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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
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.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, 14, 15), proteins (16, 17, 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 up-regulation 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, 27, 28, 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-16
-methyl-11ß,17,21-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.
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Materials and Methods |
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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 -thioglycerol in a 5% environment
at 37 C. In some studies, HMC-1 cells were treated with A23187 (500
µg/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 µg) 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 x saline-sodium citrate, 5 x 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 -70 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 µl) 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
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 µg/ml
soybean trypsin inhibitor, and 10 µM 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
µCi/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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 µg/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 activated
HMC-1 cells were similar to those observed in cultures treated with
IL-1ß for 16 h. Levels of PGHS-1 mRNA were constant, with regard
to mast cell exposure and IL-1ß 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).
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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 leukoregulin-treated 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.
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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).
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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-1ß (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.
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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 µg/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 HMC-1
cells is mediated, at least in part, through IL-4 production.
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indicate, all three strains studied
responded to both IL-1ß (10 ng/ml) and HMC-1 coculture, with regard
to increases in PGE2 synthesis. The magnitude of the
response to IL-1ß 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-1ß 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).
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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), 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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-
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 (21) and interferon- (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, 4, 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-, 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 Ca2+ 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. 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- 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- 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, 54, 55, 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-1ß (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 potentially 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.
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Acknowledgments |
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Footnotes |
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Received January 19, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is an inducer of plasminogen activator inhibitor type 1
in human orbital fibroblasts. Am J Physiol 263:C24–C29
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Evidence for post-transcriptional regulation. J Biol Chem 269:11769–11775 in cultured fibroblasts
derived from patients with Graves’ ophthalmopathy and pretibial
dermopathy. J Clin Endocrinol Metab 73:307–313This article has been cited by other articles:
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  M.-J. Shih, S.-L. Liao, K.-T. Kuo, T. J. Smith, and L.-M. Chuang Molecular Pathology of Muller's Muscle in Graves' Ophthalmopathy J. Clin. Endocrinol. Metab., March 1, 2006; 91(3): 1159 - 1167. [Abstract] [Full Text] [PDF]
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 R. Han and T. J. Smith T Helper Type 1 and Type 2 Cytokines Exert Divergent Influence on the Induction of Prostaglandin E2 and Hyaluronan Synthesis by Interleukin-1{beta} in Orbital Fibroblasts: Implications for the Pathogenesis of Thyroid-Associated Ophthalmopathy Endocrinology, January 1, 2006; 147(1): 13 - 19. [Abstract] [Full Text] [PDF]
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Y. Hiromatsu, T. Fukutani, M. Ichimura, T. Mukai, H. Kaku, H. Nakayama, I. Miyake, S. Shoji, Y. Koda, and T. Bednarczuk Interleukin-13 Gene Polymorphisms Confer the Susceptibility of Japanese Populations to Graves' Disease J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 296 - 301. [Abstract] [Full Text] [PDF]
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 B. S. Prabhakar, R. S. Bahn, and T. J. Smith Current Perspective on the Pathogenesis of Graves' Disease and Ophthalmopathy Endocr. Rev., December 1, 2003; 24(6): 802 - 835. [Abstract] [Full Text] [PDF]
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 M. B. Frungieri, S. Weidinger, V. Meineke, F. M. Kohn, and A. Mayerhofer Proliferative action of mast-cell tryptase is mediated by PAR2, COX2, prostaglandins, and PPARgamma : Possible relevance to human fibrotic disorders PNAS, November 12, 2002; 99(23): 15072 - 15077. [Abstract] [Full Text] [PDF]
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 R. Han, S. Tsui, and T. J. Smith Up-regulation of Prostaglandin E2 Synthesis by Interleukin-1beta in Human Orbital Fibroblasts Involves Coordinate Induction of Prostaglandin-Endoperoxide H Synthase-2 and Glutathione-dependent Prostaglandin E2 Synthase Expression J. Biol. Chem., May 3, 2002; 277(19): 16355 - 16364. [Abstract] [Full Text] [PDF]
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 J. Pritchard, N. Horst, W. Cruikshank, and T. J. Smith Igs from Patients with Graves' Disease Induce the Expression of T Cell Chemoattractants in Their Fibroblasts J. Immunol., January 15, 2002; 168(2): 942 - 950. [Abstract] [Full Text] [PDF]
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T. J. Smith, L. Koumas, A. Gagnon, A. Bell, G. D. Sempowski, R. P. Phipps, and A. Sorisky Orbital Fibroblast Heterogeneity May Determine the Clinical Presentation of Thyroid-Associated Ophthalmopathy J. Clin. Endocrinol. Metab., January 1, 2002; 87(1): 385 - 392. [Abstract] [Full Text] [PDF]
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 H. J. Cao and T. J. Smith Leukoregulin upregulation of prostaglandin endoperoxide H synthase-2 expression in human orbital fibroblasts Am J Physiol Cell Physiol, December 1, 1999; 277(6): C1075 - C1085. [Abstract] [Full Text] [PDF]
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