Effects of the active metabolite of leflunomide, A77 1726, on cytokine release and the MAPK signalling pathway in human rheumatoid arthritis synoviocytes

www.elsevier.com/locate/issn/10434666 Cytokine 31 (2005) 335e348

Effects of the active metabolite of leflunomide, A77 1726, on cytokine release and the MAPK signalling pathway in human rheumatoid arthritis synoviocytes Pascale Vergne-Salle a, David Yannick Le´ger b, Philippe Bertin a, Richard Tre`ves a, Jean-Louis Beneytout b, Bertrand Liagre b,* b

a Service de Rhumatologie et The´rapeutique, CHRU Dupuytren, 2 avenue Martin Luther King, 87042 Limoges Cedex, France Laboratoire de Biochimie, UPRES EA 1085, Faculte´ de Pharmacie, 2 rue du Docteur Marcland, 87025 Limoges Cedex, France

Received 8 December 2004; received in revised form 8 March 2005; accepted 1 June 2005

Abstract Inflammatory cytokines or soluble factors are essential in the pathogenesis of rheumatoid arthritis (RA). Leflunomide is an effective disease modifying antirheumatic drug (DMARD) in RA. The objective of the present study was to evaluate for the first time the effects of A77 1726 on cytokine (interleukin (IL)-8, IL-10, IL-11 secretion and tumor necrosis factor-a soluble receptor I (sTNFRI)) shedding in human RA fibroblast-like synoviocytes (FLS). At 100 mM, we observed an increase in IL-10 secretion, a decrease in IL-11 release and no effect on sTNFRI shedding and IL-8 secretion in IL-1b-stimulated human RA FLS. Furthermore, at this dose, our results also confirmed that A77 1726 decreased IL-6 and prostaglandin E2 (PGE2) synthesis while it increased IL-1 receptor antagonist secretion (IL-1Ra). The mitogen-activated protein kinases (MAPKs) represent an attractive target for RA because they can regulate cytokine expression. At 100 mM, the effect of A77 1726 on IL-10 and IL-11 secretion seemed to be associated with the status of p38 MAPK activation. Our results confirmed the immunoregulatory action of leflunomide in the cytokine network involved in RA pathogenesis. It could shift the balance from cytokine mediated inflammation to cytokine directed inhibition of the inflammatory process. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Human synoviocytes; Interleukin-10; Interleukin-11; Leflunomide; MAPK

1. Introduction

Abbreviations: COX, cyclooxygenase; DHODH, dihydroorotate deshydrogenase; DMARDs, disease modifying antirheumatic drugs; DMEM, Dulbecco’s modified Eagle’s medium; ELISA, enzyme-linked immunosorbent assay; EMSA, electromobility shift assay; FCS, fetal calf serum; FLS, fibroblast-like synoviocytes; IL, interleukin; IL-1Ra, IL-1 receptor antagonist; MAPK, mitogen-activated protein kinase; NF-kB, nuclear factor-kB; NSAIDs, non-steroidal anti-inflammatory drugs; PG, prostaglandin; RA, rheumatoid arthritis; sTNFRI, tumor necrosis factor-a soluble receptor I. * Corresponding author. Tel./fax: C33 555 43 58 39. E-mail address: [email protected] (B. Liagre). 1043-4666/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2005.06.002

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease characterized by chronic synovitis [1]. Inflammatory cytokines have been implicated as important mediators of joint destruction in RA [1e5]. However, in addition to pro-inflammatory cytokines, a compensatory anti-inflammatory response is also observed in RA. Thus, IL-1 receptor antagonist (IL-1Ra) blocks the biological activities of IL-1 by inhibiting IL-1-mediated bone resorption and PGE2 production [3,6,7]. Another inhibitory molecule is the soluble receptor of TNF (sTNFR). The recent anti-TNF

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biotherapies, spectacular in many symptoms of RA, highlight the importance of TNFa in the pathogenesis of RA and the balance between TNFa and its inhibitors [8]. IL-10, spontaneously produced in RA synovium, is an important immunoregulatory component in the cytokine network of RA, inhibiting the synthesis of pro-inflammatory cytokines such as TNFa, IL-1, IL-6 and IL-8 [9,10]. Furthermore, IL-10 stimulates the production of cytokine inhibitors (IL-1Ra, sTNFR) and downregulates a number of macrophage functions [9]. IL-11 is a cytokine that was originally identified as a stroma cell-derived lymphokine and hematopoietic factor [11]. The effects of IL-11 on connective tissue cells are diverse and include both pro- and anti-inflammatory effects. IL-11 stimulates B cell differentiation and immunoglobulin production [12]. IL-11 is produced in large amounts in RA synovial membranes and fluids [13]. Some biological activities of IL-11 are related to joint destruction such as osteoclast differentiation and bone resorption [14]. In contrast, blockade of endogenous IL-11 in FLS cultures resulted in an increase in TNFa levels, which increased dramatically if endogenous IL-10 was also blocked. Addition of exogenous IL-11 had no effect on TNFa production, but inhibited it in the presence of soluble IL-11 receptor [15]. In a randomized, placebo-controlled trial in patients with active RA, recombinant human IL-11 showed no overall therapeutic effects using the 20% American College of Rheumatology criteria [16]. Thus, the putative antiinflammatory action of IL-11 has not been confirmed in clinical practice. Leflunomide, a disease-modifying antirheumatic drug (DMARD) of the isoxazole class, has been shown to be effective in the treatment of RA, improving all clinical outcomes and delaying radiographic progression [17,18]. A77 1726, the active metabolite of leflunomide, inhibits dihydroorotate deshydrogenase (DHODH), a critical enzyme for pyrimidine synthesis in rapidly dividing cells, such as activated lymphocytes, resulting in reversible cell cycle arrest [19]. A77 1726 has been shown to inhibit PGE2 production in several models or cells and cyclooxygenase-2 (COX-2) activity in human RA synovial cells [20]. Leflunomide is also a potent inhibitor of nuclear factor-kB (NF-kB) activation induced by a wide variety of inflammatory stimuli. In particular, it blocks TNFa-mediated cellular responses in T cells [21]. A further anti-inflammatory activity exerted by leflunomide seems to be the inhibition of pro-inflammatory mediator production, especially cytokines. Cutolo et al. [22] demonstrated a decreased intracytoplasmic expression of TNFa and IL-1b and a decreased extracellular concentration of TNFa after treatment by A77 1726 in RA synovial macrophages. The production of IL-6 was inhibited by A77 1726 in human RA cultures [20] but not in human peripheral blood monocytes [23]. Palmer et al. [24] have recently demonstrated that A77 1726

increased IL-1b-induced production of IL-1Ra in human synovial fibroblasts and articular chondrocytes. The efficacy of leflunomide in the treatment of RA patients might be due to the fact that it acts at several levels of the inflammatory cascade. Indeed, in addition to the well described effects on PGE2, IL-6 and IL-1Ra production, the present study demonstrated for the first time that A77 1726 at high dose (100 mM) increased IL-10 secretion and decreased IL-11 secretion without any effect on IL-8 and sTNFRI in human RA FLS.

2. Results 2.1. A77 1726 did not cause DNA fragmentation in human RA FLS Direct observation with phase-contrast microscopy demonstrated that cells treated with 100 mM A77 1726 in 10% fetal calf serum (FCS) did not show morphological differences from control cells (Fig. 1A). Furthermore, DNA fragmentation was also studied in the same culture conditions, and we found that treatment with A77 1726 did not cause any increase in the production of mononucleosomes and oligonucleosomes in comparison with medium alone (Fig. 1B). 2.2. High doses of A77 1726 increased IL-10 secretion and decreased IL-11 secretion It has been well demonstrated that A77 1726 decreases pro-inflammatory cytokines such as TNFa, IL-1b and IL-6 in RA synovial cell cultures [20,22] and increases IL-1Ra [24,25]. It seems to favor anti- over pro-inflammatory factor productions. We chose to study the effects of A77 1726 on other anti-inflammatory factors such as sTNFRI and IL-10 and on other proinflammatory factors such as IL-8 and IL-11, although the exact role of IL-11 remains a subject of controversy. For this reason, we chose to describe the effect of A77 1726 on IL-11 secretion separately. At the same time, we confirmed the effects of A77 1726 on IL-6 and IL-1Ra secretions in our model of RA cultured cells. Our results showed that IL-1b (1 ng/ml) markedly increased IL-10 secretion (C311% versus untreated cell control; P!0.05) but also significantly enhanced IL-1Ra secretion and sTNFRI shedding (C54 andC47% respectively versus untreated cell control; P!0.05) (Fig. 2). In the presence of A77 1726, differential effects were observed depending on the concentration used. The effect of A77 1726 was dose-dependent on IL-1b induced-IL-10 secretion: low concentrations (0.01e 10 mM) provoked a large decrease in IL-10 secretion (ÿ28 to ÿ80% respectively versus IL-1b control; P!0.05) whereas high concentrations (50 and 100 mM)

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Fig. 1. (A) Representative photographs (original magnification !400) of human RA FLS treated or not with A77 1726 and (B) apoptosis quantification by ‘‘cell death’’ ELISA based on DNA fragmentation. Cells were incubated or not with 100 mM A77 1726 for 24 h. Data are expressed as meanGSD of three experiments. A representative experiment out of three is presented.

caused a dramatic increase (C137 andC359% respectively versus IL-1b control; P!0.05) (Fig. 2). Concerning the other two anti-inflammatory cytokines, IL-1Ra and sTNFRI, A77 1726 at 0.1e100 mM stimulated IL-1Ra secretion in the presence of IL-1b (C149 to C371% respectively versus IL-1b control; P!0.05). On the other hand, A77 1726 had no effect on IL-1b-induced-sTNFRI shedding for all concentrations used (Fig. 2). In addition, we investigated the effect of A77 1726 on a pro-inflammatory cytokine (IL-6) and chemokine (IL-8). As previously described, IL-1b stimulated IL-6 and IL-8 secretion in human RA FLS in our conditions. We observed that A77 1726 caused a dose-dependent inhibition of IL-6 secretion from 1 to 100 mM (ÿ11 to ÿ41% respectively versus IL-1b control; P!0.05) (Fig. 3). However, we again observed the differential effects of leflunomide because A77 1726 increased IL-8 secretion for 0.1, 1 and 10 mM (C124%, C130% and

C66% respectively versus IL-1b control; P!0.05) and had no effect for 50 and 100 mM (Fig. 3). Concerning IL-11 secretion, our results showed that IL-1b markedly increased its secretion (C274% versus untreated cell control; P!0.05) (Fig. 4). A77 1726 at low concentrations (0.1e10 mM) enhanced IL-11 secretion in the presence of IL-1b. However, high doses (50 and 100 mM) significantly decreased IL-1b-induced-IL-11 secretion (ÿ60% versus IL-1b control for two doses; P!0.05) (Fig. 4). In order to assess whether inhibition of IL-11 secretion with high doses of A77 1726 was due to the well-described in vitro inhibition of pyrimidine synthesis by A77 1726, uridine was used to reverse the inhibitory effect of A77 1726. The inhibition of IL-11 secretion by 100 mM A77 1726 was not reversed by 200 mM uridine (Fig. 5A). Furthermore, we showed that the increase in IL-10 secretion by 100 mM A77 1726 was inhibited by 200 mM uridine (ÿ41% versus A77 1726CIL-1b; P!0.05) (Fig. 5B).

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Fig. 2. Effect of A77 1726 on the secretion of anti-inflammatory cytokines (IL-10, IL-1Ra and sTNFRI) by human RA FLS. Cells were incubated or not with A77 1726 for 24 h and with IL-1b (1 ng/mL) for the last 6 h. The controls represent untreated cells and cells treated by IL-1b alone. The concentrations of IL-10, IL-1Ra and sTNFRI were determined in culture supernatants of FLS from three different patients by ELISA. Data are expressed as meanGSD of three experiments. A representative experiment out of three is presented. )P!0.05 (Fisher’s protected-least-significantdifference (PLSD) test) was considered to indicate significance compared to untreated cell control and #P!0.05 (Fisher’s PLSD test) was considered to indicate significance compared to IL-1b alone control.

2.3. A77 1726 inhibited PGE2 production but not COX-2 expression Inflammation is associated with the induction of COX-2 which controls the synthesis of PGs. It is well known that PGs, such as PGE2, are important lipid mediators that are produced at elevated levels in inflamed tissues including rheumatoid synovium. Fig. 6A showed that 10 mM A77 1726 (lane 3) did not modify COX-2 expression in comparison with stimulation by IL-1b alone (lane 2). However, high concentrations (100 mM) of A77 1726 exhibited COX-2 expression (lane 4). Then, we studied, in our conditions, the effect of A77 1726 on PGE2 production in the presence of IL-1b. We showed that, after stimulation

with IL-1b (1 ng/ml), A77 1726 induced a dosedependent inhibition of PGE2 release: ÿ68% for 10 mM, ÿ96 and ÿ98% for 50 and 100 mM respectively versus IL-1b control (P!0.05) (Fig. 6B,a). Afterwards, we only used 100 mM A77 1726 because this concentration gave the best responses with regards to the secretion of studied cytokines. Indeed, in our conditions, 100 mM A77 1726 provoked an increase in IL-10 and IL-1Ra secretion, a decrease in IL-6 and IL-11 secretion and had no effect on sTNFRI shedding and IL-8 secretion in the presence of IL-1b. Furthermore, the recommended dose of leflunomide in RA treatment is 20 mg/day. At this dose, the steady-state serum levels of A77 1726 are approximately 25e45 mg/ml (92.5e 166 mM) [26].

Fig. 3. Effect of A77 1726 on the secretion of pro-inflammatory cytokine (IL-6) and chemokine (IL-8) by human RA FLS. Cells were incubated or not with A77 1726 for 24 h and with IL-1b (1 ng/mL) for the last 6 h. The controls represent untreated cells and cells treated by IL-1b alone. The concentrations of IL-6 and IL-8 were determined in culture supernatants of FLS from three different patients by ELISA. Data are expressed as meanGSD of three experiments. A representative experiment out of three is presented. )P!0.05 (Fisher’s protected-least-significant-difference (PLSD) test) was considered to indicate significance compared to untreated cell control and #P!0.05 (Fisher’s PLSD test) was considered to indicate significance compared to IL-1b alone control.

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Fig. 4. Effect of A77 1726 on IL-11 secretion by human RA FLS. Cells were incubated or not with A77 1726 for 24 h and with IL-1b (1 ng/mL) for the last 6 h. The controls represent untreated cells and cells treated by IL-1b alone. The concentration of IL-11 was determined in culture supernatants of FLS from three different patients by ELISA. Data are expressed as meanGSD of three experiments. A representative experiment out of three is presented. )P!0.05 (Fisher’s protectedleast-significant-difference (PLSD) test) was considered to indicate significance compared to untreated cell control and #P!0.05 (Fisher’s PLSD test) was considered to indicate significance compared to IL-1b alone control.

In addition, short periods of IL-1 stimulation caused an increase in PGE2 production (Fig. 6B,b): C166, C125 and C77% after 5, 15 and 30 min versus control (medium alone) (P!0.05). Furthermore, although we did not observe this stimulation at 1 h, after 6 h of incubation with IL-1b, a dramatic increase in PGE2 secretion was seen (C532% versus untreated cell control; P!0.05). Then, we studied the effect of 100 mM A77 1726 on PGE2 synthesis during these different times of IL-1b treatment. Except after 1 h of IL-1b stimulation, 100 mM A77 1726 inhibited the production of PGE2 (ÿ72% after 5 min, ÿ98% after 15 min, ÿ81% after 30 min and ÿ85% after 6 h of IL-1b treatment; P!0.05) (Fig. 6B,b). 2.4. A77 1726 inhibited IL-1b-dependent NF-kB activation and induced p38 activation It is well known that COX-2 expression and activity but also cytokine production is correlated with the activities of intracellular signalling proteins such as ERKs, p38s or JNKs, but also with the activation of nuclear factor-kB (NF-kB). However, the correlation between the effect of A77 1726 on cytokine production and MAPK activation has not been studied much in human RA FLS. Since NF-kB activation is critical for inflammation, and as A77 1726 exhibits anti-inflammatory effects, we investigated whether A77 1726 inhibited NF-kB activation in human RA FLS. Our results showed that 100 mM A77 1726 inhibited IL-1b-induced NF-kB activation (lane 3, Fig. 7) in comparison with IL-1b (1 ng/ml) alone (lane 2, Fig. 7). Then, we verified that, in our conditions, IL-1b (1 ng/ml) activated the phosphorylation of

Fig. 5. Effect of A77 1726 on IL-11 (A) and IL-10 (B) secretion in the presence of uridine. Human RA FLS were preincubated for 30 min with 200 mM uridine before the addition of A77 1726 (100 mM) for 24 h and IL-1b (1 ng/mL) for the last 6 h. The controls represent untreated cells and cells treated by IL-1b alone. The concentration of IL-10 and IL-11 was determined in culture supernatants of FLS from four different patients by ELISA. Data are expressed as meanGSD of three experiments. A representative experiment out of three is presented. )P!0.05 (Fisher’s protected-least-significant-difference (PLSD) test) was considered to indicate significance compared to IL-1b alone control and #P!0.05 (Fisher’s PLSD test) was considered to indicate significance compared to A77 1726 with IL-1b.

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Fig. 6. Effect of A77 1726 on COX-2 expression (A) and PGE2 production by human RA FLS (B). (A) Cells were cultured with medium alone for 24 h (lane 1) or were incubated with IL-1b alone (1 ng/mL) for the last 6 h (lane 2), or with 10 or 100 mM A77 1726 for 24 h and IL-1b for the last 6 h (lanes 3 and 4 respectively). Protein extracts prepared from the cells were subjected to Western blotting and cellular expressions of COX-2 and b-actin were estimated using mouse anti-human COX-2 and b-actin antibodies respectively as described in Section 4. Quantification of each band was performed by densitometry analysis software. One of three representative experiments is shown from four different patients. (B) (a) Cells were cultured in 10% FCS medium for 48 h and then treated or not with A77 1726 for 24 h and with IL-1b (1 ng/mL) for the last 6 h. The controls represent untreated cells and cells treated by IL-1b alone. (b) Time course production of PGE2. Cells were incubated with or without A77 1726 (100 mM) for 24 h and with IL-1b for additional different times. The controls represent untreated cells and cells treated by IL-1b alone. PGE2 levels in the culture medium obtained using FLS from four different patients were measured by enzyme immunoassay. Data are expressed as meanGSD of four experiments. A representative experiment out of four is presented. )P!0.05 (Fisher’s protected-least-significant-difference (PLSD) test) was considered to indicate significance compared to untreated cell control and #P!0.05 (Fisher’s PLSD test) was considered to indicate significance compared to IL-1b alone control.

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control; P!0.05) (Fig. 8B), whereas this effect was more pronounced for IL-1b-stimulated-p38a phosphorylation which was observed at all times (1.3-fold after 5 min, 1.3-fold after 15 min, 1.6-fold after 30 min, 1.7-fold after 1 h and 1.8-fold after 6 h respectively versus IL-1b alone controls; P!0.05) (Fig. 8C). As p38 seemed to be more implicated in the effect of 100 mM A77 1726, we studied the effect of SB 203580 (a selective p38 inhibitor) on IL-10 and IL-11 secretion. Furthermore, these experiments were performed in the presence of IL-1b but also in the presence of TNFa, another potent cytokine inducer. First, we showed that TNFa did not stimulate the secretion of IL-10 and IL-11 in comparison with medium alone and in contrast to IL-1b alone (Fig. 9A and B respectively). However, 100 mM A77 1726 in the presence of TNFa increased IL-10 secretion (C107% versus TNFa alone; P!0.05) but had no significant effect on IL-11 secretion. Pretreatment with 10 mM SB 203580 totally inhibited the effect of 100 mM A77 1726 on IL-10 secretion in the presence of IL-1b or TNFa (Fig. 9A). However, 10 mM SB 203580 exhibited the inhibitory effect of 100 mM A77 1726 on IL-11 secretion only in the presence of IL-1b (Fig. 9B).

Fig. 7. Effect of A77 1726 on IL-1b-dependent NF-kB activation. Human RA FLS were cultured with 10% FCS containing medium alone for 24 h (lane 1) or were incubated with IL-1b alone (1 ng/mL) for the last 6 h (lane 2), or with A77 1726 (100 mM) for 24 h and IL-1b for the last 6 h (lane 3). Nuclear proteins were extracted and 10 mg of each sample were subjected to EMSA using NF-kB consensus site radiolabeled probe. Complexes were visualized by autoradiography. Positive controlZcontrol factor Oct2A given by the manufacturer (Roche Diagnostics); Comp 100XZ100-fold concentrated unlabeled probe. The experiments were performed three times; representative results are shown.

MAPKs for short times. We noticed that the activation of ERK1/ERK2 was maximal after 5 min IL-1b treatment (3-fold versus untreated cell control; P!0.05) and not observed after 15 min (2.5-fold versus untreated cell control; P!0.05) (Fig. 8A). However, for 30 min, 1 and 6 h IL-1b stimulation, we found a time-dependent decrease in ERK1/ERK2 phosphorylation. For IL-1bactivated-JNK and p38a phosphorylation, the maximal effect was observed after 15 min (respectively 4- and 3.5fold versus controls; P!0.05) and not observed after 30 min (respectively 2.3- and 2.2-fold versus untreated cell controls; P!0.05) (Fig. 8B and C). We then studied the effect of 100 mM A77 1726 on IL1b-stimulated-ERK1/ERK2, JNK and p38a activation for all times. A77 1726 had no effect on ERK1/ERK2 phosphorylation in the presence of IL-1b versus IL-1b alone controls (Fig. 8A). However, it seemed that 100 mM A77 1726 induced a slight increase in IL-1bstimulated-JNK phosphorylation (maximum 1.2-fold after IL-1b treatment for 30 min versus IL-1b alone

3. Discussion In the present study, we investigated for the first time the effect of the active metabolite of leflunomide, A77 1726, on the secretion of IL-8, IL-10, IL-11 and on the shedding of sTNFRI by human RA FLS. Furthermore, we also looked at its action on IL-6 and IL-1Ra secretion as previously described by other authors in synoviocytes [20,24,27] or in other cell types [25,28]. IL-6 and IL-8 participate in the pathogenesis of RA; for example, IL-6 supports the proliferation of synovial cells [29], while IL-8 promotes the formation of new blood vessels in synovial membranes [4]. Among chemokines, IL-8 is a strong inducer of neutrophil recruitment and is recognized as an important factor that contributes to neutrophil infiltration into synovial fluids in RA [4]. In our study, we showed that low concentrations but not high doses of A77 1726 increased IL-1b-stimulated-IL-8 secretion in human RA FLS. A77 1726 has only been reported to cause a dose-dependent reduction in the mRNA level of the type A receptor for IL-8 in human epidermal cell line [30]. In addition, as previously described [20], we observed a dose-dependent inhibition of IL-1b-stimulated-IL-6 secretion in our conditions. Besides inflammatory cytokines including IL-1, TNFa, IL-6 and chemokines such as IL-8, natural cytokine inhibitors such as IL-1Ra and sTNFRI are also present in the inflammatory synovial fluid of patients with RA and in cultured synovial fibroblasts [31e34]. The relative abundance of stimulatory and inhibitory

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Fig. 8. ELISA quantification of phosphorylated ERK1/ERK2 (A), JNK (B) and p38a (C) in IL-1b-stimulated human RA FLS pretreated with A77 1726. Cells were incubated with or without 100 mM A77 1726 for 24 h and with IL-1b (1 ng/mL) for additional different times. The controls represent untreated cells and cells treated by IL-1b alone. Cells were lysed and phosphorylated MAPKs were quantified with the DuoSetÒ IC assay kits (R&D Systems). The respective DuoSetÒ IC assay kits contain the basic components required for the development of sandwich ELISAs to measure natural and recombinant phospho-ERK1 (T202/Y204) and phospho-ERK2 (T185/Y187), JNK1 and JNK2 dually phosphorylated at T183/Y185 and phospho-JNK3 (T221/Y223), and phospho-p38a (T180/Y182) in cell lysates. FLS from three different patients were used for quantification. Data are expressed as meanGSD of three experiments. A representative experiment out of three is presented. )P!0.05 (Fisher’s protected-least-significantdifference (PLSD) test) was considered to indicate significance compared to untreated cell control and #P!0.05 (Fisher’s PLSD test) was considered to indicate significance compared to IL-1b alone control.

cytokines is thought to ultimately determine the activity of the disease process [35]. Pharmacological intervention should, therefore, shift the balance from cytokine mediated inflammation and tissue degradation to cytokine directed inhibition of the inflammatory process. According to the recent work of Palmer et al. [24], A77 1726 caused a dose-dependent increase of IL-1bstimulated-IL-1Ra secretion in human RA FLS with a maximum observed at 100 mM. However, we showed for all tested concentrations that A77 1726 had no effect on IL-1b-stimulated-sTNFRI shedding. On the other hand, we studied the effect of A77 1726 on IL-10 and IL-11 secretion, two cytokines implicated in the pathophysiology of RA. IL-10 is known as an anti-inflammatory cytokine on the basis of its ability to inhibit monocyte/macrophage and T cell production of pro-inflammatory cytokines [9,10,36]. IL-10 has been shown to stimulate monocyte expression of sTNFR [36] and to enhance the production of IL-1Ra [37,38]. Moreover, Morita et al. [39] demonstrated that IL-10 was able to significantly inhibit the production of IL-1b,

TNFa, IL-6, IL-8 and interferon-g in freshly isolated RA synovial tissue cells. As a result of such antiinflammatory properties, IL-10 has been suggested as a therapeutic agent for inflammatory conditions such as RA. Indeed, a very recent report showed that mycobacterial heat shock protein 70 induced IL-10 production in synoviocytes from arthritis patients which was accompanied by a decrease in TNFa production [40]. In vivo, Chang et al. [41] have demonstrated that a novel IL-1Ra/IL-10 fusion protein suppressed joint swelling and synovial inflammation in adjuvant arthritis of rats. In light of these facts, our results are interesting because we observed, for the first time, that high concentrations of A77 1726 (maximum at 100 mM) provoked a large increase in IL-10 secretion by human RA FLS. Furthermore, we demonstrated that, at 100 mM, A77 1726 enhanced the IL-1Ra secretion. A77 1726 has been shown to induce gene expression of IL-10 receptor in a human epidermal cell line [30]. Moreover, Korn et al. [42] have recently demonstrated that A77 1726 led to increased IL-10 release in a specific

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Fig. 9. Effect of p38 inhibitor (SB 203580) on IL-10 (A) and IL-11 (B) secretion by human RA FLS. Cells were preincubated for 2 h with 10 mM SB 203580 before the addition of A77 1726 (100 mM) for 24 h and IL-1b or TNFa (1 ng/mL) for the last 6 h. The concentration of IL-10 and IL-11 was determined in culture supernatants of FLS from four different patients by ELISA. Data are expressed as meanGSD of three experiments. A representative experiment out of three is presented. ),#P!0.05 (Fisher’s protected-least-significant-difference (PLSD) test) was considered to indicate significance.

T line and in cultured microglial cells. However, to our knowledge, the effect of A77 1726 on IL-10 secretion by human RA FLS has not been studied previously. On the other hand, we also observed the differential effect of A77 1726 on IL-11 secretion by human RA FLS versus IL-10 secretion: our results showed that IL-1b markedly increased IL-11 secretion (C274% versus untreated cell control) and that A77 1726 at low concentrations (0.1e10 mM) enhanced IL-1b-stimulatedIL-11 secretion whereas high doses (50 and 100 mM)

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decreased it (ÿ60% versus IL-1b control for both). The role of IL-11 in the pathogenesis of RA has been controversial. IL-1a alone [43] or IL-1a and TNFa synergistically stimulate the production of IL-11 by cultured rheumatoid synovial cells [44]. Exogenous IL-11 inhibited TNFa production in RA synovium in the presence of soluble IL-11 receptor, and it directly inhibited spontaneous metalloproteinase production and upregulated tissue inhibitors of metalloproteinases in RA synovial tissue [15]. Furthermore, IL-11 inhibited NF-kB binding activity in peritoneal macrophages [45] and decreased TNFa-induced NF-kB accumulation in human osteoarthritis synovial fibroblasts [46]. In contrast, IL-11 promoted osteoclastogenesis and might contribute to joint destruction in RA [14]. Regardless of its undetermined role in the pathogenesis of RA, IL-11 has been shown to be abundant in RA synovia and synovial fluid [13,15]. Furthermore, IL-11 is now classified as an IL-6-type cytokine based on functional similarities with IL-6 and the shared use of gp130 molecules in their receptor complexes [47,48]. As described above, A77 1726 inhibited IL-6 secretion in our conditions but also in other studies [20,28]. We demonstrated for the first time that 100 mM A77 1726 decreased IL-11 secretion in human RA FLS. The same effect of A77 1726 on the secretion of these two cytokines could be explained by the similarities between their structures. However, our results showed that the inhibition of IL-11 secretion by A77 1726 was not reversed by uridine in contrast to what has been described for IL-6 production by Burger et al. [20]. This suggests that inhibition of IL-11 secretion by A77 1726 was due to another mechanism than the inhibition of DHODH. RA is characterized by the proliferation of synoviocytes which also produce prostanoids such as PGE2. Eicosanoids and prostanoids are important lipid mediators that are produced at elevated levels in inflamed tissues including rheumatoid synovium and in cultured human RA FLS [49e53]. It was also of note that A77 1726 inhibited COX-2 activity directly and that high concentrations of A77 1726 caused an increase in the expression of COX-2 protein [54]. We showed that 100 mM A77 1726 exhibited the expression of COX-2. Our results also showed that, starting at 10 mM, A77 1726 inhibited IL-1b-stimulated-PGE2 production. This inhibition was almost total (ÿ98%) at 100 mM A77 1726 in human RA FLS. These results were in agreement with those of Burger et al. [20]. It was also of note that, for short periods of IL-1b stimulation (5, 15 and 30 min), 100 mM A77 1726 inhibited the production of PGE2. The study of Mino et al. [44] showed that IL-1 and TNFa synergistically stimulate PGE2-dependent production of IL-11 in RA FLS. Our results demonstrated that, for high doses of A77 1726 (50 and 100 mM), PGE2 production but also IL-11 secretion were inhibited.

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Furthermore, Taki et al. [43] studied the differential inhibitory effects of indomethacin and dexamethasone on IL-11 production by rheumatoid synovial cells. These authors demonstrated that indomethacin and dexamethasone, two PGE2 inhibitors, reduced IL-11 production suggesting that overexpression of IL-11 in fresh rheumatoid synovia was in part due to PGE2. Our work showed that, in the presence of low doses of A77 1726 (0.01e1 mM), the IL-1b-stimulated production of endogenous PGE2 was not abolished and that the IL-11 secretion was enhanced in a dose-dependent manner. However, when the production of endogenous PGE2 was totally inhibited by high doses of A77 1726 (50 and 100 mM), IL-11 secretion was markedly decreased compared to control in human RA FLS. Further investigations will be necessary to clarify the mechanism of inhibition of IL-11 secretion by A77 1726, using other PGE2 inhibitors than those used in the work of Taki et al. [43]. The MAPKs represent an attractive target for RA treatment because they can regulate cell proliferation, apoptosis, cytokine expression [55], and metalloproteinase production [56]. Numerous data demonstrate that pro-inflammatory cytokine synthesis is controlled by the MAPK pathway. The three major MAP kinase families, ERK, JNK and p38 kinase, differ in their substrate specificity and subsequent responses to stress depending on the cell type and the environmental influences. These MAPKs are expressed in synovial tissue and in cultured FLS [57e59]. These authors used immunohistochemistry and western blot analysis to detect the active phosphorylated forms of MAPKs whereas, in our conditions, we used a quantitative ELISA method. Our study showed that treatment with 100 mM A77 1726 had no effect on ERK activation. Furthermore, at this dose, A77 1726 induced a slight increase in IL-1bstimulated-JNK phosphorylation. This effect was more enhanced for IL-1b-stimulated-p38a phosphorylation. The correlation between the effect of A77 1726 on cytokine production and MAPK activation has not been thoroughly studied in human RA FLS. Only the recent report of Migita et al. [60] described that A77 1726 inhibited the MAPK signalling pathway and metalloproteinase synthesis in rheumatoid synovial fibroblasts. Their results were obtained using the same dose (100 mM) as we did. Regulation of cytokine production in RA was directly associated with activation of MAPKs [61]. In our study, A77 1726 caused a large increase in IL-10 and IL-1Ra secretion by human RA FLS. IL-10 has been shown to enhance IL-1Ra production in human monocytes [37,38]. Furthermore, Foey et al. [62] have shown that inhibition of p38 MAPK potently inhibited IL-10 synthesis in human monocytes. In our study, A77 1726 increased the p38a activation which could explain the large secretion of IL-10. Our results showed that

pre-treatment with a selective p38 inhibitor totally abolished the effect of A77 1726 on IL-10 secretion. Today, the connection between COX-2 expression and activation of the p38 signalling pathway is well known. Recent studies pointed out that inhibition of COX-2 expression is directly correlated with inhibition of the MAPK cascade including p38 MAPK in rheumatoid synovial cells [63,64]. In our study, 100 mM A77 1726 increased IL-1b-stimulated-p38a phosphorylation correlated with an increase in the expression of COX-2 protein. However, PGE2 synthesis was totally abolished without downregulated p38 activation. Furthermore, our results showed that A77 1726 was a potent inhibitor of NF-kB activation induced by IL-1b as previously described by other reports [21,65]. In conclusion, our study shows for the first time that a high dose of A77 1726, the active metabolite of leflunomide, causes an increase in IL-10 secretion and a decrease in IL-11 secretion in human RA FLS. These effects seemed to be associated with the regulation of p38 MAPK activation. The exact mechanism by which leflunomide brings about this phenomenon remains to be elucidated. Furthermore, A77 1726 inhibits NF-kB activation induced by IL-1b in human RA FLS.

4. Materials and methods 4.1. Materials Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS) and penicillin-streptomycin were supplied by Gibco-BRL (Cergy Pontoise, France). Collagenase was obtained from Worthington Biochemical Corporation (Halls Mill Road, Freehold, NJ, USA). Dispase, hyaluronidase, DNase I and uridine were purchased from Sigma (Saint Quentin Fallavier, France). 5B5 and JC/70A monoclonal antibodies and secondary polyclonal antibody conjugated with peroxidase were purchased from Dako (Trappes, France). RMO52 monoclonal antibody and fluorescein (DTAF)conjugated goat anti-mouse antibody were purchased from Immunotech (Marseille, France). A77 1726dthe active metabolite of leflunomidedwas provided by Aventis Pharma (Frankfurt, Germany). Recombinant human IL-1b and TNFa and QuantikineÒ human IL-6, IL-8, IL-10, IL-11, IL-1Ra and sTNFRI immunoassay kits and human phospho-ERK1/ERK2, phospho-JNK and phospho-p38a DuoSet IC ELISA were purchased from R&D Systems (Lille, France). SB 203580 (a selective inhibitor of p38 MAPK) was purchased from Calbiochem (VWR, Fontenay-sous-Bois, France). ELISA kits for PGE2 were purchased from Cayman Chemical (SpiBio, Massy, France). Cell Death Detection ELISAplus and DIG Gel Shift Kit were supplied by Roche Diagnostics (Meylan, France).

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4.2. Preparation of human synovial cells RA synoviocytes were isolated from fresh synovial biopsies obtained from six RA patients undergoing hip arthroplasty. All patients fulfilled the 1987 American Rheumatism Association criteria for RA [66]. The mean age of the patients was 61.3G3.3 years (range 58e65 years). The mean disease duration was 8.8G2.6 years. At the time of surgery, the disease activity score (DAS 28) was greater than 3.2. These activities were approved by local institutional review boards, and all subjects gave written informed consent. Synovia were minced and digested with 1.5 mg/mL collagenase-dispase, 1 mg/mL hyaluronidase and 0.15 mg/mL DNase I for 3e4 h at 37  C as previously described [51]. After centrifugation, cells were resuspended in DMEM supplemented with 10% FCS, 4.5 g/L D-glucose, 25 mM Hepes, 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco BRL) in a humidified atmosphere containing 5% (v/v) CO2 at 37  C. After 48 h, non-adherent cells were removed. Adherent cells (macrophage-like and FLS) were cultured in complete medium and, at confluence, cells were trypsinized and only FLS were passed. These cells were used between passages 4 and 8 when they morphologically resembled FLS after indirect immunofluorescence study (see Section 4.3). RA FLS were cultured 45e60 days prior to experimentation. This delay eliminated all possible interactions resulting from an eventual pre-operative treatment (NSAIDs, analgesics, DMARDs or steroids). 4.3. Culture of human RA FLS Between passages 4 and 8, RA FLS were trypsinized. Cell count and viability were determined and cells were plated in culture plates or flasks (Falcon, Oxnard, CA, USA). Viability, measured by trypan blue dye exclusion at the start and the end of culture, was always greater than 95%. RA FLS (105) from RA patients were used for indirect immunofluorescence. The following monoclonal antibodies were used: 5B5 (anti-prolyl hydroxylase) for fibroblasts at 1/50 dilution (Dako, Burlingam, CA, USA), JC/70A (anti-CD31) for endothelial cells at 1/50 (Dako), RMO52 (anti-CD14) for macrophages at 1/50 (Immunotech, Marseille, France). The negative control was a mouse antibody of the same isotype (Immunotech). Incubations were performed at room temperature for 30 min. Binding of monoclonal antibodies was visualized using fluorescein (DTAF)-conjugated goat anti-mouse antibody (Immunotech) at 1/50 dilution. RA FLS were allowed to adhere and grow for 48 h in culture medium prior to exposure to A77 1726. A stock solution of 10ÿ2 M A77 1726 was prepared in ethanol and diluted in culture medium to give a final concentration of 0.01e100 mM. The same amount of ethanol (!1%)

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was added to control cells. The culture medium was not changed during the entire study. 4.4. Light microscopy and apoptosis quantification (DNA fragmentation) For light microscopy, human RA FLS were cultured in 6-well culture plates (2!105 cells/well). After 100 mM A77 1726 treatment for 24 h, cells were fixed in phosphate-buffered saline (PBS) (pH 7.4) containing 4% paraformaldehyde for 20 min at room temperature, washed in PBS for 15 min, and observed with phasecontrast microscopy. Other experiments were performed with the same treatment (100 mM A77 1726 for 24 h) for apoptosis quantification. Apoptosis was quantified using the ‘‘cell death’’ ELISA (Cell Death Detection ELISAplus, Roche Diagnostics). Cytosol extracts were obtained according to the manufacturer’s protocol and apoptosis was measured as previously described [67]. 4.5. Cytokine quantification Human RA FLS were plated in 24-well plates (105 cells per well) for 48 h before treatment. After washing with PBS (pH 7.4), cells were incubated at 37  C with or without A77 1726 (0.01e100 mM) for 24 h and with IL-1b (1 ng/mL) for the last 6 h of incubation in DMEM containing 10% (v/v) FCS in a 5% CO2 atmosphere. Culture supernatants were collected and stored at ÿ80  C. The cytokine concentration in the medium was measured using a QuantikineÒ human cytokine immunoassay kit according to the instructions of the manufacturer (R&D Systems). For other conditions, cells were pretreated for 30 min with 200 mM uridine before the addition of 100 mM A77 1726 for 24 h and IL-1b (1 ng/mL) for the last 6 h, or cells were pretreated for 2 h with 10 mM SB 203580 (a selective p38 inhibitor) before addition of 100 mM A77 1726 for 24 h and IL-1b or TNFa (1 ng/mL) for the last 6 h. These last conditions were tested only for IL-10 and IL-11 secretion. 4.6. COX-2 expression and activity Human RA FLS were cultured in 75 cm2 tissue culture flasks for 48 h before treatment. After 10 or 100 mM A77 1726 treatment for 24 h and IL-1b (1 ng/mL) for the last 6 h, adherent cells were trypsinized and western blot analysis was performed as previously described [67] using the primary monoclonal antibodies b-actin (mouse anti-human b-actin (1:5000), Sigma), COX-2 (mouse anti-human COX-2 (1:100), Santa Cruz Biotechnology) and secondary polyclonal antibody conjugated with peroxidase (Dako). Blots were visualized using enhanced chemiluminescence reagents (Amersham Biosciences, Orsay, France) and immediately exposed to X-ray film. For PGE2 detection, human RA FLS were

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grown in 25 cm2 tissue culture flasks for 48 h before treatment. After washing with PBS (pH 7.4), cells were incubated at 37  C with or without A77 1726 (0.01e 100 mM) for 24 h and with IL-1b (1 ng/mL) for the last 6 h of incubation in DMEM containing 10% (v/v) FCS in an atmosphere of 5% CO2. Other conditions were represented by cells incubated with or without 100 mM A77 1726 for 24 h and with IL-1b (1 ng/mL) for additional different times (5 min, 15 min, 30 min, 1 h and 6 h). The PGE2 concentration in the medium was measured using the ELISA kit according to the manufacturer’s instructions (Cayman Chemical) as previously described [67]. 4.7. NF-kB nuclear translocation NF-kB nuclear translocation was evaluated by electromobility shift assay (EMSA). Human RA FLS were grown in 75 cm2 tissue culture flasks for 48 h before treatment. After washing with PBS (pH 7.4), cells were incubated at 37  C with or without 100 mM A77 1726 for 24 h and with IL-1b (1 ng/mL) for the last 6 h. EMSA experiments were performed using DIG Gel Shift Kit (Roche Diagnostics). Briefly, cells were harvested, washed in cold PBS and resuspended in lysis buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM PMSF, protease inhibitors CompleteÔ Mini, 0.5% Nonidet P-40). Nuclei were pelleted (2000!g, 10 min at 4  C) and resuspended in nuclear extraction buffer (20 mM HEPES pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 10 mM KCl, 15% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF, protease inhibitors CompleteÔ Mini). Lysates were clarified by centrifugation (13,000!g, 10 min at 4  C) and supernatants were collected. The protein level was determined by the Bradford method and 10 mg nuclear proteins were incubated with DIG labelled NF-kB probe [68] according to the manufacturer’s protocol. The samples were loaded on a 5% native polyacrylamide gel, and run in 0.5! TBE buffer. Nuclear proteins and oligonucleotidee protein complexes were then electro-blotted to Nylon membranes and incubated with anti-digoxigenin polyclonal sheep antibody conjugated with alkaline phosphatase. Gel mobility shift was visualized after incubation with CSPDÒ chemiluminescense reagent and exposition to X-ray film. NF-kB specific bands were confirmed by competition with a 100-fold excess of the respective unlabeled probe that resulted in a band of weak intensity. 4.8. Quantification of human phospho-ERK1/ ERK2, phospho-JNK and phospho-p38a Human RA FLS were grown in 25 cm2 tissue culture flasks for 48 h before treatment. After washing with PBS

(pH 7.4), cells were incubated at 37  C with or without 100 mM A77 1726 for 24 h and with IL-1b (1 ng/mL) for additional different times (5 min, 15 min, 30 min, 1 h and 6 h). After treatment, 106 cells were homogenized in lysis buffer in accordance with the manufacturer’s protocol (R&D Systems). Before assay, one plate was coated with 100 ml per well of capture antibody (4 mg/mL) overnight at room temperature. The plate was washed three times with 400 ml wash buffer and blocked by adding 300 ml blocking buffer to each well for 1e2 h at room temperature. Before use, cell lysates were centrifuged at 2000!g for 5 min and supernatants were diluted 6-fold and phospho-ERK1/ERK2, phosphoJNK or phospho-p38a detection was realized according to the manufacturer’s instructions (R&D Systems). 4.9. Statistical analysis The median and standard deviation (SD) were calculated using Excel (Microsoft Office, Version 98). Statistical analysis of differences was carried out by analysis of variance (ANOVA) using StatView Version 5.0 (SAS Institute Inc, Cary, North Carolina). A P-value of less than 0.05 (Fisher’s PLSD test) was considered to indicate significance.

Acknowledgements This study was supported by grants from Aventis Laboratory and Ministe`re de l’Education Nationale, de la Recherche et de la Technologie. The authors acknowledge Pr. Jean-Louis Charissoux for his excellent technical assistance and Dr J. Cook-Moreau for helpful discussions in the preparation of this manuscript.

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