EDITORIAL FOCUS
Leukoregulin upregulation of prostaglandin endoperoxide H synthase-2 expression in human orbital fibroblasts

H. James Cao and Terry J. Smith

Division of Molecular and Cellular Medicine, Departments of Medicine and Biochemistry and Molecular Biology, Albany Medical College and Samuel S. Stratton Veterans Affairs Medical Center, Albany, New York 12208


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Human orbital fibroblasts from patients with severe thyroid-associated ophthalmopathy are particularly susceptible to the actions of a variety of proinflammatory molecules. In this study, we demonstrate that the inductions of prostaglandin endoperoxide H synthase-2 (PGHS-2), interleukin (IL)-1alpha , and IL-1beta by leukoregulin, a product of activated T lymphocytes, are far more robust in orbital fibroblasts than those observed in dermal fibroblasts. These actions of leukoregulin are mediated through an intermediate induction of IL-1alpha . In contrast, leukoregulin also induces IL-1-receptor antagonist (IL-1ra) expression in orbital fibroblasts, but this induction is considerably greater in dermal fibroblasts (2.3- vs. 8.5-fold). Interrupting the effects of IL-1alpha , either with a neutralizing antibody or with exogenous IL-1ra, can block the induction of PGHS-2 by leukoregulin. Leukoregulin increases PGHS-2 gene transcription in orbital fibroblasts but exerts the major effect on cyclooxygenase expression by enhancing the stability of mature PGHS-2 mRNA. The cytokine triggers nuclear translocation of nuclear factor-kappa B (NF-kappa B) p50/p50 homodimers and p50/p65 heterodimers, and an inhibitor of this transcriptional factor, pyrrolidinedithiocarbamate, can attenuate the PGHS-2 induction. Thus differential inducibility of the members of the IL-1 family of genes in orbital fibroblasts would appear to underlie, at least in part, the differences in PGHS-2 induction observed in orbital and dermal fibroblasts. NF-kappa B plays an important role in mediating the effects of leukoregulin on PGHS-2 expression.

cyclooxygenase; inflammation; ophthalmopathy; Graves' disease


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HUMAN FIBROBLASTS represent a diverse population of cells that vary with regard to the anatomic region from which they are derived (47). Orbital fibroblasts, especially those derived from patients with severe thyroid-associated ophthalmopathy (TAO), exhibit exaggerated responses to several proinflammatory cytokines (29). We hypothesize that this particular susceptibility to cytokine action may underlie the often intense inflammatory response and the dramatic tissue remodeling observed in TAO (32). In that disease, there occurs a disordered accumulation of the nonsulfated glycosaminoglycan hyaluronan (32). We have reported that leukoregulin, a 50-kDa product of activated T lymphocytes (9), and interferon-gamma can dramatically upregulate the synthesis of hyaluronan in orbital fibroblast cultures (34, 39). The magnitude of increase with leukoregulin, up to 15-fold above the levels observed in untreated cultures, is unprecedented and is severalfold greater than that observed in dermal fibroblasts (39). Other cellular responses also appear to be more robust in orbital fibroblasts than in those from extra-orbital anatomic regions. Leukoregulin (16), transforming growth factor-beta (4), and interferon-gamma (31, 35) can induce the expression of the serine protease inhibitor, plasminogen activator inhibitor type 1 (PAI-1), in orbital fibroblasts, and these effects are considerably greater than those in dermal fibroblasts. Because PAI-1 modulates the pericellular proteolytic environment, the differential inducibility of the protein suggests that the extracellular matrix economy of orbital fibroblasts differs from that of its dermal counterparts.

Another response that is far more robust in orbital fibroblasts is the induction of the inflammatory cyclooxygenase prostaglandin endoperoxide H synthase-2 (PGHS-2; EC 1.14.99.1) by leukoregulin (21, 42). PGHS are a family of two bifunctional proteins that catalyze the conversion of arachidonate to PGH2 through a pair of discrete biochemical reactions involving different active sites on the enzymes (30, 41). PGHS-1 is a constitutive protein that appears to be responsible for the basal prostaglandin formation in most tissues. It is only modestly upregulated by proinflammatory molecules such as phorbol 12-myristate 13-acetate (PMA; see Ref. 45). PGHS-1 migrates as a 68-kDa protein and is encoded by a 5-kb mRNA in some tissues (10, 15) and a 2.8-kb transcript in others (12). In contrast, PGHS-2 is expressed at very low levels in most normal tissues but can be induced substantially by cytokines, growth factors, and serum (1, 18, 20, 22, 44). PGHS-2 mRNA, a 4.8-kb species, and PGHS-2 protein, usually a doublet at 72 kDa (20, 47), are undetectable in most untreated cells, but, when they are exposed to activational molecules, the expression of this cyclooxygenase is dramatically enhanced. The upregulated expression of PGHS-2 is often accompanied by increases in the production of PGE2.

With regard to cultured orbital fibroblasts, PGHS-2 is dramatically induced by leukoregulin, and the effects are mediated at the pretranslational level (42). Moreover, prostanoid generation after treatment with leukoregulin can be attenuated by the potent synthetic glucocorticoid dexamethasone and by SC-58125, a PGHS-2 selective inhibitor (42). The induction of PGHS-2 results in a twofold increase in gene transcription, as assessed by nuclear "run-on" assays, but the increase in steady-state mRNA levels is ~50-fold. Thus the cytokine is acting primarily on some other level(s) to upregulate the cyclooxygenase transcript. PGE2 production in the leukoregulin-activated orbital fibroblast, in turn, influences the morphology of the cells expressing high levels of PGHS-2 (21, 42). There is a change from typical fibroblast-like morphologies to a stellate appearance with multiple cellular processes. This same arborized appearance can be provoked by treating the orbital fibroblasts with exogenous PGE2 and with prostanoids that specifically bind to the EP2 subclass receptors (40, 43). Prostaglandins appear to exert diverse influences on immune responses and therefore condition the interplay between the immune system and other tissues. Thus, in the context of TAO, the orbital fibroblast could play an important immunomodulatory role by virtue of the prostanoids and other small molecules it elaborates and releases.

In the current paper, we report the results of studies designed at characterizing the induction by leukoregulin of PGHS-2 expression in orbital fibroblasts. We find that leukoregulin substantially upregulates interleukin (IL)-1alpha and IL-1beta synthesis and elicits a very modest increase in IL-1-receptor antagonist (IL-1ra) levels in these cells. The enhanced synthesis of IL-1alpha represents a critical intermediate step in the induction of PGHS-2. Compared with orbital fibroblasts, those from the dermis exhibit a more modest increase in IL-1alpha and -beta but a markedly more robust IL-1ra induction. We hypothesize that it is the discrepancy in the inducibility of the IL-1 family of proteins that underlies the exaggerated PGHS-2 induction in orbital fibroblasts and leads to intense orbital inflammation in TAO.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. SC-58125 was a kind gift of Dr. Peter Isakson (Searle, Skokie, IL). Dexamethasone (1,4 pregnadien-9-fluoro-16alpha -methyl-11beta ,17alpha ,21-triol-3,20-dione), pyrrolidinedithiocarbamate (PDTC), an inhibitor of nuclear factor-kappa B (NF-kappa B; see Ref. 13), and cycloheximide were from Sigma (St. Louis, MO). Leukoregulin was prepared from peripheral blood leukocytes as described (9) and was kindly supplied by Dr. Charles H. Evans (NCI, Bethesda, MD). Briefly, normal lymphocytes were stimulated with phytohemagglutinin for 48 h. The cytokine was purified by subjecting cellular material through several chromatographic steps, as described in Ref. 9. Leukoregulin has been shown to be free from contamination with other known cytokines such as IL-1alpha , IL-1beta , tumor necrosis factor (TNF)-alpha , and TNF-beta (9). PGHS-1 and PGHS-2 cDNAs were kindly provided by Dr. Donald A. Young (University of Rochester, Rochester, NY), and IL-1beta cDNA was from Dr. J. A. Melendez. Monoclonal anti-PGHS-1 and -2 antibodies were purchased from Cayman (Ann Arbor, MI), IL-1alpha and IL-1beta enzyme-linked immunosorbent assays (ELISA) were from Immunotech (Westbrook, ME), and the IL-1ra assay and IL-1alpha and IL-1beta neutralizing antibodies were from R & D Systems (Minneapolis, MN). Recombinant IL-1ra was a gift from Amgen (Boulder, CO).

Cell culture. Human orbital and dermal fibroblasts were cultivated as described previously (27, 33). Briefly, orbital tissue explants were obtained from surgical waste from individuals with severe TAO who were undergoing decompressive surgery. Dermal fibroblasts were obtained from punch biopsies of normal-appearing skin. These activities were approved by the Institutional Review Board of the Albany Medical College. The explants were allowed to adhere to plastic culture plates, and fibroblasts began to proliferate from these tissue fragments. The fibroblasts were allowed to expand, and monolayers were disrupted with gentle treatment with trypsin/EDTA, replated, and covered with Eagle's medium supplemented with 10% FBS, antibiotics, and glutamine. Cultures were maintained in a 37°C, humidified incubator in a 5% CO2 environment. Medium was changed every 3-4 days, and cultures were used for experiments between the 3rd and 12th passages.

Western blot analysis of proteins. The relative levels of proteins were assessed by immunoblot analysis using monoclonal antibodies directed against IL-1beta , PGHS-1, and PGHS-2, as described previously (42). Confluent cultures of fibroblasts, usually in 60-mm-diameter plastic culture plates, were shifted from growth medium containing 10% FBS to medium supplemented with 1% serum for 1-2 days. They were then treated with the test compounds, added at the times indicated in the text and in the figures. Monolayers were washed and harvested in ice-cold buffer containing 15 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1 mM EDTA, 20 mM Tris · HCl (pH 7.5), 10 µg/ml soybean trypsin inhibitor, and 10 µM phenylmethylsulfonyl fluoride (PMSF). Lysates were taken up in Laemmli buffer and subjected to SDS-PAGE, and the separated proteins were transferred to polyvinylidene difluoride membranes. The primary antibodies (10 µg/ml) were incubated with the membranes for 2 h at room temperature (RT). Membranes were then washed extensively and reincubated with secondary, peroxidase-labeled antibodies for 2 h. After another round of washes, the membranes were subjected to the enhanced chemiluminescence (Amersham) detection system, and the signals generated on X-Omat film were analyzed with a densitometric BioImage scanner (Milligen).

RNA extraction and Northern blot analysis of steady-state mRNA levels. Fibroblasts were cultivated in 100-mm-diameter plastic plates to a state of confluence in medium with 10% FBS, and then they were shifted to medium supplemented with 1% serum for 1-2 days. They were then treated with the test compounds indicated, the monolayers were washed, and the RNA was extracted by the method described by Chomczynski and Sacchi (6) using Ultraspec reagent (Biotecx, Houston, TX). Northern analysis was performed as described previously (42) by electrophoresing RNA on denaturing 1% agarose-formaldehyde gels. The integrity of the RNA was verified by routinely determining the 260 to 280 nm ratio and by staining the electrophoresed samples with ethidium bromide and inspecting them under ultraviolet light. Samples were transferred to a Zeta-probe membrane (Bio-Rad), and the immobilized RNA was allowed to hybridize with [32P]dCTP-labeled cDNA probes. With regard to cyclooxygenase probes, PGHS-1 was generated from a 1.6-kb cDNA, whereas PGHS-2 was synthesized from a 1.4-kb fragment. Hybridizations were allowed to proceed in a solution containing 5× saline sodium citrate, 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 autoradiography on X-Omat film (Eastman Kodak, Rochester, NY) exposed at -70°C, and the radioactive bands were scanned densitometrically. Membranes were stripped of radioactivity according to the manufacturer's instructions and were rehybridized with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe for standardization. The density of the PGHS-2 signals was normalized to that resulting from the hybridization with the GAPDH probe.

Electrophoretic mobility shift assay. Nuclei from untreated orbital and dermal fibroblasts and those treated with leukoregulin (1 U/ml) and the other test compounds indicated were prepared as follows. Two 100-mm-diameter plates of confluent cells were washed and scraped in PBS, and cell layer material was transferred to 1.5-ml microcentrifuge tubes. Cell pellets were suspended in 0.5 ml 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]. Samples were centrifuged at 750 g for 5 min. The supernatant was removed, 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 samples were subjected to centrifugation at 14,000 g for 15 min. The resultant pellet was suspended in 15 µl of buffer B [20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.5 mM DTT, 0.42 M NaCl, 0.2 mM EDTA, 25% (vol/vol) glycerol, and 0.5 mM PMSF]. After a 15-min incubation at 4°C, the suspension was centrifuged at 14,000 g for 10 min, and 15 µl of the supernatant were combined with 35 µl of buffer C (20 mM HEPES, pH 7.9, 50 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF). The nuclear pellets were frozen until assay at -80°C.

A double-stranded oligonucleotide conforming to the consensus NF-kappa B binding site (5'-AGT TGA GGG GAC TTT CCC AGG C-3') and one with a single mutation (5'-AGT TGA GGC GAC TTT CCC AGG C-3') were purchased from Santa Cruz. Complementary strands were synthesized with 5' overhanging ends. They were annealed, and double-stranded probes were end-labeled with [32P]dATP using T4 polynucleotide kinase. Aliquots of nuclear extract (2 µg protein/sample) were preincubated with 2 µl of 5× gel shift binding buffer (Promega, Madison, WI) containing 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) in a total reaction volume of 10 µl for 10 min. The reaction was stopped by adding 1 ml loading buffer (250 mM Tris · HCl, pH 7.5, 0.2% bromphenol blue, 0.2% xylene cyanol, and 20% glycerol), and the samples were loaded on native 8% polyacrylamide gels and run at 100 V for 3 h. Gels were dried and exposed to X-Omat film. Supershift assays were performed by incubating the nuclear extract with anti-p50 and anti-p65 antibodies (1 µg/ml, final concentration) for 20 min at RT.

Assays for the expression of IL-1alpha , IL-1beta , and IL-1ra. Fibroblasts were allowed to proliferate to confluence in 24-well culture plates covered with medium containing 10% FBS. Monolayers were shifted to medium with 1% serum for 1-2 days, and then the test compounds indicated were added to the medium. After the incubation period, media were removed, and the cell layers were washed and 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 PMSF, 3 µg/ml aprotinin, and 0.5% Nonidet P-40. Cellular proteins (5-10 µg/sample) were subjected to specific ELISAs for IL-1alpha , IL-1beta , and IL-1ra according to the manufacturer's instructions.

PGE2 assay. Fibroblasts were cultivated to confluence in 24-well plastic culture arrays in medium with 10% FBS and then were shifted to medium containing 1% serum for 16- 24 h. The test compounds indicated were added to the medium for the times specified. The final 30 min of the treatment periods were conducted by removing the medium and adding PBS with the respective additives. After this incubation, the PBS samples were collected, centrifuged, and subjected to a specific RIA for PGE2 (Amersham) following the manufacturer's instructions.

Statistics. Data are generally presented as means ± SE of at least three independent replicates. Where appropriate, data were subjected to Student's t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Leukoregulin treatment results in a dramatic upregulation of PGHS-2 expression in orbital fibroblasts. Orbital fibroblasts appear to play a central role in the pathogenesis of TAO by virtue of the array of biosynthetic activities they exhibit. With regard to cyclooxygenase expression, we have reported that PGHS-2 expression in these cells under control culture conditions is at a very low level (42). In contrast, PGHS-1 protein and mRNA are expressed at easily detectable levels, and a substantial fraction of basal PGE2 production can be attributed to the activity of PGHS-1 (42). Treatment of orbital fibroblasts with leukoregulin (1 U/ml) for 16 h results in a substantial enhancement of PGE2 production (Fig. 1A). The magnitude of the increase is 88-fold above control cultures (P < 0.0001). This increase is considerably greater than that observed in dermal fibroblasts (6-fold, P < 0.0002 vs. control), and the levels of PGE2 were considerably greater than those attained in the dermal fibroblasts (P < 0.001). The two types of fibroblasts were treated and cultured under the same conditions. When these fibroblasts are treated with leukoregulin and the cellular proteins were subjected to Western blot analysis for cyclooxygenase protein levels, the cytokine elicits a dramatic upregulation in PGHS-2 protein as the blot in Fig. 1B demonstrates. The magnitude of the induction is severalfold greater than that observed in dermal fibroblasts cultured under identical conditions. PGHS-2 induction by leukoregulin results from a massive increase in the levels of steady-state PGHS-2 mRNA levels (42). Figure 1 also demonstrates the remarkably constant expression of PGHS-1 protein in both orbital and dermal fibroblasts.


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Fig. 1.   Leukoregulin induces PGE2 synthesis and prostaglandin endoperoxide H synthase (PGHS)-2 expression in cultured human fibroblasts. A: orbital and dermal fibroblasts, each from a separate donor, were seeded in 24-well plates and allowed to proliferate to confluence in medium with 10% FBS. Monolayers were then shifted to medium supplemented with 1% serum without or with leukoregulin (1 U/ml) for 16 h. The final 30 min of incubation were in PBS, which was then subjected to a specific assay. Each column represents the mean ± SE of 3 replicate culture plates. B: orbital and dermal fibroblasts were seeded in 60-mm-diameter plastic dishes and grown to confluence in medium with 10% FBS and then were shifted to 1% serum-enriched medium for 16 h. Leukoregulin or nothing was added for 16 h, and the cell layers were washed and harvested as described in MATERIALS AND METHODS. Lysates were subjected to Western blot analysis using monoclonal antibodies against PGHS-1 and PGHS-2.

Leukoregulin upregulates IL-1alpha and IL-1beta in orbital fibroblasts, but the increase in IL-1ra is modest. Because we and others have demonstrated that the IL-1 family of polypeptides is expressed in many cell types, including orbital fibroblasts (5), we determined whether leukoregulin could influence the synthesis of these cytokines. As the data contained in Fig. 2 suggest, leukoregulin increases, in a time-dependent manner, the production of IL-1alpha (A), IL-1beta (B), and IL-1ra (C). The increases of IL-1alpha and IL-1beta appear to peak after 12 h of exposure to leukoregulin and begin to fall thereafter. In contrast, IL-1ra levels continue to rise so that by 48 h, the duration of the study, the cytokine-receptor antagonist is at its highest (11-fold above control values). We next compared the effects of leukoregulin on the expression of IL-1-related polypeptides in orbital and dermal fibroblasts. Figure 3 contains results of side-by-side experiments where the two cell populations were compared. As those data demonstrate, the effects of leukoregulin on IL-1alpha and IL-1beta production were considerably greater in orbital than in dermal fibroblasts (Fig. 3, A and B). The levels of IL-1alpha achieved after leukoregulin treatment for 16 h were 369 ± 36 pg/10 µg protein in orbital and 154 ± 24 pg/10 µg protein in dermal fibroblasts (P < 0.002), whereas the levels of IL-1beta achieved were 279 ± 46 and 70 ± 12 pg/10 µg protein, respectively (P < 0.002). Of great interest was the substantially higher level of IL-1ra achieved in the dermal fibroblasts treated with leukoregulin for 12 h where the levels were 181 ± 50 pg/10 µg protein in orbital cultures vs. 1,006 ± 84 pg/10 µg protein in dermal cells (P < 0.0002). The induction of IL-1beta protein was concentration dependent in the range of 0-3 U/ml (Fig. 4A) and occurred at a pretranslational level. Steady-state IL-1beta mRNA levels, migrating as a 1.7-kb transcript on Northern blot analysis, were upregulated within 4 h of the initiation of leukoregulin treatment (Fig. 4B) and were maximal at 8 h. At 12 h, the levels of IL-1beta mRNA had fallen. Leukoregulin also appears to upregulate IL-1alpha at a pretranslational level, based on increases in transcript determined by RT-PCR (data not shown).


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Fig. 2.   Leukoregulin induces the expression of interleukin (IL)-1alpha (A), IL-1beta (B), and IL-1-receptor antagonist (IL-1ra; C) in orbital fibroblasts in a time-dependent manner. Orbital fibroblasts from a patient with thyroid-associated ophthalmopathy (TAO) were cultivated to confluence in 24-well plates, shifted to medium with reduced serum as described in the legend to Fig. 1, and incubated without or with leukoregulin (1 U/ml) for the time intervals indicated along the abscissas. The monolayers were solubilized and subjected to specific assays for IL-1alpha (A), IL-1beta (B), or IL-1ra (C). Data are expressed as means ± SE of triplicate replicates from representative studies.



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Fig. 3.   Comparisons between effects of leukoregulin on IL-1alpha (A), IL-1beta (B), and IL-1ra (C) expression in orbital and dermal fibroblasts. Cultures were treated as specified in the legend to Fig. 2, except that the leukoregulin treatment lasted 16 h in samples assayed for IL-1alpha (A) and IL-1beta (B) and 12 h in those assayed for IL-1ra (C). Cell layers were processed and subjected to specific ELISA assays as described in MATERIALS AND METHODS. Each column represents the mean ± SE of 3 independent determinations.



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Fig. 4.   Induction of IL-1beta by leukoregulin is dose dependent and mediated at a pretranslational level. A: orbital fibroblast monolayers, derived from a patient with severe TAO, were incubated in medium containing 1% FBS for 16 h and then were treated with graded concentrations of leukoregulin (0-3 U/ml) for an additional 16 h. They were harvested, and the cell lysates were subjected to Western blot analysis for IL-1beta protein. B: Northern analysis of IL-1beta mRNA levels in orbital fibroblasts treated for increasing intervals with leukoregulin (1 U/ml). Monolayers were harvested after treatment with the cytokine, and RNA was extracted as described in MATERIALS AND METHODS. Northern analysis was performed with a human IL-1beta cDNA probe. After autoradiography, blot was stripped and rehybridized with a probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Bars represent the IL-1beta signals, as assessed by densitometry, normalized for their respective GAPDH signals.

IL-1 family members can induce each other and themselves (8). We therefore determined whether neutralizing either IL-1alpha or IL-1beta could influence the effects of leukoregulin on IL-1 protein expression. Addition of neutralizing anti-IL-1alpha antibodies (1 µg/ml) could block the upregulation of both IL-1alpha and IL-1beta protein (Fig. 5, A and B). In contrast, IL-1beta neutralizing antibodies failed to influence the synthesis of either IL-1 family member. Addition of exogenous IL-1ra (500 ng/ml) to leukoregulin-activated fibroblasts also blocked both IL-1alpha and IL-1beta production. These results suggest that IL-1alpha but not IL-1beta serves as a critical intermediate protein in the induction of leukoregulin-dependent IL-1 expression in orbital fibroblasts. As the Northern blot in Fig. 5C suggests, the involvement of IL-1alpha in leukoregulin's effects on IL-1beta expression is mediated at the pretranslational level. Addition of either anti-IL-1alpha or IL-1ra to leukoregulin-treated fibroblasts attenuates the induction of IL-1beta mRNA.


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Fig. 5.   Effects of anti-IL-1alpha , anti-IL-1beta antibodies (Ab), and exogenous IL-1ra on upregulation by leukoregulin of IL-1alpha and IL-1beta expression in orbital fibroblasts. Confluent monolayers of fibroblasts from an individual with TAO were treated with leukoregulin (1 U/ml) without or with anti-IL-1alpha or IL-1beta antibodies (1 µg/ml) or IL-1ra (500 ng/ml) for 16 h. Cell layers were then subjected to specific ELISA assays for IL-1alpha (A) or IL-1beta (B). C: confluent orbital fibroblast monolayers were treated with leukoregulin without or with anti-IL-1alpha antibodies or IL-1ra for 16 h, and then RNA was extracted and subjected to Northern blot analysis for IL-1beta mRNA levels. Bars represent IL-1beta transcript levels, as assessed by densitometry, corrected for their respective GAPDH mRNA signals.

Interruption of IL-1alpha but not IL-1beta appears to attenuate the induction by leukoregulin of PGHS-2 and cytokine-dependent PGE2 production in orbital fibroblasts. Because of the important role of IL-1 as an inducer of PGHS-2 and prostanoid production in many cell types, we next assessed whether either IL-1alpha or IL-1beta synthesis could be implicated in the upregulation by leukoregulin of the cyclooxygenase in orbital fibroblasts. Addition of neutralizing antibodies directed at IL-1alpha or IL-1beta (1 µg/ml each) or a high concentration of exogenous IL-1ra (500 ng/ml) to the culture medium of orbital fibroblasts treated with leukoregulin (1 U/ml) resulted in an interesting pattern of prostanoid production and PGHS-2 responses. Both anti-IL-1alpha and IL-1ra could attenuate the upregulation by leukoregulin of PGE2 synthesis (Fig. 6A). In cultures receiving leukoregulin alone, PGE2 levels were 2,030 ± 310 pg/ml compared with 617 ± 138 and 172 ± 100 pg/ml in cultures receiving leukoregulin in addition to IL-1ra or anti-IL-1alpha neutralizing antibodies, respectively. The effects of leukoregulin on PGHS-2 mRNA (Fig. 6B) and protein (Fig. 6C) were also markedly attenuated by either IL-1ra or anti-IL-1alpha antibody. In contrast, anti-IL-1beta antibodies failed to block the induction of PGHS-2 protein (Fig. 6C) or to influence cytokine-dependent PGE2 generation (data not shown). Neither anti-IL-1alpha antibodies nor IL-1ra could block the induction of PGHS-2 by PMA or TNF-alpha (data not shown). Thus it would appear that IL-1alpha is a critical intermediate protein involved in the upregulation of PGHS-2 expression by leukoregulin. This finding is also similar to our previous observation that IL-1alpha but not IL-1beta serves as an intermediate in the induction of PGHS-2 through the CD40/CD40 ligand activational bridge in orbital fibroblasts (5).


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Fig. 6.   Effects of anti-IL-1alpha antibodies and exogenous IL-1ra on the induction by leukoregulin of PGE2 production (A), PGHS-2 mRNA (B), and PGHS-2 protein (C). Monolayers of orbital fibroblasts were cultivated and treated with leukoregulin (1 U/ml), anti-IL-1alpha , and IL-1ra as specified in the legend to Fig. 5. They were then subjected to the appropriate assays, as described in MATERIALS AND METHODS. Data are expressed as means ± SE (n = 3) of triplicate culture wells in A or as individual Northern (B) or Western (C) blot determinations as described in MATERIALS AND METHODS.

Leukoregulin elicits NF-kappa B nuclear translocation in orbital fibroblasts: this activation is involved in the induction of IL-1 and PGHS-2 expression by leukoregulin. The human PGHS-2 promoter region contains at least two recognizable NF-kappa B sites (46). Moreover, genes encoding IL-1alpha and IL-1beta also contain at least one NF-kappa B site (8). We therefore next assessed whether leukoregulin could influence the nuclear translocation of NF-kappa B in orbital fibroblasts and whether this transcriptional factor was involved in the upregulation of IL-1 and PGHS-2 in these cells. We treated orbital fibroblasts with the protease inhibitor PDTC (100 µM), a selective inhibitor of the NF-kappa B pathway (13), by adding the compound to the culture medium of leukoregulin-treated cells. The inhibitor could block the induction by leukoregulin of IL-1alpha and IL-1beta (Fig. 7A). The effects of the compound were similar to those of dexamethasone. The upregulation of PGHS-2 mRNA and protein expression by leukoregulin could also be attenuated with PDTC and dexamethasone (10 nM; Fig. 7B). These effects were nearly complete, but neither compound altered PGHS-1 levels.


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Fig. 7.   Inhibition of nuclear factor-kappa B (NF-kappa B) activity can block the induction by leukoregulin of IL-1alpha , IL-1beta , and PGHS-2 expression. A: pyrrolidinedithiocarbamate (PDTC, 100 µM), a protease inhibitor, and dexamethasone (DEX, 10 nM) could block the induction of IL-1alpha and IL-1beta in orbital fibroblasts by leukoregulin (1 U/ml). Confluent orbital fibroblasts, from an individual with TAO, were cultivated in 24-well plates, shifted to medium containing 1% FBS, and treated with leukoregulin for 16 h without or with dexamethasone or PDTC. Cell layers were then analyzed for IL-1alpha (left) and IL-1beta (right) content with specific ELISAs. Data are presented as means ± SE of triplicate determinations. B: inhibitors of NF-kappa B can block the induction by leukoregulin of PGHS-2 protein and mRNA. Cell layer material was subjected to Western (top) and Northern (bottom) blot analysis as described in MATERIALS AND METHODS.

Gel shift assays were used to assess directly the binding activity exhibited by nuclear preparations from orbital and dermal fibroblasts with regard to NF-kappa B. As the autoradiograph shown in Fig. 8A suggests, there is a time-dependent increase in binding activity to the oligonucleotide corresponding to the NF-kappa B consensus site when orbital fibroblast cultures are treated with leukoregulin (1 U/ml). This activity migrates on the gels as two discrete bands that are apparent at 30 min, and this activity is sustained for 120 min, the duration of the study. Addition of PDTC (100 µM) to the culture medium resulted in a nearly complete attenuation of the NF-kappa B signal in nuclei from cultures treated with leukoregulin for 120 min. When an oligonucleotide probe representing a mutated NF-kappa B site was substituted in the assay, binding activity was virtually absent, suggesting a high degree of specificity (Fig. 8A). When nuclei from dermal fibroblasts were assayed, a single band of activity corresponding to the lower band found in the orbital fibroblast preparations was induced (Fig. 8B). Addition of a specific, unlabeled SP-1 probe in a molar excess failed to quench the binding signal in either orbital or dermal nuclear preparations, but addition of excess unlabeled NF-kappa B probe dramatically lowered the signal intensity (Fig. 8B). To determine the nature of the two bands on gel shift in orbital fibroblast nuclear preparations, antibodies specific for the p50 and p65 subunits of NF-kappa B were added to the reaction mixtures, and the preparations then were subjected to electrophoresis. As Fig. 8C indicates, the lower-molecular-weight band, present in both the orbital and dermal nuclear material, could be "super-shifted" with either the anti-p50 or the anti-p65 antibodies, suggesting that the identity of this band is the p50/p65 heterodimer. The higher-molecular-weight band, found only in orbital fibroblasts, could be shifted only with anti-p65 antibodies, suggesting that it represents the p65/p65 homodimer. Thus it would appear that orbital and dermal fibroblasts, when activated with leukoregulin, express different profiles of NF-kappa B activity.


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Fig. 8.   Electrophoretic mobility shift assay examining the effects of leukoregulin on NF-kappa B nuclear translocation in orbital and dermal fibroblasts. A: orbital cultures were treated with leukoregulin (1 U/ml) for 0-120 min. Nuclei from fibroblasts were prepared as indicated in MATERIALS AND METHODS, 2 µg/sample of the extracts were incubated with [32P]dATP-labeled NF-kappa B or mutated NF-kappa B oligonucleotide probes, and samples were subjected to SDS-PAGE. B and C: cultures were treated without or with leukoregulin for 2 h. Nuclear extracts were incubated with a [32P]dATP-labeled NF-kappa B oligonucleotide probe without or with unlabeled NF-kappa B or SP-1 probes in a 50-fold molar excess or with anti-p50 or anti-p65 antibodies (1 µg/ml). The gels were then exposed to X-Omat film at -70°C.

Leukoregulin enhances the stability of PGHS-2 mRNA in orbital fibroblasts. We have reported that, despite the 50- to 100-fold increase in steady-state PGHS-2 mRNA, levels elicited by leukoregulin in orbital fibroblasts, PGHS-2 gene transcription is increased by twofold (42). This result suggests that PGHS-2 mRNA stability might prove to be the predominant target for leukoregulin's upregulatory action on PGHS-2 expression. To assess these issues directly, we examined the decay of mature PGHS-2 mRNA by treating the cultures for 6 h with cycloheximide (10 µg/ml), a concentration associated with >90% inhibition of protein synthesis in human fibroblasts (28) and with a transient induction of PGHS-2 mRNA (42). The drug was washed out, and the inhibitor of gene transcription, 5,6-dichlorobenzimidazole (DRB, 50 µM), was added. Cultures were either untreated or treated with leukoregulin (1 U/ml) for the times indicated along the abscissa in Fig. 9. As the decay curves in Fig. 9 suggest, leukoregulin causes a marked retardation in the disappearance of hybridizable PGHS-2 mRNA in the presence of DRB. The apparent half-life for PGHS-2 mRNA in leukoregulin-treated cells is at least 4 h, whereas the half-life is 60 min in control cultures.


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Fig. 9.   PGHS-2 mRNA stability in orbital fibroblasts is greatly enhanced by leukoregulin. Orbital fibroblasts were allowed to proliferate to confluence and then were shifted to medium supplemented with 1% FBS for 16 h. They were treated with cycloheximide (10 µg/ml) for 6 h, and then the monolayers were washed extensively and all were covered with medium containing 5,6-dichlorobenzimidazole (50 µM) without or with leukoregulin (1 U/ml) for the time intervals indicated along the abscissa. At those times, cultures were harvested, and cellular RNA was subjected to Northern blot analysis with a radiolabeled PGHS-2 cDNA probe as described in MATERIALS AND METHODS. Blots were stripped and rehybridized to a GAPDH probe, and the PGHS-2 signals were normalized. Data were obtained by densitometric scanning using a BioImage system.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Orbital fibroblasts are particularly susceptible to the actions of proinflammatory cytokines such as IL-1, interferon-gamma , and leukoregulin (31, 47). Of particular potential importance is the substantial induction by cytokines of PGHS-2, the inflammatory cyclooxygenase. We hypothesize that it is the inducibility of PGHS-2 in orbital fibroblasts that underlies, at least in part, the dramatic inflammation observed in orbital connective tissue in the setting of TAO. It appears that this response is a point of divergence of the orbital fibroblast phenotype from fibroblasts derived from elsewhere in the human body. It would seem, on the basis of the results we report here, that the differential inducibility of IL-1 and IL-1ra may underlie at least some of the attributes associated with orbital fibroblasts. Our findings suggest that the local generation of IL-1 polypeptides in orbital fibroblasts could also impact cellular neighbors, since IL-1beta is released from the cell synthesizing it (8). This cytokine can function as a paracrine factor. Because IL-1alpha induction appears to be critical to the induction of PGHS-2 by both leukoregulin (Fig. 6) and the CD40/CD40 ligand activational bridge, interrupting production of this cytokine or its actions might prove to be a useful therapeutic target for disease modification.

An earlier report had attempted to demonstrate differential upregulation of both intracellular and soluble forms of IL-1ra in normal and TAO-derived orbital fibroblasts (19). Unfortunately, the studies described in that paper utilized RT-PCR to assess the levels of these IL-1ra mRNAs, and thus the data were not quantitative. The findings did suggest a considerable difference in the induction by IL-1alpha and lipopolysaccharide of soluble IL-1ra protein in normal and TAO fibroblasts as measured by an assay similar to the one used here. Thus it would appear that the substantial differences in IL-1ra inducibility in TAO orbital fibroblasts compared with dermal cultures we report are, at least in part, a consequence of the disease state in addition to the anatomic derivation of the cells.

The finding that leukoregulin represents a strong inducer of PGHS-2 in orbital fibroblasts has potential mechanistic relevance with regard to the cross talk between these cells and orbital T lymphocytes found in the tissues affected by TAO (7, 11). In addition to its effects on prostanoid biosynthesis in fibroblasts, leukoregulin can dramatically increase hyaluronan synthesis (39). This increase is very specific in that the other abundant glycosaminoglycans, all sulfated, are uninfluenced (39). Moreover, the effects on hyaluronan are anatomic-site selective in that macromolecular synthesis in dermal fibroblasts is enhanced to a far lesser degree. We have reported very recently that lymphocytes can signal orbital fibroblasts through another pathway that apparently does not involve leukoregulin, but this cross talk also results in the upregulation of multiple orbital fibroblast genes. CD40 ligand, when engaging CD40 displayed by these fibroblasts, elicits a striking induction of both PGHS-2 expression and hyaluronan synthesis (5). The expression of IL-6 and IL-8 in orbital fibroblasts can also be enhanced through the CD40/CD40 ligand bridge (24). The induction of at least some of these genes appears to be related to NF-kappa B (24). Taken together, it would appear that the orbital fibroblast can be activated in an anatomic site-selective manner by multiple molecular signals.

Another aspect that emerges from the current report is the substantial impact that leukoregulin has on the stability of mature PGHS-2 mRNA. Unlike many of the other cell types studied thus far, where the inducibility of PGHS-2 has been characterized, it is the turnover of the transcript in orbital fibroblasts rather than its rate of synthesis that represents the predominant target for cytokine action. In some experimental models, transcriptional upregulation accounts for the greatest influence on steady-state mRNA levels, whereas, in ECV304 endothelial cells, increases in both gene transcription and mRNA stability appear to contribute substantially (22). We have reported previously that the rate of PGHS-2 gene transcription in orbital fibroblasts changes only slightly after treatment with leukoregulin, approximately twofold (42). The current studies demonstrate the dramatic enhancement of the PGHS-2 mRNA half-life after leukoregulin treatment. Thus it would appear that the mechanisms involved in the upregulation of PGHS-2 and therefore of prostanoid production are cell-type specific. Leukoregulin also enhances NF-kappa B activity in orbital fibroblasts, and attenuating this pathway blocks the induction of PGHS-2. Thus the relatively small increase in PGHS-2 gene transcription might prove critical to the substantial increases in steady-state mRNA levels. Alternatively, the importance of the NF-kappa B pathway might relate to leukoregulin's upregulation of IL-1alpha or some other intermediate protein, the promoter of which contains NF-kappa B binding sites. It is of interest to note that cytokine stimulation of orbital fibroblasts results in a twofold increase in PGHS-2 promoter activity in cells transfected with a reporter gene (unpublished observations).

Orbital fibroblasts display a profile of receptors (36), gangliosides (2, 38), morphology (33), and responses to hormones (26, 33) and cytokines (14, 47) that distinguishe them from other fibroblast populations. We have suggested that it is the diverse array of molecules that fibroblasts synthesize and release that suggests broad roles for fibroblasts as sentinel cells that help coordinate complex events in wound repair and inflammation (25). The strikingly characteristic repertoire of biosynthetic activities ascribed to orbital fibroblasts underlies the unique remodeling of the orbital connective tissue observed in TAO and in other orbital inflammatory processes.

The highly inducible PGHS-2 in orbital fibroblasts implies a substantial capacity for these cells to produce large amounts of prostanoids in the context of inflammation. PGE2 has been shown to bias the differentiation of naive T lymphocytes away from the TH-1 phenotype and toward that of TH-2 lymphocytes (3). PGE2 also influences mast cell behavior and B cell differentiation (17, 23). Thus the high levels of PGE2 production found by orbital fibroblasts suggest that these cells might condition the immune responses occurring in the orbit, both under normal conditions and in disease. We have reported very recently that mast cells can directly activate gene expression in orbital fibroblasts, and at least some of this interplay between the two cell types is mediated through IL-4 (37). Thus the PGE2 generated by orbital fibroblasts might feed back on the mast cell, in turn modulating its activities. While not defining the role for prostaglandin production in TAO or in other situations of disease or health, our current findings do begin to identify the molecular events surrounding the exaggerated induction of PGHS-2 in orbital fibroblasts. Clearly, the role of PGE2 and other cyclooxygenase products in this disease will require further studies, perhaps some involving in vivo models. Should the cyclooxygenase prove to be overexpressed in TAO, therapeutic trials with the newly developed, specific PGHS-2 inhibitors would become warranted.


    ACKNOWLEDGEMENTS

We thank Julia Rozenblit and Heather Meekins for technical assistance. We are grateful to Dr. C. H. Evans for providing the leukoregulin used in these studies.


    FOOTNOTES

These studies were supported in part by National Eye Institute Grants EY-08976 and EY-11708 and by a Merit Review award from the Department of Veterans Affairs.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. J. Smith, Div. of Molecular Medicine, Dept. of Medicine, Harbor-UCLA Medical Center, 1000 West Carson St., Torrance, CA 90509.

Received 15 June 1999; accepted in final form 4 August 1999.


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RESULTS
DISCUSSION
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