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Rheumatology Advance Access originally published online on January 11, 2007
Rheumatology 2007 46(5):752-758; doi:10.1093/rheumatology/kel419
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Interleukin-1ß induced activation of nuclear factor-{kappa}b can be inhibited by novel pharmacological agents in osteoarthritis

S. N. Lauder, S. M. Carty, C. E. Carpenter, R. J. Hill1, F. Talamas1, J. Bondeson, P. Brennan2 and A. S. Williams

Rheumatology Research Laboratory, Cardiff University, Heath Park, Cardiff, CF14 4XN, UK, 1Roche, Palo Alto, CA 93404, USA and 2Medical Biochemistry and Immunology, Henry Wellcome Building, Cardiff University, Heath Park, Cardiff CF14 4XN, UK.

Correspondence to: S. Lauder, Rheumatology Research Laboratory, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. E-mail: LauderSN{at}cardiff.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. To investigate the importance of activation of the transcription factor, nuclear factor-{kappa}B (NF-{kappa}B) by interleukin-1ß (IL-1ß) and tumour necrosis factor-{alpha} (TNF-{alpha}) in the pathogenesis of osteoarthritis (OA) and assess its suitability as a target for therapy by determining its role in the induction of the cytokine IL-6 and the degenerative enzymes, matrix metalloproteinase (MMP)-1 and MMP-3 in vitro.

Methods. Three distinct cellular models, derived from primary OA tissue, were employed, namely, fibroblast-like synoviocytes (OA-SF); co-cultures containing phenotypic macrophage-like and fibroblast-like cells (OA-COCUL); and primary OA synovial tissue explants (OA-EXP). These were treated with specific inhibitors of IL-1ß, TNF-{alpha} and NF-{kappa}B to assess their differential role in the production of pathologically relevant mediators, specifically IL-6, MMP-1, MMP-3 and the tissue inhibitor of metalloproteinases-1 (TIMP-1), which were quantified by enzyme-linked immunosorbent assay.

Results. Inhibition of NF-{kappa}B by a novel agent, RO100 at a dose of 0.1 µM, exerted significant (P < 0.05) repression of IL-6, MMP-1 and MMP-3 production in OA-SF. Notably, neither TIMP-1 production nor cell viability was significantly affected at the dose tested. These data were reproduced in OA-EXP, which might be considered as having greater physiological relevance. Interestingly, comparable efficacy was noted using IL-1ß and TNF-{alpha} neutralizing antibodies in OA-COCUL.

Conclusions. We have demonstrated that a novel pharmacological inhibitor of NF-{kappa}B, RO100 inhibits pathological mediators of OA progression with equivalent efficacy as established IL-1ß and TNF-{alpha} neutralizing strategies. Our findings highlight a potential for developing NF-{kappa}B targeted therapeutics for positively regulating disease activity and improving clinical outcome in OA.

KEY WORDS: Osteoarthritis, Therapeutics, Matrix Metalloproteinases, NF-{kappa}B, Cytokines


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
As a debilitating, progressive disease affecting the joints, osteoarthritis (OA) is the most prevalent joint disorder globally. Now considered the eighth most frequent cause of disability [1], patients experience chronic pain and stiffness in the affected joint as a result of deterioration of the articular cartilage surface. Historically believed to be a consequence of ‘wear and tear’, OA was considered to involve non-inflammatory disease processes. A change in perception occurred as a result of a pioneering study in the 1970s that provided evidence that cohorts of OA patients experienced an acute inflammatory episode at disease onset [2]. The inflammation exhibited was not as severe as that seen in rheumatoid arthritis (RA) but sufficient to disprove the original belief that OA was simply an erosive process. Subsequent studies support this theory with OA now considered to be a complex multifactorial disease with sufferers classified as heterogeneous patient population, exhibiting varying degrees of inflammation, in some cases comparable with RA [3–6].

Whilst the aetiology of OA remains unclear, it appears that OA is initiated as a consequence of altered mechanical loading due to injury or excessive stress [7, 8]. In this context the chondrocytes become activated and increase levels of IL-1ß and TNF-{alpha} [9] expression in the affected joint. The upregulation of IL-1ß and TNF-{alpha} acts in both an autocrine and paracrine fashion [10] orchestrating in the formation of superficial fractures and fissures in the articular cartilage [11]. As the disease progresses, synovial hyperplasia and hypertrophy develop, and joint architecture becomes compromised.

The matrix metalloproteinase (MMP) family encompasses 23 enzymes that differentially mediate the degradation of each component of extracellular matrix. Key MMPs implicated in the pathogenesis of OA include the collagenases (MMP-1, -8 and -13), a gelatinase (MMP-9) and stromeolysin-1 (MMP-3) [12, 13]. Several studies suggest that MMP-13 is the critical MMP responsible for cartilage destruction [14, 15]. Levels of MMP-1 (a collagenase that is not as specific in its cleavage of collagen) expression is, however, 200-fold greater than MMP-13 in OA [11]. The stromeolysin MMP-3 has the ability to degrade many proteins including aggrecan, an early indicator of proteoglycan depletion in articular cartilage during the aetiopathogenesis of OA [16]. Perhaps of greater importance is the ability of MMP-3 to activate pro-MMP-1 and pro-MMP-9, which damage collagen fibrils and thereby disturb the highly organized structure of articular cartilage [17–19]. Apart from stimulating MMP production by the chondrocytes, both IL-1ß and TNF{alpha} induce IL-6 release by the chondrocytes [20] and synovial cells [21]. IL-6 is reported to be instrumental in perpetuating synovial inflammation and intensifying cartilage depletion within the joint [22].

IL-1ß and TNF-{alpha} primarily elicit their effects through inducing NF-{kappa}B activation [23]. This in turn results in the formation of the I{kappa}B kinase (IKK) complex, resulting in the phosphorylation and degradation of I{kappa}B-{alpha}. The loss of I{kappa}B{alpha} permits the translocation of NF-{kappa}B into the nucleus where it can turn on gene expression [24, 25], upregulating a plethora of cytokines and MMPs [23]. It has previously been shown that in cells derived from the osteoarthritic synovium that IL-6 production can be altered by targeting NF-{kappa}B [26]. The ERK, p38, MAPKs, AP-1 and NF-{kappa}B signalling cascades are all associated with MMP production [27–30]. It is hypothesized that inhibition of one or more of these signalling cascades may reveal a potential target for OA therapy and therefore warrant further investigation.

The aim of our present study was therefore to assess the potential of targeting NF-{kappa}B activation as a therapeutic strategy for OA. To achieve our objectives we used three distinct in vitro model systems, selected IL-6 as a relevant marker of synovial inflammation, MMP-1 and MMP-3 as relevant cartilage depleting enzymes and the endogenous regulator of MMP activity tissue inhibitor of metalloproteinases-1 (TIMP-1) as readouts that would provide a balanced perspective of efficacy.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Experimental model systems
Three in vitro model systems derived from OA synovial samples namely, fibroblast-like synoviocytes (OA-SF), synovial co-cultures (OA-COCUL) and synovial tissue explants (OA-EXP) were utilized for this study. In all cases, synovium was obtained from consenting patients diagnosed with end-stage OA who were undergoing synovectomy at the time of joint replacement surgery. Ethical approval was obtained from Bro-Taf Health Authority (Cardiff, Wales, UK) prior to commencement of the study.

Fibroblast-like synoviocytes (OA-SF)
OA-SF were generated from synovium that had been cut into small fragments and digested with collagenase (1 mg/ml) and DNAse (2000 Kunitz units) for 2 h at 37°C with mechanical shaking. Following digestion the heterogeneous population of primary OA synovial cells, referred to as OA-COCUL, were distributed into T25 tissue culture flasks (Nunc, USA) and cultured overnight before the non-adherent cells were removed. Adherent cells were grown in DMEM F12 (supplemented with 2 mM L-glutamine, 10 units/ml penicillin–streptomycin, 1% insulin-transferring-slenium and 10% heat-inactivated fetal calf serum). At confluence, cells were passaged 1 : 2. By passage 3, the cells represented a homogeneous population of OA-SF. All experimental investigations conducted used OA-SF between passages 4 and 6.

OA synovial co-cultures (OA-COCUL)
Synovial digestion (described previously) generated OA-COCUL. OA-COCUL were dispensed into 12-well plates (Nunc) at 2 x 106 cells/well for experimental analysis.

OA synovial explants (OA-EXP)
OA-EXP were excised at random from synovial tissue, and their weight recorded prior to experimental investigation. OA-EXP were dispensed into 1.5 ml of DMEM F12 (supplemented as described previously) in 12-well plates and then allowed to equilibrate for 18 h. Supernatant samples taken at this time provided baseline measurements for each mediator prior to initiation of treatment protocols. The baseline value (100% mediator production) for each well was used as the reference point against which subsequent responses to individual treatment was compared. To accommodate the intra-variability present within each synovial sample, treatment conditions were conducted in triplicate or quadruplicate depending upon the size of the original synovial specimen and the mean calculated for each treatment strategy.

Electrophoretic mobility shift assay
OA-SF were dispensed at a concentration of 1 x 106 cells/well into 6-well plates. Cells were stimulated with IL-1ß (20 ng/ml) for 30 min before terminating the stimulation using ice-cold PBS. Nuclear extracts were prepared as described previously [30]. Briefly, cells were detached using mechanical agitation, collected and centrifuged at 3000g. Cells were washed in buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl) and centrifuged at 12 000g. OA-SF were treated with 400 µl of buffer A + 0.5 mM DTT, 0.5 mM PMSF, 5 µg/ml aprotinin, 5 µg/ml pepstatin, 30 µg/ml leupeptin, 0.125% IPEGAL (equivalent to the non-ionic surfactant Nonidet-P40) on ice for 5 min. OA-SF were centrifuged at 12 000g for 5 min and the resulting pellet treated with 100 µl of buffer C + 0.5 mM DTT, 0.5 mM PMSF, 5 µg/ml aprotinin, 5 µg/ml pepstatin, 30 µg/ml leupeptin and incubated at 4°C with mechanical shaking for 60 min. OA-SF were centrifuged at 13 000g, before the supernatant was collected and 100 µl of buffer D (8 mM HEPES pH 7.9, 0.5 mM DTT, 25 mM KCl, 0.1 mM EDTA, 8% glycerol) added. The protein content of each nuclear extract was established using a BCA protein assay kit (Pierce, USA). Four micrograms of protein was incubated with 10x Binding Buffer (40% Glycerol, 10 mM EDTA, 50 mM DTT, 100 mM Tris pH 7.5, 1 M NaCl, 1mg/ml nuclease-free BSA), 2 µg of non-specific DNA competitor (polydIdC) and 1µl of radiolabelled 32P probe for NF-{kappa}B (5'-AGT TGA GGG GAC TTT CCC AGG C-3', 3'-TCA ACT CCC CTG AAA GGG TCC G-5') at room temperature for 30 min. For the cold competitor and non-self EMSA, 25-fold excess of unlabelled NF-{kappa}B oligo or 10-fold excess of AP-1 consensus oligo (5'-CGC TTG ATG ACT CAG CCG GAA-3', 3'-GCG AAC TAC TGA GTC GGC CTT- 5') or 10-fold excess of AP-1 mutant oligo (5'-CGC TTG ATG ACT TGG CCG GAA-3'3'-GCG AAC TAC TGA ACC GGC CTT-5') was added to the samples and incubated on ice for 30 min prior to addition of radiolabelled probe. Protein–DNA complexes were resolved on a 4% polyacrylamide gel for 80 min and visualized by autoradiograph.

Cell viability
OA-SF were routinely examined under the microscope and at the conclusion of each experiment an Almar Blue Assay (Biosource International, USA) was performed to assess the viability of OA-SF according to manufacturer's guidelines.

Inhibition of the NF-{kappa}B signalling pathway
Two pharmacological inhibitors developed specifically to inhibit NF-{kappa}B signalling via IKK in OA, were employed in both the OA-SF and OA-EXP model systems. RO100 and RO919 were a kind gift of Roche Palo Alto, CA USA, and were administered at a known dose or dose range. The IKK inhibitors had been previously demonstrated to be effective in HUVECs. The enzyme IC50s for IKKB activity for both RO100 and RO919 are 2 nM. The IC50s of NF-{kappa}B activation were 100 nM and 30 nM, respectively for RO100 and RO919.

Anti-cytokine strategies
OA-COCUL were treated with etanercept, a soluble TNF-receptor Ig fusion protein (100 µg/ml), a depleting anti-IL-1ß antibody (10 µg/ml), a combination of the two agents or left untreated for 48 h. OA-EXP were treated with the combination of etanercept and anti-IL-1ß for 24 h (at the doses stated previously). At the conclusion of each investigation supernatants were harvested and cytokine and MMP levels measured by specific enzyme-linked immunosorbent assay (ELISA).

Enzyme-linked immunosorbent assay (ELISA)
Cytokines and MMP levels were quantified from the supernatants harvested from experimental OA-SF, OA-COCUL and OA-EXP. IL-1ß, TNF-{alpha}, IL-6 and MMP-3 levels were measured using specific Biosource Europe ELISA kits (Human IL-1ß Cytoset-CHC1214, Human TNF-{alpha} Cytoset-CHC1754, Human IL-6 Cytoset-CHC1264 & Human MMP-3 Cytoset-CHC1544) following the manufacturer's protocol supplied with each kit. TIMP-1 levels were quantified using an R&D Systems ELISA kit (Human TIMP-1 Duoset–DY970). The levels of MMP-1 were established using a matched antibody pair system. Briefly, 96-well ELISA plates (Nunc) were coated with 100 µl of monoclonal anti-human MMP-1 antibody (MAB901, R & D Systems) at 1 µg/ml diluted in PBS overnight with mechanical shaking. Following incubation the antibody was discarded and plates were blocked for 1 h at room temperature with 300 µl of 1% BSA/PBS. Plates were washed four times in PBS with 0.05% Tween-20 (pH 7.2–7.4) added. 100 µl of diluted recombinant human MMP-1 (901-MP, R & D Systems) standards (3.125–200 ng/ml) were added in duplicate. 100 µl of each sample diluted in 1% BSA/PBS (1 : 2–1 : 100 depending on experimental investigation and in vitro model system) were added accordingly and incubated for 1.5 h at room temperature with mechanical shaking. After four washes 100 µl of biotinylated anti-human MMP-1 antibody (BAF901, R & D Systems) at 0.03 µg/ml diluted in 1% BSA/PBS was added and incubated for 1 h at room temperature with mechanical shaking. Following four washes, 100 µl of purified streptavidin–horseradish peroxidase conjugate (Biosource Europe) at 1 µg/ml was added and incubated for 30 min at room temperature with mechanical shaking. After four final washes, 100 µl of developing solution (containing 0.01% tetramethyl benzidine) was added and the colour was developed at room temperature. The reaction was terminated by the addition of 50 µl of 12.5% H2SO4. The optical density of the plates was measured at 450 nm (0.1 s) using a Wallac Victor 2 plate reader.

Statistical testing
All statistical differences determined in this study used the paired means Student's t-test. A two-tailed test was performed for Fig. 1 (A–D) and Fig. 2 (A and B), a one-tailed test was performed for Fig. 3 (A–H). P ≤ 0.05 were considered significant, with values of ≤0.01 considered highly significant. The non-parametric Wilcoxon match pairs test was additionally performed for Fig. 4 (A–D).


Figure 1
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  		FIG. 1. The effect of RO100 and RO919 upon the IL-1ß induced OA-SF model. Nuclear extracts were prepared from OA-SF treated with a dose range of RO100 and RO919 and subsequently stimulated with IL-1ß (20 ng/ml). Extracts were analysed by EMSA (A), with 25-fold excess of self and 10-fold excess of non-self competitor added to determine specificity. Densitometric analysis (B) was conducted on EMSAs derived from four individual OA patients using NIH image software with results expressed as the mean ± S.E.M., percentage of nuclear NF-{kappa}B. MMP-3 and TIMP-1 levels were measured by ELISA from the supernatants of OA-SF treated with a dose range of RO100 and RO919 (C and D). Results are given as the mean ± S.E.M., ng/ml (n = 5). (*P ≤ 0.05).

 

Figure 2
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  		FIG. 2. Effect of NF-{kappa}B inhibition upon the pathogenic mediators produced by IL-1ß stimulated OA-SF. OA-SF were treated with a 0.1 µM dose of RO100 and RO919 and subsequently stimulated with IL-1ß (20 ng/ml). The levels of IL-6, MMP-1, MMP-3 and TIMP-1 were quantified by specific ELISA. Results are expressed as mean ± S.E.M., ng/ml (n = 5 individual patients) (A and B). (*P ≤ 0.05, **P ≤ 0.01).

 

Figure 3
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  		FIG. 3. Effect of anti-cytokine strategies employed in OA-COCUL. Nuclear extracts were prepared from OA-COCUL treated with either etanercept (10 µg/ml), anti-IL-1ß antibody (100 µg/ml), the two agents in combination or left untreated (UT). The extracts were analysed by EMSA, note only the protein–DNA complexes are shown (A). Densitometric analysis was conducted on EMSAs using NIH image software, with the results given as the mean ± S.E.M. expressed as a percentage of nuclear NF-{kappa}B (B). TNF-{alpha} (C), IL-1ß (D), IL-6 (E), TIMP-1 (F), MMP-1 (G) and MMP-3 (H) levels were quantified by specific ELISA. Data are expressed as mean ± S.E.M. as a percentage. (n = 9 individual experiments). (*P ≤ 0.05 and **P ≤ 0.01).

 

Figure 4
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  		FIG. 4. OA-EXP represent a physiologically relevant model for studying disease progression in vitro. OA-EXP were prepared as described previously in the materials and methods. After an 18 h equilibration period each explant was either left untreated (UT) or treated with a combination of etanercept and anti-IL-1ß (doses previously stated) or a 0.1 µM dose of RO100. IL-6, MMP-1, MMP-3 and TIMP-1 levels were established by ELISA and adjusted for weight. Results are expressed as a percentage, with the mean of each treatment condition indicated by the solid horizontal line. (n = 10 individual, independent experiments). (*P ≤ 0.05 and **P ≤ 0.01).

 

   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
IKK inhibitors dysregulate IL-1ß induced MMP-3 and TIMP-1 production in OA-SF
IKK inhibitors, RO100 and RO919 (1, 0.1, 0.03 µM), were employed to modulate IL-1ß (20 ng/ml) induced NF-{kappa}B activation in OA-SF. EMSAs conducted on the nuclear extracts of treated OA-SF showed dose dependent inhibition of NF-{kappa}B activation (Fig. 1A). Densitometric analysis demonstrated a significant (P ≤ 0.05) reduction in NF-{kappa}B nuclear signalling at the 1 µM dose (Fig. 1B). Binding specificity was confirmed by competition assays against unlabelled oligonucleotides of identical NF-{kappa}B sequence and using unrelated AP-1 consensus or mutant sequences.

When culture supernatants were analysed both MMP-3 and TIMP-1 production (mean ± S.E.M., ng/ml) increased significantly from 1 ± 0.9 and 265 ± 53.0 to 163 ± 63.9 and 399 ± 63.3, respectively in response to IL-1ß stimulation (Fig. 1C and D). Both RO100 & RO919 elicited significant (P ≤ 0.05) dose dependent reduction of IL-1ß induced MMP-3 production, with maximum inhibition noted at the 1 µM dose for each agent (Fig. 1C and D). Interestingly, at doses below 1 µM, MMP-3 production was inhibited without affecting TIMP-1 significantly.

Amelioration of NF-{kappa}B activation inhibits IL-1ß induced cytokine and MMP expression in OA-SF
Upon IL-1ß activation, IL-6 production was significantly (P ≤ 0.05) repressed by a 0.1 µM dose of RO100 or RO919 (Fig. 2A and B). Both RO100 and RO919 also inhibited MMP-3 production, but the effect was most pronounced upon MMP-1, with a highly significant (P ≤ 0.01) 80% and 66% reduction with RO100 and RO919, respectively. Neither inhibitor elicited a notable effect upon TIMP-1 induction. These effects were not attributable to cell death, as cell viability was >95% at endpoint for each data point reported.

IL-1ß and TNF-{alpha} regulate IL-6, MMP-1 and MMP-3 in OA-COCUL
IL-1ß and TNF-{alpha} were neutralized in culture using a specific anti-IL-1ß antibody (10 µg/ml) and etanercept (100 µg/ml). Anti-IL-1ß and etanercept administered either solely or in combination resulted in marked reduction in NF-{kappa}B activation (Fig. 3A and B). Anti-IL-1ß did not significantly affect TNF-{alpha} production whilst etanercept did not elicit a significant reduction in IL-1ß levels (Fig. 3C and D). Combination therapy evoked a greater reduction in cytokine and MMP production, than either agent alone (Fig. 3E–H). These observations demonstrated that IL-6, MMP-1 and MMP-3 expression in OA-COCUL was dependent on both IL-1ß and TNF-{alpha} expression.

Anti-IL-1ß, etanercept and a novel NF-{kappa}B inhibitor reduce IL-6 and MMP-1 production in OA-EXP
Consistent with previous studies [31], histological analysis of OA-EXP revealed that the degree of inflammation and angiogenesis varied tremendously between OA patients (data not shown). OA-EXP treated with a combination of etanercept and anti-IL-1ß or a 0.1 µM dose of RO100 elicited a significant (P ≤ 0.01) reduction in IL-6 and MMP-1 production (Fig. 4A and C). However, neither MMP-3 nor TIMP-1 were significantly affected (Fig. 4B and D).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Our studies demonstrated that IL-1ß and TNF-{alpha} activation of the NF-{kappa}B signalling cascade regulate IL-6, MMP-1 and MMP-3 production in OA. We have clearly shown that the NF-{kappa}B signalling cascade can be targeted to achieve inhibition of a range of critical mediators implicated in the pathogenesis of OA, and is therefore of therapeutic interest.

Our data supports the current opinion that in OA, IL-1ß and TNF-{alpha} drive disease progression by inducing MMPs (critical mediators of cartilage destruction) [32, 33] and IL-6, unique in eliciting a significant role both in synovial inflammation and cartilage destruction within the joint [21, 22]. The present study sought to investigate the role of the NF-{kappa}B signal transduction pathway in OA, specifically employing two novel inhibitors of IKK namely, RO100 and RO919.

RO100 and RO919 repressed the production of IL-6, MMP-1 and MMP-3 in the IL-1ß stimulated OA-SF. Such observations suggest that the induction of IL-6, MMP-1 and MMP-3 in OA-SF is tightly regulated by NF-{kappa}B. Indeed, a diminutive reduction in nuclear NF-{kappa}B activation caused significant inhibition of these mediators that are so important in accelerating the progression of OA. Our findings are supported by a previous study by Wakamatsu et al. [34] who demonstrated that a small molecule inhibitor of NF-{kappa}B suppressed TNF-{alpha} induced IL-6, CCL2, CCL5 and MMP-3 production in RA fibroblast like synoviocytes (RA FLS). It has been established in both astrocytes [35] and hepatic stellate cells [36] that IL-1ß upregulates TIMP-1 production. In our study IL-1ß also caused significant upregulation of the chondroprotective mediator TIMP-1 but neither RO100 nor RO919, at concentrations up to 1 µM, significantly altered IL-1ß induced TIMP-1 production. These data suggest either, that the regulation of TIMP-1 is modestly controlled by IL-1ß induced NF-{kappa}B activation or that the IKK inhibitors (above 1 µM) elicit non-specific effects that result in the down regulation of TIMP-1 without significantly affecting cell viability. If the latter is the case, then the dramatic amelioration of MMP-3 seen at the 1 µM dose may also be a consequence of unknown non-specific effects.

We also studied the interplay between IL-1ß and TNF-{alpha} and their role in mediating pathology in OA. IL-1ß and TNF-{alpha} are spontaneously produced by synovial macrophages present in OA-COCUL [37], anti-IL-1ß and etanercept were used to neutralize each cytokines bioactivity, respectively. IL-1ß and TNF-{alpha} neutralization repressed IL-6, MMP-1 and MMP-3 production without affecting TIMP-1. However, complete inhibition was not achieved; this could be due to low levels of unidentified NF-{kappa}B activators in tissue culture media, for example, lipopolysaccharide. With hindsight the experimental design of OA-COCUL could be improved, in future studies we aim to include an overnight equilibration period prior to commencement that would allow accurate determination of baseline levels of each mediator before cytokine neutralization. It may well be that the reduction in IL-6, MMP-1 and MMP-3 elicited by the cytokine neutralization maybe masked to some extent by residual cytokine and MMP's generated before dosing with either anti-IL-1ß and/or etanercept. Our findings, however, are in accordance with a study conducted by Kobayashi et al. [38], they neutralized both IL-1ß and TNF-{alpha} activity in OA cartilage explants using recombinant IL-1ß receptor agonist (IL-1RA) and PEGylated soluble TNF receptor I (sTNFRI) respectively and showed marked reduction in MMP-1 and MMP-3 gene expression and reduced cartilage depletion. A subsequent clinical study conducted by Chevalier et al. [39] concluded that targeting IL-1ß by intraarticular administration of IL-1RA could provide a viable target for therapy in OA. Currently there are no studies published that have targeted TNF-{alpha} for treatment of OA, although, targeting TNF-{alpha} using etanercept or infliximab, for example, has revolutionized the management of RA [40, 41]. The results of our present study and those of others, detailed previously [38, 39], show that IL-1ß and TNF-{alpha} act synergistically and that a treatment strategy that targets both cytokines may be more beneficial than targeting either cytokine alone in OA. In order to clarify the role of IL-1ß and TNF-{alpha} in OA further studies are required.

We tested RO100 and RO919 in OA-COCUL but found that even at the lowest concentration (0.03 µM) both agents were extremely toxic. We attributed the high sensitivity of the cells to the aggressive collagenase and DNAse digestion required to prepare OA-COCUL. Consequently, a more robust ex vivo model was developed using OA synovial explants. Both RO100 and etanercept with anti-IL-1ß reduced IL-6 and MMP-1 production without significantly affecting TIMP-1 or MMP-3. There may be many reasons for this, firstly, several signalling pathways induce MMP expression and it would appear that MMP-3 production is not primarily dependent upon NF-{kappa}B signalling in OA-EXP. Secondly, MMP-3 is most active in early OA affecting proteoglycan depletion in cartilage, OA-EXP were derived from end-stage patients where MMP-3 levels are comparatively low [14] thus IKK inhibitors could not affect further down-regulation of this enzyme.

To conclude, our study supports the view that an intrinsic network of cytokines and MMP's exist in OA and that the subsequent interactions between them is complex. We have demonstrated, using novel IKK inhibitors (RO100 and RO919) and anti-cytokine agents (etanercept and anti-IL-1ß) the genuine potential of inhibiting pathogenic mediators by targeting the NF-{kappa}B pathway specifically. The observations have important implications with regard to developing new specific therapies for OA aimed at inhibiting cartilage depletion.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
S.N.L. and J.B.'s work was supported in part by a project grant from the Arthritis Research Campaign. S.M.C.'s work was supported by the Wellcome Trust. P.B.'s work was supported by the Leukaemia Research Fund. The authors would like to thank Dr R. Goodfellow, PhD and the orthopaedic theatre staff at the Royal Glamorgan Hospitial, Llantrisant, Wales, UK for the ethical approval and collection of synovial samples and Mr N. Amos for technical support.

The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Submitted 26 May 2006; Accepted 9 November 2006


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