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Experimental Studies |
Division of Endocrinology, Department of Medicine, University of Essen, D-45122 Essen; the Department of Medicine, University of Munich (C.S., A.E.H.), D-80336 Munich; and the Center of Internal Medicine, University of Frankfurt (P.M.S.), D-60950 Frankfurt am Main, Germany
Address all correspondence and requests for reprints to: Dr. Rudolf Hoermann, Division of Endocrinology, Department of Medicine, University of Essen, Hufelandstrasse 55, D-45122 Essen, Germany.
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Abstract |
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infusion (200 IU/24 h) for 2 days. Normal IgGs were
ineffective. Bovine TSH (bTSH) had little effect by itself, but did
enhance interferon--induced ICAM-1 expression. To assess the
specificity of their effects, experiments with Graves’ IgGs were
conducted in the presence and absence of a selective hTSHR antagonist
(asialoagalacto-hCG). Asialoagalacto-hCG nearly completely abolished
the stimulatory effect of Graves’ IgGs on ICAM-1 expression and
significantly reduced the combined bTSH/interferon- effect. It
failed, however, to affect interferon- action. In
vitro studies using human thyroid cells in primary culture
confirmed the in vivo observations; treatment with
saline resulted in 14% of cells expressing ICAM-1, with pooled normal
IgGs (500 mg/L) in 18% and with Graves’ IgGs (patient A, 448 mg/L;
patient B, 260 mg/L) in 78% and 51%, respectively. Upon exposure to
Graves’ IgGs (90 mg/L) plus asialo-hCG (350 mg/L), 25% of the cells
stained positively for ICAM-1, 29% to bTSH (10 IU/L), 31% to
recombinant human TSH (10 IU/L), and 84% to interferon- (10
IU/mL). In conclusion, stimulation of human thyroid cells, either transplanted to the nude mouse in vivo or studied under in vitro conditions, with Igs derived from patients with Graves’ disease increased the expression of ICAM-1 on the surface of the cells. The action appears to be specific and mediated by the hTSHR. This particular property of TSHR autoantibodies may be of pathophysiological relevance in Graves’ disease, as it may assist in targeting the autoimmune attack and in promoting lymphocyte recruitment to the thyroid gland.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(1, 2). Treatment of the animals with TSH antagonist
(asialoagalacto-hCG) could prevent Graves’ IgG-induced HLA-DR
expression, suggesting a specific, TSH receptor (TSHR)-mediated mode of
action (2). A role for the human TSHR (hTSHR) in the induction of HLA
class I and class II antigen expression was further corroborated by
in vitro studies demonstrating transcription and expression
of the antigens in a human thyroid epithelial cell line that was
exposed to monoclonal antibodies (mAb) raised against the hTSHR (3).
Remarkably, the effect of hTSHR mAb was comparable in magnitude to that
of interferon-.
Because these findings suggested a role for the hTSHR in immunological
stimulation of the thyroid (4, 5, 6), our present studies were designed to
further assess the putative immunomodulatory properties of Graves’
Igs. Our interest focused on the influence of Graves’ Igs and, for
comparison, other substances, such as interferon- and bovine (bTSH)
and recombinant hTSH (rhTSH), on the expression of intercellular
adhesion molecule-1 (ICAM-1), an adhesion receptor, that plays an
important role in the recruitment of lymphocytes to target tissues
(7, 8, 9, 10, 11, 12, 13). The studies were conducted in the nude mouse transplanted with
human thyroid in vivo, and the results were further
corroborated by in vitro experiments with human thyroid
cells in primary culture.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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bTSH (2 IU/mg) was purchased from Sigma Chemical Co.
(Deisenhofen, Germany), rhTSH was obtained from Genzyme (Cambridge,
MA), recombinant human interferon- (2 x 107 IU/mg)
was purchased from Boehringer Mannheim (Mannheim, Germany). The murine
IgG1 mAb against ICAM-1 (CD54) was obtained from Immunotech (Hamburg,
Germany). Radioactive iodine was purchased from Amersham Buchler
(Braunschweig, Germany), polyclonal goat anti-T4 antibody
was obtained from Bio-Yeda (Rehovot, Israel), rabbit antigoat IgG was
purchased from Renner (Dannstadt, Germany), and rabbit antimouse IgG
and horseradish peroxidase-conjugated swine antirabbit Ig were
purchased from Dakopatts (Hamburg, Germany). Thyroglobulin antibodies
(monoclonal mouse antihuman thyroglobulin; 5 µg/mL) were obtained
from Dako (Hamburg, Germany); thyroid peroxidase antibodies (mouse mAb;
0.45 g/L) were obtained from Biocytex (Marseille, France).
Immunohistochemical studies were performed using the Vectastain Elite
ABC kit (Serva, Heidelberg, Germany).
Preparation of Graves’ Igs
IgG fractions from sera of patients with Graves’ disease and healthy individuals were purified by a protein A-Sepharose method (14). Briefly, a column of protein A-Sepharose (5 mL) was loaded with approximately 4 mL patient’s serum and subsequently eluted with 10 mmol/L Tris-Cl buffer containing 50 mmol/L NaCl, pH 7.45. The column was then washed with 0.1 mol/L glycine solution, pH 3.0, to recover the IgGs that had been retained by the column. The IgGs were dialyzed, lyophilized, and appropriately diluted in saline before use. TSH binding inhibitory Igs (TBII) activity was measured by a commercially available assay (TRAK assay, Brahms, Berlin, Germany).
Preparation of asialoagalacto-hCG
The procedures used for the preparation of hCG, asialo-hCG, and
asialoagalacto-hCG were described in detail previously (1, 2). Briefly,
hCG (
13,500 IU/mg) was purified from a crude commercial preparation
(Ayerst, Rouses Point, NY) and subjected to sequential treatment with
immobilized neuraminidase from Clostridium perfringens
(Sigma; incubation of 0.2 IU enzyme and 10 mg hCG in 0.1 mol/L sodium
acetate buffer, pH 5.6, at 37 C for 30 min) and ß-galactosidase from
Aspergillus niger (Sigma; using 1 IU/mL enzyme in 0.05 mol/L
sodium acetate buffer, pH 4.6, at room temperature for 24 h) to
obtain the desialylated variant as well as a variant suitable for
in vivo use that lacked both sialic acid and galactose
residues. Removal of sialic acid and galactose residues was
approximately 90% complete, as judged from measurements of sialic acid
content [coloric method of Warren (15)] and galactose concentrations
released [copper reduction method of Somogyi and Nelson (16)]. The
properties of these hCG forms have been intensively studied in terms of
their physical behavior (by gel chromatography and SDS-PAGE),
immunological activities (by various immunoassays specific for holo-hCG
and its free subunits), and interaction with both testicular hCG
receptor and hTSHR. In this respect, the material used in the present
studies was comparable to previous lots, the properties of which were
reported (1, 2, 17, 18).
In vivo studies in the nude mouse bearing human thyroid transplants
Normal human thyroid tissue was obtained from patients without
thyroid disease undergoing neck surgery for various malignancies.
Tissue slices of 4 x 3 x 2 mm were transplanted to athymic
nude mice (strain NMRI; age, 5–6 weeks; weight, 28–30 g), with each
animal receiving two transplants (1, 2, 19, 20, 21, 22). The experiments were
performed 8 weeks after transplantation. At that time the animals were
injected iv on 2 consecutive days with single doses of the following
substances: saline (0.1 mL), bTSH (0.1 mIU), increasing doses of normal
and Graves’ Ig (0.05–1 mg), increasing doses of asialoagalacto-hCG
(0.25–1 mg), or a combination of Graves’ IgGs and asialoagalacto-hCG
(with the hCG variant given 5 min before the administration of IgGs).
For comparison as positive controls, some animals were treated with
interferon-, which, in contrast to the other agents, was supplied by
continuous infusion (Alzet 2002 minipump, London, UK) at a rate of 200
IU/24 h. Each treatment group consisted of six animals.
To determine the functional effects of the thyroid stimulators, we assessed thyroid hormone production and cellular hypertrophy of the transplant thyrocytes, as described previously (1, 2, 19, 20, 21). Briefly, thyroid hormones newly synthesized and released into the circulation were radioactively labeled by injecting 5 mCi 131I on day 2, and [131I]T4 was subsequently measured in serum with the use of a double antibody technique (goat anti-T4 antibody and rabbit antigoat antibody) 24 h later. Results are expressed as the percentage of [131I]T4 of the total radioactivity in the serum. As a measure of cellular hypertrophy, nuclear volumes of thyroid follicular cells were determined microscopically in paraffin-embedded transplant sections.
To assess ICAM-1 expression, frozen sections (10 µm) of the transplants, which were removed on day 2, were obtained, air-dried, fixed in cold acetone (15 min), and incubated with monoclonal ICAM-1 antibody (dilution, 1:100) for 1 h at room temperature, followed by rabbit antimouse IgG (dilution, 1:100; 30 min) and horseradish peroxidase-conjugated swine antirabbit IgG (dilution, 1:100; 30 min). Immunoreactivity was developed with a solution of 0.02% 3,3'-diaminobenzidine in 0.05 mol/L Tris-Cl buffer, pH 7.6, containing 0.005% hydrogen peroxide and counterstained with Meyer’s hematoxylin. Between incubations, sections were washed three times for 10 min each time with phosphate-buffered saline (PBS; pH 7.2). Control incubations, carried out in the absence of the primary antibody or an irrelevant murine mAb, failed to reveal specific immunoreactivity. Staining was evaluated in coded sections by microscopic examination at a magnification of 160-fold. Percentages of ICAM-1-positive thyroid follicular cells in each transplant were determined by examination of five sections obtained from different areas of the transplant, each comprising at least 100 follicular cells. Results were expressed as the mean \ SD (1, 2, 22).
The protocol of the present studies was approved by the local ethics committee, and they were performed in compliance with the guidelines for animal research contained in the Declaration of Helsinki.
In vitro studies with human thyroid cells in primary culture
Human thyroid cells were prepared following the method of Hinds et al. with minor modifications (23). Briefly, thyroid tissue obtained from patients undergoing thyroid surgery for euthyroid goiter was liberated from fat and connective tissue, cut into small pieces, washed three times in cold PBS (20-fold volume, centrifugation at 70 x g for 5 min), resuspended in 40 mL PBS (without calcium and magnesium) containing 5 mg/mL dispase II, and incubated at 37°C for 30 min, followed by centrifugation at 70 x g for 5 min. Incubations were repeated seven times with alternate use of collagenase (1 g/L) and dispase, finally yielding thyroid follicles and single cells. Cells were washed in RPMI 1640 containing 20 mmol/L HEPES, 10% FCS, 100 U/mL penicillin G, and 100 mg/L streptomycin, and transferred to culture flasks. Human thyroid cells were propagated in DMEM containing 10% FCS, penicillin (100 U/mL), and streptomycin (100 mg/L) in a humidified 5% CO2 incubator at 37°C.
Cultured thyroid cells were plated directly onto multichamber slides
and grown to near confluence. Graves’ Igs and normal Igs (dilutions,
1:10, 1:50, 1:100, and 1:500 in DMEM containing 2.5% FCS) were added,
and incubations were continued for 48 h. Parallel monolayers were
treated with interferon- (10 U/mL), bTSH and rhTSH (10 IU/L), and
Graves’ Igs in combination with asialo-hCG. Thereafter, monolayers
were washed with PBS and fixed in 100% methanol for 10 min at 4°C.
Air-dried slides were rehydrated in PBS and preincubated for 20 min
with normal horse serum to inhibit nonspecific binding. Cell monolayers
were then incubated with murine monoclonal ICAM-1 antibody (3.3 mg/L;
1:30) for 90 min at room temperature, washed again, and incubated with
biotin-conjugated antimouse Ig for 1 h at room temperature,
followed by incubation with preformed avidin and biotinylated
horseradish peroxidase macromolecular complex. Diaminobenzidine was
used as the chromogen and yielded a bluish-black precipitate indicative
of ICAM-1-like immunoreactivity. Slides were counterstained with
malachite green for 5 min before mounting. In addition,
immunohistochemical staining was performed on parallel monolayers using
mouse monoclonal antihuman thyroid peroxidase antibody (0.45 g/L) and
mouse monoclonal antihuman thyroglobulin antibody (5 mg/L). Parallel
monolayers with the primary and secondary antibodies replaced in turn
by isotype-matched nonimmune IgG were examined to assure specificity
and to exclude cross-reactivities between the antibodies and conjugates
employed. At least 200 individual cells in 4 randomly selected visual
fields were evaluated.
Statistical methods
For statistical analysis of the data, Wilcoxon’s test for unpaired observations was used.
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Results |
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Seven IgG preparations, obtained from patients with active untreated Graves’ disease and elevated titers (>20) in the TBII assay, were selected for the present studies. Five Graves’ IgGs were able to functionally activate the human thyroid transplants, as evidenced both by a dose-dependent increase in serum [131I]T4 concentrations and an increase in the nuclear volumes in the transplant thyrocytes, whereas normal IgGs were ineffective. T4 production increased from approximately 10–15% to 40–50%, and nuclear volumes rose from 85–95 mm3 to as much as 160 mm3 (n = 6; P < 0.01) in response to stimulation by potent Graves’ IgG preparations (1 mg/animal). For comparison, 0.1 mg of a potent Graves’ IgG was approximately equipotent to 0.1 mIU bTSH.
ICAM-1 expression in thyroid transplants
In parallel to their effects on thyroid hormone production and
cellular hypertrophy, Graves’ Igs induced expression of ICAM-1 on the
surface of thyroid follicular cells in the human transplants. This
effect was reproducible, marked, and dose dependent; a typical example
is depicted in Fig. 1
. Pooled normal IgGs were
ineffective (<5% of the cells staining positively for ICAM-1). The
percentage of ICAM-1-positive thyroid follicular cells induced by
stimulation with Graves’ IgGs was comparable to that conferred by
infusion of interferon- (Fig. 2). Correlation of
ICAM-1 expression with TBII and thyroid-stimulating antibodies was not
attempted because of the small number of sera tested and a possible
selection bias. bTSH showed little effect by itself, as did
asialoagalacto-hCG, but was capable of further enhancing ICAM-1
expression induced by interferon- (Fig. 2).
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). In contrast, administration of
the hCG variant did not affect interferon--induced ICAM-1 expression
by transplant thyrocytes (data not shown). Upon combined stimulation
with bTSH and interferon-, a significant decrease in the percentage
of ICAM-1-positive thyrocytes was observed after administration of the
hCG variant (Fig. 3b). This reduction was similar to the level of
ICAM-1 expressed after stimulation with interferon- in the absence
of bTSH.
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To corroborate the in vivo findings, in
vitro studies using human thyroid cells in primary culture were
conducted. The incubation period was the same (48 h); however, the
Graves’ IgG preparations used were different from those that had been
employed for in vivo studies. Similar to the in
vivo results, ICAM-1 expression by cultured human thyrocytes was
markedly enhanced after exposure to Graves’ IgGs (Fig. 4). Figure 5 shows the dose-related
effects of two different Graves’ IgG preparations. These Graves’ sera
were tested for possible cytokine contamination by highly sensitive
ELISAs and failed to reveal any detectable levels of interferon-,
interleukin-1
(IL-1), IL-1ß, or tumor necrosis factor-
(TNF). Moreover, coadministration of neutralizing polyclonal
antibodies directed against interferon-, IL-1, and TNF failed to
alter the level of ICAM-1 expressed by human thyroid follicular cells
exposed to Graves’ IgGs.
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). Exposure of monolayers to bTSH and rhTSH stimulated
ICAM-1 expression to a modest degree [mean \ SD,
29 \ 6% and 31 \ 7% (n = 3), respectively,
vs. 14 \ 6% of basal activity (n = 5);
P < 0.05]. As expected, interferon- markedly
enhanced ICAM-1 expression, with 84 \ 13% (n = 3;
P < 0.05) of cells staining positively after exposure
to interferon- (10 IU/mL) for 48 h.
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and 7). The inhibitory effect of 350
mg/L asialo-hCG could be overcome by the addition of higher amounts of
Graves’ IgG, suggesting a competitive mode of action of the
antagonist. In contrast, incubation with asialo-orosomucoid, an
asialo-glycoprotein capable of binding to lectins but not to hTSHR,
failed to exert any inhibitory effect on Graves’ IgG-mediated ICAM-1
expression by human thyrocytes (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß, and IL-6, but not by TSH (39, 40). It is,
however, important to note that discrepancies have been observed
between results obtained in vitro and in vivo,
and in vivo expression of ICAM-1 by thyroid epithelial cells
in Graves’ disease has been questioned (37). The present study was designed to analyze ICAM-1 expression both in the nude mouse bearing human thyroid transplants under in vivo conditions and in cultured human thyroid cells. We were particularly interested in determining whether, in addition to the well known effect of cytokines, Graves’ Ig are capable of inducing ICAM-1 and, if so, whether this effect might be related to their specific interaction with the hTSHR. The functional responsiveness of human transplants to Graves’ Ig has been well documented by 131I scintigraphy, thyroid hormone measurements, and histomorphological criteria, such as cellular hypertrophy (1, 2, 19, 20, 21, 22). Due to differences in magnitude and species specificity of the action of Graves’ Igs, greater responsiveness to TSHR antibodies of homologous human thyroid transplants compared to heterologous mouse thyroid tissue, the stimulation of which occurs inconsistently, has been noted (41, 42).
As an immunological model, the athymic nude mouse is deficient in T cell function. Immunocompetent cells, such as mononuclear cells and lymphocytes, originally present in human transplants, had disappeared at the time the experiments were conducted 8 weeks after transplantation (43, 44).
The main finding of the present study is a pronounced and
dose-dependent stimulatory effect of Graves’ IgGs on ICAM-1 expression
by human thyrocytes. The effect was demonstrated both in the nude mouse
model under in vivo conditions and in cultured thyroid
monolayer cells in vitro. The potency of Graves’ IgGs was
comparable to that of interferon-, a known potent stimulator of
ICAM-1 expression. The action of Graves’ IgGs appeared to be specific
and mediated by their ability to bind and activate hTSHR, because it
was not observed with control IgGs derived from normal subjects and was
abolished by blockade of hTSHR with selective hCG-derived TSHR
antagonists (but not other asialoglycoproteins, such as
asialo-orosomucoid). The desialylated hCG forms have previously been
shown to specifically bind to recombinant hTSHR, to inhibit
bTSH-stimulated cAMP production and T3 release in human
thyroid membranes or slices, and to antagonize the actions of both bTSH
and Graves’ Igs in the nude mouse model (1, 2, 17, 18, 45). Cytokine
contamination of IgGs used for in vitro studies was excluded
by measurements of interferon-, IL-1, IL-1ß, and TNF. Also,
contamination of IgG preparations with cytokines is unlikely to account
for the pronounced in vivo effects that were observed with
single injections of Graves’ IgG in vivo because the agents
are short lived and have to be supplied by continuous infusion to exert
their effects.
bTSH and rhTSH proved to be less potent stimulators of ICAM-1 expression than Graves’ IgGs. The discrepancy between the activities of these two stimulators was more pronounced in vivo than in vitro, which may be explained by marked differences in their plasma half-lives. The differing potencies in vitro could perhaps result from specific interactions of Graves’ IgGs and TSH with different regions of the extracellular domain of hTSHR. Divergence of cAMP-activating signaling pathways between IgGs and TSH has been documented with the use of chimeric TSH/LH/CG receptors (46). Furthermore, hTSHR has been reported to couple to numerous G proteins, although their effects and their activations by various ligands remain poorly understood (47).
In addition to their roles in mediating the functional consequence of hyperthyroidism in Graves’ disease, our present data link hTSHR activation by Graves’ Igs to another important mechanism in the evolution of autoimmune thyroid disease, namely expression of ICAM-1. Expression of adhesion molecules in concert with HLA-DR expression, which has previously been shown to be induced by TSHR antibodies (2, 3), may attract lymphocytes to the thyroid gland and facilitate immune-endocrine interactions via the delivery of important costimulatory signals. Of note, peripheral blood mononuclear cells have been reported to home to their autologous thyroid target in the severe combined immune deficiency (SCID) mouse model (48). Similarly, human intrathyroidal lymphocytes have been shown to induce thyroid hyperfunction when injected into nude mice (49).
Our data indicate that thyrocytes are induced to express both HLA class II antigens and adhesion molecules upon hTSHR activation by Graves’ Igs, and that intact T cell function or cytokines may not necessarily be required for this effect to occur. They do not answer, however, the question of whether in autoimmune thyroid disease the expression of these antigens might be a primary event rather than a secondary phenomenon related to the progression of the disease. In experiments with transgenic mice, expression of class II antigens by pancreatic islet cells was not sufficient to cause diabetes; however, to our knowledge, simultaneous expression of class II antigens and adhesion molecules has not been examined (50).
With regard to therapeutic implications, blockade of TSHR by competitive TSHR antagonists may be effective in limiting both functional hyperstimulation and immunological alterations of thyroid cells, such as expression of adhesion molecules and class II antigens, both of which are mediated by TSHR antibodies. Inhibition of expression of these molecules could perhaps down-regulate the autoimmune process in Graves’ disease, because in a rat model of experimental autoimmune thyroiditis, neutralization of ICAM-1 by mAb has recently been shown to suppress lymphocytic infiltration and inhibit cell-mediated immune mechanisms (51).
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Footnotes |
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Received February 14, 1997.
Revised February 11, 1997.
Accepted March 20, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, tissue
distribution, biochemistry and function of a natural adherence molecule
(ICAM-1). J Immunol. 137:245–254.[Abstract]
amma upregulate the
expression of intercellular adhesion molecule-1 (ICAM-1) in rat thyroid
cell line, FRTL-5. Korean J Intern Med. 9:88–92.[Medline]
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