Repatriation General Hospital, Rheumatology Unit, Adelaide, South Australia, Australia
Correspondence to: M. D. Smith. malcolm.smith{at}rgh.sa.gov.au
Rheumatoid arthritis (RA) is a chronic inflammatory disorder with a heterogeneous course. Early histological features include synovial lining hyperplasia, angiogenesis and mononuclear cell infiltrates [1]. Hyperplastic changes encompass both macrophage-like synovial cells and fibroblast-like synovial cells. The fibroblast-like synoviocytes exhibit pre-neoplastic characteristics, with invasive tendencies and expression of proto-oncogenes [2]. In the later stages of disease, synovial proliferation is reduced and often replaced by connective tissue [3]. One explanation for the synovial proliferation is an imbalance between cell proliferation and apoptosis or programmed cell death. For this reason, the induction of apoptosis has been proposed as a potential therapeutic approach (Table 1).
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Fas ligand (Fas-L) and tumour necrosis factor alpha (TNF) are classic initiators of the type I pathway. They induce clustering of their receptors (Fas, TNF receptor I and II), ligation of cell-membrane-associated death receptors and the recruitment of FADD (Fas-associated protein with death domain) and pro-caspase 8 to the C-terminus of Fas to form DISC (death-inducing signalling complex). Subsequent activation of pro-caspase 8 results in initiation of the cell death pathway. Apoptosis may then proceed down one of two paths: (i) activation of effector caspases 3 and 7 or (ii) through the mitochondrial pathway by activating Bid, a Bcl-2 related protein. Cleaved Bid ultimately results in apoptosis mediated through the type II, mitochondrial or intrinsic pathway [4]. A cellular protein known as Flip (also called Flame, CASH, Casper, Clarp, MRIT, I-Flice or Usurpin) can suppress activation of caspase 8 by competing with pro-caspases 8 and 10 for binding to FADD [5]. Flip expression has been demonstrated in RA synovial tissue, predominantly in the lining layer [6, 7]. Both type I and II synoviocytes express Flip but its role in regulating apoptosis in type II (macrophage lineage) synoviocytes is better understood.
Interest in the role of Fas and Fas-L in autoimmune disease has been stimulated by the discovery that mutations in these proteins can result in proliferative arthritis and lymphadenopathy in murine models and humans [8, 9]. In RA, Fas and Fas-L have been detected in synovial cells and, more controversially, in activated mature T cells obtained at the time of arthroplasty. These cells are susceptible to Fas-mediated apoptosis induced by an anti-Fas monoclonal antibody and this sensitivity has been limited to RA cells when directly compared with osteoarthritis (OA) controls. Much of this work has been undertaken by the authors of the article in this journal [10], although the demonstration of Fas-associated apoptosis in RA fibroblasts has been confirmed by other groups [1116]. The inflammatory milieu of the rheumatoid cells is likely to contribute to the degree of Fas-mediated apoptosis. TNF and IL-1 have been demonstrated to suppress apoptosis in vitro and TNF
has also been demonstrated to induce susceptibility to Fas-mediated apoptosis [10, 17]. Obviously, other factors must influence the effects of these key cytokines. Downstream signalling of Fas is also likely to influence cellular susceptibility to apoptosis, as evidenced by the fact that OA synoviocytes seem resistant to the actions of anti-Fas monoclonal antibody, despite detectable levels of Fas and Fas-L [3, 11]. To date, these mechanisms have been poorly understood, although Okamoto et al. [18] have previously demonstrated selective activation of the Jnk/AP-1 pathway.
The product of the tumour suppressor gene p53 is a transcription factor that plays an important role in cellular growth, DNA repair and apoptosis. In normal cells, p53 is induced in response to genotoxic stresses, genomic threat or DNA damage. Its accumulation, stabilization and activation result in cell-cycle arrest or apoptosis, thereby deleting damaged cells or allowing time for genomic repair. Cells lacking functional p53 are unable to arrest their growth after DNA injury and have reduced sensitivity to apoptotic stimuli. p53 expression has been detected in the synovial lining and sublining of RA and in cultured RA fibroblast-like synoviocytes (FLS). Mutations in p53 have been reported in only about 10% of synovial samples, suggesting that p53 abnormalities may not have a pivotal role in the pathogenesis of RA. It is uncertain whether the remaining p53 in RA synovium is functional or whether it has been suppressed by other means. In normal non-stressed cells, p53 is maintained at very low levels via its interaction with its major regulatory protein MDM2. This protein continuously targets p53 for proteasomal degradation via its capacity to ubiquitinate the p53 molecule. In addition to direct effect on p53 levels, MDM2 has been identified to bind to and inactivate p53 transcriptional targets p21 and BAX, which are p53-regulated proteins that regulate cell-cycle arrest and apoptosis respectively. In addition, Bcl-2 is known to suppress p53 apoptotic function in RA synovium. The relationship between p53 and Fas-mediated apoptosis is controversial, but the most recent reports suggest that there is a strong positive correlation between these two in RA synovium [19]. This has significance with regard to potential therapeutic modalities to alter the degree of apoptosis in RA tissue.
Despite the extensive interest in Fas-mediated apoptosis, its signal transduction pathway within the synovium has not been fully elucidated. The role of p53 in Fas-mediated apoptosis also remains unclear. Itoh et al. [10] have addressed these questions in a study published in the previous issue. Using synovial tissue obtained from 4 RA and 3 OA patients, they established cultured fibroblast cell lines. Fas-mediated apoptosis was enhanced by pre-treatment with TNF. Cultures were then treated with anti-Fas monoclonal antibody in the presence or absence of caspase inhibitors. Cytochrome c release, mitochondrial membrane potential and expression levels of activated caspase 3, caspase 9 and Bid were measured to determine activation of the mitochondrial pathway of apoptosis. Apoptotic cells were identified by DNA fragmentation assay. The role of p53 was assessed using a combination of immunoblotting to demonstrate activated p53, expression of p53-regulated apoptosis-inducing protein (p53AIP1) by real-time polymerase chain reaction (PCR), and the use of RA synovial fibroblasts stably transfected with a dominant negative p53.
Fas ligation was shown to induce Bid cleavage and subsequent changes characteristic of activation of the mitochondrial pathway of apoptosis. Treatment with a caspase-9-specific inhibitor (caspase 9 activity is limited to the mitochondrial pathway of apoptosis) almost completely inhibited Fas-mediated apoptosis. These results show the mitochondrial pathway to be the dominant Fas-mediated apoptotic pathway in cultured RA synovial fibroblasts. In addition, p53 was shown to be phosphorylated and there was subsequent activation of its target protein p53AIP1. Introduction of inactive p53 suppressed Fas-mediated apoptosis. Taken together, these results suggest that p53 activity and Fas-mediated apoptosis in RA synovial fibroblasts are closely linked.
While the findings of Itoh et al. [10] are elegantly and convincingly demonstrated, the application of these results to clinical practice and development of therapeutics must be interpreted with care. The fibroblast cultures obtained for this study were from patients at the time of joint replacement surgery. Therefore, although details of disease activity were not provided, they are likely to be from patients with long-standing and potentially less active disease. The clinical and histological features of RA alter significantly with time and synovial proliferation in the later stages of disease is less evident, often replaced by connective tissue.
Catrina et al. [20] studied 11 patients with long-standing RA and 8 with early RA and clearly showed that apoptosis was significantly higher in patients with long-standing RA. In addition, macrophage numbers were higher in the group with early disease and cytokine expression and T-cell score also varied significantly. At the clinical level, this means that the patients we wish to target, i.e. those with early, active RA who have not yet developed extensive erosive changes, are likely to have a very different histological and clinical picture from patients in Itoh's group. In early disease, synovial proliferation is much more prominent and macrophages are the major cell lineage responsible for synovial lining hyperplasia. In addition, IL-1 and TNF are detected in large quantities and these have already been shown to inhibit Fas-mediated apoptosis in vitro [17]. Before we can effectively speculate on the therapeutic role of the mitochondrial pathway in Fas-mediated apoptosis, its mechanisms must be elucidated in macrophages and fibroblasts obtained from RA patients with early disease, preferably in the presence of cytokine profiles that mimic those seen in the inflamed joint. In addition, the complexity of pathways regulating both apoptosis and cell-cycle activity, and the modulation of these pathways in RA, needs to be understood. Only then can we draw useful interpretations of the therapeutic opportunities elicited from the Fas mediated pathway of apoptosis.
Itoh et al. have demonstrated an effective method of elucidating the downstream signalling of Fas. Their findings provide further insight into the later stage inflammatory changes of RA but any conclusions about the therapeutic potential of agents targeting Fas or the mitochondrial pathway of apoptosis in today's rheumatological practice must be delayed until these studies have been extended to those with early, active disease.
The authors have declared no conflicts of interest.
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