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Endocrinology Vol. 147, No. 4 1941-1949
Copyright © 2006 by The Endocrine Society

Immunoglobulin G from Patients with Graves’ Disease Induces Interleukin-16 and RANTES Expression in Cultured Human Thyrocytes: A Putative Mechanism for T-Cell Infiltration of the Thyroid in Autoimmune Disease

Andrew G. Gianoukakis, Raymond S. Douglas, Chris S. King, William W. Cruikshank and Terry J. Smith

Divisions of Molecular Medicine and Endocrinology and Metabolism (A.G.G., R.S.D., C.S.K., T.J.S.), Harbor-UCLA Medical Center, Torrance, California 90502; Jules Stein Eye Institute (R.S.D., T.J.S.) and the David Geffen School of Medicine at the University of California Los Angeles, Los Angeles, California 90095; and the Pulmonary Center (W.W.C.), Boston University School of Medicine, Boston, Massachusetts 02118

Address all correspondence and requests for reprints to: Terry J. Smith, Division of Molecular Medicine, Building C-2, Harbor-UCLA Medical Center, Torrance, California 90502. E-mail: tjsmith{at}ucla.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mechanisms underlying lymphocyte infiltration of the thyroid gland and orbit in Graves’ disease (GD) are poorly understood. The IGF-I receptor (IGF-IR) is a newly recognized self-antigen that, when activated in GD fibroblasts by IGF-I or GD-IgGs, provokes the expression of IL-16 and RANTES (regulated upon activation, normal T cell expressed and secreted)-dependent T lymphocyte chemoattraction and hyaluronan synthesis. IL-16 is a CD4+-specific ligand, and RANTES is a C-C chemokine. Here we report that IGF-I and GD-IgG could induce IL-16 and RANTES in cultured human thyrocytes in a time-dependent manner. Importantly, human TSH failed to induce either chemoattractant. This induction could be attenuated by dexamethasone. Rapamycin, a specific inhibitor of the FRAP/mammalian target of rapamycin/p70s6k pathway, prevented GD-IgG-provoked IL-16 synthesis. IH7, a monoclonal antibody directed at IGF-IR also blocked the induction of chemoattraction as well as RANTES mRNA synthesis. Our findings suggest that thyrocytes can be activated by GD-IgG and IGF-I to express powerful T-cell chemoattractants. These actions of GD-IgG appear to be mediated through pathways independent of the TSH receptor. Thus, in GD, thyrocytes may participate directly in lymphocyte recruitment through their expression of IL-16 and RANTES.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GRAVES’ DISEASE (GD), an autoimmune disease involving the thyroid gland, is characterized by the presence of anti-TSHR (TSH receptor) antibodies. Lymphocytic infiltration predominates the histopathological picture in GD, comprising CD4+ T helper cells (1, 2). T cells trafficked to the gland are thought to orchestrate inflammation and tissue remodeling through their expression and release of cytokines. Important to the process of lymphocyte recruitment are chemoattractants that activate target cells and enhance their migration toward sites of inflammation (3). Currently, little is known about the chemoattractant molecules expressed in the thyroid, the identity of resident thyroid cells expressing them, or the mechanisms through which their production is up-regulated.

Chemokines, small chemoattractant molecules, are classified according to cysteine residue signatures conferring target-cell specificity. RANTES (regulated upon activation, normal T cell expressed and secreted) a C-C chemokine, targets CD4+ memory, activated naive T lymphocytes and monocytes, basophils, and eosinophils (4). Lymphocyte chemoattractant factor or IL-16 lacks a requisite cysteine signature and is therefore not a chemokine. Nonetheless, it is a powerful chemoattractant that specifically targets CD4+-bearing cells (5, 6, 7). IL-16 is expressed by CD8+ and CD4+ lymphocytes (8), eosinophils (9), fibroblasts (10), and thyrocytes (11). It has been implicated in the pathogenesis of asthma (12), rheumatoid arthritis (13), systemic lupus erythematosus (14), and inflammatory bowel disease (15).

Epithelial cells can play important roles as determinants of thyroid gland immunity. Thyrocytes respond to interferon-{gamma} by expressing high levels of HLA-DR and CD40 (16, 17). They produce several cytokines and display on their surface cognate receptors, which in turn can influence glandular function (18, 19, 20). Thyrocytes are capable of elaborating chemoattractants when exposed in vitro to proinflammatory cytokines (11). We have found that IL-1ß can induce IL-16 and RANTES in primary human thyrocytes (11). These cells constitutively express IL-16 mRNA, but IL-16 protein can be detected only after they are treated with IL-1ß. In contrast, the induction of RANTES involves the up-regulation of its mRNA levels. Thus, thyrocytes can express powerful T-cell chemoattractants when activated.

Pritchard et al. (21) have shown recently that Igs from patients with GD (GD-IgG) can induce IL-16 and RANTES expression in fibroblasts from these patients. This up-regulation by GD-IgG is mediated through the IGF-I receptor (IGF-IR) (21), a ubiquitous membrane tyrosine kinase. The effects of GD-IgG can be attenuated with a specific IGF-IR-blocking antibody, 1H7, or by transfecting fibroblasts with a dominant-negative mutant IGF-IR. Moreover, IGF-I and its active analogs can also induce these chemoattractants. Unlike fibroblasts from patients with GD, those from donors without the disease failed to respond to GD-IgG or IGF-I. Thus, the IGF-I/IGF-IR pathway has been identified as a mechanism for activating GD fibroblasts. In this report, we demonstrate that GD-IgG and IGF-I induce IL-16 and RANTES expression in cultured human thyrocytes. These effects occur in thyrocytes from patients with GD as well as those from individuals without autoimmune disease. Moreover, the p70s6k pathway appears to mediate at least in part, these actions. On the other hand, TSH and its receptor do not participate. Elaboration of IL-16 and RANTES by thyrocytes may represent an important mechanism for CD4+ lymphocyte recruitment to the thyroid in GD.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Eagle’s and RPMI medium, fetal bovine serum (FBS), and antibiotics were purchased from Life Technologies (Grand Island, NY). Neutralizing anti-RANTES Abs were from R&D Systems (Minneapolis, MN) and a RANTES-specific ELISA was from BioSource (Camarillo, CA). An affinity-purified polyclonal rabbit anti-recombinant IL-16 (rIL-16) Ab was prepared from rIL-16 immunized rabbit serum as described previously (21). Rapamycin was obtained from Calbiochem (La Jolla, CA), 1H7 from PharMingen (San Diego, CA), dexamethasone and TSH were from Sigma-Aldrich (St. Louis, MO). Collagenase and dispase were from Roche (Indianapolis, IN). Recombinant human TSH (rhTSH) was generously provided by Genzyme (Cambridge, MA).

Human subjects
Tissue and serum samples used for the studies reported were obtained after informed consent using a protocol approval by institutional review boards for both the Harbor-UCLA Medical Center and the Center for Health Sciences at UCLA.

Cell culture
Normal-appearing thyroid tissue was obtained as surgical waste from patients undergoing thyroidectomy for the treatment of a variety of conditions. These included GD, nodular disease, and focal well-differentiated malignancies. Tissues were processed as described previously (22). Fragments were trimmed of connective tissue, finely minced and washed with Hanks’ balanced saline solution. They were digested at 37 C in Hanks’ balanced saline solution containing collagenase type A (130 U/ml) and dispase grade I (0.5 U/ml). Liberated follicles were concentrated by differential centrifugation and sedimentation, pooled, resuspended in RPMI, and filtered through 200-µm nylon mesh. Follicles were pipetted into 25-mm plastic flasks where they attached to the surface and monolayers developed. These were covered with RPMI medium supplemented with 10% FBS, penicillin (50 U/ml), and streptomycin (50 µg/ml). Human fibroblasts were obtained from surgical waste from donors with or without GD. Monolayers were covered with Eagle’s medium supplemented with 10% FBS, antibiotics, and glutamine as described previously (21). Cultures were maintained in a humidified, 5% CO2 incubator at 37 C. They were passaged with gentle trypsin treatment and used between the second and sixth passages (thyrocytes) or third and tenth passage (fibroblasts). Experiments were performed on confluent monolayers. This was usually achieved within 1 wk of culture initiation.

IgG purification
Samples of sera were collected from donors with GD as well as healthy individuals (controls). IgG was prepared using protein A affinity chromatography as described by Hjelm et al. (23). Briefly, protein A-Sepharose (5 ml) columns were loaded with approximately 4 ml serum and eluted with 10 mmol/liter Tris-Cl buffer containing 50 mmol/liter NaCl (pH 7.45). To elute retained IgGs, columns were washed with 0.1 mol/liter glycine solution (pH 3.0). Samples were then dialyzed, lyophilized, and diluted in saline.

Chemotaxis assay
Lymphocyte chemoattraction was assayed, as described previously (24, 25, 26). In brief, primary thyrocytes were seeded in 24-well plates, grown to confluence, rinsed with PBS and shifted to medium containing 1% FBS. Test compounds such as GD-IgG (15 µg/ml) were added, cultures incubated and medium collected and stored at –80 C. Chemotaxis was examined in a modified Boyden chemotaxis chamber using human NWNA-T lymphocytes as the cellular targets. Fifty microliters of a cell suspension (107 cells/ml) were placed in the upper compartments of 48-well microchemotaxis chambers separated from 32 µl of samples by 8-µm micropore nitrocellulose filters (Neuroprobe, Cabin John, MD). These were incubated at 37 C in 5% CO2 for 3 h. Filters were fixed, stained with hematoxylin, dehydrated, mounted on glass slides, and viewed under light microscopy. Lymphocyte migration was quantified by counting the total number of cells migrating beyond a fixed depth which is set up to identify baseline migration (10–15 cells/high power field) under control conditions. Five high-power fields were counted in duplicate for each sample, and the mean ± SD was calculated and expressed as a percentage of baseline cell migration in control buffer alone (100%). Three separate experiments were performed for each set of experimental conditions. Differences between experimental and control conditions were analyzed by Student’s t test using the absolute values obtained for lymphocyte migration and statistical significance was the 95% level of confidence. To assess specificity for IL-16, neutralizing experiments were conducted by incubating culture supernatants for 15 min with neutralizing concentrations of anti-IL-16 monoclonal antibody (mAb) (clone14.1; 5 µg/ml). This neutralizes a chemotactic activity of 50 ng/ml rIL-16. Similarly, anti-RANTES mAb (5 µg/ml) with an ND50 for RANTES was added with a final concentration of 200 ng/ml.

Cytokine-specific ELISA
IL-16 protein was quantified as previously described by Lim et al. (9). rIL-16 and conditioned medium were diluted in PBS to appropriate concentrations. A standard curve was generated using serial dilutions of rIL-16. Samples of the thyrocyte culture medium (100 µl) were incubated in duplicate in a 96-well microtiter plate (Nunc, Naperville, IL) at 37 C for 1 h. Subsequent maneuvers were carried out at RT. The Ag was removed by washing with a solution of PBS/Tween 20. Nonspecific binding was minimized by blocking with 1% BSA for 1 h. After washing, 100 µl anti-IL-16 polyclonal Ab (10 ng/ml) diluted in PBS containing 0.05% Tween 20 was added to each well. IL-16/anti-IL-16 complexes were detected by incubation for 1 h with biotinylated goat antirabbit IgG diluted 1:500 in PBS. RANTES levels were determined using a commercially available ELISA (BioSource) according to the manufacturer’s recommendations. The limits of detection were 12 pg/ml and 5 pg/ml for IL-16 and RANTES, respectively. The specificity of the anti-IL-16 and anti-RANTES antibodies has been established in a number of earlier publications (26, 27, 28).

RNA isolation and RT-PCR
RNA was extracted from confluent thyrocyte monolayer cultures by the method of Chomczynski and Sacchi (29) with an RNA isolation system purchased from Biotecx (Houston, TX). RNA was isolated, quantified, and equal amounts digested with ribonuclease-free deoxyribonuclease 1 (Roche Diagnostics, Indianapolis, IN) and reverse-transcribed with oligo-deoxythymidine (Invitrogen, Carlsbad, CA) as the primer, using an Ominiscript RT kit (QIAGEN, Chatsworth, CA). PCRs were performed using Taq PCR Master Mix kit (QIAGEN). PCR was performed using the following RANTES primers: forward, 5'-GCTGTCATCCTCATTGCTACTG-3' and reverse, 5'-CTGGGGAAGGTTTTTGTAACTG-3'. Conditions for the reaction were: 94 C for 5 min, 94 C for 45 sec, 52 C for 45 sec, and 72 C for 45 sec for 35 cycles. RANTES products were normalized using ß-actin primers 5'-CCAAGGCCAACCGCGAGAAGATGAC-3' (forward) and 5'-AGGGTACATGGTGGTGCCGCCAGAC-3' (reverse) under the following conditions: 94 C for 5 min, 95 C for 1 min, 60 C for 1 min, and 72 C for 1 min for 35 cycles. Generated cDNA was subjected to electrophoresis through denaturing 2% agarose gels. Bands were visualized using ethidium bromide staining under UV light.

Flow cytometry
Thyrocytes were isolated in a single cell suspension using gentle mechanical disruption with a cell scraper. Expression of IGF-IR was determined using a phycoerythrin-conjugated IGF-IR antibody (1 µg/ 1 x 105 cells, clone 1H7; BD Biosciences). TSHR display was determined using a mouse anti-TSHR (clone 4C1; Serotech, Oxford, UK) and fluorescein isothiocyanate-conjugated rabbit antimouse IgG (BD Biosciences, San Jose, CA). In brief, cells were incubated with primary antibodies at 4 C for 30 min and washed three times with staining buffer (PBS with 2% fetal calf serum). Secondary antibody staining was performed in a similar manner. Cells were resuspended in 2% paraformaldehyde and analyzed immediately using a FACSCalibur flow cytometer (BD Biosciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Thyrocytes express and release lymphocyte chemotactic activity attributable to IL-16 and RANTES after treatment with GD-IgG
When treated with GD-IgG, primary thyrocytes release substantial lymphocyte chemotactic activity into the medium, as the data in Fig. 1AGo demonstrate. Thyrocytes, in this case from a patient undergoing thyroidectomy for multinodular goiter, were treated with GD-IgG (15 µg/ml) for the times indicated. Conditioned medium was then subjected to a lymphocyte migration assay. The effect of GD-IgG on T-cell migration is time dependent, becomes detectable by 8 h, and increases to 179% and 200% above control at 16 h and 24 h, respectively. A substantial fraction of the chemoattraction (50–75%) could be neutralized with the addition of anti-IL-16 mAb (5 µg/ml). IGF-I (10 nM) also induces T-cell chemotactic activity in a time-dependent manner (Fig. 2AGo). After 16 h, migration is 178% and at 24 h peaks at 223% above control, the duration of the study. Greater than 50% of the chemoattraction was neutralized with anti-IL-16 mAb.


Figure 1
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  		FIG. 1. GD-IgGs induce in primary human thyrocytes the expression of T-cell chemoattractant activity in a time-dependent manner. This activity derives from the synthesis of IL-16 and RANTES. Thyrocytes, in this case from a patient with a multinodular goiter, were prepared as described in Materials and Methods and allowed to proliferate to near confluence. Some were then treated with GD-IgG (15 µg/ml) for the time intervals indicated along the abscissas. Conditioned media were collected and subjected to a T-cell migration assay in the absence ({blacktriangleup}) or presence ({Delta}) of anti-IL-16 neutralizing antibodies (5 µg/ml; A). Cell migration >135% was significant at the 95% confidence level. Other aliquots of media were subjected to specific ELISAs for IL-16 (B) or RANTES (C). All data are expressed as the mean ± SD of triplicate determinations. Results are representative of three separate experiments.

 

Figure 2
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  		FIG. 2. IGF-I induces in thyrocytes the expression of T-cell chemoattractant activity and IL-16 and RANTES protein expression in a time-dependent manner. Thyrocytes, in this case from a patient with a multinodular goiter, were prepared as described in Materials and Methods and allowed to proliferate to near confluence. Some were then treated with IGF-I (10 nM) for the time intervals indicated. Conditioned media were examined in a T-cell migration assay with ({blacktriangleup}) or without ({triangleup}) the addition of anti-IL-16 neutralizing antibodies (5 µg/ml; A). Cell migration >135% was significant at the 95% confidence level. Other aliquots of media were subjected to specific ELISAs for IL-16 (B) or RANTES (C). All data are expressed as the mean ± SD of triplicate determinations. Results are representative of three separate experiments.

 
We next determined whether IL-16 and RANTES could be detected in conditioned medium from thyrocytes treated with GD-IgG. Both proteins were released in a time-dependent manner as the data in Fig. 1Go, B and C, indicate. They were undetectable by ELISA in control cultures, and after 8 h of treatment. By 12 h, levels of IL-16 reached 59 ± 12 pg/ml and increased to 79 ± 12 pg/ml at 24 h. RANTES was detectable after 12 h and reached 68 ± 14 pg/ml at 24 h (Fig. 1CGo). Assessment of the effects of IGF-I on cytokine levels revealed that the IL-16 concentration reached levels of 74 ± 9 pg/ml and 126 ± 13 pg/ml at 16 h and 24 h, respectively (Fig. 2BGo). RANTES could also be detected after 16 h (58 ± 8 pg/ml) and increased to 93 ± 9 pg/ml after 24 h (Fig. 2CGo). Thus, IGF-I and GD-IgG appear to elicit similar increases in IL-16-dependent T-cell migration as well as IL-16 and RANTES protein levels.

We examined chemoattractant activity elicited by several GD-IgG preparations, each from five different patients with GD and another from a control donor (Fig. 3Go). All donors with GD had active disease, were untreated, and were thyrotoxic. All five samples of GD-IgG increased total T-cell chemoattraction, the responses ranged from 171% to 376% above baseline. A substantial fraction of the increase was attenuated with anti-IL-16 mAb (range 50–100%). In contrast, control IgG failed to up-regulate chemotactic activity in thyrocytes. Results with control IgG are representative of three experiments with a mean chemotaxis of 107 ± 4%.


Figure 3
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  		FIG. 3. GD-IgG from several donors induce T-cell chemoattractant activity in thyrocytes. A substantial component of this activity is attributable to IL-16. Confluent thyrocyte monolayer cultures from a patient with a focal papillary thyroid cancer were treated with nothing, control IgG (15 µg/ml) or GD-IgG from five different donors. Conditioned media were subjected to a T-cell migration assay with or without the addition of anti-IL-16 neutralizing antibodies (5 µg/ml). Cell migration greater than 135% was significant at the 95% confidence level. All data are expressed as the mean ± SD of triplicate determinations.

 
Induction of IL-16-dependent chemoattractant activity and IL-16 protein in thyrocytes by GD-IgG can be blocked by glucocorticoids and rapamycin
Immunomodulatory actions attributed to glucocorticoids in the thyroid and other tissues suggest that these steroids might attenuate the activation and migration of disease-associated lymphocytes. Dexamethasone (10 µM) could completely block the induction of IL-16 by GD-IgG in thyrocytes (Fig. 4Go, A and B) and fibroblasts (Fig. 4Go, C and D). The actions of GD-IgG and IGF-I on IL-16 expression are mediated through the FKBP12-rapamycin-associated protein (FRAP)/mammalian target of rapamycin (mTOR)/p70s6k pathway in GD fibroblasts (21). We therefore determined whether rapamycin (10ng/ml), a macrolide inhibitor of that pathway, could attenuate the induction of IL-16 in thyrocytes. The compound blocked IL-16 up-regulation by greater than 50% in thyrocytes (Fig. 4Go, A and B) and fibroblasts (Fig. 4Go, C and D). Thus, it would appear that FRAP/mTOR plays an important role in signaling initiated by GD-IgG that culminates in IL-16 expression in both cell types.


Figure 4
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  		FIG. 4. The GD-IgG mediated induction of T-cell chemoattractant activity in thyrocytes and fibroblasts can be blocked by dexamethasone and rapamycin. A and B, Confluent thyrocyte monolayers from a donor with a multinodular goiter; C and D, confluent fibroblast monolayers from a patient with GD were treated with nothing (Control), control IgG (15 µg/ml), GD-IgG (15 µg/ml), alone or in combination with dexamethasone (10 µM) or rapamycin (10 ng/ml). The medium was then subjected to a T-cell migration assay with or without the addition of anti-IL-16 neutralizing antibodies (5 µg/ml). Cell migration greater than 135% was significant at the 95% confidence level. Other media aliquots were subjected to specific ELISAs for IL-16. All data are expressed as the mean ± SD of triplicate determinations. Results are representative of three separate experiments.

 
Induction of IL-16-dependent T-cell chemoattraction and IL-16 protein in thyrocytes by GD-IgG can be blocked by a specific blocking anti-IGF-IR{alpha} mAb
Thyrocytes and fibroblasts treated with GD-IgG and IGF-I for 24-h release significant T-cell chemoattractant activity. In contrast, rhTSH (2 mIU/ml) had no such effect on these cells (Fig. 5Go, A and C). Furthermore, IL-16 remained undetectable by ELISA in conditioned medium after treatment with rhTSH (Fig. 5Go, B and D). IL-16-dependent chemoattractant activity in thyrocytes was up-regulated to 189% and 184% above control by GD-IgGs from two separate donors (Fig. 5AGo), and IL-16 protein was induced from undetectable levels to 110 and 136 pg/ml, respectively (Fig. 5BGo). These effects could be variably attenuated by 1H7 (5 µg/ml), a specific blocking anti-IGF-IR{alpha} mAb that lowered chemoattraction to 127% and 174% above control and IL-16 protein to 35 and 122 pg/ml, respectively.


Figure 5
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  		FIG. 5. rhIGF-I, but not rhTSH, induces T-cell chemoattractant activity in thyrocytes and fibroblasts. A blocking anti-IGF-IR mAb attenuates the induction by GD-IgG of T-cell chemoattractant activity in thyrocytes and fibroblasts. Confluent thyrocyte and fibroblast monolayers, each from a donor with GD, were treated with nothing (Control), IGF-I (10 nM), rhTSH (2 mU/ml), GD-IgG (15 µg/ml), alone or in combination with 1H7 (5 µg/ml). The medium was then subjected to a T-cell migration assay with or without the addition of anti-IL-16 neutralizing antibodies (5 µg/ml) (A and C). Cell migration greater than 135% was significant at the 95% confidence level. Other aliquots of media were subjected to specific ELISAs for IL-16 (B and D). All data are expressed as the mean ± SD of triplicate determinations. Results are representative of three separate experiments.

 
GD-IgG induces RANTES mRNA in thyrocytes
We next determined whether the actions of GD-IgG were mediated at the pretranslational level by assessing their impact on steady-state mRNA levels. We have previously reported that human fibroblasts (10) and thyrocytes (11) express high basal levels of IL-16 mRNA that are invariant with regard to treatment with cytokines. As the results from PCR analysis of steady-state mRNA levels found in Fig. 6Go indicate, the RANTES transcript can be detected, albeit at a low level in untreated thyrocyte cultures. By 16 h, the levels have increased substantially. When 1H7 was added to the GD-IgG, the increase in RANTES mRNA was blocked, indicating that IGF-IR is mediating this cellular response.


Figure 6
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  		FIG. 6. GD-IgG induces RANTES mRNA in thyrocytes, an action mediated through IGF-IR. Confluent thyrocyte cultures from a donor with GD were shifted to 1% RPMI medium for 18 h and treated with GD-IgG (15 µg/ml) for 16 h, alone or in combination with 1H7 (5 µg/ml). Cell layers were harvested, RNA extracted, and subjected to quantitative PCR as described in Materials and Methods. Samples were then subjected to ß-actin determinations. Results are representative of two separate experiments.

 
Primary human thyrocytes display TSHR and IGF-IR
Dispersed cultured thyrocytes, in this case from a patient with GD, were subjected to flow cytometry for IGF-IR and TSHR detection. As the data in Fig. 7Go, A and B, indicate, high levels of both surface receptors could be detected in nearly 100% of the cells. Likewise, cultured orbital fibroblasts from a patient with GD and a control subject were also analyzed. GD orbital fibroblasts, like thyrocytes, express high levels of IGF-IR, whereas control orbital fibroblasts do not.


Figure 7
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  		FIG. 7. Thyrocytes display TSHR and IGF-IR on their surface. GD, but not control orbital fibroblasts, express high levels of IGF-IR. Cultured cells were dispersed by a nonenzymatic method and subjected to flow cytometric analysis using mAbs against the two receptors. B–D, Isotype control open histogram and IGF-IR expression dark histogram. A, Isotype control open histogram and TSHR expression dark histogram.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In autoimmune processes such as GD and Hashimoto’s thyroiditis, the thyroid becomes infiltrated with lymphocytes (1, 2). This intrathyroidal accumulation of T cells could underlie B cell activation associated with both diseases (30). Furthermore, lymphocyte-thyrocyte cross talk, through intercellular adhesion molecule-1/lymphocyte function-associated antigen-1 interactions, may enhance epithelial cell proliferation (31). Thyrocytes and recruited T cells might communicate through the CD40/CD154 bridge. CD40, a B-cell surface glycoprotein critical to lymphocyte cross talk, is also displayed on the surface of thyrocytes (17, 32). CD40 initiates TNF receptor-associated factor-dependent signaling when cross-linked with its cognate ligand, CD154 (also known as CD40 ligand), expressed by T cells. Thyroid epithelial cells are capable of expressing a wide array of cytokines including granulocyte-monocyte colony-stimulating factor, IL-1, IL-6, TGF-ß (33, 34) as well as chemokines like IL-8 (34, 35, 36), IP-10/CXCL10, MIG/CXCL9 (37) and MCP-1 (38). They also produce PGE2 and other immunomodulatory molecules (22). We have recently found that IL-1ß-treated thyrocytes express IL-16 and RANTES in vitro (11). The findings reported here extend our characterization of pathways through which thyroid epithelium might orchestrate tissue reactivity and remodeling. A better understanding of the mechanisms underlying trafficking of bone marrow-derived cells is critical to unraveling the nature of thyroid autoimmunity. Dexamethasone nearly completely attenuates the chemoattractant activity induced by GD-IgG and IGF-I in fibroblasts and thyrocytes. This result suggests a potentially important mechanism through which glucocorticoids might exert some of their therapeutic benefit in patients with GD.

GD fibroblasts respond to either IGF-I or GD-IgG, whereas control fibroblasts do not (21). We have also found that GD fibroblasts express and display IGF-IR at substantially higher levels than their control counterparts (21). Unlike fibroblasts, both normal and GD thyrocytes respond to GD-IgG. Moreover, the levels of IGF-IR in cultures from the two sources are similar (our unpublished observations). Thus, we hypothesize that the phenotype of fibroblasts change in GD to an elevated IGF-IR expression state that is ordinarily found in thyrocytes. The relatively low-level IGF-IR expression found in normal fibroblasts may preclude their response to IGF-I and GD-IgG.

The pattern of IL-16 expression and the mechanisms involved in its regulation are cell-type specific (9, 12, 15, 39). T cells express pro-IL-16 protein (39), whereas thyrocytes (11), much like human fibroblasts (10) and mast cells (40), express constitutive IL-16 mRNA, but IL-16 protein is undetectable until these cells are activated. The FRAP/mTOR/p70s6k pathway appears critical to the induction by IGF-I and GD-IgG of IL-16 in fibroblasts and thyrocytes (11, 21). This induction could be blocked by rapamycin. In contrast, RANTES mRNA is detectable in untreated thyrocyte cultures and substantially increased by GD-IgG treatment. Transcriptional regulation of RANTES expression has been documented in several other cell types, including astroglial cells, myofibroblasts and neoplastic cells (10, 41, 42, 43). Attenuation of the GD-IgG stimulated increase in RANTES mRNA levels by 1H7 further supports the central role of IGF-IR in mediating the actions of GD-IgG reported here.

Our findings raise the strong possibility that T-cell recruitment to the thyroid in GD may be dependent, at least in part, on the activities of IL-16 and RANTES. The former is known to influence a wide array of CD4-bearing cells, including lymphocytes, monocytes, and eosinophils (5, 6, 7). IL-16 primes lymphocytes for IL-2-dependent proliferation, protects against FAS-mediated apoptosis (44), induces cell cycle progression (6, 45) and the expression of other cytokines in these cells. Lymphocytes stimulated with IL-16 express IL-3 and GM-CSF, whereas those treated with IL-16 in combination with IL-2 appear to express INF-{gamma} rather than IL-4 or IL-5. These findings suggest that in combination with other cytokines, IL-16 may bias the differentiation of naive T cells toward the Th1 phenotype. The coordinate activation of IL-16 and RANTES production in thyrocytes might underlie the peculiar profile of T-cell recruitment in GD (11). CD4 activation by IL-16 desensitizes T cells to signaling through RANTES/CCR5 and results in the partial inhibition of T-cell migration (46). Thus, the net contribution of CCR5+ cells to the cell infiltrate in the thyroid may condition the immune responses occurring there.

Activating anti-TSHR Abs can be detected in a majority of patients with GD and result in thyroid overactivity. The role of these disease-specific Abs in extrathyroidal, inflammatory manifestations of this disease is certainly possible but has not been established. Our current findings extend previous observations concerning fibroblast activation. They elucidate a heretofore-unrecognized interaction between GD-IgG and thyrocytes, mediated through IGF-IR and productive of chemoattractant expression. It remains uncertain whether these particular responses represent the subset of a larger group of thyrocyte proteins induced as a consequence of IGF-IR activation. Despite abundant surface display of TSHR, rhTSH fails to influence the expression of either IL-16 or RANTES, whereas IGF-I mimics the actions of GD-IgG. Furthermore, the GD-IgG-induced chemoattractant expression can be attenuated by 1H7. Our findings strongly suggest that signaling through TSHR is not involved in these actions of GD-IgG and that IGF-IR displayed by thyrocytes and fibroblasts represents the self-antigen relevant to the induction of IL-16 and RANTES in these cells. The IGF-IR pathway represents a potentially attractive therapeutic target, the interruption of which might ameliorate autoimmune thyroid disease.


   Acknowledgments
 
We are indebted to the Harbor-UCLA General Clinical Research Center for assistance with patient recruitment as well as the Department of Surgery, Harbor-UCLA Medical Center for facilitating sample collection. We thank Ms. Debbie Hanaya for her expert assistance with the preparation of the manuscript.


   Footnotes
 
This work was supported in part by the National Institutes of Health Grants K23 RR017304, EY008976, EY011708, DK063121, and RR 00425.

Disclosure: A.G.G., R.S.D., and C.S.K. have nothing to declare. W.W.C. and T.J.S. are inventors in the U.S.A., patent no. 066742-10493.

First Published Online January 12, 2006

Abbreviations: FBS, Fetal bovine serum; FRAP, FKBP12-rapamycin-associated protein; GD, Graves’ disease; IGF-IR, IGF-I receptor; mAb, monoclonal antibody; mTOR, mammalian target of rapamycin; RANTES, regulated upon activation, normal T cell expressed and secreted; rhTSH, recombinant human TSH; rIL-16, recombinant IL-16; TSHR, TSH receptor.

Received October 31, 2005.

Accepted for publication December 19, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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