**Molecular Mechanisms of Rheumatoid Arthritis Revealed by Categorizing Subtypes of Fibroblast-Like Synoviocytes**

Katsuhiko Ishihara and Hideya Igarashi *Kawasaki Medical School Japan* 

#### **1. Introduction**

72 Rheumatoid Arthritis – Etiology, Consequences and Co-Morbidities

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The immune system is a highly organized defense system, which recognizes invading microorganisms and aims to exclude them. In order to do this effectively and safely, the immune system must distinguish between self- and non-self-antigens, and be tolerant of self-antigens. Autoimmune diseases develop through the breakdown of self-tolerance, as a result of immune deregulation. This is caused by the combined influence of genetic and environmental factors, including infectious microorganisms. Rheumatoid arthritis (RA) is a systemic autoimmune disease, characterized by synovial hyperplasia leading to the destruction of bones and joints. This severely impairs the life of patients. RA is a relatively common autoimmune disease, occurring in approximately 1% of the population. However, its etiology and pathophysiology are not completely understood. The incidence of RA is correlated with certain human leukocyte antigen (HLA)-DR haplotypes, and the production of autoantibodies such as rheumatoid factor and anticitrullinated protein autoantibody. Thus, the involvement of the deregulated immune system is strongly implicated. Various molecules, including type II collagen, gp39, citrullinated peptides, and glucose-6 phosphoisomerase, have been reported as potential pathogenic autoantigens. However, their involvement explains only a proportion of RA cases. Autoantigens are abundant in the body and, theoretically, the immune response to them continues indefinitely. Thus, systemic autoimmune diseases exhibit the characteristics of chronic inflammation.

In the pathological condition of RA, the joints are infiltrated with T cells, B cells, macrophages, and plasma cells, all of which are characteristic chronic inflammation cells driven by the immune system. Recently, Th17, a novel helper T-cell subset producing interleukin (IL)-17, has been recognized as a pivotal player in the local inflammation driven by acquired immunity. In addition to immune-competent cells, there is accumulating evidence for abnormalities in non-hematopoietic cells, especially fibroblast-like synoviocytes (FLSs) (Bartok & Firestein, 2010; Firestein, 2009; Mor *et al.*, 2005; Pap & Gay, 2009). The cartilage and bone are destroyed by the invasion of pannus, which is formed from proliferating FLSs and multi-nucleated osteoclasts. Osteoclasts are specialized to resolve bone, and play a major role in bone destruction in RA. However, there is strong evidence that FLSs themselves are aggressive enough to destroy bone. When cultured FLSs derived from RA or osteoarthritis (OA) were co-implanted with human cartilage under the renal

Molecular Mechanisms of Rheumatoid Arthritis

invasiveness of FLSs to the cartilage (Pap *et al.*, 2004).

**2.2.2 Infiltration with inflammatory cells** 

**2.2.1 Hyperplasia** 

Revealed by Categorizing Subtypes of Fibroblast-Like Synoviocytes 75

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 cell-

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

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.

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.

autonomous pathway results from the cell-intrinsic changes of FLSs themselves.

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 destroying bone and joints.

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 as revealed by our subtyping studies.
