Production of interleukin-1 receptor antagonist by human articular chondrocytes
© BioMed Central Ltd 2002
Received: 25 October 2001
Accepted: 11 March 2002
Published: 8 April 2002
Interleukin-1 receptor antagonist (IL-1Ra) is a natural IL-1 inhibitor possessing anti-inflammatory properties. IL-1Ra is produced as different isoforms, one secreted (sIL-1Ra) and three intracellular (icIL-1Ra1, icIL-1Ra2 and icIL-1Ra3), derived from the same gene. We examined the production of IL-1Ra species by cultured human articular chondrocytes in response to various cytokines. The levels of IL-1Ra were undetectable in culture supernatants of untreated cells, but were significantly increased by IL-1β. Cell lysates contained very low levels of IL-1Ra, even in response to IL-1β, suggesting that chondrocytes produce predominantly sIL-1Ra. IL-6, which had no effect on its own, enhanced the effect of IL-1β, while dexamethasone prevented the response. We observed by RT-PCR that IL-1β and IL-6 induced primarily the production of sIL-1Ra mRNA. Furthermore, IL-1β alone or combined with IL-6 increased the levels of nascent unspliced sIL-1Ra mRNA, suggesting that sIL-1Ra expression is regulated at the transcriptional level. Reporter gene assays in immortalized chondrocytes, C-20/A4, consistently showed increased sIL-1Ra promoter activity in response to IL-1β and IL-6. In conclusion, human articular chondrocytes produce sIL-1Ra in response to IL-1β and IL-6. The production of sIL-1Ra by chondrocytes may have a protective effect against articular inflammatory and catabolic responses.
Keywordscytokines glucocorticoids human articular chondrocytes IL-1 receptor antagonist
IL-1Ra is a member of the IL-1 family that binds to IL-1 receptors but does not induce any intracellular response. IL-1Ra prevents the interaction between IL-1 and its cell surface receptors, and thus competitively inhibits the biological effects of IL-1 [1–3]. The administration of IL-1Ra ameliorates the course of arthritis in several experimental models . In addition, therapeutic administration of IL-1Ra has beneficial effects in patients with rheumatoid arthritis (RA) .
IL-1Ra exists in four different isoforms derived from the same gene . One isoform (sIL-1Ra) is secreted, while the three others (icIL-1Ra1, icIL-1Ra2 and icIL-1Ra3) are intracellular. The sIL-1Ra and icIL-1Ra1 mRNAs are transcribed from different promoters and contain isoform-specific 5' sequences due to alternative splicing [7, 8]. The mRNA for icIL-1Ra2 is transcribed from the icIL-1Ra1 promoter, but contains an additional exon . Finally, icIL-1Ra3 is produced by alternative translation initiation from the sIL-1Ra transcript .
The expression of these various isoforms is cell-type specific and stimulus specific (see  for a review). In mice, sIL-1Ra is found predominantly in peripheral blood cells, the lungs, the spleen and the liver following lipopolysaccharide injection. icIL-1Ra1 is constitutively expressed in epithelial cells, and is inducible in monocytes and macrophages. The mRNA for icIL-1Ra2 has been detected in monocytes, neutrophils, keratinocytes and activated fibroblasts; however, the existence of a corresponding protein was never demonstrated. icIL-1Ra3 is produced in lipopolysaccharide-stimulated neutrophils and in monocytes.
The major biological role of extracellular sIL-1Ra is to modulate the effects of IL-1 at the cell surface. The intracellular isoforms may be released from cells under some circumstances, but have also been suggested to perform important regulatory roles within cells.
IL-1 plays an important role in the mechanisms leading to cartilage breakdown by increasing the production of matrix metalloproteinases and by inhibiting the production of type II collagen and aggrecan [11, 12]. Exogenous administration of IL-1Ra exerts chondroprotective effects in different in vitro and in vivo models [13–15]. However, little information is available on local IL-1Ra production in cartilage . In addition, IL-1Ra isoforms expressed in chondrocytes have not been characterized. The aim of the present study was to gain further information on the regulation of IL-1Ra isoform production by human articular chondrocytes in response to various cytokines.
Materials and methods
Cell culture reagents and DNAse I were purchased from Life Technologies AG (Basel, Switzerland). Cytokines were obtained from R&D Systems (Abingdon, Oxon, UK) and dexamethasone from Sigma (Fluka Chemie AG, Buchs, Switzerland). FuGENE 6 was purchased from Roche Molecular Biochemicals (Rotkreuz, Switzerland).
Cartilage was obtained from patients undergoing joint replacement for osteoarthritis and was cultured as previously described (see Supplementary material for details) [17, 18]. C-20/A4-immortalized human chondrocytes  were cultured in high-glucose (4.5 g/l) DMEM/F12 (1:1, v/v), supplemented with 10% FCS.
ELISA for human IL-1Ra
Freshly isolated or subcultured chondrocytes were seeded in 96-well plates (40,000 cells/well). After incubation with cytokines, culture supernatants were collected and cell lysates were obtained after three cycles of freezing and thawing. IL-1Ra concentrations in supernatants and lysates were measured by ELISA . The sensitivity of this assay is 78 pg/ml. Results shown are the mean ± SEM of three determinations in a representative experiment.
RNA isolation and RT-PCR
Total RNA was prepared using the Tripure reagent (Roche Molecular Biochemicals). RNA (3 μg) was reverse-transcribed using avian myeloblastosis virus-RT, and PCR was performed using appropriate primer pairs and conditions (see Supplementary material).
Reporter gene assays
C-20/A4 cells were transfected with 0.4 μg plasmid DNA using the FuGENE 6 reagent (see Supplementary material for details). Luciferase activity was determined with the assay system from Promega (Wallisellen, Switzerland) and was normalized for protein content measured with the protein assay reagent from BioRad (Reinach, Switzerland). Results shown are the mean ± SEM of three determinations in a representative experiment.
The significance of differences was calculated by analysis of variance.
IL-1Ra production in articular chondrocytes
IL-4, IL-10, and transforming growth factor (TGF)-beta (10 ng/ml), used either alone or in the presence of IL-1β, were devoid of any stimulatory effect on IL-1Ra production (data not shown).
Cell lysates contained very low levels of IL-1Ra, which were mostly below the detection limit of our ELISA, even after stimulation with IL-1β and IL-6/sIL-6R (data not shown). These results suggest that articular chondrocytes produce essentially the secreted isoform, sIL-1Ra.
Dexamethasone (10-7 M) specifically inhibited the stimulatory effect of IL-1β and IL-6/sIL-6R on IL-1Ra production (see Supplementary material).
Expression of IL-1Ra mRNA
Activity of the human 1680 bp sIL-1Ra promoter in chondrocytes
In the present paper, we report that human articular chondrocytes produce IL-1Ra in response to IL-1β, thereby confirming a previously published observation . In addition, IL-6 in combination with its soluble receptor sIL-6R enhanced this response, while IL-4, IL-10 and TGF-β were devoid of any stimulatory effect. Cell lysates contained very little IL-1Ra, suggesting that chondrocytes mainly produce the secreted isoform. Using specific primers, we confirmed that articular chondrocytes produce predominantly sIL-1Ra mRNA.
A similar stimulation of sIL-1Ra production by IL-1β, which was also enhanced by IL-6, was observed in hepatocytes, allowing the classification of IL-1Ra as an acute phase protein . In both hepatocytes and chondrocytes, IL-1β and IL-6 stimulated sIL-1Ra expression at the level of gene transcription. Furthermore, this response was shown to be mediated by activation of NF-κB and CCAAT/enhancer binding protein (C/EBP) in hepatocytes. Interactions between the two transcription factors NF-κB and C/EBP have been suggested to mediate the synergistic effects of IL-1 and IL-6 on acute phase protein production .
Glucocorticoids have been reported to increase or decrease IL-1Ra production depending on the cell type [20, 22]. In chondrocytes, we observed a specific inhibition of IL-1Ra expression by dexamethasone. This finding suggests that IL-1Ra does not contribute to the anti-inflammatory effect of glucocorticoids in the cartilage.
The physiologic function of endogenous IL-1Ra has been clearly demonstrated in several studies using IL-1Ra gene knockout mice, which have an earlier onset of collagen-induced arthritis and more severe synovitis, often accompanied by tissue damage . Furthermore, when bred into the BALB/cA background, IL-1Ra knockout mice develop spontaneous chronic polyarthritis, reproducing some of the clinical and biological features of RA . These findings suggest that IL-1Ra plays an important role in the modulation of articular inflammation and in subsequent cartilage breakdown. Our results show that IL-1Ra is produced by articular chondrocytes in response to IL-1 and IL-6, two cytokines present in significant amount in inflamed joints. Secreted IL-1Ra in turn then probably modulates the effects of IL-1 in the cell microenvironment. Local production of IL-1Ra might thus be part of a negative feedback mechanism initiated by these cytokines and may exert a chondroprotective effect against IL-1-mediated cartilage lesions during physiologic and pathologic processes, including RA and osteoarthritis.
Human articular chondrocytes produce sIL-1Ra in response to IL-1β and IL-6. This effect reflects increased transcription from the sIL-1Ra promoter. The local production of sIL-1Ra in cartilage may have a protective effect against articular inflammatory and catabolic responses.
Supplementary materials and methods
Cartilage was obtained from patients undergoing knee joint or hip joint replacement for osteoarthritis. Chondrocytes were isolated by collagenase digestion, as previously described [17, 18], and cultured in DMEM containing L-glutamine, streptomycin, penicillin and 10% FCS. Isolated chondrocytes were either used immediately or were expanded and subcultured several times. Cells were stimulated with cytokines 24 hours after seeding.
Proteoglycan synthesis was evaluated by measuring the aggrecan concentration in culture supernatants by enzyme-amplified sensitivity immunoassay (Biosource Europe S.A., Nivelles, Belgium). Results are presented as the mean ± SEM of three determinations.
Reverse transcription polymerase chain reaction
PCR primer location and sequence
Annealing temperature (°C)
Product length (bp)
Reporter gene assays
The 1680 bp human sIL-1Ra promoter region was recovered from plasmid psRA1680  by digestion with Hind III, and it was inserted upstream of the luciferase reporter gene in pGL3-enhancer (Promega) to yield plasmid enh-1680. Correct insertion of the promoter fragment was verified by DNA sequencing. C-20/A4 cells were seeded in 24-well plates (20,000 cells/well) and transfected 24 hours later in DMEM containing 1% FCS with 0.4 μg DNA and 1.2 μl FuGENE 6. After 4 hours, cells were switched to DMEM/F12 containing 10% FCS. Cytokines were added 24 hours after transfection.
Specific inhibition of IL-1Ra production by dexamethasone
Activity of the human 1680 bp sIL-1Ra promoter in chondrocytes
The enh-1680 reporter construct contains 1680 bp human sIL-1Ra promoter region (Supplementary Figure 1, region Ps). This region was shown previously to contain DNA cis-acting elements responsible for cell-type-specific expression of sIL-1Ra . It contains, in particular, binding sites for the transcription factors NF-κB and C/EBP, which are involved in the activation of sIL-1Ra transcription by IL-1 and IL-6 in HepG2 cells . Human osteoarthritis chondrocytes are difficult to transfect [S1] and our transfection efficiencies were not sufficient to yield detectable luciferase activities using the enh-1680 plasmid. As an alternative, we used C-20/A4-immortalized human chondrocytes for these experiments. These cells exhibit responses to IL-1 similar to those described in primary human chondrocytes .
CCAAT/enhancer binding protein
Dubecco's modified Eagle's medium
enzyme-linked immunosorbent assay
NUT.MIX.F-12 (HAM) Media
fetal calf serum
intracellular IL-1 receptor antagonist isoform
IL-1 receptor antagonist
polymerase chain reaction
secreted IL-1 receptor antagonist isoform
soluble IL-6 receptor
standard error of the mean
transforming growth factor.
Gaby Palmer and Pierre-Andre Guerne contributed equally to this work. The authors thank Cristiana Juge for her help with quantitative real-time PCR and Nathalie Pellegrinelli for her expert technical assistance. This work was supported by the Swiss National Science Foundation (grant 3100–064123.00/1 to PAG, and grants 3200–054955.98 and 3231–05454.98 to CG) and the Albert-Boeni Foundation.
- Dripps DJ, Brandhuber BJ, Thompson RC, Eisenberg SP: Interleukin-1 (IL-1) receptor antagonist binds to the 80-kDa IL-1 receptor but does not initiate IL-1 signal transduction. J Biol Chem. 1991, 266: 10331-10336.PubMedGoogle Scholar
- Granowitz EV, Clark BD, Mancilla J, Dinarello CA: Interleukin-1 receptor antagonist competitively inhibits the binding of interleukin-1 to the type II interleukin-1 receptor. J Biol Chem. 1991, 266: 14147-14150.PubMedGoogle Scholar
- Greenfeder SA, Nunes P, Kwee L, Labow M, Chizzonite RA, Ju G: Molecular cloning and characterization of a second subunit of the interleukin 1 receptor complex. J Biol Chem. 1995, 270: 13757-13765. 10.1074/jbc.270.23.13757.PubMedView ArticleGoogle Scholar
- Gabay C: IL-1 inhibitors: novel agents in the treatment of rheumatoid arthritis. Expert Opin Investig Drugs. 2000, 9: 113-127.PubMedView ArticleGoogle Scholar
- Bresnihan B, Alvaro-Gracia JM, Cobby M, Doherty M, Domljan Z, Emery P, Nuki G, Pavelka K, Rau R, Rozman B, Watt I, Williams B, Aitchison R, McCabe D, Musikic P: Treatment of rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist. Arthritis Rheum. 1998, 41: 2196-2204. 10.1002/1529-0131(199812)41:12<2196::AID-ART15>3.3.CO;2-U.PubMedView ArticleGoogle Scholar
- Arend WP, Malyak M, Guthridge CJ, Gabay C: Interleukin-1 receptor antagonist: role in biology. Annu Rev Immunol. 1998, 16: 27-55. 10.1146/annurev.immunol.16.1.27.PubMedView ArticleGoogle Scholar
- Hannum CH, Wilcox CJ, Arend WP, Joslin FG, Dripps DJ, Heimdal PL, Armes LG, Sommer A, Eisenberg SP, Thompson RC: Interleukin-1 receptor antagonist activity of a human interleukin-1 inhibitor. Nature. 1990, 343: 336-340. 10.1038/343336a0.PubMedView ArticleGoogle Scholar
- Haskill S, Martin G, Van Le L, Morris J, Peace A, Bigler CF, Jaffe GJ, Hammerberg C, Sporn SA, Fong S, Arend WP, Ralph P: cDNA cloning of an intracellular form of the human interleukin 1 receptor antagonist associated with epithelium. Proc Natl Acad Sci USA. 1991, 88: 3681-3685.PubMedPubMed CentralView ArticleGoogle Scholar
- Muzio M, Polentarutti N, Sironi M, Poli G, De Gioia L, Introna M, Mantovani A, Colotta F: Cloning and characterization of a new isoform of the interleukin 1 receptor antagonist. J Exp Med. 1995, 182: 623-628.PubMedView ArticleGoogle Scholar
- Malyak M, Guthridge JM, Hance KR, Dower SK, Freed JH, Arend WP: Characterization of a low molecular weight isoform of IL-1 receptor antagonist. J Immunol. 1998, 161: 1997-2003.PubMedGoogle Scholar
- Goldring MB, Birkhead J, Sandell LJ, Kimura T, Krane SM: Interleukin 1 suppresses expression of cartilage specific types II and IX collagens and increases types I and III collagens in human chondrocytes. J Clin Invest. 1988, 82: 2026-2037.PubMedPubMed CentralView ArticleGoogle Scholar
- Tyler JA: Articular cartilage cultured with catabolin (pig interleukin 1) synthetizes a decreased number of normal proteoglycan molecules. Biochem J. 1985, 227: 869-878.PubMedPubMed CentralView ArticleGoogle Scholar
- Baragi VM, Renkiewicz RR, Jordan H, Bonadio J, Hartman JW, Roessler BJ: Transplantation of transduced chondrocytes protects articular cartilage from interleukin 1-induced extracellular matrix degradation. J Clin Invest. 1995, 96: 2454-2460.PubMedPubMed CentralView ArticleGoogle Scholar
- Fernandes J, Tardif G, Martel-Pelletier J, Lascau-Coman V, Dupuis M, Moldovan F, Sheppard M, Krishnan BR, Pelletier JP: In vivo transfer of interleukin-1 receptor antagonist gene in osteoarthritic rabbit knee joints: prevention of osteoarthritis progression. Am J Pathol. 1999, 154: 1159-1169.PubMedPubMed CentralView ArticleGoogle Scholar
- Muller-Ladner U, Roberts CR, Franklin BN, Gay RE, Robbins PD, Evans CH, Gay S: Human IL-1Ra gene transfer into human synovial fibroblasts is chondroprotective. J Immunol. 1997, 158: 3492-3498.PubMedGoogle Scholar
- Pelletier JP, Mineau F, Ranger P, Tardif G, Martel-Pelletier J: The increased synthesis of inducible nitric oxide inhibits IL-1ra synthesis by human articular chondrocytes: possible role in osteoarthritic cartilage degradation. Osteoarthritis Cartilage. 1996, 4: 77-84.PubMedView ArticleGoogle Scholar
- Lotz M, Clark-Lewis I, Ganu V: HIV-1 transactivator protein Tat induces proliferation and TGF beta expression in human articular chondrocytes. J Cell Biol. 1994, 124: 365-371.PubMedView ArticleGoogle Scholar
- Klareskog L, Forsum U, Malmnas Tjernlund UK, Kabelitz D, Wigren A: Appearance of anti-HLA-DR-reactive cells in normal and rheumatoid synovial tissue. Scand J Immunol. 1981, 14: 183-192.PubMedView ArticleGoogle Scholar
- Goldring MB, Birkhead JR, Suen LF, Yamin R, Mizuno S, Glowacki J, Arbiser JL, Apperley JF: Interleukin-1 beta-modulated gene expression in immortalized human chondrocytes. J Clin Invest. 1994, 94: 2307-2316.PubMedPubMed CentralView ArticleGoogle Scholar
- Gabay C, Smith MF, Eidlen D, Arend WP: Interleukin 1 receptor antagonist (IL-1Ra) is an acute-phase protein. J Clin Invest. 1997, 99: 2930-2940.PubMedPubMed CentralView ArticleGoogle Scholar
- Guerne PA, Desgeorges A, Jaspar JM, Relic B, Peter R, Hoffmeyer P, Dayer JM: Effects of IL-6 and its soluble receptor on proteoglycan synthesis and NO release by human articular chondrocytes: comparison with IL-1. Modulation by dexa-methasone. Matrix Biol. 1999, 18: 253-260. 10.1016/S0945-053X(99)00021-9.PubMedView ArticleGoogle Scholar
- Sauer J, Castren M, Hopfner U, Holsboer F, Stalla GK, Arzt E: Inhibition of lipopolysaccharide-induced monocyte interleukin-1 receptor antagonist synthesis by cortisol: involvement of the mineralocorticoid receptor. J Clin Endocrinol Metab. 1996, 81: 73-79.PubMedGoogle Scholar
- Ma Y, Thornton S, Boivin GP, Hirsh D, Hirsch R, Hirsch E: Altered susceptibility to collagen-induced arthritis in transgenic mice with aberrant expression of interleukin-1 receptor antagonist. Arthritis Rheum. 1998, 41: 1798-1805. 10.1002/1529-0131(199810)41:10<1798::AID-ART11>3.3.CO;2-C.PubMedView ArticleGoogle Scholar
- Horai R, Saijo S, Tanioka H, Nakae S, Sudo K, Okahara A, Ikuse T, Asano M, Iwakura Y: Development of chronic inflammatory arthropathy resembling rheumatoid arthritis in interleukin 1 receptor antagonist-deficient mice. J Exp Med. 2000, 191: 313-320. 10.1084/jem.191.2.313.PubMedPubMed CentralView ArticleGoogle Scholar