MINIREVIEW Molecular aspects of rheumatoid arthritis: chemokines in the joints of patients Takuji Iwamoto 1,2 , Hiroshi Okamoto 1 , Yoshiaki Toyama 2 and Shigeki Momohara 1 1 Institute of Rheumatology, Tokyo Women’s Medical University, Japan 2 Department of Orthopaedic Surgery, School of Medicine, Keio University, Tokyo, Japan Introduction Rheumatoid arthritis (RA) is a chronic systemic inflam- matory disease that occurs in about 1% of the popula- tion. The inflammatory process is characterized by infiltration of inflammatory cells into the joints, leading to the proliferation of fibroblast-like synoviocytes (FLS) and the destruction of cartilage and bone. In RA syno- vial tissue, the infiltrating cells consist of macrophages, T cells, B cells, plasma cells, neutrophils, mast cells, dendritic cells and natural killer cells [1]. Migration of leukocytes into the synovium is a regulated multistep process involving interactions between leukocytes and endothelial cells and cellular adhesion molecules, as well as between leukocytes and chemokines and chemokine receptors [2]. Chemokines are small, chemoattractant cytokines that play key roles in the accumulation of inflammatory cells at the site of inflammation. There- fore, chemokines and chemokine receptors are consid- ered to be therapeutic targets in several chronic Keywords chemokine receptors; chemokines; monocyte chemoattractant protein-4 (MCP-4) ⁄ CCL13; pulmonary and activation- regulated chemokine (PARC)/CCL18; rheumatoid arthritis (RA) Correspondence H. Okamoto, Institute of Rheumatology, Tokyo Women’s Medical University, 10-22 Kawada-cho, Shinjuku, Tokyo 162-0054, Japan Fax: +81 3 5269 1726 Tel: +81 3 5269 1725 E-mail: hokamoto@ior.twmu.ac.jp (Received 14 March 2008, revised 27 May 2008, accepted 27 June 2008) doi:10.1111/j.1742-4658.2008.06580.x Rheumatoid arthritis (RA) is a chronic symmetric polyarticular joint dis- ease that primarily affects the small joints of the hands and feet. The inflammatory process is characterized by infiltration of inflammatory cells into the joints, leading to proliferation of synoviocytes and destruction of cartilage and bone. In RA synovial tissue, the infiltrating cells such as macrophages, T cells, B cells and dendritic cells play important role in the pathogenesis of RA. Migration of leukocytes into the synovium is a regu- lated multi-step process, involving interactions between leukocytes and endothelial cells, cellular adhesion molecules, as well as chemokines and chemokine receptors. Chemokines are small, chemoattractant cytokines which play key roles in the accumulation of inflammatory cells at the site of inflammation. It is known that synovial tissue and synovial fluid from RA patients contain increased concentrations of several chemokines, such as monocyte chemoattractant protein-4 (MCP-4) ⁄ CCL13, pulmonary and activation-regulated chemokine (PARC) ⁄ CCL18, monokine induced by interferon-c (Mig)⁄ CXCL9, stromal cell-derived factor 1 (SDF-1) ⁄ CXCL12, monocyte chemotactic protein 1 (MCP-1) ⁄ CCL2, macrophage inflammatory protein 1a (MIP-1a) ⁄ CCL3, and Fractalkine ⁄ CXC3CL1. Therefore, chemokines and chemokine-receptors are considered to be important molecules in RA pathology. Abbreviations CCL3L1, CCL3-like 1; GROa, growth-related oncogene a; IFN-c, interferon-c; IL, interleukin; IP-10, interferon-c-inducible protein-10; MAPK, mitogen-activated protein kinase; MCP, monocyte chemoattractant protein; Mig, monokine induced by interferon-c; MIP, macrophage inflammatory protein; MMP, matrix metalloproteinase; OA, osteoarthritis; PARC, pulmonary and activation-regulated chemokine; RA, rheumatoid arthritis; RANTES, regulated on activation, normal, T-cell expressed, and secreted; SDF, stromal cell-derived factor; TNF-a, tumor necrosis factor-a. 4448 FEBS Journal 275 (2008) 4448–4455 ª 2008 The Authors Journal compilation ª 2008 FEBS inflammatory disorders such as RA. Based on a number of recently published studies, this review focuses on the chemokines expressed in RA synovial tissues. Chemokines In humans there are more than 50 types of chemokines – small (8–10 kDa) heparin-binding proteins – that were originally identified by their chemotactic activity on bone marrow-derived cells [3]. They are classified into four families according to the location of cysteine residues. The four chemokine groups are CC, C, CXC and CX3C, where C is a cysteine and X any amino- acid residue, and their receptors are consequently clas- sified as CCR, CR, CXCR and CX3CR. The chemokine receptors are bound to the cell membrane through seven transmembrane helical segments coupled to a G-protein that transduces the intracellular signal. The two major subclasses include the CC chemokines (where the cysteines are neighboring) and the CXC chemokines (where the cysteines are separated by one amino acid). The CXC chemokines mainly act on neu- trophils and lymphocytes, whereas the CC chemokines mainly act on monocytes and lymphocytes without affecting neutrophils [4]. Lymphotactin, in the C chemokine family, is similar to members of both the CC and CXC chemokine families, but lacks two of the four cysteine residues and is a potent attractant for T cells, but not for monocytes or neutrophils [5]. Frac- talkine, in the CX3C family, is a cell-surface-bound protein, in which the first two cysteine residues are separated by three amino acids, and has potent chemo- attractant activity for T cells and monocytes [6]. One characteristic feature of chemokines is the redundancy of the system. Several chemokines bind to more than one receptor, and the majority of chemokine receptors have multiple ligands, leading to the generation of multiple pathways directing similar cellular responses. Until recently, chemokines have been named ran- domly, with no clear system being used. Some have been included with the interleukins [for example, inter- leukin (IL)-8], and others have been given names describing a function, for example, macrophage chemoattractant proteins. In an attempt to clarify the confused and complex nomenclature associated with chemokines, the nomenclature of the chemokine system has been revised. The name referring to a specific biologic function has been replaced by the chemokine subfamily name followed by a number [for example, monocyte chemoattractant protein (MCP)-1 is CCL2] [7,8]. Synovial tissue and synovial fluid from RA patients contain increased concentrations of several chemokines (Table 1) [9,10]. The inflammatory cells that infiltrate into RA synovial tissue express chemokine receptors, including CXCR3, CCR5, CCR3, CCR2 and CXCR2 [11]. Based on these data, the chemokine system is considered to be implicated in RA pathogenesis via the recruitment and retention of monocytes and T lympho- cytes into the joints [9,12]. Although macrophages and FLS are considered to be the most potent producers of chemokines in the synovial compartment, chondrocytes also have the ability to produce chemokines [13–16]. Chemokine production is known to be induced at high levels in response to inflammatory stimuli, such as lipopolysaccharide, IL-1b, tumor necrosis factor-a (TNF-a) and interferon-c (IFN-c) (Fig. 1). Chemokine expression in the RA joint CC chemokines Monocyte chemoattractant protein-1 ⁄ CCL2 (a ligand of CCR2) can attract monocytes, T cells, natural killer cells and basophils [17,18]. Monocyte chemoattractant protein-1 ⁄ CCL2 is highly expressed in synovial tissue and synovial fluid in RA patients, and synovial tissue macrophages are the dominant source of MCP-1 ⁄ CCL2 production [19]. The levels of MCP-1 ⁄ CCL2 correlate significantly with the levels of IL-1b, IL-6 and IL-8 ⁄ CXCL8 in culture supernatants of synovium from RA patients, and the expression of MCP-1 ⁄ CCL2 mRNA by cultured synovial cells is stimulated by IL-1b and TNF-a [20]. We recently found that angiotensin II activated nuclear factor-jB in FLS to induce MCP-1 ⁄ CCL2 [21]. Table 1. Chemokines and chemokine receptors expressed in the joint of RA patients. Systemic name Common name Chemokine receptors CC chemokines CCL2 MCP-1 CCR2 CCL3 MIP-1a CCR1, CCR5 CCL5 RANTES CCR1, CCR3, CCR5 CCL13 MCP-4 CCR2, CCR3 CCL18 PARC Unknown CCL20 MIP-3a CCR6 CXC chemokines CXCL1 GROa CXCR2 CXCL8 IL-8 CXCR1, CXCR2 CXCL9 Mig CXCR3 CXCL10 IP-10 CXCR3 CXCL12 SDF-1 CXCR4 C chemokine XCL1 Lymphotactin XCR1 CX3C chemokine CX3CL1 Fractalkine CX3CR1 T. Iwamoto et al. Chemokines in the joints of patients FEBS Journal 275 (2008) 4448–4455 ª 2008 The Authors Journal compilation ª 2008 FEBS 4449 Regulated on activation, normal, T-cell expressed, and secreted (RANTES) ⁄ CCL5 (a ligand of CCR1, CCR3 and CCR5) is another CC chemokine, impli- cated in RA pathogenesis, which is expressed and secreted from normal T cells that are regulated upon activation. Histological examination of affected rheu- matoid joints reveals extensive RANTES ⁄ CCL5 expression in the synovial lining and sublining layers [22]. The expression of RANTES ⁄ CCL5 in cultured FLS increases in both a time-dependent and dose- dependent manner upon stimulation with TNF-a and IL-1b [23]. Macrophage inflammatory protein (MIP)-1a ⁄ CCL3 (a ligand of CCR1 and CCR5) levels are higher in RA synovial fluid than in synovial fluid from other forms of arthritis, including osteoarthritis (OA). Isolated FLS produce MIP-1a ⁄ CCL3 mRNA and protein upon incubation with lipopolysaccharide and TNF-a [24]. Freshly isolated synovial fluid neutrophils also contain higher concentrations of MIP-1a ⁄ CCL3 protein than peripheral blood neutrophils from either RA patients or healthy controls, and incubation in the presence of TNF-a results in an increase in MIP-1a ⁄ CCL3 secretion by neutrophils in the synovial fluid of RA patients [25]. Macrophage inflammatory protein-3a ⁄ CCL20 (a ligand of CCR6) is a selective chemoattractant for leucocytes such as memory T cells, naive B cells and immature dendritic cells. Macrophage inflammatory protein-3a ⁄ CCL20 is highly expressed in synovial flu- ids and synovial tissue specimens of patients with RA, and cultured FLS derived from either RA or OA patients are capable of producing MIP-3a ⁄ CCL20 in response to IL-1b and TNF-a in vitro [26]. Increased expression of MIP-3a ⁄ CCL20 and CCR6 has also been confirmed in tissue biopsies from RA subchondral bone [27]. Our group recently found that the mRNA exp- ression of MCP-4 ⁄ CCL13 (a ligand of CCR2 and CCR3), which is the major chemoattractant for eosin- ophils, monocytes and T lymphocytes, is significantly higher in cartilage from RA patients than in cartilage from OA patients or normal controls. Furthermore, the concentration of MCP-4 ⁄ CCL13 protein in syno- vial fluid is also significantly higher in RA patients than in OA patients [28]. Monocyte chemoattractant protein-4 ⁄ CCL13 production in cultured human chon- drocytes is stimulated by IFN-c in combination with IL-1b or TNF-a [13]. We also found that pulmonary and activation-regu- lated chemokine (PARC)⁄ CCL18 is expressed more strongly in RA cartilage and synovial membrane than in OA samples. The levels of PARC ⁄ CCL18 in serum and synovial fluid are also higher in RA patients than in OA patients and normal controls. In addition, the levels of PARC ⁄ CCL18 in serum significantly correlate with the levels of rheumatoid factor [14]. Cartilage Cartilage RA joint TNF-α, IL-1β, IFN-γ A B Synoviocytes Macrophage T cell Chemokines RA joint Chemotaxis Chemokines Synovial hyperplasia MMP release Angiogenesis Pannus formation Fig. 1. Schematic representation of the role of chemokines in the joint of RA patients. (A) Synovial macrophages, T cells, synovio- cytes and also chondrocytes produce various chemokines stimu- lated mainly by inflammatory cytokines, including IL-1b, TNF-a and IFN-c. (B) Chemokines expressed in the joint recruit leukocytes into the joints. In addition to functioning in cell trafficking, several chemokines have other biological abilities. Chemokines stimulate FLS and chondrocytes to release inflammatory mediators, including cytokines and MMPs, leading to cartilage degradation and pannus formation. Furthermore, chemokines enhance cell proliferation and angiogenesis, leading to synovial hyperplasia. Chemokines released by leukocytes and FLS, or by the chondrocytes themselves, can induce autocrine ⁄ paracrine stimulation of these cells, leading to joint destruction. Chemokines in the joints of patients T. Iwamoto et al. 4450 FEBS Journal 275 (2008) 4448–4455 ª 2008 The Authors Journal compilation ª 2008 FEBS CXC chemokines Interleukin-8 ⁄ CXCL8 (a ligand of CXCR1 and CXCR2) was the first chemokine identified to be involved in leukocyte chemotaxis [29]. Interleukin-8 ⁄ CXCL8 is present in high quantities in both the synovial tissue and synovial fluid of RA patients, and synovial tissue macrophages constitutively pro- duce IL-8 ⁄ CXCL8 [30]. Interleukin-8 ⁄ CXCL8 is known to have angiogenic activity in the RA joint [31]. Strong induction of IL-8 ⁄ CXCL8 is also observed in primary cultures of articular chondro- cytes as well as in cartilage explants stimulated with IL-1b [32]. Growth-related oncogene a (GROa) ⁄ CXCL1 (a ligand of CXCR2), a chemoattractant for neutrophils, similarly to IL-8⁄ CXCL8, is highly expressed in syno- vial fluid and synovial tissue in RA patients. Further- more, the production of GROa ⁄ CXCL1 by RA FLS and chondrocytes is significantly increased upon incu- bation with TNF-a or IL-1b [16,33,34]. Interleukin-17 also induces the expression of GRO a ⁄ CXCL1 mRNA, as well as the expression of IL-8⁄ CXCL8 mRNA, which is dependent on p38 mitogen-activated protein kinase (MAPK) in RA FLS [35]. Growth-related onco- gene a ⁄ CXCL1 induces a dose-dependent decrease in the expression of interstitial collagens by rheumatoid synovial fibroblasts [36]. Interferon-c-inducible protein-10 (IP-10) ⁄ CXCL10 (a ligand of CXCR3) is also upregulated in RA synovial fluid and synovial tissue [9,37]. Immunolocalization analysis indicated that IP-10 ⁄ CXCL10 is associated mainly with infiltrating macrophage-like cells and fibroblast-like cells in the RA synovium, and the inter- action of activated leukocytes with FLS results in marked increases in the expression and secretion of IP-10 ⁄ CXCL10 [37]. Human chondrocytes also produce IP-10 ⁄ CXCL10 stimulated by the pro-inflammatory cytokines IL-1b or TNF-a [38]. Monokine induced by interferon-c (Mig) ⁄ CXCL9, also a ligand of CXCR3, is highly expressed in RA synovial fluid and synovial tissue, particularly in macrophages [9]. The expression of Mig ⁄ CXCL9 by cultured FLS is stimulated by IFN-c [39]. Stromal cell-derived factor (SDF)-1 ⁄ CXCL12, a ligand of CXCR4, is expressed in the RA synovium and is increased by CD40 stimulation [40]. Stromal cell-derived factor-1 ⁄ CXCL12 stimulates the migration of CD4 + memory T cells in the RA synovium and also inhibits activation-induced apoptosis of T cells, indicating that SDF-1 ⁄ CXCR4 interactions play important roles in CD4 + memory T-cell accumulation in the RA synovium [40]. C and CX3C chemokines The C chemokine family is represented by two chemo- kines (lymphotactin ⁄ XCL1 and SCM-1b ⁄ XCL2), whereas the CX3C chemokine family contains only one member, called fractalkine⁄ CX3CL1 [41]. The levels of lymphotactin ⁄ XCL1 are significantly higher in synovial fluid of RA patients than those in paired serum samples. Expression of XCR1, a lymphotactin ⁄ XCL1 receptor, was detected in infiltrating mono- nuclear cells and FLS of synovial tissues [42]. Fractalkine ⁄ CX3CL1, a chemoattractant for mono- cytes and lymphocytes, is significantly elevated in RA synovial fluid compared with synovial fluid from patients with OA or other forms of arthritis. The syno- vial fluid and peripheral blood of patients with RA contain a greater percentage of monocytes expressing fractalkine ⁄ CX3CL1 and CX3CR1 compared with T cells [43]. Recombinant human fractalkine⁄ CX3CL1 significantly induces the migration of human dermal microvascular endothelial cells, suggesting that it may mediate angiogenesis in RA [44]. The secretion of frac- talkine ⁄ CX3CL1 from FLS obtained from RA patients is regulated mainly by TNF-a. The role of chemokines in RA pathogenesis The chemokines listed above are implicated in RA pathogenesis via the recruitment and retention of leu- kocytes in the joints. In addition to functioning in cell trafficking, several chemokines have been shown to possess other biological abilities [45]. Chemokines are able to stimulate FLS and chon- drocytes to release inflammatory mediators, including cytokines and matrix metalloproteinases (MMPs), leading to cartilage degradation. Stimulation of RA FLS with MCP-1 ⁄ CCL2, RANTES ⁄ CCL5 and SDF- 1 ⁄ CXCL12 results in the enhanced production of IL-6 and IL-8 ⁄ CXCL8 [46]. Monocyte chemoattractant protein-1 ⁄ CCL2, SDF-1 ⁄ CXCL12, IP-10 ⁄ CXCL10, RANTES ⁄ CCL5 and Mig ⁄ CXCL9 increase, in a dose-dependent and time-dependent manner, the gela- tinase and collagenase activities in the supernatants of cultured FLS [47]. Monocyte chemoattractant pro- tein-1 ⁄ CCL2 and RANTES ⁄ CCL5 stimulate MMP-3 production by chondrocytes and are also able to inhibit proteoglycan synthesis and to enhance proteo- glycan release from the chondrocytes [48,49]. RAN- TES ⁄ CCL5 induces the expression of inducible nitric oxide synthase, IL-6 and MMP-3 in chondrocytes [50]. The release of MMP-3 is also increased by stimulating chondrocytes with SDF-1 ⁄ CXCL12 [10]. The interaction of SDF-1 ⁄ CXCL12 with CXCR4- T. Iwamoto et al. Chemokines in the joints of patients FEBS Journal 275 (2008) 4448–4455 ª 2008 The Authors Journal compilation ª 2008 FEBS 4451 positive chondrocytes results in a specific increase in the release of MMP-3 [10]. Pathological concentra- tions of SDF-1 ⁄ CXCL12 induce the death of human chondrocytes and this is dependent on the p38 MAPK activity [51]. Lymphotactin ⁄ XCL1 stimulation of RA FLS results in a marked downregulation of MMP-2 production [52]. Thus, chemokines released by mononuclear cells and FLS, or by the chondro- cytes themselves, can induce an autocrine ⁄ paracrine stimulation of these cells, leading to extracellular matrix degradation. Furthermore, chemokines have the ability to enhance cell proliferation, leading to synovial hyperplasia. Stimulation with MCP-1 ⁄ CCL2, SDF-1 ⁄ CXCL12, IP-10 ⁄ CXCL10, Mig ⁄ CXCL9 and MCP-4 ⁄ CCL13 enhances the proliferation of FLS [13,47]. Our group reported that the proliferation of FLS by MCP- 4 ⁄ CCL13 is dependent on activation of the extracellu- lar regulated kinase MAPK. The activation of MAPK is also important in regulating the RA FLS cytoskeletal structure and migration by fractalkine ⁄ CX3CL1 [53]. Fractalkine ⁄ CX3CL1 was expressed on FLS, and senescent CD28 ) T cells were positive for CX 3 CR1, the receptor for fractalkine ⁄ CX3CL1. Fractalkine ⁄ CX3CL1 was expressed on FLS costimulated T-cell- activating signals and amplified the proliferation, IFN-c production and expulsion of cytoplasmic gran- ules [54]. In addition, fractalkine ⁄ CX3CL1 not only regulated T-cell function, but directly affected FLS pro- liferation, suggesting that T-cell ⁄ FLS interactions led to an autocrine growth-promoting loop enhancing the proliferative expansion of FLS [55]. Chemokines also have either angiogenic or angio- static abilities, which are important aspects of RA synovium proliferation. Chemokines containing the ELR motif, which is the three-amino-acid sequence (Glu–Leu–Arg) near the N-terminus before the first cysteine, are thought to be angiogenic, whereas chemo- kines lacking the ELR motif mainly appear to be angiostatic [45,56]. Interleukin-8 ⁄ CXCL8 was the first chemokine identified to have angiogenic properties in addition to chemoattractant effects [31]. Continuous infusion of human recombinant IL-8 ⁄ CXCL8 into the knee joints of rabbits for 14 days led to severe arthritis characterized by apparent erythema and joint pain, accumulation of leucocytes, infiltration of mononuclear cells in synovial tissue and marked hypervascular- ization in the synovial lining layer [57]. Other ELR- containing CXC chemokines with angiogenic features include GROa ⁄ CXCL1, whereas non-ELR chemo- kines, such as Mig ⁄ CXCL9 and IP-10 ⁄ CXCL10, are angiostatic. There are some exceptions to this rule as certain chemokines lacking the ELR motif, including MCP-1 ⁄ CCL2, SDF-1 ⁄ CXCL12 and fractalkine ⁄ CX3CL1, are also known to have angiogenic proper- ties [44,58,59]. Recently, associations between the chemokine gene polymorphisms and RA have been investigated. An allelic variant in the 3¢-untranslated region of the SDF-1 gene is associated with the annual rates of radiographic progression, but not with suscep- tibility to RA. However, the functional role of these variants has not been clearly established thus far [60]. A recent meta-analysis reported a significant, negative association of a 32-bp deletion in the CCR5 gene (CCR5D32), which results in a nonfunctional receptor, with susceptibility to RA, suggesting that CCR5D32 is protective against the development of RA [61]. The gene copy number variations of CCL3-like 1 (CCL3L1), a nonallelic isoform of CCL3 encoded by different genes and a potent ligand for CCR1 and CCR5, influenced susceptibility to RA, and genetic interaction between CCL3L1 dose and CCR5D32 was also found [62]. These results indicate that the genetic variations of chemokines and chemokine receptors are associated with RA susceptibility and severity. Conclusion Chemokines have an important role in the patho- genesis of RA by recruiting leukocytes and by control- ling other important processes, such as release of mediators of inflammation, cell proliferation and angiogenesis. 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