**4. Mouse models for RA**

78 Rheumatoid Arthritis – Etiology, Consequences and Co-Morbidities

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,

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

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

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

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

*in vivo*, including experimental system using engineered mutant mice.

will be intriguing to clarify.

roles of these genes remain to be determined.

**tissues** 

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

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 immunity.

Molecular Mechanisms of Rheumatoid Arthritis

determined by the composition of heterogeneous FLSs.

**arthritis types** 

**like synoviocytes** 

**inflammation** 

Revealed by Categorizing Subtypes of Fibroblast-Like Synoviocytes 81

inflammation synovial tissues were characterized by high and low expression of genes of immune-competent cells (T cells, B cells, and APCs), respectively. Furthermore, hierarchical clustering identified 2 groups of FLSs, characterized by distinctive gene expression profiles and correlation with the inflammatory profiles of the synovial tissues. The first group correlated with the high-grade inflammation tissue, and exhibited increased expression of a TGF-/activin A-inducible gene profile, which is characteristic of myofibroblasts, a cell type involved in wound healing. The second group correlated with the low-grade inflammation tissue, and showed increased expression of the genes involved in autocrine growth regulation, cell transformation, complement activation, and oxidative stress. Reflecting the gene expression profile, an increased proportion of myofibroblast-like cells in the heterogeneous population of FLSs were immunohistochemically detected in the high-grade inflammation tissue. These data suggest that the inflammatory state of the synovium is

**6.2 Transformed fibroblast-like synoviocyte lines reveal heterogeneity irrespective of** 

The data of Kasperkovitz *et al.* (2005), Galligan *et al*. (2007) and others indicate that combining gene expression profiling with other parameters, such as clinical data or characteristics of FLS lines, constitutes a powerful tool for identifying novel disease-related genes. To identify the cell-intrinsic abnormalities of RA-FLSs, we established transformed cell lines from the synovium of RA or OA cases, by immortalization with SV40 large T Ag (unpublished data of Ishihara *et al.*). Characterization of FLSs from 2 types of arthritis revealed no significant differences in surface molecules, growth rates, patterns of tyrosine-

phosphorylated proteins, or expression of the genes related to inflammation (*IL-1*

deaminase (*AID*) (Igarashi *et al.*, 2010) and the *A20/ABIN* family.

*MMP-1, MMP-3*, etc.). Since the expression levels of these genes vary (ranges exceeding 1,000-fold) among FLS lines from each type of arthritis, we tentatively categorized them into 2 subtypes reflecting resting (r) and active (a) stages, based on the expression levels of *IL-1*

and *MMP-1*. Next, we performed a micro DNA array to obtain the gene expression profiles for 4 representative cell lines, r-OA-FLS, a-OA-FLS, r-RA-FLS, and a-RA-FLS, and obtained 10 gene clusters. Although no disease-specific clusters were obtained, 2 reciprocal, stagespecific clusters were detected, suggesting the validity of our hypothesis for the presence of subtypes in FLSs. Using these data we are presently searching for 2 types of candidate genes; master genes that determine the states of FLSs, and genes that could play a role in the pathophysiology of RA by inference based on our current understanding of FLSs. In the following sections, we will review the potential roles of activation-induced cytidine

**6.3 Ectopic expression of** *AID* **and acquisition of a tumor-like phenotype by fibroblast-**

In addition to the properties described above, the expression of the tumor-suppressor gene *p53* with somatic mutations (Firestein *et al.*, 1997; Inazuka *et al.*, 2000; Kullmann *et al.*, 1999; Reme *et al.*, 1998; Yamanishi *et al.*, 2002), and the down-regulation of the tumor suppressor *PTEN*, a protein phosphatase gene, have been demonstrated in RA-FLSs (Pap *et al.*, 2000b).

**6.3.1** *P53* **mutation in fibroblast-like synoviocytes of RA and** *AID* **expression in** 

*, IL-6,* 
