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Rheumatology 2001; 40: 302-309
© 2001 British Society for Rheumatology

Expression of interleukin-18 and its monokine-directed function in rheumatoid arthritis

B. Möller1,, N. Kukoc-Zivojnov2, U. Kessler2, S. Rehart3, J. P. Kaltwasser1,2, D. Hoelzer2, U. Kalina2 and O. G. Ottmann2

1 Centre for Rheumatic Diseases,
2 Department of Internal Medicine and
3 Department for Arthritis Surgery, University Hospital Frankfurt, Germany


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. To investigate the expression of and monokine induction by interleukin 18 (IL-18; also called interferon-{gamma} inducing factor, IGIF), in peripheral blood mononuclear cells (PBMC) and cultured synoviocytes from rheumatoid arthritis (RA) patients.

Methods. We carried out IL-18 Western blotting and semi-quantitative reverse transcription–polymerase chain reaction (RT-PCR) of cytokines in PBMC [IL-18, IL-1ß and tumour necrosis factor {alpha} (TNF-{alpha})] and long-term cultured fibroblast-like synoviocytes (FLS) [IL-18, IL-1ß, TNF-{alpha}, IL-6, interferon {gamma} (INF-{gamma}) and [granulocyte–macrophage colony stimulating factor (GM-CSF)] from RA patients and controls. FLS were isolated from RA synovial membranes (FLSSM) and RA synovial fluids (FLSSF), osteoarthritis (OA) FLSSM and FLSSF from spondyloarthropathy patients. FLS were characterized by fluorescence-activated cell sorting of the FLS. PBMC and FLS from RA patients and control subjects were stimulated with recombinant human IL-18 and IL-1ß (rHuIL-18/rHuIL-1ß), and TNF-{alpha}, IL-1ß and MMP-1 were measured by ELISA in supernatants.

Results. Constitutive expression of IL-18 mRNA was significantly reduced whereas that of TNF-{alpha} was enhanced in RA PBMC. Persistent low expression of IL-18, TNF-{alpha}, GM-CSF and IL-1ß was observed in RA and OA FLSSM as well as spondyloarthropathy FLSSF. In contrast, high constitutive expression of IL-18 in FLS (CD90/Thy-1- and CD54-positive, CD14- and CD86-negative), accompanied by persistent high levels of TNF-{alpha}, GM-CSF and IL-1ß expression, was restricted to synovial fluid-derived FLS obtained from RA patients. IFN-{gamma} was not detectable in any culture, but IL-6 mRNA was equally expressed in all FLS cultures. rHuIL-18 was effective in stimulating TNF-{alpha} and IL-1ß secretion in PBMC from healthy controls, but failed to stimulate TNF-{alpha} and IL-1ß secretion from PBMC in 11 of 12 RA patients, and all FLS cultures. rHu-IL-1ß, but not rHu-IL-18, induced interstitial collagenase (MMP-1) in FLS.

Conclusions. Persistent high production of proinflammatory cytokines in RA-FLSSF may be relevant for chronic progression in RA synovitis. Levels of TNF-{alpha} and IL-1ß expression are increased in RA-FLSSF, but are independent of IL-18. The pathological function of enhanced IL-18 expression in RA-FLSSF remains to be further elucidated.

KEY WORDS: Rheumatoid arthritis, IL-18, Fibroblast-like synoviocytes, PBMC, Cytokines.


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is a chronic inflammatory joint disease of unknown origin with several extra-articular features suggesting systemic involvement and autoimmunity. Macrophages, fibroblast-likesynoviocytes (FLS) and lymphocytes are the predominant cell types involved in RA synovitis. The macrophage-derived cytokines tumour necrosis factor {alpha} (TNF-{alpha}) and interleukin 1ß (IL-1ß) are frequently implicated mediators of cell activation in this disease [1, 2]. Like T-cell-derived inflammatory mediators [3], TNF-{alpha} and IL-1ß contribute to joint destruction by increasing the amount of FLS-released matrix metalloproteinases, e.g. the interstitial collagenase matrix metalloproteinase-1 (MMP-1) [1, 2]. The demonstration of activated macrophages in the bone marrow [4] and peripheral blood of RA patients [5] further supports the hypothesis of initially quiescent, macrophage-dependent stimulated FLS in RA.

In vitro, whole-cell cultures from dissected and minced synovial tissue specimens lose their properties of inflammatory activation. FLS further continue to proliferate during prolonged culture, while lymphocytes and macrophages disappear with increasing culture duration and number of cell passages [6]. In these long-term cultures, FLS progressively down-regulate MHC molecules, LPS receptors (CD14) and cell surface molecules such as, intercellular adhesion molecule ICAM-1. Exposure of these FLS to TNF-{alpha} and IL-1ß restores the diminished surface receptor expression and induces the release of MMPs [1, 2, 6].

Using the model of sequential activation of peripheral blood mononuclear cells (PBMC) and FLS, we investigated the expression and monokine-directed functions of IL-18, a recently identified cytokine preferentially produced in cells of the phagocytic system [7]. IL-18 as a single stimulus is able to induce IL-1ß and TNF-{alpha} in PBMC from healthy donors [8]. Its initially described interferon-{gamma} (IFN-{gamma})-inducing activity [9] requires costimulatory factors such as IL-12 [10, 11]. The ability of IL-18 to induce a Th1-type T-cell response [10], a characteristic feature of RA synovitis [12], as well as the enhanced IL-18 levels detected in the synovial fluid from RA patients [13], were additional indications of an important role for IL-18 in RA synovitis. Recently, the first evidence for a proinflammatory role of IL-18 in RA was demonstrated [14]. IL-18 expression was described as being enhanced in histological studies of RA synovial tissues. IL-18 induced a Th1 response and TNF-{alpha} production in short-term cultured cells from RA tissues and synovial fluid mononuclear cells. Furthermore, IL-18 facilitated the erosive course in the RA animal model of collagen-induced arthritis [14].

In this study we investigated the expression of IL-18 in PBMC and FLS, and its ability to induce TNF-{alpha} and IL-1ß secretion in these two cell populations. We will demonstrate that long-term cultured FLS isolated from RA synovial fluids maintain strong constitutive mRNA expression of the proinflammatory cytokines IL-18, IL-1ß, TNF-{alpha} and granulocyte–macrophage colony-stimulating factor (GM-CSF).


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
Subjects
PBMC from 11 RA patients [15] and 10 healthy control subjects were isolated to investigate their spontaneous cytokine expression profile. None of these RA patients received disease-modifying anti-rheumatic drugs (DMARDs). Six patients received prednisone at an orally administered maximal dose of 10 mg per day, and five patients received no steroids (n=5). All patients were treated with non-steroidal anti-rheumatic drugs (NSAIDs). The RA disease activity was assessed by the erythrocyte sedimentation rate (ESR), serum concentration of C-reactive protein (CRP), swollen and tender 28-joint counts, and a modified disease activity score (DAS) [16]. Detailed characteristics of these RA patients and control subjects are given in Table 1Go.


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  		TABLE 1. Characterization of the RA patients and healthy donors (controls)

 
PBMC from 12 other RA patients and five control subjects were used for stimulation experiments. Briefly, 11 of these 12 patients were rheumatoid factor (RF)-positive and one female patient with severe RA of 2 yr duration was RF-negative. There were eight females and four males, and the average age was 44.5±9.6 yr (S.D.). These patients received different treatment regimens: the DMARDs used were methotrexate in four patients, d-penicillamine in one patient and sulphasalazine in one patient. NSAIDs were given to four patients and, steroids in a daily dose equivalent to 5–10 mg prednisone to six patients. The single RF-negative patient and two RF-positive patients received no pharmacological treatment.

Synovial fluid-derived FLS (FLSSF) from 13 RA patients and three spondyloarthropathy patients [17] were obtained by arthrocentesis of the knee joint. Synovial membrane-derived FLS (FLSSM) were obtained from 14 additional RA patients at the time of synovectomy of severely inflamed wrist or metacarpophalangeal joints, and from three osteoarthritis (OA) patients undergoing joint replacement of the knee.

All blood, synovial fluid and tissue specimens from patients and healthy donors were collected after the subjects had given informed consent.

Blood cell preparation
PBMC were isolated from 25 ml heparinized blood samples by Ficoll–Hypaque (Seromed, Berlin, Germany) gradient centrifugation. Cells were washed three times with phosphate-buffered saline (PBS) and immediately used in the experiments.

Synovial cell preparation and culture
Homogenous fibroblast-like synoviocyte cell cultures were established from the synovial fluid or synovial membranes. Briefly, total synovial fluid was inoculated directly into 250-ml flasks (Falcon, Becton Dickinson, Franklin Lake, NJ, USA). Non-adherent cells and supernatant were removed after 24 h and adherent cells were cultured to confluence in Ham's F10 medium (Bio Whittaker, Verviers, Belgium) completed with 10% fetal bovine serum (FBS; Boehringer, Ingelheim, Germany), 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine in a humidified 5% carbon dioxide atmosphere at 37°C. Synovial biopsies were dissected, minced and incubated in Ham's F10 medium containing 2 mg/ml collagenase (Sigma, Deisenhofen, Germany) for 2–4 h. After digestion of the synovial tissues, cells were washed three times with PBS and cultured in flasks (Falcon) containing completed Ham's F10 medium. Cells were passaged 2–4 times. Fluorescence-activated cell sorting (FACS)-characterized fibroblast-like synoviocytes were used for experiments after a total culture duration of at least 6 weeks, and an additional experiment was performed after 6 months of culture.

Characterization of synovial cells by FACS analysis
Cells were removed from the culture dishes with trypsin-EDTA and diluted in PBS containing 1% fetal bovine serum and 0.1% NaN3. Cells (5x103) were labelled with fluorescein isothiocyanate (FITC) or peroxidase-conjugated CD14, CD54 and CD86 antibodies from Becton Dickinson or CD90/Thy-1 antibodies from Dianova (Hamburg, Germany), and analysed by FACScan (Becton Dickinson). Despite marginal morphological differences between FLSSF and FLSSM independent of the type of arthritis, FACS analysis showed an identical pattern of fibroblast-like synovial cells, which were positive for CD90 (Thy-1) and CD54 (ICAM-1) and negative for CD14 and CD86.

RNA extraction, cDNA synthesis and RT-PCR
PBMC (5x106) or 5x105 FLS were lysed with RNAzolTM B (Cinna, Cincinnati, OH, USA). Total RNA was extracted and reverse-transcribed with SuperScriptTM RT (Gibco BRL, Eggenstein, Germany) following the manufacturer's instructions. Polymerase chain reactions (PCR) were performed with Taq DNA polymerase (Gibco). The primers used were as follows (amplified length of the PCR products in parentheses): ß-actin (552 bases) sense primer TCG AGC ACG GCA TCG TCA CCA ACT, antisense-primer ACC GCT CAT TGC CAA TGG TGA TGA; IL-18 (494 bases) sense primer AGC TCG GGA TCC ATG TAC TTT GGC AAG CTT GAA TCT AAA TTA TCA, antisense primer ACT GAA TTC CTA GTC TTC GTT TTG AAC AGT GAA CAT TAT AGA; TNF-{alpha} (383 bases) sense primer ACA AGC CTG TAG CCC ATG TTG TA, antisense primer ATT GAT CTC AGC GCT GAG TCG GTC A. The other primer sequences have been published elsewhere: IFN-{gamma}, IL-1ß and IL-6 [18], granulocyte–macrophage-colony stimulating factor (GM-CSF) [19]. Primer pairs were purchased from Interactive Biotechnology (Ulm, Germany). The PCR conditions were optimized for the linear phase of the PCR reaction; the cycle number was 30–35. Ethidium bromide-stained DNA was visualized in ultraviolet light and quantified with Molecular Analyst software (Bio-Rad, Munich, Germany). Under these conditions, the IL-18 RT-PCR revealed a linear correlation (r=0.993) between cDNA input and optical density of the PCR product. We used arbitrary units (AU) to represent the ratio of optical densities of specific cytokine PCR products and ß-actin as a housekeeping gene.

Western blotting analysis
Total protein lysates of 5x106 PBMC or 3x105 FLS were prepared according to standard protocols [20]. The total protein content of each specimen was determined with a modified Lowry protein measurement DC Protein Assay Kit (Bio-Rad). We used a specific IgG mouse monoclonal IL-18 antibody that recognizes the precursor of IL-18 as well as active IL-18 [21], is cross-reactive with murine IL-18, and neutralizes IL-18 receptor binding and bioactivity (IFN-{gamma}).

Stimulation experiments
For stimulation experiments, 1x106 PBMC (or 2x104 FLS) were incubated in 24 (48)-well plates in 500 µl RPMI medium with 1% heat-inactivated autologous human serum (Ham's F10 medium with 10% FBS) for 24 (72) h. Media were completed with 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine. Experiments were performed in duplicate in a humidified 5% carbon dioxide atmosphere at 37°C. Cells were stimulated with recombinant human IL-18 (rHuIL-18; Pepro Tech, London, UK) or recombinant human IL-1ß (rHuIL-1ß; Pepro Tech). Viability of stimulated PBMC was shown by trypan blue staining. Cytokine secretion into the supernatants was measured by ELISA following the manufacturer's instructions: the IL-1ß ELISA (detection limit 5 pg/ml) and TNF-{alpha} ELISA (detection limit 10 pg/ml) were purchased from Roche Diagnostics (Mannheim, Germany), and the interstitial collagenase (MMP-1) ELISA from Amersham Pharmacia Biotech, Uppsala, Sweden (detection limit 1.7 ng/ml).

Statistics
The results are expressed as mean±S.D. or S.E.M. Differences between groups were analysed by the Mann–Whitney U-test for tailed or untailed groups. Correlations were calculated as Pearson's coefficient of correlation r with a test of two-tailed significance. For statistical analysis, we used the statistical package SPSS Base (SPSS Munich, Germany).


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
Steady-state IL-18 expression in PBMC
In order to determine whether IL-18 expression was altered in PBMC from RA patients concurrently with myeloid cell activation, we assessed mRNA levels of IL-18, IL-1ß and TNF-{alpha} in PBMC by semi-quantitative RT-PCR.

Specific IL-18, IL-1ß and TNF-{alpha} PCR products were demonstrated in PBMC from all subjects. IL-18 mRNA levels were significantly reduced in RA patients when compared with healthy donors (RA, 15.9±6.8 U; controls, 32.2±15.4 U; Mann–Whitney test of untailed groups, P<0.01) (Fig. 1aGo). Quantification of intracellular IL-18 protein by immunoblotting of PBMC lysates and subsequent densitometry was consistent with the IL-18 RT-PCR results as RA patients also showed reduced levels of intracellular IL-18 protein (S.D.) [RA, 0.84±0.32 AU (n=11); healthy controls, 1.07±0.27 AU (n=5), P<0.05].



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  		FIG. 1. IL-18 and TNF-{alpha} expression in PBMC. Semi-quantitative IL-18 mRNA levels were significantly reduced (a) and TNF-{alpha} levels were significantly increased (b) in PBMC from RA patients in comparison with healthy donors. Results are presented as arbitrary units (AU).

 

Steady-state TNF-{alpha} and IL-1ß expression in PBMC
TNF-{alpha} mRNA levels were significantly increased in RA patients (RA, 17.6±8.3 AU; controls, 10.7±4.6 AU, P<0.05) (Fig. 1BGo), while IL-1ß mRNA expression was essentially in the same range in RA patients (21.5±9.7 AU) and healthy controls (21.8±15.6 AU).

None of the three cytokines investigated displayed a significant correlation between cytokine mRNA expression in PBMC and the clinically assessed disease activity parameters ESR, CRP, DAS and the swollen or tender joint count.

Long-term cultured synoviocytes express IL-18
Multiply passaged, long-term cultured synoviocytes allowed only FLS to persist. FLS cultures from various sources differed substantially with regard to their growth characteristics: FLSSM from RA patients grew most rapidly and reached confluence within 11 days, whereas FLSSM from OA and FLSSF from RA patients did not reach confluence before day 22. FLSSF from spondyloarthropathy patients grew substantially slower, reaching confluence between 46 and 63 days after passaging. IL-18 mRNA was demonstrated by RT-PCR in all 12 investigated FLS cultures after 6 weeks. The level of expression showed substantial variability, with highest expression in RA FLSSF (Fig. 2aGo). Immunoblotting of the intracellular IL-18 protein concentrations in long-term cultures of FLS precisely reflected the results of IL-18 RT PCR (Fig. 2bGo). When an additional 10 RA-FLSSF and 11 RA FLSSM were cultured for 6 months before analysis, RA FLSSF still showed significantly higher IL-18 mRNA expression (17.7±11.8 vs 4.3±3.6 AU, P=0.002).



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  		FIG. 2. Expression of IL-18, IL-1ß, TNF-{alpha}, IL-6 and GM-CSF mRNA in FLS. (a) IL-18 mRNA was closely correlated with TNF-{alpha}, IL-1ß and GM-CSF. Expression of these four cytokines in RA FLSSF was substantially higher than in all other FLS populations. In contrast, IL-6 mRNA was expressed equally in all FLS. Western blotting for IL-18 in RA FLSSF (b) was closely correlated with IL-18 PCR results. 1Base pairs; 2negative controls.

 

Steady-state expression of IL-1ß, TNF-{alpha}, GM-CSF and IL-6 in FLS
After 6 weeks of culture, the IL-1ß, TNF-{alpha} and GM-CSF expression profile was the same as described for IL-18 in all 12 FLS cultures. Thus, high expression of IL-18 in RA FLSSF was accompanied by high, and low IL-18 expression in the other FLS was associated with low IL-1ß, TNF-{alpha} and GM-CSF expression. The obviously similar pattern of expression of these four cytokines was confirmed by densitometric analysis of their PCR products; there were significant correlations between IL-18 and IL-1ß (r=0.96, P<0.001), IL-18 and TNF-{alpha} (r=0.89, P<0.001), and IL-18 and GM-CSF (r=0.88, P<0.001). Only RA FLSSF expressed high mRNA levels of the proinflammatory cytokines IL-18, IL-1ß, TNF-{alpha} and GM-CSF (Fig. 2aGo); expression of these cytokines was significantly lower (Table 2Go) (Mann–Whitney U-test of untailed groups; P<0.05) in FLSSF from spondyloarthropathy patients and FLSSM from RA and OA patients. IL-6 mRNA was expressed most strongly in RA FLSSF and OA FLSSM, but this difference was not statistically significant. The very sensitive IFN-{gamma} RT-PCR was completely negative in all FLS cultures (Table 2Go and Fig. 2aGo).


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  		TABLE 2. Steady-state cytokine expression in FLS

 
After 6 months of culture, the IL-1ß, TNF-{alpha} and GM-CSF mRNA levels were again significantly higher in RA FLSSF than in RA FLSSM (IL-1ß, 46.4±43.7 vs 7.5±12.7 AU, P=0.002; TNF-{alpha}, 29.7±5.4 vs 8.1± 14.3 AU, P=0.015; GM-CSF, 6.1±2.8 AU vs 1.9± 1.1 AU, P=0.003). IL-6 mRNA expression was essentially the same in RA FLSSF and RA FLSSM (P>0.05).

Effects of rHuIL-18 on IL-1ß and TNF-{alpha} expression in PBMC and FLS
IL-18 is known as a potent inducer of IL-1ß and TNF-{alpha} in PBMC from healthy donors [8]. In our experiments, IL-18 stimulated TNF-{alpha} and IL-1ß secretion in PBMC from healthy donors in a dose-dependent manner. IL-1ß increased from 14.9±0.4 pg/ml in controls to 39.9±13.2 pg/ml following rHuIL-18 (10 nM) stimulation, and the TNF-{alpha} level increased from 100.2±84.5 to 789.4±317.5 pg/ml. In contrast, IL-1ß and TNF-{alpha} secretion was not induced in two RF-positive RA patients, who did not receive any drug treatment, or in nine RF-negative RA patients receiving different anti-rheumatic treatment regimens (non-responders). Moderate and dose-dependent IL-1ß and TNF-{alpha} release from PBMC following rHuIL-18 stimulation was only observed in a single RF-negative RA patient; IL-1ß increased from 12 to 27 pg/ml and TNF-{alpha} from 185 to 318 pg/ml upon 10 nM rHuIL-18 stimulation (Fig. 3Go). Intensive washing with PBS and incubation of the patient's PBMC in medium supplemented with serum from a healthy donor instead of patient's sera did not reconstitute this impaired cytokine induction in RA patients. Increasing IL-18 concentrations (2.5–10 nM) reduced the average viability of PBMC in the IL-18 responders from 97% in controls to 91, 88 and 69% in the presence of 2.5, 5 and 10 nM rHuIL-18 respectively. In contrast, non-responding RA patients failed to demonstrate IL-18-dependent reduced cell viability.



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  		FIG. 3. Impaired responsiveness of RA PBMC on IL-18 stimulation. PBMC from healthy donors and RA patients were stimulated with IL-18 at 0, 2.5, 5 and 10 nM. TNF-{alpha} and IL-1ß were determined by ELISA in the supernatants. In PBMC from healthy donors, TNF-{alpha} and IL-1ß was significantly induced by IL-18 stimulation in a dose-dependent manner (P<0.05). TNF-{alpha} and IL-1ß induction was significantly reduced in RA patients. Experiments were done in triplicate. Data are mean±S.E.M.

 
rHuIL-18 stimulation (5 and 10 nM) of FLS failed to induce release of IL-1ß or TNF-{alpha} in all 12 stimulated FLS cultures. In addition, IL-18 (10 nM) stimulation was not able to induce any MMP-1 release, whereas control experiments with rHuIL-1ß (0.1 nM) significantly increased the MMP-1 concentration in the supernatant from 20.4±19.0 to 60.9±53.7 ng/ml (Mann–Whitney U-test of tailed groups, P=0.001).


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
IL-18 expression has been demonstrated in cells of the monocyte-macrophage system [7], epithelial cells of the gut and skin [7], osteoblasts [22], chondrocytes [23], and, more recently, long-term cultured FLS [14, 24]. IL-18 has been proposed to be a new member of the IL-1 family [7], and involvement of IL-18 in joint diseases has been shown by its effects on cartilage destruction [23] and T-cell activation [14]. On the other hand, IL-18 inhibits osteoclast formation in an animal model and protects bone matrix from degradation [22].

While IL-18, GM-CSF, IL-1ß and TNF-{alpha} expression was demonstrable in all long-term cultured FLS, high expression of these proinflammatory cytokines was restricted to FLSSF from RA patients. This suggests a hitherto undefined, RA-associated type of FLS, or cell- and disease-associated, altered IL-18, IL-1ß, TNF-{alpha} and GM-CSF regulation in a subgroup of synoviocytes. Strong disease-associated differences of cytokine expression within the group of FLSSF (RA vs spondyloarthropathy patients) and FACS profiles excluded contamination with macrophages or dendritic cells from the fluids. In addition, the long culture duration (up to 6 months) should be enough to exclude DMARD effects, or artefacts resulting from the isolating procedures, on the cultured cells. The number of cell passages and the negative IFN-{gamma} PCR rule out the possibility that the results obtained might have been obtained under the influence of persistent T-cell-driven FLS activation.

In our experiments, culture initiation from synovial fluid allowed the growth of FLS populations with slower proliferation but a high level of cytokine expression, and may therefore be compatible with the spontaneous cytokine production by cloned RA FLS that was obtained by limiting dilution of cell homogenates from digested synovial membranes [6]. The finding that FLS are apparently able to maintain an inflammatory state in the absence of specific stimuli is also compatible with the results of experiments with SCID mice, which demonstrated the invasive growth of RA pannus tissue predominantly containing fibroblast-like synoviocytes [25]. In addition to the important T-cell-derived [3, 26] and macrophage-derived FLS activation [2, 27] in RA synovitis, long-term cytokine expression in FLS may be important for the chronic course of RA by ongoing activation of tissue-invading lymphocytes and macrophages. The strong and long-lasting differences between the inflammatory status of membrane- and fluid-derived FLS may suggest a peculiar cell population in the synovial fluid; alternatively, FLS cultured from the fluid are simply equivalent to detached membrane-derived FLS of the superficial lining layer, which may have a slower growth rate. The difference in proliferation rate observed between FLSSF and FLSSM may result in overgrowth by more rapidly proliferating but less inflammatory FLS of the basal synovial membrane in membrane-derived cultures. However, the role of a cellular equivalent for autochthonous articular inflammation in RA can be postulated for characterized FLS such as the FLSSF described here.

Previously published data [7–11] demonstrated IL-18 to be a pleiotropic cytokine, and its importance for the development of erosive arthritis has been shown recently [14]. The potency of IL-18 in the induction of TNF-{alpha} as well as IL-1ß [8, 14] seems to be of outstanding importance in RA pathogenesis [28, 29]. In our experiments, the failure of IL-18 to induce IL-1ß and TNF-{alpha} in PBMC from nearly all RA patients clearly distinguished these individuals from healthy donors. The ability to reproduce control experiments with PBMC from healthy donors is in agreement with the literature [8], and it clearly demonstrates the bioactivity of the recombinant IL-18 used. The failure to normalize IL-18 responsiveness by replacing patients’ sera by sera from healthy donors suggests a cellular mechanism for the inhibition of IL-18 action. Treatment-related drug effects could theoretically account for inhibited monokine induction, but appear to be unlikely because the loss of IL-18 responsiveness was also observed in untreated RA patients. The IL-18-related TNF-{alpha} enhancement in inflamed tissues and synovial fluid mononuclear cells [14] excludes a genetic basis for disease-related changes, e.g. defective receptors or receptor signalling cascades, of RA subjects.

Interestingly, the disease-associated lack of IL-18 responsiveness in RA in terms of inhibited IL-1ß and TNF-{alpha} production by mononuclear cells is limited to the peripheral blood compartment. It is known that IL-18-induced IL-1ß in PBMC is TNF-{alpha}-dependent and is produced predominantly by macrophages [8]. The induction of TNF-{alpha} by IL-18 occurs predominantly in T-cells, an effect that is substantially augmented by IFN-{gamma} [8]. In contrast to these indirect activation pathways, direct stimulation of TNF-{alpha} production by IL-18 occurs in macrophages from RA synovial fluids [14], suggesting in vivo priming of these cells by the inflammatory environment. Taking these results together, impaired stimulation of both TNF-{alpha} and IL-1ß in RA PBMC by IL-18 is likely to be attributable to the T cells [8] that might be depleted from the peripheral blood, e.g. by migration to inflammatory sites, or otherwise altered in their function. Nevertheless, additional alterations of the macrophage population cannot be excluded.

Despite the presence of an IL-18 receptor on the surface of FLS, which we have demonstrated by cross-linking experiments using biotinylated rHuIL-18 [24] and recently also by RT-PCR (data not shown), FLS did not respond to IL-18 stimulation with the release of IL-1ß or TNF-{alpha}. Moreover, the MMP-1 release from FLS following stimulation with IL-1 (rHuIL-1ß in our experiments), but not rHuIL-18, clearly distinguished IL-18 functionally from the other molecules of the IL-1 family. IL-1{alpha} and IL-1ß share the same IL-1 receptors and activate the same signalling cascades [30], resulting in MMP-1 production by FLS, whereas IL-18 binds at a different receptor and does not lead to this pathogenically important FLS response. The result was the same in RA and non-RA FLS, excluding a disease-related defective IL-18 response.

Steady-state IL-18 mRNA and protein expression was reduced in RA PBMC, in contrast to other monokines. This observation suggests a specific alteration of IL-18 regulation in PBMC of RA patients that is clearly different from that of IL-1 or TNF-{alpha}. The effects of disease and treatment on this phenomenon remain to be elucidated.

The enhanced IL-18 expression in synovial RA-FLSSF supports IL-18 involvement in RA synovitis, and the impaired effects of IL-18 on IL-1ß and TNF-{alpha} regulation in RA PBMC suggest a shift of IL-18-responding cells from the peripheral blood into inflamed synovial tissues. IL-18 is claimed to be a new member of the IL-1 cytokine family, but it obviously differs from IL-1ß with regard to its biological function in FLS. The pathophysiology of enhanced IL-18 levels in RA FLSSF as well as its function on FLS needs further elucidation.


   Acknowledgments
 
We are grateful to Roland Kurrle and Tilo Weiss for stimulating discussion and constructive help, and to Dörte Kauschat, Karin Ballas, Heike Nürnberger, Martine Pape and Sandra Wagner for technical support. This project was supported by grants from the Heinrich and Fritz Riese and Paul and Ursula Klein Foundation Frankfurt.


   Notes
 
Correspondence to: B. Möller, Medizinische Klinik III, Labor B2-19/20, Klinikum der Johann Wolfgang Goethe-Universität, auf der Binnen 2, D-64686, Lautertal, Germany Back


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 6 December 1999; Accepted 2 October 2000


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