Influence of bisphosphonates on the production of pro-inflammatory cytokines by activated human articular chondrocytes

www.elsevier.com/locate/issn/10434666 Cytokine 31 (2005) 298e304

Influence of bisphosphonates on the production of pro-inflammatory cytokines by activated human articular chondrocytes Jan F. Van Offel, Evelyne J. Dombrecht, Chris H. Bridts, Annemie J. Schuerwegh, Didier G. Ebo, Wim J. Stevens*, Luc S. De Clerck Department of Immunology, Allergology and Rheumatology, University of Antwerp, Campus Drie Eiken, Universiteitsplein 1, B-2610 Antwerp, Belgium Received 15 October 2004; received in revised form 17 February 2005; accepted 19 May 2005

Abstract Bisphosphonates have anti-inflammatory effects in rheumatoid arthritis and chondroprotective effects in animal arthritis models but their influence on chondrocytes is not known. The aim of this study is to investigate whether bisphosphonates could influence the production of pro-inflammatory cytokines by activated chondrocytes. Therefore human articular cartilage explants were incubated at 48 h with clodronate, pamidronate or risedronate (10ÿ6 and 10ÿ8 mol/L), and dexamethasone (10ÿ8 mol/L). Subsequently, cultures were stimulated with IL-1, 10 ng/mL (n Z 6) or 1 ng/mL (n Z 10) for 48 h. Co-incubation was performed with or without bisphosphonates or dexamethasone. A flow cytometric microsphere-based immunoassay was used for the detection of the pro-inflammatory cytokines IL-6, IL-8, TNF-a, IL-1 and the regulatory cytokines IL-12p70 and IL-10 in the supernatants. Stimulation with IL-1 resulted in a dose dependent induction of IL-6 and IL-8, but no production of the other cytokines could be demonstrated. This production of IL-6 and IL-8 was neither inhibited nor enhanced by bisphosphonates. Only dexamethasone caused an inhibition of IL-6 production. In conclusion, there is no evidence on the level of articular cartilage cells that bisphosphonates would suppress or enhance IL-6 and IL-8 mediated joint destruction. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Arthritis; Bisphosphonates; Cartilage; Chondrocytes; Cytokines

1. Introduction Pro-inflammatory cytokines, notably IL-1 and TNF-a, play an important role in initiating and perpetuating inflammatory and destructive processes in rheumatoid arthritis (RA) [1]. In response to these cytokines, chondrocytes secrete several mediators such as pro-

* Corresponding author. Tel.: C32 3 820 25 95; fax: C32 3 820 26 55. E-mail address: [email protected] (W.J. Stevens). 1043-4666/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2005.05.009

inflammatory cytokines [2,3], metalloproteinases [4,5], NO and reactive oxygen species [6,7] which all can promote joint inflammation and cartilage destruction. Moreover, treatment with either IL-1 [8e10] or TNF-a blockers [11e13] reduces inflammation and inhibits joint destruction in patients with active RA. Both cytokines share many activities but in some animal models of arthritis IL-1 appears to be more important than TNF-a in promoting cartilage and bone destruction [14]. IL-1 causes cartilage damage by stimulating the release of matrix metalloproteinases

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and other proteolytic enzymes that degrade proteoglycans and type 2 collagen [4,15]. IL-1 also appears to block repair processes in cartilage to a greater extent than TNF-a [16,17]. For these reasons, IL-1 is commonly used in culture models to mimic the circumstances leading to in vivo cartilage destruction [18]. In chondrocyte cell cultures and cartilage explants, IL-1 has been shown to induce other pro-inflammatory cytokines such as IL-6 [2,3,19] and IL-8 [3,20e23]. This means that the inflamed cartilage itself may contribute to inflammation and joint destruction. IL-6 is a potent growth and differentiation factor for B and T lymphocytes and appears to play an important role in osteoclast development [24]. In patients with RA, IL-6 may be involved in cartilage degradation [25,26], periarticular osteoporosis [27] and generalized bone loss [28]. IL-8 is a strong chemotactic factor for neutrophils and T lymphocytes [29] and an inducer of neutrophil degranulation facilitating cartilage destruction [30]. Different authors have reported on anti-inflammatory properties of bisphosphonates in RA [31e33] and chondroprotective effects in animal arthritis models [34e36]. In a previous study we demonstrated pamidronate treatment to down-regulate IL-1 in circulating monocytes of RA patients [37]. However, the effects of bisphosphonates on activated chondrocytes remain unknown. The aim of the present study is to investigate the influence of bisphosphonates on the in vitro production of pro-inflammatory cytokines by activated human chondrocytes.

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10ÿ8 mol/L. These concentrations are demonstrated to be non-toxic for chondrocytes [41]. In the 6 experiments using 10 ng/mL of IL-1, production of IL-6 and IL-8 was significantly higher than in unstimulated controls (Fig. 1, p ! 0.03). There was no measurable induction of TNF-a, IL-1, IL-12p70 or IL-10. Dexamethasone, 10ÿ8 mol/L, caused a significant inhibition of IL-6 production ( p ! 0.05) but not for IL-8 ( p Z 0.1). Bisphosphonates neither affect IL-1 induced IL-6 or IL-8 production ( p O 0.25 and O0.50, respectively) nor spontaneous cytokine production in unstimulated controls. In the 10 experiments using 1 ng/mL of IL-1, 1 experiment was excluded for further analysis because of outlying high values for basal IL-6 and IL-8 production. Although less than with 10 ng/mL of IL-1, IL-6 production was still significantly higher ( p Z 0.008) in the remaining 9 explants as compared to unstimulated controls (Fig. 1A). Again none of the bisphosphonates neither suppressed nor enhanced the production of IL-6 ( p O 0.1, Fig. 2). There was a clear induction of IL-8 production in 6 of the 9 experiments ( p ! 0.03, Fig. 1B)

2. Results Our experiments were performed on cartilage explants because chondrocytes remain in their extra-cellular matrix resembling most closely the in vivo situation. Two concentrations of IL-1, 10 ng/mL and 1 ng/mL, were used to stimulate the cartilage explants. These concentrations were selected from doseeresponse experiments on cytokine induction [3], apoptosis and NO production [38,39]. Although IL-1 in a concentration of 10 ng/mL was reported to induce a maximal production of IL-6 in human articular chondrocytes [3], IL-1 in a concentration of 1 ng/mL approaches the concentrations demonstrated in synovial fluid in RA [40]. Cytokine productions were measured after 48 h of stimulation. This point in time was based on a study regarding the kinetics of IL-6 production in chondrocytes after stimulation with IL-1 in a concentration of 1 ng/mL [2]. Due to limited availability of cartilage explants per patient, we performed two separate series of experiments, using 10 ng/mL of IL-1 (n Z 6) or 1 ng/mL (n Z 10), respectively. Based on former work, we used each bisphosphonate in a concentration of 10ÿ6 and

Fig. 1. IL-6 and IL-8 production before and after stimulation with two concentrations of IL-1. (A) Results of IL-6 production after stimulation with IL-1 1 ng/mL (n Z 9) and 10 ng/mL (n Z 6). (B) Results of IL-8 production after stimulation with IL-1 1 ng/mL (n Z 6) and 10 ng/mL (n Z 6).

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Fig. 2. Results of IL-6 production after stimulation with 1 ng/mL IL-1 and co-incubation with or without dexamethasone, clodronate, pamidronate or risedronate (n Z 9).

but no influence of bisphosphonates could be demonstrated ( p O 0.1, Fig. 3). In order to investigate whether these results could be related to an insufficient uptake in the cartilage explants, an additional experiment was performed on isolated articular chondrocytes using different concentrations of bisphosphonates. Again no effect on IL-6 and IL-8 production was demonstrated suggesting that insufficient penetration is unlikely.

3. Discussion The main purpose of this study was to evaluate the potential effects of bisphosphonates on activated articular chondrocytes. We could not demonstrate an inhibition by bisphosphonates on the production of IL-6 nor IL-8 by IL-1 stimulated human cartilage explants. On the other hand, no enhancement of the production of these proinflammatory cytokines was observed either. This is important as IL-6 produced in the inflamed cartilage can contribute to the activation of osteoclasts and consequently to subchondral bone loss [27] whereas IL-8 is known to induce neutrophil-mediated cartilage degradation [30]. One possible explanation for our results could be that bisphosphonates fail to reach the chondrocytes

embedded in their matrix. Some older reports demonstrated the uptake of bisphosphonates in tracheal [42] and costal [43] cartilages. As far as we know, no data are available about the uptake of bisphosphonates by normal or rheumatoid articular cartilage. Results from a similar experiment we performed with isolated chondrocytes suggest that an insufficient uptake of bisphosphonates in articular cartilage is unlikely. Although some reports mention a pro-inflammatory effect [36,44], several animal arthritis models show that bisphosphonates protect against joint destruction [34e36,45e48]. Furthermore, evidence has emerged for a down-regulating effect of bisphosphonates on inflammatory cells in RA. In an earlier study, we demonstrated a suppression of IL-1 in circulating monocytes of patients with RA treated with pamidronate and a dose related induction of apoptosis in a monocytic cell line by this bisphosphonate [37]. Other studies showed a significant decrease of serum levels of IL-1, IL-6 and TNF-a with alendronate [31], a decrease of serum IL-6 levels with etidronate [33] and a reduction of serum C-reactive protein levels (CRP) with pamidronate [32] while it is known that in RA, serum levels of CRP correlate with the levels of IL-6 [49,50]. The activity of IL-1 and TNF-a is normally balanced by several endogenous inhibitors [16]. Their importance was illustrated by the development of a polyarthropathy that resembled RA in IL-1 receptor antagonist-deficient

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Fig. 3. Results of IL-8 production after stimulation with 1 ng/mL IL-1 and co-incubation with or without dexamethasone, clodronate, pamidronate or risedronate (n Z 6).

mice [51]. The results of our study could not provide evidence for a production of IL-1 or TNF-a by the activated chondrocytes, nor for the regulatory cytokines IL-10 and IL-12p70. One possible explanation that has to be considered is the presence of inhibitors that capture the secreted cytokines. We performed an additional spiking experiment where IL-1 and TNF-a were added to supernatants from IL-1 activated chondrocytes and measured a clear reduction for both cytokines which might be an indication for the presence of inhibitors (results not shown). However, whether bisphosphonates could influence endogenous inhibitors is not known. Apart from inhibiting effects on inflammatory cells and pro-inflammatory cytokines, other mechanisms can contribute to a chondroprotective effect of bisphosphonates. In a previous study we demonstrated that aminobisphosphonates protect articular chondrocytes from apoptosis [41] which is important as chondrocytes are responsible for the production and maintenance of the cartilage matrix. Another mechanism might be at the level of metalloproteinases that are thought to be the primary mediators of matrix degradation [52]. These enzymes are upregulated by IL-1 and inhibited by tissue inhibitors of metalloproteases (TIMPs), in cartilage

notably TIMP-3 [53]. Reductions in metalloproteinases 1 and 3 have been demonstrated in patients with spondylitis ankylosans treated with pamidronate [54] whereas alendronate was shown to inhibit IL-1 induced collagenase-3 (MMP-13) [55]. A final mechanism for a chondroprotective effect of bisphosphonates could be at the level of subchondral bone metabolism. Several studies have been performed to ascertain whether bisphosphonates can modulate the synthesis of osteoclast promoting factors as IL-6 by osteoblasts. These reports show contradictory results. Alendronate was shown to increase IL-1 stimulated IL-6 production in an osteoblast cell line [56] whereas other reports mention an inhibitory effect for etidronate, clodronate, alendronate and zoledronate [57,58] or no influence at all for etidronate and alendronate [59]. In conclusion, each bisphosphonate has its proper physicochemical and biological characteristics and therefore it is impossible to extrapolate data from one compound or cell type to another. To our knowledge, our work is the first that investigates the influence of three generations of bisphosphonates on cytokine production by activated articular chondrocytes embedded in their matrix. We found no evidence on the level of articular cartilage that bisphosphonates would suppress or enhance IL-6 and IL-8 mediated joint destruction.

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4. Materials and methods 4.1. Culture of cartilage explants Human cartilage was obtained from patients undergoing total hip replacement for hip fracture. Patients with rheumatoid arthritis, destructive osteoarthritis, active infection, malignancy and treatment with bisphosphonates or glucocorticoids were excluded. The mean age of the patients was 72 years (range 53e81). All cartilages appeared normal by morphological examination. Full-thickness cartilage discs of equal size were obtained using a 4 mm biopsy punch (PFM, Ko¨ln, Germany) avoiding the calcified layer. The explants were pooled and cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Paisley, UK) supplemented with 10% fetal bovine serum (FBS, Hyclone, Utah, USA), 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen). The culture medium was enriched with CaCl2 (final concentration 8 mg/dL) [60]. 4.2. Preincubation with bisphosphonates or dexamethasone Three cartilage explants per well (48 well plates) were incubated for 48 h in medium with each bisphosphonate with a concentration of 10ÿ6 or 10ÿ8 mol/L or with dexamethasone (10ÿ8 mol/L) or in complete medium alone as a control. The bisphosphonates used were clodronate (Bonefos, Schering, Diegem, Belgium) and pamidronate (Aredia, Novartis Pharma, Vilvoorde, Belgium) both as commercially available drugs and risedronate (powder, kindly provided by Procter & Gamble Pharmaceuticals, Cincinnati, USA). Clodronate, pamidronate and risedronate were selected representing first, second and third generation of bisphosphonates, respectively. 4.3. Stimulation of cartilage discs After 48 h of incubation, the medium was replaced by culture medium without FBS and with or without bisphosphonates or dexamethasone. The cartilage discs were then stimulated with human IL-1 (IL-1b, PeproTech House, London, UK) in a concentration of 10 ng/mL for an additional 48 h (n Z 6) or with 1 ng/mL (n Z 10). Unstimulated cartilage explants incubated with medium alone or with bisphosphonates were used as controls. After culture, supernatants were collected and stored at ÿ20  C until further analysis.

Cartilage discs were incubated for 60 min with 5 mg/mL calcein AM (Molecular Probes, Leiden, The Netherlands). Thereafter 50 mg/mL propidium iodide (Sigma, St. Louis, USA) was added. Viability was visualized using a fluorescence microscope equipped with a digital camera Nikon DXM 1200 (Nikon, Tokyo, Japan) and analysed using Eclipse Net (Nikon) software. 4.5. Culture of isolated chondrocytes Isolated human chondrocytes were obtained from the femoral head of a patient who underwent total hip replacement for hip fracture. Chondrocytes were isolated as described before [38,61]. After isolation, primary chondrocytes were cultured in monolayer at 1 ! 106 cells/mL in complete medium with 10% FBS for 24 h. Thereafter the chondrocyte culture was treated in the same culture conditions as the cartilage explants. 4.6. Flow cytometric microsphere-based immunoassay Cytokine production was measured as described before [62]. Briefly, a flow cytometric microsphere-based immunoassay (Cytometric Bead Array, CBA, BD Biosciences, Erembodegem, Belgium) was used for the detection in the supernatants of the pro-inflammatory cytokines IL-6, IL-8, TNF-a, IL-1 and the regulatory cytokines IL-10 and IL-12p70. Instrument set up was performed using CaliBRITE beads and cytometer set up beads (BD), according to the manufacturer’s instructions [63]. A monomeric microsphere population was gated out on forward and sideward scatter. Two thousand five hundred beads were measured and analysed. Data were analysed in two-color fluorescence dot plots, representing the different microsphere populations and the cytokine concentration. Mean fluorescence intensity values (MFI) were measured. The assay sensitivity ranges from 1.9 to 7.2 pg/mL depending on the cytokine. The results of cytokines measured in the culture medium were expressed in pg/mL per mg of cartilage. 4.7. Statistics All statistical analyses were performed using SPSS 11.0. Differences in cytokine production were evaluated with Wilcoxon’s rank test. A p-value of !0.05 was considered significant. References

4.4. Viability of cartilage explants Viability was assessed using a calcein AM/propidium iodide staining.

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