Indications for a direct proinflammatory role of IL-18 in arthritis
IL-18 was first described as a T-cell activating, interferon-gamma- (IFN-γ)-inducing factor [S1], but it also exhibits a broader spectrum of proinflammatory effects. For example, IL-18 shows IFN-independent effects in the murine model of arthritis induced by streptococcal cell walls [S2] and directly induces TNF-α production in macrophages derived from synovial fluid . In contrast, stimulation of IL-1β and IL-8 by IL-18 in peripheral blood mononuclear cells is T-cell-dependent [S3]. IL-18 promotes collagen-induced arthritis in mice through mechanisms that may be distinct from those that operate with IL-12, another important IFN-γ-inducing molecule [S4].
Cell types expressing IL-18R
IL-18R expression is known to occur in several lymphatic and myeloid cell lines [S5], primary T and B lymphocytes, and natural killer cells [S6,S7]. IL-18 activity in unstimulated T cells is limited by low basal IL-18R expression levels; one explanation for their synergism with IL-18 is upregulated expression of IL-18R by co-stimulatory molecules IL-12 or IL-15 [1,2,3,4]. IL-18R regulation plays a pivotal role in T-cell function and decides whether T lymphocytes become polarized to either Th1 or Th2 cells [S8,S9].
Supplementary materials and methods
RNAzol™ B was purchased from CINNA, Cincinnati, OH, USA. SuperScript™ RT-II, Taq polymerase, PBS, penicillin, streptomycin, glutamine, and RPMI 1640 medium were supplied from GIBCO BRL, Eggenstein, Germany. Ham's F10 medium was purchased from Bio Whittaker, Verviers, Belgium; collagenase, protease inhibitors benzamidin, aprotinin, and phenylmethylsulfonyl fluoride (PMSF) were from Sigma, Deisenhofen, Germany. Trypsin–EDTA was from PAN Biotech, Aidenbach, Germany, FCS from Boehringer-Ingelheim, Ingelheim, Germany, and the cross-linker bis(sulfosuccimidyl) suberate was purchased from Pierce, Rockford, IL, USA. A human IL-18 GST fusion protein (molecular weight [MW] ~44 kDa) was cloned after full-length RT-PCR, inserted in a pGEX4T2 vector (Pharmacia, Freiburg, Germany) and expressed in Escherichia coli. IL-18-GST was purified in glutathione–agarose (Sigma) columns and biotinylated in accordance with standard protocols [S10]. Previous experiments have shown that this fusion protein is bioactive [S11]. Recombinant human IL-1β (1 ng/ml), IL-2 (100 ng/ml), IL-12 (1 ng/ml), IL-15 (100 ng/ml), IL-18 (10 nM), IFN-γ (5 ng/ml), and TNF-α (10 ng/ml) were purchased from Pepro Tech EC Ltd, London, UK (concentrations used are given in parentheses). IL-18 receptor antibodies and IL-18 ELISA kits were provided from R&D systems. FITC-conjugated rabbit antigoat secondary antibodies were purchased from Jackson ImmunoResearch, West Grove, PA, USA. FITC-conjugated CD90/Thy-1 and CD106/VCAM-1 antibodies were purchased from Dianova, Hamburg, Germany. FITC-conjugated CD14, phycoerythrin-conjugated CD54 (ICAM-1), phycoerythrin-conjugated CD86 antibodies, and culture flasks were from Becton Dickinson, Franklin Lane, NJ, USA.
Cells were lysed with RNAzol™ followed by RNA extraction and reverse transcription into cDNA with SuperScript™ RT-II, in accordance with the manufacturers' instructions. RT-PCR was performed for IL-18Rα using endpoint or multistep PCR kinetics (25–35 cycles), and IL-18Rβ by anchored RT-PCR under the following conditions: IL-18Rα sense GTC AAC AGC ACA TCA TTG TAT, antisense TAG AAT TCT TAT GTT TTT CCA TCT, annealing temperature 60°C, length of the RT-PCR product 670 bp. IL-18Rβ sense TAC CAG AGC AAG GAT CAG ACG C, antisense CAA TCC CAT TCC ATT GTC CAT C, optimal annealing temperature 56°C, 30 cycles, length of the PCR product 772 bp. IL-18Rβ anchored RT-PCR antisense primer: CCA GGG CTC ATT TCA CCA TTC, 20 additional cycles, length of this RT-PCR product 630 bp. Expression of the β-actin housekeeping gene was determined to ensure equivalent amounts of the extracted RNA: β-actin sense primer TCG AGC ACG GCA TCG TCA CCA ACT, antisense primer ACC GCT CAT TGC CAA TGG TGA TGA, annealing temperature 60°C, 30 cycles, length of the PCR product 552 bases. Ethidium bromide-stained DNA was visualized in ultraviolet light and quantified with Molecular Analyst software (BIORAD, Munich, Germany).
IL-18R western blotting
For IL-18R western blotting, 5 × 106 FLS was lysed on ice in buffer solution containing 20 mM TRIS (pH8.0), 137 mM NaCl, glycerol 10%, Nonidet P-40, 10 mM EDTA, 100 mM NaF, 1 mM PMSF, aprotinin, 20 μM sodium orthovanadate, and 4 μM leupeptin. Proteins were separated by electrophoresis in 15% SDS–PAGE and blotted onto a nitrocellulose membrane. Membranes were stained with an IL-18R-specific antibody and a secondary anti-goat IgG antibody (R&D Systems, Wiesbaden, Germany). Bound antibodies were visualized by chemiluminescence.
Covalent IL-18 cross-linking
RA-FLS with presence of both IL-18R chains in RT-PCR analyses were removed from the flasks using ice-cold PBS and a cell scraper. Cells were thoroughly washed in PBS containing benzamidine (10 mM), aprotinin (100 U/ml), and PMSF (1 mM). Each sample of 1 × 106 cells was either incubated with biotinylated IL-18 GST-fusion protein (final concentration 25 μg/ml) or a mixture (1:100) of biotinylated and unlabeled IL-18 GST-fusion protein for 1 hour on ice. Cross-linker BS3 (0.5 mM) was added to the cells for 10 min. Free cross-linker molecules were eliminated with 25 mM glycine and the cells were washed with PBS. Cell pellets were solubilized with Triton (1%) and protease inhibitors in PBS on ice for 20 min. Solubilisates were vortexed every 5 min before centrifugation of the cellular debris. Dissolved proteins were separated on a 15% SDS–polyacrylamide gel and visualized as biotin–conjugated protein complexes by streptavidin-peroxidase reaction.
We used the Phospho Plus IκB-α (Ser32) Antibody Kit from New England Biolabs (Beverly, MA, USA), in accordance with the manufacturer's instructions. Total IκB-α was determined by direct western blotting, and phospho-rylated IκB-α was estimated by immunoprecipitation. Each sample of 5 × 106 cells was stimulated with IL-18 for 5, 10, or 30 min. Stimulation was stopped on ice, cells were thoroughly washed, and lysates were prepared in Tris–HCl buffer (pH6.8) containing 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromphenol blue. Each sample of 20 μg protein lysate was separated by 12.5% SDS–PAGE and blotted onto a nitrocellulose membrane. Immunoprecipitation for phospho-IκB-α was performed overnight using anti-IκB-α rabbit polyclonal IgG antiserum (New England Biolabs). Antibody-IκB-α complexes were bound to protein A sepharose CL-4B (Sigma), centrifuged, and washed in TRIS–HCl buffer containing 0.2% NP-40 and 0.25 mM protease inhibitor PMSF. Sepharose beads were separated by boiling for 5 min in 10 μl SDS buffer and centrifuged. Supernatants were separated by SDS–PAGE. Membranes were blocked in triethanolamine-buffered saline solution overnight and stained with IκB-α or phospho-IκB-α (Ser32) rabbit polyclonal antiserum. Bound antibodies were detected with a secondary antirabbit antibody conjugated to horseradish peroxidase, and LumiGLO chemilu-miniscent reagent (New England Biolabs).
Additional experiments on IL-18R regulation
IL-18Rα protein levels in RA-FLS remained unaffected by IFN-γ stimulation in western blotting experiments (n = 3, data not shown), and IL-18Rα mRNA levels in U937 cells were not inducible (differences of ODQ of IL-18Rα/β-actin PCR products upon stimulation <50%) by any of the indicated stimuli.
ICAM-1 induction by IL-18 in U937 cells
Induction of ICAM-1 by IL-18 in U937 cells was identical to that observed by TNF-α stimulation, and induction by IL-18 was not further enhanced by simultaneous IL-12 challenge. In agreement with this observation, we found no upregulation of IL-18R transcripts by any of the cytokines studied (increase of ODQ <50% in all experiments).
FLS response to IL-1β
In contrast to the lack of a proinflammatory effect by IL-18 stimulation in FLS, strong prostaglandin E2 induction (>2000 pg/ml, n = 12) was observed within 18 hours in those FLS cultures exposed to IL-1β, thus excluding anergy of the studied long-term-cultured FLS to other cytokine stimuli.
Ligand binding of the IL-18Rα/β chain
IL-18R expression in FLS and IL-18 ligand binding on FLS surfaces were confirmed by western blotting and cross-linking experiments, respectively. The finding of IL-18R bands at ~55 and ~70 kDa in western blotting experiments is essentially in agreement with the wide band of published IL-18Rα molecular weights [10,S5], and IL-18 cross-linking on FLS membranes exhibited a predominant 100-kDa complex, suggesting a complex of biotinylated IL-18-GST with IL-18Rα. In contrast to the rather low binding affinity of the IL-18Rα-chain , the isolated receptor β-chain was initially reported to be ineffective for IL-18 ligand binding  but was recently shown to enhance IL-18 ligand-binding affinity together with the IL-18Rα-chain . We found two high-molecular-weight complexes, of about 150–200 kDa. In agreement with previous observations [10,11,13], quantitatively predominant ligand binding to IL-18Rα alone and, to a minor extent, binding to both IL-18R chains may be also supposed from the cross-linking experiment in FLS, assuming that the ~87-kDa MW of IL-18Rβ protein estimated by the amino acetic acid sequence is correct [S12].
IL-18Rα by IFN-γ
IL-18Rα mRNA expression in FLS was inducible by IFN-γ, whereas amounts of IL-18R protein remained unaffected by this stimulus within the first 72 hours. This discrepancy may be attributable to a missing or very slow translation of IL-18Rα transcripts. However, some enhanced FLS sensitivity to IL-18 due to longstanding influences of an inflammatory environment should be considered.