Interferon-
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
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Abstract
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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)-
markedly up-regulated IL-18BPa mRNA levels. Induction was accompanied by release of IL-18BPa immunoreactvity from FLS. Conditioned media from IFN-
-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-
, IL-18BP, Synoviocyte, Fibroblast, Rheumatoid arthritis.
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Introduction
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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)-
-inducing factor [1], but IL-18 is also capable of directly inducing expression of tumour necrosis factor-
(TNF-
) [1, 2]. Although IFN-
levels are often low, it is detectable in all histological variants of RA synovitis [3]. Data concerning the role of IFN-
in RA synovitis are conflicting. Addition of IFN-
to T cell/monocyte co-cultures enhances TNF-
production [4]. In contrast, IFN-
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-
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-
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].
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Materials and methods
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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. 1
), 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-
(5 ng/ml) or TNF-
(10 ng/ml) (Pepro Tech, London). Cells were also stimulated with the combinations IL-12+IL-18 and IL-1ß+TNF-
+IFN-
.
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-
(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-
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-
-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-
(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-
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.
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Results
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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-
strongly up-regulated IL-18BPa mRNA (Fig. 1A
). 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-
. TNF-
, 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-
did not significantly enhance IFN-
-induced IL-18BPa mRNA levels (Fig. 1B
). Quantitative real-time PCR confirmed induction of IL-18BPa by IFN-
in FLS (Fig. 1C
). Similar results were obtained using human dermal fibroblasts (data not shown).
IFN-
-induced mRNA in RA-FLS was accompanied by secretion of IL-18BPa immunoreactivity (Fig. 2A
). Immunoreactivity appeared with a molecular mass of about 45 kDa, which is in keeping with previous reports [11, 15]. As shown in Fig. 2B
, compared with conditioned media obtained from unstimulated cells, conditioned media from IFN-
-stimulated FLS reduced production of IFN-
in PBMC exposed to IL-12/IL-18.
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Discussion
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IL-18 can activate both T cells and macrophages in the microenviroment of RA synovitis. IFN-
, IL-1ß and TNF-
are supposed to be induced in RA joints [13] and production of TNF-
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-
. 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-
in PBMC. IFN-
-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-
. A murine model of septic antigen-induced arthritis illustrates that IFN-
can display proinflammatory properties when given in the initiation phase. In contrast, IFN-
ameliorates joint inflammation when given later on [20]. Detrimental functions of IFN-
may include a more effective antigen presentation at the onset of disease, whilst inhibition of IL-18 through IFN-
-induced IL-18BPa could represent a negative feedback mechanism upon established inflammation. Such a feedback principle concurs with reported overexpression of IFN-
in IFN-
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-
-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-
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].
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Acknowledgments
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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.
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Notes
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Correspondence to: B. Möller, Rheumazentrum Rhein-Main, Marienburgstraße 2, D-60528 Frankfurt/M., Germany. E-mail: b.moeller{at}em.uni-frankfurt.de 
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Submitted 9 November 2001;
Accepted 4 September 2002