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Division of Molecular Medicine (T.J.S.), Harbor-UCLA Medical Center, Torrance, California 90502, and School of Medicine, University of California, Los Angeles, Los Angeles, California 90095; Cancer Center and Department of Microbiology and Immunology (L.K., G.D.S., R.P.P.), University of Rochester School of Medicine and Dentistry, Rochester, New York 14642; and Ottawa Health Research Institute and Departments of Medicine, Biochemistry, Microbiology, and Immunology (A.G., A.B., A.S.), University of Ottawa, Ottawa, Canada K1Y 4E9
Address all correspondence and requests for reprints to: Terry J. Smith, M.D., Division of Molecular Medicine, Building C-2, Harbor-UCLA Medical Center, 1124 West Carson Street, Torrance, California 90502. E-mail: tjsmith{at}ucla.edu
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, suggesting that differences in the potential for differentiation may reside with phenotypic attributes downstream from this receptor/adipogenic transcription factor. These observations enhance our understanding of orbital adipogenesis and define previously unrecognized differences between fibroblasts from the extraocular muscle and connective tissue. |
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Orbital fibroblasts from the connective/adipose depots are heterogeneous with regard to the expression and surface display of Thy-1 (19), a glycoprotein found on a variety of cell types (20). Although the function of Thy-1 as a receptor is unknown, a thoughtful investigation into the functional consequences of segregating fibroblasts on the basis of Thy-1 display has been undertaken. In murine lung fibroblasts, Phipps et al. (21) reported that Thy-1- cells express IL-1
, but Thy-1+ failed to express either IL-1 or IL-1ß. Moreover, the expression of immunologically relevant molecules such as HLA-DR class II appears to differ in the two subpopulations. The full scope of cellular differences imposed by the bimodal distribution of Thy-1 on orbital fibroblasts is not yet understood but may relate to discrete functions of the two populations, as has been proposed previously (22).
Some patients with TAO present with muscle enlargement as the dominant feature of their orbital disease. The muscles of other patients appear normal in volume, but some of these individuals present with evidence of enlargement of the posterior orbital connective/adipose tissue depot and develop optic neuropathy (23). Although the connective tissue-derived fibroblasts of the orbit have been studied extensively by several groups of investigators, those from the extraocular muscles have received little attention. Kiljanski et al. (24) have long focused their efforts on defining the primary importance of the muscle fibers in the pathogenesis of TAO. But most of the evidence generated to date would support the notion that the muscle fibers are spared until very late in the disease process (16) when they may become the casualty of fibrosis. Thus, most authorities now attribute the muscle dysfunction associated with TAO to the misdeeds of perimysial fibroblasts. To our knowledge, few studies have appeared comparing these fibroblasts with those from the connective/adipose tissue of the orbit. Potential differences in the two populations of fibroblasts, such as their adipogenic potential, could underlie the differences among patients with regard to the anatomic distribution of orbital disease manifestations.
In this article, we report that perimysial fibroblasts derived from the extraocular muscles represent a population of cells that are distinct from those originating in the adipose/connective tissue depot of the orbit. These cells are homogeneous with regard to the surface display of Thy-1 and, unlike their counterparts from connective tissue, do not appear to possess in vitro the capacity for differentiation into adipocytes. Adipogenic differentiation in the connective tissue derived fibroblasts occurs in nearly half of the cells when the cultures are incubated in a medium containing the PPAR agonist, rosiglitazone. These current findings have direct implications with regard to the distribution of disease manifestations within the orbital contents associated with TAO. They more precisely define the highly specialized phenotypes of fibroblasts investing orbital tissues.
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Materials and Methods |
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Anti-Thy-1 antibody (F15–421–5) was kindly provided by Dr. J. W. Fabre, University of London (UK). Dexamethasone was from Steraloids (Wilton, NH). Carbaprostacyclin (cPGI2) was from Cayman Chemical Co. (Ann Arbor, MI). Rosiglitazone was from Zen-Bio (Research Triangle Park, NC). Other reagents were of the highest purity commercially available. Anti-PPAR antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) or Calbiochem (San Diego, CA).
Cell culture
Human orbital and dermal fibroblasts were cultivated as reported previously (25). Tissue explants were obtained from individuals undergoing surgical decompression for severe TAO or orbital surgery for some other, noninflammatory problem. Dermal tissues were obtained from punch biopsies of normal-appearing skin, following informed consent. These activities have been approved by the institutional review boards of Albany Medical College and Harbor-UCLA Medical Center. Some of the fibroblast strains were generously provided by Dr. R. S. Bahn (Mayo Clinic, Rochester, MN). The array of culture strains examined in these studies included fibroblasts from patients with inactive orbital disease. A total of 10 different strains were examined in these studies. Donors were euthyroid at the time of culture harvest. Explants were allowed to attach to the bottom of culture plates and were covered with Eagle’s medium containing 10% FBS, antibiotics, and glutamine. They were incubated in a 37 C, 5% CO2, humidified environment. The resulting fibroblast monolayers were passaged serially by gentle treatment with trypsin/EDTA. Strains were stored in liquid N2 until needed and were used between the 2nd and 12th passage. They were determined to not express Factor VIII, cytokeratin, or smooth muscle-specific actin.
Immunofluorescence and flow cytometric analysis of Thy-1 display by fibroblasts
Indirect immunofluorescence was used to determine the levels of Thy-1 expression in individual cells and to screen for its display in various strains of human fibroblasts. Cell-free supernatant prepared from a mouse hybridoma (F15–421-5) was used as the primary antihuman Thy-1 antibody. For flow cytometric studies, cultures were disrupted with gentle treatment with trypsin (0.5%, Worthington Biochemical Corp., Freehold, NJ) for 30 sec and suspended in PBS containing BSA (1%) and sodium azide (0.01%). This solution was used as a staining medium. We have documented that trypsin treatment under these circumstances does not cleave Thy-1 from the surface of fibroblasts. Individual samples were incubated with F15–421-5 or an isotype control antibody for 45 min at 4 C. Samples were then washed three times with staining medium and incubated with goat antimouse immunoglobulin conjugated with fluorescein (Organon Teknika Corp., West Chester, AL) for 45 min at 4 C. After three washes, cell pellets were resuspended in 0.5 ml staining medium and subjected to flow cytometric analysis using a Coulter Profile analyzer (Coulter Electronics, Hialeah, FL). Forward and side-scatter gates were set to exclude nonviable cells and 5000 cellular events were collected for each sample analyzed. For staining of Thy-1 in situ, fibroblasts were cultured on chamber slides (Nunc, Naperville, IL). They were stained under identical conditions as those used in the flow cytometric studies. Slides were removed from the chambers, coverslips applied, and cell monolayers visualized with a BX 40 microscope (Olympus Corp. America, Lake Success, NY) fitted with a UPlan-FL (Ph2) X 40 objective.
Induction of fibroblast differentiation into adipocytes
Orbital fibroblasts between passage 3 and 12 were allowed to proliferate to near-confluence in DMEM containing 10% FBS and antibiotics, usually on 6-well plastic culture arrays (Costar, Cambridge, MA). Some cultures were then shifted to fresh medium and underwent one of two differentiation protocols. Protocol A was conducted essentially as described previously (18). It consisted of shifting cultures to serum-free medium containing DMEM:F12 (1:1, vol/vol) supplemented with 33 µmol/liter biotin, 17 µmol/liter pantothenate, 10 µg/ml transferrin, 0.2 nmol/liter triiodothyronine, 1 µmol/liter insulin, 0.2 µmol/liter cPGI2, 10 nmol/liter TSH, and for the first 4 d only, 1 µmol/liter dexamethasone and 0.1 mmol/liter isobutylmethylxanthone (IBMX). This protocol was conducted for 2 wk. Protocol B involved shifting cultures to a medium consisting of DMEM:F-10 (1:1) (vol/vol) supplemented with 3% FBS, 100 nmol/liter insulin, 1 µmol/liter dexamethasone, and for the first 3 d only, 1 µmol/liter rosiglitazone and 0.2 mmol/liter IBMX. Cultures were maintained in this medium for 18 d. Control cultures were prepared in parallel, as described previously (18).
The extent of differentiation was assessed morphologically by phase contrast microscopy using a 1x-70 microscope (Olympus Corp.) linked to a dual-color charge-coupled device camera (Sony, Tokyo, Japan). The fraction of cells undergoing differentiation was quantified by the methods of Crandall et al. (26) and Sottile and Seuwen ( 27) with the following modifications. Following fixation in 0.5% paraformaldehyde, cells were incubated for 10 min at room temperature (RT) in a solution containing 0.1 µg/ml Nile Red to stain cytoplasmic triacylglycerol. An excitation light of 488 nm generated a forward linear scatter signal depicted along the ordinates, and fluorescence emission was detected at 565 nm shown along the abscissas of the relevant figures.
Western blot and immunohistochemistry analysis of PPAR expression
For immunoblots, cultures of orbital fibroblast subsets were allowed to proliferate to confluence in standard culture conditions. They were then subjected to differentiation protocol B and the monolayers were solubilized in a buffer containing 15 mmol/liter Tris-HCl (pH 8.3), 15 mmol/liter CHAPS, 1 mmol/liter EDTA, 10 µg/ml soybean trypsin inhibitor, and 10 µmol/liter phenylmethylsulfonyl fluoride. An aliquot of each sample was used for protein determination using the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL). Equal amounts of protein were loaded onto a 4.5% stacking gel and run on a 10% polyacrylamide gel at 150 V for 80 min. The gel was transferred to Hybond-C nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ), blocked with 10% nonfat dry milk and incubated with anti-PPAR polyclonal antibody (1:2000, Calbiochem) for 1 h at RT. Following washes, the membrane was incubated with goat antirabbit IgG horseradish peroxidase (HRP) secondary antibody (1:2000, Caltag Laboratories, Inc., Burlingame, CA) for 1 h at RT. Blots were developed using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech) and exposed to x-ray film (Kodak, Rochester, NY).
For immunohistochemical analysis of PPAR expression, monolayers of fibroblasts were stained with anti-PPAR (Santa Cruz Biotechnology) or isotype control mouse IgG1 (Caltag Laboratories, Inc.) at 4 µg/ml overnight at 4 C. Biotinylated horse antimouse IgG (heavy and light chain, 1:200, Vector Laboratories, Inc., Burlingame, CA) was added as the secondary antibody for 2 h at 4 C. Cells were washed three times and then streptavidin-HRP (1:1000, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was added as the substrate for 1 h at RT. Samples were visualized by adding an aminoethyl-carbachol chromogen (Zymed Laboratories, Inc., South San Francisco, CA) and cover-slips added using Immu-mount (Shandon, Pittsburgh, PA).
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Orbital connective/adipose tissue-derived fibroblasts proliferate as a monolayer and have a morphology that is very similar to that of perimysial fibroblasts. Both exhibit fusiform and angular shapes with multiple dendritic processes. In general, both types of orbital fibroblast exhibit a rather granular-appearing cytoplasm. Dermal fibroblasts, in contrast, are predominantly angular appearing and are more homogeneous and less granular than those from either orbital source. No differences in cellular appearance or proliferative pattern were observed with regard to whether strains were harvested from diseased or normal tissues.
Perimysial fibroblasts uniformly express Thy-1
We have found that orbital fibroblasts from the connective/adipose tissue depots exhibit a bimodal distribution with regard to the expression and surface display of Thy-1. Approximately 50% of these cells, when cultivated in vitro, stain positively for the antigen. Importantly, this proportion remains stable through many population doublings and passage to daughter cultures. The flow cytometric analysis of Thy-1 surface expression shown in Fig. 1D
exhibits this bimodal distribution of cells in parent strains from the orbital connective tissue. In contrast, those from the extraocular muscles appear to uniformly display Thy-1. This is the case whether the patient from whom the cultures derived had been diagnosed with TAO (Fig. 1B) or some other condition not affecting the orbit (Fig. 1A). In this regard, perimysial fibroblasts resemble dermal fibroblasts. Pretibial fibroblasts also express Thy-1 uniformly (Fig. 1C). A number of fibroblast strains from the connective tissue and perimysium were assessed by flow cytometry for Thy-1 expression and the results are summarized in Table 1. Those derived from muscle are uniformly homogeneous. Moreover, treatment with glucocorticoids and a wide array of proinflammatory cytokines failed to influence Thy-1 expression in any of the fibroblasts thus far examined (data not shown).
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A subpopulation of connective/adipose tissue-derived orbital fibroblasts can be induced to differentiate in vitro into adipocytes. The fraction of cells undergoing such conversion is small under conditions we first reported (about 5–10% after 18 d) (18). Using protocol A, monolayers were incubated in medium containing insulin, cPGI2, dexamethasone, triiodothyronine, and IBMX. As the photomicrographs contained in Fig. 2 indicate, cells undergoing adipogenic differentiation under these conditions uniformly failed to exhibit Thy-1 expression as determined by immunofluorescence. With regard to their morphology, they take on large cytoplasmic inclusions that stain positively with Oil Red O and represent accumulations of triglycerides. It would appear that a subset of orbital connective/adipose tissue fibroblasts not displaying Thy-1 in culture represent preadipocytes.
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ligand, rosiglitazone, was added to the differentiation mixture, the yield of cells undergoing adipogenesis was increased dramatically (Fig. 3). These cells accumulate droplets of triglyceride that stain with Oil Red O. Unlike fibroblasts from orbital connective tissue, those from the perimysium fail to accumulate triglyceride droplets and continue to exhibit their native morphologies. In this regard, too, they resemble those from the skin. Dermal fibroblasts, derived from the pretibium and abdominal wall, are also refractory to the effects imposed by the differentiation medium. It would appear from our studies to date that the level of disease activity and the duration of TAO when the tissues were obtained play little or no role in determining the adipogenic potential of the fibroblast strains. Moreover, cultures initiated from normal and TAO tissues behaved in a very similar manner with regard to their response to the differentiation protocols.
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indicate, in a typical study, nearly 50% of the fibroblasts from the connective/adipose tissue depot differentiate after 18 d. In contrast, there was no evidence of adipocyte differentiation in dermal fibroblast cultures subjected to the same protocol (Fig. 5).
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PPAR is a key molecular trigger for the differentiation of preadipocytes into mature adipocytes (28). We thus determined whether orbital fibroblasts express this receptor/adipogenic transcription factor. As the immunofluorescence photomicrographs in Fig. 6 demonstrate, the protein is easily detectable in both Thy-1+ and Thy-1- connective tissue fibroblasts under culture conditions not favoring the differentiation of the cells. The cellular distribution of PPAR staining is similar in both Thy-1+ and Thy-1- subsets and contains a prominent nuclear component. These observations were then quantified by subjecting cell lysates to Western blot analysis for PPAR. The receptor protein is abundantly expressed in cells from both subsets and the levels are very similar (Fig. 7). This result suggests that the inability of the Thy-1+ subset to undergo adipogenic differentiation following exposure to a PPAR ligand is a consequence of phenotypic differences downstream from receptor occupancy. When parent strains of the fibroblasts are subjected to the differentiation protocol B, the levels of PPAR were slightly enhanced (50%, data not shown), consistent with the findings in other differentiating cell types (29).
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Discussion |
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Disease manifestations within the orbit appear to be distributed differently in old and young patients. In those over 60 yr, severe eye muscle involvement in the absence of adipose tissue expansion is often found. In contrast, patients younger than 40 yr often present with relatively little inflammation or involvement of the muscle but substantial adipose/connective tissue expansion (23). These differences in the distribution of orbital tissue involvement could be related to the distinct phenotypes of muscle and connective tissue-derived fibroblasts. It is possible that the adipogenic potential of orbital fibroblasts diminishes as a consequence of aging, as has been demonstrated in abdominal preadipocytes (32). Clearly, greater insight into the cellular divergence existing in the two fibroblast populations will be necessary before this hypothesis can be tested adequately.
The current findings raise important questions concerning the role of fibroblasts as participants in both physiological and pathological states. Previously it was thought that these cells were relatively inert and played limited reactive roles. Instead, they were viewed in the narrow context of producing extracellular matrix molecules such as collagen. We now know that fibroblasts are sentinel cells, capable of reacting rapidly to a wide array of molecular cues and orchestrating complex interactions with immunocompetent cells (1). Fibroblasts are the source of many proinflammatory molecules such as cytokines and growth factors and release chemoattractant molecules involved in the recruitment and activation of lymphocytes and other bone marrow-derived cells (1, 2).
The extent of phenotypic divergence exhibited by fibroblasts from the adipose and muscle depots of the orbit had not been appreciated previously. It will be necessary to assess the capacity of these different cell populations to elaborate collagen and other profibrotic molecules and to define cell type-specific signaling pathways that might be interrupted. These could represent important therapeutic targets for modifying the course of TAO. Of substantial relevance to this discussion are observations made concerning the extraocular muscles of patients with TAO. Some muscles exhibit, with magnetic resonance and computer tomography imaging, areas that are consistent with fatty infiltration (33). These findings have led to speculation that under certain conditions, endomysial and perimysial fibroblasts might differentiate in situ into adipocytes. It is possible that in these individuals, some contribution to the muscle-associated fibroblast population might derive from the Thy-1- subset.
Orbital fibroblasts, when activated with cytokines, synthesize substantial levels of hyaluronan, a nonsulfated glycosaminoglycan (7, 34). This enhanced synthesis can be attributed to a coordinated induction of the uridine 5'-diphosphate-glucose dehydrogenase (35) and hyaluronan synthase genes (36). Hyaluronan accumulation is an important component in the volume expansion seen in TAO. This is thought to result from the rheologic properties of these complex sugars, which occupy extraordinary volumes when hydrated. From the findings we report here, it would appear that the orbital expansion could result from a greater adipose tissue mass as well.
Our current findings differ somewhat from those reported recently by Jeney et al. (37). Those investigators found widespread, spontaneous adipogenic changes in fibroblasts from the skin and orbit that were allowed to propagate on glass. Their observations are interesting and represent potentially important findings. They may underscore the critical physical/chemical nature of the substratum in defining, at least in part, the behavior of fibroblast lineage cells. On the other hand, that study failed to provide important negative controls demonstrating examples of cells not undergoing adipogenic differentiation. Just how their findings can be reconciled with our results concerning the specialized differentiation of orbital connective tissue-derived fibroblasts will require further investigation.
We now report the important contribution of the PPAR agonist, rosiglitazone, to the adipogenesis of orbital fibroblasts. PPAR functions as a transcriptional factor that has been implicated in adipogenic differentiation (38). This receptor recognizes a number of ligands that elicit such cellular changes through its ability to activate a constellation of key downstream genes involved in adipogenesis and insulin sensitization (39). PPAR possesses biological properties that are distinct from those of PPAR and PPAR
, the latter of which does not exhibit adipogenic activity (40). A possible endogenous ligand for PPAR is 15-deoxy- 12, 14-PGJ2 (38), although considerable debate remains concerning the relationship existing between that prostanoid and PPAR. Studies involving the interruption of the PPAR gene strongly suggest the central importance of this receptor in the development of fat as well as heart and placenta (41). This contention was strengthened by the demonstration that PPAR overexpression drives adipogenic differentiation in cells not ordinarily considered preadipocytes (42). With regard to our current findings, a concern arises regarding the potential worsening of fat expansion in TAO, should patients ingest PPAR agonists as therapy for concomitant diabetes mellitus. Conversely, the PPAR antagonists currently under development may prove of therapeutic value in modifying the adipogenic potential of orbital tissue in TAO. Such a strategy might limit expansion of the fat compartment in TAO.
Recent reports have appeared suggesting that adipogenesis of orbital fibroblasts results in increased expression of the TSH receptor (TSHr) (43, 44). The authors of these studies have concluded that differentiation also results in a substantial enhancement of cAMP generation provoked by the ligation of the TSHr with TSH. The second of these reports documented that a majority of cells undergoing differentiation display the receptor (44). The enhanced TSHr expression they observed in orbital fibroblasts is consistent with earlier studies in 3T3-L1 cells (45) and rat adipocytes ( 46, 47). These are potentially important observations. They may have identified an important consequence of adipocyte differentiation, namely that the abundance of a putative orbital self-antigen is increased. It is also important to note that TSHr expression has been documented in a wide array of fibroblast lineage cells (48, 49), including those from anatomic regions ordinarily not manifesting Graves’ disease. Whether the levels of TSHr might be enhanced in those depots following adipogenesis as well remains to be determined.
The observations we report here extend substantially our earlier work in which we introduced the concept of heterogeneity exhibited by fibroblasts from the orbital connective tissue (19). The current findings imply important functional consequences from the cellular diversity and provide evidence suggesting divergent biological roles for Thy-1+ and Thy-1- fibroblast subsets. It would appear that Thy-1- fibroblasts represent preadipocytes that, under certain conditions, can be induced to differentiate. When prompted by the constellation of growth factors and cytokines that are expressed as a consequence of TAO, these cells may undergo differentiation into adipocytes and thus contribute to the increased tissue volume associated with the disease. The Thy-1- fibroblast must now be singled out as a potential target for disease-modifying therapeutics designed to retard adipogenesis in TAO.
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Acknowledgments |
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Footnotes |
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This work was supported in part by NIH Grants EY08976, EY11708, DE11390, H150002, and ES01247; a Merit Review award from Research Service of the Department of Veterans Affairs; Heart and Stroke Foundation of Canada Grant T-4740. A.G. was supported by a CDA postdoctoral research award. A.B. was supported by a CIHR/HSFC doctoral research award. A.S. is a Career Investigator of the Heart and Stroke Foundation of Ontario.
Abbreviations: cPGI2, Carbaprostacyclin; HRP, horseradish peroxidase; IBMX, isobutylmethylxanthone; RT, room temperature; TAO, thyroid-associated ophthalmopathy; TSHr, TSH receptor.
Received June 14, 2001.
Accepted October 1, 2001.
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