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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 2 734-742
Copyright © 2000 by The Endocrine Society

Original Studies

Interleukin-1 Receptor Antagonist Ribonucleic Acid and Protein Expression by Cultured Graves’ and Normal Orbital Fibroblasts Is Differentially Modulated by Dexamethasone and Irradiation1

Tina Mühlberg, Werner Joba, Christine Spitzweg, Hermann-Dieter Schworm, Hans-Jürgen Heberling and Armin E. Heufelder

Division of Endocrinology, Philipps University (W.J., C.S., A.E.H.), D-35033 Marburg; University Eye Hospital, Klinikum Innenstadt, Ludwig Maximilians Universität (H.-D.S.), D-80336 Munich; and Städtisches Klinikum Leipzig-West (T.M., H.-J.H.), D-04177 Leipzig, Germany

Address all correspondence and requests for reprints to: Armin E. Heufelder, M.D., Division of Gastroenterology, Endocrinology and Metabolism, Department of Internal Medicine, Philipps University, Baldingerstrasse, D-35033 Marburg, Germany. E-mail: heufeld{at}mailer.uni-marburg.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent data have indicated that orbital fibroblasts (OF) can be stimulated to produce marked quantities of interleukin-1 receptor antagonist (IL-1RA), a powerful inhibitor of the proinflammatory activities of interleukin-1 in the orbital tissues in Graves’ ophthalmopathy (GO). We examined whether the beneficial effects of dexamethasone or irradiation, the two main therapeutic modalities applied in patients with active GO, may be related to their capacity to alter IL-1RA ribonucleic acid (RNA) and protein expression in OF. Early passages of cultured OF were obtained from orbital connective tissue and extraocular muscle of patients with severe active GO and five control subjects. Modulation of the two variants of IL-1RA, intracellular IL-1RA (icIL-1RA) and soluble IL-1RA (sIL-1RA), was studied after exposure of OF to increasing concentrations of dexamethasone (10-10-10-6 mol/L), the glucocorticoid receptor antagonist RU 38486 (10-3 mol/L), or combinations thereof. Alternatively, cell monolayers were exposed to increasing doses of UV irradiation (0.1–1 J/cm2) or ionizing irradiation (0.2–2 Gy). The IL-1RA gene and protein variants were analyzed by RT-PCR, immunocytochemistry, immunoblotting, and enzyme-linked immunosorbent assay. Dexamethasone inhibited IL-1RA RNA steady state levels in GO OF and control OF in a dose-dependent manner. Combined exposure of OF to dexamethasone and RU 38486 completely restored baseline levels of IL-1RA RNA. By contrast, low doses of UV and ionizing irradiation dose dependently up-regulated IL-1RA-specific transcripts in GO OF and control OF, whereas higher doses were less effective. Immunoblotting and enzyme-linked immunosorbent assay revealed suppression of IL-1RA immunoreactivity after treatment with dexamethasone and enhanced expression of IL-1RA by GO OF and normal OF after low doses of UV and ionizing irradiation. Our results indicate that, in contrast to dexamethasone, low doses of irradiation stimulate expression of the IL-1RA gene and protein variants in OF. Induction by irradiation of IL-1RA expression in target cells of the orbital immune process represents an as yet unrecognized mechanism by which orbital radiotherapy may exert some of its beneficial therapeutic effects in patients with active GO.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INFILTRATION of orbital connective, adipose, and extraocular muscle tissue by T lymphocytes and macrophages represents a prominent histological feature of Graves’ ophthalmopathy (GO) (1, 2, 3, 4). Various cytokines have been detected within the orbital tissue in GO, including interleukin-1{alpha} (IL-1{alpha}), tumor necrosis factor-{alpha}, interferon-{gamma}, and insulin-like growth factor I. These factors are mainly released from infiltrating immunocompetent cells and, in addition, are produced locally in an autocrine and paracrine manner by residential cells such as orbital fibroblasts (OF) (3, 5, 6, 7). A prominent role for IL-1 is suggested by its capacity to stimulate cell proliferation, glycosaminoglycan synthesis, and PG production, and to induce the expression of various adhesion molecules, immunomodulatory proteins, and metalloproteinases in OF (1, 7, 8, 9). IL-1 receptor antagonist (IL-1RA), a naturally occurring inhibitor of IL-1-mediated activities that recognizes the two IL-1 cell surface receptors, acts as a competitive inhibitor of IL-1{alpha} and IL-1ß and blocks IL-1-mediated cellular activities without inducing detectable cellular responses (10, 11, 12, 13). Two structural variants of IL-1RA, soluble (s) and intracellular (ic) IL-1RA, have been reported previously and are regulated and produced differentially in various cell types (14, 15, 16, 17). Recently, it was demonstrated that exogenous addition of recombinant IL-1RA inhibits IL-1-induced stimulation of glycosaminoglycan synthesis in cultured human OF, suggesting that IL-1-mediated effects may be counteracted by IL-1RA (18). We have reported expression by cultured human OF of both icIL-1RA and sIL-1RA messenger ribonucleic acid (mRNA) and protein (19). Compared to normal OF, GO OF produced significantly lower levels of icIL-1RA and sIL-1RA, suggesting that an imbalance between IL-1 receptor agonist and antagonist may facilitate IL-1 receptor-mediated proinflammatory and fibrogenic actions in GO (19).

Glucocorticosteroids and orbital radiotherapy represent the two major therapeutic modalities employed in patients with active GO, and both forms of therapy are commonly applied in combination with superior results (20, 21). We hypothesized that these therapeutic modalities may influence agonist stimulation of the IL-1 receptor by altering IL-1RA expression in human OF. In support of this concept, we have recently observed a rise of IL-1RA serum levels in patients with GO who responded well to orbital radiotherapy (22). Therefore, we designed a series of experiments to examine in vitro whether dexamethasone and irradiation may act at least in part by modulating IL-1RA RNA levels and protein expression in human OF. We report that, in contrast to the suppressive effect of dexamethasone, low doses of UV and ionizing irradiation stimulate IL-1RA RNA and protein expression in Graves’ and normal OF.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Human recombinant IL-1{alpha} was obtained from Roche Molecular Biochemicals (Mannheim, Germany). Lipopolysaccharide (LPS), dexamethasone, and alkaline phosphatase-conjugated rabbit antigoat Ig were purchased from Sigma (Deisenhofen, Germany). RU 38486 was provided by Roussel-UCLAF (Romainville, France). Antihuman IL-1RA antibody and recombinant IL-1RA were obtained from R&D Systems, Inc. (Minneapolis, MN). The RT kit was obtained from Life Technologies, Inc. (Gaithersburg, MD), and Thermus aquaticus DNA polymerase (Taq DNA polymerase) was from Perkin-Elmer Corp./Cetus (Vaterstetten, Germany). The 3'-end labeling kit and DIG Luminescent Detection Kit were purchased from Roche Molecular Biochemicals.

Cell culture

Orbital connective tissue and extraocular muscle biopsies were obtained from three patients during transantral orbital decompression surgery for severe active GO. These patients had previously received glucocorticosteroids (not within 3 months before surgery), but had failed to respond or had experienced intolerable side-effects. Normal orbital connective tissue was derived from five individuals undergoing orbital surgery for refractory glaucoma (n = 2) and strabism surgery (n = 3). OF were isolated and propagated as previously described (23). All cell strains were used between the second and fifth passages. Cell monolayers were switched to medium 199 containing 1% FCS, antibiotics and amphothericin B for 24 h before study. Inducers of IL-1RA were added to the medium at predetermined optimal concentrations: IL-1{alpha}, 10 U/mL; and LPS, 50 ng/mL (19). In some experiments, IL-1{alpha}- or LPS-stimulated OF monolayers were treated with increasing doses of dexamethasone (10-10–10-6 mol/L) or combinations of 10-6 mol/L dexamethasone and RU 38486 (10-3 or 10-6 mol/L) for 44 h. Further, OF monolayers were treated with IL-1{alpha} or LPS for 44 h and then exposed to increasing doses of UV irradiation (0.1–1 J/cm2) or ionizing irradiation (0.2–2 Gy) generated by a 5-meV linear accelerator. All irradiation exposures were administered only once as single doses. After irradiation, cells were cultured in medium 199 containing 10% FCS for 36 h before harvesting or immunocytochemical staining. Cell counting before and after irradiation revealed no significant differences in cell numbers. Cell viability was monitored by trypan exclusion staining and standard lactate dehydrogenase release assay. In addition, RNA and protein gels were routinely run to rule out treatment-related changes in total RNA and protein levels. Terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling staining and DNA ladder analysis were performed by standard techniques to assess apoptosis in cells subjected to UV and ionizing irradiation.

RNA isolation, PCR, and Southern hybridization

After cell counting, equal amounts of total RNA was extracted and quantitated using UV spectroscopy (absorbance at 260 nm) as well as by comparing the fluorescence of ethidium bromide-stained 16S ribosomal RNA after agarose gel electrophoresis using NIH Image 1.55 software. Quantities of total RNA were determined to differ less than 5%. Aliquots (2 µg) of heat-denatured RNA were subjected to RT using an oligo(deoxythymidine) primer. Aliquots of the resulting complementary DNA (cDNA) were amplified using icIL-1RA-specific primer (5'-TGTTGTGACGCCTTCTGAGGGTCC-3') and sIL-1RA-specific primer (5'-CGGGCTG-CAGTCACAGAATGGA-3'), each paired with an antisense primer (5'-AGCTCCACCCT-GGGAGGGACTG-3'), respectively (24). IL-1RA variant-specific primers were designed to span at least one intron and to generate products of 750 bp (icIL-1RA) and 700 bp (sIL-1RA), respectively (15, 16, 17). Amplifications were performed with 5 µL of each cDNA template, 5 µL 10 x PCR reaction buffer [20 mmol/L Tris-HCl (pH 8.0), 100 mmol/L KCl; 0.1 mmol ethylenediamine tetraacetate, 1 mmol/L dithiothreitol, 50% glycerol, 0.5% Tween-20, and 0.5% Nonidet P-40], 1 µL 10 mmol/L deoxy-NTP mix, 1 µL of each oligonucleotide primer (20 µmol/L), and 2 U Taq DNA polymerase in a final volume of 50 µL. Amplifications were conducted with 5 min of denaturation at 94 C, followed by 35 cycles (1 min at 94 C, 2 min at 65 C, 3 min at 72 C), and a final 10-min extension at 72 C. PCR conditions were chosen to assure that amplifications were within the linear range. To control for the integrity of the cDNA templates and to rule out DNA contamination of the samples, all templates were amplified with primers spanning an intron of the ß-actin gene (5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' and 5'-CTAGAAGCATTGCGGTGG-ACGATGGAGGG-3'). The expected ß-actin product generated by these primers from a cDNA template is 661 bp in length. Reaction conditions were as described above. Amplified PCR products were resolved by electrophoresis on 1% agarose gels with 10 mg/mL ethidium bromide, visualized under UV light, and compared with a 123-bp molecular size control ladder (Life Technologies, Inc.). In addition, equal amounts of total RNA were reverse transcribed into cDNA, and PCR was performed to amplify ß-actin-specific transcripts. Using predetermined PCR conditions, these experiments were repeated three times with similar results, assuring that amplifications increased exponentially and that the plateau phase of the reaction had not been reached. Quantities of resulting ß-actin-specific transcripts were determined by separating an aliquot of each reaction on an ethidium bromide-stained agarose gel and comparing the fluorescence of ethidium bromide intercalated into dsDNA using the NIH Image 1.55 software. Differences in calculated quantities of ß-actin transcripts were less than 5%, which is well within the known range of variation of Taq amplification efficiency. Southern hybridization was performed to verify the specificity of PCR products and to improve the sensitivity of fragment detection. Membranes containing immobilized DNA were prehybridized by standard procedures, hybridized with digoxigenin-labeled oligonucleotide probes (25 ng) specific for icIL-1RA gene or sIL-1RA gene (24), respectively, washed, and incubated with anti-dioxygenin-alkaline phosphatase conjugate and lumigen-purified protein derivative substrate solution before exposure to x-ray film for 10 min at room temperature.

PAGE and immunoblotting

OF monolayers were extracted in 0.5% SDS in the presence of protease inhibitor complex. Cell lysates and culture supernatants were concentrated and subjected to SDS-PAGE under reducing conditions, as previously described (25). For quantitation of IL-1RA reactivity, immunoblots were subjected to scanning densitometry using a dual wave length densitometer (Shimadzu Scientific, Columbia, MD). Intensities are expressed in arbitrary units, reflecting the relative areas under the curves of the stained bands. The assay was linear with respect to protein concentrations within the range used.

Immunocytochemistry

OF were plated directly onto multichamber slides and grown to near confluence. Monolayers were incubated for 44 h in the presence or absence of cytokines, subjected to increasing doses of UV or ionizing irradiation, and propagated for 36 h. Slides were then washed, fixed in 100% methanol, and rehydrated in PBS, and nonspecific binding was blocked with 1% BSA. Goat antihuman IL-1RA antibody was applied at a concentration of 1.5 µg/mL for 2 h. Washed cell monolayers were incubated with alkaline phosphatase-conjugated antigoat Ig (1 µg/mL) for 1 h, rinsed with TBS, and incubated in developing buffer using nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) chromogenic substrates, and counterstained with malachite green. A brown precipitate was indicative of IL-1RA immunoreactivity. Parallel monolayers with the primary and secondary antibody replaced, in turn, by phosphate-buffered saline and isotype-matched nonimmune IgGs and irrelevant IgGs, respectively, were examined to assure specificity and to exclude cross-reactivities between the antibodies and conjugates employed.

Human IL-1RA immunoassay

Quantitation of human IL-1RA in cell culture supernatants was performed using a commercially available quantitative sandwich enzyme immunoassay (Quantakine, R&D Systems). This immunoassay is calibrated against highly purified recombinant human IL-1RA and detects IL-1RA concentrations as low as 10 pg/mL. The monoclonal antihuman IL-1RA used in this assay recognizes both natural and recombinant human IL-1RA, with no significant cross-reactivity or interference by a broad range of cytokines tested. Intra- and interassay variabilities were determined for three samples of known IL-1RA concentrations, and the coefficient of variance was generally less than 6.5%. Data represent the mean ± SD of duplicate measurements derived from at least three separate experiments.

Statistical analysis

Intraassay variabilities of measurements of IL-1RA reactivity on immunoblots were determined in untreated and IL-1{alpha}-stimulated samples run in quadruplicate, and the coefficients of variance were 9.4% and 6.4%, respectively. For determination of the interassay variabilities, equal amounts of protein (100 µg) from the same sample were loaded onto two gels run on different days, and the resulting immunoblots were quantitated by densitometry. Pair plotting (day 1 vs. day 2) showed that all data points were within ±10% of the line of identity, and five of seven data points were within ±5% of the line of identity. Student’s t test for analysis of paired and unpaired data was used to establish levels of significance (P < 0.05). Investigations adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from the subjects after the nature and possible consequences of the study were explained to them. The research protocol was approved by the institutional human experimentation committee.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Modulation of IL-1RA RNA expression by dexamethasone and irradiation

Exposure of IL-1{alpha}-treated OF monolayers derived from patients with active GO to increasing concentrations of dexamethasone (10-10–10-6 mol/L) dose dependently suppressed the abundance of icIL-1RA RNA steady state levels (Fig. 1Go). Near-complete suppression of icIL-1RA RNA steady state levels was observed upon exposure of IL-1{alpha}-stimulated cell monolayers to dexamethasone at 10-6 and 10-7 mol/L, respectively. In contrast, when exposed simultaneously to a combination of dexamethasone (10-6 mol/L) and the glucocorticoid receptor antagonist RU 38486 (10-3 mol/L), the inhibitory effect of dexamethasone (10-6 mol/L) on icIL-1RA gene expression by IL-1{alpha}-stimulated OF was fully reversed. Further, when RU 38486 was used at a concentration of 10-6 mol/L, suppression by dexamethasone (10-6 mol/L) of IL-1{alpha}-stimulated IL-1RA mRNA levels was partially (~70%) inhibited (data not shown). Treatment of cell monolayers with RU 38486 (10-3 mol/L) alone had no effect on the expression of icIL-1RA-specific transcripts by IL-1{alpha}-stimulated GO OF. Further, similar dose-dependent inhibition by dexamethasone was detected with respect to sIL-1RA RNA expression (data not shown). Treatment of OF monolayers with dexamethasone alone in the absence of IL-1{alpha} or LPS (a nonspecific inducer of IL-1RA), respectively, had no effect on IL-1RA expression by OF. Moreover, when OF were pretreated with LPS instead of IL-1{alpha}, similar, but less pronounced, effects on icIL-1RA and sIL-1RA RNA steady state levels by OF were obtained (data not shown). Further, although GO OF produced significantly lower quantities of IL-1RA, no apparent differences between GO OF and normal OF were noted with respect to their responses to dexamethasone under the conditions tested (data not shown). In addition, no differences were observed between OF derived from connective/fatty tissue and those derived from extraocular muscle with respect to IL-1RA RNA steady state levels under the experimental conditions tested. All experiments were repeated at least twice with comparable results. Cell viability was monitored by trypan blue exclusion staining and lactate dehydrogenase release assay and was not altered during any of these experiments. In addition, total cellular RNA and protein levels were routinely checked and were not altered by exposure to dexamethasone, cytokines, or irradiation, respectively, during the course of this study.



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  		Figure 1. Representative Southern blot (top) and ethidium bromide-stained agarose gel (bottom) demonstrating modulation of intracellular IL-1RA RNA steady state levels by dexamethasone in cultured OF derived from a patient with GO. Confluent OF monolayers were stimulated with cytokines, increasing concentrations of dexamethasone, RU 38486, or combinations thereof. RT-PCR was performed using icIL-1RA-specific (top) and ß-actin-specific (bottom) primers, and membranes with immobilized transcripts were hybridized with a digoxigenin-labeled oligonucleotide probe specific for icIL-1RA before exposure to x-ray film. Experiments were repeated twice with similar results, using OF derived from two other patients with GO and two normal individuals. Experimental conditions for treatment of OF monolayers were as follows: lane 1, IL-1{alpha} (10 U/mL); lane 2, IL-1{alpha} and dexamethasone (10-6 M); lane 3: IL-1{alpha}, dexamethasone (10-7 M); lane 4: IL-1{alpha}, dexamethasone (10-8 mol/L); lane 5, IL-1{alpha} and dexamethasone (10-9 mol/L); lane 6, IL-1{alpha} and dexamethasone (10-10 mol/L); lane 7, IL-1{alpha} and dexamethasone (10-6 mol/L), RU 38486 (10-3 mol/L); lane 8, IL-1{alpha} and RU 38486 (10-3 mol/L); lane 9, medium 199 control.

 
In a second series of experiments, OF monolayers derived from patients with GO and control individuals were stimulated with IL-1{alpha} and exposed to increasing doses of UV irradiation. Single radiation doses of 0.1, 0.2, and 0.4 J/cm2 dose dependently stimulated icIL-1RA RNA steady state levels in IL-1{alpha}-treated GO OF (Fig. 2Go, top panel) and normal OF. In contrast, higher doses of UV irradiation (0.6–1.0 J/cm2) suppressed the expression of icIL-1RA-specific transcripts in GO OF and normal OF. Similar dose-dependent up-regulation of IL-1RA RNA steady state levels by low doses of UV irradiation and suppression by higher doses of UV irradiation were detected for the soluble IL-1RA form (Fig. 2Go, middle panel). When OF were pretreated with LPS instead of IL-1{alpha}, similar effects on icIL-1RA and sIL-1RA RNA steady state levels were obtained (data not shown).



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  		Figure 2. Representative ethidium bromide-stained agarose gel demonstrating modulation of intracellular (top) and soluble (middle) IL-1RA RNA steady state levels by UV irradiation in cultured OF derived from a patient with GO. Confluent OF monolayers were treated as indicated below. RT-PCR was performed with icIL-1RA-specific primers (top), sIL-1RA-specific primers (middle), and ß-actin-specific primers (bottom). Experiments were repeated twice with similar results, using OF from two other patients with GO and two normal individuals. Experimental conditions for treatment of OF monolayers were as follows: lane 1, IL-1{alpha} (10 U/mL) and 0.1 J/cm2 UV irradiation; lane 2, IL-1{alpha} and 0.2 J/cm2 UV irradiation; lane 3, IL-1{alpha} and 0.4 J/cm2 UV irradiation; lane 4, IL-1{alpha} and 0.6 J/cm2 UV irradiation; lane 5, IL-1{alpha} and 0.8 J/cm2 UV irradiation; lane 6, IL-1{alpha} and 1.0 J/cm2 UV irradiation; lane 7, IL-1{alpha}; lane 8, medium 199 control.

 
In a third series of experiments, we assessed whether ionizing radiation exerts effects similar to those observed with UV irradiation. GO OF and normal OF monolayers were pretreated with IL-1{alpha} or LPS and then exposed to ionizing irradiation using a 5-meV linear accelerator. Single radiation doses of 0.5, 0.8, and 1.0 Gy markedly enhanced the expression of the sIL-1RA gene variant and, to a lesser degree, of the icIL-1RA variant in IL-1{alpha}-treated GO OF (Fig. 3Go). Similar results were obtained in LPS-treated GO OF and in IL-1{alpha}- or LPS-treated normal OF (data not shown). At higher radiation doses (1.5–2 Gy), suppression of both icIL-1RA and sIL-1RA RNA steady state levels was observed. Equal loading of all samples and comparable efficiencies of amplification were confirmed by RT-PCR amplification using ß-actin-specific primers (Fig. 3Go). Although IL-1RA RNA levels were generally lower in GO OF compared to those in normal OF, no apparent differences were observed between GO OF and normal OF or between OF derived from connective/fatty and that from extraocular muscle tissue, respectively, with respect to their IL-1RA responses to UV irradiation or ionizing irradiation. Cell viability and total cellular RNA and protein levels were not altered during the time course of any of these experiments.



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  		Figure 3. Representative ethidium bromide-stained agarose gel demonstrating modulation of IL-1RA RNA steady state levels by ionizing irradiation of cultured OF derived from a patient with GO. Confluent OF monolayers were treated with IL-1{alpha} before irradiation and RNA extraction. RT-PCR was performed using icIL-1RA-specific (top), sIL-1RA-specific (middle), and ß-actin-specific primers (bottom). Experiments were repeated twice with similar results, using OF derived from two other patients with GO and two normal individuals. Experimental conditions for treatment of OF monolayers were as follows: lane 1, IL-1{alpha} (10 U/mL) and 0.2 Gray; lane 2, IL-1{alpha} and 0.5 Gray; lane 3, IL-1{alpha} and 0.8 Gray; lane 4, IL-1{alpha} and 1.0 Gray; lane 5, IL-1{alpha} and 1.5 Gray; lane 6, IL-1{alpha} and 2.0 Gray; lane 7, 1.0 Gray; lane 8, IL-1{alpha}; lane 9, negative control.

 
Modulation of icIL-1RA expression and sIL-1RA production by dexamethasone and irradiation

IL-1RA protein expression in cell lysates (icIL-1RA) and culture supernatants (sIL-1RA) was assessed by immunoblot analysis, using recombinant IL-1RA (17 kDa) as a positive control. Treatment of IL-1{alpha}-stimulated GO OF (Fig. 4Go) and normal OF with increasing concentrations of dexamethasone (10-10–10-6 mol/l) for 44 h revealed dose-dependent inhibition of IL-1RA immunoreactivity in cell lysates. Simultaneous exposure of cell monolayers to dexamethasone (10-6 mol/L) and RU 38486 (10-3 mol/L) completely reversed this inhibitory effect (Fig. 4Go). By contrast, when GO OF and normal OF were exposed to dexamethasone (10-6 and 10-10 mol/L) alone without IL-1{alpha} or LPS pretreatment, no effect on IL-1RA protein expression was detected (data not shown). As we have recently shown (19), the molecular mass of icIL-1RA protein (22 kDa) differs from the protein sizes of sIL-1RA (23 and 26 kDa) and from the molecular mass of recombinant IL-1RA (17 kDa) depending on their degrees of N-glycosylation. In contrast, exposure of cell monolayers to UV irradiation at doses of 0.2–0.4 J/cm2, respectively, enhanced the abundance of IL-1RA immunoreactivity in IL-1{alpha}-stimulated GO OF (Fig. 5Go) and normal OF, whereas irradiation doses of 0.6 J/cm2 or greater failed to reveal a significant effect. Similar effects were noted when LPS-treated GO OF and normal OF were subjected to UV irradiation (data not shown). Analysis of total cell counts and total protein levels in OF monolayers did not reveal any changes before or after UV irradiation.



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  		Figure 4. Representative immunoblot of IL-1RA protein expression after dexamethasone treatment of OF monolayers obtained from a patient with GO. Confluent OF monolayers were treated for 44 h as indicated below. Cell lysates derived from identical numbers of cells were concentrated 10-fold, and aliquots (100 µg) of total protein were subjected to SDS-PAGE under reducing conditions. Gels were blotted onto nitrocellulose membranes, incubated with antihuman IL-1RA antibody, and visualized using NBT and BCIP substrates. Similar results were obtained in OF derived from two other patients with GO and two other normal individuals. Experimental conditions were as follows: lane 1, IL-1{alpha} (10 U/mL) and dexamethasone (10-6 mol/L); lane 2, IL-1{alpha}, dexamethasone (10-6 mol/L), and RU 38486 (10-3 mol/L); lane 3, IL-1{alpha} and dexamethasone (10-8 mol/L); lane 4, IL-1{alpha}; lane 5, medium 199 alone.

 


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  		Figure 5. Representative immunoblot of IL-1RA expression after UV irradiation of OF monolayers derived from a patient with GO. Confluent OF monolayers were treated for 44 h as indicated below. Cell lysates derived from identical number of cells were concentrated 10-fold, and aliquots (100 µg total protein) were subjected to SDS-PAGE under reducing conditions. Gels were blotted onto nitrocellulose membranes, incubated with antihuman IL-1RA antibody, and visualized using NBT and BCIP substrates. Similar results were obtained in OF derived from two other patients with GO and two other normal individuals. Experimental conditions were as follows: lane 1, IL-1{alpha} (10 U/mL); lane 2, IL-1{alpha} and 0.6 J/cm2 UV irradiation; lane 3, IL-1{alpha} and 0.4 J/cm2 UV irradiation; lane 4, IL-1{alpha} and 0.2 J/cm2 UV irradiation; lane 5, medium 199 alone.

 
Soluble IL-1RA concentrations in culture supernatants produced by GO OF and normal OF monolayers were quantitated using a highly sensitive sIL-1RA enzyme-linked immunosorbent assay. Treatment of IL-1{alpha}- or LPS-stimulated GO OF and normal OF with dexamethasone (10-6 mol/L) significantly suppressed the release of sIL-1RA into the supernatants (Table 1Go). In contrast, exposure of IL-1{alpha}- or LPS-stimulated GO OF and normal OF to UV irradiation dose dependently increased the levels of sIL-1RA production. Similarly, low doses (0.2–0.8 Gy) of ionizing irradiation markedly enhanced sIL-1RA protein levels in culture supernatants of IL-1{alpha}-treated GO OF and normal OF, whereas higher doses (1.0–2.0 Gy) of ionizing irradiation consistently stimulated a lesser degree of sIL-1RA production (Table 2Go). Similar, but less pronounced, effects were obtained in LPS-treated GO OF and normal OF (Table 2Go).


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  		Table 1. Modulation by dexamethasone or UV irradiation of soluble IL-1RA levels in conditioned medium derived from IL-1{alpha}- or LPS-stimulated Graves’ and normal orbital fibroblast monolayers

 

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  		Table 2. Modulation by ionizing irradiation of soluble IL-1RA levels in conditioned medium derived from IL-1{alpha}- or LPS-stimulated Graves’ and normal orbital fibroblast monolayers

 
Immunocytochemical staining of untreated GO OF (Fig. 6AGo) and normal OF monolayers with monoclonal anti-human IL-1RA antibody revealed low levels of cytoplasmic IL-1RA immunoreactivity. Treatment of GO OF and normal OF with IL-1{alpha} markedly enhanced the level of IL-1RA expression (Fig. 6BGo). By contrast, IL-1RA immunoreactivity was strongly suppressed by treatment of IL-1{alpha}-stimulated GO OF (Fig. 6CGo) and normal OF with dexamethasone (10-6 mol/L) for 44 h, and this effect was partially reversed by cotreatment with RU 38486 (10-6 mol/L; Fig. 6DGo). In addition, both UV irradiation and ionizing irradiation (Fig. 6EGo) further enhanced IL-1RA expression in IL-1{alpha}-treated OF beyond the level already present after pretreatment with IL-1{alpha}. IL-1RA immunoreactivity was lost when using IL-1RA antibody preabsorbed with recombinant human IL-1RA (Fig. 6FGo). In addition, staining of OF monolayers with isotype-matched nonimmune IgG and antithyroglobulin antibody, respectively, failed to produce specific immunoreactivity (data not shown). Cell counts and cell viability were not altered during the course of any of these experiments. Moreover, both terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling and DNA ladder analysis failed to detect any evidence of apoptosis for up to 36 h after radiation exposure (not shown).



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  		Figure 6. Localization of IL-1RA immunoreactivity in cultured OF derived from a patient with GO using monoclonal anti-human IL-1RA antibody. Experimental conditions were as follows: A, untreated OF monolayers; B, IL-{alpha}-pretreated OF monolayer; C, IL-1{alpha}-pretreated OF monolayers exposed to dexamethasone (10-7 mol/L); D, IL-1{alpha}-pretreated OF monolayers exposed simultaneously to dexamethasone (10-7 mol/L) and RU 38486 for 44 h; E, IL-1{alpha}-pretreated OF monolayers exposed to ionizing irradiation (0.8 Gy). Similar results were obtained in OF derived from two other patients with GO and two other normal individuals. IL-1RA immunoreactivity in IL-1{alpha}-stimulated OF monolayers was not detected with IL-1RA antibody after preabsorption with recombinant human IL-1RA (F) or with IL-1RA antibody replaced by an isotype-matched nonimmune IgG or antithyroglobulin antibody, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Biological relevance of IL-1RA as an antagonist of IL-1 in GD and GO has been implicated by several studies both in vitro (18, 19, 27) and in vivo (22). In view of these results, this study was designed to assess whether dexamethasone and ionizing irradiation, the two main therapeutic modalities routinely applied in patients with active GO (20, 21), act by modulating the expression of IL-1RA in cultured OF derived from patients with active GO and healthy individuals. As demonstrated by several complementary techniques, therapeutic doses of dexamethasone inhibited, to a similar extent, icIL-1RA and sIL-1RA RNA steady state and IL-1RA protein levels in GO OF and normal OF. The antiglucocorticoid agent RU 38486 was capable of inhibiting this effect, suggesting that the glucocorticoid receptor is involved in the inhibitory effect of dexamethasone. These data are in agreement with those of Arzt et al. (28), who observed dose-dependent suppression of IL-1RA levels in endotoxin-stimulated human monocytes after dexamethasone treatment, and with those of Cominelli et al. (29), who reported suppression of IL-1RA expression in inflamed rabbit colon tissue after corticosteroid treatment. Further, Sauer et al. (30) recently demonstrated that cortisol, the major physiological human glucocorticoid, diminished LPS-stimulated IL-1RA synthesis in cultured peripheral blood monocytes. Moreover, Joyce et al. (31) reported that dexamethasone antagonizes IL-4- and IL-10-induced release of IL-1RA by monocytes. In addition, treatment of peripheral blood mononuclear cells induced soluble type II IL-1 receptor expression, but suppressed IL-1R, suggesting that dexamethasone differentially modulates two competitive inhibitors of IL-1 binding to the signaling IL-1R (32). Thus, glucocorticoids may act not only to counteract inflammatory mediators (33), but also to physiologically balance agonist and antagonist systems and to prevent overactivity of the immune system. Treatment of OF monolayers with dexamethasone alone in the absence of IL-1{alpha} or LPS failed to affect IL-1RA RNA steady state levels and protein expression in our study. In agreement with this observation, Arzt et al. (28) reported the absence of an inhibitory effect of dexamethasone on basal IL-1RA expression by unstimulated monocytes. Although the reason for this difference between stimulated and unstimulated cells remains to be determined, it is possible that expression of an increasing number of glucocorticoid receptors after stimulation with cytokines may render these cells more susceptible to the effects of endogenous or exogenous glucocorticoids (34).

An important result of our study is the demonstration of increased levels of IL-1RA RNA and protein in cytokine-stimulated OF subjected to low doses of UV and ionizing irradiation. Although orbital radiotherapy in patients with active GO involves ionizing irradiation rather than UV irradiation, these two modalities of irradiation had similar effects on IL-1RA expression by OF in vitro. Up-regulation of IL-1RA synthesis in GO OF and normal OF was observed in a dose-dependent manner at low doses ranging from 0.1–0.4 J/cm2 after UV irradiation and from 0.2–0.6 Gy after ionizing irradiation. Recently, we reported significantly higher sIL-1RA levels in sera derived from patients with active GO after orbital radiotherapy compared to those obtained before orbital radiotherapy (22). Collectively, these results suggest that low doses of irradiation alter icIL-1RA and sIL-1RA production both locally within the orbital tissue and systemically. Further, in addition to other cell types, such as macrophages, OF appear to contribute to the antiinflammatory effects of orbital radiotherapy through their local production of substantial quantities of IL-1RA. Our data are in agreement with those of Schwarz et al. (35), who demonstrated that UV irradiation of epidermal cells promotes the release of a specific inhibitor of IL-1 activity. Further, an increase in IL-1RA production has been observed in UVB-irradiated stratum corneum of human skin (36, 37). Moreover, UVB irradiation of human keratinocytes has been reported to exert several regulatory effects on cytokine expression, such as augmented release of interleukin-8 and melanoma growth-stimulating activity (38). Similarly, in a recent study designed to examine modulation of IL-1RA expression after laser irradiation, a 4- to 5-fold increase in IL-1RA production by human monocytes was observed (39). Although the molecular details of how irradiation stimulates the expression and release of IL-1RA remain to be examined, Akashi et al. (40) recently reported that posttranscriptional stabilization of repetitive AUUUA sequences in short-lived cytokine transcripts represents a candidate mechanism by which irradiation may increase levels of cytokine RNAs.

In conclusion, we have demonstrated that, in contrast to dexamethasone, low doses of either UV or ionizing irradiation stimulate icIL-1RA and sIL-1RA production in cultured human OF derived from patients with active GO and control individuals. Although these data were derived from experiments using cultured OF monolayers in vitro and therefore are not directly applicable to the situation in vivo, our results may prompt further clinical studies comparing the benefits of conventional orbital irradiation (20 Gy in 2 weeks) with those of lower doses of irradiation administered over a prolonged period of time. Enhancement by irradiation of IL-1RA expression in target cells of the orbital immune process (orbital fibroblasts, lymphocytes, or macrophages) appears to represent an as yet unrecognized antiinflammatory mechanism by which orbital radiotherapy exerts some of its beneficial therapeutic effects in patients with active GO. It is possible that a shift toward a more favorable IL-1RA/IL-1{alpha}-ratio contributes at least in part to the superior clinical results obtained when combining glucocorticosteroids (which suppress IL-1RA) and orbital irradiation (which stimulates IL-1RA) for treatment of GO.


   Footnotes
 
1 This work was supported by a postdoctoral fellowship (to T.M.) from Städtisches Klinikum-West (Leipzig, Germany), a grant from Deutsche Forschungsgemeinschaft (Bonn, Germany; He 1485/5–2, Gerhard-Hess program), and a grant from Forum Schilddrüse e.V., Germany. Back

Received July 20, 1999.

Revised August 12, 1999.

Accepted August 19, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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