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Original Studies |
Division of Endocrinology, Mayo Clinic/Foundation (R.W.V., D.Z.E., D.A.H., C.M.D., S.C.J., R.S.B.), Rochester, Minnesota 55905; and Medizinische Klinik, Klinikum Innenstadt, University of Munich (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
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Abstract |
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GO and normal orbital preadipocyte fibroblasts, cultured under conditions known to stimulate adipocyte differentiation, showed evidence of adipogenesis and positive immunostaining for TSHr protein. In addition, significantly more cAMP was produced in response to TSH stimulation in the differentiated cultures than in undifferentiated cultures derived from the same individuals’ cells. Other studies demonstrated relatively greater TSHr gene expression in GO than in normal orbital tissue specimens.
These results indicate that orbital preadipocyte fibroblasts increase their TSHr expression with differentiation and suggest that these cells play an important role in the pathogenesis of GO. Furthermore, our studies support the concept that TSHr may be an important target antigen in this condition. Factors that stimulate adipocyte differentiation and TSHr expression in the orbit in GO have yet to be defined.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adipocyte precursor cells have been isolated from the stromal-vascular fraction of neonatal and adult human adipose/connective tissues from several regions of the body (4). These cells are thought to be a subpopulation of fibroblasts, termed preadipocyte fibroblasts, having the potential to undergo adipocyte differentiation when cultured in appropriate medium (5, 6). Indeed, a recent report by Sorisky and colleagues showed that human orbital fibroblasts contain such a subpopulation capable of in vitro adipogenesis (7). Orbital fibroblasts also produce increased quantities of glycosaminoglycans in vitro after treatment with cytokines known to be present in the orbit in GO (8). Thus, it appears that functional changes in orbital fibroblasts might account for the histological changes characteristic of GO orbital tissues.
Although the autoantigen involved in Graves’ hyperthyroidism is known to be the TSH receptor (TSHr), whether this antigen plays a primary role in the ocular manifestations of the disease is unclear. Several laboratories have detected the presence of TSHr messenger ribonucleic acid (mRNA) or protein in various human orbital cell preparations (9, 10, 11, 12, 13, 14, 15). However, the physiological relevance of this mRNA and the precise nature of the orbital cells expressing this receptor have not been clarified. In the current study, we subjected human orbital preadipocyte fibroblasts from GO patients and normal individuals to adipogenic culture conditions and sought to determine whether the differentiated cells express immunoreactive and functional TSHr. In addition, we examined relative levels of TSHr gene expression in GO and normal orbital adipose/connective tissue specimens.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Orbital adipose/connective tissue specimens were obtained in the course of orbital decompression surgery for severe GO (n = 23). Normal orbital and abdominal adipose/connective tissue specimens were collected at early autopsy from patients with no history of thyroid disease (n = 2). Tissue specimens were stored frozen at -70 C until processed for RNA isolation.
Total RNA was isolated directly from tissue specimens using the Totally RNA Kit (Ambion, Inc., Austin, TX). Orbital tissue samples (0.5–1.0 mg each) from 11–12 different GO patients were combined before RNA extraction to obtain sufficient material to perform each ribonuclease protection assay (RPA). Thus, 2 different orbital tissue pools were examined, each containing specimens from 11–12 different GO patients. In contrast, each single piece of normal orbital adipose/connective tissue or normal abdominal adipose/connective tissue was large enough to supply sufficient RNA for analysis. Positive control RNA was prepared in the same manner from cultured Chinese hamster ovary cells that had been transfected with plasmid containing the human TSHr (JPO9 line) or from a negative control counterpart (JPO2 line) (16).
RPA
As described previously (9), the antisense RNA probe for TSHr RPA was transcribed from a 320-bp PCR product with a T7 phage promotor at its 3'-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 complementary DNA. The resulting high specific activity probe encompassed nucleotides 576–873 (exons 6–9) of the human TSHr complementary DNA sequence, as reported by Nagayama (17), and was designed to detect both the 2.4-kb intact TSHr (protecting a product of 298 bp) and the 1.3-kb variant form (protecting a product of 217 bp). The antisense RNA probe for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RPA was generated from pTRI-GAPDH human antisense control template (Ambion, Inc.), which was digested with restriction enzyme DdeI to generate a 154-nucleotide GAPDH probe.
Total RNA (63–80 µg) was combined with 300,000 cpm TSHr probe and 3,000 cpm GAPDH probe 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). Hybridized samples were 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).
Cell culture and adipocyte differentiation
Orbital adipose/connective tissue explants were obtained from patients undergoing orbital decompression surgery for severe GO (n = 7). All patients had been treated previously for Graves’ hyperthyroidism with 131I; none was currently taking antithyroid drugs. One of the patients was currently taking corticosteriods, whereas none of the others had ever been prescribed this medication for treatment of eye disease. Control cells were obtained in the course of orbital surgery for other conditions from patients with no history of GO or Graves’ disease (n = 4). GO or normal tissue samples were minced and placed directly in plastic culture dishes, allowing preadipocyte fibroblasts to proliferate as described previously (18). Cells were propagated in medium 199 containing 20% FBS (HyClone Laboratories, Inc., Logan, UT), penicillin (100 U/mL), and gentamicin (20 µg/mL) in a humidified 5% CO2 incubator at 37 C and maintained in 80-mm2 flasks with medium 199 containing 10% FBS and antibiotics.
To initiate adipocyte differentiation, orbital cells were grown to confluence in six-well plates. Differentiation was carried out as reported previously (7, 19) with minor modifications; cultures were changed to serum-free DMEM-Ham’s F-12 (1:1; Sigma Chemical Co., St. Louis, MO) supplemented with biotin (33 µmol/L), pantothenic acid (17 µmol/L), transferrin (10 µg/mL), T3 (0.2 nmol/L), insulin (1 µmol/L), carbaprostacyclin (cPGI2; 0.2 µmol/L; Calbiochem, La Jolla, CA), and, for the first 4 days only, dexamethasone (1 µmol/L) and isobutylmethylxanthine (IBMX; 0.1 mmol/L). The differentiation protocol was continued for 10 days, during which time the media were replaced every 3–4 days. Control fibroblasts derived from the same patients’ orbital tissues were cultured similarly, except for the omission in the medium of cPGI2, dexamethasone, and IBMX, as cells cultured in this way do not undergo complete differentiation into mature adipocytes (7). As another control, orbital fibroblasts were cultured for the same period of time in medium 199 with 10% FBS without being switched to differentiation medium.
Oil Red O staining
Orbital preadipocyte fibroblast cultures were plated in one-well culture chamber slides (Nalge Nunc International, Rochester, NY) in medium 199 containing 10% FBS, grown to confluence, and subjected to the differentiation protocol or control conditions. Cells were washed twice with 1 x PBS, fixed in 10% formalin overnight at room temperature, and rinsed in 60% isopropanol before staining with filtered 0.21% Oil Red O in isopropanol-water for 1 h (19). Washed cells were exposed to Mayer’s hematoxylin solution (Sigma Chemical Co., MHS-32) for 5 min and rinsed with tap water before being visualized and photographed at x40 and x100 using an Olympus Corp. BX60 light microscope (Olympus Corp., Melville, NY).
TSHr immunocytochemistry
Orbital preadipocytes were seeded onto glass chamber slides (Nalge Nunc International, Naperville, IL), grown to confluence as monolayers, and subjected to the differentiation protocol as described above. These cells as well as control JPO2 and JPO9 cells were chilled on ice, washed, and fixed in methanol for 15 min at -20 C. Nonspecific binding was blocked using an avidin-biotin blocking kit (SP-2001, Vector Laboratories, Inc., Burlingame, CA) and incubating the cells in 5% sheep serum for 30 min. Monoclonal mouse anti-TSHr antibody directed against TSHr extracellular domain amino acids 32–41 (1:1000 dilution; MA3–217; Affinity BioReagents, Inc., Golden, CO) was applied for 2 h at room temperature. Slides were washed several times with Tris-buffered saline (TBS)-Tween-20 and incubated with biotinylated antimouse IgG (1:300 dilution; RPN 1001, Amersham International, Arlington Heights, IL) at room temperature for 30 min. After multiple washings, slides were exposed to avidin-biotinylated enzyme complex alkaline phosphatase reagent (Vectastain ABC-AP kit, AK 5000, Vector Laboratories, Inc., Burlingame, CA) for 30 min at room temperature. The reaction product was visualized after incubation in the dark for 30 min with an alkaline phosphatase substrate kit (SK-5100; Vector Laboratories, Inc.) containing levamisole to block endogenous alkaline phosphatase activity. Slides were counterstained with Mayer’s hematoxylin solution. Parallel slides with secondary or primary antibodies replaced, in turn, by TBS-Tween-20 and isotype-matched nonimmune mouse IgG were processed to assure specificity and exclude cross-reactivities between the antibodies and conjugates used. Cells were examined at x40 and x100 using an Olympus Corp. BX60 light microscope.
cAMP measurement
Differentiated or control orbital cells in six-well culture plates were preincubated in medium containing IBMX (1 mmol/L; 1 mL/well) for 2 h at 37 C. Recombinant human TSH (rhTSH; 3 x 10-7 mol/L; Genzyme, San Carlos, CA) was added to duplicate wells, and incubation was continued for 1 and 2 h until it was terminated with the addition of hydrochloric acid (0.1 mol/L; 1 mL/well) for 10 min. The culture media were subjected to centrifugation (600 x g), and cAMP production was measured using a commercially available kit (R&D Systems, Minneapolis, MN). Each data point represents the mean of duplicate determinations.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 RPA gel lanes corresponding to GO
orbital adipose/connective tissue (Fig. 1
). In contrast, only weakly positive
protected bands were present in all lanes containing mRNA from normal
orbital tissue samples. No TSHr mRNA was detected in lanes
corresponding to normal abdominal adipose/connective tissue samples.
The intact receptor transcript (2.4 kb) was present in positive control
JPO9 lanes, whereas negative control JPO2 lanes contained no bands.
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These studies were carried out to assess and document the morphological changes in orbital cells undergoing adipogenesis. Confluent GO or normal preadipocyte fibroblasts were subjected to the differentiation protocol for 4–10 days. Cells were then either stained with Oil Red O and examined under light microscopy or were left unstained and examined using phase contrast. Control cells from the same patients or normal individuals were maintained in medium 199 with 10% FBS or in control medium (i.e. differentiation medium lacking cPGI2, dexamethasone, and IBMX). During the process of differentiation, orbital cells lost their stellate (fibroblastic) morphology and converted to a spherical (adipocytic) shape. Discrete vacuoles were apparent under phase contrast in the cytoplasm of the differentiating cells (not shown). The morphology of the differentiated GO or normal cultures was clearly distinguishable from that of control undifferentiated cultures, with cytoplasmic inclusions apparent in approximately 10–20% of the cells. However, some of the normal cultures had evidence of adipocyte differentiation in a greater proportion of cells containing particularly large cytoplasmic inclusions. This feature was notable despite the fact that there was significant individual to individual variation in the proportion of cells (whether GO or normal) acquiring these inclusions and in the average size of the inclusions.
TSHr immunostaining
TSHr-specific staining was clearly evident in GO orbital
preadipocyte fibroblasts that had been subjected to the adipocyte
differentiation protocol (Fig. 2C). This
immunoreactivity was most intense in the perinuclear and nuclear
regions. In contrast, GO preadipocytes from the same patients cultured
for the same period of time in control medium 199 with 10% FBS showed
no staining of TSHr protein (Fig. 2D), whereas only very faint
reactivity was apparent in cells cultured in control differentiation
medium (not shown). Differentiated normal orbital preadipocytes
exhibited similar, but somewhat more intense, staining than was seen in
the differentiated GO cultures. Normal preadipocytes grown in control
medium 199 with 10% FBS or in the control differentiation medium
showed only very faint TSHr immunoreactivity (not shown). Positive
control JPO9 cells exhibited strong TSHr immunoreactivity (Fig. 2A),
whereas the negative control JPO2 cells showed no staining for this
protein (not shown). Further, JPO9 cells or differentiated GO or normal
preadipocytes, processed with the primary or secondary antibody
replaced, in turn, with TBS-Tween-20 or isotype-matched nonimmune mouse
IgG, showed no TSHr staining (Fig. 2B). The method of fixation used for
these studies did not allow for assessment of cell surface staining
independent of intracellular staining.
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cAMP production in response to rhTSH stimulation was measured in
GO or normal preadipocyte fibroblasts that had been subjected to the
adipocyte differentiation protocol. Paired control cultures of cells
derived from the same GO patient or normal individual were grown in
control differentiation medium that lacked cPGI2,
dexamethasone, and IBMX. At baseline, both GO and normal differentiated
orbital cells produced barely measurable quantities of cAMP. However,
after the addition of 3 x 10-8 mol/L rhTSH
(corresponding to 
1000-fold the normal serum TSH concentration),
cAMP production increased in these cells; maximal values were measured
with 3 x 10-7 mol/L rhTSH (Fig. 3). At 2 h after the addition of
rhTSH, cAMP production in differentiated GO cultures was 16- to 63-fold
greater than that in paired undifferentiated cultures (Fig. 4). In normal cultures, the
differentiated cells produced 2.0- to 10-fold more cAMP than did paired
undifferentiated cultures (Fig. 5).
Although the increase in cAMP production noted in the undifferentiated
control GO cultures after rhTSH stimulation was barely detectable (Fig. 4), there was a modest rise in cAMP measured in the undifferentiated
control normal cultures (Fig. 5). A marked individual to individual
variation was apparent in the maximal cAMP responses in normal cultures
(range, 212- to 9305-fold over baseline) and GO orbital cultures
(range, 4- to 183-fold over baseline; Fig. 6). However, the magnitude of the maximal
responses was generally greater in differentiated normal cultures
(mean, 2654-fold over baseline) than in differentiated GO cultures
(mean, 72-fold; Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a previous report, we demonstrated the presence of mRNA corresponding to TSHr extracellular domain in uncultured GO orbital adipose/connective tissue specimens using RPA, but were unable to detect TSHr mRNA in normal orbital fatty connective tissue samples (9). This apparent acquisition of TSHr expression in GO orbital tissues was the first direct evidence that this antigen may play an important role in the pathogenesis of the disease. In the current study, we again demonstrated TSHr RNA in uncultured orbital tissues from patients with GO. However, this time we used an even more sensitive RPA with longer exposure times and were able to detect faint TSHr expression in normal orbital tissues as well. These mRNA levels were consistently lower than those seen in the GO tissues. In contrast, even with this ultrasensitive method, we could not detect TSHr in normal abdominal adipose/connective tissues. Because normal orbital tissue appears to express TSHr to a greater degree than does normal abdominal fatty tissue, it may be that orbital tissue is more sensitive to local or circulating factors that stimulate TSHr expression than is adipose stromal tissue from other regions of the body.
In earlier studies, we demonstrated TSHr mRNA and protein expression in primary cultures of GO preadipocyte fibroblasts that were grown in medium 199 with 10% FBS (9). These early passage cultures contained mature adipocytes, as determined by Oil Red O staining. In contrast, neither adipocytes nor TSHr expression was detected in late (fourth to fifth) passage cultures of these cells. Because cell passaging and the particular culture conditions used appeared to selected against adipocytes and cells expressing TSHr, we reasoned that the adipocytes in these cultures might be the TSHr-bearing cells.
In the current study, we began to explore the potential link between adipogenesis and TSHr expression in human orbital fibroblasts. After exposure in vitro to adipogenic conditions, TSH-dependent cAMP production and TSHr protein expression increased in GO and normal cultures. Therefore, both TSHr expression and adipogenesis appeared to be stimulated by the same culture conditions. This finding is similar to that reported in rat epididymal, subcutaneous, and perirenal preadipocytes by Haraguchi and colleagues (24). Whether TSHr and adipocyte-specific genes are coregulated in these cells remains to be determined. Of note is that the EC50 of the cAMP response in the orbital cells was higher than that generally observed in either thyroid cells or TSHr-transfected nonthyroid cells. This finding is compatible with the low levels of TSHr mRNA and protein present in these orbital cells.
A striking finding was that the cAMP response to rhTSH in the differentiated normal cultures was, on the average, 40-fold greater than that in the differentiated GO cultures. Likewise, our immunocytochemical studies revealed more intense TSHr staining in differentiated normal than in differentiated GO orbital cells. A related observation was that normal orbital cells cultured in the control differentiation medium (lacking cPGI2, dexamethasone, and IBMX) did, in fact, show a modest increase in cAMP after rhTSH stimulation. In contrast, similarly undifferentiated GO cells did not respond to rhTSH. Thus, differentiated or undifferentiated normal orbital cells were found to express significantly more TSHr than did GO orbital cells exposed to the same culture conditions.
On the surface, these observations may appear to contradict our finding that uncultured GO orbital tissue specimens express TSHr to a greater extent than do normal orbital specimens. However, in the setting of GO, the subpopulation of orbital preadipocytes most sensitive to TSHr-stimulating (and perhaps adipogenic) factors may encounter and respond to such factors in vivo. If terminally differentiated into adipocytes, these cells would not passage in culture. However, they would serve to increase the level of TSHr expression measured in uncultured orbital adipose/connective tissue specimens. In contrast, this same subpopulation of orbital cells from normal individuals would not have been exposed to these factors in vivo. They would therefore remain as adipocyte precursor cells able to be passaged in culture and capable of mounting a vigorous response to in vitro stimuli for TSHr expression and adipocyte differentiation. In further support of this hypothesis is the fact that in some differentiated normal (but not GO) orbital cell cultures there is evidence of adipocyte differentiation in a particularly large proportion of cells containing especially large inclusions.
In summary, these studies support the concept that a subpopulation of orbital fibroblasts may be target cells in GO. In the setting of Graves’ disease, these orbital preadipocytes may be stimulated by unknown factors to differentiate into mature adipocytes that express increased levels of TSHr. As a result, the orbital tissue volume might expand, and TSHr-reactive T cells might infiltrate the orbit. The local release of inflammatory cytokines, TSHr-directed antibodies, or other factors could result in further adipogenesis, stimulation of glycosaminoglycan synthesis, and the expression of immunomodulatory proteins within the orbit. It is also possible that some of these factors might act as counterbalancing inhibitors of adipogenesis or TSHr expression. However, if the net effect of these changes were to significantly increase the volume of the adipose and connective tissues within the orbit, then proptosis, extraocular muscle dysfunction, and congestion would ensue.
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Footnotes |
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Received January 19, 1999.
Revised March 4, 1999.
Accepted March 24, 1999.
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