**2. Fibroblast-like synoviocytes**

#### **2.1 Fibroblast-like synoviocytes in the normal synovium**

The syovium is a membranous structure that extends from the margins of articular cartilage and lines the capsule of diarthrodial joints. The synovium supports the joint structure, provides nutrition to the cartilage and lubricates the surface. The synovial membrane has 2 compartments: the initimal lining layer and the sublining layer. The initimal lining layer is the superficial layer that faces the intra-articular cavity, and produces synovial fluid as lubricant. This lining layer is normally 2 to 3 cells thick and consists of 2 types of synovial cells: macrophage-like synoviocytes (Type A synoviocytes), and fibroblast-like synoviocytes (Type B synoviocytes). Type A synoviocytes are hematopoietic in origin, bone marrowderived, and terminally differentiated, as are other tissue-resident macrophages. Type B synoviocytes are mesenchymal cells with vimentin in the cytoskeleton, and Thy-1 (CD90) on their surface. Type B synoviocytes display many characteristics of fibroblasts, such as the production of extracellular matrix, and collagen type IV and V. Specific characteristics for FLSs in the intimal lining layer include expression of cadherin-11 for homotypic aggregation (Lee *et al.*, 2007) and uridine diphosphoglucose dehydrogenase for synthesis of hyaluronic acid, an essential joint lubricant. Expression of decay accelerating factor, CD55, and adhesion molecules (VCAM-1 and ICAM-1) is also characteristic.

#### **2.2 Fibroblast-like synoviocytes in the synovium of RA**

In the synovium of RA, the histopathological characteristics are hyperplasia of FLSs, and infiltration with inflammatory cells. The pathophysiological reactions are joint destruction and perpetuation of inflammation.

#### **2.2.1 Hyperplasia**

74 Rheumatoid Arthritis – Etiology, Consequences and Co-Morbidities

capsule of a severe combined immunodeficiency (SCID) mouse, the FLSs derived from RA, but not from OA, destroyed the cartilage (Muller-Ladner *et al.*, 1996; Pierer *et al.*, 2003). In RA, cytokines produced by surrounding cells in the inflamed joints, such as basic fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-, tumor necrotizing factor (TNF)-, and IL-1 are thought to be responsible for the hyperplasia of FLSs. On the other hand, activated FLSs produce TNF-, IL-1, IL-6, chemokines, and matrix metalloproteinases (MMPs), thereby establishing the chronic and destructive inflammatory circuit driven by cellular interaction. Thus, it appears that some passively activated FLSs may be changed to be in a distinctly activated state, autonomously

The critical roles of inflammatory cytokines are evidenced by the effectiveness of cytokineblockade therapies for RA, using anti-TNF- or anti-IL-6 receptor antibodies (Brennan & McInnes, 2008; Nishimoto & Kishimoto, 2006). In spite of the promising effects shown by these anti-cytokine therapies, several problems remain, such as suppression of the normal immunity and substantial numbers of resistant cases (Firestein, 2007). To overcome these difficulties, increased knowledge of the molecular mechanisms involved in the complex and multi-factorial pathophysiology of RA is required. In this context, our research on the disease-associated genes of RA is based on the theory that FLSs are heterogeneous in physiological, and also in pathological situations. In this chapter, we briefly overview the current understanding of FLSs in RA, and introduce the pathophysiological natures of FLSs

The syovium is a membranous structure that extends from the margins of articular cartilage and lines the capsule of diarthrodial joints. The synovium supports the joint structure, provides nutrition to the cartilage and lubricates the surface. The synovial membrane has 2 compartments: the initimal lining layer and the sublining layer. The initimal lining layer is the superficial layer that faces the intra-articular cavity, and produces synovial fluid as lubricant. This lining layer is normally 2 to 3 cells thick and consists of 2 types of synovial cells: macrophage-like synoviocytes (Type A synoviocytes), and fibroblast-like synoviocytes (Type B synoviocytes). Type A synoviocytes are hematopoietic in origin, bone marrowderived, and terminally differentiated, as are other tissue-resident macrophages. Type B synoviocytes are mesenchymal cells with vimentin in the cytoskeleton, and Thy-1 (CD90) on their surface. Type B synoviocytes display many characteristics of fibroblasts, such as the production of extracellular matrix, and collagen type IV and V. Specific characteristics for FLSs in the intimal lining layer include expression of cadherin-11 for homotypic aggregation (Lee *et al.*, 2007) and uridine diphosphoglucose dehydrogenase for synthesis of hyaluronic acid, an essential joint lubricant. Expression of decay accelerating factor, CD55, and

In the synovium of RA, the histopathological characteristics are hyperplasia of FLSs, and infiltration with inflammatory cells. The pathophysiological reactions are joint destruction

destroying bone and joints.

as revealed by our subtyping studies.

**2. Fibroblast-like synoviocytes** 

**2.1 Fibroblast-like synoviocytes in the normal synovium** 

adhesion molecules (VCAM-1 and ICAM-1) is also characteristic.

**2.2 Fibroblast-like synoviocytes in the synovium of RA** 

and perpetuation of inflammation.

Hyperplasia of FLSs exhibits features of stable activation—the so-called tumor-like transformation. Features of tumor-like transformation include anchorage–independent growth, adhesion to the extracellular matrix of cartilage, resistance to apoptotic signaling, and invasiveness to cartilage and bone. Tumor-like transformation may be cell-autonomous or non-cell-autonomous. The non-cell-autonomous pathway is indirectly driven by factors produced by autoimmune-competent cells in the microenvironment. These include cytokines, growth factors, lipid mediators, and reactive oxygen species. By contrast, the cellautonomous pathway results from the cell-intrinsic changes of FLSs themselves.

Reflecting cell-intrinsic changes, FLSs in RA have a characteristic morphology, i.e., an abundant cytoplasm; a dense, rough endoplasmic reticulum; and large, pale nuclei with several prominent nucleoli (Pap & Gay, 2009). One of the important molecular characteristics of FLSs in RA is the expression of proto-oncogenes (Bartok & Firestein, 2009), including c-fos, (Aikawa et al., 2008), *ras, raf, sis, myb*, and *myc* (Roivainen *et al.*, 1999). Interestingly, proto-oncogenes are predominantly expressed by FLSs attached to cartilage and bone (Muller-Ladner *et al.*, 2000). Furthermore, some of these proto-oncogenes regulate gene expression of MMPs or cathepsin L. Thus, in the SCID mouse, inhibition of c-Raf-1 or c-Myc significantly reduced the expression of *MMP-1* and *MMP-3*, resulting in decreased invasiveness of FLSs to the cartilage (Pap *et al.*, 2004).

Among the various cells in the inflamed synovium, macrophages and T cells are thought to be most responsible for producing various stimuli for stable activation of FLSs. Various combinations of PDGF, TGF-, TNF-, IL-1, and the arachidonic acid metabolites induce the proliferation of FLSs (Konttinen *et al.*, 1999). On the other hand, FLSs in RA have been shown to exhibit defective apoptosis, rather than enhanced proliferation (Jacob *et al.*, 1995; Korb *et al.*, 2009). Apoptosis was rapidly induced in RA-derived FLSs by retroviral transduction of a combination of dominant-negative c-Raf-1 and dominant-negative c-Myc (Pap *et al.*, 2004), indicating that some proto-oncogenes are involved. Death receptor Fas is expressed and is functional in FLSs *in vitro*. However, apoptosis induced by anti-Fas antibody was prevented by TNF-, IL-1 and IL-6, suggesting that FLSs in the inflamed joints are resistant to apoptosis (Ohshima *et al.*, 2000; Wakisaka *et al.*, 1998). The antiapoptotic function of nuclear factor (NF)-B activated by TNF-signaling, and the induction of the anti-apoptotic molecule Bcl-xL by IL-1 are involved (Jeong *et al.*, 2004). In addition to the effects of cytokines, the adhesion molecule VLA-5 (integrin 51), upon ligation with fibronectin, is involved in this resistance to Fas-mediated apoptosis (Kitagawa *et al.*, 2006). Under conditions of genotoxic stress, the tumor-suppressor p53 induces cell-cycle arrest, followed by either DNA repair or apoptosis, depending on the degree of DNA damage (Gudkov & Komarova, 2010). A main effector of p53-dependent apoptosis, PUMA (p53 upregulated modulator of apoptosis) is present in very low concentrations in the synovium. Adenovirus-mediated transfer of the *p53* gene into FLSs induced production of the p53 protein, leading to p21 expression; however, PUMA expression was not enhanced and apoptosis was not induced (Cha *et al.*, 2006). This suggests that, under conditions of genotoxic stress, the FLSs in RA tend to undergo cell-cycle arrest rather than apoptosis.

#### **2.2.2 Infiltration with inflammatory cells**

Infiltration with inflammatory cells mainly involves chemokines, cytokines, lipids of chemical mediators, and adhesion molecules. It comprises the mutual activation of interacting cells of distinct lineages, leading to the perpetuation of inflammation.

Molecular Mechanisms of Rheumatoid Arthritis

MMP-9.

bone (Min *et al.*, 2004).

**2.2.4 Perpetuation of inflammation** 

Revealed by Categorizing Subtypes of Fibroblast-Like Synoviocytes 77

elevated serum levels of MMP-3 are correlated with systemic inflammation at the clinical and also the serologic level (Manicourt *et al.*, 1995; Yoshihara *et al.*, 1995). Although expression of MMP-13/collagenase-3 correlates with elevated levels of systemic inflammatory markers, this is not specific to RA (Lindy *et al.*, 1997; Westhoff *et al.*, 1999). MT1-MMT/MMP-14 degrades the extracellular matrix, and activates MMP-2/gelatinase A and MMP-13 (Pap *et al.*, 2000a). The expression of MMPs in synovial cells is regulated by several extracellular signals, including inflammatory cytokines, growth factors, and molecules of the extracellular matrix, such as collagen and fibronectin (Pap & Gay, 2009). Among these, IL-1 is the most potent inducer of MMPs, including MMP-1, MMP-3, MMP-8, MMP-13, and MMP-14. FGF and PDGF also act as potent inducers for MMPs, by enhancing the effects of IL-1. TNF and TGF- induce MMP-1 and MMP-13, respectively, while IL-17 induces MMP-1 and

Another group of proteinases involved in joint destruction is the cathepsins, which cleave cartilage types II, IX, and XI, and proteoglycan. The expression of the cysteine proteases, cathepsins B and L, was increased in the synovium of RA, especially at the sites of cartilage invasion (Keyszer *et al.*, 1995, 1998). Similarly to MMPs, the production of cathepsins is induced by proto-oncogene, IL-1, and TNF- (Joseph *et al.*, 1987; Huet *et al.*, 1993; Lemaire *et al.*, 1997). Cathepsin K, which plays an important role in bone resorption by osteoclasts, is also expressed by FLSs and macrophages at the site of synovial invasion into the articular

When cells producing soluble factors or expressing ligands on their surfaces are located close to cells that receive signals through the specific receptor, a circuit of chronic inflammation may be generated through an exchange of cell roles. For example, activated Th1 cells produce IFN, which activates macrophages. The activated macrophages produce IL-1 and TNF-, which in turn activate T cells. In cases of RA, IL-1 and TNF- from macrophages can activate FLSs, creating another circuit with a distinct cellular combination. Although one can easily imagine the operation of such a circuit at a certain time point of autoimmune diseases, it is difficult to demonstrate the mechanism by a suitable model system. Recently, Ogura *et al.* (2008) proposed that IL-17 secreted from Th17 cells induces fibroblasts to produce more IL-6, in a manner dependent on the transcription factor NF-B, and the signal transducer and activator of transcription (STAT) 3. The mechanism, designated as "IL-17A-triggered positive-feedback loop of IL-6 signaling", is thought to amplify the inflammatory responses mediated by interactive cytokines. Enhancement of this loop was shown to be involved in the development of RA-like arthritis or experimental autoimmune encephalomyelitis in knock-in mice gp130F759, which are defective in the negative regulation of signaling through a common receptor subunit of IL-6 family cytokines, gp130. The identification of such a powerful circuit, specific to each autoimmune

disease, will facilitate the development of a critical target point for effective therapy.

The risk of developing RA and the severity of the disease are significantly affected by genetics. It has long been recognized that certain HLA alleles, especially HLA-DR4, are

**3. Progress in the study of RA by the molecular genetic approach** 

**3.1 Genome-wide screening for disease-related genes** 

Inflammatory mediators produced by FLSs include IL-15 (Miranda-Carus *et al.*, 2004), IL-16 (Pritchard *et al.*, 2004), IL-18 (Gracie *et al.*, 1999), TNF-, TGF-(Pohlers *et al.*, 2007), NO, and prostagrandin E2 (Kojima *et al.*, 2003). Various chemokines are reported to be produced by FLSs in RA (Iwamoto *et al.*, 2008). The production of IL-8/CXCL8 and GRO/CXCL1, which recruit neutrophils, is induced by stimulation of FLSs with IL-1, IL-1, TNF-, or IL-17 (Hosaka *et al.*, 1994; Kehlen *et al.*, 2002; Koch *et al.*, 1991, 1995). Neutrophils are abundant in the synovial fluid of RA, but rare in the synovial tissue. The levels of lymphotactin/XCL1 are elevated in the synovial fluid and tissues of RA. Moreover, infiltrating mononuclear cells and FLSs in the tissues of RA express XCR1, a receptor for lymphotactin/XCL1 (Wang *et al.*, 2004). The levels of macrophage inflammatory protein (MIP)-1/CCL3 (a ligand of CCR1 and CCR5) are higher in the synovial fluid of RA. Furthermore, upon stimulation with lipopolysaccharide and TNF- isolated FLSs produce MIP-1/CCL3 (Koch *et al.*, 1994). The migration of CD4+ memory T cells to the synovium of RA, and the inhibition of activationinduced apoptosis of T cells, are induced by stromal cell-derived factor (SDF)-1/CXCL12. Thus, the accumulation of CD4+ memory T cells in the synovium plays an important role in RA (Nanki *et al.*, 2000). The production of RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted)/CCL5 is histologically detected in the synovial lining and sublining layers of affected rheumatoid joints (Robinson *et al.*, 1995). All these suggest that, in microenvironments rich with TNF- and IL-1 FLSs themselves recruit monocytes, neutrophils, Th1 cells, eosinophils, and basophils (Rathanaswami *et al.*, 1993).

In addition to the regulation of migration, stimulation with MCP-1/CCL2, SDF-1/CXCL12, IP-10/CXCL10, Mig/CXCL9, and MCP-4/CCL13 enhances the proliferation of FLSs, leading to synovial hyperplasia (Garcia-Vicuna *et al.*, 2004; Iwamoto *et al.*, 2007). Furthermore, continuous infusion of human IL-8/CXCL8 into the knee joints of rabbits for 14 days led to severe arthritis, characterized by erythema, joint pain, infiltration of leucocytes and mononuclear cells in the synovial tissue, and hypervascularization in the synovial lining layer (Endo *et al.*, 1994). Thus, the angiogenic properties of chemokines, such as IL-8/CXCL8, GRO/CXCL1, MCP-1/CCL2, SDF-1/CXCL12, and fractalkine/CX3CL1 (Koch *et al.*, 1992; Salcedo *et al.*, 1999, 2000; Volin *et al.*, 2001) may play an important role in the development of RA. Angiogenic factors, including FGF (Thomas *et al.*, 2000), vascular endothelial growth factor (VEGF) (Cho *et al.*, 2002), IL-18, and angiopoietin (Scott *et al.*, 2002), are also produced by FLSs. This suggests that FLSs are involved in neovascularization, and may cause critical pathological changes to sustain pannus formation in RA (Szekanecz & Koch, 2007).

#### **2.2.3 Joint destruction**

Proteinases, such as MMPs and cathepsins, are produced by FLSs attached to cartilage and bone, and play an important role in joint destruction. The expression of *MMP-1/interstitial collagenase* and *MMP-3/stromelysin* correlates with the invasive growth of FLSs in RA (Tolboom *et al.*, 2002). MMP-1 is found in the synovial membranes of all RA patients. Moreover, the levels of MMP-1 in the synovial fluid, but not in the sera, correlate with the degree of synovial inflammation (Konttinen *et al.*, 1999; Maeda *et al.*, 1995; Sorsa *et al.*, 1992). MMP-3 plays a key role in joint destruction, not only by degrading matrix molecules, but also by activating other pro-MMPs into their active forms (Okada, 2009). The major source of MMP-3 is FLSs in the lining layer (Tetlow *et al.*, 1993). High concentrations of MMP-3 have been detected in the synovial fluid and sera of RA patients (Beekman *et al.*, 1997; Taylor *et al.*, 1994). Moreover,

Inflammatory mediators produced by FLSs include IL-15 (Miranda-Carus *et al.*, 2004), IL-16 (Pritchard *et al.*, 2004), IL-18 (Gracie *et al.*, 1999), TNF-, TGF-(Pohlers *et al.*, 2007), NO, and prostagrandin E2 (Kojima *et al.*, 2003). Various chemokines are reported to be produced by FLSs in RA (Iwamoto *et al.*, 2008). The production of IL-8/CXCL8 and GRO/CXCL1, which recruit neutrophils, is induced by stimulation of FLSs with IL-1, IL-1, TNF-, or IL-17 (Hosaka *et al.*, 1994; Kehlen *et al.*, 2002; Koch *et al.*, 1991, 1995). Neutrophils are abundant in the synovial fluid of RA, but rare in the synovial tissue. The levels of lymphotactin/XCL1 are elevated in the synovial fluid and tissues of RA. Moreover, infiltrating mononuclear cells and FLSs in the tissues of RA express XCR1, a receptor for lymphotactin/XCL1 (Wang *et al.*, 2004). The levels of macrophage inflammatory protein (MIP)-1/CCL3 (a ligand of CCR1 and CCR5) are higher in the synovial fluid of RA. Furthermore, upon stimulation with lipopolysaccharide and TNF- isolated FLSs produce MIP-1/CCL3 (Koch *et al.*, 1994). The migration of CD4+ memory T cells to the synovium of RA, and the inhibition of activationinduced apoptosis of T cells, are induced by stromal cell-derived factor (SDF)-1/CXCL12. Thus, the accumulation of CD4+ memory T cells in the synovium plays an important role in RA (Nanki *et al.*, 2000). The production of RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted)/CCL5 is histologically detected in the synovial lining and sublining layers of affected rheumatoid joints (Robinson *et al.*, 1995). All these suggest that, in microenvironments rich with TNF- and IL-1 FLSs themselves recruit monocytes,

neutrophils, Th1 cells, eosinophils, and basophils (Rathanaswami *et al.*, 1993).

in RA (Szekanecz & Koch, 2007).

**2.2.3 Joint destruction** 

In addition to the regulation of migration, stimulation with MCP-1/CCL2, SDF-1/CXCL12, IP-10/CXCL10, Mig/CXCL9, and MCP-4/CCL13 enhances the proliferation of FLSs, leading to synovial hyperplasia (Garcia-Vicuna *et al.*, 2004; Iwamoto *et al.*, 2007). Furthermore, continuous infusion of human IL-8/CXCL8 into the knee joints of rabbits for 14 days led to severe arthritis, characterized by erythema, joint pain, infiltration of leucocytes and mononuclear cells in the synovial tissue, and hypervascularization in the synovial lining layer (Endo *et al.*, 1994). Thus, the angiogenic properties of chemokines, such as IL-8/CXCL8, GRO/CXCL1, MCP-1/CCL2, SDF-1/CXCL12, and fractalkine/CX3CL1 (Koch *et al.*, 1992; Salcedo *et al.*, 1999, 2000; Volin *et al.*, 2001) may play an important role in the development of RA. Angiogenic factors, including FGF (Thomas *et al.*, 2000), vascular endothelial growth factor (VEGF) (Cho *et al.*, 2002), IL-18, and angiopoietin (Scott *et al.*, 2002), are also produced by FLSs. This suggests that FLSs are involved in neovascularization, and may cause critical pathological changes to sustain pannus formation

Proteinases, such as MMPs and cathepsins, are produced by FLSs attached to cartilage and bone, and play an important role in joint destruction. The expression of *MMP-1/interstitial collagenase* and *MMP-3/stromelysin* correlates with the invasive growth of FLSs in RA (Tolboom *et al.*, 2002). MMP-1 is found in the synovial membranes of all RA patients. Moreover, the levels of MMP-1 in the synovial fluid, but not in the sera, correlate with the degree of synovial inflammation (Konttinen *et al.*, 1999; Maeda *et al.*, 1995; Sorsa *et al.*, 1992). MMP-3 plays a key role in joint destruction, not only by degrading matrix molecules, but also by activating other pro-MMPs into their active forms (Okada, 2009). The major source of MMP-3 is FLSs in the lining layer (Tetlow *et al.*, 1993). High concentrations of MMP-3 have been detected in the synovial fluid and sera of RA patients (Beekman *et al.*, 1997; Taylor *et al.*, 1994). Moreover, elevated serum levels of MMP-3 are correlated with systemic inflammation at the clinical and also the serologic level (Manicourt *et al.*, 1995; Yoshihara *et al.*, 1995). Although expression of MMP-13/collagenase-3 correlates with elevated levels of systemic inflammatory markers, this is not specific to RA (Lindy *et al.*, 1997; Westhoff *et al.*, 1999). MT1-MMT/MMP-14 degrades the extracellular matrix, and activates MMP-2/gelatinase A and MMP-13 (Pap *et al.*, 2000a).

The expression of MMPs in synovial cells is regulated by several extracellular signals, including inflammatory cytokines, growth factors, and molecules of the extracellular matrix, such as collagen and fibronectin (Pap & Gay, 2009). Among these, IL-1 is the most potent inducer of MMPs, including MMP-1, MMP-3, MMP-8, MMP-13, and MMP-14. FGF and PDGF also act as potent inducers for MMPs, by enhancing the effects of IL-1. TNF and TGF- induce MMP-1 and MMP-13, respectively, while IL-17 induces MMP-1 and MMP-9.

Another group of proteinases involved in joint destruction is the cathepsins, which cleave cartilage types II, IX, and XI, and proteoglycan. The expression of the cysteine proteases, cathepsins B and L, was increased in the synovium of RA, especially at the sites of cartilage invasion (Keyszer *et al.*, 1995, 1998). Similarly to MMPs, the production of cathepsins is induced by proto-oncogene, IL-1, and TNF- (Joseph *et al.*, 1987; Huet *et al.*, 1993; Lemaire *et al.*, 1997). Cathepsin K, which plays an important role in bone resorption by osteoclasts, is also expressed by FLSs and macrophages at the site of synovial invasion into the articular bone (Min *et al.*, 2004).

## **2.2.4 Perpetuation of inflammation**

When cells producing soluble factors or expressing ligands on their surfaces are located close to cells that receive signals through the specific receptor, a circuit of chronic inflammation may be generated through an exchange of cell roles. For example, activated Th1 cells produce IFN, which activates macrophages. The activated macrophages produce IL-1 and TNF-, which in turn activate T cells. In cases of RA, IL-1 and TNF- from macrophages can activate FLSs, creating another circuit with a distinct cellular combination. Although one can easily imagine the operation of such a circuit at a certain time point of autoimmune diseases, it is difficult to demonstrate the mechanism by a suitable model system. Recently, Ogura *et al.* (2008) proposed that IL-17 secreted from Th17 cells induces fibroblasts to produce more IL-6, in a manner dependent on the transcription factor NF-B, and the signal transducer and activator of transcription (STAT) 3. The mechanism, designated as "IL-17A-triggered positive-feedback loop of IL-6 signaling", is thought to amplify the inflammatory responses mediated by interactive cytokines. Enhancement of this loop was shown to be involved in the development of RA-like arthritis or experimental autoimmune encephalomyelitis in knock-in mice gp130F759, which are defective in the negative regulation of signaling through a common receptor subunit of IL-6 family cytokines, gp130. The identification of such a powerful circuit, specific to each autoimmune disease, will facilitate the development of a critical target point for effective therapy.

#### **3. Progress in the study of RA by the molecular genetic approach**

#### **3.1 Genome-wide screening for disease-related genes**

The risk of developing RA and the severity of the disease are significantly affected by genetics. It has long been recognized that certain HLA alleles, especially HLA-DR4, are

Molecular Mechanisms of Rheumatoid Arthritis

platelets, and shorter disease durations (van Baarsen *et al.*, 2010).

facilitating the development of effective therapy with a clear target point.

inducing receptors (*IL-2R*

**4. Mouse models for RA** 

*et al.*, 2002; Ohtani *et al.*, 2000).

immunity.

Revealed by Categorizing Subtypes of Fibroblast-Like Synoviocytes 79

expression signature (RAlow), common to the tissues from patients with OA, and characterized by increased expression of genes involved with tissue remodeling activity. Importantly, the cluster of RAhigh showed an increased expression of STAT1-pathway related genes;STAT1-

*1, and IRF-1*), suggesting a prominent role for this pathway. Furthermore, patients with the high-grade inflammation tissue type had higher Disease Activity Scores in 28 joints, higher C-reactive protein levels, higher erythrocyte sedimentation rates, increased numbers of

Several trials have profiled gene expression in the synovial tissues of RA patients undergoing molecular-targeting therapy (Lindberg *et al.*, 2006; Wijbrandts *et al.*, 2008). Further analysis is expected to yield valuable data on cytokine activity in the human body,

The generation of RA-like joint diseases in engineered mutant mice appears to reflect the heterogeneous and complicated mechanisms of human arthritis, diagnosed simply as rheumatoid arthritis. In contrast to previous years, when animal models for human diseases rarely emerged by point mutations in nature, current research on autoimmune diseases such as RA benefits from the existence of various engineered mutant mice models. For example, mechanisms for RA-like disease revealed by murine models include, the abnormal T-cell receptor (TCR) signaling by a natural mutant ZAP70 in SKG mouse (Sakaguchi *et al.*, 2003), an autoantibody to glucose-6-phosphoisomerase in K/BxN TCR transgenic mouse (Korganow *et al.*, 1999), defective autoantigen clearance in *DNaseII*-/- moue (Kawane *et al.*, 2006), overexpression of the viral gene in HTLV-1 *pX* transgenic mouse (Iwakura *et al.*, 1991), and excessive amounts or activity of cytokines. RA-like disease developed in TNF transgenic mice (Keffer *et al.*, 1991) and IL-1 transgenic mice (Niki *et al.*, 2001) with overproduction of inflammatory cytokines and in TNF AU-rich elements-deficient (ARE) mice (Kontoyiannis *et al.*, 1999) with increased stability of cytokine messenger RNA. Excessive activities of arthritogenic cytokines were evoked in IL-1 receptor antagonist knock-out mouse (Horai *et al.*, 2000) lacking a physiological negative feedback molecule, and in gp130F759 with a defective, intracellular negative-regulatory signaling pathway (Atsumi

These wide variety of murine arthritis models with a defined genetic defect will be useful for analyzing the mechanisms for the synergistic action of genetic and environmental factors in RA development (Ishihara *et al.*, 2004), and also the mechanisms for initiation or perpetuation of joint inflammation (Murakami *et al.*, 2011; Ogura *et al.*, 2008). Furthermore, bone marrow transplantation experiment revealed a unique feature of gp130F759 that nonhematopoietic cells with a point mutation Y759F in gp130 are sufficient to induce passive but arthritogenic activation of wild type CD4+T cells (Sawa *et al.*, 2006). In human TNF transgenic mouse, arthritogenic FLSs showed increased expression of *MMP-1* and *MMP-9*, and also diminished adhesion to extracellular matrix components. These changes could induce increased proliferation and migration, which are critical for the spread of hyperplasia in the joints (Aidinis *et al.*, 2003). Dispensable roles for *RAG* in arthritis have been observed in TNFARE mouse (Kontoyiannis *et al.*, 1999) and *DNaseII*-/- mouse (Kawane *et al.*, 2010), indicating that synovial hyperplasia may develop independently of acquired

*, CCR5*), and STAT1 target genes (*MMP-1, MMP-3, caspase-1, TAP-*

associated with increased risk of onset and severity of RA (Weyand *et al.*, 1992). A shared epitope on certain HLA haplotypes is thought to affect the binding of peptides derived from self-antigens, leading to autoimmune responses by T cells (Wordsworth *et al.*, 1989). To identify non-HLA genes that regulate the development and severity of RA, human genomewide studies have been performed. Some of these studies have used a combined approach, with factors such as microsatellites (Tamiya *et al.*, 2005), or disease subsets; serum autoantibody alone (Stahl *et al.*, 2010) or combined with a shared epitope (Sugino *et al.*, 2010); race, or nation (Freudenberg *et al.*, 2011; Martin *et al.*, 2010); correlation with other autoimmune diseases (Cui *et al.*, 2009; Zhernakova *et al.*, 2011); or responsiveness to therapies targeting specific cytokines (Liu *et al.*, 2008; Plant *et al.*, 2011). Single nucleotide polymorphisms that may be involved in the development of RA include protein tyrosine phosphatase, nonreceptor-type 22 (*PTPN22*), cytotoxic T-lymphocyte antigen 4 (*CTLA4)*, *STAT4*, and peptidylarginine deiminase type 4 (*PADI4)*. Among these, *PADI4* has been identified by genome-wide screening (Suzuki *et al.*, 2003) as being able to modify selfantigens by citrullination. Moreover, the presence of anti-cyclic citrullinated antibody in the serum is highly specific to RA and has a high diagnostic value. The role of PADI4 in the pathogenesis of RA, especially with respect to "autoimmunity" to modified self-antigens, will be intriguing to clarify.

Large-scale, genome-wide association studies, based firmly on statistics, have provided valuable information on the candidate genes for RA. Nevertheless, to understand the complex pathophysiology of RA, data from studies on additional aspects must be integrated. Such studies should include molecular and cell-biological analyses of clinical materials from individual RA cases, and functional analyses of candidate genes *in vitro* and *in vivo*, including experimental system using engineered mutant mice.

#### **3.2 Transcription profiling reveals disease-specific genes and heterogeneity in RA tissues**

Gene expression profiling of FLSs, comparing RA and OA, has revealed disease-specific genes. The genes highly and exclusively expressed in RA were *HOXD10, HOXD11, HOXD13, CCL8*, and *LIM homeobox 2*. Further analysis of the relationships between gene expression on RA-FLSs and clinical disease parameters revealed specific and unique correlations as follows; *HLA-DQA2* with Health Assessment Questionnaire (HAQ) score; *Clec12A* with rheumatoid factor; *MAB21L2*, *SIAT7E*, *HAPLN1*, and *BAIAP2L1* with Creactive protein level; and *RGMB* and *OSAP* with erythrocyte sedimentation rate (Galligan *et al.*, 2007). The data indicated the heterogeneity of gene expression in patients with the same disease. These RA-specific or clinical state-related genes differ from those identified by genome-wide screening, indicating that the complete pathophysiology of RA, as a multifactorial disease, involves genomic and also epi-genomic regulation of genes. The functional roles of these genes remain to be determined.

Evidence for the heterogeneity of gene expression in synovial tissues from erosive RA cases has been demonstrated by large-scale profiling studies. Systemic characterization of the differentially expressed genes highlighted the existence of at least 2 molecularly distinct forms of RA tissues (van der Pouw Kraan *et al.*, 2003). The first is RA tissue with high-grade inflammation (RAhigh), which exhibits abundant expression of gene clusters indicative of adaptive immune responses, such as genes expressed by T cells, B cell, and antigenpresenting cell (APC). The second form of RA tissue is a low-grade inflammatory gene expression signature (RAlow), common to the tissues from patients with OA, and characterized by increased expression of genes involved with tissue remodeling activity. Importantly, the cluster of RAhigh showed an increased expression of STAT1-pathway related genes;STAT1 inducing receptors (*IL-2R, CCR5*), and STAT1 target genes (*MMP-1, MMP-3, caspase-1, TAP-1, and IRF-1*), suggesting a prominent role for this pathway. Furthermore, patients with the high-grade inflammation tissue type had higher Disease Activity Scores in 28 joints, higher C-reactive protein levels, higher erythrocyte sedimentation rates, increased numbers of platelets, and shorter disease durations (van Baarsen *et al.*, 2010).

Several trials have profiled gene expression in the synovial tissues of RA patients undergoing molecular-targeting therapy (Lindberg *et al.*, 2006; Wijbrandts *et al.*, 2008). Further analysis is expected to yield valuable data on cytokine activity in the human body, facilitating the development of effective therapy with a clear target point.
