Clin Exp Med (2015) 15:277–283 DOI 10.1007/s10238-014-0307-4
ORIGINAL ARTICLE
Combined effects of infliximab and methotrexate on rheumatoid arthritis osteoblastic cell metabolism Addolorata Corrado • Anna Neve • Arcangela Marucci Annamaria Gaudio • Francesco Paolo Cantatore
•
Received: 2 May 2014 / Accepted: 5 August 2014 / Published online: 23 August 2014 Ó Springer-Verlag Italia 2014
Abstract The goal of this study is to investigate the in vitro effects of two disease-modifying anti-rheumatic drugs, largely used in the treatment of rheumatoid arthritis (RA), [infliximab (IFX) and methotrexate (MTX)], in RA primary osteoblast cell cultures. MTX inhibited proliferation and metabolic activity in RA osteoblasts was able to increase apoptosis. Conversely, IFX increased the proliferation, osteocalcin production and the alkaline phosphatase activity. Interestingly, IFX appeared to antagonise the negative effect exerted by MTX. Both drugs significantly reduced the IL-6 production in osteoblasts when used alone, and the combination of the two agents resulted in a significant additional reduction of IL-6 synthesis, with an apparent additive effect. The present study suggests that MTX exerts negative direct effects on bone metabolism in RA patients, but the combined treatment with anti-TNF-a can be beneficial for the interaction of MTX with bone cells. Keywords Osteoblasts Rheumatoid arthritis Disease modifying anti-rheumatic drugs (DMARDs)
Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised by joint inflammation associated with structural bone damage represented by generalised
A. Corrado A. Neve A. Marucci A. Gaudio F. P. Cantatore (&) Rheumatology Clinic, Department of Medical and Surgical Sciences, University of Foggia, Ospedale ‘‘Col. D’Avanzo’’, V.le degli Aviatori 1, 71100 Foggia, Italy e-mail: [email protected]
osteoporosis and localised bone loss, which includes erosions and juxta-articular osteopenia of affected joints. Recent literature suggests that these three types of bone loss are at least in part mediated by common pathogenic mechanisms [1, 2] that converge toward an alteration of bone remodelling processes. Among the factors that regulate bone remodelling and osteoclast activity, TNF-a plays a central role as it is involved both in local erosive processes and in systemic osteoporosis. Further, TNF-a is among the most important cytokines sustaining the chronic inflammation that characterises RA, thus representing a possible link between inflammation and bone loss. Besides TNF-a, there are various cytokines involved both in the maintenance of inflammatory state and in the pathogenesis of systemic bone loss and structural joint damage, such as IL-6, IL-1, IL-11 and IL-17 [3]. Indeed, chronic inflammation and bone loss are strictly linked in RA, and the control of inflammation appears to be one of the most important strategies for the prevention of bone loss in this disease [2]. These observations suggest that disease modifying anti-rheumatic drugs (DMARDs) can have a beneficial systemic effect on bone metabolism. Many data consistent with a positive effect of TNF-a blockade on bone mass in RA patients have been reported in the last years [4, 5]; interestingly, the benefit on bone mineral density (BMD) induced by anti-TNF-a treatment appears to be independent of the clinical response in terms of effect on disease activity [6]. Conversely, methotrexate (MTX), whose mechanism of action is independent of TNF-a blockade, in spite of its ability to control systemic and local joint inflammation, not only does not exert any direct positive effect on bone metabolism but also could impair bone formation, particularly when it is used at high doses [7], or for long term [8, 9]. It has been shown that low dose of MTX is able to inhibit osteoblast function [10] when it is
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given to normal rats. But in rats with adjuvant-induced arthritis, bone mass was maintained in the low dose MTXtreated animals compared with untreated controls [11, 12]. To date, the exact mechanisms of the direct effects of DMARDs on bone cells in inflammatory joint diseases are poorly understood and little is known about the effects of the combined treatment on cells involved in bone remodelling and bone reparation processes. The aim of this study was to evaluate the direct effect of the anti-TNF-a drug, infliximab (IFX) and MTX, alone or in combination, on proliferation, viability, metabolic activity and IL-6 synthesis in primary human osteoblast cultures derived from trabecular bone samples of patients with RA.
Patients and methods RA bone specimens were obtained from 11 subjects (2 men, 9 women) aged 55.12 ± 9.6 years (mean ± SD, range 39–67), fulfilling the 2010 ACR/EULAR diagnostic criteria for RA, undergoing total joint replacement of hip or knee. None of the enrolled subjects was affected by osteoporosis or other metabolic bone diseases and none received medication, including corticosteroids, which could interfere with bone metabolism for 3 months prior to the bone biopsy. Further, none of the selected subjects were treated with methotrexate or antiTNF-a drugs for 3 months prior the bone biopsy. Normal bone fragments were obtained from 9 adult subjects (4 men and 5 women), aged 58 ± 12.9 years (mean ± SD, range 35–61 years) undergoing surgery for traumatic fractures of the distal femoral epiphysis, who served as controls. Appropriate informed consent was obtained from each patient and the study was approved by Institutional Ethics Committee.
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Cell cultures treatment MTX was provided by Hansermann; IFX, a chimeric antihuman TNF–a monoclonal antibody, was provided by Schoering-Plough. When reaching confluence, osteoblasts were seeded in 24-well plates, in DMEM containing 10 % FCS and antibiotics, at a density of 6 9 104 cells for every plate until semi-confluence was reached. Cells were then treated with MTX (100 lg/ml) and IFX 0.1, 1 and 10 lg/ml, either alone and combined, for 24 h. The concentration of each agent used in the experiments was determined from the available data concerning IFX and MTX serum concentrations in RA patients after administration at therapeutic dosage [13, 14]. After drugs exposure, osteocalcin levels, alkaline phosphatase activity (ALP), apoptosis, cell proliferation and IL6 production were determined in cell cultures. All experiments were performed in triplicate. Osteocalcin synthesis and ALP activity Osteocalcin release into the culture medium was measured using an intact human osteocalcin ELISA (MetraOsteocalcin EIA kit, San Diego, CA). Osteocalcin secretion rates were expressed as nanogram/mg of intracellular proteins. The intracellular protein content of each well was determined by the Bradford method (Bio-Rad protein assay, Bio-Rad Laboratories, Richmond, CA). In parallel, ALP activity was evaluated in cell lysate, obtained through the solubilization of cell monolayer with 0.1(vol/vol) Triton X-100. The colorimetric determination of ALP activity was carried out with the MetraBap EIA kit (San Diego, CA) that uses p-nitrophenylphosphate (pNPP) as a substrate and evaluates the release of p-nitrophenol derived from its hydrolysis at 405 nm. The results were expressed as UI and were normalised per mg/intracellular proteins.
Primary subchondral bone osteoblast cell cultures Cell proliferation assay Bone marrow biopsies were performed using a bone biopsy needle to obtain very small fragments of cancellous bone. Each fragment was digested with 0.5 mg/ml type II collagenase (PAA, Austria) for 1 h at 37 °C and then cultured in sterile flasks in DMEM supplemented with antibiotics (penicillin 100 IU/ml and streptomycin 100 mg/ml) and 20 % foetal calf serum (FCS) at 37 °C in a water-saturated atmosphere containing 5 % CO2. After cells were observed in the flasks, the culture medium was replaced every 3 days with a fresh medium containing 10 % FCS. Osteoblasts began to grow out from the bone specimens after approximately 1 week and proliferated on the flask surface, reaching confluence within 3–4 weeks.
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Three hundred microlitre of MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) (stock solution 50 mg/ml, Sigma, St. Louis, MO) was added to each well and cultures were continued for 2 h at 37 °C to allow the conversion of MTT into insoluble formazan. The cells were then lysed and the formazan was solubilized with acidic propanol at room temperature for 24 h. Colorimetric changes in 200 ll of supernatant were quantified in a microplate reader at an OD of 540 nm (Multiskan EX, Thermo Electron Corporation, Finland). The percentage of viable cells was expressed as percent ratio between the OD of samples and OD of control (untreated normal cells).
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Apoptosis
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Cell apoptosis was assessed by the measurement of caspase-3 activity using a colorimetric assay kit (APOCYTO Caspase-3 Colorimetric Assay Kit, MBL, Woburn, MA) according to the manufacturer’s protocol. The sub-confluent cells were lysed; the Caspase 3 labelled substrate (DVED-pNA) was added to cell lysate and the mixture was incubated at 37 °C for 1–2 h. The concentration of p-NA released from the Caspase 3 substrate was measured reading the OD at 405 nm, according to the manufacturer’s recommendations. The results were expressed as nmol/ml pNA production.
not show significant differences compared with untreated RA cells. Concerning the ALP activity in RA osteoblasts, the effects of both treatments were similar to those observed for osteocalcin synthesis. Thus, levels of ALP activity were reduced by MTX (§p \ 0.05) and they were enhanced by infliximab treatment (**p \ 0.001), at any used concentrations and with an apparent dose-effect; this positive effect was significantly reduced by combined treatment of drugs. Nevertheless, even if MTX treatment reduced the effect of IFX in increasing ALP activity, the ALP activity levels after combined treatment remain significantly higher compared with untreated RA cells (§§p \ 0.01, Fig. 1b).
IL-6 synthesis
Cell proliferation
The production of IL-6 was evaluated with an ELISA kit (R&D Systems, Minneapolis, MN). According to the manufacturer’s instructions, the results, expressed as pg/ml, were directly determined from the calibration curve. The concentrations of samples were normalised to the milligram intracellular protein. All measurements were performed in triplicate for each sample, and the mean values were calculated.
MTX treatment induced a significant reduction of cell proliferation (*p \ 0.01, Fig. 2). Conversely, osteoblasts treated with infliximab alone, at any used concentrations, showed a higher proliferation compared with untreated RA cells (**p \ 0.05 vs. untreated RA osteoblasts). The combined treatment of drugs does not modify the negative effect exerted by MTX on cell proliferation; the proliferating activity of osteoblast treated with IFX and MTX was reduced when compared with untreated cells and was comparable to proliferating activity of cell treated with MTX alone.
Statistical analysis Results were expressed as mean ± SD. The differences in alkaline phosphatase activity, osteocalcin and IL-6 synthesis between the different cell populations were assessed with the non-parametric Kruskal–Wallis test, using the Dunn’s test for multiple comparisons. Differences in proliferating activity and caspase activity were assessed using the Bonferroni’s test. A p value \0.05 was considered as significant.
Cell apoptosis (Caspase 3 activity)
Results
Compared with the untreated cells, Caspase 3 activity was enhanced by MTX (**p \ 0.001), while it was significantly reduced after infliximab treatment, with an apparent dose-dependent effect (Fig. 3). MTX appears to inhibit the effect exerted by IFX in reducing osteoblast apoptosis, as the combined treatment with IFX and MTX resulted in a lesser reduction of Caspase 3 activity compared with the cell treated with IFX alone (§p \ 0.05).
Osteocalcin production and ALP activity
IL-6 production
In basal condition (untreated cell cultures), both osteocalcin and ALP production were significantly lower in RA osteoblasts compared with the normal ones (°p \ 0.05, Fig. 1 a, b). In RA osteoblasts, MTX induced a significant reduction of osteocalcin synthesis (§p \ 0.05); conversely, infliximab induced a significant increase at any used concentrations, with a maximum effect observed at 1 lg/ml (*p \ 0.01; **p \ 0.001). MTX appears to inhibit the stimulating effect of infliximab on osteocalcin production, as in cells treated with both MTX and infliximab, the osteocalcin production was lower compared with cells treated with infliximab alone (Fig. 1a) and did
IL-6 production was significantly higher in RA osteoblasts compared with the normal ones (°p \ 0.001). MTX significantly inhibited IL-6 synthesis in RA osteoblasts (*p \ 0.05). Similarly, osteoblasts treated with the higher concentrations of IFX showed a significant reduction of IL6 production, compared with the untreated cells (*p \ 0.05; §p \ 0.001 vs. RA-untreated osteoblasts). The combined treatment (IFX ? MTX) induced a further reduction of IL-6 production compared with the cell treated with the single drugs (**p \ 0.001; §§p \ 0.0001) with an apparent additive effect on the inhibition of the synthesis of this cytokine (Fig. 4).
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Fig. 1 Osteocalcin production and Alkaline phosphatase (ALP) activity in RA osteobasts and after treatment with MTX and infliximab, both alone and combined. Both osteocalcin and ALP activity are reduced in RA osteoblasts compared with normal osteoblasts (°p \ 0.05, a, b). a Osteocalcin synthesis is significantly further reduced in RA osteoblasts by MTX (§p \ 0.05 vs. RAuntreated osteoblasts). IFX significantly increases osteocalcin synthesis (*p \ 0.01; **p \ 0.001 vs. RA-untreated osteoblasts). IFX appears to inhibit the negative effect by MTX exerted on osteocalcin
synthesis. b Levels of ALP activity are significantly reduced in RA osteoblast by MTX (§p \ 0.05 vs. RA-untreated osteoblasts), whereas they are significantly increased by IFX (**p \ 0.001 vs. RA-untreated osteoblasts). The positive effect on ALP activity exerted by IFX is significantly reduced by MTX treatment when used in combination with IFX, as in RA osteoblasts treated with both drugs. ALP activity levels are lower compared with IFX alone treated cells, even if they remain higher compared with untreated cells (§§p \ 0.01)
Fig. 2 Effect of MTX and infliximab, both alone and combined, in RA osteoblasts. The percentage of viable cells was measured using MTT assay. MTX treatment significantly reduces cell proliferation (*p \ 0.01 vs. untreated RA osteoblasts), whereas cell proliferation is significantly increased by IFX infliximab alone, at any used concentrations (**p \ 0.05 vs. untreated RA osteoblasts). IFX treatment does not modify the negative effect of MTX on cell proliferation
Fig. 3 Caspase-3 activity assay in primary RA osteoblasts after treatment with MTX and infliximab, both alone and combined. Apoptosis is increased in RA osteoblasts compared with normal ones (°p \ 0.01). Compared with the RA-untreated cells, Caspase 3 activity is enhanced by MTX (**p \ 0.001), while it is reduced by infliximab (*p \ 0.01; **p \ 0.001 vs. untreated RA osteoblasts), with an apparent dose-dependent effect. The combined treatment with IFX and MTX results in a partial inhibition of the IFX effect in reducing apoptosis, as it results in a lesser reduction of Caspase 3 activity compared with the cell treated with IFX alone (§p \ 0.05)
Discussion It is well known that systemic inflammation is associated with bone loss and represents an independent risk factor for osteoporotic fractures, as chronic inflammation is able to
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modify the balance between bone formation and bone resorption processes, resulting in a net loss of bone mass [15]. The relationship between systemic inflammation and bone loss is based on the interaction between bone cells
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Fig. 4 IL-6 production in RA osteoblasts treated with MTX and infliximab, both alone and combined. IL-6 production is significantly higher in RA osteoblasts compared with normal osteoblasts (°p \ 0.001). Both MTX and IFX significantly reduced the IL-6 production when used alone; concerning IFX, the greater effect is observed with the higher concentrations (*p \ 0.05; §p \ 0.001 vs. RA-untreated osteoblasts). The combination of the two agents resulted in a significant additional reduction of IL-6 synthesis (**p \ 0.001; §§p \ 0.0001 vs. IFX and MTX alone treated RA osteoblasts)
and immune cells, the latter producing molecules with negative effect on bone homeostasis. The key feature of inflammatory bone loss is represented by the enhanced osteoclast differentiation and activity, which are supported by various cytokines, such as TNF-a, IL-1, IL-6, produced by both immune and mesenchymal cells. These cytokines induce the expression of nuclear factor jB ligand (RANKL) and macrophage colony-stimulating factor (MCSF) that are essential factors for the differentiation and activation of osteoclasts. In physiological conditions, the enhanced osteoclast activity, resulting in increased bone resorption, is balanced by an equivalent increase of bone formation that is sustained by the differentiation and activation of osteoblasts. Chronic inflammation can interfere with the physiological mechanisms that promote the anabolic bone response of osteoblasts to the increased bone resorption. TNF-a, for instance, is able to reduce the proper differentiation and the metabolic activity of osteoblasts, with downregulation of bone formation, by promoting the osteocytes expression of Dickkopf (Dkk-1) and sclerostin [16, 17], which in turn inhibit the Wnt signalling system, one of the most prominent factors that regulate the differentiation and the anabolic activity of osteoblasts. In RA patients, the reduction of inflammation and an adequate control of disease activity with subsequent prevention of joint structural damage are the main therapeutic goals of the DMARDs. DMARDs can be distinguished in traditional DMARDs, such as methotrexate (MTX), Cyclosporine A, Sulfasalazine, and the more recent
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biological drugs, such as anti-TNF-a, anti-CD20, anti-IL6 agents. Amongst the traditional DMARDs, the most and extensively used is MTX, whereas amongst the new biological drugs, the most diffusely used in clinical practice are the TNF-a blockers. However, even it is clearly established that the control of inflammation obtained with DMARDs plays an essential role in preventing both generalised and local bone loss in RA, there is very little knowledge regarding the direct effect of these drugs on bone-healing and bone-forming cells. In this study, we evaluate in vitro the direct effect of MTX and the anti-TNF-a, infliximab, alone and in combination, on proliferation, apoptosis, metabolic activity and IL-6 production of osteoblasts derived from trabecular bone of patients affected by RA. The concentrations of each agent used in the experiments were chosen from the available data concerning IFX and MTX serum concentrations in RA patients after administration at therapeutic dosages [13] [14]. The results of our study clearly show that MTX exerts an inhibitory effect in RA osteoblasts proliferation and metabolic activity; further, MTX is able to increase apoptosis. Conversely, IFX induces a positive direct effect in RA osteoblasts, increases the proliferation and the production of ALP and osteocalcin and reduces apoptosis. The most interesting results are observed after combined treatment of MTX and IFX. Indeed, IFX appears to antagonise the negative effect exerted by MTX on ALP and osteocalcin osteoblast synthesis, as illustrated in Fig. 1, showing that osteocalcin and ALP synthesis in RA osteoblasts treated with MTX alone is significantly lower than normal or untreated RA osteoblasts, whereas in cultures treated with both MTX and IFX, the observed levels of osteocalcin and ALP are higher, although they are lower compared with the IFX alone treated cells. Similarly, MTX and IFX appear to exert an opposite effect on RA osteoblasts proliferation, as in MTX-treated cells, a significant decrease in proliferating activity is observed, whereas in IFX-treated cultures, cell proliferation is significantly increased. However, IFX does not modify the inhibitory effect of MTX on proliferation, as the proliferating activity of cells treated with both drugs remains lower and it is similar to those observed in cells treated with MTX alone (Fig. 2). Similar opposite effects of MTX and IFX are observed concerning cell apoptosis, evaluated by Caspase 3 activity. In untreated RA osteoblasts, a significant increase in Caspase 3 activity is observed compared with normal osteoblasts that is significantly further increased by MTX treatment; conversely, IFX significantly reduces Caspase 3 activity, with a dose-dependent effect. Interestingly, when the drugs are used in combination, the reduction of
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apoptosis induced by IFX appears to be partially inhibited by MTX (Fig. 3). Both drugs significantly reduce the IL-6 production in osteoblasts when used alone, and their combination results in a significant additional reduction of IL-6 synthesis, with an additive effect (Fig. 4). The beneficial effect of TNF-a blockade on bone metabolism in RA has been shown in several studies. In RA patients treated with infliximab for 12 months, the BMD was preserved at lumbar spine and at femoral neck, whereas in patients treated with methotrexate alone, a reduction of BMD in the same site, amounting, respectively, to 3.9 and 2.5 %, was observed [18]. Interestingly, this beneficial effect of anti-TNF-a appears to be independent of the effect on disease activity [19, 20]. In another case series, a stable spine and hip BMD was observed in 102 patients treated with infliximab, with a concomitant decrease of serum levels of CTX, a bone resorption marker [20]. Other authors reported a significant gain in BMD at lumbar spine (?2.7 %) and femoral neck (?13 %) in patients with RA treated with infliximab [19]. Of particular interest, an increase of BMD during infliximab treatment appears to occur independently of the clinical response and the activity disease control. Other reports consistent with effects of TNF blockade on BMD have begun to emerge in recent years [4, 19, 20]. Conversely, despite its established effectiveness in controlling joint inflammation, pain, stiffness and to prevent the development of erosive changes, several clinical and experimental evidences suggest that MTX could exert a direct negative effect on bone metabolism. The severe adverse reactions induced in bone by MTX are almost exclusively observed with the high-dose regimens used in chemotherapy, which entail a reduction of bone turnover and impair osteoblastic activity, resulting in an increased risk of osteoporosis and fractures [21, 22]. The relatively low doses of MTX used for the treatment of RA act as an immune-modulator and/or inflammation suppressor by interfering with leukocyte activity and inflammatory cytokine production, not as cytostatic drugs [12]. However, it is not known whether the low-dose pulse MTX regimens used for RA can have any negative repercussion on bone. Some data based on radiological and histological findings show that low doses of MTX for prolonged periods negatively affect bone mass, especially in post-menopausal women [23], with enhanced risk of osteoporosis and fractures. In an arthritis animal model, MTX induced a reduction of mineral apposition rate and the number of osteoblastic cells [24, 25, 26]. Several in vitro and in vivo studies have shown the direct negative effect that MTX exerts on osteoblastic cells. The predominant effect of MTX is to inhibit osteoblast proliferation and osteoblast metabolic activity, in terms of ALP and
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osteocalcin production [26], even when it is used in the RA therapeutic low-dose range [12]. The results of the presented study seem to confirm the potential negative effects of MTX on osteoblast metabolism and proliferation. Interestingly, we found that infliximab exerts a positive direct effect on osteoblast metabolism and proliferation, and it antagonises the negative effect induced by MTX on ALP and osteocalcin synthesis and reduces osteoblast apoptosis. Conversely, infliximab does not prevent the reduction of proliferating activity induced by MTX on osteoblasts, probably because the anti-proliferative action of MTX is mediated by mechanisms that are independent of anti-TNF-a blockade. Further studies are necessary to better clarify the exact cellular responses in bone environment to MTX and antiTNF-a agents, including the effects on osteoclasts development and activity, and to identify the real physiological and clinical relevance of these effects, especially concerning the structural bone damage outcome in RA patients. Our results imply a negative direct effects of MTX on bone metabolism in RA patients, but the combined treatment with an anti-TNF–a drug that is very common in clinical practice, can be beneficial for the interaction of MTX with bone cells. Further, both drugs are able to reduce inflammation by inhibiting the production of pro-inflammatory cytokines, with a possible additive effect, and consequently their overall effect on local and systemic bone metabolism is probably a protective effect, especially when these drugs are administered together. Conflict of interest
None.
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ORIGINAL ARTICLE
Combined effects of infliximab and methotrexate on rheumatoid arthritis osteoblastic cell metabolism Addolorata Corrado • Anna Neve • Arcangela Marucci Annamaria Gaudio • Francesco Paolo Cantatore
•
Received: 2 May 2014 / Accepted: 5 August 2014 / Published online: 23 August 2014 Ó Springer-Verlag Italia 2014
Abstract The goal of this study is to investigate the in vitro effects of two disease-modifying anti-rheumatic drugs, largely used in the treatment of rheumatoid arthritis (RA), [infliximab (IFX) and methotrexate (MTX)], in RA primary osteoblast cell cultures. MTX inhibited proliferation and metabolic activity in RA osteoblasts was able to increase apoptosis. Conversely, IFX increased the proliferation, osteocalcin production and the alkaline phosphatase activity. Interestingly, IFX appeared to antagonise the negative effect exerted by MTX. Both drugs significantly reduced the IL-6 production in osteoblasts when used alone, and the combination of the two agents resulted in a significant additional reduction of IL-6 synthesis, with an apparent additive effect. The present study suggests that MTX exerts negative direct effects on bone metabolism in RA patients, but the combined treatment with anti-TNF-a can be beneficial for the interaction of MTX with bone cells. Keywords Osteoblasts Rheumatoid arthritis Disease modifying anti-rheumatic drugs (DMARDs)
Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised by joint inflammation associated with structural bone damage represented by generalised
A. Corrado A. Neve A. Marucci A. Gaudio F. P. Cantatore (&) Rheumatology Clinic, Department of Medical and Surgical Sciences, University of Foggia, Ospedale ‘‘Col. D’Avanzo’’, V.le degli Aviatori 1, 71100 Foggia, Italy e-mail: [email protected]
osteoporosis and localised bone loss, which includes erosions and juxta-articular osteopenia of affected joints. Recent literature suggests that these three types of bone loss are at least in part mediated by common pathogenic mechanisms [1, 2] that converge toward an alteration of bone remodelling processes. Among the factors that regulate bone remodelling and osteoclast activity, TNF-a plays a central role as it is involved both in local erosive processes and in systemic osteoporosis. Further, TNF-a is among the most important cytokines sustaining the chronic inflammation that characterises RA, thus representing a possible link between inflammation and bone loss. Besides TNF-a, there are various cytokines involved both in the maintenance of inflammatory state and in the pathogenesis of systemic bone loss and structural joint damage, such as IL-6, IL-1, IL-11 and IL-17 [3]. Indeed, chronic inflammation and bone loss are strictly linked in RA, and the control of inflammation appears to be one of the most important strategies for the prevention of bone loss in this disease [2]. These observations suggest that disease modifying anti-rheumatic drugs (DMARDs) can have a beneficial systemic effect on bone metabolism. Many data consistent with a positive effect of TNF-a blockade on bone mass in RA patients have been reported in the last years [4, 5]; interestingly, the benefit on bone mineral density (BMD) induced by anti-TNF-a treatment appears to be independent of the clinical response in terms of effect on disease activity [6]. Conversely, methotrexate (MTX), whose mechanism of action is independent of TNF-a blockade, in spite of its ability to control systemic and local joint inflammation, not only does not exert any direct positive effect on bone metabolism but also could impair bone formation, particularly when it is used at high doses [7], or for long term [8, 9]. It has been shown that low dose of MTX is able to inhibit osteoblast function [10] when it is
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given to normal rats. But in rats with adjuvant-induced arthritis, bone mass was maintained in the low dose MTXtreated animals compared with untreated controls [11, 12]. To date, the exact mechanisms of the direct effects of DMARDs on bone cells in inflammatory joint diseases are poorly understood and little is known about the effects of the combined treatment on cells involved in bone remodelling and bone reparation processes. The aim of this study was to evaluate the direct effect of the anti-TNF-a drug, infliximab (IFX) and MTX, alone or in combination, on proliferation, viability, metabolic activity and IL-6 synthesis in primary human osteoblast cultures derived from trabecular bone samples of patients with RA.
Patients and methods RA bone specimens were obtained from 11 subjects (2 men, 9 women) aged 55.12 ± 9.6 years (mean ± SD, range 39–67), fulfilling the 2010 ACR/EULAR diagnostic criteria for RA, undergoing total joint replacement of hip or knee. None of the enrolled subjects was affected by osteoporosis or other metabolic bone diseases and none received medication, including corticosteroids, which could interfere with bone metabolism for 3 months prior to the bone biopsy. Further, none of the selected subjects were treated with methotrexate or antiTNF-a drugs for 3 months prior the bone biopsy. Normal bone fragments were obtained from 9 adult subjects (4 men and 5 women), aged 58 ± 12.9 years (mean ± SD, range 35–61 years) undergoing surgery for traumatic fractures of the distal femoral epiphysis, who served as controls. Appropriate informed consent was obtained from each patient and the study was approved by Institutional Ethics Committee.
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Cell cultures treatment MTX was provided by Hansermann; IFX, a chimeric antihuman TNF–a monoclonal antibody, was provided by Schoering-Plough. When reaching confluence, osteoblasts were seeded in 24-well plates, in DMEM containing 10 % FCS and antibiotics, at a density of 6 9 104 cells for every plate until semi-confluence was reached. Cells were then treated with MTX (100 lg/ml) and IFX 0.1, 1 and 10 lg/ml, either alone and combined, for 24 h. The concentration of each agent used in the experiments was determined from the available data concerning IFX and MTX serum concentrations in RA patients after administration at therapeutic dosage [13, 14]. After drugs exposure, osteocalcin levels, alkaline phosphatase activity (ALP), apoptosis, cell proliferation and IL6 production were determined in cell cultures. All experiments were performed in triplicate. Osteocalcin synthesis and ALP activity Osteocalcin release into the culture medium was measured using an intact human osteocalcin ELISA (MetraOsteocalcin EIA kit, San Diego, CA). Osteocalcin secretion rates were expressed as nanogram/mg of intracellular proteins. The intracellular protein content of each well was determined by the Bradford method (Bio-Rad protein assay, Bio-Rad Laboratories, Richmond, CA). In parallel, ALP activity was evaluated in cell lysate, obtained through the solubilization of cell monolayer with 0.1(vol/vol) Triton X-100. The colorimetric determination of ALP activity was carried out with the MetraBap EIA kit (San Diego, CA) that uses p-nitrophenylphosphate (pNPP) as a substrate and evaluates the release of p-nitrophenol derived from its hydrolysis at 405 nm. The results were expressed as UI and were normalised per mg/intracellular proteins.
Primary subchondral bone osteoblast cell cultures Cell proliferation assay Bone marrow biopsies were performed using a bone biopsy needle to obtain very small fragments of cancellous bone. Each fragment was digested with 0.5 mg/ml type II collagenase (PAA, Austria) for 1 h at 37 °C and then cultured in sterile flasks in DMEM supplemented with antibiotics (penicillin 100 IU/ml and streptomycin 100 mg/ml) and 20 % foetal calf serum (FCS) at 37 °C in a water-saturated atmosphere containing 5 % CO2. After cells were observed in the flasks, the culture medium was replaced every 3 days with a fresh medium containing 10 % FCS. Osteoblasts began to grow out from the bone specimens after approximately 1 week and proliferated on the flask surface, reaching confluence within 3–4 weeks.
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Three hundred microlitre of MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) (stock solution 50 mg/ml, Sigma, St. Louis, MO) was added to each well and cultures were continued for 2 h at 37 °C to allow the conversion of MTT into insoluble formazan. The cells were then lysed and the formazan was solubilized with acidic propanol at room temperature for 24 h. Colorimetric changes in 200 ll of supernatant were quantified in a microplate reader at an OD of 540 nm (Multiskan EX, Thermo Electron Corporation, Finland). The percentage of viable cells was expressed as percent ratio between the OD of samples and OD of control (untreated normal cells).
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Apoptosis
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Cell apoptosis was assessed by the measurement of caspase-3 activity using a colorimetric assay kit (APOCYTO Caspase-3 Colorimetric Assay Kit, MBL, Woburn, MA) according to the manufacturer’s protocol. The sub-confluent cells were lysed; the Caspase 3 labelled substrate (DVED-pNA) was added to cell lysate and the mixture was incubated at 37 °C for 1–2 h. The concentration of p-NA released from the Caspase 3 substrate was measured reading the OD at 405 nm, according to the manufacturer’s recommendations. The results were expressed as nmol/ml pNA production.
not show significant differences compared with untreated RA cells. Concerning the ALP activity in RA osteoblasts, the effects of both treatments were similar to those observed for osteocalcin synthesis. Thus, levels of ALP activity were reduced by MTX (§p \ 0.05) and they were enhanced by infliximab treatment (**p \ 0.001), at any used concentrations and with an apparent dose-effect; this positive effect was significantly reduced by combined treatment of drugs. Nevertheless, even if MTX treatment reduced the effect of IFX in increasing ALP activity, the ALP activity levels after combined treatment remain significantly higher compared with untreated RA cells (§§p \ 0.01, Fig. 1b).
IL-6 synthesis
Cell proliferation
The production of IL-6 was evaluated with an ELISA kit (R&D Systems, Minneapolis, MN). According to the manufacturer’s instructions, the results, expressed as pg/ml, were directly determined from the calibration curve. The concentrations of samples were normalised to the milligram intracellular protein. All measurements were performed in triplicate for each sample, and the mean values were calculated.
MTX treatment induced a significant reduction of cell proliferation (*p \ 0.01, Fig. 2). Conversely, osteoblasts treated with infliximab alone, at any used concentrations, showed a higher proliferation compared with untreated RA cells (**p \ 0.05 vs. untreated RA osteoblasts). The combined treatment of drugs does not modify the negative effect exerted by MTX on cell proliferation; the proliferating activity of osteoblast treated with IFX and MTX was reduced when compared with untreated cells and was comparable to proliferating activity of cell treated with MTX alone.
Statistical analysis Results were expressed as mean ± SD. The differences in alkaline phosphatase activity, osteocalcin and IL-6 synthesis between the different cell populations were assessed with the non-parametric Kruskal–Wallis test, using the Dunn’s test for multiple comparisons. Differences in proliferating activity and caspase activity were assessed using the Bonferroni’s test. A p value \0.05 was considered as significant.
Cell apoptosis (Caspase 3 activity)
Results
Compared with the untreated cells, Caspase 3 activity was enhanced by MTX (**p \ 0.001), while it was significantly reduced after infliximab treatment, with an apparent dose-dependent effect (Fig. 3). MTX appears to inhibit the effect exerted by IFX in reducing osteoblast apoptosis, as the combined treatment with IFX and MTX resulted in a lesser reduction of Caspase 3 activity compared with the cell treated with IFX alone (§p \ 0.05).
Osteocalcin production and ALP activity
IL-6 production
In basal condition (untreated cell cultures), both osteocalcin and ALP production were significantly lower in RA osteoblasts compared with the normal ones (°p \ 0.05, Fig. 1 a, b). In RA osteoblasts, MTX induced a significant reduction of osteocalcin synthesis (§p \ 0.05); conversely, infliximab induced a significant increase at any used concentrations, with a maximum effect observed at 1 lg/ml (*p \ 0.01; **p \ 0.001). MTX appears to inhibit the stimulating effect of infliximab on osteocalcin production, as in cells treated with both MTX and infliximab, the osteocalcin production was lower compared with cells treated with infliximab alone (Fig. 1a) and did
IL-6 production was significantly higher in RA osteoblasts compared with the normal ones (°p \ 0.001). MTX significantly inhibited IL-6 synthesis in RA osteoblasts (*p \ 0.05). Similarly, osteoblasts treated with the higher concentrations of IFX showed a significant reduction of IL6 production, compared with the untreated cells (*p \ 0.05; §p \ 0.001 vs. RA-untreated osteoblasts). The combined treatment (IFX ? MTX) induced a further reduction of IL-6 production compared with the cell treated with the single drugs (**p \ 0.001; §§p \ 0.0001) with an apparent additive effect on the inhibition of the synthesis of this cytokine (Fig. 4).
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Fig. 1 Osteocalcin production and Alkaline phosphatase (ALP) activity in RA osteobasts and after treatment with MTX and infliximab, both alone and combined. Both osteocalcin and ALP activity are reduced in RA osteoblasts compared with normal osteoblasts (°p \ 0.05, a, b). a Osteocalcin synthesis is significantly further reduced in RA osteoblasts by MTX (§p \ 0.05 vs. RAuntreated osteoblasts). IFX significantly increases osteocalcin synthesis (*p \ 0.01; **p \ 0.001 vs. RA-untreated osteoblasts). IFX appears to inhibit the negative effect by MTX exerted on osteocalcin
synthesis. b Levels of ALP activity are significantly reduced in RA osteoblast by MTX (§p \ 0.05 vs. RA-untreated osteoblasts), whereas they are significantly increased by IFX (**p \ 0.001 vs. RA-untreated osteoblasts). The positive effect on ALP activity exerted by IFX is significantly reduced by MTX treatment when used in combination with IFX, as in RA osteoblasts treated with both drugs. ALP activity levels are lower compared with IFX alone treated cells, even if they remain higher compared with untreated cells (§§p \ 0.01)
Fig. 2 Effect of MTX and infliximab, both alone and combined, in RA osteoblasts. The percentage of viable cells was measured using MTT assay. MTX treatment significantly reduces cell proliferation (*p \ 0.01 vs. untreated RA osteoblasts), whereas cell proliferation is significantly increased by IFX infliximab alone, at any used concentrations (**p \ 0.05 vs. untreated RA osteoblasts). IFX treatment does not modify the negative effect of MTX on cell proliferation
Fig. 3 Caspase-3 activity assay in primary RA osteoblasts after treatment with MTX and infliximab, both alone and combined. Apoptosis is increased in RA osteoblasts compared with normal ones (°p \ 0.01). Compared with the RA-untreated cells, Caspase 3 activity is enhanced by MTX (**p \ 0.001), while it is reduced by infliximab (*p \ 0.01; **p \ 0.001 vs. untreated RA osteoblasts), with an apparent dose-dependent effect. The combined treatment with IFX and MTX results in a partial inhibition of the IFX effect in reducing apoptosis, as it results in a lesser reduction of Caspase 3 activity compared with the cell treated with IFX alone (§p \ 0.05)
Discussion It is well known that systemic inflammation is associated with bone loss and represents an independent risk factor for osteoporotic fractures, as chronic inflammation is able to
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modify the balance between bone formation and bone resorption processes, resulting in a net loss of bone mass [15]. The relationship between systemic inflammation and bone loss is based on the interaction between bone cells
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Fig. 4 IL-6 production in RA osteoblasts treated with MTX and infliximab, both alone and combined. IL-6 production is significantly higher in RA osteoblasts compared with normal osteoblasts (°p \ 0.001). Both MTX and IFX significantly reduced the IL-6 production when used alone; concerning IFX, the greater effect is observed with the higher concentrations (*p \ 0.05; §p \ 0.001 vs. RA-untreated osteoblasts). The combination of the two agents resulted in a significant additional reduction of IL-6 synthesis (**p \ 0.001; §§p \ 0.0001 vs. IFX and MTX alone treated RA osteoblasts)
and immune cells, the latter producing molecules with negative effect on bone homeostasis. The key feature of inflammatory bone loss is represented by the enhanced osteoclast differentiation and activity, which are supported by various cytokines, such as TNF-a, IL-1, IL-6, produced by both immune and mesenchymal cells. These cytokines induce the expression of nuclear factor jB ligand (RANKL) and macrophage colony-stimulating factor (MCSF) that are essential factors for the differentiation and activation of osteoclasts. In physiological conditions, the enhanced osteoclast activity, resulting in increased bone resorption, is balanced by an equivalent increase of bone formation that is sustained by the differentiation and activation of osteoblasts. Chronic inflammation can interfere with the physiological mechanisms that promote the anabolic bone response of osteoblasts to the increased bone resorption. TNF-a, for instance, is able to reduce the proper differentiation and the metabolic activity of osteoblasts, with downregulation of bone formation, by promoting the osteocytes expression of Dickkopf (Dkk-1) and sclerostin [16, 17], which in turn inhibit the Wnt signalling system, one of the most prominent factors that regulate the differentiation and the anabolic activity of osteoblasts. In RA patients, the reduction of inflammation and an adequate control of disease activity with subsequent prevention of joint structural damage are the main therapeutic goals of the DMARDs. DMARDs can be distinguished in traditional DMARDs, such as methotrexate (MTX), Cyclosporine A, Sulfasalazine, and the more recent
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biological drugs, such as anti-TNF-a, anti-CD20, anti-IL6 agents. Amongst the traditional DMARDs, the most and extensively used is MTX, whereas amongst the new biological drugs, the most diffusely used in clinical practice are the TNF-a blockers. However, even it is clearly established that the control of inflammation obtained with DMARDs plays an essential role in preventing both generalised and local bone loss in RA, there is very little knowledge regarding the direct effect of these drugs on bone-healing and bone-forming cells. In this study, we evaluate in vitro the direct effect of MTX and the anti-TNF-a, infliximab, alone and in combination, on proliferation, apoptosis, metabolic activity and IL-6 production of osteoblasts derived from trabecular bone of patients affected by RA. The concentrations of each agent used in the experiments were chosen from the available data concerning IFX and MTX serum concentrations in RA patients after administration at therapeutic dosages [13] [14]. The results of our study clearly show that MTX exerts an inhibitory effect in RA osteoblasts proliferation and metabolic activity; further, MTX is able to increase apoptosis. Conversely, IFX induces a positive direct effect in RA osteoblasts, increases the proliferation and the production of ALP and osteocalcin and reduces apoptosis. The most interesting results are observed after combined treatment of MTX and IFX. Indeed, IFX appears to antagonise the negative effect exerted by MTX on ALP and osteocalcin osteoblast synthesis, as illustrated in Fig. 1, showing that osteocalcin and ALP synthesis in RA osteoblasts treated with MTX alone is significantly lower than normal or untreated RA osteoblasts, whereas in cultures treated with both MTX and IFX, the observed levels of osteocalcin and ALP are higher, although they are lower compared with the IFX alone treated cells. Similarly, MTX and IFX appear to exert an opposite effect on RA osteoblasts proliferation, as in MTX-treated cells, a significant decrease in proliferating activity is observed, whereas in IFX-treated cultures, cell proliferation is significantly increased. However, IFX does not modify the inhibitory effect of MTX on proliferation, as the proliferating activity of cells treated with both drugs remains lower and it is similar to those observed in cells treated with MTX alone (Fig. 2). Similar opposite effects of MTX and IFX are observed concerning cell apoptosis, evaluated by Caspase 3 activity. In untreated RA osteoblasts, a significant increase in Caspase 3 activity is observed compared with normal osteoblasts that is significantly further increased by MTX treatment; conversely, IFX significantly reduces Caspase 3 activity, with a dose-dependent effect. Interestingly, when the drugs are used in combination, the reduction of
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apoptosis induced by IFX appears to be partially inhibited by MTX (Fig. 3). Both drugs significantly reduce the IL-6 production in osteoblasts when used alone, and their combination results in a significant additional reduction of IL-6 synthesis, with an additive effect (Fig. 4). The beneficial effect of TNF-a blockade on bone metabolism in RA has been shown in several studies. In RA patients treated with infliximab for 12 months, the BMD was preserved at lumbar spine and at femoral neck, whereas in patients treated with methotrexate alone, a reduction of BMD in the same site, amounting, respectively, to 3.9 and 2.5 %, was observed [18]. Interestingly, this beneficial effect of anti-TNF-a appears to be independent of the effect on disease activity [19, 20]. In another case series, a stable spine and hip BMD was observed in 102 patients treated with infliximab, with a concomitant decrease of serum levels of CTX, a bone resorption marker [20]. Other authors reported a significant gain in BMD at lumbar spine (?2.7 %) and femoral neck (?13 %) in patients with RA treated with infliximab [19]. Of particular interest, an increase of BMD during infliximab treatment appears to occur independently of the clinical response and the activity disease control. Other reports consistent with effects of TNF blockade on BMD have begun to emerge in recent years [4, 19, 20]. Conversely, despite its established effectiveness in controlling joint inflammation, pain, stiffness and to prevent the development of erosive changes, several clinical and experimental evidences suggest that MTX could exert a direct negative effect on bone metabolism. The severe adverse reactions induced in bone by MTX are almost exclusively observed with the high-dose regimens used in chemotherapy, which entail a reduction of bone turnover and impair osteoblastic activity, resulting in an increased risk of osteoporosis and fractures [21, 22]. The relatively low doses of MTX used for the treatment of RA act as an immune-modulator and/or inflammation suppressor by interfering with leukocyte activity and inflammatory cytokine production, not as cytostatic drugs [12]. However, it is not known whether the low-dose pulse MTX regimens used for RA can have any negative repercussion on bone. Some data based on radiological and histological findings show that low doses of MTX for prolonged periods negatively affect bone mass, especially in post-menopausal women [23], with enhanced risk of osteoporosis and fractures. In an arthritis animal model, MTX induced a reduction of mineral apposition rate and the number of osteoblastic cells [24, 25, 26]. Several in vitro and in vivo studies have shown the direct negative effect that MTX exerts on osteoblastic cells. The predominant effect of MTX is to inhibit osteoblast proliferation and osteoblast metabolic activity, in terms of ALP and
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osteocalcin production [26], even when it is used in the RA therapeutic low-dose range [12]. The results of the presented study seem to confirm the potential negative effects of MTX on osteoblast metabolism and proliferation. Interestingly, we found that infliximab exerts a positive direct effect on osteoblast metabolism and proliferation, and it antagonises the negative effect induced by MTX on ALP and osteocalcin synthesis and reduces osteoblast apoptosis. Conversely, infliximab does not prevent the reduction of proliferating activity induced by MTX on osteoblasts, probably because the anti-proliferative action of MTX is mediated by mechanisms that are independent of anti-TNF-a blockade. Further studies are necessary to better clarify the exact cellular responses in bone environment to MTX and antiTNF-a agents, including the effects on osteoclasts development and activity, and to identify the real physiological and clinical relevance of these effects, especially concerning the structural bone damage outcome in RA patients. Our results imply a negative direct effects of MTX on bone metabolism in RA patients, but the combined treatment with an anti-TNF–a drug that is very common in clinical practice, can be beneficial for the interaction of MTX with bone cells. Further, both drugs are able to reduce inflammation by inhibiting the production of pro-inflammatory cytokines, with a possible additive effect, and consequently their overall effect on local and systemic bone metabolism is probably a protective effect, especially when these drugs are administered together. Conflict of interest
None.
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