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Endocrinology Vol. 148, No. 1 54-62
Copyright © 2007 by The Endocrine Society

Hyaluronan Accumulation in Thyroid Tissue: Evidence for Contributions from Epithelial Cells and Fibroblasts

Andrew G. Gianoukakis, Timothy A. Jennings, Chris S. King, Christine E. Sheehan, Neil Hoa, Paraskevi Heldin and Terry J. Smith

Divisions of Molecular Medicine (A.G.G., C.S.K., T.J.S.) and Endocrinology and Metabolism (A.G.G., T.J.S.), Harbor-UCLA Medical Center, Torrance, California 90502; the David Geffen School of Medicine at the University of California Los Angeles (A.G.G., T.J.S.), Los Angeles, California 90095; Department of Pathology (T.A.J., C.E.S.), Albany Medical College, Albany, New York 12208; the Veterans Affairs Medical Center (N.H.), Long Beach, California 90822; and the Ludwig Institute for Cancer Research Biomedical Center (P.H.), Uppsala S-75124, Sweden

Address all correspondence and requests for reprints to: Terry J. Smith, M.D., 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
 
Graves’ disease (GD) and Hashimoto’s thyroiditis (HT) are autoimmune processes often associated with hyperthyroidism and hypothyroidism, respectively. Despite their diverging clinical presentations, immune activation drives both diseases and results in connective tissue accumulation of the nonsulfated glycosaminoglycan, hyaluronan. The hydrophilic property of hyaluronan contributes to the pathogenesis of thyroid-associated ophthalmopathy, dermopathy and hypothyroid myxedema. Whether hyaluronan accumulates in the thyroid and plays a role in goiter formation in GD and HT remains unknown. We report here that levels of hyaluronan are increased in thyroid tissue from individuals with both diseases compared with glands uninvolved with autoimmune disorders. The transcript encoding hyaluronan synthase (HAS)-3, one of three mammalian HAS isoforms, was detected in thyroid tissue. Isolated thyrocytes in primary culture express all three HAS isoforms when treated with IL-1ß. Thyrocytes and thyroid fibroblasts produce hyaluronan under basal culture conditions and IL-1ß enhances levels of this molecule in both cell types. On a per-cell basis, fibroblasts produce more hyaluronan than do thyrocytes under basal conditions and after cytokine treatment. Synthesis in thyrocytes can also be altered by increasing serum concentration in the medium and by modifying culture density. Our findings suggest that hyaluronan accumulation in thyroid tissue might derive from thyrocytes and fibroblasts. Moreover, this glycosaminoglycan becomes more abundant as a consequence of autoimmune disease. It may therefore contribute to increased thyroid volume in GD and HT. Coupled with the newly identified influence exerted by hyaluronan on immunocompetent cells, our findings represent potentially important insights into the pathogenesis of autoimmune thyroid diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AUTOIMMUNE THYROID DISEASES such as Graves’ disease (GD) and Hashimoto’s thyroiditis (HT) disrupt glandular architecture and function. GD is often associated with hyperthyroidism as a consequence of TSH receptor (TSHR) activation by antibodies (Abs) specific to the disease (1, 2). In contrast, HT most commonly presents as hypothyroidism resulting from immune destruction of the thyroid and generation of antithyroid Abs (1, 2). Despite their divergent presentations, both diseases result in characteristic lymphocytic infiltration (3), enlargement, and altered function of the thyroid.

Hyaluronan, an abundant nonsulfated glycosaminoglycan, was first implicated in the peripheral manifestations of thyroid disease more than 60 yr ago (4). It comprises linear disaccharides consisting of alternating D-glucuronic acid and N-acetyl-D-glucosamine residues. Unlike many other complex carbohydrates, it lacks a core protein. Hyaluronan exhibits a remarkable avidity for water. When hydrated, each molecule can occupy a volume 10,000 times greater than that of an equivalent mass of albumin (5). Thus, tissues accumulating hyaluronan often expand greatly. It plays important roles in cell migration, signaling, and the trafficking of lymphocytes (6). Hyaluronan fragments can induce gene expression in a variety of cell types and may initiate inflammatory responses (6, 7). Synthesis of this molecule occurs at the cell surface (8) and involves the activities of three synthetic enzymes, hyaluronan synthases (HAS) 1, 2, and 3. Each isoform is encoded by a separate gene localizing to a different human chromosome (9). Moreover, each exhibits a characteristic pattern of tissue and cellular expression, role in development, and product size (9, 10, 11).

Hyaluronan accumulates in connective tissues in both GD and HT, although the mechanism for deposition and tissue distribution differs in each. In GD, hyaluronan accumulates in the orbit and pretibial skin, resulting from the underlying autoimmune process (12, 13, 14). In the orbit, it promotes edema, congestion, and contributes to the anterior displacement of the globe, also known as proptosis (5). These processes characterize thyroid-associated ophthalmopathy. In contrast, hyaluronan accumulation in HT is a direct result of the thyroprivic state (15, 16). Whereas hyaluronan accumulates in connective tissues in both diseases, little is known about its levels in the thyroid. Van Dessel et al. (17) first described the presence of hyaluronan in porcine thyroid. Proteoglycans have been demonstrated in normal and malignant thyroid tissue (18, 19). Hyaluronan production is TSH dependent in a porcine thyrocyte model (20, 21). By virtue of its high abundance in the stroma of malignant tumors (22, 23, 24), including differentiated thyroid carcinoma (19), and the correlation with an unfavorable outcome, hyaluronan is thought to play an important role in tumor growth and metastasis. The relationship between thyroid involvement in autoimmune disease and hyaluronan deposition has not been examined previously.

Here we present the results of a systematic examination of hyaluronan content in human thyroid tissue from patients with GD and HT and compare these with control tissue. We find evidence of excess deposition in both diseases. Furthermore, thyrocytes as well as thyroid fibroblasts produce hyaluronan in vitro. This synthesis is influenced by cell density and the medium concentration of fetal bovine serum (FBS) and can be up-regulated by IL-1ß. Our findings suggest that accumulation of hyaluronan might contribute to the increased volume of the thyroid in GD and HT.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Eagle’s medium, FBS, and antibiotics were purchased from Life Technologies (Grand Island, NY). A hyaluronan-specific ELISA was from Echelon Biosciences Inc. (Salt Lake City, UT). IGF-I was purchased from R&D (Minneapolis, MN). IL-1ß was supplied by Biosource International (Camarillo, CA). Dexamethasone was from Sigma-Aldrich (St. Louis, MO). rhTSH was generously provided by Genzyme (Cambridge, MA). Hyaluronan binding protein (HABP) was isolated from bovine cartilage extract using affinity chromatography on a hyaluronan-EAH Sepharose 4B column (Amersham Bioscience, Piscataway, NJ) as described by Tengblad (25). The purified HABP was then biotinylated with biotin-aminocarpoic acid sulfo-NHS (Sigma; catalog no. B1022). Collagenase Type A and dispase Grade I were purchased from Roche (Indianapolis, IN). Mouse anti-TTF-1 (thyroid transcription factor-1) (Clone 8G7G3/1) and antithyroglobulin abs (Clone 1D4) were purchased from Invitrogen (San Jose, CA) and Ventanna Medical Systems (Tuscon, AZ), respectively.

Tissue acquisition and cell culture
Tissue samples used for the studies reported here were obtained after informed consent using a protocol approved by the institutional review boards for both the Harbor-UCLA Medical Center and the Center for Health Sciences at UCLA. Control thyroid tissues and those from patients with GD and HT were obtained as surgical waste from therapeutic thyroidectomy. Controls were excised from normal appearing thyroid tissue remote from conditions including nodular goiter, and focal, well-differentiated neoplasms. Tissues were processed as described previously (26). Briefly, fragments were trimmed of connective tissue, finely minced and washed with Hanks’ balanced saline solution (HBSS). They were digested at 37 C in HBSS containing collagenase (130 U/ml) and dispase (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 transferred into 25-mm plastic flasks where they attached to the culture surface and monolayers developed. These were covered with RPMI supplemented with 10% FBS, penicillin (50 U/ml), and streptomycin (50 µg/ml). Thyroid fibroblasts were isolated from surgical waste tissue as described previously (27) from donors without or with GD. Monolayers were covered with Eagle’s medium supplemented with 10% FBS, antibiotics, and glutamine. Cultures were maintained in a humidified, 5% CO2 incubator at 37 C and passaged gently with trypsin/EDTA. Thyrocytes were used between the second and sixth passages, whereas fibroblasts were used between the third and tenth passage. Experiments were performed on confluent monolayers. This was usually achieved within 1 wk of culture initiation.

Hyaluronan detection by ELISA
Experiments were performed in replicates as indicated in the figure legends. Serum concentration in the medium was reduced as indicated. Before experimental manipulations, monolayers were washed and fresh medium was added. Following the incubations specified, conditioned medium was collected and hyaluronan concentrations were determined using the ELISA, according to the manufacturer’s instructions. Monolayers were solubilized in a buffer containing [50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 10% glycerol, 0.5% Triton X-100, 100 mM sodium fluoride, 1 mM sodium vanadate, 10 mM sodium pyrophosphate, 1 mM phenylmethylsufonyl fluoride, 10 µg/ml leupeptin, 9 µg/ml aprotinin], and an aliquot subjected to protein analysis. Alternatively, monolayers were gently treated with trypsin/EDTA, disrupted with a rubber policeman, resuspended and manually counted. Results are normalized to either cell number or protein.

RNA isolation and RT-PCR
RNA was extracted from thyroid tissue or confluent thyrocyte monolayer cultures by the method of Chomczynski and Sacchi (28) 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, Valencia, CA). PCRs were performed using Taq PCR Master Mix kit (QIAGEN) in the linear phase. PCR was performed using the following HAS primers: HAS 1, 5'-TGTGTATCCTGCATCAGCGGT-3'(forward) and 5'-CTGGAGGTGTACTTGGTAGCATAACC-3' (reverse) under the following conditions: 94 C for 5 min, 94 C for 45 sec, 55 C for 45 sec, and 72 C for 45 sec for 39 cycles, HAS2, 5'-GTGTTATACATGTCGAGTTTACTTCC-3' (forward) and 5-GTCATATTGTTGTCCCTTCTTCCGC-3' (reverse), HAS3, 5'-GGTACCATCAGAAGTTCCTAGGCAGC-3' (forward) and 5'-GAGGAGAATGTTCCAGATGCG-3' (reverse) under the following conditions: 94 C for 5 min, 94 C for 45 sec, 55 C for 45 sec, and 72 C for 45 sec for 35 cycles. HAS mRNA levels were normalized using the following primers for ß-actin: 5'-CCAAGGCCAACCGCGAGAAGATGAC-3' (forward) and 5'-AGGGTACATGGTGGTGCCGCCAGAC-3' (reverse) under the following conditions: 95 C for 5 min, 95 C for 1 min, 60 C for 1 min, and 72 C for 1 min for 35 cycles or glyceraldehyde-3-phosphate dehydrogenase primers 5'-ATCACTGCCACCCAGAAGACT-3' (forward) and 5'-CATGCCAGTGAGCTTCCCGTT-3' (reverse) under the following conditions: 94 C for 5 min, 94 C for 45 sec, 57 C for 1 min, and 72 C for 45 sec for 37 cycles. The resultant cDNAs were subjected to electrophoresis through denaturing 2% agarose gels. Bands were visualized using ethidium bromide staining under UV light.

Immunofluorescent staining of primary thyrocytes
Thyroglobulin staining.
A thyrocyte monolayer cultured on eight-well glass slides (ICN, Costa Mesa, CA) was washed in PBS, air dried at room temperature, and fixed in ice-cold acetone for 10 min. Slides were rehydrated in PBS for 5 min, incubated in 0.1% saponin in PBS for 30 min, blocked in 5% nonfat milk in PBS for 30 min, and incubated with primary mouse antithyroglobulin monoclonal antibody for 1 h at room temperature. They were washed in PBS for 10 min, incubated with Ab binding solution containing goat-antimouse IgG (A-11032) coupled to red-fluorescent Alexa-Fluor 594 (both from Molecular Probes, Eugene, OR) for 1 h, washed and mounted with 95% glycerol in PBS. Negative control slides were run in the absence of the primary Ab.

TTF-1 staining.
Adherent thyrocytes were cultured overnight on a poly-D-lysine-coated sterile cover glass and fixed in 3.75% paraformaldehyde solution (pH 7.4) for 30 min. To enhance epitope retrieval, thyrocytes were boiled in 0.01 M citrate buffer for 15 min. Cover glasses were rinsed with PBS, permeated in 0.05% Triton X-100 buffer for 5 min, rinsed and incubated with 10% horse blocking serum in PBS for 30 min followed by a 60-min incubation with primary mouse anti-TTF-1 monoclonal antibody. After rinsing, they were incubated with a horse antimouse fluorescein isothiocyante Ab for 30 min at room temperature. Cover glasses were mounted on glass slides with Prolong Gold antifade reagent (Invitrogen, San Jose, CA). Negative control slides were run in the absence of the primary Ab. Images were captured using Nikon confocal Imaging System on an Eclipse 800 microscope at x40 magnification.

Immunohistochemical staining of thyroid tissue for hyaluronan.
Surgical pathology files of the Albany Medical Center Hospital were reviewed. Representative cases of GD and HT were randomly selected following an institutional review board-approved research protocol. Similarly, we reviewed cases of papillary carcinoma for which near-total thyroidectomy was performed. The contralateral, uninvolved lobe of normal appearing tissue was sectioned. Sixteen cases were selected for this study, including four normal, seven GD, and five HT. Immunohistochemical staining for hyaluronan was performed by a manual method on 4-µm formalin-fixed paraffin-embedded sections from a representative block in each case. After removal of paraffin in EZ-Common solution (EZ-Retrieval System, BioGenex, San Ramon, CA), endogenous peroxidase activity was blocked with 3% H2O2 in methanol. The sections were then incubated with 24 µg/ml biotinylated-HABP for 60 min, followed by streptavidin-horseradish peroxidase for 30 min at room temperature. After color development with diethylaminobenzidine, slides were counterstained with hematoxylin, dehydrated, and mounted. Similarly processed formalin-fixed paraffin-embedded tissue sections from human umbilical cord were used as positive controls.

Statistics
Data were analyzed using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hyaluronan accumulation in cultured human thyrocytes is FBS and cell density dependent
Initial studies examined the production of hyaluronan in cultured primary thyrocytes. As the data in Fig. 1Go demonstrate, thyrocytes from individuals without autoimmune disease produce quantifiable levels of hyaluronan under basal culture conditions. To assess the effects of serum on hyaluronan accumulation, cultures were exposed to medium with FBS concentrations ranging from 0–20% for 24 h (Fig. 1Go, A and B). Medium was replaced for an additional 24 h incubation and then collected and hyaluronan concentrations assayed. As shown in Fig. 1BGo, hyaluronan accumulation, normalized to protein content, is dependent on FBS concentration. Thyrocytes cultured in 20% serum accumulated 3-fold higher hyaluronan levels (1.3–3.9 ng/µg) and those in 10% serum produced 2-fold higher levels (1.3–2.8 ng/µg) when compared with cultures in serum-free medium (P < 0.001). Of note, hyaluronan could not be detected in medium supplemented with 20% serum that had not been used to incubate cells. Similar results were obtained when hyaluronan expression was calculated on a per-cell basis (1A). Control thyrocytes were then plated at graded cell densities of 3,000, 6000, 24,000 and 96,000 cells/cm2 in 10% serum. As Fig. 1DGo indicates, hyaluronan accumulation, normalized to protein content, is dependent on cell density. The thyrocytes plated at the lowest density produced 2.5-fold more hyaluronan (2.1–5.3 ng/µg) compared with those plated at the highest (P < 0.001). Furthermore, production by each group differed significantly from each other (P < 0.05) except for that produced by the cells plated at a density of 6000 vs. 24,000 cells/cm2 (P = 0.87). Similar results were seen when results were calculated on a per-cell basis (Fig. 1CGo). Secreted hyaluronan was 3-fold greater than cell-associated hyaluronan when expressed on a per-cell basis (data not shown).


Figure 1
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  		FIG. 1. Hyaluronan production in thyrocytes is serum and cell density dependent. Control thyrocytes were cultured in 24-well plates. A and B, Confluent monolayers were cultured for 24 h in medium supplemented with the concentrations of FBS indicated. C and D, Thyrocytes were plated at the densities indicated, in medium supplemented with 10% serum for 24 h. The medium was replaced and the monolayers cultured for an additional 24 h. Hyaluronan concentration in the medium was determined by ELISA and expressed on a per-cell basis or normalized to protein content. All data are expressed as the mean ± SD of triplicate determinations.

 
Hyaluronan accumulation by thyrocytes can be up-regulated by cytokines and growth factors
Control and GD thyrocytes responded to treatment with IL-1ß (10 ng/ml) for 24 h with a powerful up-regulation of hyaluronan accumulation (Fig. 2Go). Levels increased 5-fold in control thyrocytes while increasing 4.6-fold in those from patients with GD. In both, the IL-1ß-dependent hyaluronan production could be attenuated with dexamethasone (10 µM). Interestingly, dexamethasone also attenuated cytokine-independent hyaluronan production by GD thyrocytes (Fig 2AGo). Notably, treatment with IGF-I (10 nM) significantly increased hyaluronan production in thyrocytes, which could be blocked by dexamethasone. Other cytokines known to be present in autoimmune thyroid glands such as TNF-{alpha} (10 ng/ml) and TGF-ß (5 ng/ml) as well as rhTSH (2–10 mIU/ml) failed to affect thyrocyte hyaluronan production (Fig. 2Go). We next compared hyaluronan production by thyrocytes with that of thyroid fibroblasts. Both cell types, derived from the same donor, were treated with IL-1ß for 24 h. All cells examined exhibited greater than a 50% increase in hyaluronan accumulation when stimulated with the cytokine compared with basal levels. Thyrocytes produced significantly less hyaluronan on a per-cell basis than did fibroblasts (Fig. 3Go). The basal and stimulated hyaluronan produced by thyrocytes and thyroid fibroblasts was found to be independent of the type of medium (RPMI, DMEM) in which they were cultured. Unstimulated thyrocytes and thyroid fibroblasts cultured in RPMI elaborated hyaluronan at a level of 27.4 ± 4.8 ng/1000 cells (mean ± SD) and 49.6 ± 2.8 ng/1000 cells, respectively, whereas those grown in DMEM elaborated 31.4 ± 3.4 ng/1000 cells and 54.6 ± 8.5 ng/1000 cells. After stimulation with IL-1ß (10 ng/ml), thyrocytes and thyroid fibroblasts grown in RPMI secreted hyaluronan at levels of 71.6 ± 8.9 ng/1000 cells and 104.2 ± 13.3 ng/1000 cells, respectively, whereas those grown in DMEM produced 73.6 ± 6.4 ng/1000 cells and 90.8 ± 7.5 ng/1000 cells.


Figure 2
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  		FIG. 2. Cytokines induce hyaluronan production in thyrocytes. Confluent monolayers were treated with nothing (control), IL-1ß (10 ng/ml), TNF-{alpha} (10 ng/ml), TGF-ß (5 ng/ml), IGF-I (10 nM), or rhTSH (2 mIU/ml) without or with dexamethasone (10 µM) for 24 h. Hyaluronan was assayed in the medium and normalized to protein content. All data are expressed as the mean ± SD of triplicate determinations.

 

Figure 3
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  		FIG. 3. Thyrocytes and thyroid fibroblasts produce hyaluronan in response to IL-1ß. Thyrocytes and thyroid fibroblasts derived from the same gland were grown to confluence in medium with 10% FBS and treated with nothing (control) or IL-1ß (10 ng/ml) for 24 h. Hyaluronan concentrations were determined and expressed on a per-cell basis. All data are expressed as the average ± range of duplicate determinations.

 
Induction of HAS 1, 2, and 3 mRNAs in cultured thyrocytes
mRNAs encoding HAS 2 and 3 were detectable in thyrocytes cultured under basal conditions, unlike that for HAS 1 (Fig. 4Go). Treatment of GD thyrocytes with IL-1ß (10 ng/ml) induced a significant increase of all three HAS transcripts (Fig. 4Go). HAS 2 and 3 mRNA levels were elevated within 2 h, effects that were maintained for 24 h, whereas HAS 1 levels first became detectable at 16 h. All three mRNAs were undetectable in untreated thyroid fibroblasts but HAS 3 mRNA was induced by IL-1ß after 16 h. rhTSH (2 mIU/ml) rapidly increased the transcript levels for all three HAS within 2 h. Notably, HAS 1 levels returned to baseline at 6 h, whereas the induction of HAS 2 and 3 mRNA remained elevated for 24 h, the duration of the study (Fig. 4Go). IGF-I (10 nM) increased HAS 2 and 3 mRNA levels at 6 h followed by a decline at 16 h, but HAS 1 remained undetectable at all time points.


Figure 4
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  		FIG. 4. HAS 1,2,3 transcripts are inducible by IL-1ß, TSH, and IGF-I in thyroid epithelial cells and HAS 3 mRNA is detectable in situ in thyroid tissue. Thyrocytes and fibroblasts were seeded in 100-mm plates and grown to confluence in medium with 10% FBS. They were then shifted to serum-free medium for 16 h and treated with IL-1ß (10 ng/ml), rhTSH (2 mIU/ml), or IGF-I (10 nM). Cell layers were harvested, RNA extracted and subjected to quantitative PCR as described in Materials and Methods. Results are representative of three separate experiments.

 
HAS 1, 2, and 3 mRNA levels in thyroid tissue
Thyroid tissue (0.5–1 g) from control, GD, and HT glands was homogenized, RNA extracted and subjected to RT-PCR using specific HAS 1, 2, and 3 primers. HAS 3 mRNA could be detected in all three tissues examined (Fig. 4Go), whereas HAS 1 and HAS 2 transcripts were undetectable (data not shown).

Primary thyroid epithelial cells stain strongly for thyroglobulin and TTF-1
Thyrocytes exhibited a uniform morphology in culture, which was maintained after multiple passages. We next determined whether they retained aspects of differentiated thyroid function. As seen in Fig. 5Go, A and C, most cells from normal thyroid tissue stained strongly for thyroglobulin and TTF-1. To confirm the specificity of the primary Ab, negative control slides were run in the absence of the primary Ab (Fig. 5Go, B and D). Thyroglobulin staining was undetectable in HeLa cells (data not shown).


Figure 5
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  		FIG. 5. Immunostaining of thyrocytes and thyroid tissue. Thyrocytes express thyroglobulin and TTF-1. A, Thyrocytes in monolayer culture show strong cytoplasmic staining for thyroglobulin. C, Nuclear staining for TTF-1. B and D, Negative controls. Thin sections from GD and HT thyroid exhibit increased hyaluronan staining. E, Negative staining in normal thyroid (x40); inset shows faint focal cytoplasmic positivity (x400). F, Diffuse staining for hyaluronan in HT (x40); inset highlights stromal staining (x100). G, Diffuse reactivity in GD (x40); membranous localization in follicular epithelium, inset (x400). H, Diffuse reactivity in GD (x40); predominance of stromal staining, inset (x600). I, Patchy reactivity in GD (x40), cytoplasmic localization in follicular epithelium, inset (x400). J, From a GD gland with focal patchy staining, an area of essentially negative staining is shown (x40; inset, x400).

 
Elevated hyaluronan levels in thyroid tissue from patients with GD and HT
Thyroid tissue sections from patients with GD, HT, and those without autoimmune disease were examined for hyaluronan content. Reactivity for hyaluronan was interpreted without knowledge of clinical parameters. Intensity and distribution of staining were considered in a semiquantitative assessment of immunohistochemical results. The distribution of staining in stroma and cells was graded as focal (≤10%), regional (11–50%) or diffuse (>50%). The intensity of cytoplasmic staining was subjectively graded as negative (0+), weak (1+), moderate (2+), or intense (3+) (Table 1Go).


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  		TABLE 1. Distribution and intensity of hyaluronan staining in thyroid tissue sections

 
Staining was observed in stroma and in the cytoplasm of follicular epithelium, and to a far lesser extent in lymphoid cells. In normal samples, the staining was limited to rare foci of weakly positive cytoplasm within the follicular epithelium (Fig. 5EGo and Table 1Go). Cases of GD exhibited variable staining distribution (focal to diffuse) and staining intensity (1–3+) although the staining did appear to be predominantly epithelial (Fig. 5Go, G–J, and Table 1Go). Cases of HT demonstrated strong (2–3+) staining, which was similarly variable in distribution (Table 1Go). Diffuse hyaluronan staining in a HT gland is shown in Fig. 5FGo. The follicular epithelium staining appears increased as the volume of cytoplasm increases, a phenomenon observed in both GD and HT. Epithelial staining appears to be stronger in GD than in HT. There also appears to be increased stromal staining in association with increased numbers of lymphoid cells (Table 1Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Localized hyaluronan accumulation in orbital and dermal tissues represents a hallmark of GD (5, 29), whereas generalized accumulation in many tissues is characteristic of the hypothyroidism associated with HT. Whether levels of this complex carbohydrate become altered in the human thyroid as a consequence of autoimmune disease had not been explored previously. Burgi-Saville et al. (30) studied several molecules comprising the extracellular matrix in normal and adenomatous thyroid but failed to assess hyaluronan levels. Shishiba et al. (18) examined total glycosaminoglycan levels in human thyroid and found them to be elevated in adenomas and adenocarcinomas. Stromal hyaluronan abundance, as assessed by immunostaining intensity, was increased in differentiated thyroid carcinomas (19). From the data presented here, hyaluronan is undetectable in control thyroid tissue, yet can be detected uniformly and is frequently abundant in autoimmune thyroid glands albeit with variable patterns of distribution. Hyaluronan represents a normal constituent of many tissues including the skin, myocardium, cerebral cortex, kidney (31, 32), vasculature (33), gastric mucosa (34), and tracheal cartilage (35). During thyroid hormone deprivation, it is thought to accumulate in these tissues due to a reduced clearance rate (16) as well as an increased synthetic rate (36). After correction of the hypothyroid state, tissue content normalizes (15, 31, 34, 35, 37). In contrast, the molecular basis for hyaluronan accumulation in the orbit and pretibial skin associated with GD is less well understood. Resident fibroblasts may increase synthesis in response to circulating Abs and/or cytokines elaborated by infiltrating immune cells (12, 13, 14). Furthermore, reversal of hyperthyroidism fails to correct the pathologic process. Because HT and GD are associated with goiter development and thyroid dysfunction, our findings warrant further examination into the potential relationship between elevated levels of hylauronan and gland enlargement.

Increased serum and urine glycosaminoglycans in GD had been observed some time ago (38, 39). However, Martins et al. (40) only recently attempted to correlated levels of hyaluronan with the clinical activity of thyroid-associated ophthalmopathy. Whether these elevated levels reflect the generalized activation of fibroblasts and other cells generating the molecule or represent synthesis occurring in the thyroid is uncertain.

The results presented here suggest that thyrocytes produce hyaluronan under basal culture conditions. The rate of accumulation is dependent on cell density and FBS concentration. Like thyrocytes, thyroid fibroblasts also synthesize hyaluronan, and the production is substantially greater on a per-cell basis than that found in epithelial cells. Hyaluronan production in thyroid fibroblasts can be up-regulated by IL-1ß and IGF-I. Both have been shown to enhance hyaluronan synthesis previously in other types of fibroblasts (14, 41) and cells of mesenchymal origin (42). In the context of inflammation, cytokines might drive the increased hyaluronan production occurring in autoimmune diseases. The effects of IL-1ß were robust and specific in that other proinflammatory cytokines including, TNF{alpha} and TGF-ß failed to significantly alter hyaluronan production. Wegrowski et al. (21) and Giraud et al. (20) examined porcine thyrocytes arranged as follicles and found that those chronically cultured in medium containing bovine TSH (1 and 5 mIU/ml, respectively) exhibited increased 3H-glucosamine incorporation into hyaluronan compared with cells cultured without TSH (20, 21). TSH (1 mIU/ml) treatment for 24 h had no effect on 3H-glucosamine incorporation in thyrocytes (21). Shishiba et al. (43) studied FRTL-5 cells and found a dose-dependent increase in 3H-glucosamine and 35S-sulfate incorporation into chondroitin and heparan sulfate proteoglycans in response to bovine TSH for 72 h. In our studies, rhTSH treatment for 48 h failed to alter hyaluronan production in human thyrocytes arranged in monolayers. Differences between experimental protocols, inherent divergence between human and animal thyrocyte phenotypes, or the different TSH preparations used could account for these variable results.

Glycosaminoglycans have been implicated in the pathogenesis of rheumatoid arthritis (44). Extracellular matrix hyaluronan plays a role in cell aggregation and intercellular adhesion (45), as well as endothelial cell attachment to basement membranes (46). It can induce maturation and migration of dendritic cells and macrophages as well as T cell activation and trafficking (6, 7). These effects are mediated through high-affinity association with cellular proteins, including CD44, RHAMM, and the toll-like receptor 4 complex (6). Interestingly, hyaluronan molecules of different chain-length, ranging from oligosaccharides to intermediate (80–200 kDa) and high molecular mass (600–1000 kDa), may act through distinct receptors and exert different biological influence (6).

We have previously shown that IL-1ß induces HAS mRNA in orbital fibroblasts (47). Jacobson et al. (11) examined HAS transcripts in human mesothelial cells and found differential induction of each transcript by various stimuli suggesting separate functional roles for each. All three HAS isoforms were detected in cultured thyrocytes and were up-regulated differentially by IL-1ß, TSH, and IGF-I. HAS mRNA induction did not correlate well with hyaluronan production, suggesting that other factors might be necessary for the expression of HAS proteins and for the biosynthesis of the macromolecule. Our current findings indicate increased intra-glandular hyaluronan levels in autoimmune thyroid disease. Thyrocytes and fibroblasts both produce the complex sugar and therefore might contribute to its accumulation in disease. Whether increased hyaluronan content in the thyroid plays any role in goiter formation or in other aspects of glandular dysfunction remains unclear.


   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 for facilitating sample collection. We thank Elizabeth Paunovich and Shweta Kamat for their expert technical assistance and Debbie Hanaya for her assistance with preparation of the manuscript.


   Footnotes
 
This work was supported in part by the National Institutes of Health Grants K23 RR17304, M01 RR00425, EY 08976, EY 11708, and DK 063121.

Disclosure: A.G.G., T.A.J., and C.S.K. have nothing to declare. T.J.S. is an inventor in U.S. Patent No. 066742-10493.

First Published Online October 26, 2006

Abbreviations: Ab, Antibody; FBS, fetal bovine serum; GD, Graves’ disease; HABP, hyaluronan binding protein; HAS, hyaluronan synthase; HT, Hashimoto’s thyroiditis; TSHR, TSH receptor; TTF-1, thyroid transcription factor-1.

Received June 2, 2006.

Accepted for publication October 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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