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
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 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
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 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
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 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}.



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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
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 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.



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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
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 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 [13] 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
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 9 November 2001; Accepted 4 September 2002