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Endocrinology, doi:10.1210/en.2003-0199
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Endocrinology Vol. 144, No. 7 2856-2864
Copyright © 2003 by The Endocrine Society

Cytokine-Induced Lymphocyte Chemoattraction from Cultured Human Thyrocytes: Evidence for Interleukin-16 and Regulated upon Activation, Normal T Cell Expressed, and Secreted Expression

Andrew G. Gianoukakis, Leon J. Martino, Noah Horst, William W. Cruikshank and Terry J. Smith

Department of Medicine (A.G.G., L.J.M., T.J.S.), Albany Medical College, Albany, New York 12208; Pulmonary Center (N.H., W.W.C.), Boston University School of Medicine, Boston, Massachusetts 02118; and Divisions of Endocrinology and Metabolism and Molecular Medicine (A.G.G., T.J.S.), Harbor-University of California Los Angeles Medical Center, Torrance, California 90502; and University of California Los Angeles School of Medicine, Los Angeles, California 90095

Address all correspondence and requests for reprints to: Terry J. Smith, M.D., Division of Molecular Medicine, Harbor-University of California Los Angeles Medical Center, Building C-2, 1124 West Carson Sreet, Torrance, California 90502-2064.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mediators of lymphocyte infiltration in inflammatory thyroid disease have yet to be identified. Here we examine the ability of IL-1ß to enhance the production of chemoattractants by human thyrocytes. Primary cultures, when treated with the cytokine, release T lymphocyte chemotactic activity. The effect of IL-1ß is time dependent, and the chemoattraction activity can be partially attenuated by the addition of either anti-IL-16 or anti-regulated upon activation, normal T cell expressed, and secreted (RANTES) neutralizing antibodies. IL-16 is a CD4+-specific ligand, and RANTES is a C-C type chemokine that targets monocytes and lymphocytes. These chemoattractants could be detected by specific ELISAs in conditioned medium from IL-1ß treated thyrocytes. Northern analysis revealed that thyrocytes express high constitutive levels of IL-16 mRNA, which were invariant with regard to IL-1ß (10 ng/ml) or glucocorticoid treatment. RANTES mRNA was not detected in control cultures but was strongly induced by the cytokine. IL-16 but not RANTES expression was dependent on the activity of caspase-3. Pro-IL-16 protein could be detected in homogenates of thyroid tissue from patients with multinodular goiter and Graves’ disease. Thus, human thyrocytes, through the expression of chemoattractants, may participate in the recruitment of lymphocytes to the thyroid in inflammatory states.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LYMPHOCYTE INFILTRATION IS a prominent feature of thyroid autoimmune disease (1, 2). T cells are thought to orchestrate thyroid inflammation through their secretion of cytokines. The T cell phenotypes that predominate in inflammatory states determine the profile of cytokines generated and thereby the characteristics of tissue remodeling that result. Important components of lymphocyte recruitment are chemoattractants that activate target cells and enhance their accumulation at sites of inflammation. Currently little is known about the chemoattractant molecules expressed in the thyroid, the identity of the cell-type expressing them, or the mechanisms through which their expression is regulated.

The precise role of epithelial cells in determining the immunity of the thyroid is uncertain, but recent evidence suggests that they can interact directly with immune cells. For instance, thyrocytes, although not expressing unprovoked class II molecules, respond to interferon-{gamma} by expressing high levels of HLA-DR (3). They respond to a wide variety of cytokines, which in turn can modify the normal function of the gland (4, 5, 6, 7). Recently, CD40, a B cell surface glycoprotein, critical to lymphocyte cross-talk, was shown to be displayed on the surface of thyrocytes in situ (8) and in culture (9). Furthermore, CD40 was competent to signal when cross-linked with its cognate ligand, CD154, and its expression can be regulated by immunomodulatory molecules (9). In addition, thyrocytes produce the immunomodulatory molecule, prostaglandin E2, and express a characteristic profile of cyclooxygenases under basal and cytokine-provoked conditions (7). It is likely therefore that the epithelium plays an important role in defining the immunity and conditioning inflammatory responses of the thyroid gland.

Chemokines are small molecules that activate target cells and promote migration toward sites of inflammation. They are classified by the presence of particular cysteine residue signatures. It appears that the specificity with which they target cells is dependent on the arrangement of these cysteine residues. Those with two cysteines interrupted by a dissimilar residue are termed C-X-C, and those with adjacent cysteines are classified as C-C chemokines. The latter group contains regulated upon activation, normal T cell expressed, and secreted (RANTES), a molecule using GTP-protein-coupled receptors such as CCR1 and CCR5 expressed on monocytes; resting CD4+ memory; and activated naive T lymphocytes, basophils, and eosinophils (10). Other chemoattractants do not belong to a chemokine family because they lack the requisite cysteine signatures. Lymphocyte chemoattractant factor or IL-16 is a nonchemokine, immunomodulatory molecule that specifically targets CD4+-bearing cells (11, 12, 13). CD8+ and CD4+ lymphocytes were the first cells found to express IL-16 (14). Subsequently, eosinophils (15), fibroblasts (16), and mast cells (17) have been found capable of IL-16 production. Expression and regulation of IL-16 appears to be cell type specific and has been implicated in the pathogenesis of human diseases such as asthma (18), rheumatoid arthritis (19), systemic lupus erythematosus (20), and inflammatory bowel disease (21).

In this article, we demonstrate that human thyrocytes can produce and release T lymphocyte chemoattractant activity when treated with IL-1ß. Much of this activity can be attributed to IL-16 and RANTES. Moreover, both chemoattractants can be detected in conditioned medium by specific ELISAs. Thus, elaboration of IL-16 and RANTES by thyrocytes may represent an important mechanism for CD4+ lymphocyte recruitment to the thyroid during inflammation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
IL-ß, IL-4, TGF-ß, TNF-{alpha}, and interferon-{gamma} were from BioSource Technologies, Inc. (Camarillo, CA). Eagle’s medium, fetal bovine serum (FBS), and antibiotics were purchased from Life Technologies, Inc. (Grand Island, NY). Specific inhibitors of caspase-1 and caspase-3, acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-Ald) and Ac-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), respectively, were obtained from Bachem (Torrance, CA). Neutralizing anti-RANTES antibodies (Abs) were purchased from R&D Systems (Minneapolis, MN) and RANTES-specific ELISA was obtained from BioSource Technologies, Inc. An affinity-purified polyclonal rabbit anti-recombinant (r)IL-16 Ab was prepared from rIL-16 immunized rabbit serum as described previously (22). Anticaspase-3 antibodies were purchased from R&D Systems. IL-16 cDNA was cloned as previously described (23), and a RANTES cDNA clone was generously provided by Dr. A. M. Krensky (Stanford University).

Primary thyrocyte isolation
Thyroid tissue was obtained as surgical waste from patients undergoing thyroidectomy for the treatment of a variety of conditions. Tissues were processed as described previously (7). Briefly, they were trimmed of connective tissue, finely minced, and washed with Hanks’ buffered salt solution. This was digested at 37 C in Hanks’ buffered 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 medium and filtered through 200-µm nylon mesh. Follicles were then pipetted into 25-mm plastic flasks to which they attached and monolayer cultures were propagated.

Cell culture
Primary thyrocytes were maintained in a humidified, 5% CO2 incubator at 37 C covered with RPMI supplemented with 10% FBS, penicillin (50 U/ml), and streptomycin (50 µg/ml). Medium was changed every 3–4 d, and cells were passaged with gentle trypsin treatment and used between the second and sixth passages. Experiments were performed on confluent monolayers, in most cases, within 1 wk of passage.

Northern blot analysis
To determine the steady-state levels of IL-16 and RANTES mRNA, confluent thyrocyte monolayers in 100-mm-diameter plates were shifted to medium supplemented with 1% FBS for 16 h before experimental manipulation. Plates were then rinsed and cellular RNA was extracted, precipitated, and solubilized in diethyl pyrocarbonate-treated water using the RNA isolation system (ULTRASPEC, Biotecx, Houston, TX) as described previously (24). Equal amounts of RNA (usually 30 µg) were electrophoresed in 1% agarose (Life Technologies, Inc.)-formaldehyde gels and transferred to Zetaprobe membrane (Bio-Rad Laboratories, Inc., Hercules, CA). Before transfer, ethidium bromide staining was performed to verify the integrity of the electrophoresed RNA. A [32P]-labeled 2-kb IL-16 cDNA probe was generated using a random primer labeling kit (Bio-Rad Laboratories, Inc.). The membrane was incubated with the probe in a hybridization solution [20x saline sodium citrate, 100x Denhardt’s solution (2% BSA, 2% polyvinyl pyrrolidone, 2% ficoll 400), 50% formamide, 1 M phosphate buffer, 10% sodium dodecyl sulfate, and 0.25 mg/ml sheared, denatured salmon sperm DNA (Amresco, Solon, OH)] at 42 C overnight. After high-stringency washing, membranes were exposed to X-OMAT AR film (Kodak, Rochester, NY) at -20 C. To normalize the amount of RNA transferred, the membranes were stripped according to manufacturer’s guidelines and rehybridized with a radiolabeled human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe.

Chemotaxis assay
Lymphocyte chemoattraction was assayed, as described previously (22, 23). 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 IL-1ß (10 ng/ml) were added for the indicated periods, at the end of which the culture medium was collected and stored at -80 C until use. Chemotaxis was then examined in a modified Boyden chemotaxis chamber using human NWNA-T lymphocytes as the cellular targets, as described previously. In brief, 50 µl 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. The differences between experimental and control conditions were analyzed by 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 antibodies (clone14.1, 5 µg/ml), which neutralizes the chemotactic activity of 50 ng/ml rIL-16. Similarly, anti-RANTES monoclonal antibodies (5 µg/ml) with an ND50 of 200 ng/ml for rRANTES were used to neutralize that cytokine.

ELISA assays for IL-16 and RANTES
IL-16 was measured as previously described by Lim et al. (15). 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 room temperature. The antigen was removed by washing extensively with a solution of PBS/Tween 20. Nonspecific binding was minimized by blocking with 1% BSA for 1 h. After washing, 100 µl of a rabbit anti-IL-16 polyclonal Ab (10 ng/ml) diluted in PBS containing 0.05% Tween 20 were 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 Technologies, Inc.) 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 (25, 26, 27).

Western blot analysis
Standard immunoblot techniques were used to determine protein levels. Confluent monolayers in 60-mm-diameter plates were shifted to medium containing 1% FBS for 16 h before initiation of experimental treatments. Following incubation with test compounds, plates were rinsed with PBS and harvested in a buffer containing 15 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1 mM EDTA, 20 mM Tris, 10 µg/ml soybean trypsin inhibitor, 5 µl/ml Nonidet P-40, 0.1 µl/ml 100 mM phenylmethylsulfonyl fluoride). Lysates were taken up in Laemmli buffer and subjected to SDS-PAGE, and the separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). The membrane was incubated with primary antibodies at room temperature for 2 h, washed, reincubated with peroxidase-labeled secondary antibodies for 2 h at room temperature and rewashed. The ECL-plus system (Amersham Pharmacia Biotech, Piscataway, NJ) was then used to generate the specific signals, which were captured on X-OMAT film (Kodak) and quantified densitometrically with a BioImage scanner (Milligen, Ann Arbor, MI).

Caspase-3 inhibition
To address the potential role of caspase-3 in the processing of IL-16, a specific peptide inhibitor of the enzyme, Ac-DEVD-CHO (100 µM), was added to the culture medium of near-confluent thyrocytes treated with IL-1ß (10 ng/ml). As a control, the peptide Ac-YVAD-Ald (100 µM), a specific caspase-1 inhibitor, was added to other IL-1ß-treated cultures. The medium was then subjected to specific ELISA for IL-16 or RANTES and the lymphocyte migration assay. With regard to detection of caspase-3 in thyrocytes, cell lysates from control and IL-1ß-treated cultures were subjected to Western blot analysis using an anti-caspase-3 (R&D Systems) as the primary Ab.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Thyrocytes produce and release lymphocyte chemotactic activity after treatment with IL-1ß
Primary thyrocytes proliferate to confluence in culture medium supplemented with 10% FBS. When treated with IL-1ß, they release substantial levels of lymphocyte chemotactic activity into the medium, as the data in Fig. 1Go demonstrate. Thyrocytes, in this case from a patient undergoing thyroidectomy for the removal of a multinodular goiter, were allowed to proliferate to confluence, and some were then treated with IL-1ß (10 ng/ml) for the times indicated. The medium was subjected to a lymphocyte migration assay. As suggested in Fig. 1Go, A and B, the effect of IL-1ß on thyrocyte-derived T cell migration-stimulating activity is time dependent and becomes substantial after 16 h of cytokine treatment. This activity reaches its peak at 24 h when it is 3-fold above control levels and remains elevated for the duration of the study (48 h). A substantial fraction of the chemoattraction (up to 75%) can be neutralized with the addition of anti-IL-16 (5 µg/ml, A). Another component of T cell migration provoked by IL-1ß could be attributed to RANTES because neutralizing antibodies (5 µg/ml) directed at that protein could also block 50% of the cytokine-dependent activity (Fig. 1BGo).



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  		Figure 1. IL-1ß up-regulates the expression of T cell chemoattractant activity in cultured human thyrocytes. This activity is derived from the synthesis of IL-16 and RANTES. Thyrocytes, in this case from a patient with multinodular goiter, were prepared as described in Materials and Methods and allowed to proliferate to near-confluence. Some were then treated with IL-1ß (10 ng/ml) for the time intervals indicated along the abscissas. Conditioned media were collected and subjected to a T cell migration assay in the absence or presence of anti-IL-16 neutralizing antibodies (5 µg/ml) (A); anti-RANTES neutralizing antibodies (5 µg/ml) (B). Cell migration greater than 135% was significant at the 95% confidence level. Other aliquots of media were subjected to specific ELISA assays for IL-16 (C) or RANTES (D). All data are expressed as the mean ± SD of triplicate determinations.

 
When thyrocytes derived from multiple glands were examined, including those from individuals with autoimmune thyroid disease and others from normal-appearing tissue distant from neoplasms, very low basal expression of T cell chemoattractant activity was found. IL-1ß induced lymphocyte chemotaxis was clearly evident in three of the four strains studied (Fig. 2Go) and could be attributed largely to IL-16. The strain in which no detectable chemotaxis could be detected (strain 1) derived from a multinodular goiter.



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  		Figure 2. Thyrocyte strains derived from glands affected by autoimmune and nonautoimmune disease can be provoked by IL-1ß to express IL-16- and RANTES-dependent T cell chemotaxis. Thyrocyte strains, including those from multinodular goiters (strains 1 and 3), Graves’ disease (strain 2), and normal thyroid tissue distant from a hyperplastic nodule (strain 4) were prepared and some cultures treated with IL-1ß (10 ng/ml), for 24 h. Conditioned media were collected and subjected to a T cell migration assay. All data are expressed as the mean ± SD of triplicate determinations. Cell migration greater than 135% was significant at the 95% confidence level.

 
IL-1ß induces IL-16 and RANTES protein release by thyrocytes
Because IL-16- and RANTES-dependent chemoattractant activity was detected in thyrocytes, we next determined whether these cytokines could be detected in the conditioned medium. Both were released from culture in a time-dependent manner as suggested by the data in Fig. 1Go, C and D. With regard to IL-16, the cytokine was undetectable by ELISA in control cultures or after 6 h of IL-1ß treatment. At 16 h, IL-16 levels had reached 15 ± 9 pg/ml, increased to 78 ± 16 pg/ml at 24 h, and remained elevated at 48 h when the concentration was 105 ± 20 pg/ml. RANTES was also undetectable under control conditions, but the levels of this chemokine were elevated after 8 h of exposure to IL-1ß to 177 ± 5 pg/ml. The concentration of RANTES reached 223 ± 21 pg/ml and 287 ± 32 pg/ml at 24 h and 48 h, respectively.

Caspase-3 activity is essential for the release of IL-16 but not RANTES from thyrocytes
Caspase-3 is a cysteine protease that plays a critical role in the proteolytic processing of mature IL-16 from its precursor protein (16, 28). This has been demonstrated in COS cells transfected with a cDNA encoding the 50-kDa form of pro-IL-16 (28). Subsequently, caspase-3 was implicated in IL-16 release from IL-1ß-treated fibroblasts (16). We therefore determined whether the enzyme played a similar role in thyrocytes. Addition of a caspase-3 inhibitory peptide, Ac-DEVD-CHO (100 µM), into the medium of thyrocytes in combination with IL-1ß (10 ng/ml) resulted in a near-complete blockade of IL-16 release from the cells (Fig. 3AGo). In contrast, inhibition of caspase-1 with the peptide Ac-YVAD-Ald (100 µM) failed to alter the IL-16 release from these cultures. Unlike IL-16, RANTES release was unaffected by either caspase inhibitor, suggesting that its processing in thyrocytes is independent of either enzyme. Caspase-3 could be detected in IL-1ß-treated thyrocyte cultures. As the Western blot in Fig. 3BGo demonstrates, both pro-caspase-3 and the mature protease could be detected in cytokine-activated cultures, the latter in a time-dependent manner. These results are entirely consistent with those reported in fibroblasts (16).



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  		Figure 3. Caspase-3 activity is essential for the release of mature, active IL-16 from IL-1ß-activated thyrocytes. A, Confluent thyrocytes, in this case from a patient with multinodular goiter, were treated with nothing (control) or IL-1ß (10 ng/ml) alone or in combination with specific caspase-1 (Ac-YVAD-Ald, 100 µM) or caspase-3 (Ac-DEVD-CHO, 100 µM) inhibitory peptides. Conditioned medium was collected and subjected to specific ELISA assays as described in Materials and Methods. Data are expressed as the mean ± SD of triplicate determinations. B, Confluent thyrocytes, in this case from a Graves’ gland, were treated with IL-1ß (10 ng/ml) for the times indicated. Cell lysates were examined by Western analysis for the presence of caspase-3 and its active fragments.

 
The induction of IL-16 and RANTES proteins in thyrocytes by IL-1ß can be blocked by glucocorticoids
The immunomodulatory actions associated with glucocorticoid therapy in the thyroid and elsewhere suggest that these steroids might attenuate the activation and migration of disease-relevant lymphocytes. We next determined whether the synthetic glucocorticoid, dexamethasone, could alter the expression of either IL-16 or RANTES induced by IL-1ß. As indicated by the data in Fig. 4Go, dexamethasone (10 µM) could block the induction of both chemoattractant molecules in cytokine-provoked thyrocytes. This result is consistent with the effects of dexamethasone on chemoattractant expression in human fibroblasts (16) and defines a potential target for this class of steroids in attenuating thyroid inflammation.



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  		Figure 4. The induction of IL-16 and RANTES protein expression in thyrocytes can be blocked by dexamethasone. Confluent thyrocyte monolayers were treated with nothing (control) or IL-1ß (10 ng/ml) alone or in combination with dexamethasone (10 µM), and the medium was then subjected to specific ELISAs for IL-16 and RANTES. Data are expressed as the means ± SD of triplicate determinations. Absence of a bar indicates that a measurement fell below the limits of detection for the assays, as stated in Materials and Methods.

 
Thyrocytes express unprovoked IL-16 mRNA
The pattern of IL-16 mRNA expression varies among the cell types in which the cytokine has been found to be expressed. In lymphocytes, basal levels are undetectable but are increased as a consequence of the factors that elicit IL-16 expression (29). In fibroblasts, high constitutive steady-state levels of mature IL-16 transcript are found, and these do not appear to be altered following treatment with compounds that induce protein expression (16). Thyrocytes also express high basal IL-16 mRNA levels, as the Northern blot analysis in Fig. 5Go indicates. The transcript appears as a single 2.2-kb band, consistent with the mRNA detected in fibroblasts (16). Levels are uninfluenced by IL-1ß (10 ng/ml) or dexamethasone (10 µM) treatment for 12 h. It appears therefore that the up-regulation of IL-16 expression by thyrocytes after treatment with IL-1ß is mediated through effects on translation of constitutively expressed mRNA. In contrast, RANTES mRNA is undetectable in control cultures but highly induced following IL-1ß treatment. Dexamethasone attenuates this induction of RANTES mRNA by 60%. Thus, the up-regulation of RANTES expression in thyrocytes appears to occur at a pretranslational level.



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  		Figure 5. Effects of IL-1ß and dexamethasone on the expression of IL-16 and RANTES mRNA levels in cultured thyrocytes. Thyroid epithelial cells, in this case from a patient with multinodular goiter, were allowed to proliferate to near-confluence in RPMI medium containing 10% FBS. They were then shifted to medium with 1% serum for 16 h and then nothing (control), IL-1ß (10 ng/ml), or dexamethasone (10 µM) alone or in combination with IL-1ß were added for 6 h. Monolayers were rinsed and cellular RNA extracted as described in Materials and Methods and subjected to Northern hybridization with probes generated from IL-16 or RANTES cDNAs and labeled by random primer synthesis. RNA/DNA hybrids were analyzed with autoradiography, the membranes stripped, and rehybridized with a GAPDH probe. The bar graphs represent IL-16 and RANTES signals normalized to the respective GAPDH densities.

 
IL-16 and RANTES expression in thyrocytes can be induced by several cytokines
IL-1ß acts in the context of a cytokine milieu rather than as an isolated factor. Moreover, substantial redundancy exists with regard to the impact most cytokines exert on their molecular and cellular targets. We therefore assessed the ability of other cytokines to induce lymphocyte chemoattractive activity in thyrocytes. Interferon-{gamma} (100 U/ml), IL-4 (10 ng/ml), TNF{alpha} (10 ng/ml), and TGFß (5 ng/ml) were added to the culture medium of confluent thyrocyte cultures for 24 h, and the conditioned medium was subjected to the lymphocyte migration assay. These cytokines were chosen because they represent cytokines produced by TH1 and TH2 lymphocytes and have been detected in autoimmune thyroid glands (30, 31). As the data in Fig. 6Go suggest, IL-4, TNF{alpha}, and TGFß induced the generation of chemotactic activity, and a substantial fraction of this could be attributed to IL-16. It would appear that IL-4 exerted its effects almost entirely through a modest up-regulation of IL-16. The cytokine failed to induce detectable levels of RANTES-dependent migration activity or protein (Fig. 6Go). TNF{alpha} and TGFß, like IL-1ß induced both IL-16 and RANTES proteins as assessed by ELISA. Interferon-{gamma} appears to have increased T cell chemotaxis, but this activity could not be attributed to either IL-16 or RANTES.



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  		Figure 6. Effects of several cytokines on lymphocyte migration activity, IL-16, and RANTES expression in thyrocytes. Several proinflammatory cytokines were examined for their effects on the expression of IL-16 and RANTES in thyrocyte cultures. Near-confluent monolayers were treated with nothing, IL-1ß (10 ng/ml), IL-4 (10 ng/ml), TGFß (5 ng/ml), TNF{alpha} (10 ng/ml), or interferon-{gamma} (100 U/ml) for 24 h. The conditioned medium was subjected to a lymphocyte migration assay or a specific ELISA for IL-16 and RANTES, as described in Materials and Methods. The data are expressed as the mean ± SD of studies performed in triplicate. Absence of a bar indicates that a measurement fell below the limits of detection for the assay as stated in Materials and Methods.

 
Pro-IL-16 can be detected in thyroid tissue
An important issue concerning the finding that thyrocytes express IL-16 in vitro relates to whether the cytokine can be detected in organized thyroid tissue in situ. As the Western blot in Fig. 7Go indicates, pro-IL-16 protein is abundant in thyroid from individuals with Graves’ disease and multinodular goiter. Mature IL-16 protein was not detected in any of the specimens thus far examined, suggesting a low level of abundance. This is consistent with previous studies examining IL-16 protein levels in lung tissues from asthmatic patients.



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  		Figure 7. Pro-IL-16 is detectable in situ in thyroid tissue. Samples (0.5–1 g) from two multinodular goiters and a gland affected by Graves’ disease were subjected to Western analysis using a monoclonal antihuman IL-16 antibody as described in Materials and Methods. Equivalent amounts of solubilized protein were loaded in each lane.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In autoimmune processes such as Graves’ disease and Hashimoto’s thyroiditis, the thyroid gland becomes infiltrated with CD4+ and CD8+ lymphocytes (1, 2). Moreover, the accumulation of intrathyroidal CD4+ and CD29+ cells could underlie B cell activation in Graves’ disease (32). Recent observations shed some light on the nature of lymphocyte cross-talk occurring in Graves’ disease. Arao et al. (33) have suggested that lymphocytes enhance thyrocyte proliferation through intercellular adhesion molecule-1/lymphocyte function-associated antigen-1 interactions. Factors proximate to the trafficking of bone marrow-derived cells to the thyroid in disease states are as yet unidentified. Thus, defining these mechanisms is critical to an understanding of thyroid autoimmunity.

Epithelial cells produce chemokines but the patterns of expression vary with cell type (34, 35). Bronchial epithelial cells express RANTES, IL-8, and eotaxin when activated by cytokines such as TNF{alpha}, and these inductions can be modified by TH2 cytokines such as IL-4 and IL-13 (34). Enterocyte-like CaCO-2 cells express high levels of IL-8 and monocyte chemotactic protein-1 (MCP-1) when challenged with bacteria (35). Thyrocytes are capable of expressing a wide array of cytokines including granulocyte-macrophage-colony-stimulating factor, IL-1, IL-6, and TGFß (36, 37). Moreover, the other cells resident in the thyroid, including endothelial cells and fibroblasts, can also produce many small molecules capable of influencing the activation and migration of immunocompetent cells. Several reports have appeared recently demonstrating that thyrocytes express chemokines such as IL-8 (37, 38, 39), IP-10/CXCL10, MIG/CXCL9 (40), and MCP-1 (41). We now present evidence that thyroid epithelial cells are capable of expressing the T cell chemoattractants, IL-16, and RANTES in vitro when treated with proinflammatory cytokines such as IL-1ß. It is noteworthy that cytokine-induced chemoattractant expression was similar in thyrocytes obtained from patients with autoimmune disease and in those without inflammatory thyroid disease. This finding suggests that IL-16 and RANTES production is inherent to the phenotype of human thyrocytes. Moreover, the equivalence demonstrated in cultures from autoimmune and nonautoimmune sources suggests that in the autoimmune thyroid, the molecular neighborhood, in addition to intrinsic thyrocyte factors may represent important determinants of chemoattractant expression and thus T cell recruitment.

The pattern of IL-16 expression and the mechanisms involved in its regulation appear to be cell type specific. CD4+ and CD8+ lymphocytes express pro-IL-16 protein (15, 29). Bronchial epithelial cells from patients with asthma have been shown to express IL-16 following their activation (18). Intestinal epithelial cells can express mature IL-16 (21), and this production has been implicated in the pathogenesis of inflammatory bowel disease. Here we demonstrate that thyrocytes express constitutive IL-16 mRNA. In contrast, IL-16 protein is undetectable until these cells are activated by cytokines, a similar finding to that in human fibroblasts (16). Untreated mast cells also express unprovoked IL-16 mRNA, but activation of IL-16 gene transcription by phorbol-12-myristate-13-acetate or C5a is necessary for the expression and release of mature IL-16 protein in these cells (17). Despite these differences among cells, the requirement for caspase-3 in the processing of precursor IL-16 into mature cytokine may prove universal (16, 28). With regard to RANTES, thyrocytes fail to express detectable levels of the transcript until provoked by IL-1ß. Transcriptional regulation of RANTES expression has been documented in a number of cell types (16, 42, 43, 44). Whether the induction of RANTES by IL-1ß involves the up-regulation of gene transcription or reflects enhanced mRNA stability will require further studies.

IL-16 influences a wide array of CD4-bearing cells, including lymphocytes, monocytes, and eosinophils (11, 12, 13). IL-16 primes lymphocytes for IL-2-dependent proliferation, an effect that is at least partially related to an up-regulation of the IL-2 receptor (12). IL-16 protects these cells against FAS-mediated apoptosis (45) and induces cell cycle progression (12, 46) and the expression of other cytokines in CD4+ cells (46). CD4+ cells stimulated with IL-16 express IL-3 and granulocyte-macrophage-colony-stimulating factor (46). Those treated with IL-16 in combination with IL-2 appear to express interferon-{gamma} rather than IL-4 or IL-5. These results suggest that in combination with other cytokines, IL-16 may bias the differentiation of naïve T cells toward the TH1 phenotype (46). The coordinate activation of IL-16 and RANTES production in thyrocytes might underlie the recruitment of a particular T cell population. CD4 activation by IL-16 desensitizes T cells to signaling through RANTES/CCR5 and results in the partial inhibition of T cell migration (47). Thus, the net contribution of CCR5+ cells to the cell infiltrate in the thyroid may bias the nature of the immune responses occurring there.

Signaling initiated through the binding of IL-16 to CD4 is complex and involves the use of multiple intracellular pathways. RANTES exhibits an even broader repertoire of cellular targets and acts through its association with at least four GTP-coupled protein chemokine receptors, including CCR5 (48). RANTES engagement of CCR5 activates Janus kinase, p38 MAPK, and multiple downstream signaling pathways (48). Although the mechanisms involved in the induction of IL-16 and RANTES are quite different, it would appear that these molecules are induced in thyrocytes in a well-coordinated manner. These activational molecules, perhaps in combination with other factors such as prostaglandin E2 (7), can help define the molecular microenvironment and therefore condition the thyroid’s response to immune events such as those occurring in autoimmune disease. If this proves to be the case, these signals may represent therapeutic targets through which the autoimmune disease process might be interrupted.


   Acknowledgments
 
We are indebted to Dr. Larry Robinson, Department of Surgery, Albany Medical College, for his help in obtaining thyroid tissue.


   Footnotes
 
This work was supported in part by NIH Grants RR-17303 (to A.G.G.), EY-08976, EY-11708 (to T.J.S.), and HL-32802 (to W.W.C.); a Merit Review award from the Medical Research Service of the Department of Veterans Affairs (to T.J.S.); and the Willard B. Warring memorial fund (to A.G.G.).

Abbreviations: Ab, Antibody; Ac-DEVD-CHO, Ac-Asp-Glu-Val-Asp-aldehyde; Ac-YVAD-Ald, acetyl-Tyr-Val-Ala-Asp-CHO; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MCP-1, monocyte chemotactic protein-1; r, recombinant; RANTES, regulated upon activation, normal T cell expressed, and secreted.

Received February 11, 2003.

Accepted for publication March 26, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tezuka H, Eguchi K, Fukuda T, Otsubo T, Kawabe Y, Ueki Y, Matsunaga M, Shimomura C, Nakao H, Ishikawa N, Ito K, Nagataki S 1988 Natural killer and natural killer-like cell activity of peripheral blood and intrathyroidal mononuclear cells from patients with Graves’ disease. J Clin Endocrinol Metab 66:702–707[Abstract]
  2. Totterman TH, Andersson LC, Hayry P 1979 Evidence for thyroid antigen-reactive T lymphocytes infiltrating the thyroid gland in Graves’ disease. Clin Endocrinol 11:59–68[Medline]
  3. Weetman AP, Volkman DJ, Burman KD, Gerrard TL, Fauci AS 1985 The in vitro regulation of human thyrocyte HLA-DR antigen expression. J Clin Endocrinol Metab 61:817–824[Abstract]
  4. Rasmussen AK 2000 Cytokine actions on the thyroid gland. Dan Med Bull 47:94–114[Medline]
  5. Rasmussen AK, Bendtzen K, Feldt-Rasmussen U 2000 Thyrocyte-interleulin-1 interactions. Exp Clin Endocrinol Diabetes 108:67–71[CrossRef][Medline]
  6. Giordano C, Stassi G, De Maria R, Todaro M, Richiusa P, Papoff G, Ruberti G, Bagnasco M, Testi R, Galluzo A 1997 Potential involvement of FAS and its ligand in the pathogenesis of Hashimoto’s thyroiditis. Science 275:960–963[Abstract/Free Full Text]
  7. Gianoukakis AG, Cao HJ, Jennings TA, Smith TJ 2001 Prostaglandin endoperoxide H synthase expression in human thyroid epithelial cells. Am J Physiol Cell Physiol 280:C701–C708
  8. Smith TJ, Sciaky D, Phipps RP, Jennings TA 1999 CD40 expression in human thyroid tissue: evidence for involvement of multiple cells types in autoimmune and neoplastic diseases. Thyroid 9:749–755[Medline]
  9. Metcalfe RA, McIntosh RS, Marelli-Berg F, Lombardi G, Lechler R, Weetman AP 1998 Detection of CD40 on human thyroid follicular cells: analysis of expression and function. J Clin Endocrinol Metab 83:1268–1274[Abstract/Free Full Text]
  10. Schall TJ, Bacon K, Toy KJ, Goeddel DV 1990 Selective attraction of monocytes and T lymphocytes of the memory phenotype by the cytokine RANTES. Nature 347:669–671[CrossRef][Medline]
  11. Berman JS, Cruikshank WW, Center DM, Theodore AC, Beer DJ 1985 Chemoattractant lymphokines specific for the helper/inducer T-lymphocyte subset. Cell Immunol 95:105–112[CrossRef][Medline]
  12. Cruikshank WW, Berman JS, Theodore AC, Bernardo J, Center DM 1987 Lymphokine activation of T4+ T lymphocytes and monocytes. J Immunol 138:3817–3823[Abstract]
  13. Rand TH, Cruikshank WW, Center DM, Weller PF 1991 CD4-mediated stimulation of human eosinophils: lymphocyte chemoattractant factor and other CD4-binding ligands elicit eosinophil migration. J Exp Med 173:1521–1528[Abstract/Free Full Text]
  14. Laberge S, Cruikshank WW, Kornfeld H, Center DM 1995 Histamine-induced secretion of lymphocyte chemoattractant factor from CD8+ T cells is independent of transcription and translation: evidence for constitutive protein synthesis and storage. J Immunol 155:2902–2910[Abstract]
  15. Lim KG, Wan HC, Bozza PT, Resnick MB, Wong DT, Cruikshank WW, Kornfeld H, Center DM, Weller PF 1996 Human eosinophils elaborate the lymphocyte chemoattractants. IL-16 (lymphocyte chemoattractant factor) and RANTES. J Immunol 156:2566–2570[Abstract]
  16. Sciaky D, Brazer W, Center DM, Cruikshank WW, Smith TJ 2000 Cultured human fibroblasts express constitutive IL-16 mRNA: cytokine induction of active IL-16 protein synthesis through a caspase-3 dependent mechanism. J Immunol 164:3806–3814[Abstract/Free Full Text]
  17. Rumsaeng V, Cruikshank WW, Foster B, Prussin C, Kirshenbaum AS, Davis TA, Kornfeld H, Center DM, Metcalfe DD 1997 Human mast cells produce the CD4+ T lymphocyte chemoattractant factor, IL-16. J Immunol 159:2904–2910[Abstract]
  18. Bellini A, Yoshimura H, Vittori E, Marini M, Mattoli S 1993 Bronchial epithelial cells of patients with asthma release chemoattractant factors for T-lymphocytes. J All Clin Immunol 92:412–424[CrossRef][Medline]
  19. Franz JK, Kolb SA, Hummel KM, Lahrtz F, Neidhart M, Aicher WK, Pap T, Gay RE, Fontana A, Gay S 1998 Interleukin-16, produced by synovial fibroblasts, mediates chemoattraction for CD4+ T lymphocytes in rheumatoid arthritis. Eur J Immunol 28:2661–2671[CrossRef][Medline]
  20. Lee S, Kaneko H, Sekigawa I, Tokano Y, Takasaki Y, Hashimoto H 1998 Circulating interleukin-16 in systemic lupus erythematosus. Br J Rheumatol 37:1334–1337[Abstract/Free Full Text]
  21. Seegert D, Rosenstiel P, Pfahler H, Pfefferkorn P, Nikolaus S, Schreiber S 2001 Increased expression of IL-16 in inflammatory bowel disease. Gut 48:326–332[Abstract/Free Full Text]
  22. Cruikshank WW, Greenstein JL, Theodore AC, Center DM 1991 Lymphocyte chemoattractant factor (LCF) induces CD4-dependent intracytoplasmic signaling in lymphocytes. J Immunol 146:2928–2934[Abstract]
  23. Cruikshank WW, Center DM, Nisar N, Wu M, Natke B, Theodore AC, Kornfeld H 1994 Molecular and functional analysis of a lymphocyte chemoattractant factor: association of biologic function with CD4 expression. Proc Natl Acad Sci USA 91:5109–5113[Abstract/Free Full Text]
  24. Smith TJ, Jennings TA, Sciaky D, Cao HJ 1999 Prostaglandin-endoperoxide H synthase-2 expression in human thyroid epithelium. J Biol Chem 274:15622–15632[Abstract/Free Full Text]
  25. Cruikshank WW, Long A, Tarpy RE, Kornfeld H, Carroll MP, Teran L, Holgate ST, Center DM 1995 Early identification of interleukin-16 (lymphocyte chemoattractant factor) and macrophage inflammatory protein 1{alpha} (MIP1{alpha}) in bronchoalveolar lavage fluid of antigen-challenged asthmatics. Am J Respir Cell Mol Biol 13:738–747[Abstract]
  26. Mashikian MV, Tarpy RE, Saukkonen JJ, Lim KG, Fine GD, Cruikshank WW, Center DM 1998 Identification of IL-16 as the lymphocyte chemotactic activity in the bronchoalveolar lavage fluid of histamine-challenged asthmatic patients. J Allergy Clin Immunol 101(6 Pt 1):786–792
  27. Bandeira-Melo C, Sugiyama K, Woods LJ, Phoofolo M, Center DM, Cruikshank WW, Weller PF 2002 IL-16 promotes leukotriene C(4) and IL-4 release from human eosinophils via CD4- and autocrine CCR3-chemokine-mediated signaling. J Immunol 168:4756–4763[Abstract/Free Full Text]
  28. Zhang Y, Center DM, Wu DM, Cruikshank WW, Yuan J, Andrews DW, Kornfeld H 1998 Processing and activation of IL-16 by caspase-3. J Biol Chem 273:1144–1149[Abstract/Free Full Text]
  29. Wu DM, Zhang Y, Parada NA, Kornfeld H, Nicoll J, Center DM, Cruikshank WW 1999 Processing and release of IL-16 from CD4+ but not CD8+ T cells is activation dependent. J Immunol 162:1287–1293[Abstract/Free Full Text]
  30. Watson PF, Pickerill AP, Davies R, Weetman AP 1994 Analysis of cytokine gene expression in Graves’ disease and multinodular goiter. J Clin Endocrinol Metab 79:355–360[Abstract]
  31. Hamilton F, Black M, Farquharson MA, Stewart C, Foulis AK 1991 Spatial correlation between thyroid epithelial cells expressing class II MHC molecules and interferon-gamma-containing lymphocytes in human thyroid autoimmune disease. Clin Exp Immunol 83:64–68[Medline]
  32. Goto Y, Itoh M, Ohta Y, Ogawa N, Goto Y, Ohashi H 1997 Increased production of B-cell growth factor by T lymphocytes in Graves’ thyroid: possible role of CD4+ CD29+ cells. Thyroid 7:567–573[Medline]
  33. Arao T, Morimoto I, Kakinuma A, Ishida O, Zeki K, Tanaka Y, Ishikawa N, Ito K, Ito K, Eto S 2000 Thyrocyte proliferation by cellular adhesion to infiltrating lymphocytes through the intercellular adhesion molecule-1/lymphocyte function-associated antigen-1 pathway in Graves’ disease. J Clin Endocrinol Metab 85:382–389[Abstract/Free Full Text]
  34. Fujisawa T, Kato Y, Atsuta J, Terada A, Iguchi K, Kamiya H, Yamada H, Nakajima T, Miyamasu M, Hirai K 2000 Chemokine production by the BEAS-2B human bronchial epithelial cells: differential regulation of eotaxin, IL-18 and RANTES by TH2- and TH1-derived cytokines. J All Clin Immunol 105:126–133[CrossRef][Medline]
  35. Haller D, Bode C, Hammes WP, Pfeifer AM, Schiffrin EJ, Blum S 2000 Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leukocyte co-cultures. Gut 47:79–87[Abstract/Free Full Text]
  36. Kasai K, Banba N, Motohashi S, Fukuda H, Manaka K, Matsumura M, Sekiguchi Y, Hattori Y 1997 Production of granulocyte/macrophage colony-stimulating factors by human thyrocytes in culture. Biochem Biophys Res Commun 238:191–196[CrossRef][Medline]
  37. Aust G, Scherbaum WA 1996 Expression of cytokines in the thyroid: thyrocytes as potential cytokine producers. Exp Clin Endocrinol Diabetes 104:64–67
  38. Fiore L, Pollina LE, Fontanini G, Casalone R, Berlingieri MT, Giannini R, Pacini F, Miccoli P, Toniolo A, Fusco A, Basolo F 1997 Cytokine production by a new undifferentiated human thyroid carcinoma cell line, FB-1. J Clin Endocrinol Metab 82:4094–4100[Abstract/Free Full Text]
  39. Watson PF, Pickerill AP, Davies R, Weetman AP 1995 Semi-quantitative analysis of interleulin-1 alpha, interleukin-6 and interleukin-8 mRNA expression by human thyrocytes. J Mol Endocrinol 15:11–21[Abstract/Free Full Text]
  40. Romagnani P, Rotondi M, Lazzeri E, Lasagni L, Francalanci M, Buonamano A, Milani S, Vitti P, Chiovato L, Tonacchera M, Bellastella A, Serio M 2002 Expression of IP-10/CXCL10 and MIG/CXCL9 in the thyroid and increased levels of IP-10/CXCL10 in the serum of patients with recent-onset Graves’ disease. Am J Pathol 161:195–206[Abstract/Free Full Text]
  41. Kasai K, Banba N, Motohashi S, Hattori Y, Manaka K, Shimoda SI 1996 Expression of monocyte chemoattractant protein-1 mRNA and protein in cultured human thyrocytes. FEBS Lett 394:137–140[CrossRef][Medline]
  42. Andoh A, Takaya H, Saotome T, Shimada M, Hata K, Araki Y, Nakamura F, Shintani Y, Fujiyama Y, Bamba T 2000 Cytokine regulation of chemokine (IL-8, MCP-1, and RANTES) gene expression in human pancreatic periacinar myofibroblasts. Gastroenterology 119:211–219[CrossRef][Medline]
  43. Li QQ, Bever CT 2001 Th1 cytokines stimulate RANTES chemokine secretion by human astroglial cells depending on de novo transcription. Neurochem Res 26:125–133[CrossRef][Medline]
  44. Nelson PJ, Kim HT, Manning WC, Goralski TJ, Krensky AM 1993 Genomic organization and transcriptional regulation of the RATES chemokine gene. J Immunol 151:2601–2612[Abstract]
  45. Cruikshank WW, Lim K, Theodore AC, Cook J, Fine G, Weller PF, Center DM 1996 IL-16 inhibition of CD3-dependent lymphocyte activation and proliferation. J Immunol 157:5240–5248[Abstract]
  46. Parada NA, Center DM, Kornfeld H, Rodriguez WL, Cook J, Vallen M, Cruikshank WW 1998 Synergistic activation of CD4+ T cells by IL-16 and IL-2. J Immunol 160:2115–2120[Abstract/Free Full Text]
  47. Mashikian MV, Ryan TC, Seman A, Brazer W, Center DM, Cruikshank WW 1999 Reciprocal desensitization of CCR5 and CD4 is mediated by IL-16 and macrophage-inflammatory protein-1ß, respectively. J Immunol 163:3123–3130[Abstract/Free Full Text]
  48. Wong M, Uddin S, Majchrzak B, Huynh T, Proudfoot AEI, Platanias LC, Fish EN 2001 RANTES activates Jak2 and Jak3 to regulate engagement of multiple signaling pathways in T cells. J Biol Chem 276:11427–11431[Abstract/Free Full Text]



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