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Thyrotropin Receptor Expression in Graves’ Orbital Adipose/Connective Tissues: Potential Autoantigen in Graves’ Ophthalmopathy¹

Division of Endocrinology, Mayo Clinic/Foundation (R.S.B., C.M.D., N.N.), Rochester, Minnesota 55905; and Medizinische Klinik, Klinikum Innenstadt, University of Munich (W.J., C.S., A.E.H.), 80336 Munich, Germany

Address all correspondence and requests for reprints to: Rebecca S. Bahn, M.D., Division of Endocrinology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905. E-mail: bahn.rebecca{at}mayo.edu

	Abstract

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It is acknowledged that the TSH receptor (TSHr) on thyroid follicular cells is the autoantigen involved in the hyperthyroidism of Graves’ disease. However, whether this receptor is expressed in extrathyroidal tissues, and whether it participates directly in the pathogenesis of Graves’ ophthalmopathy (GO) are unclear. We sought to detect the expression of TSHr messenger ribonucleic acid (mRNA) and protein in orbital adipose/connective tissue specimens and in human orbital preadipocyte fibroblast cultures using liquid hybridization analysis and immunohistochemical methods. We demonstrated intact and variant TSHr mRNA transcripts and TSHr-like immunoreactivity in orbital adipose/connective tissue specimens from patients with GO. In addition, TSHr-like immunoreactivity was detected in early passage GO preadipocyte fibroblast cultures that were shown to include some adipose cells. In contrast, neither TSHr mRNA nor protein was detected in normal orbital adipose/connective tissue specimens or in late passage GO orbital fibroblast cultures containing no lipid-laden adipose cells.

In conclusion, we showed that TSHr is expressed in the adipose/connective tissue of the diseased orbit in GO. In addition, TSHr is demonstrable in early passage GO preadipocyte orbital fibroblast cultures that contain a subpopulation of adipocytes. Subsequent passaging of these cells results in the loss of both TSHr expression and adipocyte-specific staining. These results suggest that both the expression of this receptor and the accumulation of adipose tissue in the orbit in GO may be induced in vivo by a humoral factor(s) not present in the cell culture environment.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
IT IS WELL accepted that the TSH receptor (TSHr) on thyroid follicular cells is the autoantigen involved in the hyperthyroidism of Graves’ disease (1). The close clinical association between Graves’ hyperthyroidism and Graves’ ophthalmopathy (GO) has led to the concept that there may be an autoantigen common to thyroid and orbital tissues that is recognized by circulating lymphocytes in Graves’ disease. Infiltration of the orbit by activated lymphocytes, a process known to occur in GO, results in the local release of inflammatory cytokines and leads to the characteristic histologic and clinical features of GO (2). If the TSHr, or a variant thereof, were present in affected orbital tissues, this receptor might serve as the putative shared autoantigen and constitute the link between the thyroid and the eye in Graves’ disease.

Several groups of investigators have sought evidence for TSHr expression in various orbital tissues. Attempts to identify TSHr messenger ribonucleic acid (mRNA) in these tissues using Northern blotting have proved largely unsuccessful (3, 4, 5), although a recent study showed positive bands in a single specimen after a long exposure period (6). In contrast, several laboratories have detected TSHr mRNA or a variant TSHr transcript in human orbital tissues and cell cultures using RT-PCR (3, 7, 8, 9, 10, 11). However, RNA transcripts that can be detected only by PCR-based amplification of complementary DNA (cDNA) may have little physiological relevance (12). Therefore, we sought to clarify the issue using the sensitive and direct methods of liquid hybridization analysis (LHA) and immunohistochemistry to detect this low abundance mRNA and protein in multiple orbital tissue specimens and fibroblast cultures.

Recent studies by Smith and colleagues showed that a subpopulation of orbital fibroblasts is capable of differentiating into lipid-filled adipocytes when treated with particular hormones and other supplements (13). Based on these observations, the term preadipocyte fibroblasts was used to acknowledge that at least some orbital fibroblasts (14) are capable of undergoing adipocyte differentiation. We have noted that some early passage GO orbital fibroblasts are rounded, granular, and appear to contain lipid droplets (unpublished observation). In this study, we examined both early and late passage preadipocyte fibroblasts (cultured without supplementation) to determine whether a subpopulation of these cells contains triacylglycerol, and whether there appears to be a correlation between TSHr expression and the presence of adipocytes in these fibroblast cultures.

	Materials and Methods

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Cell culture

Orbital adipose/connective tissue explants were obtained from patients during orbital decompression surgery for severe GO or in the course of orbital surgery for other conditions in patients with no history of Graves’ disease. Graves’ (n = 14) or normal (n = 3) tissue samples were minced and placed directly in plastic culture dishes, allowing preadipocyte fibroblasts to proliferate as described previously (14). Cultures were propagated in medium 199 containing 20% FBS (HyClone, Logan, UT), penicillin (100 U/mL), and gentamicin (20 µg/mL) in a humidified 5% CO₂ incubator at 37 C. Cultures were maintained in 75-mm² flasks with medium 199 containing 10% FBS and used between the first (early) and fifth (late) passages. All cells in culture established in this fashion show immunoreactivity with antibodies directed against fibroblast antigen, vimentin, and collagen and do not react with antidesmin and antimyosin antibodies (Dakopatts Corp., Santa Barbara, CA). OCT specimens were obtained in the course of orbital decompression surgery for severe GO (GO OCT; n = 19) or from a patient with no history of thyroid disease who underwent ocular enucleation for malignant disease (NL OCT). These specimens were stored frozen at -70 C until processed for RNA isolation.

RNA preparation

Total RNA was isolated directly from approximately 10 x 10⁶ preadipocyte fibroblasts in culture or from uncultured OCT samples using the Totally RNA Kit (Ambion, Austin, TX). GO OCT samples (0.5–1.0 mg each) from 2–10 individual GO patients were combined before RNA extraction to obtain sufficient material for LHA (n = 4 different GO OCT sample pools examined). A single NL OCT sample supplied sufficient RNA for analysis. Positive control RNA was prepared in the same manner from cultured Chinese hamster ovary (CHO) cells that had been transfected with plasmid containing the human TSHr (JPO9 line) or from a negative control counterpart (JPO₂ line) (15).

LHA

The antisense RNA probe for TSHr LHA was transcribed from a 320-bp PCR product with a T7 phage promoter at its 3-prime end, in the presence of T7 RNA polymerase (10 U) and [³²P]UTP (50 µCi) for labeling. The DNA template used for the PCR was a pBluescript II (SK⁺) plasmid containing TSHr cDNA. The resulting high specific activity probe encompassed nucleotides 576–873 (exons 6–9) of the human TSHr cDNA sequence as reported by Nagayama (16) and was designed to detect both the 2.4-kilobase (kb) intact TSHr (protecting a product of 298 bp) and the 1.3-kb variant form (17) (protecting a product of 217 bp). The antisense RNA probe for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) LHA was generated from pTRI-GAPDH human antisense control template (Ambion, Austin, TX). This probe was designed to detect a 316-bp fragment of GAPDH mRNA.

Total RNA (80 µg) was combined with probe (300,000–500,000 cpm) in hybridization buffer, denatured at 95 C, and hybridized at 45 C for 16 h. Nonhybridized total RNA and probe were digested for 1 h at 37 C with ribonuclease A (RNase A; 0.175 U) and RNase T1 (25 U; RNase Protection Kit, Boehringer Mannheim, Indianapolis, IN). Samples were subsequently digested with proteinase K (50 µg) in the presence of 0.5% SDS and extracted with phenol-chloroform-isoamyl alcohol. The resulting ethanol-precipitated protected fragments were resuspended in loading buffer and resolved on a denaturing polyacrylamide gel (5% acrylamide-8 mol/L urea). A standard curve was generated concurrently using in vitro synthesized full-length sense strand TSHr RNA in quantities ranging from 0.05–1.0 fmol.

Immunohistochemistry

Cryostat-cut, 7-µm sections of frozen GO OCT (n = 6) and NL OCT (n-4) sections were washed with phosphate-buffered saline (PBS), and nonspecific binding was blocked with 5% sheep serum (Sigma, Deisenhofen, Germany). Early (first and second) passage to late (fifth and higher) passage normal (n = 4) and GO (n = 7) orbital preadipocyte fibroblasts were plated onto glass slides, fixed with 2% paraformaldehyde, and blocked with 5% sheep serum. Mouse monoclonal hTSHr antibody (1 mg/mL) directed against C-terminal amino acid residues 604–764 of recombinant human TSHr (18) (TRANSBIO S.A.R.L., Boulogne, France) was applied to slides for 2 h at room temperature. Slides were washed and incubated with biotinylated antimouse Ig (1:200 dilution; Amersham, Braunschweig, Germany) for 1 h at room temperature, then washed with PBS, rinsed with Tris-buffered saline (100 mmol/L Tris and 150 mmol/L NaCl), and incubated with streptavidin-alkaline-phosphatase conjugate (dilution, 1:150; Amersham) for 30 min. Slides were washed with Tris-buffered saline before incubation in developing buffer [100 mmol/L Tris (pH 9.5), 100 mmol/L NaCl, and 50 mmol/L MgCl₂] for 10 min, and the reaction product was visualized with chromogenic substrates in developing buffer. Slides were counterstained with malchite green for 5 min before mounting. A brown precipitate signified the presence of hTSHr-like immunoreactivity. Parallel slides with the primary and secondary antibodies, replaced, in turn, by PBS and isotype-matched nonimmune Ig (Sigma), were examined to assure specificity and to exclude cross-reactivities between the antibodies and the conjugates employed.

Oil Red O staining

First and late (greater than fourth) passage GO orbital fibroblast cultures were plated in 35-mm plastic culture dishes and maintained in medium 199 containing 10% FBS in a humidified 5% CO₂ incubator at 37 C. Cells were washed, fixed in 10% formalin, and exposed to filtered 0.35% Oil Red O in isopropanol/water (1:1 dilution) for 2 h. Washed cells were then incubated overnight with Giemsa stain before being visualized and photographed.

	Results

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Liquid hybridization analysis

Strongly positive protected bands at 298 and 217 bp, indicating the presence of intact (2.4 kb) and variant (1.3 kb) TSHr mRNA, respectively, were apparent in LHA gel lanes corresponding to GO OCT (n = 4 different explant pools; Fig. 1). In contrast, no TSHr mRNA was detected in the uncultured NL OCT sample obtained from a patient during ocular enucleation surgery for malignant disease. The intact receptor transcript (at 2.4 kb) was present in positive control JPO9 lanes and in lanes corresponding to the standard curve. Negative control JPO₂ lanes showed no bands. In addition, no protected bands were apparent in preparations of either normal (n = 3) or GO (n = 14) late passage orbital fibroblasts. It is not possible to perform LHA for TSHr on early passage NL or GO fibroblasts because the quantity of mRNA required for this assay (80 µg per sample) necessitates repeated passaging of cells. LHA of GAPDH mRNA using the same RNA samples showed equal protected bands in all lanes (data not shown).

View larger version (77K): [in this window] [in a new window]   Figure 1. Liquid hybridization analysis of TSHr mRNA. Lane 1, Undigested probe (321 bp); lane 2, digested probe; lane 3, NL OCT; lane 4, GO OCT; lanes 5–7, late passage GO orbital preadipocyte fibroblasts; lanes 8–12, standard curve; lane 13, JPO2 (negative control); lane 14, JPO9 (positive control); lane 15, molecular standards. Positive protected bands at 298 bp (corresponding to 2.4-kb intact TSHr) and 217 bp (corresponding to a 1.3-kb variant form TSHr) are apparent in lane 4 containing GO OCT.

TSHr-like immunoreactivity was detected in uncultured GO OCT (n = 6; Fig. 2A). In contrast, immunohistochemical analysis of NL OCT (n = 4) and various control human tissues, including uterus, bladder, and skeletal tissues, was negative (data not shown). In addition, TSHr-like immunoreactivity was detected strongly in first and second passage GO orbital preadipocyte fibroblast cultures (n = 5; Fig. 2B) and was also present, albeit somewhat less strongly, in the third passage of these cells (n = 4; Fig. 2C). However, no TSHr-like immunoreactivity was detected in late (fifth and higher) passage GO orbital preadipocyte fibroblast cultures (n = 7; Fig. 2D) or in early or late passage normal orbital fibroblast cultures (n = 4; data not shown). Parallel control OCT sections or cultured orbital fibroblasts that were processed with the primary and secondary antibodies replaced, in turn, by PBS and isotype-matched nonimmune Ig were also negative.

View larger version (99K): [in this window] [in a new window]   Figure 2. Immunohistochemical analysis to detect the presence of TSHr-like immunoreactivity in a frozen section of uncultured orbital/connective tissue from a patient with GO OCT (A) and in a representative first passage GO orbital preadipocyte fibroblast culture showing dark staining (B), a third passage culture showing light staining (C), and a fifth passage culture showing no staining (D). A monoclonal antibody directed against TSHr C-terminal amino acids 604–764 was used in these studies. Parallel control sections and plated slides with the primary and secondary antibodies replaced, in turn, by PBS and isotype-matched nonimmune Ig were negative.

A subpopulation of early passage GO orbital preadipocyte fibroblasts appeared rounded and granular and stained positively for Oil Red O, indicating the presence of traicylglycerol (Fig. 3). These cells tended to cluster together and constituted approximately 5% of the total cell population. Late passage GO orbital fibroblasts were more uniformly elongated, and cultures did not include cells that stained positively for lipid.

View larger version (147K): [in this window] [in a new window]   Figure 3. Photomicrograph of Oil Red O staining of human cultured preadipocyte fibroblasts obtained from a patient with severe Graves’ ophthalmopathy (magnification, x40). The arrows indicate the presence of triacylglycerol inclusions in a subpopulation of these cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we used LHA to demonstrate the presence of mRNA corresponding to TSHr extracellular domain in uncultured GO OCT and its absence in NL OCT samples. Because LHA is a method that does not depend on antibody specificity or RNA amplification, these results are the most convincing to date showing the presence of TSHr mRNA in orbital adipose/connective tissues in GO. The specificity of the particular TSHr antibody used in these studies (18) has been fully examined by several laboratories (18, 35), including our own (36), further supporting the concept that TSHr protein is present in these tissues. In addition, we repeated several of these studies using antibodies directed against other epitopes of the extracellular domain of TSHr, with similar results (data not shown). When these LHA and immunohistochemical studies are considered together, a total of 10 of 10 GO OCT samples showed evidence of TSHr RNA or protein, whereas 5 of 5 NL OCT samples were negative for TSHr expression. These combined results allow us to conclude that TSHr (both RNA and protein) is present in GO OCT, but is not detectable in NL OCT. The TSHr-like immunoreactivity demonstrated in early, but not in late, passage GO orbital preadipocyte fibroblast cultures supports our assertion that it is these cells within the GO OCT, rather than macrophages or other resident or infiltrating cells, that contain the TSHr mRNA and protein.

We found no evidence of TSHr mRNA in cultured late passage normal or GO orbital preadipocyte fibroblasts using LHA. However, we and others have demonstrated the presence of TSHr mRNA in these late passage cells using PCR (6, 7, 8, 9, 10, 11). Because our LHA can detect TSHr at 0.005 fmol, our negative LHA results suggest that less than this quantity of TSHr is present in these cells. The positive PCR results of previous studies, also using late passage cells, suggest either that less than 0.005 fmol TSHr RNA is present in late passage GO orbital fibroblasts or that illegitimate transcription of cDNA was being detected.

Our studies examining cultured orbital preadipocyte fibroblasts for the presence of triacylglycerol inclusions compliment the previous studies by Smith and colleagues (13). However, instead of exposing the cells to factors known to induce adipocyte differentiation, we cultured the cells under the standard conditions used for our orbital fibroblast cell cultures (14). We were interested in determining whether some of these cells, obtained for culture from GO orbital adipose/connective tissue, retain an adipocyte phenotype in early passage. We found that a subpopulation of orbital fibroblasts in early passage culture are indeed adipocytes, and that cells expressing this particular phenotype are not present in late passage cultures. Of particular interest was our observation that fibroblast cultures containing adipocytes (early passage GO orbital cells) tended to be the ones in which TSHr expression could be detected.

In conclusion, TSHr mRNA and protein are present in uncultured GO OCT and early passage GO orbital preadipocyte fibroblasts, but are not demonstrable in NL OCT specimens or late passage fibroblasts. These findings suggest that TSHr expression in the orbit in GO may be induced in this disease by a humoral factor that is not present in the cell culture environment. Because the orbit in GO is known to contain excess adipose tissue (37), it is possible that factors involved in the stimulation of TSHr expression in the orbit also play a role in the accumulation of orbital adipose tissue in this disease. The identity of the putative humoral TSHr-stimulating and adipocyte-differentiating factor(s) in GO awaits further study.

	Footnotes

 
¹ This work was supported in part by NIH Grant EY-O8819 (to R.S.B.) from the National Eye Institute and Grant He 1485/5–2 (to A.E.H.) from Deutsche Forschungsgemeinschaft (Bonn, Germany).

Received September 19, 1997.

Revised November 11, 1997.

Accepted December 5, 1997.

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				K. Starkey, A. Heufelder, G. Baker, W. Joba, M. Evans, S. Davies, and M. Ludgate Peroxisome Proliferator-Activated Receptor-{gamma} in Thyroid Eye Disease: Contraindication for Thiazolidinedione Use? J. Clin. Endocrinol. Metab., January 1, 2003; 88(1): 55 - 59. [Abstract] [Full Text] [PDF]

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				R. W. Valyasevi, D. A. Harteneck, C. M. Dutton, and R. S. Bahn Stimulation of Adipogenesis, Peroxisome Proliferator-Activated Receptor-{gamma} (PPAR{gamma}), and Thyrotropin Receptor by PPAR{gamma} Agonist in Human Orbital Preadipocyte Fibroblasts J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2352 - 2358. [Abstract] [Full Text] [PDF]

				R. Metcalfe, N. Jordan, P. Watson, S. Gullu, M. Wiltshire, M. Crisp, C. Evans, A. Weetman, and M. Ludgate Demonstration of Immunoglobulin G, A, and E Autoantibodies to the Human Thyrotropin Receptor Using Flow Cytometry J. Clin. Endocrinol. Metab., April 1, 2002; 87(4): 1754 - 1761. [Abstract] [Full Text] [PDF]

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				B. E. Busuttil and A. G. Frauman Extrathyroidal Manifestations of Graves' Disease: The Thyrotropin Receptor Is Expressed in Extraocular, But Not Cardiac, Muscle Tissues J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 2315 - 2319. [Abstract] [Full Text]

				R. W. Valyasevi, S. C. Jyonouchi, C. M. Dutton, N. Munsakul, and R. S. Bahn Effect of Tumor Necrosis Factor-{{alpha}}, Interferon-{{gamma}}, and Transforming Growth Factor-{beta} on Adipogenesis and Expression of Thyrotropin Receptor in Human Orbital Preadipocyte Fibroblasts J. Clin. Endocrinol. Metab., February 1, 2001; 86(2): 903 - 908. [Abstract] [Full Text]

				A. P. Weetman Graves' Disease N. Engl. J. Med., October 26, 2000; 343(17): 1236 - 1248. [Full Text] [PDF]

				M. Crisp, K. J. Starkey, C. Lane, J. Ham, and M. Ludgate Adipogenesis in Thyroid Eye Disease Invest. Ophthalmol. Vis. Sci., October 1, 2000; 41(11): 3249 - 3255. [Abstract] [Full Text]

				A. Bell, A. Gagnon, L. Grunder, S. J. Parikh, T. J. Smith, and A. Sorisky Functional TSH receptor in human abdominal preadipocytes and orbital fibroblasts Am J Physiol Cell Physiol, August 1, 2000; 279(2): C335 - C340. [Abstract] [Full Text] [PDF]

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				R. W. Valyasevi, D. Z. Erickson, D. A. Harteneck, C. M. Dutton, A. E. Heufelder, S. C. Jyonouchi, and R. S. Bahn Differentiation of Human Orbital Preadipocyte Fibroblasts Induces Expression of Functional Thyrotropin Receptor J. Clin. Endocrinol. Metab., July 1, 1999; 84(7): 2557 - 2562. [Abstract] [Full Text]

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