Original Paper
HORMONE Horm Res 2009;71:350–358
RESEARCH DOI: 10.1159/000223420
Interaction of Lymphocytes and Thyrocytes
in Graves’ Disease and Nonautoimmune
Thyroid Diseases in Immunohistochemical and
Ultrastructural Investigations
Iwona Ben-Skowronek a Jadwiga Sierocinska-Sawa b Leszek Szewczyk a
Elzbieta Korobowicz b
Departments of a Paediatric Endocrinology and Neurology, and b Pathomorphology, Lublin Medical University,
Lublin, Poland
Key Words
Lymphocyte subsets ⴢ Graves’ disease ⴢ Nodular goiter ⴢ
Simple goiter
Abstract
Background: Graves’ disease is the archetype for organ-spe-
cific autoimmune disorders. It is very important for our un-
derstanding of the mechanisms responsible for progression
of autoimmunity. The aim of this study was to present inter-
actions of lymphocytes and thyrocytes in the thyroid tissue
in Graves’ disease and nonautoimmune thyroid diseases.
Methods: The study involved 30 children with Graves’ dis-
ease, 30 children with nodular goiter, 30 with simple goiter
and 30 healthy children. After thyroidectomy, T cells were
detected in the thyroid specimens by CD3, CD4, CD8 anti-
bodies, B cells by CD79␣ antibodies and the antigen-pre-
senting dendritic cells with CD1a antibodies (DakoCyto-
mation) and were examined in the EM 900 Zeiss Germany
Electron Microscope. Results: The most enhanced immune
reaction was observed in the thyroid from children with
Graves’ disease. The cells of the immune system infiltrated
the thyroid follicles and interfollicular compartments; they
also formed lymph follicles. Conclusion: The immune reac-
tion in Graves’ disease and migration of lymphocytes T and
© 2009 S. Karger AG, Basel
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E-Mail karger@karger.ch Accessible online at:
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Received: January 11, 2008
Accepted: August 1, 2008
Published online: June 9, 2009
B between thyrocytes results in the thickening of the basal
membrane of the thyroid follicle. No cytotoxic effect of T cy-
totoxic/suppressor CD8+ cells on thyrocytes was observed
in Graves’ disease, while a mild cytotoxic effect was observed
in non-autoimmune thyroid disease.
Copyright © 2009 S. Karger AG, Basel
Introduction
Autoimmune diseases in children occur much less fre-
quently than in adults, and most often they are not ac-
companied by any other illnesses. The syndromes com-
prising autoimmune thyroid disease (AITD) are the three
intimately related illnesses: Graves’ disease with goiter,
hyperthyroidism and, in many patients, associated oph-
thalmopathy, Hashimoto thyroiditis with goiter and eu-
thyroidism or hypothyroidism. The syndromes are bound
together by their similar thyroid pathology, similar im-
mune mechanism, co-occurrence in family groups, and
transition from one clinical picture to another within the
same individual over time [1].
The immune response proceeds via T-cell receptor
antigen recognition, followed by activation of the T cell
through the combined effect of antigen recognition and
Iwona Ben-Skowronek
Department of Paediatric Endocrinology and Neurology
Lublin Medical University
PL–20-093 Lublin DSK, ul. Chodzki 2 (Poland)
Tel. +48 81 718 5440, Fax +48 81 718 4373, E-Mail skowroneki@interia.pl
 Table 1. Description of patients

Diagnosis Children TSH, mU/l fT4, ng/dl TPO Ab, U/l TG Ab, U/l TRAB, U/l

Normal ranges 0.27–4.2 0.8–2.4 <12 <35 <1.1

Control group 30 0.27–4.2 – – – –
Struma colloides 30 0.21–3.9 0.8–2.3 0–14 0–36 –
Struma nodosa 30 0.2–4.1 0.8–2.1 0–16 0–40 –
Graves’ disease 30 0.007–0.001 3.3–5.1 21–6,663 25–13,351 7–462

costimulatory signals, including interleukin (IL)-1 action
leading to T cell IL-2 secretion and IL-2 receptor expres-
sion and, subsequently, to proliferation of the T cell into
an active clone [2]. The antigen can be presented to CD4+
cells by conventional antigen-presenting cells, particu-
larly dendritic cells (DCs) [3] and also by B cells and ac-
tivated T cells, and less effectively by a variety of other
cells (thyrocytes, fibroblasts) [4]. Antigen presentation to
T cells leads to a variety of responses, which include pro-
liferative or suppressive functions, development of cell
cytotoxic responses, control of immunoglobulin secre-
tion, and many more. Responding lymphocytes can be
segregated into groups based on whether they are naive
or memory cells, CD4+ helper cells, or CD8+ cytotoxic/
suppressor cells. CD8+ T cell activation requires addi-
tional costimulatory pathways to the B7-dependent path-
way largely used by CD4+ T cells, and ICAM-1 is par-
ticularly important [5, 6]. The CD4+ T cells are catego-
rized into Th1 and Th2 cells. Th1 cells produce IL-2, INF
and TNF and are predominant in ‘delayed hypersensitiv-
ity’ type of reactions, whereas Th2 cells produce IL-4, IL-
5, stimulate B cells to immunoglobulin synthesis, and are
involved especially in antibody-mediated reactions. Cy-
tokines produced by Th1 cells enhance the activity of
these subsets but inhibit Th2 cells and vice versa [2]. Some
CD4+ and CD8+ T cells appear to provide suppressor
signals. Most recently, attention has been focused on al-
ternates of local secretion TGF-␤ and direct cell contact,
which involves binding of CTLA-4 to the T regulatory
cell [7]. In addition to the standard T cell function de-
scribed above, other cells participate in immune respons-
es. Macrophages may destroy cells having immune com-
plexes on their surface. Other cells which do not bear the
CD3 marker of T cell lineage exist (K and NK cells) and
have the ability to spontaneously kill other cells (espe-
cially those expressing HLA antigens). NK cells can be
detected by specific monoclonal antibodies such as anti-
CD16 and are recognized phenotypically as large gran-
ular lymphocytes (LGLs) [8].
Numerous investigations evaluated lymphocyte sub-
sets in the peripheral blood [9–15], but the reactions in
the thyroid tissue are very important in a situation when
new experimental methods of treatment by B-lympho-
cyte depletion during monoclonal anti-CD20 antibody
therapy are elaborated [11, 12].
The aim of the study was to present interactions of
lymphocytes and thyrocytes in the thyroid tissue in
Graves’ disease and nonAITDs in children.
Materials and Methods
Patients
The study concerned 90 children: 30 children aged 10–19
(mean 16 8 2.3) years, affected with Graves’ disease, 30 children
with nodular goiter, 30 children with simple goiter. The children
were treated in the Department of Pediatric Endocrinology and
Neurology in Lublin and in the Pediatric Department in Rzeszow
in the years 1994–2007 (table 1).
The TSH, fT4 and TT3 hormones were assayed by MEIA (Ab-
bott Kit). The levels of TSH receptor antibodies were measured by
RIA (TRAK assay, BRAHMS Diagnostica GmbH, Berlin, Ger-
many). TPO and TG antibodies were assayed by LIA (Lumitest,
BRAHMS Diagnostica GmbH).
In the patients with Graves’ disease, symptoms of thyrotoxi-
cosis and an increase in fT4 (mean 3.8 8 0.7 ng/dl) and TT3
(mean 363 8 175.3 ng/dl) were observed, and also a decrease in
TSH (mean 0.004 8 0.003 mU/l). The levels of antibodies against
TSH receptor (TRAB; 7–462 U/ml) and usually the levels of TPO
antibodies (21–6,663 U/ml) and TG antibodies (25–13,351 U/ml)
were increased.
The patients were treated with methimazole at initial doses of
0.9–0.5 mg/kg body weight/day during 4–6 weeks, and after that
time, when in euthyreosis, they received maintenance doses of
approx. 0.1 mg/kg body weight/day (mainly 5 mg/day) in combi-
nation with a low dose of L-thyroxin (25 ␮g/day) during 18–24
months. Children with Graves’ disease in whom early relapses of
hyperthyreosis were observed and led to surgery treatment (thy-
roidectomy) after 18–36 months were included in the investiga-
tion.
The patients with nodular goiter and simple goiter were euthy-
roid or hypothyroid at the beginning of the treatment. They were
treated with L-thyroxin at a dose of 25–100 ␮g/day.

Lymphocytes and Thyrocytes in GD and Horm Res 2009;71:350–358 351

Non-AITD
Table 2. Subsets of lymphocytes as percentage of cells in vision fields in thyroid tissue
Group Subsets of lymphocytes
CD3+ CD4+
T p T helper p T suppressor/ p
Control group 1.0280.01 0.1980.28
Simple goiter 6.3680.45 0.063 1.0282.71* 0.049
Nodular goiter 14.982.82* 0.032 2.185.5 0.051
Graves’ disease 17.73817.9* 0.003 2.9582.51* 0.034
* p < 0.05 vs. control.
Specimens from thyroids of children who had died in acci-
dents or who had been operated due to thyroglossal cysts, neck
injuries and during surgery of parathyroid glands were investi-
gated as controls.
Immunohistochemical Investigations
After thyroidectomy, 4-␮m thyroid slices were histologically
examined with hematoxylin-eosin. Immunohistochemical reac-
tions in paraffin specimens with monoclonal antibodies against
T-cell markers CD3, CD4, CD8 as well as against B-cells –
CD79- ␣ and the antigen presenting DCs – CD1a antibodies (Da-
koCytomation Denmark) were performed. The specimens were
dewaxed in xylene, hydrated by rinsing with numerous alcohols
and distilled water. Next, antigens were revealed in citrate buffer
at pH 6.0. After the specimens had been washed in distilled water
and TBS buffer, endogenous peroxidase was blocked with 3%
H2O2, which was followed by a subsequent wash of the specimens
in TBS buffer. Thus prepared thyroid specimens were incubated
with a primary antibody and then with dextran marked with
horseradish peroxidase (DakoCytomation EnVision + Peroxidase
kit) and with the chromogene of DAB horseradish. When the im-
munohistochemical reaction was finished, cell nuclei were stained
with Mayer’s hematoxylin; the specimens were dehydrated in al-
cohol, exposed to xylene and embedded in Canada balsam.
The specimens were estimated in Axiostar plus microscope.
The lymphocytes were counted in Sony Color Camera Exwave-
HAD and the lymphocyte subsets were analyzed in MultiScan5
software and hardware with Show Time Plus, S-VHS frame grab-
ber using an IBM Pentium computer. We determined the number
of lymphocyte subpopulations in the thyroid tissue by counting
lymphocytes marked with CD3+, CD4+, CD8+, CD79␣+ and
CD1a+ monoclonal antibodies in every 1,000 cells present in 10
vision fields of the microscope and by estimating their content
percentage.
The results were expressed as means 8 SD. Comparison of the
percentage of lymphocytes expressing receptors to monoclonal
antibodies was carried out using the t test confirmed by the Mann-
Whitney U test. T value was considered statistically significant at
p ! 0.05. For correlation analysis, Pearson’s and Spearman’s tests
were used. Mathematical and statistical calculations were per-
formed with Statistica 5.0.
352 Horm Res 2009;71:350–358
CD8+ CD79␣+ CD1a+
B p dendritic p
cytotoxic cells
0.6580.001 4.0780.67 0.1683.62
4.3885.54* 0.038 0.2880.67 0.071 0.2880.69 0.084
985.5* 0.034 5.9811.6* 0.030 0.180.3 0.066
6.4986.93* 0.035 23.21823.6* 0.001 0.3780.3* 0.047
Ultrastructural Investigations
Specimens for ultrastructural investigations were obtained
during thyroidectomy or from paraffin-dewaxed preparations.
Small segments of the thyroid were cut into 0.5-mm3 pieces and
fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 for
24 h at 4 ° C, postfixed in 2% OsO4 in the same buffer for 1 h at
room temperature, dehydrated in the graded series (up to 100%)
of ethanol and embedded in 812 Epon. They were then polymer-
ized at 60 ° C. Epon blocks were cut with ultramicrotome RMC
MT-7 (RMC, Tucson, Ariz., USA). Ultrathin sections were con-
trasted with uranyl acetate and lead citrate and examined with the
EM 900 Zeiss Germany Electron Microscope.
The investigation was approved by the local Ethical Commit-
tee of the Medical University in Lublin.
Results
In the thyroid specimens of the control group, lym-
phatic cells constituted an insignificant percentage. They
were dispersed among the thyroid follicles and were often
present in the proximity of blood vessels. The most prev-
alent were CD79␣+ B lymphocytes, their contents being
approximately 4.07%. T lymphocytes with a CD3+ phe-
notype comprised 1.02% of the cells in the thyroid speci-
mens. CD8+ suppressor/cytotoxic T lymphocytes were
more frequently observed (0.65%) than T helper cells
(0.19%). The least frequently occurring were the CD1a+
antigen-presenting cells (0.16%; table 2). In ultrastructur-
al investigations, we most often observed T lymphocytes
(with poor cytoplasm and the nucleus filling the cell) in
the basal membrane of thyroid follicles. The basal mem-
brane itself was thin. The thyrocytes had a follicular cell
nucleus. The mitochondria were concentrated in the bas-
al pole, and in the apical pole there were the Golgi appa-
ratus and secretion follicles containing colloid. The cell
membrane formed microvilli in the lumen of the thyroid
Ben-Skowronek /Sierocinska-Sawa /
Szewczyk /Korobowicz
Fig. 1. Control group. Cuboidal thyrocytes with a round nucleus,
numerous mitochondria in the basal pole and Golgi apparatus,
colloid vesicles and microvilli in the apical pole. Thin basal mem-
brane. !10,000. N = Nucleus; V = vesicles with colloid; RER =
rough endoplasmatic reticulum; M = mitochondrion; R = ribo-
some; BM = basal membrane; Lu = lumen of the thyroid follicle.
follicles (fig. 1). In the thyroid vesicle epithelium, sporad-
ically there appeared dying cells with apoptotic features:
a pycnotic cell nucleus, electron-dense cytoplasm and in-
distinct structural membranes of the cell organelles.
Immunohistochemical investigations of the lympho-
cyte subpopulation in the simple colloidal goiter indicate
an insignificant contribution of lymphatic cells to the
goiter structure. The percentage of B CD79␣+ lympho-
cytes was merely 0.28%; they were far less numerous than
in the control group. The number of CD3+ lymphocytes
did not exceed 6.36%. The most dominant were the CD8+
suppressor/cytotoxic T lymphocytes (4.38%) compared
with T-helper lymphocytes (1.02%). Antigen-presenting
cells were similarly scarce (0.28%; table 2). Morphologi-
cal interrelations of thyrocytes and lymphocytes in the
ultrastructural investigations were similar to those in the
control group (fig. 2).
Immunological reactions in the nodular colloidal goi-
ter were slightly more intense. CD79␣+ B lymphocytes
constituted 5.9% of the cells. CD3+ T lymphocytes were
the most abundant (14.9%). They were present in the bas-
al membrane of the thyroid follicles and in the vicinity of
Lymphocytes and Thyrocytes in GD and
Non-AITD
Fig. 2. Colloid goiter. Cuboidal thyrocytes with a round nucleus,
mitochondria in the basal pole and small colloid vesicles and mi-
crovilli in the apical pole. Thin basal membrane. Lymphocyte in
the interstitium. !10,000.
blood vessels. They sometimes formed inconsiderable in-
filtrations, but no lymphatic follicles. Among them, CD8+
suppressor/cytotoxic T lymphocytes were prevalent
(9.0%), while CD4+T-helper lymphocytes constituted
1.02% of the observed cells. Antigen-presenting cells were
very scarce (table 2; fig. 3).
Electron microscope investigations revealed that the
thyrocytes within the thyroid nodules frequently had a
different shape than that in the control group. In the api-
cal region, there were fewer colloidal granules; the cell
membrane projected into the lumen of the vesicles with
scarce microvilli. More frequently, there appeared dying,
apoptotic cells with dark cytoplasm and a pycnotic nucle-
us. Small lymphocytes were present in the basal mem-
brane of the thyroid follicles, especially in the sites where
thyroid epithelium cells were dying. Single T lympho-
cytes (small lymphocytes with poor cytoplasm) or NK
cells (big, granular lymphocytes) were observed in the lu-
men of thyroid vesicles (fig. 4).
The most enhanced immunological reaction was ob-
served in the thyroid specimens from children with
Graves’ disease. The cells of the immune system infil-
Horm Res 2009;71:350–358 353
Fig. 3. Nodular goiter. Cuboidal thyrocytes with the folded nucle-
ar membrane, small mitochondria in basal pole and numerous
colloid follicle and microvilli in apical pole. Enlarged rough en-
doplasmatic reticulum and spaces between thyrocytes. Lympho-
cyte in interstitium near the blood vessel. !10,000. Mv = Micro-
villi.
Fig. 4. Nodular goiter. Thyrocytes: one thyrocyte with extremely
enlarged rough endoplasmatic reticulum and pycnotic nucleus
characteristic of thyrocyte in cytolysis stage. In the lumen of thy-
roid follicle, a small T lymphocyte and LGL lymphocyte, pheno-
typically cytotoxic lymphocyte, are seen. !10,000. Ly = Lyso-
some.

trated the thyroid follicles and interfollicular compart-

ments; they also formed lymph follicles with typical pro-
liferation centers. T lymphocytes constituted 17.73% of
the cells in the specimens. 6.49% of T lymphocytes were
suppressor/cytotoxic T lymphocytes (CD8+) and 2.95%
T-helper cells (CD4+). The other T lymphocytes did not
positively react with either CD8 or CD4 antibodies. CD8+
lymphocytes were located in the basal membranes of the
thyroid follicles and among the thyrocytes. They were
also present in the concentration regions of lymph nod-
ules. CD4+ lymphocytes occurred in proliferation cen-
ters, at the peripheries of lymph nodules and in the inter-
follicular space. Lymphocytic infiltrations were domi-
nated by CD79␣+ B lymphocytes, which constituted
23.21% of the observed cells. They were visible in the bas-
al membranes of the thyroid follicles, and also infiltrated
the walls as well as the interior part of the follicles. They Fig. 5. Graves’ disease. Lymphocyte between the thyrocytes in the
constituted a remarkable percentage of lymph nodule wall of the thyroid follicle. Ultrastructure of the lymphocyte char-
cells. They were found both in the proliferation centers acteristic for T lymphocytes. !10,000. CM = Cell membrane;
RBC = red blood cell.
and at the peripheries of the nodules. CD1a+ antigen-pre-
senting cells appeared more frequently with statistical
significance than in the other examined groups, still they

354 Horm Res 2009;71:350–358 Ben-Skowronek /Sierocinska-Sawa /

Szewczyk /Korobowicz

Fig. 6. Graves’ disease. a Lymphocyte be-
tween the thyrocytes in the wall of the thy-
roid follicle. Ultrastructure of the lympho-
cyte characteristic of T lymphocytes.
!10,000. C = Cytoplasm; CG = cytoplasm
granules. Lymphocyte connected with
thyrocyte with immunological synapse
(b) and direct (c). !20,000. a
were not numerous in the investigated thyroid specimens
(0.37%). In the apical region of some thyrocytes in pa-
tients with Graves’ disease, a positive reaction with CD1a
in the form of CD1a+ granules was observed (table 2).
The analysis of the thyroid ultrastructure in patients
with Graves’ disease demonstrated numerous lympho-
cytes near the blood vessels (fig. 5). They were T lympho-
cytes – small cells with poor cytoplasm and with a big cell
nucleus filled with dark chromatin, or B lymphocytes –
plasmatic cells with characteristic layers of rough endo-
plasmic reticulum around the nucleus. We also observed
cells with a dark nucleus, cytoplasm with vacuoles filled
with dark electron-dense substance and single projec-
tions of the cell membrane morphologically related to
LGLs of the NK type. T lymphocytes were also observed
beneath the basal membrane of the thyroid follicles. They
were also often present among the thyrocytes and in the
thyroid follicle lumen (fig. 5–7). The lymphocytes formed
processes binding with thyrocytes through adhesive pro-
tein substances or directly; the structure of this binding
resembled a tight junction (fig. 6b). It should be empha-
sized that the basal membrane was thickened, with in-
creased electron density compared with that in the nodu-
lar or simple goiter or in the control. In some regions of
the basal membrane, there were electron-dense deposits,
which coincided with plasmocytes (fig. 8). The thyro-
cytes did not show any traits of destruction, they were in
fact fairly active: they had big, light cell nuclei, numerous
Lymphocytes and Thyrocytes in GD and
Non-AITD
b
c
c
secretion vacuoles and well-developed microvilli in the
apical region. In some places, we could observe mitotic
cell division. Symptoms of thyroid cell death were ob-
served when they were in contact with LGLs; the cyto-
plasm in the thyrocytes was filled with electron-dense
structures, the endoplasmic reticulum was enlarged and
the mitochondria and pycnotic cell nuclei were swollen.
In some specimens, there were B lymphocytes with a de-
veloped rough endoplasmic reticulum surrounding the
cell nucleus. B lymphocytes were frequently in contact
with T lymphocytes.
Correlation between the levels of TRAB and CD19+
subset B lymphocytes was observed only in Graves’ dis-
ease patients (r = 0.62, p = 0.002). No correlation was ob-
served between the levels of TPO Ab and TG Ab and lym-
phocyte subsets. In the other groups, no correlation was
noticed between TRAB, TPO AB and TG AB levels and
the percentage of lymphocyte subsets in the thyroid tis-
sue.
Discussion
In our investigations, a small number of DCs present-
ing CD1a, which characteristic of immature DCs, was ob-
served in the thyroids without AITD, but it was signifi-
cantly higher in the thyroids from Graves’ disease pa-
tients. The DCs were immature – antigen CD1a had the
Horm Res 2009;71:350–358 355
Fig. 7. Graves’ disease. Lymphocyte and plasmocyte in contact
with each other in the lumen of the thyroid follicle. Lymphocyte
in the interstitium between the thyroid follicles. Many collagen
fibers are seen in the interstitium. Basal membrane is partially
thickened. !10,000. CF = Fibers of collagen.
structure of an ␣-chain connected with ␤-microglobu-
lins [15, 16]. Similar observations were described by
Quadbeck et al. [16], who suggested that the phenotype
of intrathyroidal DCs in Graves’ disease supports their
role as potential costimulators of thyroid immunity [16].
Other authors observed a higher number of DC in healthy
thyroid (2–3% interstitial cell population) [17]. There are
indications that such DCs are able to proliferate [18],
which means that not all of the thyroid DCs need to have
recently immigrated with the bloodstream. Thyroidal
DCs are often in close contact with thyrocytes, are clear-
ly in immature state and often show monocyte marker
characteristics [19]. It is thought that soluble factors pro-
duced by TSH-stimulated thyrocytes such as GM-CSF,
TGF-␤, IL-6 keep intrathyroidal DCs in their immature
state [18]. The presence of positive reaction to CD1a pro-
tein in the granules of the apical part of some thyrocytes
suggested that the thyrocytes probably initiated thyroid
autoimmune reactivity [20]. The investigations in trans-
genic mice by Kimura et al. [21] suggested that expression
of class II MHC molecules on epithelial thyroid cells is
not required for the initiation of an autoimmune attack
on the thyroid. The initiation, then, seems to be mainly
356 Horm Res 2009;71:350–358
Fig. 8. Plasmocyte in contact with thyrocytes. Thick basal mem-
brane of the thyroid follicle with protein deposits (asterisk) –
probably immunological complexes. !30,000. R = Ribosome.
mediated by the professional antigen-presenting cells in
secondary lymphoid organs.
In nontoxic simple and nodular goiter, only lympho-
cytes CD4+ and CD8+ are observed in bigger numbers
in the interstitium of the thyroid, and they occasionally
form mononuclear cell infiltrations. In ultrastructure in-
vestigations, thyrocytes of different morphology than
that in the control group were observed. Probably, it was
the effect of somatic mutations of the thyroid [22]. Small
T lymphocytes were present mainly in the interstitium or,
sporadically, in the follicle lumen.
Our immunohistochemical investigations and other
observations in Graves’ disease suggested that T and B
lymphocytes accumulating in the thyroid do not form
destructive infiltrates, but they are organized as a periph-
eral lymphatic tissue. Graves’ disease patients seem to
have mixed Th1/Th2 profiles [23]. In the in vitro investi-
gations, Estienne et al. [24] indicated the possibility of
forming a so-called immunological synapse of a tight
junction between lymphocytes and thyrocytes with par-
ticipation of adhesive proteins. This physical contact may
result in the establishment of an immunological synapse
[25] able to stimulate intrathyroid T lymphocyte prolif-
Ben-Skowronek /Sierocinska-Sawa /
Szewczyk /Korobowicz
eration and differentiation [24]. In our immunohisto-
chemical investigations, it was the CD8+ lymphocytes
that most frequently entered the basal membrane of thy-
roid follicles. The latest investigations [26–28] suggested
that a crucial role in peripheral tolerance or autoreactive
T cells is played by T regulatory subsets (Tregs) divided
into two populations: naturally occurring and inducible
[29, 30]. Tregs so far identified as participating in the
pathogenesis of Graves’ disease include naturally occur-
ring CD4+CD25+T cells [26], C8+CD122+T cells [27, 28]
and NK cells [31]. The comparison of immunohisto-
chemical localization of CD4+ T cells with ultrastruc-
tural investigations showed that CD4+T lymphocytes
were small cells with large nuclei and that a small amount
of cytoplasm was in contact with thyrocytes and other
lymphocytes. We observed T lymphocytes in close con-
tact with plasmocytes, which suggested the regulation/
helper function of the cells stimulating plasmocytes to
production of autoantibodies.
It is known, and we observed it too, that patients with
Graves’ disease have an increased number of circulating
B cells [32–35]. The close contact with T cells (probably
Th2 cells) and plasmocytes was frequently observed only
in Graves’ disease and sporadically in the nodular goi-
ter.
The other subset of T cells, T suppressor/cytotoxic
cells, was detected in immunohistochemical investiga-
tions between the basal membrane of the thyroid follicle
and thyrocytes and in lymphatic infiltrations. Rifa’i et al.
[30] and Endharti et al. [31] described the subsets of nat-
urally occurring Tregs CD8+CD25+. It is possible that
when T CD8+ cells, which we observed in our study,
come into contact with thyrocytes, they play the role of
Tregs in the pathogenesis of Graves’ disease. The investi-
gations of Negrini et al. [36] characterized a subpopula-
tion of CD8 T suppressor lymphocytes able to inhibit
both cell proliferation and cytotoxicity, and observed
that glucocorticoid-induced TNF-like receptor is ex-
pressed on such CD8 T suppressor cells and that its acti-
vation by specific antibody inhibits generation, but not
function of these cells [36]. The papers of Nakano et al.
[37] and Nagayama [38] suggested a preventive role of
Tregs in autoimmunological reaction in the thyroid with
AITD.
In Graves’ disease, the immunological deposits ob-
served in the basal membrane of the thyroid follicles lead
to the thickening of this membrane and probably chang-
es in polarization of cell membranes. The thyrocytes in
this region were columnar, with signs of increased activ-
ity (big nuclei, active, enlarged mitochondria, big amount
Lymphocytes and Thyrocytes in GD and
Non-AITD
of granules in the apical pole, long microvilli). It is known
that B cells are involved in the production of thyroid au-
toantibodies [34, 39–41], and their depletion leads to im-
provement of thyroid function in Graves’ disease [11, 12].
The migration of lymphocytes into thyroid follicles fa-
cilitates direct contact with the antigens of the thyrocyte
apical pole – especially TPO – and production of anti-
TPO antibodies as well as formation of LGL phenotype
NK cells, which destroy thyrocytes [1, 42–44]. The mech-
anism of migration of lymphocytes into thyroid follicles
is unknown, but is probably dependent on chemokines
[44] and expression of HLA molecules on the surface of
thyrocytes [45].
Conclusions
The immune reaction in Graves’ disease and migra-
tion of lymphocytes T and B between thyrocytes results
in the thickening of the basal membrane of the thyroid
follicle. Lymphocytes T and B can migrate into the thy-
roid follicle lumen.
No cytotoxic effect of T cytotoxic/suppressor CD8+
cells on thyrocytes was observed in Graves’ disease, while
a mild cytotoxic effect was observed in non-AITD.
Acknowledgements
This work was supported by grant 2P05E04327 from the Pol-
ish Ministry of Science and Higher Education.
1 Weetmann AP, De Groot LJ: Autoimmunity
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