1 Division of Molecular and Cellular Medicine, Department of Medicine, Department of Biochemistry and Molecular Biology, Albany Medical College, Samuel S. Stratton Veterans Affairs Medical Center, Albany, New York 12208; 2 Division of Molecular Medicine, Harbor-UCLA Medical Center, Los Angeles 90502; 3 David Geffen School of Medicine, University of California, Los Angeles 90095; and 4 Veterans Affairs Medical Center, Long Beach, California 90822
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ABSTRACT |
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Human orbital
fibroblasts are more susceptible to some actions of proinflammatory
cytokines than are fibroblasts from other anatomic regions. These cells
produce high levels of PGE2 when activated by cytokines.
Here we report that they express high levels of
prostaglandin-endoperoxide H synthase (PGHS)-2, the inflammatory
cyclooxygenase, when treated with IL-1. This induction results from
enhanced PGHS-2 mRNA stability and small increases in gene promoter
activity. The enhanced transcript stability is a result of actions of
the cytokine on the 3'-untranslated region. Orbital fibroblasts, unlike
those from skin, fail to express high levels of IL-1 receptor
antagonist (IL-1ra) when treated with IL-1
, leading to loss of
modulation of IL-1 action. This can be overcome by transiently
transfecting cells with IL-1ra. Thus a decreased level of IL-1ra
expression in orbital fibroblasts may underlie the exaggerated
responses to IL-1 observed in those cells and, therefore, the
susceptibility of the orbit to inflammation.
ophthalmopathy; inflammation; Graves' disease; cytokine
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INTRODUCTION |
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ORBITAL
TISSUE is particularly susceptible to the inflammatory
manifestations of Graves' disease, termed thyroid-associated ophthalmopathy (TAO). We hypothesize that this propensity is directly related to the orbital fibroblast phenotype (23).
In particular, proinflammatory cytokines, such as IL-1, exhibit
striking activity in cultured orbital fibroblasts. IL-1 represents a
small family of cytokines, including IL-1, IL-1
, and IL-1
receptor antagonist (IL-1ra) (8). Occupation of the IL-1
receptor by IL-1ra blocks the binding and action of both IL-1
and
IL-1
. It is the balance between IL-1 and IL-1ra binding that
determines the net biological impact of IL-1 on the target cell.
IL-1 has been shown to regulate the synthesis of prostaglandin E2 (PGE2) in a variety of cells, including those from various depots of connective tissue. These include dermal (18) and lung fibroblasts (19), osteoblasts (7), and rheumatoid synoviocytes (6). Prostaglandin-endoperoxide H synthase (PGHS; EC 1.14.99.1) catalyzes the two rate-limiting steps in the synthesis of prostaglandin H2 (PGH2) from arachidonic acid. Two isoforms of PGHS have been identified. PGHS-1 is expressed in most tissues and cell types under unprovoked conditions and appears to be responsible for the production of prostanoids involved in physiological functions (28). PGHS-2 becomes induced during inflammation, and its expression is upregulated by various cytokines, growth factors, and serum and is attenuated by glucocorticoids (16). PGHS-2 has been cloned, and the deduced amino acid sequence is 61% identical to PGHS-1. The human PGHS-2 transcript contains at least 22 AUUUA sequence motifs in the 3'-untranslated region (UTR), perhaps accounting for its high degree of instability (15).
We reported previously (31) that leukoregulin, a 50-kDa product of mitogen-activated T lymphocytes, can upregulate dramatically steady-state levels of PGHS-2 mRNA in human orbital fibroblasts. This massive induction of PGHS-2 was associated with a marked increase in PGE2 production. In contrast, PGHS-2 expression in dermal fibroblasts was induced more modestly by leukoregulin. The rate of PGHS-2 gene transcription is increased modestly, strongly suggesting that transcript stability is being enhanced substantially by the cytokine. CD40 is displayed by orbital fibroblasts (21) and, when ligated with CD154, results in a substantial induction of PGHS-2 and an increase in PGE2 production (3). Enhanced PGHS-2 mRNA stability probably accounts for this increase in prostanoid generation.
Orbital fibroblasts exhibit a set of phenotypic attributes that
distinguish them from cells derived elsewhere
(25-27). Activation of these fibroblasts appears to
lead to extensive tissue remodeling and accumulation of
glycosaminoglycans in TAO (24). In addition, the
inflammatory reaction often associated with TAO may result from the
local generation by orbital fibroblasts of disease mediators (14). In the current study, we examined the ability of
IL-1 to influence PGHS-2 expression and PGE2 production in
these fibroblasts. IL-1 increases substantially the levels of PGHS-2
mRNA and protein. In contrast, PGHS-1 expression in orbital fibroblasts
is not influenced by either of these cytokines. The induction of PGHS-2
is blocked by dexamethasone, resulting in decreased PGE2
synthesis. Both IL-1 gene products can upregulate the expression of
endogenous IL-1 and IL-1
far more in orbital than dermal
fibroblasts. Exogenous IL-1 can induce IL-1ra, but this is
substantially greater in dermal than in orbital fibroblasts. We
conclude that a "defective" IL-1ra response to IL-1 may underlie,
at least in part, the exaggerated PGHS-2 induction in orbital fibroblasts.
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METHODS |
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Materials.
Human recombinant IL-1 and IL-1
were purchased from BioSource
International (Camarillo, CA). PGHS-1 and PGHS-2 Abs were kindly
supplied by Dr. J. MacLouf (CEA, Gif-sur-Yvette, France) or
purchased from Cayman Chemical (Ann Arbor, MI). IL-1ra was kindly
supplied by Amgen (Boulder, CO). PGE2 assay kits were
purchased from Amersham, and IL-1
and IL-1
ELISA kits were from
Immunotech (Westbrook, ME); those for IL-1ra were from R&D Systems
(Minneapolis, MN). PGHS-1 and PGHS-2 cDNA plasmids were kindly provided
by Drs. D. A. Young and Kerry O'Banion (University of Rochester,
Rochester, NY). Anti-IL-1
and IL-1
antibodies were
purchased from R&D. SC-58125 was a gift from G. D. Searle (Skokie,
IL). Dexamethasone and pyrrolidinedithiocarbamate (PDTC) were purchased
from Sigma (St. Louis, MO). NF-
B oligonucleotide
(5'-AGTTGAGGGGACTTTCCCAGGC-3') and its complement, as well as SP1
oligonucleotide (5'-ATTCGATCGGGGCGGGGGCGATGC-3') and its complement,
were obtained from Promega (Madison, WI). A fragment of the
3'-UTR of the human PGHS-2 sequence spanning +1885 to +2395 was
generated by PCR using the following primers: 5'-CTAAATACGTAGAACGTTCGACTGAACTG-3' and
5'-GAAATTACTCGAGCTGGTAATGTCTAAT-TTAAATAT-3'.
Cell culture. Fibroblast cultures were initiated from tissue explants obtained during orbital surgery. Dermal fibroblast cultures were obtained from punch biopsies of normal-appearing skin. The Institutional Review Boards of Albany Medical College and Harbor-UCLA Medical Center have approved these activities. Procedures for the cell culture have been described in detail previously (22). Briefly, tissue specimens were minced and covered with Eagles' MEM (GIBCO) containing 10% fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin, and nystatin. When fibroblast confluence was reached, the explant was removed and monolayers were disrupted by treatment with trypsin-EDTA. All experiments were performed with fibroblasts within 12 passages of culture initiation. Seven fibroblast strains, each from a different donor, including four from the orbit and three from skin, were analyzed.
Western blot analysis. Levels of cyclooxygenase proteins were determined by Western immunoblot analysis using Abs directed against PGHS-1 and PGHS-2, as previously described (31). Confluent 60-mm plate cultures were shifted to medium containing 1% FBS for 48 h. Cells were then treated with IL-1 (10 ng/ml) or the test compounds indicated, usually for 16 h. Cell layers were washed three times with phosphate-buffered saline (PBS) and taken up in lysis buffer {20 mM Tris · HCl, pH 7.5, 15 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), 1 mM EDTA, 10 µM phenylmethylsulfonyl fluoride (PMSF), and 10 U/ml soybean trypsin inhibitor}. Cellular protein (20 µg) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membrane (Bio-Rad). After incubation with primary and then secondary peroxidase-labeled antibodies (10 µg/ml), signals were detected with enhanced chemiluminescence (ECL; Amersham). The resulting bands were scanned densitometrically with a BioImage densitometer (Milligen).
Isolation and quantification of mRNAs in human fibroblasts.
Fibroblasts were cultivated to confluence in 100-mm plates, shifted to
medium containing 1% FBS for 16 h, and then treated with IL-1 (10 ng/ml) for the times indicated. Cellular RNA was extracted by the
method of Chomczynski and Sacchi (4) with the use of
guanidium isothiocyanate (Ultraspec RNA isolation systems; Biotecx,
Houston, TX), precipitated from the aqueous phase by the addition of
isopropanol, washed with 75% ethanol, and solubilized in diethyl
pyrocarbonate-treated water. Equal amounts of RNA (10 µg) were
electrophoresed in 1% agarose formaldehyde gels and transferred to
Zetaprobe (Bio-Rad). [32P]dCTP random-primed (Bio-Rad)
PGHS probes were hybridized in 5× SSC, 5× 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
washed under high-stringency conditions and exposed to X-OMAT AR film
(Kodak) at 70°C. To normalize the amount of RNA transferred,
membranes were stripped and rehybridized with a human GAPDH cDNA probe.
Radioactive DNA/RNA hybrids were quantified by subjecting
autoradiographs to densitometric analysis.
PGE2 assay. Fibroblasts were cultured to confluence in 24-well plates covered with medium containing 10% FBS. Monolayers were then shifted to medium with 1% FBS for 16 h. IL-1 without or with other test compounds was added at the times indicated. Before assay, the medium was removed and replaced with 150 µl of PBS in the presence of the treatment compounds for the final 30 min of incubation. PBS was collected and subjected to radioimmunoassay as previously described (31) to determine PGE2 release from cultured cells.
Assay of IL-1ra proteins. Fibroblasts were cultured in 24-well plates to confluence in medium supplemented with 10% FBS. Cultures were shifted to 1% FBS for the final 16 h of incubation. Test compounds were added at the times indicated. After treatment, the monolayers were washed extensively with PBS and the cells were taken up in lysis buffer. Both medium (200 µl) and cellular protein (10 µg) were subjected to an ELISA assay (R&D).
Isolation of nuclear proteins and EMSA assays.
Nuclear proteins were prepared essentially as described by Andrews and
Faller (1). Confluent fibroblasts, grown in
100-mm-diameter dishes, were treated with IL-1 (10 ng/ml) for up to
2.0 h. Cells were then washed and scraped in PBS, and then
microcentrifuged. The cell pellet was suspended in 0.5 ml of
buffer A [10 mM HEPES (pH 7.8), 15 mM KCl, 2 mM
MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), and 1 mM
PMSF] and centrifuged at 750 g for 5 min. The pellet was
resuspended in 200 µl of buffer A and incubated at 4°C
for 10 min. Nonidet P-40 was added to a final concentration of 0.5%,
and the suspension was centrifuged at 14,000 g for 15 min.
The resultant nuclear pellet was suspended in 15 µl of 20 mM HEPES
(pH 7.9), 1.5 mM MgCl2, 0.5 mM DTT, 0.42 M NaCl, 0.2 mM
EDTA, 25% glycerol, and 0.5 mM PMSF, incubated for 15 min at 4°C,
and centrifuged at 14,000 g for 10 min. The supernatant (15 µl) was diluted with 35 µl of 20 mM HEPES (pH 7.9), 50 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF and stored at
80°C.
Transfection of fibroblasts with gene promoter/reporter constructs and IL-1ra cDNA. Some cultures were transiently transfected with an 1,800-bp fragment of the PGHS-2 promoter, kindly supplied by Dr. Stephen Prescott (University of Utah) fused to a luciferase reporter gene. Others were transfected with a fragment of the PGHS-2 3'-UTR spanning nt +1885 to +2395 and fused to a CAT reporter. Control cultures were transfected with an unrelated yeast gene sequence fused to the same reporter. Fibroblasts 60%-80% confluent in 60-mm-diameter plates were transfected with 2 µg of plasmid DNA with LipofectAMINE PLUS reagent and allowed to incubate for 5 h. Medium containing 10% FBS was then added. CAT assays were performed on cultures after 48-h incubations with or without the test agents indicated by using a kit purchased from Promega.
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RESULTS |
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IL-1 enhances PGE2 synthesis in human orbital
fibroblasts, which is associated with a dramatic induction of PGHS-2.
Confluent orbital and dermal fibroblasts synthesize low levels of
PGE2 (31). Treatment of orbital fibroblasts
with IL-1 (10 ng/ml) increases prostanoid production. Levels are
enhanced 50- to 70-fold. In contrast, PGE2 synthesis is
upregulated more modestly (10-fold) in dermal fibroblasts treated under
identical culture conditions (Fig.
1A). We examined seven
different strains of fibroblasts, each from a different donor. These
included individuals with Graves' disease and those without any
evidence of autoimmune disease. Four of the strains were from the
orbit, whereas three derived from the skin. From these studies, it
appears that orbital fibroblasts, regardless of whether they are from
patients with Graves' disease or from normal connective tissue,
exhibit substantially greater increases in PGE2 synthesis
when activated by IL-1 than do their dermal counterparts.
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PGHS-2 protein and steady-state mRNA levels are increased by
IL-1 in fibroblasts.
The effect of IL-1 on PGHS-2 expression in orbital fibroblasts was
determined by incubating cultures in the absence or presence of IL-1
(10 ng/ml) for the times indicated in Fig.
2. Cellular proteins and RNA were
harvested and subjected to Western and Northern blot analysis,
respectively, as indicated in Fig. 2. PGHS-2 protein was undetectable
at time 0 and peaked at 12 h, when levels were at least
10-fold above controls. In contrast, PGHS-1 protein levels were
uninfluenced by IL-1
(data not shown). A concentration curve was
generated using graded doses of IL-1
(Fig.
3). The data from that study demonstrate
that the induction of PGHS-2 protein is near maximal at an
IL-1
concentration of ~1 ng/ml in orbital fibroblasts. PGHS-2
induction was far less substantial in dermal cultures than that found
in orbital fibroblasts (Fig. 2). The protein was readily detectable
only at the 16-h time point.
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IL-1 activates NF-
B in orbital fibroblasts.
The effects of IL-1
on PGE2 production and PGHS-2
expression in orbital fibroblast cultures could be attenuated by
treatment with PDTC, a relatively specific inhibitor of NF-
B
(30). The human PGHS-2 promoter contains two NF-
B
binding sites at
214 to
204 and
447 to
437, and it has been
shown previously that IL-1 can activate NF-
B in other cell types.
Therefore, the impact of IL-1
on NF-
B binding activity in orbital
fibroblasts was determined. Cultures were treated with nothing or
IL-1
(10 ng/ml), and nuclear proteins were harvested after 2 h.
There was negligible NF-
B binding activity in the control nuclear
extract (Fig. 4A), but a
substantial increase occurred after addition of the cytokine. Binding
could be quenched with excess unlabeled NF-
B but not by the SP1
consensus sequence oligonucleotide, indicating that the binding of the
nuclear proteins is specific to NF-
B (Fig. 4B).
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IL-1 increases steady-state PGHS-2 mRNA levels primarily through
stabilization of the transcript.
IL-1
influences target gene transcription and is also known to
influence the stability of mature transcripts encoding
inflammation-related proteins. Its impact on levels of PGHS-2
expression in orbital fibroblasts is considerable, and thus the effect
of IL-1
on PGHS-2 promoter activity was assessed. As Fig.
6 indicates, the cytokine fractionally
increased the activity of an 1,800-bp fragment of the human PGHS-2
promoter fused to a luciferase reporter and transiently transfected
into orbital and dermal fibroblasts. The magnitude of increase in
promoter activity was modest in both cultures (2- to 3-fold),
suggesting that the levels of PGHS-2 gene transcription may not differ
in IL-1
-treated orbital and dermal fibroblasts.
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Exogenous IL-1 upregulates IL-1ra expression in orbital and dermal
fibroblasts.
We have shown that treatment of orbital fibroblasts with
exogenous IL-1 results in an upregulation of PGHS-2 expression that is
far greater than that in dermal cultures. We next determined whether
IL-1 is inducing the expression of other members of its cytokine family
in fibroblasts, as has been shown in other cell types (9).
Treatment of orbital fibroblasts with either IL-1 (10 ng/ml) or
IL-1
(10 ng/ml) resulted in substantial induction of IL-1
(30- to
60-fold above control) (Fig.
8A). The peak levels for
IL-1
expression were 408 ± 45 and 211 ± 35 pg/10 µg
protein when cells were treated with IL-1
and IL-1
for 12 h,
respectively. IL-1
was also dramatically induced in these cells. The
levels were undetectable under control conditions but increased to
97.5 ± 8 and 116 ± 3.5 pg/10 µg protein after 12 h
of IL-1
and IL-1
treatment, respectively.
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Overexpressing IL-1ra in orbital fibroblasts or interrupting its
expression in dermal fibroblasts alters PGHS-2 induction by IL-1.
To determine whether differential induction by IL-1 of PGHS-2
expression in orbital and dermal fibroblasts was related to IL-1ra
expression, levels of the cytokine antagonist were altered and the
impact on enzyme induction assessed. As the data in Fig. 9A indicate, treating dermal
fibroblasts with IL-1
in addition to neutralizing anti-IL-1ra
antibodies could enhance the induction of PGHS-2. Interrupting IL-1ra
expression in dermal cultures using an IL-1ra anti-sense
oligonucleotide could produce an equivalent upregulation in PGHS-2
induction with IL-1
(Fig. 9B). Orbital fibroblasts were
then transiently transfected with IL-1ra. As the Western blot in Fig.
9C, left, indicates, levels of IL-1ra protein
were increased dramatically after the transfection. When these cells
were then challenged with IL-1
(10 ng/ml), the PGHS-2 induction was
substantially attenuated (Fig. 9C, right) and the levels were similar to those in dermal fibroblasts. Thus it
would appear that the relative level of IL-1ra protein expressed by fibroblasts after cytokine activation represents an important determinant of PGHS-2 inducibility in human fibroblasts.
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DISCUSSION |
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Exogenous IL-1 can dramatically enhance PGHS-2 expression and
PGE2 production in orbital fibroblasts. Similar actions of IL-1 on prostanoid biosynthesis have been reported in a wide array of
cell types, including primary and established endothelial cells, synovial cells, and astrocytes (28). The induction of
PGHS-2 by IL-1 is considerably less robust in dermal fibroblasts. We have reported previously that leukoregulin can also substantially upregulate PGHS-2 expression in orbital fibroblasts (31).
Those effects were also anatomic site selective.
A potentially important insight into the basis for differential PGHS-2 inducibility in orbital and dermal fibroblasts relates to the differences in levels of cytokine-inducible IL-1ra protein observed in the two cell types. A major determinant of fibroblast phenotype underlying its potential to participate in inflammation may relate to the usage of the IL-1 family of cytokines and the magnitude of the IL-1ra response to a particular proinflammatory signal.
With regard to PGHS-2, IL-1 has been shown to exert effects on
gene transcription and on mRNA stability in endothelial cells in
culture (19). Consistent with those findings, we show that induction of PGHS-2 by IL-1 in orbital fibroblasts is a consequence of both a modest increase in gene transcription and enhanced PGHS-2 mRNA stability. Morrison and colleagues (5, 29)
demonstrated the critical importance of the 3'-UTR in PGHS-2 mRNA to
cytokine regulation of cyclooxygenase expression.
Our finding that PGHS-2 expression in orbital fibroblasts can be
dramatically upregulated by IL-1 is of considerable clinical relevance.
The cytokine milieu associated with TAO remains poorly defined. IL-1
as well as TNF-
and interferon-
have been detected with
immunohistological techniques in the orbital tissues from patients with
TAO (12). Though that study failed to examine IL-1
expression or to establish convincingly the identity of the particular
cell type expressing these cytokines, it does suggest that IL-1 may
play a role in TAO.
The dramatic increases in PGE2 production found in cytokine-activated orbital fibroblasts suggest that orbital connective tissue might generate high levels of the prostanoid. PGE2 exerts important influences on T and B cells (2, 10, 20). For instance, the development of naive T cells is biased from TH0 to TH2 cells at the expense of the TH1 phenotype. Moreover, PGE2 influences B cell development. Mast cells also react to PGE2, and the molecule plays a role in their behavior. Thus high levels of prostanoid generation in the orbit might condition the immune responses occurring there (17).
Our current findings advance the understanding of prostanoid generation in fibroblasts and suggest that the expression of IL-1 and IL-1ra in human cultures may provide an important phenotypic signature for potential involvement in inflammation. On the basis of our results to date, it would appear that orbital fibroblasts represent a cell population particularly poised to engage in inflammatory activities. Substantial debate remains concerning the nature of PGE2 effects on inflammation and immune function (17). Clearly, the realization that orbital fibroblasts produce unusually high levels of this prostanoid when treated with proinflammatory cytokines suggests that the connective tissues from which they derive might be exposed to PGE2 as a consequence of disease.
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ACKNOWLEDGEMENTS |
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We thank Heather Meekins for expert technical support.
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FOOTNOTES |
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This work was supported in part by National Eye Institute Grants EY-08976 and EY-11708 (to T. J. Smith) and by a Merit Review Award from the Department of Veterans Affairs Medical Research Service (to T. J. Smith).
Address for reprint requests and other correspondence: T. J. Smith, Division of Molecular Medicine, Bldg. C-2, Harbor-UCLA Medical Center, 1124 W. Carson St., Torrance, CA 90504 (E-mail: tjsmith{at}ucla.edu).
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.
First published January 8, 2003;10.1152/ajpcell.00354.2002
Received 1 August 2002; accepted in final form 7 January 2003.
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