HMC-1 Mast Cells Activate Human Orbital Fibroblasts in Coculture ...

0013-7227/99/$03.00/0 Endocrinology Copyright © 1999 by The Endocrine Society

Vol. 140, No. 8 Printed in U.S.A.

HMC-1 Mast Cells Activate Human Orbital Fibroblasts in Coculture: Evidence for Up-Regulation of Prostaglandin E2 and Hyaluronan Synthesis* TERRY J. SMITH

AND

SONIA J. PARIKH

Division of Molecular and Cellular Medicine, Department of Medicine, Department of Biochemistry and Molecular Biology, Albany Medical College and Samuel S. Stratton Veterans Affairs Medical Center, Albany, New York 12208 ABSTRACT The purpose of this study was to determine the effects of mast cell coculture on human orbital fibroblasts. Thyroid-associated ophthalmopathy is characterized by infiltration of lymphocytes and mast cells and connective tissue activation in the orbit, leading to a disordered accumulation of hyaluronan and intense inflammation. Here, we report that HMC-1, an established human mast cell line, can activate human orbital fibroblasts to produce increased levels of prostaglandin E2 (PGE2) and hyaluronan when cocultured. HMC-1 cells up-regulate, in these fibroblasts, the expression of PG endoperoxide H synthase-2 (EC 1.14.99.1, PGHS-2), the inflammatory cyclooxygenase. This induction, at a pretranslational level, underlies the increase in PGE2 synthesis. The up-regulation can be attenuated with dexamethasone

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HE PATHOGENESIS OF thyroid-associated ophthalmopathy (TAO) is characterized by a dramatic activation of orbital connective tissue and is associated with Graves’ disease. This tissue activation leads to an accumulation of the nonsulfated glycosaminoglycan, hyaluronan (1), in the perimysial tissue of the extraocular muscles and in the orbital connective/adipose tissue. The extremely hydrophilic nature of hyaluronan leads to increases in tissue volume and to the anterior displacement of the eye, culminating in proptosis. In addition, the orbital tissue can become intensely inflamed and this can lead to scar formation and extraocular muscle disfunction. Little is currently understood about the factors that initiate the earliest inflammatory events or activate orbital connective tissue in TAO. Moreover, how the dramatic tissue remodeling in the orbit relates to disease of the thyroid is uncertain. A hallmark histopathological feature of TAO is the presence of lymphocytes and mast cells in affected tissue (2). The T lymphocyte population in the diseased orbit has been characterized as containing both CD41 and CD81 cells, and several putative lymphocyte/fibroblast interactions have been hypothesized (3, 4). Moreover, the B lymphocyte population infiltrating the orbit in TAO and its Ig-encoding genes have also been studied (5). Received January 19, 1999. Address all correspondence and requests for reprints to: Terry J. Smith, M.D., Division of Molecular and Cellular Medicine (A-175), Department of Medicine, Albany Medical College, 47 New Scotland Avenue, Albany, New York 12208. * These studies were supported, in part, by National Institutes of Health Grants EY 08976 and EY 11708 and by a Merit Review award from the Research Service of the Department of Veterans Affairs.

(10 nM), and the increase in PGE2 production can be inhibited by SC 58125, a specific PGHS-2 inhibitor. Moreover, anti-interleukin-4 receptor antibodies can block prostanoid production in the fibroblasts elicited by HMC-1 cells, suggesting that this cytokine might represent a molecular conduit for mast cell/fibroblast cross-talk. HMC-1 cells also increased hyaluronan synthesis, as was evidenced by a 2-fold increase in [3H]glucosamine incorporation into the macromolecule. To our knowledge, these findings are the first demonstrating the ability of mast cells to activate orbital fibroblasts, and the findings suggest a potential role for these cell-cell interactions in the pathogenesis of thyroid-associated ophthalmopathy. (Endocrinology 140: 3518 –3525, 1999)

However, the significance of the numerous mast cells infiltrating orbital tissues has not been investigated in detail, despite their description in experimental models of exophthalmos nearly 50 yr ago (6). In fact, Asboe-Hansen hypothesized that mast cells were a source of hyaluronan production (7). The impact of mast cells on orbital tissue function or their influence on orbital fibroblasts in culture has not been examined previously. Mast cells play diverse roles in allergic and nonallergic reactions. They are currently believed to participate in tissue fibrosis. When cocultured with human dermal fibroblasts, mast cells enhance proliferation, through their expression of interleukin (IL)-4 (8). Moreover, coculture with mast cells up-regulates the surface expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 on dermal fibroblasts, through increases in steady-state levels of the respective messenger RNAs (mRNAs), leading to enhanced adhesion of T lymphocytes (9). The impact of coculturing mast cells with fibroblasts on hyaluronan and prostaglandin E2 (PGE2) synthesis has not yet been explored, but recent findings concerning other aspects of fibroblast activation suggest that mast cells might alter the expression of multiple fibroblast genes and their products. Fibroblasts are critical components of the early inflammatory response and participate actively in tissue remodeling and fibrosis (10). Orbital fibroblasts constitute a heterogeneous population of cells that can be subdivided on the basis of Thy-1 surface display (11). They exhibit a phenotype that is very different from fibroblasts found in other anatomic regions of the human body, by virtue of the profile of cell surface receptors (12), hormone responses (13–15), proteins

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HMC-1 MAST CELLS AND HUMAN ORBITAL FIBROBLASTS

(16 –18), and gangliosides (19, 20) they express. They manifest a particular susceptibility to several actions of proinflammatory cytokines. The molecular basis for these exaggerated responses is as yet uncertain, but we hypothesize that they underlie the orbit’s involvement in Graves’ disease. With particular relevance to TAO, we have reported that the T lymphocyte-derived cytokine, leukoregulin, can induce hyaluronan synthesis in orbital fibroblasts and that this upregulation is considerably more robust than that observed in dermal fibroblasts (21). The cyclooxygenases or PG endoperoxide H synthases (EC 1.14.99.1, PGHS) belong to a family consisting of two genes that catalyze the rate-limiting steps in the synthesis of prostanoids from arachidonate (22, 23). PGG2 is generated from arachidonate through oxygenase activity and is subsequently converted to PGH2 by virtue of the peroxidase activity of these bifunctional enzymes. PGHS-1 is a constitutively expressed enzyme that is found widely in states of health (24, 25). It is currently believed that the basal PG production in the stomach and kidney, maintaining epithelial integrity, derives from the unprovoked activity of PGHS-1. PGHS-2, the inflammatory cyclooxygenase, is ordinarily not expressed in most tissues and cell types; but when cells are exposed to inflammatory cytokines, growth factors, and mitogens, it is expressed at high levels (26 –29). PGHS-2 is massively induced in orbital fibroblasts by leukoregulin (30). The magnitude of PGHS-2 induction in orbital fibroblasts from patients with TAO is substantially greater than that observed in dermal fibroblasts. It is associated with a dramatic increase in the synthesis of PGE2 that can be blocked by SC 58125, a PGHS-2-selective inhibitor (30). Moreover, the induction of PGHS-2 mRNA and protein by leukoregulin in orbital fibroblasts can be attenuated by dexamethasone (1,4-pregnadien-9-fluoro-16a -methyl11b,17a,21-triol-3,20-dione). It is the unusually great magnitude of the PGHS-2 induction elicited by proinflammatory cytokines such as leukoregulin that, we believe, contributes to the inflammation of TAO. In this paper, we report, for the first time, that the coculture of HMC-1 mast cells (31) with human orbital fibroblasts results in substantial increases in PGE2 production and hyaluronan synthesis. The increase in PG synthesis can be attributed to the induction of PGHS-2. The effects of coculture are blocked by dexamethasone and are time-dependent. Thus, we have determined that mast cells can activate two biosynthetic pathways in orbital fibroblasts that we believe are responsible for the manifestations of TAO. Our findings suggest that fibroblast/mast cell interactions may participate in orbital tissue remodeling and define potentially important therapeutic targets for disrupting the disease. Materials and Methods Materials IL-1b and IL-4 were purchased from Biosource, Camarillo, CA. AntiIL-4R monoclonal antibodies were from Genzyme (Cambridge, MA). Complementary DNAs (cDNAs) for PGHS-1 and -2 were kindly provided by Dr. D. A. Young (Rochester, NY). Hyaluronan synthase (HAS)2 cDNA was a gift of Dr. Y. Yamaguchi (Burnham Institute, La Jolla, CA). PGE2 RIA was purchased from Amersham Pharmacia Biotech (Chicago, IL). SC 58125 was a generous gift from Dr. Peter Isakson (Searle, Skokie,

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IL), and dexamethasone and A23187 were purchased from Sigma Chemical Co. (St. Louis, MO). Monoclonal antibodies directed against human PGHS-1 and PGHS-2 were purchased from Cayman Chemical (Ann Arbor, MI).

Cell culture HMC-1 cells were kindly provided by Dr. J. H. Butterfield (Mayo Clinic, Rochester, MN) (31). Human orbital fibroblasts were prepared from surgical waste tissue obtained during transantral decompression for severe TAO or from patients undergoing procedures for nonorbital diseases and without known thyroid disease. These activities were approved by the Institutional Review Board of the Albany Medical College. The fibroblasts were allowed to proliferate from the surgical explants, as described (32), and were serially passaged with gentle treatment with trypsin/EDTA. Monolayers were covered with Eagle’s medium supplemented with 10% FBS, glutamine, and antibiotics. Cells were used between the 2nd and 12th passage from culture initiation and were maintained in a 37 C, 5% CO2, humidified incubator. Mast cells were allowed to proliferate in Iscoves’s medium enriched with 10% FBS, antibiotics, and a-thioglycerol in a 5% environment at 37 C. In some studies, HMC-1 cells were treated with A23187 (500 mg/ml) and then washed with medium before their introduction into coculture. Cocultures were initiated by introducing HMC-1 cells to confluent cultures of fibroblasts. The cocultures were maintained in Eagle’s medium, and the ratio of mast cells to fibroblasts was varied but was usually 1:1 unless otherwise specified in the figure legends. At the end of the coculture incubation, HMC-1 cells were washed away with gentle rinsing. The completeness of this removal was carefully monitored by microscopic inspection.

Northern analysis of PGHS and HAS mRNAs Fibroblasts were allowed to proliferate to confluence and were then shifted to medium with 1% FBS. After the treatments indicated in the figure legends, HMC-1 cells were removed completely, and fibroblast RNA was harvested essentially by the method of Chomczynski and Sacchi (33) using Ultraspec reagent (Biotecx, Houston, TX). Solubilized material was precipitated from the aqueous phase by the addition of isopropanol; the precipitate was washed with ethanol (75%) and solubilized in DEPC-treated water. Equal amounts of RNA (usually 10 mg) were electrophoresed in 1% agarose formaldehyde gels and then transferred to Zetaprobe (Bio-Rad Laboratories, Inc., Hercules, CA) membrane. [32P]-PGHS and HAS probes were hybridized to the immobilized RNA in a solution containing 5 3 saline-sodium citrate, 5 3 Denhardt’s solution, 50% formamide, 50 mm phosphate buffer (pH 6.5), 1% SDS, and 0.25 mg/ml sheared, denatured salmon sperm DNA at 48 C overnight. Membranes were then washed under high stringency conditions and exposed to X-OMAT AR film (Eastman Kodak Co., Rochester, NY) at 270 C. To normalize the amounts of RNA on the membranes, blots were stripped according to the manufacturer’s instructions and rehybridized with a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe. Radioactive DNA/RNA hybrids were quantitated by subjecting the autoradiographs to densitometric analysis using a BioImage system (Milligen).

PGE2 Assay Fibroblasts were cultured to confluence in 24-well plates covered with medium containing 10% FBS. Cultures were then shifted to medium containing 1% FBS for 24 h; and HMC-1 cells, without or with the test compounds, were added, usually in the ratio of 1:1. The coculture was continued for the intervals indicated; and 30 min before the end of the period, medium and HMC-1 cells were removed, and PBS was used to cover the cells. At the end of the incubation, an aliquot of the PBS (150 ml) was collected and subjected to an RIA (Amersham Pharmacia Biotech), as described previously (30).

Western analysis of PGHS protein Relative levels of PGHS proteins were determined by immunoblot analysis using monoclonal antibodies specifically directed against PGHS-1 and PGHS-2, as described previously (30). Fibroblast cultures

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were grown to confluence in 60-mm-diameter plates covered with medium supplemented with 10% FBS. They were then shifted to medium with 1% serum, and some were cocultured with HMC-1 cells, without or with test compounds, as described in the figure legends. After these treatments, the monolayers were washed and monitored for the completeness of HMC-1 cell removal, and the fibroblast proteins were solubilized in a solution containing 15 mm CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 1 mm EDTA, 20 mm Tris/HCl (pH 7.5), 10 mg/ml soybean trypsin inhibitor, and 10 mm phenylmethylsulfonyl fluoride. Lysates were taken up in Laemmli buffer and subjected to SDS-PAGE, and the separated proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.). Nonspecific binding was blocked by incubating membranes in PBS, to which 0.05% polyoxyethylene-sorbitan monolaurate (Sigma Chemical Co.) and 10% nonfat dry milk were added at room temperature for 1 h. The primary antibodies were then added at a 1:500 dilution for 3 h at room temperature. Membranes were washed and incubated with the secondary, peroxidase-labeled antibodies for 2 h, and then the ECL (Amersham Pharmacia Biotech) chemiluminescence detection system was used to generate signals.

[3H]Hyaluronan assay The accumulation of [3H]hyaluronan in cultures was assessed essentially as described previously by Smith, et al. (34). Briefly, confluent fibroblast monolayers in 60-mm-diameter plastic culture plates were shifted to medium supplemented with 1% FBS for 24 h; and then HMC-1 cells, without or with the test compounds indicated, were added to the fibroblast cultures. Mast cells were removed, and fibroblast monolayers were labeled with [3H]glucosamine (specific activity 21.6 Ci/mmol; NEN Life Science Products, Boston, MA; 3 mCi/ml). After the incubation, media were collected quantitatively, the monolayers were washed, and the cellular material was solubilized in 0.2 n NaOH. An aliquot of cellular material was taken for protein determination, and the medium and cellular material were combined, adjusted to pH 8.0 with 100 mm Tris, and the mixture was subjected to pronase (1 mg/ml) at 50 C for 16 h. The reaction was terminated by lowering the sample temperature to 4 C and precipitating proteins with trichloroacetic acid (5% final concentration). The acid-soluble material was then dialyzed extensively against cold H2O, lyophilized, re-solubilized in 0.15 m NaCl, and subjected to liquid scintillation counting.

Results Coculture of HMC-1 mast cells with orbital fibroblasts results in a substantial up-regulation of steady-state PGHS-2 mRNA

The incubation of human orbital fibroblasts in culture under conditions that do not include cytokines, serum, or growth factors results in relatively low levels of PGE2 synthesis, a large part of which can be attributed to the activity of PGHS-1 (30). In contrast, fibroblasts cultured under basal conditions express very low levels of PGHS-2 mRNA and protein. When these cells are exposed to proinflammatory molecules such as leukoregulin or IL-1b, the levels of PGHS-2 are increased substantially (30). We added HMC-1 cells to confluent cultures of orbital fibroblasts to determine whether these mast cells could elicit increases in fibroblast PGHS-1 and -2 mRNA expression. As the Northern blot analysis contained in Fig. 1 suggests, the steady-state levels of PGHS-2 mRNA are up-regulated by the presence of HMC-1 cells for 3 h. The predominant transcript is 4.8 kb, consistent with that reported in a number of cell types, including fibroblasts (30) and endothelial cells (35). As the data suggest, HMC-1 cells, whether preactivated with the calcium ionophore A23187 (500 mg/ml) or not, elicit a substantial increase in the PGHS-2 transcript. Thus, for most subsequent studies, we used nonactivated HMC-1 cells. The levels achieved by

FIG. 1. Northern blot analysis of steady-state PGHS-1 and PGHS-2 mRNA levels in orbital fibroblasts treated with IL-1b or cocultured with HMC-1 mast cells. Confluent 100-mm-diameter cultures of orbital fibroblasts (in this case, from a patient with severe TAO) were treated with nothing (control), or with IL-1b (10 ng/ml) for 16 h, or were cocultured with unactivated or ionophore-activated HMC-1 cells for 6 h. After respective incubations, monolayers were rinsed of cytokine or HMC-1 cells, and fibroblast cellular RNA was extracted as described in Materials and Methods. Immobilized RNA was hybridized with [32P] labeled PGHS-1 and -2 probes synthesized from the respective cDNAs. Membranes were exposed to film, and the resulting bands were densitometrically analyzed with a BioImage scanner. Membranes were stripped of radioactivity and were rehybridized with a GAPDH probe. The PGHS signals were normalized for their respective GAPDH signals.

activated HMC-1 cells were similar to those observed in cultures treated with IL-1b for 16 h. Levels of PGHS-1 mRNA were constant, with regard to mast cell exposure and IL-1b treatment, consistent with our previous observations (30). The transcript migrates as a 5-kb band that is similar to the mRNA expressed by monocytes and endothelial cells (25, 36) but different from other human and animal tissues expressing predominately a 2.8-kb species (24). The increase in orbital fibroblast PGHS-2 mRNA elicited by HMC-1 cells is time-dependent and transient (Fig. 2). The transcript is apparent after 3 h of coincubation, consistent with the induction pattern observed in other cell types treated with cytokines and serum and in leukoregulintreated orbital fibroblasts (30). This signal is maximal at 3 h, when it is severalfold higher than control values. The induction has decayed rapidly by 6 h, and after 12 h of exposure to HMC-1, has returned to the level observed in the control cultures. Coculture of HMC-1 mast cells with orbital fibroblasts results in an increase in PGHS-2 protein

We next determined whether the up-regulation of steadystate PGHS-2 mRNA levels, observed following HMC-1 cell addition to the orbital fibroblasts, resulted in an increase in PGHS-2 protein. As the Western blot displayed in Fig. 3


FIG. 2. Time-dependence of the induction of PGHS-2 mRNA in orbital fibroblasts by HMC-1 cells in coculture. Orbital fibroblasts were cocultured with HMC-1 cells for the duration of time indicated along the abscissa. Total cellular RNA was extracted and subjected to Northern blot analysis with PGHS-2 and GAPDH cDNA probes, as described in the legend to Fig. 1.

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Fig. 4 suggest, orbital fibroblasts cultured under control conditions produce relatively low levels of PGE2, as we have reported previously (30). When they are treated with IL-1b (10 ng/ml), the levels of PGE2 are dramatically increased (12.4-fold above levels observed in the control cultures) after 16 h of cytokine exposure. Treatment with dexamethasone (10 nm) or with SC 58125, a highly specific inhibitor of PGHS-2 (37), lowered the basal levels of PGE2 by 78% and 50%, respectively. When HMC-1 cells were added to confluent fibroblast monolayers for 4 h, there was a robust increase in the levels of PGE2 synthesized (10-fold above the levels of control fibroblasts). A substantial fraction of this increase could be blocked with SC 58125 (82%), suggesting that the increase in prostanoid levels derived from the activity of PGHS-2. Dexamethasone could also attenuate the HMC-1 induction by 31% (Fig. 4). The up-regulation of PGE2 production elicited by HMC-1 coculture was time dependent, as the data in Fig. 5 suggest. At 3 h of coculture, the levels had increased approximately 7-fold, and the elevation was sustained for at least 24 h, the duration of the study. IL-4 is synthesized by human mast cells. Thus, we examined whether this cytokine could influence the production of PGE2 in orbital fibroblasts. Exogenous IL-4 (10 ng/ml) was added to the medium and, as the data in Fig. 6 suggest, could increase PGE2 production in the fibroblasts. We then sought to determine whether IL-4 released from the HMC-1 cells was driving the increase of PGE2 synthesis in the coculture. Addition of anti-IL-4 receptor antibodies (10 mg/ml) to the coculture medium resulted in a near-complete blockade of the increase in PGE2 production (Fig. 6). Thus, it would seem that the increase in fibroblast PGE2 production provoked by

FIG. 3. Western analysis of PGHS-2 protein levels in orbital fibroblasts cocultured with HMC-1 mast cells. Fibroblast were allowed to proliferate to confluence in 60-mm plates. They were then incubated with HMC-1 cells, at a ratio of 1:1, for the intervals indicated in the abscissa. The mast cells were removed and fibroblast protein extracted as described in Materials and Methods. Proteins were subjected to Western blot analysis, and the ECL system was used to generate the signals. The resulting bands were densitometrically analyzed.

demonstrates, the protein is undetectable in control cultures but is abundant after 3 h of coculture. The protein migrates as a single band of 72 kDa. This increase is sustained for at least 6 h, the duration of the study. The maximal induction is approximately 16-fold above baseline levels. In contrast, the level of PGHS-1 protein, which appears as a 68-kDa band, was uninfluenced by the addition of the mast cells (data not shown). HMC-1 cell coculture with orbital fibroblasts results in a substantial increase in PGE2 production

We next examined the impact of HMC-1/fibroblast coculture on the generation of PGE2. As the data contained in

FIG. 4. Coculture of orbital fibroblasts with HMC-1 mast cells results in an up-regulation of PGE2 synthesis. Orbital fibroblasts (in this case, from a patient with severe TAO) were allowed to proliferate to confluence in 24-well plates. They were then either treated with nothing (control), with IL-1b (10 ng/ml) for 16 h, with dexamethasone (Dex, 10 nM) for 12 h, with SC 58125 (5 mM) for 1 h, or with HMC-1 (at a ratio of 1:1) for 4 h, alone or with dexamethasone or SC 58125. Thirty minutes before the end of the incubation, HMC-1 cells were removed from those cultures with them, and all monolayers were washed, and PBS was added. After 30 min, the PBS was removed, and an aliquot was subjected to analysis of PGE2 levels, as described in Materials and Methods. Data are expressed as the mean SEM of triplicate replicates from a representative experiment.

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FIG. 5. Time-course of effects of HMC-1 cell coculture on orbital fibroblast PGE2 synthesis. Orbital fibroblasts (in this case, those from a patient with severe TAO) were allowed to proliferate to confluence in 24-well cluster plates. They were then cocultured with HMC-1 cells at a ratio of 1:1 for the duration of time indicated in the abscissa. The final 30 min of the incubation involved removal of the medium and mast cells and replacement of PBS without HMC-1 cells. An aliquot of the PBS was subjected to assay for PGE2, as indicated in Materials and Methods.

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FIG. 7. Assessment of the impact of HMC-1 coculture on PGE2 production in several strains of orbital fibroblasts. Three strains of fibroblasts were plated in 24-well culture plates and allowed to proliferate to confluence. They were then treated with nothing (control), or with IL-1b (10 ng/ml) for 16 h, or were seeded with HMC-1 mast cells at a cell ratio of 1:1 and allowed to incubate for 3.5 h. Medium with the HMC-1 cells was removed and replaced with PBS for the final 30 min of incubation. The fibroblasts were obtained from donors with the following medical conditions: Strain 1, severe TAO; Strain 2, severe TAO; Strain 3, normal orbital tissue in an individual without known thyroid disease. Data are expressed as the mean 6 SEM of triplicate replicates from a single, representative experiment.

of the response to IL-1b was remarkably similar in all three strains and ranged from 5- to 9-fold. The responses to HMC-1 were somewhat less brisk than those observed with IL-1b and ranged from 2- to 6-fold. Whereas strains designated 1 and 2 were from individuals with severe TAO, strain 3 derived from an individual without orbital or apparent thyroid disease. It is interesting to note that, in preliminary studies, dermal and thyroid fibroblasts failed to exhibit increases in PGE2 production after exposure to HMC-1 cells, under experimental conditions identical to those used here (data not shown). FIG. 6. The effects of IL-4 and IL-4 neutralization on orbital fibroblast PGE2 production. Orbital fibroblasts were allowed to proliferate to confluence in 24-well cluster plates. Some of the wells received nothing (control), IL-4 (10 ng/ml), or HMC-1 cells (cell ratio 1:1) without or with IL-4 receptor antibodies (10 mg/ml). The fibroblast cultures were then washed, and HMC-1 cells were completely removed, and PBS was replaced over the monolayers for 30 min. An aliquot was subjected to assay for PGE2, as described in Materials and Methods.

HMC-1 cells is mediated, at least in part, through IL-4 production. To assess whether the activation of the PGE2 synthetic pathway by HMC-1 cells in orbital fibroblasts was a common attribute or was limited to certain strains, we next examined fibroblasts from three different donors in side-by-side studies. As the results in Fig. 7 indicate, all three strains studied responded to both IL-1b (10 ng/ml) and HMC-1 coculture, with regard to increases in PGE2 synthesis. The magnitude

Coculture of orbital fibroblasts with HMC-1 cells results in the up-regulation of [3H]hyaluronan synthesis

Hyaluronan production in orbital fibroblasts is particularly susceptible to the influence of proinflammatory cytokines (21). To determine whether coculture of orbital fibroblasts with HMC-1 cells results in the enhancement of hyaluronan synthesis, confluent cultures of fibroblasts were exposed to HMC-1 cells for 3.5 h, the fibroblast monolayers were washed, mast cells were removed, and fibroblast monolayers were labeled with [3H]glucosamine (3 mCi/ml) for 30 min. As Fig. 8 indicates, the presence of HMC-1 cells resulted in a 2-fold increase in [3H]glycosaminoglycan accumulation. We have demonstrated that more than 70% of the labeled macromolecular material is sensitive to streptomyces hyaluronidase digestion (21), indicating that the macromolecule is hyaluronan. Moreover, we have reported that hyaluronan degradation in orbital fibroblast cultures is nil (14, 15, 21),


FIG. 8. HMC-1 mast cells cocultured with orbital fibroblasts up-regulates the synthesis of [3H]glycosaminoglycan. Confluent 60-mmdiameter culture plates of orbital fibroblasts were seeded with HMC-1 mast cells at a cell ratio of 1:1 for 3.5 h. The mast cells were removed, and the monolayers were washed, medium containing [3H]glucosamine (3 mCi/ml) was added and the fibroblasts were radiolabeled for 30 min. The medium and cell layers were combined and analyzed for [3H]glycosaminoglycan content, as described in Materials and Methods. Data are presented as the mean 6 SEM, n 5 3.

indicating that the effects of mast cells relate to macromolecular synthesis. We have reported, in preliminary form, that the most abundant HAS mRNA expressed by orbital fibroblasts is HAS2 (38). Our initial studies suggest that HAS2 mRNA expression in orbital fibroblasts is up-regulated by HMC-1 coculture (data not shown). Discussion

Here we report that the coculture of orbital fibroblasts with HMC-1 mast cells leads to substantial increases in fibroblastic PGHS-2 expression. This induction results in marked enhancement of PGE2 synthesis. In addition, HMC-1 coculture with orbital fibroblasts substantially up-regulates hyaluronan synthesis. Thus, two of the features dominating the pattern of tissue remodeling seen in TAO can be attributed mechanistically to mast cell/fibroblast interactions. Although other cells recruited to the orbit in this disease, most notably lymphocytes, are also likely to be involved, interactions between mast cells and fibroblasts could contribute substantially to the fibroblast activity associated with TAO. Moreover, they might support the molecular events, mediated through the biosynthetic repertoire of orbital fibroblasts, that underlie tissue remodeling in that disease process. Fibroblasts residing in the human orbit are a population of diverse cells that seem to be particularly susceptible to several proinflammatory molecules that activate a number of genes and their products. For instance, the serine protease inhibitor, plasminogen activator inhibitor type-1, is induced in orbital fibroblasts by interferon-g and by leukoregulin but is down-regulated or only modestly up-regulated by these same cytokines in dermal fibroblasts (39, 40). PGE2 elicits a dramatic shape alteration in orbital fibroblasts but not in those from the skin (41, 42). Cytokines such as leukoregulin

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(21) and interferon-g (43) up-regulate glycosaminoglycan synthesis in orbital fibroblasts in an anatomic site-selective manner. Thus, there is a substantial body of evidence to suggest that orbital fibroblasts are phenotypically distinct from those emanating from the skin and other regions of the body. We believe that it is the peculiar phenotype of these fibroblasts that underlies the characteristic pattern of tissue remodeling seen in TAO. It is currently unknown what links the pretibial tissue, which manifests the dermopathy of Graves’ disease, with the orbit. However, Young et al. (18) have reported very recently that a set of cytokine-responsive gene products are expressed in orbital and pretibial dermal fibroblasts but not in those from an irrelevant anatomical area, such as the abdominal skin, which ordinarily does not manifest Graves’ dermopathy. Though the molecular basis for the differences in fibroblasts from distant body regions is uncertain, it will be important to examine potential pretibial fibroblast/mast cell interactions to determine whether inductions observed in orbital cultures occur in those fibroblasts as well. We became interested in studying potential mast cell/ orbital fibroblast interactions because of the presence of both cell types in the inflamed orbital connective tissue in the context of TAO. The role of mast cells in the pathogenesis of TAO has been largely ignored, with most attention focused on activated T lymphocytes (3–5). It is very likely that these lymphocytes are intimately involved in the pathogenesis of TAO, but mast cells are capable of cross-talk with both lymphocytes and fibroblasts. Interactions between mast cells and nonimmune cells have only recently been examined in detail. It would seem that the diverse repertoire of activities exhibited by mast cells makes them candidates for facilitating complex processes such as wound healing and fibrosis. Interactions between mast cells and dermal fibroblasts have been shown recently to be mediated through the actions of several mast cell products. Their presence in a wide array of both allergic and nonallergic tissue reactions suggests that they are multifunctional. Indeed, the high levels of expression by mast cells of IL-4 (8), for example, implies that these cells might be important modulators of proinflammatory cytokine actions, such as those associated with interferon-g, the actions of which IL-4 antagonizes (44). Mast cells also express tryptase, a neutral serine protease currently believed to be mast cell-specific (45). Tryptase has been shown to enhance the rate of fibroblast proliferation (46). In addition, the protease can up-regulate expression of collagen mRNA and enhance fibroblast chemotaxis (47). On the other hand, it would seem that tryptase fails to elicit Ca21 mobilization in fibroblasts, which is mediated through the proteolytic activity on protease-activated receptors observed in keratinocytes (48). The involvement of mast cells in the connective tissue manifestations of thyroid disease was hypothesized by Asboe-Hansen and colleagues (6) nearly 50 yr ago. In a series of papers, they demonstrated that mast cells were abundant in orbital connective tissue from experimental guinea pigs rendered ophthalmopathic by administering daily sc injections of purified thyrotrophin. The material surrounding the mast cells stained metachromatically with toluidine blue but failed to concentrate stain after hyaluronidase treatment.

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They concluded that the frequent finding of mast cells in tissues with hyaluronan deposition implied that these cells might have a proximate role in the accumulation of this macromolecule. Despite these important and provocative findings, little has appeared in the literature since, that explores a potential role for mast cells in autoimmune thyroid disease. Mast cells express high levels of CD40 ligand or CD154, a member of the tumor necrosis factor-a superfamily (49). We have reported very recently that orbital fibroblasts, as well as those from the lung, express high levels of surface-displayed CD40, a member of the tumor necrosis factor-a receptor superfamily originally found on B cells (50). When the CD40 on these fibroblasts is engaged with its ligand, the cells exhibit a substantial increase in hyaluronan and PGE2 synthesis (51). The up-regulation in prostanoid production is mediated through increases in the expression of PGHS-2 and can be abolished with nonsteroidal antiinflammatory drugs such as indomethacin and SC 58125 (51, 52). T lymphocytes also express high levels of CD40 ligand, and thus, the CD40/ CD40 ligand bridge represents a potentially important conduit for the activation of fibroblasts by both mast cells and lymphocytes. It would seem, therefore, that orbital fibroblasts can be activated by the immune system through a number of orthodox and nontraditional pathways. We are currently examining the potential utilization of the CD40/ CD40 ligand bridge in some of the interactions between orbital fibroblasts and mast cells. The increase in hyaluronan synthesis after the addition of HMC-1 cells to orbital fibroblast cultures suggests an important mechanism through which the accumulation of that glycosaminoglycan might occur in the context of TAO. The most abundant glycosaminoglycan synthesized by activated human orbital fibroblasts is hyaluronan (33). Unlike the other abundant glycosaminoglycans, hyaluronan is not sulfated and lacks a core protein (1). Despite these differences, the rheologic properties of hyaluronan resemble those of the other glycosaminoglycans. It is the extraordinarily hydrophilic nature of hyaluronan that accounts for its bulky nature when hydrated. This results in substantial increases in the volumes of tissues accumulating the glycosaminoglycan, such as is the case in the orbital tissues involved in TAO. Important insights into the pathways involved in the synthesis of hyaluronan have recently evolved. A family of three mammalian HAS genes has been cloned (53–56) and each encoded enzyme has been characterized partially. It would seem, from preliminary studies, that HAS2 is the most abundant synthase mRNA expressed in human fibroblasts, although we have found that HAS1 and HAS3 mRNAs can also be detected in at least some strains of human fibroblasts (unpublished observations of the authors). Moreover, the mammalian UDP glucose dehydrogenase has been cloned in mouse and human tissue and has been shown to be regulated in orbital fibroblasts with IL-1b (57). We have begun to examine HAS2 expression in orbital fibroblasts and its response to HMC-1 coculture. To date, we have been able to detect a substantial induction of the HAS2 mRNA after exposure to HMC-1 cells (data not shown). We are currently examining the expression of all three HAS genes, as well as other po-

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tentially relevant enzymes in the glycosaminoglycan biosynthetic cascades, in our coculture model. It would seem, from these studies, that orbital fibroblast activation by mast cells could represent an important mechanism for the tissue remodeling that occurs in TAO. Future studies will be directed at defining the precise pathways through which these interactions occur and establishing their role in vivo in the pathogenesis of the disease. Considering the array of molecules the synthesis of which is up-regulated by mast cells and their products, it would be of particular interest to examine other aspects of the orbital fibroblast phenotype that are potentially relevant to inflammation and wound healing. Moreover, the impact of coculture on reciprocal mast cell activity will need to be studied before the implications of mast cell/fibroblast interactions can be fully evaluated. Acknowledgments The authors are grateful to Ms. Heather Meekins for expert technical assistance and to Dr. H. J. Cao for advice. We thank Dr. Peter Isakson, of Searle, for the provision of SC 58125; and Dr. J. H. Butterfield, of the Mayo Clinic, for kindly providing the HMC-1 cells.

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