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J Biol Chem, Vol. 273, Issue 45, 29615-29625, November 6, 1998
From the Human orbital fibroblasts play a
putative role in the pathogenesis of thyroid-associated ophthalmopathy
(TAO). We hypothesize that the hyaluronan accumulation and inflammation
in TAO derive from enhanced biosynthetic activities of orbital
fibroblasts. CD40, a member of the tumor necrosis factor- Fibroblasts derived from the connective tissue investing the orbit
possess a distinctive phenotype in vitro. Orbital
fibroblasts are heterogeneous with regard to the surface display of
Thy-1 (1), exhibit characteristic profiles of receptor (2), ganglioside (3, 4), and plasminogen activator inhibitor type-1 expression (5-7),
and respond distinctively to cytokines and hormones (8-12) when
compared with other types of human fibroblasts. They have attracted
substantial attention recently because of their putative role in the
pathogenesis of thyroid-associated ophthalmopathy (TAO)1 (13). A small fraction
of individuals with Graves' disease develop TAO, which involves
remodeling of the orbital tissue, including the perimysial connective
tissue. Molecular events underlying the relatedness of the orbital
tissue activation with the thyroid glandular disease process have yet
to be identified. The cardinal features of orbital tissue remodeling
include a disordered accumulation of hyaluronan, a non-sulfated
glycosaminoglycan, and intense inflammation. Histopathological
examination reveals an infiltration of lymphocytes and mast cells (14).
Lymphocyte populations infiltrating the orbit have been characterized
partially, and both CD4+ and CD8+ cells are
present (15, 16). The mechanism whereby bone marrow-derived cells are
recruited to the orbit in TAO is currently not understood. Moreover,
the proximate conduit(s) for molecular cross-talk between resident
cells of the orbital tissues and recruited immunocompetent cells has
yet to be identified. We hypothesize that bone marrow-derived cells
drive the activation of orbital fibroblasts through several cell-signaling pathways. A variety of inflammatory mediators can up-regulate orbital fibroblast target genes and alter the biosynthetic activities of these cells (2, 5, 6, 10-12).
A recently recognized pathway through which fibroblasts can be
activated is the CD40/CD40 ligand bridge (17). CD40 is a member of the
TNF- Cyclooxygenases also known as prostaglandin endoperoxide-H synthases
(PGHS) are enzymes that catalyze the conversion of arachidonate to
prostaglandins and other prostanoids (25). They are bifunctional, heavily glycosylated proteins possessing heme prosthetic groups. Two
isoforms of PGHS have been identified recently and their cDNAs cloned (26-29). PGHS-1 is a constitutively expressed enzyme, located on human chromosome 9, that is thought to catalyze the production of
prostaglandins involved in normal physiological homeostasis. In
contrast, PGHS-2 localizes to chromosome 1, is expressed ordinarily at
extremely low levels but is inducible by serum, mitogens, and cytokines. PGHS-2 is an early immediate response gene and represents the inflammatory cyclooxygenase; its activities generate prostanoids believed to be involved in the inflammatory response. The up-regulation of PGHS-2 expression by cytokines is rapid and transient and can involve both increased gene transcriptional activity and an enhancement of mature mRNA stability (30, 31). The profound attenuation of
prostaglandin production observed following glucocorticoid treatment is
mediated, at least in part, through the blockade of PGHS-2 expression
(28, 32). We have found that orbital fibroblasts are particularly
susceptible to the up-regulation of PGHS-2 expression by certain
pro-inflammatory cytokines such as leukoregulin, a 50-kDa product of
activated T lymphocytes and IL-1 Another aspect of cell biology evolving rapidly and of proximate
relevance to TAO relates to the regulation, synthesis, and action of
hyaluronan. This non-sulfated glycosaminoglycan possesses rheologic
properties similar to those of the other abundant complex carbohydrates
but lacks a core protein. The extraordinary hydrophilic nature
exhibited by hyaluronan suggests that its accumulation in the orbit
underlies the anterior displacement of the eye in TAO. Hyaluronan
accumulates in the orbital fat pad and infiltrates the perimysial
connective tissue of the extraocular muscles (13). Cellular receptors
for hyaluronan have been identified and include CD44 and RHAM (34, 35).
The recent discovery of three distinct mammalian hyaluronan synthases
(HAS), each encoded by a separate gene, has allowed rapid advancement
of insight into the mechanisms involved in hyaluronan synthesis
(36-42). We have reported previously that hyaluronan synthesis in
orbital fibroblasts is particularly vulnerable to up-regulation by
cytokines such as IL-1 In this paper, we report studies examining the consequences of CD40
engagement by CD40 ligand in human orbital fibroblasts. When incubated
with CD40 ligand, these cells exhibit substantial increases in
hyaluronan and PGE2 synthesis. The effects on prostanoid production, but not those involving hyaluronan, are mediated through an
enhanced expression of PGHS-2 and are partially dependent upon an
intermediate up-regulation of IL-1 Materials--
Human recombinant CD40 ligand in insect membranes
was prepared as described previously (45) and kindly provided by Dr.
Marilyn Kehry (Boehringer Ingelheim, Ridgefield, CT). SC 58125, a
PGHS-2-selective cyclooxygenase inhibitor (46), was a generous gift of
Searle, and IL-1ra was provided by Amgen (Boulder, CO). Dexamethasone (1,4-pregnadien-9-fluoro-16 Cell Culture--
Orbital fibroblast cultures were initiated
from tissue explants obtained during decompression surgery for severe
TAO or from normal orbital tissue derived from surgical waste during
procedures to correct non-orbital disease. These activities have been
approved by the Institutional Review Board of the Albany Medical
College. Tissue specimens were disrupted mechanically and covered with Eagle's medium to which fetal bovine serum (FBS, Life Technologies, Inc) was added. Medium also contained glutamine (435 µg/ml) and penicillin/streptomycin. The disrupted explants were allowed to attach
to the bottom of the culture plates as described previously. Cultures
were maintained in a 37 °C, humidified incubator with 5%
CO2-enriched air as described previously (47). Medium was changed routinely every 3-4 days. When fibroblasts were outgrown, the
explants were removed, the fibroblast monolayer disrupted with
trypsin/EDTA, and the cells re-plated. Culture strains were utilized
between the 2nd and 12th passage and had reached a state of confluence
when all experimental manipulations were undertaken. We have determined
that these cells fail to express factor VIII or smooth muscle-specific
actin (1), and thus the cultures are not contaminated with endothelial
or smooth muscle cells.
Hyaluronan Assay--
Hyaluronan was quantitated by measuring
the incorporation of [3H]glucosamine into
glycosaminoglycans. Details concerning this assay have been published
by us previously (8, 44, 47, 48). Briefly, confluent cultures of
orbital fibroblasts were shifted to medium containing 1% FBS for
72 h without or with interferon-
Hyaluronan was quantitated essentially as reported previously (44, 48).
Samples that had been dialyzed were lyophilized to dryness and
rehydrated in 0.15 M NaCl, 0.02 M sodium
acetate buffer, pH 6.0. An aliquot was subjected to digestion with
Streptomyces hyaluronidase (50 units/ml; Calbiochem) at
37 °C for 48 h and then dialyzed against sodium acetate buffer.
Samples were diluted in 10 mM Tris/HCl, pH 8.4, and layered
on to 1.5 × 15 cm DEAE-Sephacel columns. These were washed with
30 ml of the same buffer, and the radiolabeled material was eluted with
50 ml of a 0-0.6 M NaCl linear gradient in Tri/HCl buffer,
pH 8.4. One-ml fractions were collected and counted for radioactivity.
PGE2 Assay--
Fibroblasts were seeded in 24-well
plastic dishes and allowed to proliferate to confluence in medium
containing 10% FBS. Medium was removed, and fresh medium supplemented
with 1% FBS was added for 16 h, and then cultures were treated
with CD40 ligand without or with other test compounds as described in
the legends to the figures. The final 30 min of the incubation was
conducted by removing the medium and adding 100 µl of PBS containing
the additives. At the end of the incubation, the PBS was collected,
centrifuged, and subjected to a specific radioimmunoassay or an ELISA
for PGE2.
Western Blot Analysis of Fibroblast PGHS Proteins--
Relative
levels of PGHS proteins were determined by immunoblot analysis using
monoclonal antibodies specifically directed against PGHS-1 and PGHS-2.
Fibroblast cultures were allowed to proliferate to confluence in 60-mm
plastic dishes covered with medium supplemented with 10% FBS and then
were shifted to medium with 1% FBS for 16-24 h. CD40 ligand was added
without or with other test reagents as described in the legends to the
figures. At the time of harvest, medium was removed, and monolayers
were washed with PBS and harvested in an ice-cold buffer containing 15 mM CHAPS, 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 sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the separated proteins transferred to polyvinylidene difluoride membrane (Bio-Rad).
Nonspecific binding sites were blocked by incubating the membranes in
PBS to which 0.05% polyoxyethylene-sorbitan monolaurate (Sigma) 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 extensively and incubated with
the secondary, peroxidase-labeled antibodies for 2 h. Following three washes, the ECL (Amersham Pharmacia Biotech) chemiluminescence detection system was used to generate the signal. Resulting bands were
analyzed densitometrically with a BioImage scanner (Milligen).
Immunocytochemistry--
Orbital fibroblasts were seeded in
eight-well chamber slides and cultured as indicated in the legend to
the figure. Cells were fixed in 2% paraformaldehyde, treated with 3%
H2O2, blocked with 2% horse serum, and
incubated overnight at 4 °C with 10 µg/ml of either anti-PGHS-1 or
anti-PGHS-2 monoclonal antibodies. Sister cultures were incubated with
isotype control antibodies to assess nonspecific staining. Biotinylated
horse anti-mouse or anti-goat IgG (heavy + light chain, 1:200; Vector
Labs, Inc., Burlingame, CA) was used as a secondary antibody followed
by incubation with streptavidin-horseradish peroxidase (1:1000; Jackson
ImmunoResearch Labs, Inc., West Grove, PA).
Isolation of Fibroblast RNA and Northern
Hybridizations--
Levels of the relevant transcripts were determined
with standard Northern blotting techniques. Cultures were allowed to
proliferate to confluence in 100-mm plastic dishes covered with medium
containing 10% FBS. They were then shifted to medium with 1% serum
for 16 h, and then some plates were pretreated with interferon- Assays for the Expression of IL-1 Immunoprecipitation and Western Blot Analysis of Phosphorylated
Mitogen-activated Protein Kinase (MAPK)--
Nuclear protein extracts
from orbital fibroblasts were prepared as described (50) and
immunoprecipitated using a polyclonal anti-phosphotyrosine antibody
(Transduction Labs, Lexington, KY) overnight. Protein A-agarose was
added and samples incubated for 1 h at 4 °C. Complexes were
washed with hypotonic buffer containing 0.2% Nonidet P-40, and
immunoprecipitated proteins were separated by discontinuous 9% PAGE
and transferred to Immobilon membranes (Millipore, Bedford, MA). After
blocking with 5% powdered milk in Tris-buffered saline containing 1%
Tween, membranes were incubated with 1:1000 monoclonal anti-MAPK
antibody (anti-ERK2, Upstate Biotechnology Inc., Lake Placid, NY)
overnight at room temperature and washed, and the secondary, rabbit
anti-mouse IgG was added at 1:1000. The signals were visualized with
the ECL detection kit.
Statistical Analysis--
Data are expressed as the mean ± S.E. of replicate determinations unless indicated otherwise.
Statistical significance was determined by Student's t test.
CD40 Engagement with CD40 Ligand on Orbital Fibroblasts Results in
a Substantial Up-regulation of Hyaluronan Synthesis--
Orbital
fibroblasts under basal culture conditions synthesize hyaluronan, as
assessed by the incorporation of [3H]glucosamine into
macromolecular material. In general the rate of hyaluronan synthesis in
untreated orbital fibroblast cultures is lower than that observed in
dermal fibroblasts (8, 44, 47). When the fibroblast monolayers were
treated with interferon-
The radiolabeled macromolecular material was subjected to digestion
with Streptomyces hyaluronidase (50 units/ml) for 48 h. This enzyme has an absolute specificity for hyaluronan (51). As the
DEAE-Sephacel ion-exchange column chromatographic profile shown in Fig.
2 indicates, the radiolabeled material
elutes as a single peak, consistent with our previous findings in
orbital fibroblasts treated with leukoregulin (44). Approximately 95% of the material in the interferon-
We next attempted to implicate the recently cloned members of the HAS
family of enzymes in the up-regulation of hyaluronan synthesis elicited
by CD40 ligand. We were unable to detect by Northern analysis any of
the three transcripts in control orbital fibroblast cultures or those
treated with CD40 ligand for 8 h (Fig.
3). Moreover, pretreatment of cultures
for 72 h with interferon-
Glucocorticoids can influence the synthesis of hyaluronan in human
fibroblasts (8). We have reported previously that dexamethasone can
block the induction of hyaluronan production by cytokines in orbital
fibroblasts (44). We therefore examined whether the glucocorticoid
could affect the impact of CD40 engagement on hyaluronan synthesis in
these cells. In an experiment where incorporation of
[3H]glucosamine into macromolecules was 94 ± 20 cpm/µg protein in control cultures, addition of CD40 ligand for
22 h increased incorporation to 381 ± 29 cpm/µg protein
(p < 0.001 versus control). When
dexamethasone (10 nM) was added with the CD40 ligand,
incorporation was attenuated to 218 ± 48 cpm/µg protein
(p < 0.02 versus CD40 ligand alone), representing a 57% decrease in the response to CD40 ligand. In contrast, addition of SC 58125 (5 µM) failed to alter the
response to CD40 ligation (377 ± 27 cpm/µg protein, not
significant compared with CD40 ligand alone). Thus, the effects of CD40
ligation on hyaluronan synthesis are unrelated to increases in
PGE2 synthesis. This is entirely consistent with our
findings concerning the up-regulation of hyaluronan synthesis by
leukoregulin (44).
CD40 Engagement in Orbital Fibroblasts Elicits a Dramatic Increase
in Prostanoid Production--
Orbital fibroblasts, like most other
cells in culture, fail to express high levels of PGHS-2 under
un-stimulated conditions. We have reported that these fibroblasts
produce lower basal levels of PGE2 than do dermal
fibroblasts under identical conditions (31). When they are treated with
pro-inflammatory cytokines such as leukoregulin and IL-1
The up-regulation of PGE2 production by CD40 ligand is
time-dependent, detectable within 4 h, and continues
to increase so that at 24 h the levels are approximately 240-fold
above control levels (control, 34 ± 20 pg/ml; 24 h CD40
ligand, 8190 ± 850 pg/ml, Fig. 4, panel B). The
increase in prostanoid levels can be blocked by approximately 80% when
dexamethasone (10 nM) is added concomitantly with CD40
ligand (Fig. 4, panel C). SC 58125 (5 µM) can
also inhibit the PGE2 synthesis elicited by CD40 ligand
(Fig. 4, panel C), supporting the role of an enhanced level
of PGHS-2 expression in the up-regulation of prostanoid biosynthesis.
We have demonstrated previously that this concentration of inhibitor
used has near-absolute specificity for PGHS-2 in orbital fibroblasts
(31).
CD40 Engagement Enhances PGE2 Production through an
Increase in PGHS-2 Protein Levels--
We next assessed the level of
PGHS-1 and PGHS-2 protein expressed by orbital fibroblasts without and
with CD40 ligand treatment. As the Western blot in Fig.
5 indicates, PGHS-1 is constitutively expressed as a single 68-kDa band and is largely unaffected by CD40
ligation, consistent with our previous findings concerning cytokine
treatment in these cells (31). However, PGHS-2 protein is not detected
under basal conditions but appears as an abundant 72-kDa band after
8 h of CD40 ligand treatment. The induction is transient in that
the PGHS-2 signal has returned nearly to control levels by 16 h.
The up-regulation of PGHS-2 by CD40 ligand could be attenuated by
concomitant addition of dexamethasone (10 nM) to the
culture medium. Orbital fibroblasts are heterogeneous, and thus we
assessed whether the induction of PGHS-2 by CD40 ligand uniformly
involved the fibroblast population. We treated cultures with CD40
ligand for 20 h, and the cells were then fixed and stained with
anti-PGHS-1 and anti-PGHS-2 antibodies. As the photomicrographs in Fig.
6 demonstrate, a majority of the CD40
ligand-treated cells exhibit marked PGHS-2 staining which appears to be
both perinuclear and cytoplasmic, consistent with the localization
studies previously reported (54) and our findings in lung fibroblasts
(33). In contrast, control monolayers failed to stain for PGHS-2
although they exhibit strong staining for PGHS-1, which is unaffected
in the cells treated with CD40 ligand. Pretreatment of the cells with
interferon- CD40 Ligand Induces PGHS-2 Expression at a Pretranslational
Level--
Our next studies were directed at assessing whether PGHS-2
up-regulation following CD40 engagement was mediated through an increase in the steady-state levels of PGHS-2 mRNA. Cultures were pretreated with nothing or interferon- CD40 Engagement Results in a Substantial Up-regulation of IL-1 The Induction of PGHS-2 Expression but Not Hyaluronan Production
Elicited by CD40 Engagement Is Dependent upon Intermediate IL-1 CD40 Engagement in Orbital Fibroblasts Enhances MAPK
Activation--
MAPK has been implicated in the signal transduction
pathway utilized by certain cytokines, such as IL-1, in their induction of PGHS-2 expression (58). We therefore determined whether the engagement of CD40 on orbital fibroblasts by CD40 ligand could influence the activation of this important kinase pathway. As the data
contained in Fig. 10 (panel
A) indicate, CD40 ligand up-regulates nuclear MAPK activation in a
time-dependent manner in orbital fibroblasts that is
substantial at 8 h, maximal at 16 h, and had decreased to
near base line at 24 h. Because dexamethasone can block the
induction by CD40 ligand of PGHS-2 expression, we added the
glucocorticoid (10 nM) at the same time as CD40 ligand
treatment was initiated and found that the increase in nuclear MAPK
activation was entirely blocked after 16 h (Fig. 10, panel
B). Apigenin (25 nM), a specific inhibitor of MAPK
(59), could also substantially attenuate the effect of CD40 engagement
on MAPK activation, as the figure attests. To determine whether these
increases in MAPK activation were related to the induction of PGHS-2,
we assessed whether inhibitors of two enzymes in the MAPK cascade
influenced cyclooxygenase induction. Addition of geldanamycin (10 nM) or apigenin (25 nM) to the culture medium
at the time of initiation of CD40 ligand treatment results in a
dramatic attenuation of PGHS-2 induction (Fig. 10, panel C).
Geldanamycin inhibits the action of Raf1 by causing its dissociation
from Hsp90 (60). Thus, interruption of the MAPK pathway at two
different levels attenuates PGHS-2 induction mediated by CD40
engagement.
Human orbital fibroblasts can be activated through the CD40/CD40
ligand bridge to express substantially greater levels of hyaluronan and
PGE2, as the current studies have demonstrated. These two
features of the cellular response to CD40 engagement are consonant with
the remodeling known to occur in orbital connective tissue in TAO. They
potentially represent the molecular basis for fibroblast activation in
that disease process and explain multiple aspects of the complex
interplay between the immune system and connective tissue. The
up-regulation of hyaluronan by CD40 ligation is apparently independent
of the induction of PGHS-2 expression and PGE2 production
as is evidenced by the lack of effect of SC 58125 on that cellular
response. We have reported previously that CD40 engagement on orbital
fibroblasts results in dramatic increases in the expression of IL-6 and
IL-8 (24), and thus it would appear that the expression of a number of
potentially important genes and their products is enhanced through the
CD40/CD40 ligand bridge. This previously unrecognized pathway for the
activation of orbital fibroblasts suggests a pathway through which CD40
ligand-displaying cells such as T lymphocytes and mast cells could
directly and specifically cross-talk with orbital fibroblasts. We
hypothesize that the CD40/CD40 ligand bridge is a widespread conduit
utilized for the activation of connective tissue by the immune system. Both lymphocytes and mast cells have been found in the orbital connective tissue in patients with TAO (14). This has led us and others
to hypothesize that bone marrow-derived cells are responsible for
driving the profound tissue remodeling in this disease.
We have reported that lung (61), gingival (62), and thyroid-derived (4)
fibroblasts express CD40 and that this expression can be substantially
induced by interferon- Fibroblasts from the human orbit exhibit distinctive phenotypic
attributes that render the connective tissue susceptible to as yet
unidentified pathogenic factors associated with TAO. We have shown
previously that orbital fibroblasts are capable of an extraordinary
capacity to synthesize hyaluronan when treated with a variety of
pro-inflammatory cytokines, including interferon- The mechanisms involved in the enhancement by CD40 engagement of
hyaluronan synthesis are not completely defined. Unlike the CD40 ligand
actions on PGHS-2 expression, the induction of hyaluronan is
independent of IL-1 Orbital fibroblasts express extraordinarily high levels of PGHS-2
expression and produce PGE2 in response to pro-inflammatory cytokines such as leukoregulin and IL-1 (31). The elevated levels of
prostanoid result in a dramatic alteration in cellular morphology, which appears to be mediated through EP2 receptors (67).
With regard to earlier studies involving cytokines, it appeared that alterations in PGHS-2 mRNA stability account for the majority of
the increases in enzyme levels as PGHS-2 gene transcriptional rates
were increased only modestly (2-3-fold) (31). From the results
reported here, it would appear that CD40 engagement represents another
potentially important pathway through which prostanoid production can
be up-regulated in orbital fibroblasts. Steady-state PGHS-2 mRNA
and protein levels are rapidly elevated, and the up-regulation of
PGE2 production can be abolished with SC 58125, a
PGHS-2-selective cyclooxygenase inhibitor (46) and by dexamethasone.
The mechanism involved in the PGHS-2 induction elicited by CD40 ligand
has been partially identified in these studies. It would appear that
the intermediate synthesis of IL-1 The signal transduction pathways utilized by CD40 have been examined
recently, and the issues appear to be complex and cell type-specific.
In B lymphocytes, CD40 is coupled to both ERK and Jun kinase pathways
(70-72). Nuclear MAPK activation is enhanced by CD40 engagement in
orbital fibroblasts (Fig. 10A), and the interruption of the
MAPK cascade can partially block PGHS-2 up-regulation (Fig. 10C). This was accomplished with geldanamycin which depletes
the cell of Raf-1 and therefore acts at a level up-stream of MAPK (59)
and with apigenin, a compound which inhibits MAPK directly (60). We
have proposed a pathway through which the CD40/CD40 ligand bridge might
activate orbital fibroblasts in a schematic (Fig.
11). Our findings are entirely
consistent with a very recent report demonstrating the role of MAPK in
the induction by IL-1 of PGHS-2 in fibroblasts and human umbilical vein
endothelial cells (58). In contrast to the effects of CD40 engagement
on PGHS-2 expression and PGHS-2-dependent PGE2
production, PGHS-1 levels appeared to be invariant with regard to CD40
engagement.
The molecular trigger(s) that drives the hyaluronan synthesis and
inflammation in TAO is uncertain but is presumed to emanate from the
immunocompetent cells that are trafficked to the orbit (14).
Lymphocytes and mast cells have been shown to express high levels of
CD40 ligand (19, 73), and thus our finding that orbital fibroblasts
display surface CD40 (24) and can be activated by CD40 ligand is of
particular mechanistic importance. Whether the CD40 bridge constitutes
a functionally important component of fibroblast signaling in
situ under normal physiologic conditions or in disease states will
require further studies, such as those utilizing conditional disruption
of CD40 or CD40 ligand gene expression. Interruption of this
intercellular signaling conduit may represent an important therapeutic
target for modifying the natural course of TAO.
We are grateful for helpful discussions with
Drs. Paul J. Davis and Faith B. Davis. We thank Dr. Peter Isakson,
Searle, for the kind provision of SC 58125. The expert technical
assistance of Heather Meekins is acknowledged.
*
This work was supported in part by National Institutes of
Health Grants EY 08976, EY 11708, CA 11198, DE 11390, and HL 56002; by
Merit Review funding from the Research Service of the Department of
Veterans Affairs; by the National Science Council of the Republic of
China Grant NSC 87-2314-B-010-066, and by the Rochester Area Pepper
Center.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence and reprint requests should be addressed:
Division of Molecular and Cellular Medicine (A-175), Albany Medical
College, 47 New Scotland Ave., Albany, NY 12208. Tel.: 518-262-5266;
Fax: 518-262-5304.
The abbreviations used are:
TAO, thyroid-associated ophthalmopathy; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; dexamethasone, 1,4-pregnadien-9-fluoro-16 2
L. A. Kaback and T. J. Smith,
manuscript in preparation.
Activation of Human Orbital Fibroblasts through CD40 Engagement
Results in a Dramatic Induction of Hyaluronan Synthesis and
Prostaglandin Endoperoxide H Synthase-2 Expression
INSIGHTS INTO POTENTIAL PATHOGENIC MECHANISMS OF
THYROID-ASSOCIATED OPHTHALMOPATHY*
§,
,,, and§**
Division of Molecular and Cellular Medicine, University of Rochester
Cancer Center and Departments of Microbiology and Immunology,
Pediatrics and Environmental Medicine, Rochester, New York 14642
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
receptor
superfamily, is a critical signaling molecule expressed by B
lymphocytes. Engagement of CD40 with CD154 or CD40 ligand results in
the activation of target genes. Orbital fibroblasts also display CD40.
Here we report that CD40 engagement leads to substantial increases in
hyaluronan synthesis in orbital fibroblasts. The increase is
approximately 5-fold above control values, is comparable to the
induction elicited by IL-1
and could be attenuated with
dexamethasone but not by SC 58125, a prostaglandin endoperoxide H
synthase-2 (PGHS-2)-selective inhibitor. PGHS-2 is also induced by CD40
engagement in a time-dependent manner, and this is mediated
through increases in levels of steady-state mRNA. The induction of
PGHS-2 leads to a dramatically enhanced prostaglandin
E2 production that can be blocked by SC 58125 and dexamethasone. CD40 ligand up-regulates the synthesis of IL-1, and
blocking this cytokine with exogenous IL-1 receptor antagonist (IL-1ra)
or with IL-1 neutralizing antibodies partially attenuates the
induction of PGHS-2. In contrast, CD40 ligand up-regulation of
hyaluronan synthesis is unaffected by IL-1ra. CD40 cross-linking enhances mitogen-activated protein kinase activation, and interrupting this pathway attenuates the PGHS-2 induction. Thus the CD40/CD40 ligand
bridge represents a potentially important activational pathway for
orbital fibroblasts that may underlie the cross-talk between these
cells and leukocytes. These findings may be relevant to the
pathogenesis of TAO and provide insights into previously unrecognized, potential therapeutic targets.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
receptor superfamily that was initially identified on B
lymphocytes (18). It functions as a crucial conduit for the activation
of lymphocytes when ligated with gp39, also known as CD40 ligand or
CD154. CD40 ligand is a member of the TNF- family and is expressed
on T lymphocytes and mast cells (19, 20). Fibroblasts from a variety of
anatomic regions express CD40 (17), and other cell types also have been
shown to display this glycoprotein, including epithelial (21) and
endothelial cells (22) and keratinocytes (23). We reported recently
that orbital fibroblasts, derived either from normal tissue or from that affected with TAO, display surface CD40 that can be up-regulated with interferon-
(24). Moreover, the CD40 expression on orbital fibroblasts is functional. When engaged with CD40 ligand, there is a
nuclear translocation of nuclear factor-
B and the activation of IL-6
and IL-8 expression (24). Of particular interest is our finding that
thyroid fibroblasts also express functional CD40 (4).
(31). This induction, mediated
through both transcriptional and post-transcriptional mechanisms,
results in dramatic increases in the production of PGE2.
Moreover, the cytokine-dependent PGE2 synthesis
can be inhibited with PGHS-2-selective inhibitors such as SC 58125 (31). We recently reported that PGE2 production in lung
fibroblasts is enhanced through the ligation of CD40, an effect
mediated through the induction of PGHS-2 (33). This action of CD40
ligand on PGHS-2 expression has not been characterized until now.
Moreover, whether orbital fibroblasts are similarly activated through
CD40 has not been examined, but this could be of critical importance to
the pathogenesis of orbital inflammatory states such as TAO.
, interferon-, and leukoregulin (43, 44).
In contrast, the accumulation of abundant sulfated glycosaminoglycans
is not influenced by these cytokines. Thus the up-regulation of
hyaluronan biosynthetic activity in orbital fibroblasts may be an
important target for the molecular signals derived from activated
immunocompetent cells recruited to the orbit in TAO.
synthesis. Thus we have identified a previously unrecognized mechanism for the activation of
orbital fibroblasts through the CD40/CD40 ligand bridge that can
explain the participation of these fibroblasts in situ in the tissue remodeling observed in TAO. This fibroblast-signaling pathway may represent a highly efficient and specific means by which
the immune system can activate connective tissue cells in the setting
of an inflammatory response. We hypothesize that the disruption of the
CD40/CD40 ligand bridge, either directly or through the attenuation of
the IL-1 autocrine loop, may represent an important therapeutic target
relevant to TAO and other lymphocyte-driven forms of inflammation
involving connective tissue.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-methyl-11,17, 21-triol-3,20-dione) was from Sigma. Recombinant IL-1 and human interferon were from
Biosource (Camarillo, CA) and Genzyme (Cambridge, MA), respectively. [3H]Glucosamine hydrochloride (specific activity 1369 GBq/mmol) was supplied by NEN Life Science Products. cDNAs encoding
human PGHS-1 and PGHS-2 were kindly provided by Dr. D. A. Young
(University of Rochester). HAS1 cDNA was a gift from Dr. M. J. Briskin (LeukoSite, Cambridge, MA); HAS2 cDNA was kindly provided
by Dr. Y. Yamaguchi (Burnham Institute, La Jolla, CA); and HAS3
cDNA was from Drs. A. Spicer and J. McDonald (Mayo, Scottsdale,
AZ). Anti-PGHS-1 and PGHS-2 antibodies were purchased from Cayman
Chemical (Ann Arbor, MI), and anti-IL-1 neutralizing antibody was
purchased from R & D Systems (Minneapolis, MN). PGE2
radioimmunoassay was from Amersham Pharmacia Biotech;
PGE2 ELISA was from Cayman; the IL-1 and IL-1 ELISA
assays were from Immunotech (Westbrook, ME), and the IL-1ra assay was
from R & D Systems. Geldanamycin was kindly provided by the Drug
Synthesis & Chemistry Branch, Developmental Therapeutics program,
Division of Cancer Treatment, National Cancer Institute (Bethesda), and
apigenin was purchased from Calbiochem (San Diego).
(100 units/ml). After this
pretreatment period, medium of all cultures was changed, and fresh
medium without or with interferon- alone or in combination with CD40
ligand was added for 16 h. All plates were then shifted to fresh
medium with respective additives and labeled with
[3H]glucosamine (1 µCi/ml) for 6 h. At the time of
culture harvest, media samples were aspirated quantitatively and
reserved, and the cell layers were rinsed well with phosphate-buffered
saline (PBS) that was added to the corresponding medium samples. Cell layers were solubilized in 0.2 N NaOH, removed from the
substratum with a rubber policeman, and the cells disrupted by
sonication. After an aliquot was removed from the cell layer material
for protein determination using bovine serum albumin as standard, the
medium and cell layer material were combined and subjected to Pronase
(1 mg/ml) digestion in 100 mM Tris buffer, pH 8.0, at
50 °C overnight. Samples were cooled to 4 °C, and trichloroacetic acid was added to a final concentration of 5% (w/v). After complete precipitation on ice, samples were centrifuged, and acid-soluble material was subjected to exhaustive dialysis against cold water. The
retained samples were subjected to liquid scintillation counting and
defined as total glycosaminoglycan.
(500 units/ml) for 72 h. Both naive and interferon-pretreated
cultures were then either treated with nothing or with CD40 ligand in
the presence or absence of interferon- for the intervals indicated in the legends to the figures. Monolayers were rinsed and total cellular RNA extracted by the method of Chomczynski and Sacchi (49)
using Tri-Reagent (Molecular Research Center, Cincinnati, OH) or
ULTRASPEC (Biotecx, Houston, TX). Northern analysis was conducted as
described previously (31) by electrophoresing RNA on denaturing 1%
agarose, formaldehyde gels. RNA integrity was verified by routinely
ascertaining 260/280 spectroscopic ratio and by staining the
electrophoresed sample with ethidium bromide and inspecting it under UV
light. Samples were transferred to Zeta-probe membrane (Bio-Rad), and
the immobilized RNA was allowed to hybridize with
[32P]dCTP-labeled PGHS-1 and PGHS-2 cDNA probes.
These were generated from 1.6- and 1.4-kb human cDNAs,
respectively. Hybridizations were allowed to proceed in a solution
containing 5× SSC, 50% formamide, 5× Denhardt's solution, 50 mM phosphate buffer, pH 6.5, 1% SDS, and 0.25 mg/ml salmon
sperm at 48 °C overnight. Membranes were washed under high
stringency conditions, and then radioactive hybrids were visualized by
radioautography on X-Omat film (Eastman Kodak Co.) exposed at
70 °C, and the radioactive bands were scanned densitometrically.
Membranes were stripped of radioactivity following the manufacturer's
instructions and rehybridized with probes complementary to GAPDH for standardization.
, IL-1, and IL-1 Receptor
Antagonist (IL-1ra)--
Fibroblasts were allowed to proliferate to
confluence in 24-well plastic culture plates covered with medium
containing 10% FBS. Monolayers were shifted to medium with 1% serum
for 16-24 h and then test compounds, including CD40 ligand, were added
at the times indicated in the figure legends. Medium was removed, and
cellular material was harvested in a buffer containing 15 mM CHAPS, 1 mM EDTA, 20 mM
Tris/HCl, pH 7.5, 10 µg/ml soybean trypsin inhibitor, 10 µM phenylmethylsulfonyl fluoride, 3 µg/ml aprotinin and
0.5% Nonidet P-40. Cellular proteins (5 or 10 µg) were subjected to
specific ELISA assays for IL-1, IL-1, and IL-1ra.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
for 94 h, the final 6-h period of
which was incubated in the presence of [3H]glucosamine (1 µCi/ml), there was no increase in [3H]hyaluronan
accumulation (Fig. 1). This treatment of
orbital fibroblasts results in a substantial increase in the surface
display of CD40 (24). Addition of recombinant CD40 ligand-containing membranes to naive, non-interferon- pretreated fibroblasts for 22 h increases hyaluronan accumulation by 3.4-fold
(p < 0.001). When fibroblasts were pretreated with
interferon- for 72 h and then incubated with CD40 ligand for an
additional 22 h, the magnitude of the increase in hyaluronan
accumulation is 4.8-fold above control levels (p < 0.001). This increase was comparable to that observed when the cultures
were treated with IL-1 (10 ng/ml) for 22 h (Fig. 1).
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Fig. 1.
CD40 engagement in orbital fibroblasts
results in a substantial up-regulation of hyaluronan accumulation.
Orbital fibroblasts were allowed to proliferate to confluence, and then
some cultures were pretreated with interferon- (500 units/ml) for
72 h. Medium was then changed in all cultures and some received
CD40 ligand containing membranes (1:100) for an additional 16 h
without or with interferon- as indicated. Cultures were then labeled
with [3H]glucosamine (1 µCi/ml) for 6 h in the
presence of the respective additives and were harvested as described
under "Experimental Procedures." Monolayers and media were added
together and analyzed for total glycosaminoglycan accumulation. Each
data point represents the mean ± S.E. n = 3-4
replicate cultures from a representative experiment.
-pretreated, CD40 ligand-treated cultures was digested with the hyaluronidase, indicating that the
induced macromolecule was comprised almost entirely of hyaluronan. We
have reported that hyaluronan degradation in cultured human orbital and
dermal fibroblasts under these culture conditions is undetectable (8,
44, 47, 48). Our findings are consistent with the absence of
hyaluronidase activity in human fibroblast cultures reported by
Arbogast et al. (52). Thus the increases in macromolecular
accumulation elicited by CD40 ligand reflect an up-regulation in
synthesis.
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Fig. 2.
DEAE-Sephacel ion-exchange chromatographic
analysis of the CD40 ligand-induced macromolecules incorporating
[3H]glucosamine in orbital fibroblasts. The
non-dialyzable macromolecular radiolabeled material was lyophilized,
solubilized, and subjected to treatment with Streptomyces
hyaluronidase (50 units/ml) as described under "Experimental
Procedures." Both untreated and digested materials were then
subjected to ion-exchange chromatography. The material was layered on a
DEAE-Sephacel column and eluted with a 0-0.6 M NaCl
gradient. Squares represent undigested macromolecules, and
circles refer to hyaluronidase-treated material.
(500 units/ml) failed to elicit a
response of any HAS transcripts to CD40 ligation. We have been able to
demonstrate an induction of HAS2 in several strains of orbital
fibroblasts with IL-1 (10 ng/ml) within 6-8 h of cytokine treatment
(53). This transcript appears as two discrete bands of 4.4 and 2.5 kb.
Thus it would appear that the induction of HAS mRNAs may not be a
prominent mechanism for the up-regulation of hyaluronan synthesis
elicited by CD40 ligation in orbital fibroblasts.
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Fig. 3.
Northern analysis of HAS mRNA
inducibility by CD40 ligation in human orbital fibroblasts.
Orbital fibroblasts from a patient with severe TAO were allowed to
proliferate to confluence in 100-mm culture plates in medium
supplemented with 10% FBS. They were then shifted to medium with 1%
serum without or with interferon (500 units/ml) for 72 h. A
group of naive cultures as well as interferon- pretreated cultures
received CD40 ligand (1:100) for 8 h. At the end of the treatment
period, total cellular RNA was extracted from the monolayers, and 50 µg of each sample was subjected to electrophoresis, transferred to
membranes, and allowed to hybridize with cDNA probes for HAS1,
HAS2, and HAS3. Membranes were stripped and rehybridized to a GAPDH
probe to verify gel loading. The integrity of the HAS cDNA probes
was established by demonstrating hybridization to appropriately sized
transcripts in orbital fibroblasts treated with IL-1 (data not
shown).
, orbital
fibroblasts exhibit substantial increases in PGE2
production, which are considerably greater than those observed in the
dermal cultures. We therefore determined whether CD40 ligation would
increase prostanoid production. As the data in Fig.
4, panel A, demonstrate, there
is a dramatic increase in PGE2 levels following addition of
CD40 ligand to the culture medium of TAO-derived orbital fibroblasts
for 16 h. PGE2 levels in control cultures were
122 ± 2.8 pg/ml (mean ± S.E.) and had increased to
1277 ± 42 pg/ml in the cultures treated with CD40 ligand. A very
similar level of prostanoid was found in cultures pretreated with
interferon- (500 units/ml) for 72 h and then treated with CD40
ligand (1312 ± 49 pg/ml). Thus there is an absence of an apparent
contribution from pretreatment with interferon- to the
PGE2 level achieved. We have found previously that in lung fibroblasts, interferon- pretreatment enhances PGE2
production in response to CD40 ligand substantially (3-4-fold) (33).
Fig. 4, panel A, also demonstrates a similar induction of
PGE2 synthesis by CD40 ligation in orbital fibroblasts from
normal tissue.
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Fig. 4.
CD40 engagement in orbital fibroblasts
results in a dramatic increase in PGE2 synthesis.
Orbital fibroblasts were allowed to proliferate to confluence in
24-well plates in medium supplemented with 10% FBS. Monolayers were
then shifted to 1% FBS-enriched medium. Panel A, some
cultures were pretreated with interferon- (500 units/ml) for 72 h, and then some received CD40 ligand (1:100) without or with
interferon- for 16 h. For the final 30 min of incubation, PBS
with the respective additives was added and then collected for
PGE2 determinations as described under "Experimental
Procedures." Fibroblasts used in this study were from a patient with
severe thyroid-associated ophthalmopathy (TAO) or from a
donor without orbital disease (normal). Panel B,
cell layers from a patient with TAO were treated as in panel
A, except cultures were not pretreated with interferon-,
and the duration of treatment with CD40 ligand was varied as indicated
along the abscissa. Panel C, cultures from a
patient with severe TAO received CD40 ligand alone or in combination
with dexamethasone (10 nM) or SC 58125 (5 µM). Data are presented as the mean ± S.E. of
triplicate wells from representative studies.
(500 units/ml) for 96 h appears to have failed to
increase the intensity of the PGHS-2 staining elicited by CD40 ligand.
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Fig. 5.
Western blot analysis of the effects of CD40
engagement on PGHS-1 and PGHS-2 protein expression in orbital
fibroblasts. Confluent cultures of orbital fibroblasts, in this
case from an individual with severe TAO, were shifted to medium
supplemented with 1% FBS without or with CD40 ligand (1:100) for the
intervals indicated along the abscissa. Monolayers were
harvested and proteins separated by PAGE and subjected to Western
analysis of PGHS-1 and PGHS-2 proteins as described under
"Experimental Procedures." The resulting signals were analyzed
densitometrically and are shown in the bottom panel.
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Fig. 6.
Immunocytochemical detection of PGHS-1 and
PGHS-2 in orbital fibroblasts. Orbital fibroblasts from a patient
with TAO were cultured for 72 h in medium supplemented with 10%
FBS without or with interferon- (500 units/ml) and then replaced
with serum-free medium for 20 h containing nothing (panel
A), interferon- (panel B), CD40 ligand (1:100,
panel C), or interferon- with CD40 ligand (panel
D). Cells were then fixed and stained with PGHS-1
(bottom) and PGHS-2-specific (top) Abs. Original
magnification × 400.
for 72 h to up-regulate CD40 display, and then some cultures received CD40 ligand for 8 h.
As the Northern blot analysis contained in Fig.
7 demonstrates, PGHS-2 transcript is not
detectable under control conditions. CD40 ligand elicits a substantial
increase in PGHS-2 mRNA levels. This transcript appears as a 4.8-kb
band, similar to the pattern we have found previously following
treatment with pro-inflammatory cytokines (31). Moreover, the
interferon- pretreatment fails to induce the transcript alone or to
augment the effects of CD40 ligand on PGHS-2 mRNA expression.
PGHS-1 mRNA, migrating as a 5.0-kb transcript, is expressed at
similar levels under control, interferon-, and CD40 ligand treatment
conditions, as expected. We have reported previously that the
predominant PGHS-1 transcript expressed by human fibroblasts is 5.0 kb
(31), similar to that observed in endothelial cells and monocytes (55,
56) but different from some other human and animal cells that express a
2.8-kb PGHS-1 mRNA (26).
View larger version (16K):
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Fig. 7.
Northern analysis of PGHS-1 and PGHS-2
expression in orbital fibroblasts treated with CD40 ligand.
Confluent cultures of orbital fibroblasts, in this instance from a
patient with severe TAO, were shifted to medium supplemented with 1%
FBS to which nothing or interferon- (500 units/ml) was added for
72 h of pretreatment. Then some of the pretreated and control
(naive) plates were either treated with nothing or with CD40 ligand
(1:100) for 8 h. Following washes, monolayers were solubilized,
and total cellular RNA was extracted and subjected to electrophoresis,
transferred to membrane, and allowed to hybridize with probes
synthesized from PGHS-1 and PGHS-2 cDNAs. The resulting signals,
denoting RNA/DNA hybrids, were captured by autoradiography and were
analyzed densitometrically with a BioImager scanner. Membranes were
stripped according to the manufacturer's instructions and re-probed
for GAPDH. The normalized PGHS-2 signal is plotted in the bottom
panel of the figure.
Expression in Orbital Fibroblasts--
We have found that orbital
fibroblasts can express high levels of both IL-1 and IL-1 when
treated with pro-inflammatory cytokines such as leukoregulin and IL-1
itself (57). In contrast, the magnitude of IL-1ra induction by
exogenous IL-1 was substantially less than that found in dermal
fibroblasts. Moreover, IL-1 is a potent inducer of PGHS-2 in orbital
fibroblasts as well as in many other cell types. Thus, we examined the
effects of CD40 engagement on IL-1 expression. As the data in Fig.
8 (panel A) indicate, IL-1
expression is increased by CD40 engagement. The increase is
time-dependent (control, <15.6 pg/10 µg of protein,
n = 3) and maximal at 8 h when it reaches 110 ± 12 pg/10 µg of protein. By 16 h, the levels of IL-1 have
begun to decline. IL-1 expression in response to CD40 ligation was
assessed in four orbital strains, three from patients with TAO and one
from normal tissue. All expressed undetectable basal IL-1 levels but
achieved a level of 48 ± 9 pg/10 µg (mean ± S.E.,
n = 3) after 8 h of treatment. In contrast, the
levels of IL-1 appear uninduced or are up-regulated only slightly by
CD40 ligation, suggesting a divergence in these cells away from the use
of IL-1 (Fig. 8, panel B). Orbital fibroblasts are
capable of expressing IL-1 when treated with other stimuli. When
cultures from this same cell strain were treated with exogenous IL-1
(10 µg/ml), the level of IL-1 increased to 28 ± 1 pg/10 µg
of protein from undetectable levels. This failure of CD40 ligand to
elicit a substantial increase in IL-1 production was verified in
four orbital fibroblast strains. The mean IL-1 level achieved after
8 h of CD40 ligand treatment was <15.6 pg/10 µg of protein compared with 106 ± 50 pg/10 µg in cultures treated with
exogenous IL-1 (10 ng/ml). As the data in Fig. 8 (panel
C) indicate, CD40 engagement elicits a modest increase in IL-1ra
levels, which begin to increase at 16 h and have tripled by
24 h. The up-regulation of IL-1 and IL-1ra following CD40
engagement were completely blocked with dexamethasone (10 nM). In an experiment where IL-1 levels were <15.6 and
90 ± 15 pg/10 µg of protein in control and CD40-treated
cultures, respectively, addition of dexamethasone resulted in an
IL-1 level below the limits of detectability (<15.6 pg/10 µg of
cellular protein). With regard to IL-1ra expression, the control levels
were 476 ± 33 pg/10 µg of protein and achieved a level of
1292 ± 11.4 pg/10 µg (p < 0.0001 versus control) following CD40 ligation. Concomitant
addition of dexamethasone with CD40 ligand resulted in IL-1ra levels of
400 ± 10 pg/10 µg of protein.
View larger version (13K):
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Fig. 8.
CD40 ligand elicits a substantial,
time-dependent increase in IL-1 synthesis in orbital
fibroblasts. Confluent 24-well culture plates were shifted to
medium supplemented with 1% FBS for 16 h, and then CD40 ligand
(1:100) was added to some of the wells for the durations of time
indicated along the abscissas. Media samples were removed,
and the cell monolayers were solubilized as described under
"Experimental Procedures," and 10 µg from each sample was
subjected to specific ELISA for Il-1 (panel A), IL-1
(panel B), or IL-1ra (panel C). The data are the
means ± S.E. (n = 3) from representative
experiments.
Production--
Because we have found that IL-1 induces PGHS-2
expression and hyaluronan synthesis in orbital fibroblasts, we examined
the impact of neutralizing the action of the cytokine on
PGE2 production and PGHS-2 expression elicited by CD40
ligand. That up-regulation of PGE2 and PGHS-2 was
substantially attenuated with the addition to the culture medium of
either anti-IL-1 antibodies or exogenous IL-1ra (Fig.
9, panels A and B).
Addition of IL-1 Abs (1 µg/ml) or IL-1ra (500 ng/ml) attenuated
the increase in PGE2 by 63 and 73%, respectively, and
PGHS-2 expression by 78 and 79%, respectively. These findings strongly
implicate the intermediate induction of IL-1 in the up-regulation by
CD40 ligand of PGHS-2 expression and PGE2 synthesis. In
contrast, when IL-1ra was added to cultures that were then CD40 engaged
and analyzed for [3H]hyaluronan synthesis, the cytokine
receptor antagonist exerted no influence on the production of the
macromolecule (control 94 ± 20 cpm/µg of protein, CD40 ligand;
381 ± 29 cpm/µg of protein, CD40 ligand + IL-1ra; 491 ± 103 cpm/µg of protein). Thus it would appear that some but not all of
the actions of CD40 ligation in orbital fibroblasts are dependent upon
an intermediate induction of IL-1.
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Fig. 9.
Effect of neutralizing IL-1 Abs and
exogenous IL-1ra on the up-regulation of PGE2 production
(panel A) and PGHS-2 protein expression (panel
B) elicited by CD40 ligand in orbital fibroblasts. Confluent
orbital fibroblasts from a patient with TAO were treated for 16 h
with CD40 ligand (1:100) without or with neutralizing IL-1 Abs (1 µg/ml), IL-1ra (500 ng/ml), or dexamethasone (10 nM). The
cultures were then processed for PGE2 assay (panel
A) or Western blot analysis of PGHS-1 and PGHS-2 protein
expression (panel B) as described under "Experimental
Procedures."
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Fig. 10.
CD40 engagement in orbital fibroblasts
results in an increase in nuclear MAPK activation. Interruption of the
MAPK signal transduction cascade results in an attenuation of PGHS-2
induction by CD40 ligand. Orbital fibroblasts from a patient with
TAO were allowed to proliferate to confluence, and then they were
shifted to medium containing 1% FBS and supplemented without or with
CD40 ligand (1:100) as well as the additives indicated along the
abscissas. Panel A, cultures were treated with
CD40 ligand for the times indicated, and the monolayers were harvested
and nuclear proteins extracted as described under "Experimental
Procedures." These were subjected to immunoprecipitation with an
anti-phosphotyrosine antibody, subjected to PAGE, transferred to
Immobilon membrane, and Western blot analysis with anti-MAPK (ERK2)
antibody. The signal was generated with the ECL system, and the
relative densities were scanned. Panel B, cells were treated
as in panel A, except that the CD40 ligand treatment time
was 16 h and was added without or with dexamethasone (10 nM) or apigenin (25 nM). Panel C,
confluent cultures were treated with nothing or the compounds
indicated. The concentration of apigenin used was 25 nM and
that of geldanamycin was 10 nM. After 16 h, monolayers
were harvested and cellular protein subjected to PAGE followed by
Western blot analysis of PGHS-2 protein levels. The data shown are from
representative experiments.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
. Moreover, PGHS-2 expression and
PGE2 production are substantially up-regulated by CD40
ligation in lung fibroblasts (33). There appear to exist important
differences between fibroblasts in the orbit and those from the lung
with regard to CD40-mediated signaling. In lung fibroblasts, the
induction of PGHS-2 by CD40 ligand in cultures not pretreated with
interferon- is modest (33). When the cells are pretreated with the
cytokine and treated with CD40 ligand, the magnitude of PGHS-2
induction in lung fibroblasts is enhanced substantially. In orbital
fibroblasts, the up-regulation by CD40 ligand of PGHS-2 mRNA and
PGE2 synthesis is near maximal in cultures not pretreated
with interferon- (Figs. 4, panel A and Fig. 7). It is
possible that interferon- is enhancing the CD40 induction of PGHS-2
in lung fibroblasts by increasing the number of CD40 ligand-binding
sites on the surfaces of these cells, as we have demonstrated
previously (24, 61). Alternatively, the cytokine might be enhancing the
cyclooxygenase induction through an up-regulation of relevant
components of intracellular signaling pathways utilized by CD40
engagement in lung fibroblasts. In any event, it would appear that
orbital fibroblasts are inherently more susceptible to the
up-regulation by CD40 ligation of PGHS-2 expression than are lung
fibroblasts. This finding further supports our view that fibroblasts
represent a heterogeneous population of cells (1, 2, 5, 7, 11).
, IL-1, and
leukoregulin (43, 44). These cytokine-dependent increases
in hyaluronan synthesis exhibited by orbital fibroblasts are
considerably more robust than those observed in dermal fibroblasts under the same experimental conditions. Glucocorticoids such as dexamethasone can attenuate the up-regulation of hyaluronan synthesis in orbital fibroblasts elicited by cytokines (44) and can inhibit basal
hyaluronan synthesis in dermal fibroblasts (8). Our finding that CD40
engagement can also enhance hyaluronan synthesis in orbital fibroblasts
and that this up-regulation can be blocked partially by glucocorticoids
suggests that regulation of glycosaminoglycan production in orbital
fibroblasts is complex. Coupled with the finding that dexamethasone can
block CD40 ligand-dependent PGHS-2 expression, it would
appear that the interruption of the molecular consequences of the
CD40/CD40 ligand bridge by glucocorticoids could account, at least in
part, for the appreciable therapeutic benefit associated with their use
in TAO (63). We have reported previously that the actions of
glucocorticoids on glycosaminoglycans in human fibroblasts are mediated
through orthodox glucocorticoid receptors (64).
, as is suggested by the inability of IL-1ra to
influence the response. We have been unable to demonstrate a consistent
impact of CD40 ligation on the expression of any of the three HAS
mRNAs (Fig. 3) in orbital fibroblasts. In our studies to date, the
predominant, inducible hyaluronan synthase isoform mRNA expressed
in confluent, adult human fibroblasts is HAS2, and this transcript is
up-regulated severalfold by both leukoregulin and IL-1 (53). In some
fibroblast strains, we have been able to detect induction by IL-1 of
HAS1 and HAS3 mRNAs as well, although the abundance of these
transcripts, on the basis of Northern analysis, appears considerably
lower than that of HAS2.2 It
is possible that some other step(s) in the hyaluronan biosynthetic cascade is being induced/activated as a consequence of CD40 engagement. For instance, Spicer et al. (66) have reported very recently the cloning of the human UDP-glucose dehydrogenase and the induction of
its mRNA by IL-1 in cultured orbital fibroblasts from patients with severe TAO. Thus it is possible that a step upstream from HAS,
such as the UDP-glucose dehydrogenase, might act as the target for
CD40-dependent up-regulation of hyaluronan biosynthesis. On the other hand, we may have as yet failed to define the proper conditions under which some HAS induction may be occurring. Our findings are consistent with those reported recently in microvascular endothelial cells where IL-1, TNF-, and bacterial
lipopolysaccharide failed to increase steady-state HAS mRNA levels
despite their up-regulation of hyaluronan in those cells (65). Clearly,
additional studies are now warranted to define the molecular basis for
the up-regulation of hyaluronan we report here.
is important for the full impact of CD40 engagement on PGHS-2 expression because neutralizing the IL-1, with either a specific antibody or with exogenous IL-1ra, can
substantially attenuate the induction (Fig. 9). A recent study has
demonstrated that CD40 engagement in vascular smooth muscle and
endothelial cells results in the activation of caspase-1 and the
synthesis of IL-1 (68). Thus, there is precedent for an up-regulatory role for CD40 engagement on IL-1 production in other cell
types. Despite the capacity of orbital fibroblasts to express IL-1,
the CD40-dependent pathway appears to utilize only IL-1. We have reported previously that murine lung fibroblasts express high
levels of IL-1 but fail to synthesize IL-1 when activated with
TNF- (69). It may be that different fibroblast subpopulations utilize members of the IL-1 family of genes selectively, with regard to
particular activational triggers. Our demonstration of an autocrine
loop comprised of IL-1 mediating, at least in part, the induction of
PGHS-2 in orbital fibroblasts represents a potentially powerful new
insight concerning the mechanism through which CD40 ligand exerts its
actions in non-lymphoid tissues. These findings suggest that disruption
of IL-1 expression and/or action at the level of the fibroblast could
attenuate the impact of CD40 ligation in these cells. On the other
hand, the inability of IL-1ra to influence the impact of CD40 ligation
on hyaluronan synthesis suggests a divergence of intermediate pathways
relevant to the CD40 activational bridge in fibroblasts.
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Fig. 11.
Schematic of the proposed pathways utilized
by CD40/CD40 ligand bridge in the activation of PGHS-2 expression and
hyaluronan synthesis in human orbital fibroblasts. Uncertain is
the target(s) for CD40 activation of the hyaluronan biosynthetic
pathway.
ACKNOWLEDGEMENTS
FOOTNOTES
-methyl-11,17,21-triol-3,20-dione; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; IL, interleukin; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; PGE2, prostaglandin
E2; PGHS, prostaglandin endoperoxide H synthase; ra, receptor antagonist; TNF-, tumor necrosis factor-; ELISA, enzyme-linked immunosorbent assay; Ab, antibody; PAGE, polyacrylamide
gel electrophoresis; kb, kilobase pairs; HAS, hyaluronan synthases.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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D. El Fassi, C. H Nielsen, H. C Hasselbalch, and L. Hegedus The rationale for B lymphocyte depletion in Graves' disease. Monoclonal anti-CD20 antibody therapy as a novel treatment option. Eur. J. Endocrinol., May 1, 2006; 154(5): 623 - 632. [Abstract] [Full Text] [PDF]
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M. Salvi, G. Vannucchi, I. Campi, S. Rossi, P. Bonara, F. Sbrozzi, C. Guastella, S. Avignone, G. Pirola, R. Ratiglia, et al. Efficacy of rituximab treatment for thyroid-associated ophthalmopathy as a result of intraorbital B-cell depletion in one patient unresponsive to steroid immunosuppression. Eur. J. Endocrinol., April 1, 2006; 154(4): 511 - 517. [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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S. E. Feldon, D. J. J. Park, C. W. O'Loughlin, V. T. Nguyen, S. Landskroner-Eiger, D. Chang, T. H. Thatcher, and R. P. Phipps Autologous T-Lymphocytes Stimulate Proliferation of Orbital Fibroblasts Derived from Patients with Graves' Ophthalmopathy Invest. Ophthalmol. Vis. Sci., November 1, 2005; 46(11): 3913 - 3921. [Abstract] [Full Text] [PDF]
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B. Chen, S. Tsui, and T. J. Smith IL-1{beta} Induces IL-6 Expression in Human Orbital Fibroblasts: Identification of an Anatomic-Site Specific Phenotypic Attribute Relevant to Thyroid-Associated Ophthalmopathy J. Immunol., July 15, 2005; 175(2): 1310 - 1319. [Abstract] [Full Text] [PDF]
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R. Han and T. J. Smith Induction by IL-1{beta} of Tissue Inhibitor of Metalloproteinase-1 in Human Orbital Fibroblasts: Modulation of Gene Promoter Activity by IL-4 and IFN-{gamma} J. Immunol., March 1, 2005; 174(5): 3072 - 3079. [Abstract] [Full Text] [PDF]
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 O. Beloqui, J. A. Paramo, J. Orbe, A. Benito, I. Colina, A. Monasterio, and J. Diez Monocyte cyclooxygenase-2 overactivity: a new marker of subclinical atherosclerosis in asymptomatic subjects with cardiovascular risk factors? Eur. Heart J., January 2, 2005; 26(2): 153 - 158. [Abstract] [Full Text] [PDF]
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 T. J. Smith and N. Hoa Immunoglobulins from Patients with Graves' Disease Induce Hyaluronan Synthesis in Their Orbital Fibroblasts through the Self-Antigen, Insulin-Like Growth Factor-I Receptor J. Clin. Endocrinol. Metab., October 1, 2004; 89(10): 5076 - 5080. [Abstract] [Full Text] [PDF]
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J. Pritchard, S. Tsui, N. Horst, W. W. Cruikshank, and T. J. Smith Synovial Fibroblasts from Patients with Rheumatoid Arthritis, Like Fibroblasts from Graves' Disease, Express High Levels of IL-16 When Treated with Igs against Insulin-Like Growth Factor-1 Receptor J. Immunol., September 1, 2004; 173(5): 3564 - 3569. [Abstract] [Full Text] [PDF]
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 A K Eckstein, B Quadbeck, S Tews, K Mann, C Kruger, C H Mohr, K-P Steuhl, J Esser, and R K Gieseler Thyroid associated ophthalmopathy: evidence for CD4+ {gamma}{delta} T cells; de novo differentiation of RFD7+ macrophages, but not of RFD1+ dendritic cells; and loss of {gamma}{delta} and {alpha}{beta} T cell receptor expression Br. J. Ophthalmol., June 1, 2004; 88(6): 803 - 808. [Abstract] [Full Text] [PDF]
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Y. Inoue, T. Otsuka, H. Niiro, S. Nagano, Y. Arinobu, E. Ogami, M. Akahoshi, K. Miyake, I. Ninomiya, S. Shimizu, et al. Novel Regulatory Mechanisms of CD40-Induced Prostanoid Synthesis by IL-4 and IL-10 in Human Monocytes J. Immunol., February 15, 2004; 172(4): 2147 - 2154. [Abstract] [Full Text] [PDF]
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J. Kaufman, P. J. Sime, and R. P. Phipps Expression of CD154 (CD40 Ligand) by Human Lung Fibroblasts: Differential Regulation by IFN-{gamma} and IL-13, and Implications for Fibrosis J. Immunol., February 1, 2004; 172(3): 1862 - 1871. [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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 C M Gelbmann, S N Leeb, D Vogl, M Maendel, H Herfarth, J Scholmerich, W Falk, and G Rogler Inducible CD40 expression mediates NF{kappa}B activation and cytokine secretion in human colonic fibroblasts Gut, October 1, 2003; 52(10): 1448 - 1456. [Abstract] [Full Text] [PDF]
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L. Koumas, T. J. Smith, S. Feldon, N. Blumberg, and R. P. Phipps Thy-1 Expression in Human Fibroblast Subsets Defines Myofibroblastic or Lipofibroblastic Phenotypes Am. J. Pathol., October 1, 2003; 163(4): 1291 - 1300. [Abstract] [Full Text] [PDF]
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 E. Eriksson, L. Dons, A. G. Rothfuchs, P. Heldin, H. Wigzell, and M. E. Rottenberg CD44-Regulated Intracellular Proliferation of Listeria monocytogenes Infect. Immun., July 1, 2003; 71(7): 4102 - 4111. [Abstract] [Full Text] [PDF]
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J. Pritchard, R. Han, N. Horst, W. W. Cruikshank, and T. J. Smith Immunoglobulin Activation of T Cell Chemoattractant Expression in Fibroblasts from Patients with Graves' Disease Is Mediated Through the Insulin-Like Growth Factor I Receptor Pathway J. Immunol., June 15, 2003; 170(12): 6348 - 6354. [Abstract] [Full Text] [PDF]
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 H. J. Cao, R. Han, and T. J. Smith Robust induction of PGHS-2 by IL-1 in orbital fibroblasts results from low levels of IL-1 receptor antagonist expression Am J Physiol Cell Physiol, June 1, 2003; 284(6): C1429 - C1437. [Abstract] [Full Text] [PDF]
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 L. Fuentes, M. Hernandez, F. J. Fernandez-Aviles, M. S. Crespo, and M. L. Nieto Cooperation Between Secretory Phospholipase A2 and TNF-Receptor Superfamily Signaling: Implications for the Inflammatory Response in Atherogenesis Circ. Res., October 18, 2002; 91(8): 681 - 688. [Abstract] [Full Text] [PDF]
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 R. Han and T. J. Smith Cytoplasmic Prostaglandin E2 Synthase Is Dominantly Expressed in Cultured KAT-50 Thyrocytes, Cells That Express Constitutive Prostaglandin-endoperoxide H Synthase-2. BASIS FOR LOW PROSTAGLANDIN E2 PRODUCTION J. Biol. Chem., September 20, 2002; 277(39): 36897 - 36903. [Abstract] [Full Text] [PDF]
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 E. Nemoto, H. Tada, and H. Shimauchi Disruption of CD40/CD40 ligand interaction with cleavage of CD40 on human gingival fibroblasts by human leukocyte elastase resulting in down-regulation of chemokine production J. Leukoc. Biol., September 1, 2002; 72(3): 538 - 545. [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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 M. Kazim, R. A. Goldberg, and T. J. Smith Insights Into the Pathogenesis of Thyroid-Associated Orbitopathy: Evolving Rationale for Therapy Arch Ophthalmol, March 1, 2002; 120(3): 380 - 386. [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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 J. Kaufman, B. A. Graf, E. C. Leung, S. J. Pollock, L. Koumas, S. Y. Reddy, T. M. Blieden, T. J. Smith, and R. P. Phipps Fibroblasts as Sentinel Cells : Role of the CD40-CD40 Ligand System in Fibroblast Activation and Lung Inflammation and Fibrosis Chest, July 1, 2001; 120(2007): 53S - 55S. [Full Text] [PDF]
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R. F. Schwabe, B. Schnabl, Y. O. Kweon, and D. A. Brenner CD40 Activates NF-{{kappa}}B and c-Jun N-Terminal Kinase and Enhances Chemokine Secretion on Activated Human Hepatic Stellate Cells J. Immunol., June 1, 2001; 166(11): 6812 - 6819. [Abstract] [Full Text] [PDF]
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A. G. Gianoukakis, H. J. Cao, T. A. Jennings, and T. J. Smith Prostaglandin endoperoxide H synthase expression in human thyroid epithelial cells Am J Physiol Cell Physiol, March 1, 2001; 280(3): C701 - C708. [Abstract] [Full Text] [PDF]
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J. Padilla, K. Kaur, H. J. Cao, T. J. Smith, and R. P. Phipps Peroxisome Proliferator Activator Receptor-{gamma} Agonists and 15-Deoxy-{Delta}12,1412,14-PGJ2 Induce Apoptosis in Normal and Malignant B-Lineage Cells J. Immunol., December 15, 2000; 165(12): 6941 - 6948. [Abstract] [Full Text] [PDF]
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A. Bell, A. Gagnon, L. Grunder, S. J. Parikh, T. J. Smith, and A. Sorisky Functional TSH receptor in human abdominal preadipocytes and orbital fibroblasts Am J Physiol Cell Physiol, August 1, 2000; 279(2): C335 - C340. [Abstract] [Full Text] [PDF]
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D. Sciaky, W. Brazer, D. M. Center, W. W. Cruikshank, and T. J. Smith Cultured Human Fibroblasts Express Constitutive IL-16 mRNA: Cytokine Induction of Active IL-16 Protein Synthesis Through a Caspase-3-Dependent Mechanism J. Immunol., April 1, 2000; 164(7): 3806 - 3814. [Abstract] [Full Text] [PDF]
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Y.-i. Hwang, M. H. Nahm, D. E. Briles, D. Thomas, and J. M. Purkerson Acquired, but Not Innate, Immune Responses to Streptococcus pneumoniae Are Compromised by Neutralization of CD40L Infect. Immun., February 1, 2000; 68(2): 511 - 517. [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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L. A. Kaback and T. J. Smith Expression of Hyaluronan Synthase Messenger Ribonucleic Acids and Their Induction by Interleukin-1{beta} in Human Orbital Fibroblasts: Potential Insight into the Molecular Pathogenesis of Thyroid-Associated Ophthalmopathy J. Clin. Endocrinol. Metab., November 1, 1999; 84(11): 4079 - 4084. [Abstract] [Full Text]
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T. J. Smith and S. J. Parikh HMC-1 Mast Cells Activate Human Orbital Fibroblasts in Coculture: Evidence for Up-Regulation of Prostaglandin E2 and Hyaluronan Synthesis Endocrinology, August 1, 1999; 140(8): 3518 - 3525. [Abstract] [Full Text]
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T. J. Smith, T. A. Jennings, D. Sciaky, and H. J. Cao Prostaglandin-endoperoxide H Synthase-2 Expression in Human Thyroid Epithelium. EVIDENCE FOR CONSTITUTIVE EXPRESSION IN VIVO AND IN CULTURED KAT-50 CELLS J. Biol. Chem., May 28, 1999; 274(22): 15622 - 15632. [Abstract] [Full Text] [PDF]
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