Skip Navigation


Rheumatology Advance Access originally published online on February 28, 2003
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
42/3/442    most recent
keg146v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (11)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Möller, B.
Right arrow Articles by Mühl, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Möller, B.
Right arrow Articles by Mühl, H.
Related Collections
Right arrow Osteoarthritis and Cartilage
Right arrow Spondylarthropathies
Right arrow Rheumatoid Arthritis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Rheumatology 2003; 42: 442-445
© 2003 British Society for Rheumatology

Interferon-{gamma} induces expression of interleukin-18 binding protein in fibroblast-like synoviocytes

B. Möller, J. Paulukat1, M. Nold1, M. Behrens2, N. Kukoc-Zivojnov, J. P. Kaltwasser, J. Pfeilschifter1 and H. Mühl1

Rheumazentrum Rhein-Main/Medizinische Klinik III,
1 Pharmazentrum Frankfurt and
2 Institut für Kardiovaskuläre Physiologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. To investigate expression of the endogenous antagonist of interleukin 18 (IL-18) bioactivity, IL-18 binding protein isoform a (IL-18BPa), in fibroblast-like synoviocytes (FLS).

Methods. Long-term cultured FLS from rheumatoid arthritis (RA), osteoarthritis (OA) and spondylarthropathy patients were analysed for spontaneous and cytokine-induced IL-18BPa expression. Messenger RNA and release of IL-18BPa were assessed by semi-quantitative and quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) as well as immunoblot analysis, respectively.

Results. All investigated FLS cultures expressed low amounts of IL-18BPa transcripts. However, there was no detectable release of IL-18BPa from unstimulated synoviocytes. Of the investigated cytokines, only interferon (IFN)-{gamma} markedly up-regulated IL-18BPa mRNA levels. Induction was accompanied by release of IL-18BPa immunoreactvity from FLS. Conditioned media from IFN-{gamma}-stimulated FLS cultures reduced IL-12/IL-18-dependent IFN- production by peripheral blood mononuclear cells.

Conclusion. The present data imply that IFN--activated synoviocytes mediate a negative feedback loop via IL-18BPa, which may limit IL-18 biological activity in arthritis.

KEY WORDS: IFN-{gamma}, IL-18BP, Synoviocyte, Fibroblast, Rheumatoid arthritis.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The proinflammatory cytokine interleukin 18 (IL-18) is one current focus of research on rheumatoid arthritis (RA) pathogenesis [1, 2]. IL-18 was first identified as an interferon (IFN)-{gamma}-inducing factor [1], but IL-18 is also capable of directly inducing expression of tumour necrosis factor-{alpha} (TNF-{alpha}) [1, 2]. Although IFN-{gamma} levels are often low, it is detectable in all histological variants of RA synovitis [3]. Data concerning the role of IFN-{gamma} in RA synovitis are conflicting. Addition of IFN-{gamma} to T cell/monocyte co-cultures enhances TNF-{alpha} production [4]. In contrast, IFN-{gamma} can down-regulate parameters of joint destruction such as IL-1 [5], matrix metalloproteinases and proliferation of fibroblast-like synoviocytes (FLS) [6]. Contradicting results were also obtained in clinical trials evaluating the therapeutic potential of IFN-{gamma} in RA [7, 8].

Here we investigated expression of IL-18 binding protein (IL-18BP), a decoy receptor of IL-18 [9]. Serum levels of IL-18BP are augmented in septic patients [10], and injection of human IL-18BP inhibits lipopolysaccharide-induced IFN-{gamma} in mice by 90% [9]. In the present study on FLS cultures, we evaluated expression of IL-18BPa, the most abundant splice variant of human IL-18BP, which exhibits the highest affinity for IL-18 [11].


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Patients
IL-18BPa expression was analysed in long-term cultured FLS from 20 RA [12], three OA, and four spondylarthropathy patients [13]. All patient materials were obtained after receiving informed consent.

FLS cultures
FLS were isolated either from dissected synovial membrane tissues or from synovial fluids [14]. A total of 5x105 FLS from five RA patients was cultured for 24 h in duplicate (culture A and B, Fig. 1Go), as unstimulated control, or exposed to IL-1ß (1 ng/ml), IL-2 (100 ng/ml), IL-12 (1 ng/ml), IL-15 (100 ng/ml), IL-18 (200 ng/ml), IFN-{gamma} (5 ng/ml) or TNF-{alpha} (10 ng/ml) (Pepro Tech, London). Cells were also stimulated with the combinations IL-12+IL-18 and IL-1ß+TNF-{alpha}+IFN-{gamma}.



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 1. IL-18BPa gene expression by IFN-{gamma} in FLS. (A) Each two FLS cultures (denoted A, B) from two RA patients remained unstimulated (top lane), or were stimulated (bottom lane) for 48 h with IFN-{gamma} (5 ng/ml). In unstimulated FLS cultures, over 30 PCR cycles were needed to detect IL-18BPa. The IL-18BPa mRNA detection limit was remarkably reduced in corresponding, IFN-{gamma}-stimulated FLS cultures (less than 25 cycles). (B) Other cytokines investigated did not enhance IL-18BPa expression, as shown by RT-PCR remaining negative after 30 cycles, similar to unstimulated FLS. The combination IFN-{gamma}+TNF-{alpha}+IL-1ß showed a minor increase in IL-18BPa mRNA compared with IFN-{gamma} alone. One representative RT-PCR of two independently performed experiments is shown. (C) IL-18BPa induction by IFN-{gamma} was confirmed by quantitative real-time PCR on total RNA from two patients.

 

IL-18BP reverse transcriptase-polymerase chain reaction (RT-PCR)
IL-18BP primers were appropriate for amplification of IL-18BPa and c; forward: 5'-tgccactgcctcagttagaagc-3'; reverse: 5'-acgagcacacaggagaagttgg-3'; annealing temperature 56°C; cycles of PCR as indicated; length of IL-18BPa amplicon: 342 bp. ß-Actin gene: forward: 5'-tcgagcacggcatcgtcaccaact-3'; reverse: 5'-accgctcattgccaatggtgatga-3'; annealing temperature: 60°C; 30 cycles of PCR; length of the amplicon 552 bases.

Analysis of IL-18BPa expression by quantitative real-time PCR analysis
Primers and probe for IL-18BPa were: forward 5'-acctcccaggccgactg-3'; reverse 5'-ccttgcacagctgcgtacc-3'; probe 5'-caccagccgggaacgtggga-3'. For GAPDH, we used a pre-developed assay (Applied Biosystems, Weiterstadt). PCR was performed on the AbiPrism 7700 Sequence Detector (Applied Biosystems) as follows: one initial step at 50°C for 2 min and 95°C for 10 min was followed by 40 cycles at 95°C for 15 s and 60°C for 1 min.

Detection of IL-18BPa by immunoblotting
A total of 2x106 FLS from four RA patients was cultured in serum-free supplemented Ham's F10 medium (Gibco, Karlsruhe, Germany), as unstimulated control or stimulated with IFN-{gamma} (5 ng/ml). After 48 h, cell-free supernatants were precipitated with trichloroacetic acid, separated by 10% SDS-PAGE (sodium dodecyl sulphate, polyacrylamide gel electrophoresis) and IL-18BPa was detected using a rabbit polyclonal antiserum [15].

Interferon-{gamma} production by IL-12/IL-18-stimulated peripheral blood mononuclear cells (PBMC) cultivated in FLS-derived conditioned medium
To investigate whether conditioned media from IFN-{gamma}-stimulated FLS may contain IL-18BPa activity, the following protocol was performed [15]: 106 FLS/ml culture medium from four different RA patients were kept as control or stimulated with IFN-{gamma} (20 ng/ml, 16 h). Thereafter, FLS were washed with phosphate-buffered saline (3x) and incubated in control medium (48 h, serum free). Cell-free supernatants were concentrated 5-fold using Ultra-free-4 Biomax 10K centrifugal filters (Millipore, Bedford). Concentrated conditioned media were pre-incubated for 30 min without stimuli or with IL-12/IL-18 (20 ng/ml each). PBMC were isolated as described, resuspended in conditioned media and production of IFN-{gamma} was determined after 24 h by enzyme-linked immunosorbent assay (ELISA) (Pharmingen, Hamburg). Experiments were performed in duplicate, statistics were done by Wilcoxon test of two-tied groups.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Constitutive IL-18BPa mRNA expression was detected by RT-PCR (35 cycles) in all 27 unstimulated FLS cultures investigated (data not shown). Only one amplicon with the length representing IL-18BPa was detected. IFN-{gamma} strongly up-regulated IL-18BPa mRNA (Fig. 1AGo). In unstimulated FLS, more than 30 cycles were needed to detect IL-18BPa amplicons, whereas the IL-18BPa mRNA detection limit was reduced below 25 cycles in cultures challenged with IFN-{gamma}. TNF-{alpha}, IL-1ß, IL-2 (data not shown), IL-12, IL-15 (data not shown), IL-18, IL-12+IL-18 showed no IL-18BPa-inducing effect, and IL-1ß+TNF-{alpha} did not significantly enhance IFN-{gamma}-induced IL-18BPa mRNA levels (Fig. 1BGo). Quantitative real-time PCR confirmed induction of IL-18BPa by IFN-{gamma} in FLS (Fig. 1CGo). Similar results were obtained using human dermal fibroblasts (data not shown).

IFN-{gamma}-induced mRNA in RA-FLS was accompanied by secretion of IL-18BPa immunoreactivity (Fig. 2AGo). Immunoreactivity appeared with a molecular mass of about 45 kDa, which is in keeping with previous reports [11, 15]. As shown in Fig. 2BGo, compared with conditioned media obtained from unstimulated cells, conditioned media from IFN-{gamma}-stimulated FLS reduced production of IFN-{gamma} in PBMC exposed to IL-12/IL-18.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 2. IFN-{gamma} mediates release of IL-18BPa from FLS. (A) FLS cultures from RA patients were stimulated with IFN-{gamma} (5 ng/ml). After 48 h, secreted IL-18BPa was detected by immunoblotting. Representative results from FLS cultures of two patients are shown. Right-hand panel shows negative control (pORF-control) and positive control (p-ORF IL-18BPa overexpressed in DLD-1 cells) for IL-18BPa immunoreactivity [15]. (B) Control- and IFN-{gamma}-conditioned media from four FLS cultures were used to resuspend IL-12/IL-18-PBMC cultures (see Materials and methods). Data are expressed as percentage of IFN-{gamma} production by IL-12/IL-18-stimulated PBMC resuspended in control conditioned FLS medium (*n=8, P=0.012).

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
IL-18 can activate both T cells and macrophages in the microenviroment of RA synovitis. IFN-{gamma}, IL-1ß and TNF-{alpha} are supposed to be induced in RA joints [1–3] and production of TNF-{alpha} and IL-1ß is of paramount relevance, as highlighted by successful anti-cytokine therapies in RA [16, 17]. IL-18 bioactivity in RA might be controlled by IL-18BPa and this was addressed in this investigation. Expression of IL-18BPa mRNA in FLS and in dermal fibroblasts was markedly augmented by IFN-{gamma}. Gene induction was accompanied by release of IL-18BPa and coincided with release of an activity from FLS that reduced IL-12/IL-18-induced IFN-{gamma} in PBMC. IFN-{gamma}-induced IL-18BPa has been recognized in several different cell types [15, 18, 19]. Here we present the first data on induction in resident synoviocytes. Up-regulation of IL-18BPa underscores the importance of these cells in the control of IL-18 bioactivity in synovitis. The present data suggest that FLS contribute to the complex network of immunoregulation that ensures control of primarily macrophage-derived IL-18 during synovitis. Induction of IL-18BPa was characteristic for IFN-{gamma}. A murine model of septic antigen-induced arthritis illustrates that IFN-{gamma} can display proinflammatory properties when given in the initiation phase. In contrast, IFN-{gamma} ameliorates joint inflammation when given later on [20]. Detrimental functions of IFN-{gamma} may include a more effective antigen presentation at the onset of disease, whilst inhibition of IL-18 through IFN-{gamma}-induced IL-18BPa could represent a negative feedback mechanism upon established inflammation. Such a feedback principle concurs with reported overexpression of IFN-{gamma} in IFN-{gamma} receptor-deficient mice evaluated in collagen-induced arthritis [21]. In patients with adult-onset Still's disease [22], in Crohn's disease [19] and in patients with sepsis [10], levels of IFN-{gamma}-inducing IL-18 appear to correlate with expression of IL-18BP. These observations suggest a close association of IL-18 with expression of its decoy receptor in inflammatory diseases.

Induction of IL-18BPa by IFN-{gamma} could prove to be a crucial endogenous corrective that counterregulates IL-18-mediated leucocyte activation in rheumatoid arthritis. Although this feedback loop is functional in RA materials, there is still IL-18 bioactivity demonstrable in RA synovial fluids, despite the presence of an IL-18 inhibitory activity [23]. Therefore, therapeutic administration of IL-18BP in patients may modulate the cytokine balance, thereby ameliorating established arthritis, as recently observed in a RA animal model [24].


   Acknowledgments
 
This work was supported by the Riese- and the Klein-Foundation. We thank S. Rehart for providing synovial material. We thank J. Bauer, S. Garkisch and S. Höfler for technical assistance.


   Notes
 
Correspondence to: B. Möller, Rheumazentrum Rhein-Main, Marienburgstraße 2, D-60528 Frankfurt/M., Germany. E-mail: b.moeller{at}em.uni-frankfurt.de Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Dinarello CA, Novick D, Puren AJ et al. Overview of interleukin-18: more than an interferon-{gamma}-inducing factor. J Leukoc Biol 1998;63:658–64.[Abstract]
  2. Gracie JA, Forsey RJ, Chan WL et al. A proinflammatory role for IL-18 in rheumatoid arthritis. J Clin Invest 1999;104:1393–401.[Web of Science][Medline]
  3. Klimiuk PA, Goronzy JJ, Bjornsson J, Beckenbaugh RD, Weyand CM. Tissue cytokine patterns distinguish variants of rheumatoid synovitis. Am J Pathol 1997;151:1311–9.[Abstract]
  4. Sebbag M, Parry SL, Brennan FM, Feldmann M. Cytokine stimulation of T lymphocytes regulates their capacity to induce monocyte production of tumor necrosis factor-{alpha}, but not interleukin-10: possible relevance to pathophysiology of rheumatoid arthritis. Eur J Immunol 1997;27:624–32.[Web of Science][Medline]
  5. Ghezzi P, Dinarello CA. IL-1 induces IL-1. III. Specific inhibition of IL-1 production by IFN-{gamma}. J Immunol 1988;140:4238–44.[Abstract]
  6. Alvaro-Gracia JM, Zvaifler NJ, Firestein GS. Cytokines in chronic inflammatory arthritis. Mutual antagonism between interferon-{gamma} and tumor necrosis factor-{alpha} on HLA-DR expression, proliferation, collagenase production, and granulocyte–macrophage colony-stimulating factor production by rheumatoid arthritis synoviocytes. J Clin Invest 1990;86:1790–8.[Medline]
  7. Lemmel EM, Brackertz D, Franke M et al. Results of a multicenter placebo-controlled double-blind randomized phase III clinical study of treatment of rheumatoid arthritis with recombinant interferon-{gamma}. Rheumatol Int 1988;8:87–93.[CrossRef][Web of Science][Medline]
  8. Cannon GW, Pincus SH, Emkey RD et al. Double-blind trial of recombinant {gamma}-interferon versus placebo in the treatment of rheumatoid arthritis. Arthritis Rheum 1989;32:964–73.[Web of Science][Medline]
  9. Novick D, Kim SH, Fantuzzi G, Reznikov LL, Dinarello CA, Rubinstein M. Interleukin-18 binding protein: a novel modulator of the TH1 cytokine response. Immunity 1999;10:127–36.[CrossRef][Web of Science][Medline]
  10. Novick D, Schwartsburd B, Pinkus R et al. A novel IL-18bp ELISA shows elevated serum IL-18bp in sepsis and extensive decrease of free IL-18. Cytokine 2001;69:5025–30.
  11. Kim SH, Eisenstein M, Reznikov L et al. Structural requirements of six naturally occurring isoforms of the IL-18 binding protein to inhibit IL-18. Proc Natl Acad Sci USA 2000;97:1190–5.[Abstract/Free Full Text]
  12. Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24.[Web of Science][Medline]
  13. Dougados M, van der Linden S, Juhlin R et al. The European Spondylarthropathy Study Group preliminary criteria for the classification of spondylarthropathy. Arthritis Rheum 1991;34:1218–27.[Web of Science][Medline]
  14. Möller B, Kukoc-Zivojnov N, Kessler U et al. Interleukin-18 expression and its monokine directed function in rheumatoid arthritis. Rheumatology 2001;40:302–9.[Abstract/Free Full Text]
  15. Paulukat J, Bosmann M, Nold M et al. Expression and release of IL-18 binding protein in response to interferon-{gamma}. J Immunol 2001;167:7038–43.[Abstract/Free Full Text]
  16. Feldmann M, Charles P, Taylor P, Maini RN. Biological insights from clinical trials with anti-TNF therapy. Springer Semin Immunopathol 1998;20:21–8.
  17. Jiang Y, Genant HK, Watt I et al. A multicenter, double-blind, dose-ranging, randomized, placebo-controlled study of recombinant human interleukin-1 receptor antagonist in patients with rheumatoid arthritis: radiologic progression and correlation of Genant and Larsen scores. Arthritis Rheum 2000;43:1001–9.[CrossRef][Web of Science][Medline]
  18. Mühl H, Kämpfer H, Bosmann M, Frank S, Radeke H, Pfeilschifter J. Interferon-{gamma} mediates gene expression of IL-18 binding protein in nonleukocytic cells. Biochem Biophys Res Commun 2000;267:960–3.[CrossRef][Web of Science][Medline]
  19. Corbaz A, ten Hove T, Herren S et al. IL-18-binding protein expression by endothelial cells and macrophages is up regulated during active Crohn's disease. J Immunol 2002;168:3608–16.[Abstract/Free Full Text]
  20. Puliti M, von Hunolstein C, Bistoni F, Mosci P, Orefici G, Tissi L. Influence of interferon-{gamma} administration on the severity of experimental group B streptococcal arthritis. Arthritis Rheum 2000;43:2678–86.[CrossRef][Medline]
  21. Matthys PK, Vermeire T, Mitera T, Heremans H, Huang S, Billiau A. Anti-IL-12 antibody prevents the development and progression of collagen-induced arthritis in IFN-{gamma} receptor-deficient mice. Eur J Immunol 1998;28:2143–51.[CrossRef][Web of Science][Medline]
  22. Kawashima M, Yamamura M, Taniai M et al. Levels of interleukin-18 and its binding inhibitors in the blood circulation of patients with adult-onset Still's disease. Arthritis Rheum 2001;44:550–60.[CrossRef][Web of Science][Medline]
  23. Yamamura M, Kawashima M, Taniai M et al. Interferon-{gamma}-inducing activity of interleukin-18 in the joint with rheumatoid arthritis. Arthritis Rheum 2001;44:275–85.[CrossRef][Web of Science][Medline]
  24. Plater-Zyberk C, Joosten LA, Helsen MM et al. Therapeutic effect of neutralizing endogenous IL-18 activity in the collagen-induced model of arthritis. J Clin Invest 2001;108:1825–32.[CrossRef][Web of Science][Medline]
Submitted 9 November 2001; Accepted 4 September 2002


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Ann Rheum DisHome page
T Rooney, E Murphy, M Benito, P Roux-Lombard, O FitzGerald, J-M Dayer, and B Bresnihan
Synovial tissue interleukin-18 expression and the response to treatment in patients with inflammatory arthritis
Ann Rheum Dis, November 1, 2004; 63(11): 1393 - 1398.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
42/3/442    most recent
keg146v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (11)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Möller, B.
Right arrow Articles by Mühl, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Möller, B.
Right arrow Articles by Mühl, H.
Related Collections
Right arrow Osteoarthritis and Cartilage
Right arrow Spondylarthropathies
Right arrow Rheumatoid Arthritis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?