Robust induction of PGHS-2 by IL-1 in orbital fibroblasts results from low levels of IL-1 receptor antagonist expression

H. James Cao1, Rui Han1,2,3, and Terry J. Smith1,2,3,4

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-1beta . 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-1beta , 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-1alpha , IL-1beta , 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-1alpha and IL-1beta . 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-1alpha and IL-1beta 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Human recombinant IL-1alpha and IL-1beta 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-1alpha and IL-1beta 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-1alpha and IL-1beta 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-kappa 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-1beta (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.

Nuclear extracts (2 µg) were incubated with 2 µl of 20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris · HCl (pH 7.5), and 0.25 mg/ml poly(dI-dC) · poly (dI-dC) for 10 min at room temperature, followed by the addition of 0.04 pmol of 32P-labeled double-strand NF-kappa B oligonucleotide with or without a 50-fold excess of cold NF-kappa B or SP1 competitor. After incubation at room temperature for 20 min, the reaction was stopped by the addition of 1 µl of 250 mM Tris · HCl (pH 7.5), 0.2% bromphenol blue, 0.2% xylene cylanol, and 20% glycerol and then electrophoresed in 8% Tris-borate-EDTA (TBE) polyacrylamide gels. Gels were dried and exposed to X-OMAT AR film overnight. When the supershift assay was performed, either rabbit anti-human NF-kappa B p50 or NF-kappa B p65 polyclonal antibody (Santa Cruz, Santa Cruz, CA) was added to a final concentration of 1 µg/ml before the addition of dye. The antibodies were incubated with the nuclear protein-oligonucleotide complex for an additional 20 min, after which the reaction was stopped and the complexes were electrophoresed.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   A: effects of IL-1alpha and IL-1beta on levels of PGE2 production in human orbital and dermal fibroblasts (B). These effects can be attenuated by dexamethasone (Dex) and pyrrolidinedithiocarbamate (PDTC), an inhibitor of NF-kappa B activity. Confluent fibroblasts in 24-well plates were treated without (control) or with IL-1alpha (10 ng/ml) or IL-1beta (10 ng/ml), in the absence or presence of Dex (10 nM) or PDTC (100 µM), for 16 h. Medium was replaced with PBS for the final 30 min of incubation. The PBS (100 µl) was subjected to PGE2 analysis. Values are means ± SE of triplicate determinations from representative experiments.

Glucocorticoids can block the generation of PGE2 provoked by cytokines in a number of cell types. A hallmark of prostanoid production in inflammation is its susceptibility to glucocorticoid inhibition. We therefore tested dexamethasone in these cultures (Fig. 1B). The glucocorticoid (10 nM) blocked the upregulation of PGE2 by IL-1beta . SC-58125 (5 µM), a selective inhibitor of PGHS-2, also attenuated the response to IL-1beta (data not shown), suggesting that induction of PGE2 synthesis derives from the induction of PGHS-2. PDTC, an inhibitor of NF-kappa B, also attenuated the cytokine-provoked PGE2 production (Fig. 1B).

PGHS-2 protein and steady-state mRNA levels are increased by IL-1beta 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-1beta (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-1beta (data not shown). A concentration curve was generated using graded doses of IL-1beta (Fig. 3). The data from that study demonstrate that the induction of PGHS-2 protein is near maximal at an IL-1beta 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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Fig. 2.   IL-1beta upregulates the expression of prostaglandin-endoperoxide H synthase-2 (PGHS-2) mRNA (A) and protein (B) in orbital and dermal fibroblasts. Confluent cultures of orbital fibroblasts from a patient with severe thyroid-associated ophthalmopathy (TAO) and abdominal wall dermal fibroblasts were treated with IL-1beta (10 ng/ml) for the times indicated. Monolayers were processed as described in METHODS. Cellular RNA was electrophoresed and subjected to Northern blot hybridization with a probe generated from PGHS-2 cDNA and rehybridized with a GAPDH probe. For Western blot analysis, 15 µg of cellular protein were subjected to SDS-PAGE. Separated proteins were transferred to polyvinylidene difluoride membrane and immunoblotted with an anti-human PGHS-2 antibody (10 µg/ml).



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Fig. 3.   Induction by IL-1beta of PGHS-2 protein in orbital fibroblasts is concentration dependent. Confluent cultures were treated with the graded concentrations of IL-1beta indicated. Cell lysates were then subjected to Western blot analysis for PGHS-2 protein. Data are shown as relative density of protein expressed.

The impact of IL-1beta on steady-state PGHS-2 mRNA levels was compared in orbital and dermal fibroblasts. The transcript was undetectable under control conditions, regardless of the tissue of origin (Fig. 2). Addition of IL-1beta for 8 h resulted in substantial upregulation in the steady-state levels of a 4.8-kb transcript in orbital cultures. Peak levels were found after 12 h, and the transcript abundance had declined at 24 h. Substantially lower levels of the mRNA were achieved in dermal cultures at all times tested. Dexamethasone (10 nM) attenuated the induction of PGHS-2 mRNA levels by IL-1beta (data not shown). Neither IL-1beta nor dexamethasone influenced PGHS-1 mRNA levels. We have reported recently (31) that human orbital fibroblasts predominantly express a 5.2-kb PGHS-1 transcript, similar to that expressed in human endothelial cells (13) and different from the 2.8-kb transcript found in other cells (11).

IL-1beta activates NF-kappa B in orbital fibroblasts. The effects of IL-1beta on PGE2 production and PGHS-2 expression in orbital fibroblast cultures could be attenuated by treatment with PDTC, a relatively specific inhibitor of NF-kappa B (30). The human PGHS-2 promoter contains two NF-kappa B binding sites at -214 to -204 and -447 to -437, and it has been shown previously that IL-1 can activate NF-kappa B in other cell types. Therefore, the impact of IL-1beta on NF-kappa B binding activity in orbital fibroblasts was determined. Cultures were treated with nothing or IL-1beta (10 ng/ml), and nuclear proteins were harvested after 2 h. There was negligible NF-kappa 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-kappa B but not by the SP1 consensus sequence oligonucleotide, indicating that the binding of the nuclear proteins is specific to NF-kappa B (Fig. 4B).


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Fig. 4.   NF-kappa B binding activity is increased by IL-1beta in orbital fibroblasts. A: confluent orbital fibroblasts were treated with nothing (control) or IL-1beta (10 ng/ml) for 2 h. Nuclear proteins were harvested and subjected to EMSA as described in METHODS. B: EMSA competition assay showing binding buffer (without nuclear extract) and nuclear extract in the absence or presence of unlabeled NF-kappa B or SP1 oligonucleotides, as indicated. C: supershift assay. EMSA was performed without (left lane) antibodies directed against NF-kappa B or with antibodies against p50 (middle lane) or p65 (right lane).

The components of the NF-kappa B dimer activated by IL-1beta were identified by a supershift assay (Fig. 4C). Antibodies directed against either p50 or p65 shifted the NF-kappa B oligonucleotide complex, indicating that the complex consisted of p50/p65 heterodimer. Thus the activity profile of NF-kappa B dimeric partners associated with substantial transcriptional activity is enhanced by IL-1beta in orbital fibroblasts. When orbital and dermal fibroblasts were treated with IL-1beta under identical conditions, a similar NF-kappa B activation pattern was demonstrated in both cell types (Fig. 5). Thus the more robust PGHS-2 induction found in orbital fibroblasts does not appear to result from differences in NF-kappa B activity.


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Fig. 5.   NF-kappa B binding activity in IL-1beta -treated orbital and dermal fibroblasts. Cultures were treated with IL-1beta (10 ng/ml) for 2 h. Nuclear extracts were then processed as described in METHODS.

IL-1beta increases steady-state PGHS-2 mRNA levels primarily through stabilization of the transcript. IL-1beta 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-1beta 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-1beta -treated orbital and dermal fibroblasts.


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Fig. 6.   Effect of IL-1beta on PGHS-2 gene promoter activity in orbital (A) and dermal (B) fibroblasts. Fibroblasts were transiently transfected with the +1840 to +123 bp fragment of the human PGHS-2 promoter fused to a luciferase (Luc) reporter gene. Cells were treated for 3 h with IL-1beta (10 ng/ml). Luciferase activity was determined as described in METHODS. Data are expressed as means ± SE of triplicate cultures.

IL-1beta increases PGHS-2 expression through rapid and transient elevations in steady-state mRNA levels. Because enhancement of PGHS-2 promoter activity does not fully account for this increase in orbital fibroblasts, effects of IL-1beta on mRNA stability were examined. Addition of the cytokine (10 ng/ml) to the culture medium increased PGHS-2 mRNA half-life from less than 1 h to well over 5 h in orbital cultures (Fig. 7A). To determine whether IL-1beta was influencing some element of the PGHS-2 3'-UTR, a fragment of that sequence was cloned, fused to a reporter gene, and transfected transiently into orbital fibroblasts. IL-1beta substantially increased the activity of the CAT reporter (Fig. 7B). The magnitude of the effect was eight-fold after a 3-h exposure to the cytokine. Activity of the same reporter fused to an SV40 promoter failed to change with IL-1beta treatment. Thus the dominant action of IL-1beta on PGHS-2 expression in orbital fibroblasts appears to be mediated through effects on the PGHS-2 3'-UTR.


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Fig. 7.   Effect of IL-1beta on PGHS-2 mRNA stability and 3'-untranslated region (UTR) of PGHS-2. A: orbital fibroblast cultures were pretreated with cycloheximide (10 µg/ml) for 3 h, and then all were shifted to fresh medium containing 5,6-dichlorobenzimidazole (50 µM) without (control) or with IL-1beta , and monolayers were harvested at the times indicated. Cellular RNA was subjected to Northern blot hybridization with a 32P-labeled PGHS-2 cDNA probe and subsequently with GAPDH, the later signal used to normalize the RNA transfer. B: orbital fibroblasts were transiently transfected with SV40 CAT, an unrelated yeast gene control sequence, or a fragment (+1885/+2395) of the PGHS-2 3'-UTR fused to a CAT reporter gene. Cultures were treated with nothing or IL-1beta for 3 h. CAT activity was determined as described in METHODS. Data are expressed as means ± SE of triplicate cultures.

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-1alpha (10 ng/ml) or IL-1beta (10 ng/ml) resulted in substantial induction of IL-1alpha (30- to 60-fold above control) (Fig. 8A). The peak levels for IL-1alpha expression were 408 ± 45 and 211 ± 35 pg/10 µg protein when cells were treated with IL-1alpha and IL-1beta for 12 h, respectively. IL-1beta 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-1alpha and IL-1beta treatment, respectively.


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Fig. 8.   Effect of IL-1alpha and IL-1beta on IL-1alpha expression (A) and the effect of IL-1beta on IL-1 receptor antagonist (IL-1ra) protein levels (B) in fibroblasts. Confluent cultures in 24-well plastic plates were incubated without (control) or with IL-1alpha or IL-1beta (10 ng/ml) for 12 h in A or for the times indicated in B. The cellular proteins were harvested, and 10 µg of protein were subjected to the respective ELISA assays. Results are expressed as means ± SE of triplicate determinations.

Balance between the actions of IL-1 and IL-1ra can provide modulation of cytokine action. We therefore determined whether differences in either basal or cytokine-inducible levels of IL-1ra could explain the disparity observed with regard to the magnitude of induction by IL-1 of PGHS-2 in orbital and dermal fibroblasts. Both fibroblasts express low basal levels of IL-1ra (16 ± 4 vs. 37.5 ± 6 pg/10 µg protein) (Fig. 8B). When treated with IL-1beta (10 ng/ml), the levels of IL-1ra achieved in dermal fibroblasts were considerably greater. In a time-course study, IL-1ra expression began to increase in dermal but not orbital cultures after 8 h (Fig. 8B). At 24 and 48 h, IL-1ra levels associated with the dermal cell layer were 581 ± 90 and 1,514 ± 250 pg/10 µg protein vs. 40 ± 11 and 55 ± 0.4 pg/10 µg protein in orbital cultures, respectively. Thus levels in dermal culture were 30-fold greater than those in orbital fibroblasts.

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-1beta 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-1beta 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-1beta (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-1beta (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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Fig. 9.   Effect of interrupting or augmenting IL-1ra activity in orbital and dermal fibroblasts. A: orbital and dermal fibroblast monolayers were treated without (control) or with IL-1beta without or with neutralizing anti-IL-1ra antibodies (Ab) for 16 h. Cultures were solubilized and subjected to Western blot analysis for PGHS-2 protein expression. B: orbital and dermal fibroblasts were treated with an antisense oligonucleotide for 5 h. Fibroblasts were then treated without (control) or with IL-1beta for 16 h, and PGHS-2 protein expression was determined by Western blot analysis. C: orbital fibroblasts were transiently transfected with an empty vector or with one containing IL-1ra cDNA. IL-1ra protein was assayed (left) to confirm expression, and then Western blot analysis of PGHS-2 protein was performed on the transfected cells after a 16-h treatment without (control) or with IL-1beta . Data are shown as relative density of protein expressed.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Exogenous IL-1beta 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-1beta 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-1alpha as well as TNF-alpha and interferon-gamma have been detected with immunohistological techniques in the orbital tissues from patients with TAO (12). Though that study failed to examine IL-1beta 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.


    ACKNOWLEDGEMENTS

We thank Heather Meekins for expert technical support.


    FOOTNOTES

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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ABSTRACT
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METHODS
RESULTS
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