Regulation of Intercellular Adhesion Molecule-1 Expression in Human ...

0021-972X/97/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1997 by The Endocrine Society

Vol. 82, No. 7 Printed in U.S.A.

Regulation of Intercellular Adhesion Molecule-1 Expression in Human Thyroid Cells in Vitro and Human Thyroid Tissue Transplanted to the Nude Mouse in Vivo: Role of Graves’ Immunoglobulins and Human Thyrotropin Receptor* R. HOERMANN, C. SPITZWEG, S. POERTL, K. MANN, A. E. HEUFELDER, AND P. M. SCHUMM-DRAEGER 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 ABSTRACT To further explore a potential role for the human TSH receptor (hTSHR) in the propagation of thyroid autoimmune disease, we examined immunomodulatory effects in response to its stimulation by Graves’ Igs both in human thyroid tissue transplanted to the nude mouse and in primary cultures of human thyrocytes. We injected nude mice bearing transplants derived from normal human thyroid with protein A-Sepharose-purified Graves’ IgGs (0.05–1 mg) on 2 days and assessed, in addition to functional stimulation, the expression of intercellular adhesion molecule-1 (ICAM-1) by transplant thyrocytes. In parallel to functional stimulation, as demonstrated by thyroid follicular cell hypertrophy in the transplants and increased T4 production, Graves’ IgGs induced a marked dose-dependent expression of ICAM-1 by transplanted thyrocytes, which exceeded that of a continuous interferon-g infusion (200 IU/24 h) for 2 days. Normal IgGs were ineffective. Bovine TSH (bTSH) had little effect by itself, but did enhance interferon-g-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-g effect. It failed, however, to affect interferon-g 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-g (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. (J Clin Endocrinol Metab 82: 2048 –2055, 1997)

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against the hTSHR (3). Remarkably, the effect of hTSHR mAb was comparable in magnitude to that of interferon-g. Because these findings suggested a role for the hTSHR in immunological stimulation of the thyroid (4 – 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-g 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 (7–13). 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.

E PREVIOUSLY demonstrated that Graves’ Igs are capable of inducing expression of HLA class II antigen (HLA-DR) in normal human thyroid tissue transplanted to the nude mouse, as is interferon-g (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

Received February 14, 1997. Revision received February 11, 1997. Accepted March 20, 1997. 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. * Portions of these results have been presented in abstract form at the 11th International Thyroid Congress, Toronto, Canada, 1995, and at the 39th Symposium of the German Society of Endocrinology (DGE), Leipzig, Germany, 1995.

Materials and Methods Materials bTSH (2 IU/mg) was purchased from Sigma Chemical Co. (Deisenhofen, Germany), rhTSH was obtained from Genzyme (Cambridge,

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ICAM-1 EXPRESSION AND TSHR MA), recombinant human interferon-g (2 3 107 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 mg/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 b-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 3 3 3 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 –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-g, 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 –21). Briefly,

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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 mm) 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% 3,39-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 phosphatebuffered 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 6 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 3 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 3 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-g (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.

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Statistical methods For statistical analysis of the data, Wilcoxon’s test for unpaired observations was used.

Results Thyroid hormone production in the nude mouse and cellular morphometry in human thyroid transplants

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 5 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-g (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.

FIG. 2. ICAM-1 expression by human thyroid follicular cells in human thyroid transplants of nude mice after injection of various agents: saline, asialoagalacto-hCG (asag-hCG; 1 mg/animal), bTSH (0.1 mIU/animal), interferon-g (infusion of 200 IU/24 h), and a combination of interferon-g and bTSH. Results are expressed as percentages of thyroid follicular cells staining positively for ICAM-1 (mean 6 SD; n 5 6).

bTSH showed little effect by itself, as did asialoagalacto-hCG, but was capable of further enhancing ICAM-1 expression induced by interferon-g (Fig. 2). Treatment of animals with the TSHR antagonist asialoagalacto-hCG almost completely prevented induction of ICAM-1 on transplant thyrocytes by Graves’ IgGs (Fig. 3a). In contrast, administration of the hCG variant did not affect interferon-g-induced ICAM-1 expression by transplant thyrocytes (data not shown). Upon combined stimulation with bTSH and interferon-g, 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-g in the absence of bTSH. ICAM-1 expression by human thyrocytes in primary culture

FIG. 1. ICAM-1 expression by thyroid follicular cells in human thyroid transplants in response to treatment of animals with Graves’ Igs. Mice were injected on 2 consecutive days with the various doses of a protein A-purified IgG preparation obtained from a patient with untreated active Graves’ disease. The data shown represent the mean 6 SD of experiments conducted with six animals per group. *, P , 0.01.

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-g, interleukin-1a (IL-1a), IL-1b, or tumor necrosis factor-a (TNFa). Moreover, coadministration of neutralizing polyclonal antibodies directed against interferon-g, IL-1, and TNFa failed to alter the level of ICAM-1 expressed by human thyroid follicular cells exposed to Graves’ IgGs.

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FIG. 3. a and b, Inhibition by asialoagalacto-hCG of ICAM-1 expression in transplanted human thyroid follicular cells induced by Graves’ IgGs (0.1 mg/animal; a) and a combination of bTSH (0.1 mIU/animal) and interferon-g (200 IU/24 h; b). Data represent the mean 6 SD obtained in six animals per group. *, P , 0.01 compared to IgGor TSH only values.

In contrast, normal IgGs, tested both as individual samples and after pooling of sera obtained from 50 healthy blood donors, failed to significantly increase ICAM-1 expression over basal values (Fig. 6). Exposure of monolayers to bTSH and rhTSH stimulated ICAM-1 expression to a modest degree [mean 6 sd, 29 6 6% and 31 6 7% (n 5 3), respectively, vs. 14 6 6% of basal activity (n 5 5); P , 0.05]. As expected, interferon-g markedly enhanced ICAM-1 expression, with 84 6 13% (n 5 3; P , 0.05) of cells staining positively after exposure to interferon-g (10 IU/mL) for 48 h. Coincubation of Graves’ IgGs at approximately the halfmaximum stimulatory concentration together with increasing concentrations of asialo-hCG resulted in a dose-dependent decrease in ICAM-1 expression. At a concentration of 350 mg/L asialo-hCG, nearly total inhibition of the stimulatory effect of Graves’ IgGs on ICAM-1 expression was observed (Figs. 4 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 asialoorosomucoid, 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). Discussion

Lymphocytic infiltration of the thyroid gland is a histopathological hallmark of autoimmune thyroid disease. To improve our understanding of the mechanisms that facilitate recruitment of lymphocytes, targeting the autoimmune attack to the thyroid gland has, therefore, become an important challenge. Members of the Ig superfamily, termed adhesion molecules, have been identified to play an important role in the recruitment and homing of lymphocytes to target tissues;

ICAM-1 is one of these (7, 9, 10, 13, 24). ICAM-1 has been reported to be induced in many different cell types upon exposure to various cytokines (8, 11, 12). Compared to controls, increased levels of ICAM-1 expression have been reported by several investigators in both thyroid and retroocular connective tissue specimens derived from patients with Graves’ disease and Hashimoto’s thyroiditis (25–31). Moreover, some, but not all, researchers reported ICAM-1 expression by thyroid epithelial cells and orbital fibroblasts in addition to leukocytes and endothelial cells, and certain differences have been noted between Graves’ disease and Hashimoto’s thyroiditis (32–38). ICAM-1 expression may also occur spontaneously in a proportion of Graves’ and normal thyrocytes under conditions of monolayer culture and may be readily induced by exposure to various cytokines (25, 32, 37). In FRTL-5 cells, expression of a rat homolog of ICAM-1 was low under basal conditions and markedly enhanced by cytokines, such as interferon-g, IL-1b, 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,

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FIG. 4. Monolayer culture of thyroid cells. Staining of cells with thyroid peroxidase mAb (a) and ICAM-1 mAb (b– e). Cells had been exposed for 48 h to medium alone (a and b), interferon-g (10 IU/mL; c), Graves’ IgGs (90 mg/L; d), and a combination of Graves’ IgGs (90 mg/L) and asialo-hCG (350 mg/L; e).

2, 19 –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 in-

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FIG. 5. ICAM-1 expression by thyroid follicular cells in monolayer culture in response to stimulation by IgGs obtained from two patients (patients A and B) with active Graves’ disease. Results are the means of triplicate (basal values, 5-fold) determinations 6 SD. *, P , 0.05 compared to basal values.

FIG. 6. ICAM-1 expression by thyroid follicular cells in vitro after exposure to pooled normal IgGs (500 mg/L), bTSH (10 IU/L), rhTSH (10 IU/L), and interferon-g (10 IU/mL). Results are the means of triplicate or 5-fold (basal values) determinations 6 SD. *, P , 0.05 compared to basal values.

terferon-g, 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

FIG. 7. Inhibition of ICAM-1 expression induced by IgGs from patients A (90 mg/L) and B (52 mg/L) with Graves’ disease in human thyroid cells in monolayer culture by coincubation of the cells with asialo-hCG (350 mg/L). Results are the means of quadruplicate or 5-fold (basal-values) determinations 6 SD.

excluded by measurements of interferon-g, IL-1a, IL-1b, and TNFa. 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 inter-

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actions 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). References 1. Hoermann R, Schumm-Draeger PM, Rehbach K, Mann K. 1991 Asialoagalacto-human chorionic gonadotropin, a carbohydrate-modified variant of human chorionic gonadotropin, antagonizes the stimulatory actions of bovine thyroid-stimulating hormone on thyroid function and HLA-DR expression in human thyroid in vitro and in vivo. J Clin Invest. 88:1947–1954. 2. Hoermann R, Schumm-Draeger PM, Mann K. 1993 Inihibition of functional and immunological responses to thyroid-stimulating antibodies from patients with Graves’ disease by blockade of the thyrotropin receptor. Thyroid. 4:273–278. 3. Ropars A, Marion S, Takorabet L, Braun J, Charreire J. 1994 Antibodies specific for human thyrotropin receptor induce MHC antigen expression in thyroid cells. J Immunol. 153:3345–3352. 4. Smith BR, McLachlan SM, Furmaniak J. 1988 Autoantibodies to the thyrotropin receptor. Endocr Rev. 9:106 –121. 5. Volpe´ R. 1991 Autoimmunity causing thyroid dysfunction. Endocrinol Metab Clin North Am. 20:565–587.

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