease with unknown etiology. TSHR Ab plays the most important role for the pathogenesis of Graves' disease. Recently, the role of cytokines for the pathogenesis of Graves' disease has been studied extensively. Royal jelly (RJ) is a creamy product secreted by young nurse worker bees (Apis mellifera), and it is synthesized in the hypopharyngeal and mandibular glands. RJ has been reported to have such pharmacological characteristics as antitumor, antibacterial, antihypercholesterolemic, antiallergic, antiinflammatory, and immunomodulatory properties. The major aim of the present study is to evaluate the effect of RJ on autoimmunity in peripheral lymphocyte culture and to establish the therapeutic doses.
In the first phase, lymphocyte cell isolation from four voluntary healthy subjects was performed to find the effective concentration of RJ on immunity. Serial dilutions of the RJ were prepared (0-5 mg/mL). All isolated lymphocyte cells were treated with the above diluted samples. MTT test was carried out after incubation of 72 h. In the second phase, six patients with Graves' disease, newly diagnosed by clinical and laboratory methods and admitted to my hospital and untreated were identified. RJ samples of 0 and 4 mg/mL were incubated in a culture medium for 72 h with isolated lymphocytes obtained from the patients. After incubation, MTT test in lymphocyte cell culture, Th1 cytokines IFN-gamma, TNF-alpha, and IL-12, and Th2 cytokines IL-4 and IL-10 levels by the enzyme amplified sensitivity immunoassay (EASIA) method and TSHR Ab by the radioreceptor method were determined.
The concentration causing lymphocytes to proliferate was found to be 4 mg/mL by MTT test after incubation of 72 h in cell culture medium. Of the cytokines produced and secreted from lymphocytes, IFN-gamma increased, whereas, other cytokines decreased in RJ concentration of 4 mg/mL. Significant differences were found only for IFN-gamma and TNF-alpha. IL-4 concentrations were kept near the level of significancy. Of Th1/Th2 ratios, IFN-gamma/IL-4 and IFN-gamma/IL-10 ratios also exhibited significant differences between 0 and 4 mg/mL. RJ treatment in lymphocytes from patients with Graves' disease shifted the Th1/Th2 cytokine ratio to the side of Th1 cytokine. Therefore, RJ using the treatment and establishing a remission of Graves' disease may be effective as an antithyroid drug treatment. TSHR Ab levels of lymphocyte cell culture supernatants treated with RJ showed significant decreases. Also, the result may suggest that RJ may exert an effect similar to an antithyroid drug for decreasing TSHR Ab levels.
RJ may be effective as an immunomodulatory agent in Graves' disease.

Full text

Received May 30, 2006; Revised July 21, 2006; Accepted October 1, 2006.
Author to whom all correspondence and reprint requests should be addressed:
Prof. Dr. Cihangir Erem, K.T.Ü. Tip Fakültesi, Iç Hastaliklari Anabilim Dali,
61080, Trabzon, Turkey. E-mail: cihangirerem@hotmail.com or cihangir
erem@netscape.net
The Effects of Royal Jelly on Autoimmunity in Graves’ Disease
Cihangir Erem,
1
Orhan Deger,
2
Ercüment Ovali,
3
and Yasam Barlak
2
1
Department of Internal Medicine Division of Endocrinology and Metabolism,
3
Division of Hematology,
and
2
Department of Biochemistry, Karadeniz Technical University Faculty of Medicine, Trabzon, Turkey
Endocrine, vol. 30, no. 2, 175–183, October 2006 0969–711X/06/30:175–183/$30.00
ENDO (Online) ISSN 1559-0100 © 2006 by Humana Press Inc. All rights of any nature whatsoever reserved.
175
Objective. Graves’ disease is an organ-specific autoim-
mune disease with unknown etiology. TSHR Ab plays
the most important role for the pathogenesis of Graves’
disease. Recently, the role of cytokines for the patho-
genesis of Graves’ disease has been studied extensively.
Royal jelly (RJ) is a creamy product secreted by young
nurse worker bees (Apis mellifera), and it is synthe-
sized in the hypopharyngeal and mandibular glands.
RJ has been reported to have such pharmacological
characteristics as antitumor, antibacterial, antihyper-
cholesterolemic, antiallergic, antiinflammatory, and
immunomodulatory properties. The major aim of the
present study is to evaluate the effect of RJ on autoim-
munity in peripheral lymphocyte culture and to estab-
lish the therapeutic doses.
Research Design and Methods. In the first phase,
lymphocyte cell isolation from four voluntary healthy
subjects was performed to find the effective concen-
tration of RJ on immunity. Serial dilutions of the RJ
were prepared (0–5 mg/mL). All isolated lymphocyte
cells were treated with the above diluted samples.
MTT test was carried out after incubation of 72 h. In
the second phase, six patients with Graves’ disease,
newly diagnosed by clinical and laboratory methods
and admitted to my hospital and untreated were iden-
tified. RJ samples of 0 and 4 mg/mL were incubated in
a culture medium for 72 h with isolated lymphocytes
obtained from the patients. After incubation, MTT test
in lymphocyte cell culture, Th1 cytokines IFN-
γγ
γγ
γ, TNF-
αα
αα
α,
and IL-12, and Th2 cytokines IL-4 and IL-10 levels by
the enzyme amplified sensitivity immunoassay (EASIA)
method and TSHR Ab by the radioreceptor method
were determined.
Results. The concentration causing lymphocytes to
proliferate was found to be 4 mg/mL by MTT test after
incubation of 72 h in cell culture medium. Of the cyto-
kines produced and secreted from lymphocytes, IFN-
γγ
γγ
γ
increased, whereas, other cytokines decreased in RJ con-
centration of 4 mg/mL. Significant differences were
found only for IFN-
γγ
γγ
γ and TNF-
αα
αα
α. IL-4 concentrations
were kept near the level of significancy. Of Th1/Th2
ratios, IFN-
γγ
γγ
γ/IL-4 and IFN-
γγ
γγ
γ/IL-10 ratios also exhib-
ited significant differences between 0 and 4 mg/mL. RJ
treatment in lymphocytes from patients with Graves’
disease shifted the Th1/Th2 cytokine ratio to the side
of Th1 cytokine. Therefore, RJ using the treatment and
establishing a remission of Graves’ disease may be effec-
tive as an antithyroid drug treatment. TSHR Ab levels
of lymphocyte cell culture supernatants treated with RJ
showed significant decreases. Also, the result may sug-
gest that RJ may exert an effect similar to an antithy-
roid drug for decreasing TSHR Ab levels.
Conclusions. RJ may be effective as an immunomod-
ulatory agent in Graves’ disease.
Key Words: Autoimmunity; Graves’ disease; lympho-
cyte cell culture; royal jelly; cytokines.
Introduction
Hyperthyroidism affects approx 2% of women and 0.2%
of men (1). Among autoimmune diseases, Graves’ disease
(GD) is the most frequent cause seen in hyperthyroidism.
GD is an organ-specific autoimmune disease with unknown
etiology (2).
Humoral and cellular immune responses are involved in
the pathogenesis of GD, as demonstrated by the presence
of autoantibodies to the thyrotropin (TSH) receptor (TSHR
Ab) responsible for Graves’ hyperthyroidism, as well as by
the finding of activated T cell and B cell infiltration in thy-
roid tissue from patients with GD (3). TSHR Abs are respon-
sible for hyperthyroidism and goiter by overstimulating the
TSHR.
Cytokines are a group of polypeptides produced mainly
by inflammatory cells, and have a key role in triggering
and coordinating inflammatory and immune reactions (4).
Because they have a pivotal role in the generation and per-
petuation of immune and inflammatory responses, it has
been suggested that the polypeptide mediators may also be
involved in the development and perpetuation of autoim-
mune diseases (5). Mosmann et al. reported that mouse helper
cells could be divided into two subpopulations, Th1 and
Th2 cells, according to differences in their cytokine expres-
sion profiles (6). Th1 cells, predominantly secrete interferon-

Royal Jelly in Graves’ Disease / Erem et al.
176
Endocrine
γ (IFN-γ), produce tumor necrosis alpha (TNF-α), TNF-β,
IL-1, IL-2, IL-8, IL-12, and IL-18, and promote cellular
immune responses. In contrast, Th2 cells produce mainly
IL-4 including IL-3, IL-5, IL-6, IL-10, IL-13, and TGF-β,
and are responsible for B-cell differentiation and antibody
production (2,4,7). IFN-γ is known to induce differentia-
tion of Th0 to Th1 cells and to inhibit the proliferation of
Th cells. On the other hand, IL-4 and IL-10, secreted from
Th2 cells, have been known to induce the differentiation of
Th0 to Th2 cells and to inhibit the function of Th1 cells (8).
Thyroid is a major site of TSHR Ab synthesis. Peripheral
blood and thyroidal lymphocytes obtained from patients
with GD may produce in vitro TSHR Abs. Thionamide
drugs, such as propylthiouracil, carbimazole, and methima-
zole, control the hyperthyroidism of GD primarily by block-
ing iodine organification (9). Thionamide therapy is also
associated with reduction in circulating levels of thyroid
autoantibodies (10,11), including the TSHR Abs that appear
to be responsible for the hyperthyroidism (2). Because meth-
imazole inhibits synthesis of thyroid autoantibodies by lym-
phocytes in vitro (11,12), the fall in titers of thyroid auto-
antibodies in Graves’ patients treated with antithyroid drugs
(ATDs) may be due to the immunosuppressive action of the
drug on thyroid lymphocytes.
Royal jelly (RJ), which is secreted from the hypopharyn-
geal and mandibular glands of worker honeybees (Apis mel-
lifera), is the exclusive principal food source of the queen
honeybee and larvae. It directs the development of honey-
bee larvae into queen bees (13–15). RJ is composed of pro-
teins (12–15%), sugars (10–16%), lipids (3–6%), vitamins,
and free amino acids, and has been used for medical and
nutritional purpose in folk medicine (13). In in vitro and in
vivo studies, RJ has been reported to have such pharmaco-
logical characteristics as antimicrobial and antioxidative
activities (16,17), insulin-like effect (18), antitumor activity
(19), vasodilatotary activity (20), antihypercholesterolemic
(21), antihypertensive (22), antiallergic (23), antifatigue
(24), wound-healing properties (25), and protective activity
against hematopoietic dysfunction in X-irradiated mice
(26) and endogenous sepsis in X-irradiated mice, through
activation of macrophages and hematopoietic stem cells (26,
27). Furthermore, RJ was reported to enhance Th1 responses
in aged mice (28). Sver et al. first reported that RJ exhibited
immunomodulatory properties by stimulating antibody pro-
duction and immunocompetent cell proliferation in mice or
depressing humoral immune functions in rats (14). Oka et
al. studied the immunomodulatory effects of RJ in immu-
nized mice, and they reported that RJ suppressed antigen-
specific IgE production and histamine release from mast
cells in association with the restoration of macrophage func-
tion and improvement of Th1/Th2 cell responses in immu-
nized mice (15). Majtan et al. (29) showed that immuno-
stimulatory effect of RJ on TNF-α release is explained by
a protein named apalbumin-1.
Despite these investigations, which did not completely
establish a biological desirable (protective but not toxic)
activity for RJ, it is certain that the immunopharmacolog-
ical (30) or therapeutic (31) effects can be ascribed to 10-
hydroxy-2-decenoic acid (10-HDA) or to RJ, respectively.
In particular, several substances contained in RJ, including
10-HDA, royalisin, and apisin, have been found to exhibit
these pharmacological activities. In recent studies, RJ has
been confirmed to have antiallergic, anti-inflammatory and
immunumodulatory effects (32–35).
In the literature, there is no experimental and clinical
study regarding to use of RJ in GD. The major aim of the
present study is to evaluate the effect of RJ on autoimmu-
nity in peripheral lymphocyte culture and to establish the
therapeutic doses in patients with GD.
Results
Determination of Effective Concentration
in the Establishment of the Effect (Stimulation,
Inhibition, or Immunomodulation) of Royal Jelly
on Immunity
The results of the MTT test performed in lymphocyte
cell cultures belonging to healthy individuals incubated
with royal jelly in 0–5 mg/mL concentrations prepared as
described previously with the aim of determining the effec-
tive concentration of royal jelly to be used in our experi-
ments are given in Table 1. As shown in Table 1, the 0,
0.025, 0.05, and 1 mg/mL results are close to one another.
There is a decreasing shift in absorbance values of 0.1, 0.25,
and 0.5 mg/mL concentrations according to the lowest
concentration. The maximum absorbances were obtained
between 2 and 5 mg/mL. However, because cell viability
was partially lost at a concentration of 5 mg/mL, it was
Table 1
Findings of the MTT Test Performed
in Lymphocyte Cell Cultures Belonging to Healthy Individuals
Following 72 h Incubation with Royal Jelly (Absorbance)
RJ concentration (mg/mL) Absorbance [n = 4, X (±SD)]*
0 0.182 (0.05)
0.025 0.177 (0.06)
0.050 0.170 (0.04)
0.10 0.164 (0.06)
0.25 0.166 (0.02)
0.50 0.162 (0.02)
1.0 0.188 (0.03)
2.0 0.215 (0.05)
4.0 0.270 (0.02)
5.0 0.300 (0.03)
*Arithmetic mean (±standard deviation).

Royal Jelly in Graves’ Disease / Erem et al.
Vol. 30, No. 2
177
decided to use 0 and 4 mg/mL concentrations in subsequent
experiments.
The Tests in Cell Culture and Its Supernatants
MTT Test
The MTT test results obtained as a result of 72 h of
incubation with 0 and 4 mg/mL of royal jelly in lymphocyte
cell cultures isolated from Graves’ disease patient speci-
mens are given in Table 2. A significant difference at each
concentration was found in the general means given in the
table below and obtained using Friedman test with post hoc
Wilcoxon test. The 4 mg/mL concentration was found to be
significantly different from 0 concentration (Wilcoxon w =
21.0, p = 0.004). It was therefore determined that RJ of 4
mg/mL affected the proliferation ability of lymphocytes. A
microscopic image obtained as a result of 72 h of incuba-
tion of 4 mg/mL of royal jelly with lymphocytes is given in
Fig. 1.
Cytokine Levels
The Th1 marker cytokines (IFN-γ, TNF-α, IL-12) and
Th2 marker cytokines (IL-4, IL-10) levels determined in
supernatants obtained as a result of 72 h lymphocyte incu-
bation with royal jelly are shown in Table 3. The results
obtained and statistical comparisons when the statistics in
Table 3 for each concentration and each patient are com-
bined in groups are given in Table 4. As shown in Table 4,
IFN-γ concentrations showed a tendency to increase and all
the other cytokine concentrations displayed a tendency to
decrease between concentrations of 0 and 4 mg/mL. Among
the cytokines, significant differences were determined in
IFN-γ and TNF-α. IL-4 concentrations decreased and re-
mained at the significance threshold (p = 0.05).
Table 2
MTT Test Findings with Graves’ Disease
(Absorbance × 10
3
) [n = 10, X (±SD)]
Patient Control* 0 4
C.K. 96.5 (10.6) 114.9 (13.6) 174.4 (9.4)
S.T. 119.8 (10.5) 104.1 (9.5) 222.9 (20.2)
S.H. 126.5 (16.6) 121.8 (17.0) 222.8 (10.5)
E.T. 138.2 (10.6) 106 (12.3) 170 (17.5)
S.K. 89.5 (4.1) 72.1 (8.8) 178.9 (11.8)
Y.A. 88.7 (6.6) 81.4 (7.6) 183.2 (15.8)
X (±SD) 109.9 (21.1) 100.1 (19.4) 192.0 (24.3)**
*RPMI was used instead of PBS in control specimens.
**Post-hoc Wilcoxon test by Friedman test (w = 21.0, p = 0.004).
Fig. 1. Microscopic image following 72 h of incubation of lympho-
cytes treated with a 4 mg/mL concentration of royal jelly (×100).
Table 3
Cytokine Levels Obtained from Cell Culture Supernatants [n = 2, X (±SD)]
Th1 cytokines Th2 cytokines
Patient Concentration IFN-γ (IU/mL) TNF-α (pg/mL) IL-12 (pg/mL) IL-4 (pg/mL) IL-10 (pg/mL)
C.K. 0 2.45 (0.44) 40.0 (2.83) 15.8 (0.84) 13.0 (1.02) 10.0 (2.82)
4 2.85 (0.51) 8.84 (2.08) 11.6 (0.36) 0.10 (0.02) 8.5 (1.16)
S.T. 0 2.23 (0.65) 9.4 (0.29) 12.7 (0.64) 28.2 (1.08) 15.4 (1.10)
4 3.62 (0.46) 3.2 (0.18) 22.4 (1.02) 0.12 (0.02) 9.5 (0.84)
S.H. 0 1.81 (0.50) 10.8 (1.20) 22.6 (0.36) 30.4 (0.84) 9.5 (0.80)
4 2.88 (0.35) 0.10 (0.03) 8.9 (0.22) 14.8 (0.32) 3.7 (0.74)
E.T. 0 1.85 (0.34) 31.5 (0.80) 19.3 (0.24) 1.8 (0.16) 12.5 (2.16)
4 2.16 (0.74) 19.4 (0.96) 18.5 (0.21) 0.12 (0.02) 10.4 (1.32)
S.K. 0 0.95 (0.13) 26.9 (1.32) 3.16 (0.24) 11.1 (1.20) 5.9 (0.96)
4 5.14 (0.74) 8.3 (0.26) 2.43 (0.33) 2.9 (0.45) 0.7 (0.19)
Y.A. 0 2.08 (0.55) 32.2 (0.94) 13.9 (0.66) 13.7 (0.82) 10.2 (2.64)
4 3.85 (0.24) 12.4 (0.38) 2.43 (0.20) 5.6 (0.44) 6.1 (0.94)

Royal Jelly in Graves’ Disease / Erem et al.
178
Endocrine
Th1/Th2 cytokine level ratios were also investigated in
our study. These ratios are shown in Table 5. A significant
group difference was determined for IFN-γ/IL-4 and IFN-
γ/IL-10 from the Th1/Th2 ratios.
TSHR Ab Levels
The TSHR Ab levels measured in supernatants obtained
as a result of 72 h of incubation of lymphocyte cell culture
with royal jelly are shown in Table 6. When patient results
were evaluated individually, the lowest Ab levels for each
patient were found with treatment with royal jelly in a 4
mg/mL concentration.
Discussion
It is known that the MTT test is used to define prolifera-
tion ability and number of living cells with continuing
mitochondrial activity (37). We also determined that royal
jelly increased absorbance significantly in all Graves’ dis-
ease patients at a concentration of 4 mg/mL as a result of
72 h of incubation with royal jelly in lymphocyte cell cul-
ture isolated from Graves’ disease patient specimens. To put
it another way, royal jelly at a concentration of 4 mg/mL
increases living lymphocyte numbers and proliferation capa-
bility. The antigenic property of royal jelly and therefore its
increasing lymphocyte proliferation as a foreign protein is
an expected finding. Kamakura et al. reported that a 57-kDa
protein (now called as apalbumin-1) in RJ enhances prolif-
eration of primary cultured rat hepatocytes (38).
In different studies, it was reported that serum IFN-γ
levels in patients with GD increased (39), decreased (5),
and/or did not change (40,41). IFN-γ, IL-2, and TNF-α all
suppressed the production of antithyroid autoantibodies
by thyroid B cells in vitro (42). Although the evidence in
somewhat conflicting, the majority opinion is that GD is
promoted by type 2 cytokines and regulated by type 1 cyto-
kines. It has been reported that serum IFN-γ levels were
increased after ATD or RAI treatments, whereas Th1/Th2
ratios were lower than those of healthy subjects, i.e., were
in favor of Th2 (1,43).
There are a few studies that evaluate the effect of RJ on
IFN-γ. Oka et al. studied the immunomodulatory effects of
RJ (15). They reported that, in immunized mice, IFN-γ
production from T4 cells was suppressed and IL-4 produc-
tion from T4 cells was increased as compared to normal
mice. On the other hand, RJ (1 g/kg, po) improved the bal-
ance of Th1/Th2 cell responses from Th2-dominant to Th1-
dominant (an increase in IFN-γ and a decrease in IL-4).
Taniguchi et al. reported that, in experimental mice model–
induced atopic dermatitis-like skin lesions, oral adminis-
tration of RJ suppresses the development of these skin
lesions in immunized mice (44). They suggested that the
mechanism of this result is a decreased IFN-γ production
by spleen cells and increased inducible nitric oxide (NO)
synthase (iNOS) expression in the dorsal skin lesions of
immunized mice (44). Thus, IFN-γ may play pivotal roles
in the accumulation of inflammatory cells in lesional skin of
chronic atopic dermatitis. Moreover, Okamoto et al. reported
that major royal jelly protein 3 (MRJP3), purified 70-kDa
glycoprotein, markedly inhibited IFN-γ, IL-12, and IL-4
production by stimulated purified splenic T cells (35). IFN-
γ and TNF-α inhibit thyroid follicular cell (TFC) growth
and proliferation (45).
In the present study, IFN-γ levels of cell culture super-
natants obtained after incubation of peripheral blood lym-
phocyte cell culture with RJ of 4 mg/mL for 72 h increased.
The result shows that RJ for GD may change Th1/Th2
ratio in favor of Th1. In view of the above positive effects
(growth and differentiation of supressor T cells, inhibition
of IgE response, and in vitro supression of antithyroidal Ab
production by thyroid B cells), IFN-γ increasing effect of
RJ may be evaluated as a beneficial effect.
Table 4
Group Means of Cytokines on Table 3 [n = 6, X (±SD)]
Parameter 0 4 mg/mL
IFN-γ (IU/mL) 1.90 (0.52) 3.42 (1.04)*
TNF-α (pg/mL) 25.13 (12.39) 8.70 (6.83)**
IL-12 (pg/mL) 14.58 (6.67) 11.04 (8.22)
IL-4 (pg/mL) 16.36 (10.92) 3.89 (5.80)
IL-10 (pg/mL) 10.58 (3.18) 6.49 (3.74)
*Wilcoxon w = 25, p = 0.010.
**w = 25, p = 0.025.
Table 5
Group Means of Th1/Th2 Cytokine Ratios
Obtained from Cell Culture Supernatants [n = 6, X (±SD)]
Parameter 0 4
IFN-γ/IL-4 0.27 (0.38) 13.22 (14.15)*
IFN-γ/IL-10 0.18 (0.04) 1.61 (2.82)**
TNF-α/IL-4 4.34 (6.55) 46.97 (65.48)
TNF-α/IL-10 2.67 (1.56) 2.86 (4.48)
IL-12/IL-4 2.40 (4.09) 76.45 (86.01)
IL-12/IL-10 1.37 (0.65) 1.96 (1.05)
*w = 23.5, p = 0.013; **w = 26.5, p = 0.045.
Table 6
TSHR Ab Levels
Obtained from Cell Culture Supernatants
TSHR Ab (U/L)
Patient 0 4
C.K. 29 16
S.T. 34 19
S.H. 28 14
E.T. 36.5 17.5
S.K. 31 16
Y.A. 33 15.0
X (±SD) 31.9 ± 3.2 16.6 ± 1.8*
*w = 21, p = 0.004.

Royal Jelly in Graves’ Disease / Erem et al.
Vol. 30, No. 2
179
The TNF-α system might be a role in the regulation of
the pituitary–thyroid axis. TNF-α receptors have been dem-
onsrated on human thyroid cells (46).
In vivo production of TNF-α has been demonstrated by
both intrathyroidal lymphocytes and TFCs in patients with
GD (8). In different studies performed in patients with GD,
serum levels of TNF-α have been reported to be elevated
(47–49) or in normal ranges (50). ATDs exert an immuno-
suppressive effect by blocking production of some inflam-
matory mediators such as TNF-α, IL-1, and IL-6 (4,9). Diez
et al. showed that TNF-α concentrations were increased in
relation to controls, and these levels normalized by ATD,
radioactive therapy, or surgical treatment (47). However,
plasma TNF-α has a short half-life and tissue levels of TNF-
α are more closely related to pathophysiological conditions.
There are a few studies that evaluate the effect of RJ
on TNF-α. Kohno et al. have examined the inflammatory
actions of RJ at a cytokine level, and when supernatants of
RJ suspensions were added to a culture of mouse peritoneal
macrophages stimulated with polysaccharide and IFN-γ, the
production of proinflammatory cytokines, such as TNF-α,
IL-6, and IL-1, was efficiently inhibited in a dose-dependent
manner without having cytotoxic effects of macrophages
(32). They have suggested that RJ has anti-inflammatory
actions through inhibiting proinflammatory cytokine pro-
duction by activated macrophages, and it is an effective
dietary supplement for the improvement of quality of life in
the autoimmune diseases. Simuth et al. reported that apalbu-
min-1 (monomeric form, 55 kDa), the most abundant pro-
tein of RJ, and apalbumin-2 (49 kDa), stimulate mouse mac-
rophages to release TNF (33). They suggested that TNF-α
might play a role in cytokine-induced activation of genes
important for immune response of honeybees and humans,
and it could play a pivotal role as the factor participating on
regulation of important cellular processes such as cell pro-
liferation and inflammation.
In our study we found that TNF-α levels we measured
in cell culture supernatants obtained after 72 h of incuba-
tion of peripheral blood lymphocyte cell cultures with RJ
of 4 mg/mL decreased. Reduction in TNF-α in Graves’ dis-
ease patients may show remission of the disease or, as stated
above, a reduction in disease activity. For that reason, this
effect of royal jelly in peripheral blood lymphocyte cell
culture in our patients may be considered one that is thera-
peutic and provides remission.
In various studies, it was reported that serum IL-12 lev-
els in patients with GD increased (51,52) and did not change
(41). Kocjan et al. reported that the mononuclear cell (MNC)
cultures from the peripheral blood of patients with newly
diagnosed GD before treatment produced significantly less
IL-12 and significantly more IL-10 and IL-4 than normal
lymphocytes from healthy donors (40). Also, all calculated
ratios Th1 against Th2 cytokines in MNC cultures from
patients with GD were significantly lower than in MNC cul-
tures from healthy controls. They showed a systemic shift
of cytokine production in patients with GD toward the Th2
cytokine response, thus confirming the key role of TSHR
Abs and humoral immunity in the pathogenesis of GD. Jones
et al. reported that peripheral blood MNC cultures from
patients with GD before treatment with RAI are produced
significantly less IFN-γ and IL-4 compared with healthy
controls (1). IL-12 production was normal. Tamaru et al.
reported that the serum IL-12 levels in GD were signifi-
cantly increased in the hyperthyroid state, and were decreased
during treatment with methimazole or propylthiouracil (in
euthyroid state) (53).
There is an only one study that evaluates the effect of RJ
on IL-12. In the study, Oka et al. reported that RJ suppressed
antigen-specific IgE production and histamine release from
mast cells in association with the restoration of macrophage
function (increased IL-12 p40 mRNA expression and NO
production, and decreased PGE
2
production) and improve-
ment of Th1/Th2 cell responses in immunized mice (15).
But, in this study, IL-12 production in lymphocytes as Th1
cytokine was not investigated.
In our study we found that IL-12 levels decreased as RJ
concentrations increased. But, this decrease was not statis-
tically significant.
In different studies, it was found that serum IL-4 levels in
patients with GD increased (1,39,43). Kocjan et al. reported
that IL-4 levels in the MNC culture supernatants in patients
with newly diagnosed GD were higher than those of the
normal lymphocytes from healthy subjects (40). Mysliwiec
et al. found that IL-4 and IL-12 levels are elevated in patients
with GD, and that there is an increase in the ratios of IL-4/
IFN-γ, IL-4/TNF-α, IL-10/ IFN-γ, and IL-10/TNF-α after
steroid therapy in patients with Graves’ ophthalmopathy.
They suggested that these cytokines may have a role in dis-
ease remission. In that study, the cytokine levels did not
correlate with severity, remission and recurrence of the
disease (54).
The effects of RJ on Th1/Th2 cell responses have first
been investigated by Oka et al. (15). They reported that
IFN-γ production from Th cells in immunized mice in-
creased as compared to normal mice. Kataoka et al. found
that intraperitoneal administration of RJ into immunized
mice resulted in the inhibition of both antigen-specific IgG1
and IgE production, and IL-4, IL-5, and IL-10 production
by antigen-stimulated spleen cells (23). Recently, Okamoto
et al., using a series of column chromatographies, purified
a 70-kDa glycoprotein, MRJP3, that suppresses IL-4 pro-
duction. In this study, MRJP3 suppressed the production of
not only IL-4 but also that of IL-2 and IFN-γ by T cells
concomitant with inhibition of proliferation (35). Interest-
ingly, in spite of the antigenicity, MRJP3 inhibited serum
antigen-specific IgE and IgG1 levels in immunized mice.
In our study we found that IL-4 levels decreased with RJ
of 4 mg/mL. Although not statistically significant, this de-
crease was nevertheless very close to significance thresh-
olds (p = 0.06). This result is probably due to our patient

Royal Jelly in Graves’ Disease / Erem et al.
180
Endocrine
numbers being small (n = 6). Royal jelly suppression of
manufacture of Th2 cytokine IL-4 from lymphocytes may
be regarded as a beneficial and therapeutic effect. That is
because in relapsed Graves’ patients Th2 cytokines such as
IL-6, IL-10, and IL-13 increase in direct relation to disease
activity (55,56).
IL-10 is likely to have a major influence on autoanti-
body production in GD. In various studies, it was reported
that serum IL-10 levels in patients with GD increased
(39,54,57). Kocjan et al. reported that IL-10 levels in the
MNC culture supernatants are elevated in patients with GD
compared with controls. The ratios IFN-γ/IL-10 and IL-12/
IL-10 in MNC cultures from patients with GD were signifi-
cantly lower than in MNC cultures from healthy controls
(40). Takeoka et al. found that serum IL-10 levels were sig-
nificantly higher in patients with seriously intractable GD
than in patients with GD in remission, although serum IL-4
levels did not differ significantly between these two groups
(58). They suggested that IL-10, but not IL-4, may play a
major role in GD intractability. Mysliwiec at al. reported that
serum IL-10 was elevated significantly in patients with GD
in comparison to the control group. In this study, serum IL-
10 levels increased significantly after glucocorticoids (56).
In the literature, there is an only one study that evaluated
the effects of RJ on IL-10 (23). In this study, Kataoka et al.
reported that intraperitoneal administration of RJ into im-
munized mice resulted in the inhibition of IL-10 production
by antigen-stimulated spleen cells (23). But, in this study,
IL-10 production as Th2 cytokine was not investigated.
In our study we found that IL-10 levels decreased as royal
jelly concentrations increased. But, this decrease was not
statistically significant.
As already stated, Th cell cytokine response in Graves’
disease has been found to be different in various studies per-
formed. In this autoimmune disease Th2 cytokine response
generally increases, and Th1 cytokine response decreases,
increases, or remains unchanged (40,41,43). In these patients,
however, the Th1/Th2 ratio may generally be regarded as
decreasing (shifting toward the Th2 cytokine), and follow-
ing ATD therapy this ratio is regarded as either falling (55)
or remaining unchanged (43). Very recently, Kocjan et al.
evaluated the balance shift in Th1/Th2 cytokines in the
PBMC culture supernatants from patients with GD after 1
yr of methimazole treatment, when compared to the same
balance in patients with newly diagnosed GD before treat-
ment and in healthy controls (43). They reported that PBMC
from patients with GD after treatment produced signifi-
cantly more IFN-γ and IL-4 than PBMC from patients with
GD before treatment, but there were no significant differ-
ences in calculated ratios of Th1 against Th2 cytokines
between these two groups. When compared to PBMC from
healthy controls, PBMC from patients with GD after treat-
ment produced significantly more IL-4 and significantly
less IL-2. The calculated IL-12/IL-4 ratio after treatment
was significantly lower than the same ratio from healthy
controls. In conclusion, they reported that there is no sig-
nificant change in the ratio between Th1 and Th2 cytokines
produced by PBMC from patients with GD after 1 yr of
methimazole treatment, when compared to the ratio before
treatment.
In our study we also evaluated the effect of royal jelly on
the Th1/Th2 ratio. As the concentration increased from 0
mg/mL toward 4 mg/mL we observed a change in the Th1/
Th2 ratio in favor of Th1. This change was found to be sta-
tistically significant in IFN-γ/IL-4 and IFN-γ/IL-10 ratios.
This finding with royal jelly is compatible with that in
Graves’ disease patients entering remission based on ATD
therapy.
The stimulatory effect of the Th2 cytokines (especially
IL-4, IL-10, and IL-13) on thyroid B lymphocytes, in such
a way that causes synthesis and secretion of TSHR Ab, is
very important. TSHR Abs are responsible for hyperthy-
roidism and goiter by overstimulating the TFCs (59,60).
There is a positive correlation between serum TSHR Ab
concentrations and disease activity. In patients who have
highly increased antibody levels, clinical progression is more
serious and response to treatment is delayed, therefore,
relapse is seen more frequently (2,10,11). Serum TSHR Ab
concentrations decline in most patients after long-term
ATD therapy. Propylthiouracil decreases release of Ig from
B lymphocytes and increases a number of suppressor cells
(61). Methimazole blocks the increase in serum TSHR Ab
concentrations that occurs in patients with GD treated with
RAI, suggesting that an organ-specific effect, rather than
generalized immunosuppression, is of primary importance
(2). In another study, patients treated with either PTU or car-
bimazole had identical decrements in serum thyroid hor-
mone concentrations, but the carbimazole-treated patients
had greater decreases in serum TSHR Ab concentrations
and increases in the number of suppressor T cells, suggest-
ing, indirectly, an effect on the immune system independent
of thyroid function (62). Serum TSHR Ab concentrations
tend to decrease during ATD therapy because of an immu-
nosuppressive effect of the drug, amelioration of thyrotox-
icosis, spontanenous remission, or a combination of these
factors. The failure of serum TSH Ab to become undetect-
able during ATD therapy signifies almost certain relapse
after discontinuation of therapy (10). If serum TSHR Abs
do disappear, there is still a 30–50% change of relapse
(63). Thus, detectable serum TSHR Ab activity, but not its
absence, has prognostic value (9).
Decreasing TSHR Ab level is most probably linked to
direct inhibition of antibody production in B lymphocytes
by royal jelly or else to a decrease in the stimulant effect of
IL-4 and IL-10 on B lymphocytes under the effect of royal
jelly in a cell culture environment. In addition, it has been
reported that increasing Th1 cytokine IFN-γ under the ef-
fect of royal jelly suppresses the production of thyroid anti-
bodies by in vitro thyroid B cells (64). This result is most
important because imbalance in the Th1/Th2 cytokine ratio

Royal Jelly in Graves’ Disease / Erem et al.
Vol. 30, No. 2
181
leads to defects seen in Graves’ disease by way of humoral
immunity. The most important role in humoral immunity
belongs to TSHR Ab.
Another of the striking results from our study is that the
levels of cytokine released from lymphocyte cell cultures
at a 0 and 4 mg/mL concentrations show a wide range of var-
iation among patients. When we formed groups of patients
with the same royal jelly concentration, this had a negative
effect on significance levels among the groups. For exam-
ple, although the IL-4 level decreased as royal jelly concen-
tration increased, statistical significance remained within
the threshold only because the initial levels among patients
were very different (IL-4 level at 0 mg/mL concentration
was 30.4 pg/mL in patient S.H., but 1.8 pg/mL in patient
E.T.). How is this to be explained? It is known that analysis
of the peripheral blood T cell population, particularly after
mitogen stimulation, will clearly be biased by the inclusion
of the majority of lymphocytes that do not have specificity
for thyroid autoantigens and even the intrathyroidal popula-
tion will not be free from such biases. In vitro culture after
cell fractionation, with measurement of cytokine release
into culture supernatant, has clear advantages over RT-PCR
methods in terms of directly quantifying cytokine-as-pro-
tein but requires large numbers of cells for purification of
population such as the CD4
+
cells, and may not be free from
the possible artefacts of any in vitro system (7).
One of the interesting findings in our study is that TSHR
Ab increased in only half the Graves’ disease patients, and
that while antibody levels were normal in patients 2, 3, and
6, the TSHR Ab levels we measured in these patients’ PBLCs
were high. This finding shows that TSHR Ab levels mea-
sured in lymphocyte culture supernatants are much more
important, reliable, and valuable in the diagnosis of Graves’
disease than serum THSR Ab levels. In practice, however,
TSHR Ab levels may still be prepared given their ease of use.
In conclusion, RJ in lymphocyte cell culture obtained
from GD patients decreased TNF-α, Th1 cytokine, and in-
creased IFN-γ, Th1 cytokine, changed Th1/Th2 ratio in
favor of Th1; therefore, RJ may be effective as an immuno-
modulatory agent in Graves’ disease.
Materials and Methods
In the first phase of the present study, lymphocyte cell
isolation from four voluntary healthy subjects without any
known autoimmune, allergic, or infectious disease was per-
formed to find effective concentration of RJ on immunity
(stimulation, inhibition, or immunomodulation). The periph-
eral blood lymphocyte cells (PBLC) were isolated from the
peripheral venous blood samples with centrifugation on
Ficoll-Paque (Pharmacia, Sweden) density gradient (36).
The sample of RJ used in the study were collected fresh
from Trabzon in the Turkey was provided by Trabzon Agri-
cultural Development Cooperative. It was kept frozen at
−85°C until used.
RJ was suspended in sterile phosphate-buffered saline
(PBS) at a concentration of 500 mg/mL. The supernatant of
the RJ suspension was collected by centrifugation at 10,000g
for 10 min. From the samples serial dilutions of 500, 400,
200, 100, 50, 25, 10, 5, and 2.5 mg/mL were prepared. PBS
solution was used for zero concentration. RJ samples were
passed through 0.2 µm filter unit in a laminar airflow to
sterilize them; 240 µL of lymphocyte sample isolated from
each healthy subjects and 60 µL of RJ sample at various
concentrations (1:5 final dilution) were added to cell cul-
ture wells. Final volume of wells was completed to 3 mL
by RPMI-1640. Therefore, final RJ concentrations were 5,
4, 2, 1, 0.5, 0.25, 0.10, 0.05, and 0.0025 mg/mL, respec-
tively. The PBLC cultures were incubated in the CO
2
incu-
bator with 5% CO
2
and 95% humidity for 72 h.
MTT Test (Tetrazolium Dye-Reduction Assay) (37)
PBLCs were seeded in 96-well plates (100 µL/well at
density of 1 × 10
5
/mL) and exposed to different concentra-
tions of RJ for 72 h. The cell-survival fraction was deter-
mined with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide] dye reduction test assay. In brief,
after incubation with RJ, 10 µL MTT solution (2 mg/mL)
was added to the well plates and further incubated for 4 h
at 37°C. The formazan crystals formed were dissolved by
addition of 200 µL isopropanol /well. Absorbtion was mea-
sured by ELISA reader (Bio-Tek Instruments, USA) at 540
nm, reference filter 620 nm. The experiments were per-
formed four times on all concentrations and the means of the
results were used for final analysis. An increase in absor-
bance indicated a greater proliferating activity.
Selection of the Patients with GD
In the second phase, patients with GD who untreated and
newly diagnosed by clinical and laboratory methods admit-
ted to Endocrinology and Metabolic Diseases Clinic of Med-
ical Faculty, Karadeniz Technical University. Table 7 shows
demographic and laboratory characterictics of patients with
GD. Each patient was clinically and biochemically hyper-
thyroid, defined as having increased serum thyroid hormone
levels, a suppressed TSH concentration (<0.1 µU/mL). The
diagnosis of GD was defined as the presence of biochemi-
cal hyperthyroidism (raised serum total T
4
, total T
3
, free T
4
,
and free T
3
concentrations and suppressed TSH) together
with the presence of two of the following: a palpable dif-
fuse goiter, a significant titer of thyroid peroxidase, Tg auto-
antibodies and/or TSH receptor antibodies, and/or the pres-
ence of ophthalmopathy. At the time of the study, patients
were neither taking drugs nor had diseases known to affect
immunity.
Blood was drawn in the morning 0:800/09:00 h after an
overnight fast. Serum total and free triiodothyronine (TT
3
and FT
3
), total and free thyroxine (TT
4
and FT
4
), and TSH
concentrations were measured by automated chemilumines-
cence system (Roche, E-170, Switzerland). Normal ranges

Royal Jelly in Graves’ Disease / Erem et al.
182
Endocrine
are 0.8–2.0 ng/mL for TT
3
, 5.1–14.1 µg/dL for TT
4
, 1.8–
4.6 pg/mL for FT
3
, 0.9–1.7 ng/dL for FT
4
, and 0.27–4.2
µU/mL for TSH.
PBLC isolation from patients with GD were performed
as explained previously.
RJ samples of 0 and 4 mg/mL were incubated in a cul-
ture medium for 72 h with isolated lymphocytes obtained
from the patient such as explained above. After incubation,
the MTT test in lymphocytic cell culture was performed.
For each patient and control samples, 10 experiments were
performed.
After the incubation period, the culture supernatants were
removed for the measurement of cytokines and TSHR Abs.
Th1 cytokines IFN-γ (cat no. KAC1231), TNF-α (cat no.
KAC1751), and IL-12 (cat no. KAC1561), and Th2 cyto-
kines IL-4 (cat no. KAC1281) and IL-10 (cat no. KAC1321)
levels were measured by an immunoenzymometric assay
(EASIA) using commercially available kits (Biosource,
Belgium) in the culture supernatants. Two experiments was
performed for each concentration. The ratios of Th1 against
Th2 cytokines were calculated. TSHR Ab levels by radio-
receptor method were determined in the culture superna-
tants using commercially available kits (Brahms, USA).
Statistical Analysis
All statistical analyses were performed using SPSS/PC
statistical program (version 11.0 for Windows; SPSS, Inc.,
Chicago, USA). Nonparametric Friedman test with a signed
Wilcoxon post-hoc test was used to find the differences in
the groups. Results were shown as chi-square and p value
on the tables. Wilcoxon test was used to compare the con-
centrations of groups. Results were calculated as w and p
Table 7
Demographic and Laboratory Characteristics of Patients with Graves’ Disease
Patient no 1 2 3 4 5 6
Age (yr) 26 31 26 40 45 51
Gender M M F F F M
TT
3
6.5 6.4 4.1 6.5 5.4 4.7
(normal: 0.8–2.0 ng/mL)
TT
4
22.8 18.6 17.5 24.9 16.9 21.2
(normal: 5.1–14.1 µg/dL)
FT
3
23.1 21.2 13.7 32.6 24.5 22.6
(normal: 1.8–4.6 pg/mL)
FT
4
7.8 4.3 4.3 6.4 4.1 5.4
(normal: 0.9–1.7 ng/dL)
TSH 0.01 0.01 0.01 0.01 0.01 0.01
(normal: 0.27–4.2 µU/mL)
Anti-TPO 643 235 864 16.0 16 44.1
(normal: <34 IU/mL)
Anti-Tg 41.8 238 46.1 294 <20 938
(normal: <115 IU/mL)
TSH R Ab 25.5 8.8 9.30 14.0 58 2.5
(normal: 0–10 U/L)
values on the tables. p < 0.05 was considered statistically
significant.
Acknowledgments
This study was supported by a research grant from the
Karadeniz Technical University (Project No. 2002.114.001.
6). We are grateful to Murat Topbas for statistical advice.
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