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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 10 5076-5080
Copyright © 2004 by The Endocrine Society

Immunoglobulins from Patients with Graves’ Disease Induce Hyaluronan Synthesis in Their Orbital Fibroblasts through the Self-Antigen, Insulin-Like Growth Factor-I Receptor

Terry J. Smith and Neil Hoa

Division of Molecular Medicine (T.J.S.), Department of Medicine, Harbor-University of California Los Angeles Medical Center, Torrance, California 90502; The Jules Stein Eye Institute (T.J.S.), David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90095 (T.J.S.); and Long Beach Veterans Administration Healthcare System (T.J.S., N.H.), Long Beach, California 90822

Address all correspondence and requests for reprints to: Terry J. Smith, M.D., Division of Molecular Medicine, Building C-2, Harbor-University of California Los Angeles Medical Center, 1124 West Carson Street, Torrance, California 90502. E-mail: tjsmith{at}ucla.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A distinctive histopathological feature associated with thyroid-associated ophthalmopathy is the disordered accumulation of the glycosaminoglycan, hyaluronan, in orbital connective tissues. This often occurs in the context of dramatic inflammation and tissue remodeling. Orbital fibroblasts exhibit a novel phenotype including exaggerated responses to cytokines. Here, we report for the first time the ability of IgG isolated from the sera of patients with Graves’ disease (GD-IgG) to provoke in orbital fibroblasts the synthesis of hyaluronan. The effect of GD-IgG can be reproduced by IGF-I, appears to be mediated through the IGF-I receptor, and is abolished with glucocorticoid treatment. TSH failed to influence the synthesis of hyaluronan. In contrast to the effects in GD fibroblasts, cultures derived from donors without known thyroid disease fail to respond to GD-IgG or IGF-I. The observation that hyaluronan production is induced by GD-IgG in fibroblasts suggests that the IGF-I receptor and its activating antibodies may represent a key pathway through which important pathogenic events in thyroid-associated ophthalmopathy are mediated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID-ASSOCIATED ophthalmopathy (TAO) is a component of Graves’ disease (GD), in which orbital connective tissues undergo an often dramatic remodeling, including intense inflammation and accumulation of the glycosaminoglycan, hyaluronan (1, 2). Net increases in hyaluronan levels in conjunction with fat expansion are currently believed to underlie much of the tissue dysfunction associated with TAO. Important rheological properties of hyaluronan include its profound hydrophilic nature and extreme molecular bulk when hydrated (2). Increased tissue volume in the orbit leads to the partial expulsion of the eye and results in proptosis. Unfortunately, the morbidity directly associated with tissue volume expansion does not appear particularly amenable to therapies for TAO thus far developed, including external beam radiation and steroids. We believe that the development of successful remedies for TAO awaits additional insight into its pathogenesis.

Hyaluronan production in orbital fibroblasts has been a focus of several groups over the past few years because it has been presumed that the high tissue levels of the glycosaminoglycan occurring in TAO result from enhanced macromolecular synthesis in these cells. Moreover, it was appreciated that proinflammatory cytokines such as IL-1ß, CD154, and leukoregulin, as well as growth factors such as plateletderived growth factor and IGF-I can substantially enhance the rate of hyaluronan and/or proteoglycan synthesis in orbital fibroblasts (3, 4, 5). Increases in production are generally inhibited with glucocorticoids and in most instances are associated with induction of the hyaluronan synthases (6) and UDP-glucose dehydrogenase (7). The latter enzyme is immediately upstream from the terminal hyaluronan synthases. The magnitude of enhanced hyaluronan synthesis was appreciably greater in orbital fibroblasts than that found in their dermal counterparts (3).

Patients with TAO invariably present with circulating autoantibodies [IgG isolated from the sera of patients with GD (GD-IgG)], including those directed against the TSH receptor (TSHR) (8). These antibodies are known to bind to the receptor and activate signaling through G protein-coupled pathways. The roles of TSHR and the IgGs directed against it in the pathogenesis of extraglandular GD are uncertain but have been the topic of considerable debate in recent years (9, 10). TSHR has been detected in orbital connective tissues (11, 12) and cultured fibroblasts derived from those tissues (13). Subsequently, tissues from all body regions examined have been found to express TSHR (14, 15). Levels of TSHR are currently thought to be considerably lower in connective tissue than those found in thyroid and receptor expression may be the consequence of illegitimate transcription (16).

We recently reported that patients with GD have evidence for another potentially pathogenic GD-IgG that can recognize the IGF-I receptor (IGF-IR) on fibroblasts and activate that receptor (17). IGF-IR activation caused by treatment of cells with either GD-IgG or IGF-I results in the induction of two powerful T cell chemoattractants (18). IL-16 is a CD4-specific ligand, whereas RANTES (regulated on activation normal T cell expressed and secreted) belongs to the C-C class of chemokines (19, 20). Both are substantially upregulated by GD-IgG, albeit through apparently different mechanisms, in fibroblasts from patients with GD (18). In contrast, GD-IgG and IGF-I have no such effects in control fibroblasts (18). By interfering with IGF-IR expression or function, the signaling provoked by GD-IgG can be completely attenuated, suggesting that this receptor is mediating the cellular responses. Moreover, whereas IGF-I can mimic the effects of GD-IgG, TSH had no impact on IL-16 or RANTES expression (18).

We report here for the first time that GD-IgG, like IGF-I, can up-regulate the production of hyaluronan in cultured GD orbital fibroblasts. We find that interfering with the association between GD-IgG and IGF-IR can block this effect. Our findings, for the first time, offer a potential explanation for how GD-IgG can provoke hyaluronan accumulation in orbital connective tissues in TAO.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

An ELISA kit for hyaluronan was purchased from Echelon Biosciences Inc. (Salt Lake City, UT). Recombinant human IGF-I was from R&D Systems (Minneapolis, MN). Dexamethasone (9{alpha}-fluoro-16{alpha}-methyl-11ß,17{alpha}, 21-trihydroxy-1,4-pregnadiene-3,20-dione) and recombinant human TSH were obtained from Sigma (St. Louis, MO). Recombinant IL-1ß and 1H7 were purchased from Biosource International (Camarillo, CA) and BD PharMingen (San Diego, CA), respectively. Human control and GD sera as well as surgical waste for initiating fibroblast cultures were obtained after informed consent using a protocol approved by the institutional review board of the Harbor-University of California Los Angeles Medical Center (Torrance, CA). GD sera were collected from patients without or with TAO. Patients were newly diagnosed and had not yet been treated for their hyperthyroidism. Those with TAO exhibited active disease of recent onset and were not on steroids. Control sera were obtained from individuals without autoimmune disease or from patients with systemic lupus erythematosus or Hashimoto’s thyroiditis. GD-IgG was prepared as described (21). Reagents used in these studies were of the highest purity commercially available.

Cell culture

Human orbital and dermal fibroblasts were cultivated in T-75 culture flasks. Orbital fibroblasts were obtained from patients undergoing decompressive surgery for TAO or some other surgical procedure to treat a noninflammatory eye disease, after informed consent. Dermal fibroblasts were initiated from normal appearing skin or were purchased from The American Type Culture Collection (Rockville, MD). Fibroblasts were cultured in DMEM containing 10% fetal bovine serum, antibiotics, and glutamine. Cells were passaged by gentle treatment with trypsin/EDTA in PBS. They were incubated in a 37 C, 5% CO2, humidified environment and allowed to reach confluence in 24-well plastic dishes. Cultures were then shifted to DMEM containing 1% fetal bovine serum overnight. They were then treated with the test compounds indicated in the legends to the figures.

Hyaluronan assay

Hyaluronan was quantified with a specific ELISA. After the treatments described, media were aspirated and cell layers rinsed with PBS. Monolayers were solubilized in 0.1 N NaOH for protein determination or trypsinized for cell count. The remainder of the cellular material was removed from the substratum with a rubber policeman and disrupted by sonication. Cell layers and media were combined, centrifuged, and the supernatants were subjected to the hyaluronan assay according to the manufacturer’s instructions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human fibroblasts in culture synthesize relatively high levels of hyaluronan (22, 23). They apparently fail to express hyaluronidase activity in vitro (24), suggesting that disposal of the macromolecule in situ may involve other cell types. Confluent cultures of dermal and orbital fibroblasts were treated with IGF-I (10 nmol/liter) for graded intervals. Media and cell layer material were subjected to an ELISA specific for hyaluronan. As the data in Fig. 1Go demonstrate, hyaluronan levels are increased by IGF-I in GD orbital fibroblasts in a time-dependent manner. They reached levels nearly twice those of controls at 48 and 72 h after initiation of treatment. In contrast, IGF-I failed to exert an appreciable effect on hyaluronan accumulation in orbital fibroblasts from a patient without known thyroid disease and in control dermal fibroblasts. The effect of IGF-I treatment for 48 h was compared with that of GD-IgG (100 ng/ml) in the three types of fibroblast cultures. Although both could induce hyaluronan production in GD orbital cultures, neither influenced glycosaminoglycan accumulation in dermal or control orbital cultures (Fig. 2Go). This result is entirely congruent with that found earlier concerning the effect of IGF-I and GD-IgG on IL-16 and RANTES expression in fibroblasts (17).



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  		FIG. 1. Time-dependent effects of IGF-I on the accumulation of hyaluronan in orbital fibroblasts from patients with GD, orbital fibroblasts from donors without known thyroid disease (control), and dermal fibroblasts. Confluent cultures were treated with IGF-I (10 nmol/liter) for the times indicated along the abscissa. Cell layer material was harvested, an aliquot reserved for protein determination, and the remainder combined with media and subjected to an ELISA specific for hyaluronan. Results were corrected for cell layer protein content and are expressed as the mean ± SD of triplicate determinations.

 


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  		FIG. 2. Effects of IGF-I and GD-IgG on hyaluronan production in GD and control orbital fibroblasts and dermal fibroblasts. Confluent cultures were treated with nothing (control), IGF-I (10 nmol/liter), or GD-IgG (100 ng/ml) for 48 h. Medium was collected, cell layers disrupted, and cell counts conducted. The monolayer and medium material were combined and subjected to the specific hyaluronan ELISA. Results were corrected for respective cell counts and are shown as the mean ± SD of triplicate determinations.

 
We next determined whether we could implicate the IGF-IR in the mediation of the GD-IgG effects on hyaluronan production in GD orbital fibroblasts because we were able to do so concerning its effects on T cell chemoattractant expression in these cells (17). Using the specific IGF-IR-blocking monoclonal antibody 1H7, the induction of hyaluronan by GD-IgG was completely attenuated (Fig. 3AGo). This blocking antibody binds to the receptor but fails to initiate signaling and prevents IGF-IR activation (25). Moreover, IGF-I could induce hyaluronan production selectively in the GD orbital cultures, and this could also be blocked with 1H7. The blocking monoclonal antibody had no effect on the induction of hyaluronan by IL-1ß, suggesting its specificity (Fig. 3BGo). In contrast to GD-IgG, IgG from individuals without known thyroid disease (control IgG) failed to alter hyaluronan accumulation. Multiple GD sera were tested for their effect on hyaluronan synthesis in GD fibroblasts. Of the six tested, including three from patients with GD without TAO and three from patients with eye involvement, all could upregulate hyaluronan synthesis (Fig. 3CGo). In addition, sera from patients with systemic lupus erythematosus or Hashimoto’s thyroiditis failed to induce hyaluronan production. Importantly, rhTSH (1 mU/ml) also failed to influence synthesis of the macromolecule (Fig. 3BGo). To determine whether the action of GD-IgG on hyaluronan production was common to multiple fibroblast strains, we tested several cultures, each initiated from a different donor. As the data in Fig. 4Go demonstrate, all three strains of GD orbital fibroblasts exhibited a substantial induction of hyaluronan when treated with GD-IgG, whereas the control orbital and dermal cultures failed to respond. Thus, it would appear that the action of GD-IgG may be restricted to fibroblasts derived from patients with GD.



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  		FIG. 3. Effects of GD-IgG, IGF-I, rhTSH, IL-1ß, or sera from donors with other autoimmune diseases on hyaluronan accumulation in GD orbital fibroblasts. Confluent cultures were treated with control IgG or GD-IgG (100 ng/ml), IGF-I (10 nmol/liter), rhTSH (1 mU/ml), sera from various autoimmune diseases (1%), or IL-1ß (10 ng/ml) without or with dexamethasone (Dex; 10 nmol/liter) or 1H7 (5 µg/ml) for 48 h. Hyaluronan was quantified using a specific ELISA and normalized to cell counts. Each datum point represents the mean ± SD of triplicate determinations (A) or mean ± range of duplicates (B and C).

 


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  		FIG. 4. Effect of control IgG and GD-IgG on hyaluronan accumulation in several strains of dermal and orbital fibroblasts. Confluent cultures were treated for 48 h with nothing (control) or 100 ng/ml of either IgG preparation. Samples were processed as described in the legend to Fig. 2Go. Each datum point represents the mean ± SD of triplicate determination.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have recently reported that GD fibroblasts, when treated with GD-IgG, express high levels of IL-16 and RANTES (18) and that IGF-IR mediates those effects (17). The observations we report here coupled with those earlier findings strongly suggest that IGF-IR represents an important self-antigen relevant to the pathogenesis of GD. When activated by GD-IgG, multiple biological consequences can be detected in fibroblasts. From the current findings, GD-IgG appears to induce hyaluronan in orbital fibroblasts, potentially explaining, at least in part, the accumulation of that glycosaminoglycan in GD. On the other hand, expression of powerful T cell chemoattractants might underlie lymphocytic infiltration found in thyroid and connective tissues. It would appear that these responses are unified by virtue of the apparent involvement of IGF-IR and the IgGs recognizing the receptor. The responses in fibroblasts to GD-IgG reported earlier from other laboratory groups, including an induction of collagen production (26) and the up-regulation of intercellular adhesion molecule expression (27), suggest the broader implication of the interactions between IgGs associated with GD and fibroblasts. Neither of these earlier studies provided insight into the identity of the relevant antigen; thus, it is tempting to speculate whether IGF-IR might have played some role in those responses.

IGF-IR expression and activity in GD fibroblasts appear to differ from those in control cells derived from individuals without known autoimmune disease. Levels of IGF-IR{alpha} and IGF-IRß are substantially higher in the former (17). Moreover, neither GD-IgG nor IGF-I or its highly specific analog Des 1–3 can induce IL-16 expression in control fibroblasts (17). Thus, the findings reported here that hyaluronan production in control fibroblasts is also unaffected would suggest that fundamental differences in GD fibroblasts may exist with regard to IGF-IR display and the activation of relevant signal transduction pathways.

Orbital fibroblasts exhibit a phenotype that sets them apart from cells derived from other anatomic regions. For instance, they express extraordinarily high levels of the inflammatory cyclooxygenase, prostaglandin endoperoxide H synthase-2, when activated by proinflammatory cytokines such as leukoregulin, CD154, and IL-1ß (5, 28, 29, 30). Moreover, these same cytokines up-regulate hyaluronan production in orbital fibroblasts to levels considerably greater than those found in dermal cultures (3). TGF-1ß, leukoregulin, interferon-{gamma}, and IL-1ß induce plasminogen activator inhibitor 1 dramatically in orbital fibroblasts (31, 32, 33), suggesting powerful effects on extracellular matrix economy. The results we report here strongly suggest that potentially important differences in the regulation of macromolecular synthesis might also exist between orbital fibroblasts from patients with GD and those from control donors. Whether dermal fibroblasts from patients with dermopathy will respond to GD-IgG remains uncertain. Unfortunately, access to fibroblasts from these lesions is limited, in part because of concerns for healing of the biopsy sites.

It is unclear from the current studies whether the same signaling pathways used by GD-IgG in up-regulating IL-16 and RANTES are relevant to the induction of hyaluronan. GD-IgG has been shown to activate p70s6k in GD fibroblasts, and blocking the FRAP/mTOR/p70s6k pathway with the specific macrolide inhibitor rapamycin can completely attenuate the IL-16 induction (18). Whether this pathway proves relevant to the effects we report here on hyaluronan accumulation will necessarily await further studies. Of considerable mechanistic importance is the current observation that TSH fails to influence hyaluronan production. Thus, the TSHR does not appear to be involved in these activities of GD-IgG. We have reported previously that the induction of hyaluronan by cytokines involves the up-regulation of key components of the biosynthetic pathway, including all three isoforms of hyaluronan synthase (6) and UDP-glucose dehydrogenase (7). In future studies, we will need to assess the levels of these proteins and the activities of their respective encoding genes to fully understand the mechanisms involved in the actions of GD-IgG on hyaluronan synthesis in fibroblasts. In any event, the current findings further demonstrate the potential importance of interactions between GD-IgGs and these fibroblasts. From the results of studies thus far performed, the IGF-IR pathway emerges as a potentially important pathogenic conduit for many of the actions of activating GD-IgGs on fibroblasts. Therefore, it is possible that specific therapy for the autoimmune components of GD (TAO and pretibial dermopathy) might involve the interruption of IGF-IR signaling in fibroblasts and perhaps in other cell types as well.


   Acknowledgments
 
We thank Ms. Debbie Hanaya for expert help in preparing this manuscript.


   Footnotes
 
This work was supported in part by National Institutes of Health Grants EY008976, EY011708, and DK063121 and by a Merit Review Award from the Research Service of the Department of Veterans Affairs.

Abbreviations: GD, Graves’ disease; GD-IgG, IgG isolated from the sera of patients with GD; IGF-IR, IGF-I receptor; RANTES; regulated on activation normal T cell expressed and secreted; TAO, thyroid-associated ophthalmopathy; TSHR, TSH receptor.

Received April 15, 2004.

Accepted June 29, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hufnagel TJ, Hickey WF, Cobbs WH, Jakobiec FA, Iwamoto T, Eagle RC 1984 Immunohistochemical and ultrastructural studies on the exenterated orbital tissues of a patient with Graves’ disease. Ophthalmology 91:1411–1419[Medline]
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  29. Cao HJ, Smith TJ 1999 Leukoregulin up-regulation of prostaglandin endoperoxide H synthase-2 expression in human orbital fibroblasts. Am J Physiol Cell Physiol 277:C1075–C1085
  30. Han R, Tsui S, Smith TJ 2002 Up-regulation of prostaglandin E2 synthesis by interleukin-1ß in human orbital fibroblasts involves coordinate induction of prostaglandin-endoperoxide H synthase-2 and glutathione-dependent PGE2 synthase expression. J Biol Chem 277:16355–16364[Abstract/Free Full Text]
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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals