Role for interleukin-6 in structural joint damage and systemic bone loss in rheumatoid arthritis

Joint Bone Spine 77 (2010) 201–205

Review

Role for interleukin-6 in structural joint damage and systemic bone loss in rheumatoid arthritis Benoit Le Goff a,∗,b, Frédéric Blanchard b, Jean-Marie Berthelot a,b, Dominique Heymann b, Yves Maugars a a b

Service de rhumatologie, Hôtel-Dieu, 1, place Alexis-Ricordeau, 44093 Nantes cedex 1, France Laboratoire de physiopathologie de la résorption osseuse, Inserm UMR-S 957, 1, rue Gaston-Veil, 44035 Nantes cedex 1, France

a r t i c l e

i n f o

Article history: Accepted 21 August 2009 Available online 4 May 2010 Keywords: Interleukin-6 Osteoclasts Osteoblasts Osteoporosis Rheumatoid arthritis

a b s t r a c t Interleukin-6 (IL-6) plays a key role in the local and systemic manifestations of rheumatoid arthritis (RA). IL-6 is not only a proinflammatory cytokine, but also interacts in complex ways with the cells involved in bone remodeling. In RA, IL-6 may indirectly promote osteoclastogenesis by increasing the release of RANK-L by osteoblasts and synovial cells. However, IL-6 inhibits osteoclastogenesis in vitro, via a direct mechanism. The effects of IL-6 on osteoblasts may vary with the cell differentiation stage: thus, IL-6 may promote the differentiation of pre-osteoblasts to mature osteoblasts while also diminishing the proliferation of osteoblasts at late differentiation stages. Thus, the effects of IL-6 on bone remodeling are complex and may occur in opposite directions depending on the model or experimental conditions. Nevertheless, results from studies in animal models and humans support a negative effect of IL-6 on bone. Thus, in patients with RA, blocking IL-6 may be effective both in diminishing the inflammatory manifestations and in preventing the bone complications of the disease. © 2010 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.

1. Introduction Interleukin-6 (IL-6) is a key proinflammatory cytokine in rheumatoid arthritis (RA). Other cytokines belonging to the same family include IL-11, leukemia inhibitory factor (LIF), oncostatin M, cardiotrophin-1, and ciliary neutrophic factor (CNF). All these cytokines act via the same gp130dependent intracellular signaling pathway [1]. Studies of animal models have established that, among these cytokines, IL-6 plays the main role in the pathogenesis of RA. Thus, IL6 knockout protects mice against adjuvant-induced polyarthritis, an effect not seen with deficiencies in IL-11 or oncostatin M, for instance [2]. The efficacy in RA of the anti-IL-6 antibody tocilizumab further illustrates the crucial role for IL-6 in this disease. Systemic bone loss and structural joint damage are two manifestations of major concern in patients with RA. Both involve osteoclasts, the only cell type capable of resorbing bone minerals. In RANK-L knockout mice, polyarthritis induction causes joint inflammation but no bone erosions [3]. Furthermore, the osteoblast response is impaired in RA [4]. Thus, erosion repair is rare in RA but occurs in other conditions such as psoriatic arthritis. Osteoblast activation and differentiation are dependent on the Wnt pathway.

∗ Corresponding author. Tel.: +33 240 084 848; fax: +33 240 084 830. E-mail address: [email protected] (B. Le Goff).

Recent data indicate a role for Dickkopf-1, a Wnt pathway inhibitor, in the osteoblastic response impairment seen in RA [5]. The effects of IL-6 on osteoclasts, osteoblasts, and bone remodeling are complex and vary across diseases [6]. IL-6, together with other proinflammatory cytokines, probably plays a key role in the joint damage and osteoporosis seen in RA. The objective of this review is to discuss recent data about IL-6 with special attention to effects on cells involved in bone remodeling and on the genesis of erosions and systemic bone loss during the course of RA. 2. Structure and functions of IL-6 2.1. Structure of IL-6 IL-6 is a monomeric protein arranged into four long helical chains. This pleiotropic cytokine is released by many cell types (T cells, monocytes, epithelial cells, fibroblasts. . .) and plays a crucial role in both the specific antigen response and the inflammatory response [7,8]. IL-6 was first identified 25 years ago as a cytokine capable of stimulating the maturation of B cells and plasma cells [9]. Since then, the list of known IL-6 effects has grown steadily. IL-6 is a key proinflammatory cytokine that induces the production of C-reactive protein and fibrinogen by the hepatocytes. Effects on T cells include increased proliferation and differentiation to cytotoxic T-cells and Th17 cells [10]. IL-6 stimulates the proliferation of macrophages and megakaryocytes. It induces the expression of adhesion molecules at the endothelial cell surface

1297-319X/$ – see front matter © 2010 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.jbspin.2010.03.002

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tion, thereby activating the intracellular signaling pathways. The gp130 protein can act via two intracellular signaling pathways, the JAK/STAT pathway and the mitogen-activated protein (MAP) kinase pathway (Erk1/2, p38, and JNK). Migration of these transcription factors to the nucleus induces the expression of target genes involved in numerous physiological processes (e.g., proliferation and differentiation). The JAK/STAT pathway plays a pivotal role in controlling the immune response, as demonstrated in models of arthritis and other inflammatory diseases [14]. Nevertheless, IL-6 overactivity may produce deleterious effects. Thus, a negative feedback system is needed. This system involves the suppressors of cytokine signaling (SOCS) proteins, whose expression is induced by STATs. More specifically, SOCS-3 can inhibit the binding of JAK to STAT, thus blocking the IL-6 signaling pathway. The production of mice with genetically engineered modifications in gp130 signaling (activation of STAT1/3 or MAPK in a mutually exclusive manner) has shed light on the respective roles for the STAT1/3 and MAPK signaling pathways on the cells involved in bone remodeling. The STAT1/3 pathway seems to activate the osteoblasts and osteoclasts, whereas the MAPK pathway may inhibit osteoclastogenesis and bone resorption [15]. Thus, the biological effects of IL-6 on bone may depend on which intracellular signaling pathway is predominantly activated.

3. IL-6: a key cytokine in the pathophysiology of rheumatoid arthritis Fig. 1. Binding of IL-6 to its receptor induces gp130 dimerization, which activates the Janus protein-tyrosine kinases (JAKs), thereby activating the JAK/STAT and MAP kinase intracellular signaling pathways to induce the expression of target genes involved in numerous physiological processes (proliferation, differentiation. . .).

and the production of monocyte chemoattractant protein-1 (MCP1), thereby enhancing cell migration to sites of inflammation [11]. IL-6 also influences the differentiation and activity of osteoclasts and osteoblasts, as discussed later on. 2.2. IL-6 receptor (Fig. 1) The IL-6 receptor is composed of two polypeptide chains [12], an 80-kDa chain specific of IL-6 (IL-6R) and the intracellular signal transducer gp130. IL-6R has a short intracytoplasmic segment that binds to gp130 when stimulated by IL-6, thereby producing a dimer and triggering intracellular signal transduction. IL-6R exists in both transmembrane and soluble forms. Thus, the IL-6 signal can be transmitted either via the conventional pathway involving association with the membrane receptor or via trans-signaling involving binding to the soluble receptor. The soluble receptor is found in many body fluids including serum and joint fluid [13]. The conventional signal transduction pathway, in contrast, is available only in the limited number of cell types that produce the transmembrane form of IL-6R (hepatocytes, monocytes, macrophages, and some lymphocytes). Thus, the trans-signaling pathway plays a major role in IL-6 effects on joints (as synovial cells do not normally produce transmembrane IL-6R) and on endothelial cells. IL-6 stimulates endothelial cells to express adhesion molecules and cytokines, which attract inflammatory cells to the synovial membrane [11]. 2.3. Intracellular signaling pathway The gp130 protein is expressed ubiquitously in all cell types [10] and serves as the shared signaling pathway for all cytokines belonging to the IL-6 family. Binding of IL-6 to its receptor leads to gp130 dimerization and Janus protein-tyrosine kinase (JAK) activa-

3.1. IL-6 and animal models of polyarthritis IL-6 knockout mice are partly protected against the development of experimentally induced polyarthritis [16–18]. This protective effect is not associated with decreases in the local levels of TNF␣ or IL-1, indicating a direct role for IL-6. It varies across animal models. Thus, although IL-6 knockout mice are protected against collagen- and adjuvant-induced arthritis [16], the absence of IL-6 does not modify the clinical expression of polyarthritis in K/BxN mice or mice transgenic for human TNF␣ [19,20]. Soluble IL-6R also plays a crucial role. Thus, when injected intraarticularly, the IL-6/IL-6R fusion protein, but not IL-6 alone, induces inflammatory lesions [21]. These results are ascribable to the absence of membrane IL-6R expression by synovial pannus cells. Human IL-6 overexpression in mice does not induce polyarthritis, in contrast to human TNF␣ overexpression [22]. In contrast, studies of mice carrying gp130 mutations have established the crucial importance of the gp130 protein, which is involved in intracellular cell signaling. Interestingly, an activating mutation in the mouse gp130 gene causes the spontaneous development of polyarthritis with autoantibody production [23]. This polyarthritis is dependent on CD4+ T cells and involves overactivation of the STAT3 signaling pathway.

3.2. Role for Il-6 in the inflammatory manifestations of human polyarthritis IL-6 is involved in the systemic manifestations of RA. First, IL-6 is a proinflammatory cytokine that activates the production of acutephase proteins such as C-reactive protein, fibrinogen, and serum amyloid A [1]. IL-6 levels are significantly elevated in patients with RA [24,25] and are correlated with clinical variables (e.g., morning stiffness duration and number of affected joints) and laboratory variables (e.g., erythrocyte sedimentation rate, C-reactive protein, and rheumatoid factor titer) [24–26]. Serum IL-6 levels decrease with disease-modifying treatment and correlate with the clinical treatment response [27].

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IL-6 is found in large amounts in synovial membrane and joint fluid specimens from patients with RA [28]. Thus, IL6 contributes to pannus development and to the other local manifestations of RA. IL-6 can activate many cell types including immune system cells and synovial fibroblasts. Together with TGF␤ and IL-1, IL-6 may be involved in the differentiation of naive lymphocytes to Th17 lymphocytes, although this remains to be confirmed in humans [29]. Recent data support a crucial role for Th17 lymphocytes in the pathophysiology of RA. Thus, IL17 produced in the rheumatoid synovium perpetuates the local inflammatory process [30,31]. 4. Complex effects of IL-6 on bone metabolism Bone remodeling is a continuous process that maintains bone formation and bone resorption in equilibrium by coordinating osteoblast and osteoclast activity. In RA, the equilibrium is disrupted, with both inadequate bone formation and increased bone resorption by the osteoclasts [4,32]. The effects of proinflammatory cytokines on bone tissue probably contribute to mediate the bone loss. IL-6 exerts complex effects on bone tissue, whose net in vitro result varies across models. 4.1. In vitro effects of IL-6 on osteoblasts Osteoblasts are among the cells that express the transmembrane IL-6 receptor [33,34]. However, although the level of expression increases during osteoblast differentiation [35], it remains low, and soluble IL-6R seems required for IL-6 to exert its full effects on osteoblasts. IL-6 coupled to its soluble receptor may promote both the differentiation and the activation of osteoblasts. IL-6 can increase the expression of osteoblast differentiation markers such as alkaline phosphatase or osteocalcin in various in vitro models of osteoblast differentiation (e.g., calvaria cells and human mesenchymatous cells) [34,36]. IL-6 may also exert indirect effects on osteoblast differentiation and proliferation by controlling the local production of factors that influence osteoblastogenesis. For instance, IL-6 combined to its soluble receptor increases the expression of insulin growth factor 1 (IGF-1) and bone morphogenetic protein-6 (BPM-6) by osteoblasts from rat calvaria [37,38]. Similarly, IL-6 can stimulate the expression of parathyroid hormone (PTH)-related peptide by osteoblasts from human trabecular bone [39]. The effect of IL-6 on osteoblast proliferation seems more modest [40], and two studies even found that IL-6 decreased osteoblast proliferation and induced osteoblast apoptosis [35,41]. In both studies, these effects occurred late in the osteoblast differentiation process. In an in vitro rat osteogenesis model, IL-6 inhibited the formation of mineralized bone nodules in calvaria cell cultures when administered throughout osteoblast differentiation [41]. Thus, the effects of IL-6 may differ according to the stage of osteoblast differentiation, with activation of pre-osteoblast differentiation to mature osteoblasts but decreased proliferation of osteoblasts at late differentiation stages. 4.2. In vitro effects of IL-6 on osteoclastogenesis (Fig. 2) The effect of IL-6 on osteoclastogenesis is also complex but seems chiefly mediated by an indirect mechanism. Osteoclasts are cells of monocytic lineage whose differentiation to mature osteoclasts depends mainly on two cytokines, M-CSF and RANK-L. IL-6 acts indirectly on osteoclastogenesis by stimulating the release of RANK-L by bone tissue cells including osteoblasts [42]. Thus, adding IL-6 and its soluble receptor to mouse calvaria bone tissue cultures (containing both stromal cells/osteoblasts and osteoclastic precursor cells) stimulates osteoclastogenesis by increasing the

Fig. 2. Role for IL-6 in rheumatoid erosions. IL-6 is produced by various cell types found in the synovial pannus (T cells, monocytes, and fibroblasts), osteoblasts, and synovial fibroblasts. IL-6 indirectly stimulates osteoclastogenesis by activating RANK-L release by osteoblasts and synoviocytes.

release of RANK-L by osteoblasts. Recent data establish that this effect is mediated by the STAT-3 signaling pathway [43]. STAT3 may act not only via RANK-L, but also by increasing the release by osteoblasts of other pro-osteoclastic factors such as PTH-related peptide and prostaglandins [44]. Another mechanism of action may be involved in the stimulating effect of IL-6 on osteoclastogenesis. As indicated above, IL-6, together with TGF␤ and IL-1, may be among the key cytokines involved in T-cell differentiation to Th17 cells. Th17 cells act as powerful osteoclastogenesis inductors by increasing the release of both RANK-L and IL17, which directly stimulate osteoclast differentiation [2,45]. Interestingly, a direct effect of IL-6 on osteoclasts was found in a study of cultured osteoclast progenitors derived from human CD14+ cells or mouse bone marrow CD14+ cells [46]. In these cultures, the absence of other cells enables the demonstration of a direct effect of IL-6 on osteoclast differentiation. Paradoxically, IL6 under these conditions can inhibit osteoclastogenesis by inducing differentiation of the osteoclast progenitors to macrophages [47]. However, these models do not taken into account the potential effects of other cytokines and cells found at bone resorption sites. Consequently, whether IL-6 exerts similar inhibitory effects in vivo is unclear, although the in vitro data might explain the apparently paradoxical effect of IL-6 in several animal models [48,49]. 4.3. Animal models: effects of IL-6 on bone tissue Animal models provide insight into the in vivo effects of IL-6 on bone tissue. IL-6 knockout mice [50] exhibit only minor bone status changes consisting in an increase in bone turnover. The absence of major changes in osteoclast or osteoblast counts indicates that the role for IL-6 in controlling physiological bone remodeling is modest [51]. After oophorectomy to induce estrogen deprivation, however, IL-6 knockout mice are protected from the development of osteoporosis [51], because the absence of IL-6 is associated with a decrease in osteoclast bone resorption capabilities. Thus, IL-6 promotes bone resorption in disease states. Similarly, bone marrow cells from IL-6 knockout mice are less prone to osteoclast differentiation, compared to cells from wild-type animals [2]. This proosteoclastic effect of IL-6 is not always deleterious. Thus, IL-6-

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deficient mice show delayed callus formation and bone fracture healing related to a decreased number of osteoclasts at the fracture site. After a fracture, IL-6 promotes bone healing by stimulating osteoclastogenesis, thereby enhancing bone repair and callus formation [52]. IL-6 overexpression from birth in transgenic mice, in contrast, is associated with increased bone resorption and with a 50% to 70% decrease in growth [53]. Bone mass is diminished at both trabecular and cortical sites. Bone resorption is increased via stimulation of osteoclastogenesis and, concomitantly, the activity of mature osteoblasts is decreased [54]. The data from these animal models demonstrate that IL-6 can modulate bone remodeling in vivo by stimulating osteoclastogenesis and inhibiting osteoblast function. However, as shown in vitro, IL-6 has the opposite effect in some animal models. Thus, in a titanium particle-induced osteolysis model, IL-6 knockout mice exhibit greater degrees of bone resorption around the particles as a result of increases in osteoclast counts and bone resorption [48]. In a mouse model of human IL-6 overexpression, histomorphometry showed decreased bone turnover and reduced osteoblast counts concomitantly with decreases in bone resorption and osteoclast counts [49]. In some disease states, and for poorly understood reasons, IL-6 may therefore decrease osteoclastogenesis. These in vivo data illustrate the complexity of cytokine effects on bone tissue. The exact role for IL-6 in bone tissue remains to be determined. 5. Role for IL-6 in systemic bone loss and bone erosions in rheumatoid arthritis 5.1. Effects of IL-6 on systemic bone loss Patients with RA exhibit systemic bone loss related both to conventional risk factors for osteoporosis and to RA-related systemic inflammation and treatments [55]. Proinflammatory cytokines such as TNF␣ play a central role in this manifestation, and TNF␣ antagonists improve the bone status of patients with RA [56]. IL-6 may be involved via effects on bone remodeling. This possibility is supported by animal studies establishing a role for IL-6 in postmenopausal osteoporosis [57]. In humans with RA, periarticular bone loss (assessed on bone biopsies) correlated with local IL-6 overexpression but not with the expression of TNF␣ or IL1 [58]. Two studies found associations linking the Z-score and bone remodeling markers to IL-6 production by monocytes or to serum IL-6 levels in patients with RA [59]. Finally, assays in patients treated with tocilizumab showed increases in bone formation markers (type 1 collagen N-propeptide [PINP]) and decreases in bone resorption markers (Cross laps) [60], suggesting a negative effect of IL-6 on bone mass. Studies of bone mineral density changes in tocilizumab-treated RA patients can be expected to provide interesting information. 5.2. Effects of IL-6 on structural joint damage (Fig. 2) The osteoclast is the main cell involved in bone erosions in RA. Within the inflamed synovial membrane, osteoclast differentiation is activated by many cytokines, most notably RANK-L and TNF␣. IL-6 is expressed in large amounts at sites of synovial membrane inflammation in patients with RA. IL-6 can stimulate osteoclastogenesis. This effect may be related chiefly to an indirect mechanism involving increased RANK-L release by inflammatory pannus cells or increased IL-17 production via differentiation of Th17 lymphocytes [2]. Synovial fibroblasts also produce RANK-L after stimulation by IL-6 [61]. Data from animal models support this hypothesis. Thus, experimental arthritis is less severe in IL6 knockout mice, which have fewer osteoclasts and erosions [18].

The structural effect of IL-6 may be independent from proinflammatory? effect. In mice transgenic for human TNF␣, a model in which IL-6 does not modulate the inflammatory response, treatment with an IL-6R antagonist decreases the joint osteoclast count and the number of erosions without modifying the degree of local inflammation [62]. In humans, IL-6 levels in serum or joint fluid correlate with disease activity and with the development of erosions [13,24]. A study of RA patients showed that joint fluid levels of IL-6 and IL-6R correlated with the severity of the radiological joint damage. Adding the joint fluid to osteoclast cultures activated osteoclastic differentiation, which was inhibited by an antibody to IL-6R [28]. Further support for a structural effect of IL-6 comes from the SAMURAI study, in which radiological progression was slower in tocilizumab-treated patients compared to methotrexate-treated patients [63]. 6. Conclusions and future prospects Current treatments for RA seek both to decrease disease activity and to prevent systemic bone loss and joint erosions. Proinflammatory cytokines are crucial to both objectives, since they act both on the immune system and on bone tissue. IL-6 exerts complex effects on bone cells, with variations across models. IL-6 stimulates osteoclastogenesis via an indirect effect involving RANK-L release by osteoblasts and synovial cells. Th17 cells may contribute to the proosteoclastic effect. The effect of IL-6 on osteoblasts is more controversial. Whereas prolonged IL-6 exposure seems to diminish osteoblast proliferation, IL-6 promotes osteoblast differentiation in vitro. Overall, available data from human studies and the marked efficacy of anti-IL-6 on both the inflammatory manifestations and the bone lesions seen in RA patients support a net deleterious effect of IL-6 on bone. However, the present review highlights the complexity of cytokine effects on bone tissue. Further studies in this area are needed to elucidate the paradoxical effects seen with some treatments and to identify new treatment targets. Conflict of interest The authors have no conflict of interest to declare. References [1] Kishimoto T. Interleukin-6: discovery of a pleiotropic cytokine. Arthritis Res Ther 2006;8:S2. [2] Wong PK, Quinn JM, Sims NA, et al. Interleukin-6 modulates production of T lymphocyte-derived cytokines in antigen-induced arthritis and drives inflammation-induced osteoclastogenesis. Arthritis Rheum 2006;54:158–68. [3] Pettit AR, Ji H, von Stechow D, et al. TRANCE/RANKL knockout mice are protected from bone erosion in a serum transfer model of arthritis. Am J Pathol 2001;159:1689–99. [4] Walsh NC, Reinwald S, Manning CA, et al. Osteoblast Function is Compromised at Sites of Focal Bone Erosion in Inflammatory Arthritis. J Bone Miner Res 2009;24:1572–85. [5] Diarra D, Stolina M, Polzer K, et al. Dickkopf-1 is a master regulator of joint remodeling. Nat Med 2007;13:156–63. [6] Blanchard F, Duplomb L, Baud’huin M, et al. The dual role of IL-6-type cytokines on bone remodeling and bone tumors. Cytokine Growth Factor Rev 2009;20:19–28. [7] Nishimoto N, Kishimoto T. Interleukin 6: from bench to bedside. Nat Clin Pract Rheumatol 2006;2(11):619–26; Nishimoto N, Kishimoto T. Nat Clin Pract Rheumatol 2006;2:691. [8] Lipsky PE. Interleukin-6 and rheumatic diseases. Arthritis Res Ther 2006;8:S4. [9] Kishimoto T, Akira S, Narazaki M, et al. Interleukin-6 family of cytokines and gp130. Blood 1995;86:1243–54. [10] Hibi M, Murakami M, Saito M, et al. Molecular cloning and expression of an IL-6 signal transducer, gp130. Cell 1990;63:1149–57. [11] Romano M, Sironi M, Toniatti C, et al. Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment. Immunity 1997;6:315–25. [12] Taga T, Hibi M, Hirata Y, et al. Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp130. Cell 1989;58:573–81.

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