Apoptosis in rheumatoid arthritis: friend or foe

Rheum Dis Clin N Am 30 (2004) 603 – 625

Apoptosis in rheumatoid arthritis: friend or foe Hongtao Liu, MD, PhD, Richard M. Pope, MD* Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611, USA

Apoptosis is an evolutionarily conserved, multistep process by which the cell undergoes an orderly demise. In contrast to cells undergoing necrosis, the contents of those undergoing apoptosis are not released, and the apoptotic cell is cleared by phagocytosis. Although necrotic cell death promotes inflammation, apoptotic cell death, following phagocytosis, results in the release of anti-inflammatory mediators. Apoptosis may be initiated through death receptor (DR)- or mitochondria-dependent pathways (Fig. 1).

The death receptor pathway Apoptosis or programmed cell death may be initiated through ligation of specific DRs, including tumor necrosis factor receptor 1 (TNFR1), Fas (CD95, apoptis antigen [APO]1), TNF-related apoptosis inducing ligand-receptor 1 (TRAIL-R1; death receptor [DR]4), TRAIL-R2 (DR5), and TRAMP (TNFreceptor related apoptosis-mediated protein; DR3, APO-3). Fas is the most potent, and best characterized, DR. The execution phase initiated by Fas ligation is associated with the activation of caspase 8, leading ultimately to the activation of caspase 3. Ligation of Fas by Fas ligand or agonistic anti-Fas antibody results in oligomerization and aggregation of the cytoplasmic tail of Fas, which contains one death domain (DD). Upon Fas aggregation, the adapter molecule Fasassociated via death domain (FADD) binds to Fas by means of their DDs and recruits caspase 8 to the complex. Caspase 8 (also called FLICE) binds to FADD through their death effector domains (DEDs). The complex of Fas, FADD, and caspase 8 is called the death-inducing signal complex (DISC). The FLICE inhibitory protein (FLIP) is capable of inhibiting the binding of caspase 8 to FADD, preventing the autolytic cleavage and activation of caspase 8. This work was supported by the National Institutes of Health (Grants AR048269 and AR049217). * Corresponding author. E-mail address: [email protected] (R.M. Pope). 0889-857X/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.rdc.2004.04.010

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The mitochondria-dependent pathway Apoptosis may also be initiated through a mitochondria-dependent pathway, which is independent of caspase activation and can be induced by DNA damage, cytotoxic drugs, or by the forced reduction of antiapoptotic Bcl-2 family members. Mitochondrial damage mediates apoptosis through the disruption of mitochondrial transmembrane potential (Dym), which results in the release of cytochrome c. The association of cytochrome c with adenosine triphosphate (ATP) induces apoptotic protease-activating factor 1 (Apaf-1) to bind and activate caspase 9, which then activates the effector caspases 3 and 7. The B-cell leukemia/lymphoma-2 (Bcl-2) family consists of multiple members that contain Bcl-2 homology domains and exhibit either pro- or antiapoptotic effects. The activation of proapoptotic proteins (eg, Bax, Bik, Bak, or Bad) may disrupt Dym, even without caspase activation. By protecting mitochondrial integrity, the antiapoptotic proteins (eg, Bcl-2, Bcl-xL, A1/Bfl-1, and Mcl-1) protect against apoptosis initiated by, or dependent on, loss of Dym. Thus, the Bcl-2 family

Fig. 1. Apoptosis induction and suppression in RA synovial cells. Apoptosis may be induced by Fas – Fas ligand interaction in the absence of FLIP expression. In some cell types, especially macrophages, growth factor may activate the PI3K pathway, which may be suppressed by PTEN. The phosphorylation and activation of Akt by PI3K regulates Mcl-1 expression. The activation of NFkB contributes to the regulation of the expression of another Bcl-2 family member, A1. The forced down-regulation of Bcl-2 and Mcl-1 or A1 resulted in apoptosis in fibroblasts or macrophages, respectively. Cytokines inside the rheumatoid joint may also activate the STAT3 pathway, which contributes to the maintenance of survival of RA synovial fibroblasts. Mutated p53 in the rheumatoid joint may also suppress the induction of apoptosis induced by DNA damage. Up-regulation of SUMO-1 in the joint might inhibit apoptosis by blocking the signal transduction through Fas and TNFR ligation.

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regulates apoptosis mediated through the mitochondria-dependent pathway. In certain cell types, communication between the death receptor and mitochondrial pathway may be induced by the caspase-8 – mediated cleavage of the proapoptotic molecule Bid, which triggers the loss of Dym and the release of cytochrome c through the action of the proapoptotic molecules Bax and Bak (reviewed in reference [1]).

How frequently is apoptosis detected in active rheumatoid arthritis? Apoptosis is a form of programmed cell death that is essential for normal development, cancer prevention, and the resolution of acute inflammation. Apoptosis also has been implicated in the development of autoimmunity. Therefore, rheumatoid arthritis (RA) is a natural target for study to determine if there is too much or too little apoptosis in RA, and if it even matters? There are various tests to identify apoptosis and distinguish it from other forms of cell death, such as necrosis and autophagy. Apoptosis is an orderly programmed cell death that does not result in the release of the intracellular contents and the rapid clearance of the dead cell. The cell dies quietly, without commotion. A hallmark of apoptosis is the degradation of DNA by cleavage between nucleosomes. This characteristic can be identified in several ways, including electrophoresis of cell lysates identifying the nucleosomal ladder, an ELISA assay measuring DNA fragments, or by flow cytometry quantitating the percentage of cells with subdiploid DNA, which occurs because of DNA fragmentation. To detect DNA strand breaks in tissues, however, a technique called terminal-deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) is commonly used. Although this technique is very sensitive to the conditions used, early studies in RA identified an increased frequency of TUNEL-positive or in situ end-labeling using Klenow polymerase (ISEL)– positive cells in the joints of patients with RA, compared with patients with osteoarthritis [2 –6]. These observations were initially interpreted as evidence of ongoing apoptosis. Not all investigators found evidence of TUNEL-positive cells in RA synovial tissue, however [2]. Although this finding might have been caused by the differences in patients studied, it also may have been caused by technical differences, because incubation for too long with the enzymes used to perform TUNEL or ISEL assays can generate positive results. At least three groups have examined RA synovial tissue by electron microscopy, the gold standard for defining apoptosis in tissues, because it can accurately identify changes in nuclear morphology that define apoptosis, without the use of an enzyme that might confound the results. None of these studies could detect a significant increase of apoptotic cells in the joints of patients with RA [2,4,7], even when TUNELpositive cells were identified within the synovial tissues. Additional observations support the lack of apoptosis in RA synovial tissue. When cells undergo apoptosis, they are phagocytosed by macrophages, which are abundant in the joints of patients with RA. Of the many studies published examining RA synovial

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tissue, to the current authors’ knowledge, none has identified phagocytosed cells within macrophages in RA synovial tissues. In contrast, in an animal model of RA, when apoptosis of neutrophils occurred during the course of the disease, phagocytosed cells within macrophages were readily identified [8]. The basis for the TUNEL-positive cells in RA synovial tissues and their significance for the pathogenesis of RA remain to be clarified. One explanation is that oxidant damage, caused by reactive oxygen (ROS) and nitrogen species produced as a result of the chronic inflammation, may result in DNA damage [9 –11], which may then be detected as TUNEL-positive cells. In support of this possibility, evidence of oxidant damage, identified as mutations of the proapoptotic molecule p53 specifically caused by oxidant injury, has been identified in the joints of patients with RA [12]. In addition, nitrosylated proteins, resulting from reactive nitrogen species, are enriched in the joints of patients with RA [9] . Damaged DNA is a potent stimulus for the induction of apoptosis. Therefore, the presence of damaged DNA, in the absence of apoptosis, would suggest that the environment of the RA joint is strongly directed toward protection against apoptosis. Furthermore, recent studies have characterized various mechanisms in the RA joint that protect against the induction of apoptosis, and these are reviewed later.

Role of apoptosis in the resolution of acute inflammation The accumulation of neutrophils is the hallmark of acute inflammation. Apoptosis of neutrophils is important in the resolution of acute inflammation. In response to a localized stimulus, such as a microbial infection, neutrophils accumulate. The phagocytosis of the pathogen induces an apoptosis differentiation program in neutrophils that results in the up-regulation of proapoptotic molecules, such as Bax, whereas some genes encoding antiapoptotic genes are down-regulated [13]. The induction of this program results in neutrophil apoptosis. The regulation of the apoptosis differentiation program in neutrophils is mediated in part by the generation of ROS, which occurs in response to pathogen phagocytosis. Cells undergoing apoptosis or programmed cell death proceed in an orderly fashion that does not result in the release of the cellular contents, including the organism that is being killed within the cell. Another form of cell death, necrosis, which can also follow apoptosis if the dying cells are not cleared by phagocytosis, results in the release of the cell contents. This process does not result in the death of an ingested pathogen, and promotes inflammation. Cells undergoing apoptosis express several receptors that are recognized by macrophages, a process that causes the cells undergoing apoptosis to become phagocytosed and further processed. Among the receptors on macrophages that identify apoptotic cells as altered and in need of removal are the scavenger receptor class A (SRA) and class B (CD36) receptors, which recognize oxidized low density lipoproteins (LDL)-like sites, and receptors recognizing phosphatidylserine, which are exposed on apoptotic cells [14]. Once ingested,

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apoptotic cells are capable of inducing the production of transforming growth factor b1 (TGF-b1) by the macrophages. TGF-b1 is capable of suppressing macrophage activation, such as the secretion of tumor necrosis factor a (TNF-a) [15]. In support of the relevance of the anti-inflammatory effects of apoptotic cells, the intra-articular injection of apoptotic leukocytes suppressed immune complex (IC) –mediated arthritis in experimental animals [16]. Therefore, apoptosis promotes the resolution of an acute inflammatory response not only by reduction in the number of neutrophils but also by the release of anti-inflammatory mediators from macrophages.

Apoptosis as a mechanism of action of current therapy in rheumatoid arthritis Several effective therapies currently in use to treat patients with RA may work, at least in part, through the induction of apoptosis (Table 1). Methotrexate Various mechanisms for the action of methotrexate have been identified, including the induction of apoptosis. Methotrexate binds with high affinity to dihydrofolate reductase, interfering with the purine nucleotide synthesis pathway in rapidly dividing cells. This process results in the release of adenosine, which binds adenosine cell surface receptors, and induces the suppression of cytokines and other mediators of inflammation (reviewed in reference [17]). The in vitro exposure to methotrexate sensitized proliferating lymphocytes to undergo apoptotic cell death that was not dependent on Fas– Fas ligand interactions [17]. Furthermore, T cells removed from patients with RA who were treated with methotrexate demonstrated increased apoptosis following stimulation. This effect of methotrexate was independent of adenosine. Methotrexate was also effective at inducing apoptosis of proliferating monocytic cells in vitro [18]. In contrast, methotrexate failed to induce apoptosis of nonproliferating synovial macrophages from the RA joint [18,19]. Methotrexate also induced apoptosis of proliferating RA synovial fibroblasts [20] and induced TUNEL-positive staining, which sugTable 1 Induction of apoptosis by current pharmaceutic therapies used in patients with rheumatoid arthritis Drug

Induction of apoptosis

Methotrexate

Proliferating lymphocytes, especially T cells Proliferating monocytic THP-1 cells Proliferating RA synovial fibroblasts Nonproliferating macrophages Peripheral blood T cells Circulating monocytes Lamina propria lymphocytes in Crohn’s disease

Sulfasalasine Infliximab

Abbreviations: RA, rheumatoid arthritis; THP-1, human monocytic leukemia cell line.

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gested apoptosis in RA synovial tissue implanted in severe combined immunodeficient (SCID) mice that were treated with methotrexate in vivo [20]. These observations suggest that methotrexate, at the low doses used to treat patients with RA, may work, in part, by the induction of apoptosis, particularly of proliferating cells. Sulfasalasine In contrast to methotrexate, sulfasalasine may induce apoptosis of certain nonproliferating cells. When added to macrophages, sulfasalasine suppresses lipopolysaccharide-induced nuclear factor kB (NF-kB) activation and the secretion of TNF-a, whereas methotrexate is ineffective [19]. In vivo and in vitro studies demonstrated that these effects of sulfasalasine are mediated by caspasedependent apoptosis, at concentrations achieved in patients with RA [19]. Because macrophages depend on the constitutive activation of NF-kB for survival [21], it is possible that the inhibition of NF-kB by sulfasalasine induces apoptosis, which results in suppression of the secretion of cytokines, such as TNF-a. T lymphocytes also depend on a basal activation of NF-kB for survival. Sulfasalasine induces apoptosis of peripheral blood T cells by a mechanism that depends on the loss of mitochondrial integrity [22]. Similarly, following the inhibition of NF-kB, macrophage apoptosis is induced, because of the suppression of the Bcl-2 family member A1 [21] . In contrast, in nonhematopoetic cells, such as normal or RA synovial fibroblasts, neither sulfasalasine nor direct inhibition of NF-kB activation results in apoptosis [21,22]. These observations suggest that, although both methotrexate and sulfasalasine may induce apoptosis, they target different cell types and use different mechanisms. Anti –TNF-a antibody Infliximab, a monoclonal antibody directed against TNF-a, is commonly used to treat patients with RA and those with Crohn’s colitis. A recent study examined the synovial tissue of patients with RA before and after therapy with infliximab [7]. A significant reduction in the number of macrophages in the synovial lining was observed 48 hours after treatment. In contrast, no reduction of B cells, T cells, or synovial fibroblasts was observed. Although TUNEL-positive cells were present in the synovial tissues, no increase of DNA fragmentation identified by TUNEL was observed in response to infliximab. Furthermore, electron microscopic analysis demonstrated that apoptosis was virtually absent. This study not only documents the absence of apoptosis, it also supports the fact that TUNEL is not a reliable measure of apoptosis in RA synovial tissue. What then might account for the reduction of macrophages in the rheumatoid joint following therapy? Studies of patients with Crohn’s disease demonstrated the induction of caspase-dependent apoptosis of circulating monocytes within 4 hours of infusion [23]. Even though apoptosis was independent of the Fas– Fas ligand pathway, caspase 8 was activated, suggesting the involvement of a DR

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pathway, such as TNR1, TRAIL-R1, or TRAIL-R2. Monocytes do not express high levels of FLIP, which protects against DR-mediated apoptosis, whereas normal macrophages and those from the joints of patients with RA express high levels of FLIP [24,25]. Therefore, it is possible that infliximab induces DRmediated apoptosis of monocytes, which reduces the numbers of cells capable of migrating into the actively inflamed joint. In contrast to the lack of apoptosis locally in the RA joint, in patients with Crohn’s disease, infliximab induces caspase-dependent apoptosis of lamina propria lymphocytes, and the effect is dose dependent and sustained [26,27]. Apoptosis of lamina propria T cells was not observed following treatment with etanercept, possibly accounting for the difference in the effectiveness of these agents in Crohn’s disease [27]. These observations suggest that the effects of treatment with anti-TNF agents may in part be from the induction of apoptosis.

The mechanisms of resistance to apoptosis—road to develop future therapy in rheumatoid arthritis The mechanisms that contribute to the persistence of chronic inflammation, such as that observed in the joints of patients with RA, are poorly characterized. The proliferation of synovial fibroblasts and resistance of synovial macrophages, fibroblasts, lymphocytes, neutrophils, and osteoclasts to apoptosis may be contributing factors (reviewed in reference [28]). This section reviews the potential mechanisms involved in the resistance to apoptosis in the different cell types obtained from the joints of patients with RA (Table 2). Synovial T cells RA is characterized by T-lymphocyte accumulation within the inflamed joint. The lack of T-cell apoptosis in the joint may be one of the contributing factors for the persistence of these cells [29]. The lack of apoptosis of T cells in the rheumatoid joint may in part be from the cognate interaction between synovial fibroblasts and T cells, which results in the enhanced expression of the antiapoptotic molecule Bcl-xL in T cells [29]. In addition, stromal cell –derived factor 1 (SDF-1), secreted by fibroblast-like synoviocytes in the rheumatoid joint, binds to CXC chemokine receptor 4 (CXC CR4) on T cells, thus inhibiting apoptosis by activation-induced cell death, which occurs following T-cell activation [30]. SDF-1 is highly expressed in RA synovial tissues, compared with those from patients with osteoarthritis. In addition, CXC CR4 on T cells is up-regulated by interleukin 15 (IL-15), which is also highly expressed in the rheumatoid joint [31]. Further supporting the contribution of T cell resistance to apoptosis in the pathogenesis of RA, a subset of CD4+ T cells deficient in CD28 expression, CD4+ CD28
T cells, is frequently detected in peripheral blood of patients with RA. These T cells frequently undergo clonal expansion, persist in vivo for long periods, and are characterized by autoreactivity [32]. This T-cell subset expresses

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Table 2 Mechanisms contributing to the resistance to apoptosis in the joints of patients with rheumatoid arthritis Cell type

Mechanisms contributing the resistance of apoptosis

Synovial T cells

" Bcl-xL by interaction with synovial fibroblasts " Bcl-2 in CD4+ CD28
T cells " FLIP in anti-proteoglycan-reactive CD4+ T cells Failure of deletion of autoreactive T cells caused by mutation of ZAP-70 or TRAIL pathway " FLIP " Bcl-2 family members regulated by NFkB activation, possibly A1 " Mcl-1, possibly regulated by PI3K/Akt-1 activation " Mcl-1 " A1 Fas-FasL – mediated apoptosis " FLIP TNF-a – mediated apoptosis " FLIP " XIAP " Activation of PI3K/Akt-1 " Activation of STAT3 " Sentrin/SUMO-1 " Synoviolin/Hrd1 " Bcl-2 family member " Bcl-2 " Mcl-1 Mutation of p53 Activation of NF-kB by RANK – RANKL interaction plus TNF-a

Synovial macrophages

Neutrophils Synovial fibroblasts

Osteoclasts

increased Bcl-2 compared with CD4+ CD28+ T cells, rendering them resistant to activation-induced cell death or apoptosis induced by IL-2 withdrawn [33]. Data from animal models of inflammatory arthritis also support the role of T-cell resistance to apoptosis in disease pathogenesis. CD4+ T cells from mice with proteoglycan-induced arthritis display an activated phenotype. Apoptosis induced by activation-induced cell death is defective in these anti-proteoglycan-reactive CD4+ T cells because of the aberrantly high expression of the antiapoptotic molecule FLIP [34], which made these T cells resistant to the Fas-induced apoptosis. Together, these observations suggest that the failure of deletion of autoreactive T cells because of apoptosis resistance, which may be mediated through DR or mitochondrial pathways, may contribute to the pathogenesis of RA. Studies in animal models also suggest that the regulation of T cell apoptosis may be important in preventing the development of arthritis. The DR TRAIL may protect against the development of arthritis by deleting autoreactive T cells. Data from TRAIL-deficient mice demonstrate that TRAIL controls negative selection of T cells in the thymus [35]. TRAIL-deficient mice demonstrate increased sensitivity to developing collagen-induced arthritis, possibly because of their failure to delete autoreactive T cells or to properly silence activated T cells [35].

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In addition, defects in pathways that control T cell tolerance may cause or promote the development of arthritis. Mice with a spontaneous point mutation of ZAP-70, a key signal transduction molecule in T cells, display enhanced thresholds for selection of T cells in the thymus, which lead to the failure to induce apoptosis and delete autoimmune T cells [36]. Mice with this ZAP-70 point mutation develop spontaneous arthritis resembling RA [36]. These studies suggest that failure to delete activated T cells, which may be caused by resistance to apoptosis, may be important in the pathogenesis of RA, contributing to the induction or persistence of the inflammation. Synovial macrophages Synovial tissue macrophages are critical to the destruction of cartilage and bone in RA (reviewed in reference [37]). Synovial macrophages express high levels of mediators of inflammation and joint destruction, including cytokines, chemokines, and matrix metalloproteinases. Various mechanisms have been characterized that may contribute to the resistance of these macrophages to apoptosis, which might be exploited to develop new therapies. The Fas death receptor pathway and the role of FLIP Increased concentrations of soluble Fas (sFas) and sFas ligand have been detected in the serum and synovial fluid from patients with RA [38 – 40]. sFas protected cells from Fas-mediated apoptosis [41], and 30% (10 of 33 patients) of patients with large, granular lymphocyte leukemia with high-level serum sFas developed RA [42]. Together, these observations suggest that inhibition of Fasmediated apoptosis by sFas or sFas ligand might contribute to the pathogenesis of RA. In contrast to circulating monocytes, differentiated macrophages, as found in the rheumatoid joint, are resistant to Fas-mediated apoptosis, even though Fas and Fas ligand are strongly expressed [24]. FLIP, which is up-regulated during macrophage differentiation, confers macrophage resistance to Fas-mediated apoptosis in vitro [24] and in vivo [43]. In certain areas, particularly in the synovial lining, macrophages and fibroblasts are in intimate contact with one another, yet there is little evidence for ongoing apoptosis (reviewed in reference [1]). The current authors recently have documented a potential role of FLIP in RA [44]. FLIP, an inhibitor of DR-mediated caspase-8 activation, is more highly expressed in RA synovial tissue, compared with osteoarthritis, particularly in the lining [44]. In these experiments, FLIP was identified by two-color immunofluorescence in macrophages and synovial fibroblasts. FLIP expression was also detected in RA synovial tissue by in situ hybridization, which demonstrated that FLIP mRNA is expressed in lining and sublining regions, and at the site of cartilage invasion and bone destruction [45]. In the adjuvant-induced arthritis animal model of RA, FLIP was also highly expressed at sites of erosion and in the pannus and in areas of the synovial tissue where apoptosis was not observed [8]. The expression of FLIP was increased in CD14+ monocytes/macrophages

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obtained from the joints of patients with RA, compared with RA peripheral blood CD14+ cells [44]. The potential importance of FLIP in macrophages in the RA joint is supported by the recent findings that FLIP is highly expressed in early RA [46]. This finding was associated with an increased number of macrophages and a low level of apoptosis. In patients with longstanding RA, who had been treated with various medications, however, there was a marked reduction of FLIP, increased apoptosis, and a reduced detection of macrophages [46]. These observations suggest the possibility that therapy results in reduced FLIP, which leads to increased macrophage apoptosis. In summary, FLIP is important for protecting RA synovial macrophages from DR-mediated apoptosis. Because Fas- and Fas ligand – expressing cells are in intimate contact in the synovial lining and pannus, the therapeutic reduction of FLIP is an attractive target for future treatment. NFjB activation and TNF-a Although NF-kB may be activated by various mechanisms, initiation through TNFR and IL-1 receptor type 1, or IL-1R1, is highly relevant to RA. NF-kB may be activated following TNFR1 ligation and mediated by the recruitment of TRADD (TNFR1-associated DD protein) and the subsequent binding of receptorinteracting protein (RIP) and TNFR-associated factor 2 (TRAF2) or TRAF5. TRAF2 or TRAF5 is necessary for recruiting IkB kinase (IKK) to the complex [47], and RIP is essential for TNFR1-induced NF-kB activation [48], which is mediated through the phosphorylation of IKK [47]. The phosphorylation of IkBa by IKK results in the degradation of IkBa, the nuclear localization of complexes containing NF-kB p65/p50, and the activation of NF-kB –regulated genes. NFkB activation has been readily detected in synovial tissues from patients with RA and from mice with experimentally induced collagen-induced arthritis [49,50]. Data from experimental animals suggest that NF-kB activation is important in the collagen-induced arthritis model of RA [49,50]. One study reported that inhibition of NF-kB activation resulted in apoptosis that was associated with clinical improvements, although the cell types were not identified [51]. Although monocytes in the circulation display minimal activation of NF-kB, NF-kB p65/p50 heterodimers are constitutively activated in normal, in vitro– differentiated macrophages, as determined by electrophoretic mobility shift assay [21]. In studies on the in vitro –differentiated primary human macrophages, the activation of NF-kB contributed to the survival of differentiated macrophages. The current authors demonstrated that inhibition of NF-kB activation in in vitro – differentiated primary human macrophages by various methods, including a superrepressor IkBa, in the absence of an additional death-inducing signal, results in macrophage apoptosis [21]. The apoptosis resulted from the rapid reduction of the Bcl-2 family member A1. A unique characteristic of the mechanism responsible for the induction of apoptosis in this system is that, even though the macrophages experience a loss of mitochondrial transmembrane potential and activation of caspase 9, no activation of caspase 3 was observed [21].

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These observations are relevant to RA because NF-kB is activated within the synovial tissue of these patients, primarily in macrophages, as determined by the nuclear localization of NF-kB [52]. Furthermore, with macrophages isolated from the joints of patients with RA, the inhibition of the constitutive activation of NF-kB using the super-repressor IkBa resulted in apoptosis (L.J. Pagliari, R.M. Pope, unpublished observations, 2001). Therefore, the activation of NF-kB locally within the rheumatoid joint, in addition to promoting the expression of inflammatory mediators [53], may also protect macrophages from apoptosis. When NF-kB is inhibited, the addition of TNF-a to normal macrophages results in a marked increase in apoptotic cell death [54]. The apoptosis observed in macrophages demonstrates several unique characteristics. Unexpectedly, neither cell death nor apoptosis depend on caspase-8 activation. Furthermore, inhibition of caspase activation using the pan-caspase inhibitor zVAD.fmk converts apoptotic cell death to necrotic cell death. The inhibition of caspase activation does not protect the mitochondria from the mitochondrial membrane permeability, which is responsible for the cell death. It is possible that in the rheumatoid joint, where TNF-a is already present, the inhibition of NF-kB may result in TNF-a– induced apoptosis of macrophages. The PI3K/Akt-1 pathway The PI3K/Akt-1 pathway is also important for macrophage survival. Akt-1 is constitutively activated in normal, in vitro– differentiated macrophages [55]. The suppression of Akt-1 using a PI3K inhibitor or a dominant-negative Akt-1, in the absence of an additional death-inducing signal, induced apoptosis that was mediated through the loss of mitochondrial integrity from the reduction of the Bcl-2 family member Mcl-1 [55]. In support of the importance of this pathway in the rheumatoid joint, apoptosis was induced in macrophages obtained from the synovial fluid of patients with RA following inhibition of the PI3K pathway [44]. These observations suggest that the PI3K/Akt-1/Mcl-1 pathway may be important for the survival of macrophages in the rheumatoid joint. Therapeutic induction of macrophage apoptosis Macrophage apoptosis has been used therapeutically. Etoposide, a topoisomerase-2 antagonist, selectively induces monocyte apoptosis in mice and rabbits [56,57]. Mice given etoposide before immunization with collagen do not develop collagen-induced arthritis, and mice treated with etoposide at the onset of clinical arthritis show reduced frequency of their disease by 50% [58]. In addition, the collagen-specific B-cell responses in the draining lymph nodes were highly suppressed. Another interesting approach to target macrophages is to conjugate a toxin to an anti-CD64 antibody, directed against the high-affinity receptor for IgG (FcgR1) [59]. The fusion protein selectively eliminates macrophages in the synovial fluid from patients with RA by induction of apoptosis. This molecule also inhibits TNF-a and IL-1b production and the cartilage-degrading activity of RA synovial tissue explants [59]. These observations suggest that targeting

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synovial tissue macrophages for apoptosis is feasible and may be a realistic therapeutic goal. Neutrophils The resistance of neutrophils to apoptosis might also contribute to the joint inflammation in the rheumatoid joint. Neutrophil apoptosis is a well-characterized mechanism for the resolution of acute inflammation, and neutrophils are abundant in the synovial fluids of patients with RA, suggesting that resistance to apoptosis may promote chronic inflammation. TNF-a and the protein synthesis inhibitor cycloheximide induced apoptosis of circulating neutrophils [60]. In contrast, these mediators fail to induce significant apoptosis in neutrophils isolated from the synovial fluid of patients with RA. The resistance to TNF-a –induced apoptosis is because of the enhanced activation of NF-kB [60]. It has been demonstrated that expression of antiapoptotic molecules, especially Mcl-1 and A1, is important in protecting neutrophils from apoptosis (reviewed in reference [61]). Although not examined in the rheumatoid joint, Mcl-1 expression level correlates with the reduced apoptosis in neutrophils obtained from patients with sepsis [62]. Neutrophils are plentiful in the joints of patients with RA, and they contribute to disease pathogenesis through the release of inflammatory mediators and degradative enzymes. Similar to the results observed with pathogenic microorganisms, ICs, as occur in the joints of patients with RA, are capable of inducing ROS and neutrophil apoptosis. Synovial fluids from patients with RA, however, despite the presence of ICs, inhibit neutrophil apoptosis, which may contribute to the persistence of neutrophils in the joints of patients who are not adequately treated [63]. These data suggest that apoptosis resistance by neutrophils might contribute to the chronic inflammation observed in the rheumatoid joint. Synovial fibroblasts RA synovial fibroblasts are present in the lining, the pannus, and sublining. They produce cytokines such as the following: IL-6; matrix metalloproteinases, including collagenase and stromelysin; and chemokines, such as IL-8, in response to the TNF-a and IL-1b produced by adjacent macrophages. In RA, synovial fibroblasts also display a transformed phenotype, capable of invading and destroying adjacent bone and cartilage (reviewed in reference [37]). Multiple mechanisms contribute to the resistance of RA synovial fibroblasts to apoptosis (see Fig. 1). Fas death receptor– mediated apoptosis FLIP. RA synovial fibroblasts express Fas receptor, but not Fas ligand [3,64]. Despite differences observed concerning apoptosis induced by agonistic anti-Fas antibody (CH-11) [3,64 –66], RA synovial fibroblasts are sensitive to apoptosis induced when cell surface Fas ligand is overexpressed using an adenoviral vector [66,67]. Nonetheless, in vivo, synovial fibroblast apoptosis is rarely observed. Recent observations suggest that FLICE inhibitory protein (FLIP) [65] or sentrin

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[68] may protect RA synovial fibroblasts from Fas-mediated apoptosis in vivo, because Fas receptor – positive fibroblasts are in intimate contact with Fas ligand – expressing macrophages in vivo in the RA synovial lining. Real-time polymerase chain reaction (PCR) demonstrates that FLIP mRNA is expressed in RA synovial fibroblasts, and its expression is increased by TNF-a [45]. The role of FLIP in RA synovial fibroblasts in the protection against DR -mediated apoptosis has yet to be published. Studies on human dermal fibroblasts, however, support the role of FLIP in the protection of fibroblasts from Fas-induced apoptosis [69]. The down-regulation of FLIP by cycloheximide or FLIP antisense oligonucleotides sensitizes the fibroblasts to Fas-induced apoptosis. Furthermore, the expression of FLIP is up-regulated by TNF-a and Th1 cytokines, which are abundant in the rheumatoid joint [69]. These findings suggest that up-regulation of FLIP may contribute to the resistance to cell death, which permits the persistent proliferation of synovial fibroblasts, and the formation of the pannus within the rheumatoid joint. Fas. The Fas DR has been examined as a potential therapeutic target. The ectopic expression of Fas ligand using an adenoviral vector results in extensive apoptosis of RA synovial fibroblasts in vitro and RA synovial cells in vivo [67,70]. Therapy with Fas ligand also ameliorates collagen-induced arthritis in DBA/1 mice [67,70]. In addition, in an ex vivo model, the injection of Fas ligand – transfected fibroblasts locally into joints eliminated synoviocytes and mononuclear cells from RA synovial tissue implanted in SCID mice [71]. Gene transfer of FADD, the downstream adopter molecule mediating Fas activation, also induces apoptosis of synoviocytes both in vitro and in vivo in the arthritis model of human rheumatoid synovium engrafted into the SCID mouse [72], suggesting that enhancement of Fas-mediated apoptosis might be effective in the treatment of RA. The direct therapeutic use of this pathway, however, may be limited by the increased expression of antiapoptotic molecules, such as FLIP, and because the Fas ligand therapy may induce acute inflammation. TRAIL and TRAIL receptors. Studies in experimental models of RA suggest that TRAIL and its receptors might contribute to disease and might be effective therapeutic targets. Recent studies that examined the expression of TRAIL and its receptors on the human synovial fibroblasts have been controversial, however. Using flow cytometry, data from the current authors’ laboratory did not show TRAIL and TRAIL receptors on the cultured synovial fibroblasts, whereas the same monoclonal antibodies worked well in the positive cell line [73]. Consistent with the lack of TRAIL receptors, the ectopic expression of human TRAIL failed to induce apoptosis of human RA synovial fibroblasts [73]. In addition, the authors did not find TRAIL or the TRAIL death-inducing receptors on macrophages from the joints of patients with RA. In contrast, a decoy TRAIL receptor was found on these synovial fluid macrophages. In contrast to the authors’ observations, another group, using a different antibody, detected high levels of TRAIL DR 2 (DR5) in RA synovial tissue and synovial fibroblasts [74]. This anti-

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DR5 antibody also induced apoptosis in a synovial fibroblast cell line [74]. Further studies are needed to define the reason for the observed differences and to determine if TRAIL receptors might be good therapeutic targets in patients with RA. NFjB- and TNF-a– induced apoptosis RA synovial fibroblasts are not sensitive to TNF-a– induced apoptosis, which instead induces them to proliferate. These cells may become sensitive to the proapoptotic effects of TNF-a under certain experimental conditions, however. The inhibition of NF-kB alone, with a nondegradable, super-repressor IkBa, in contrast to observations with macrophages, does not induce apoptosis of isolated, in vitro –cultured human RA synovial fibroblasts [53]. The inhibition of NF-kB activation using either a proteasome inhibitor or ectopically expressed IkBa, however, sensitizes in vitro– cultured human RA synovial fibroblast to TNF-a– induced apoptosis [75 – 77]. The inhibition of NF-kB using a proteasome inhibitor also sensitizes the vast majority of in vitro – cultured human RA synovial fibroblasts to Fas-mediated apoptosis [76]. The mechanism responsible for the TNF-a– induced apoptosis of in vitro – cultured human RA synovial fibroblasts remains to be fully elucidated, although antiapoptotic protein XIAP may be a contributing factor [75]. The PI3K/Akt-1 pathway Immunohistochemical staining, immunoblot analysis, and Akt kinase assays demonstrate that the levels of phosphorylated or activated Akt-1 are higher in RA compared with osteoarthritis synovial fibroblasts and that activated Akt-1 is increased following treatment with TNF-a [78]. When the PI3K pathway is inhibited, TNF-a induces synovial fibroblast apoptosis [78], which is inhibited by the ectopic expression of PTEN (phosphatase and tensin homolog deleted from chromosome 10), an inhibitor of PI3K activity. These data are consistent with previous findings that there is a lack of expression of the tumor suppressor PTEN at sites of invasive fibroblast growth and joint destruction in RA tissue [79]. Therefore, in vivo, suppressed PTEN expression may promote destruction in RA by permitting the enhanced activation of Akt-1, which promotes synovial fibroblast survival, even in a hostile environment. Recent findings also reveal that the PI3K/Akt pathway is involved in the TGF-b– mediated growth and antiapoptotic effects observed with RA synovial fibroblasts [80]. These observations suggest that the PI3K/Akt-1 pathway is a viable therapeutic target in RA treatment. Role of STAT3 High levels of IL-6 are present in the rheumatoid joint. A downstream target of IL-6, STAT3, is constitutively activated in the synovial tissues of patients with RA [81]. Activated STAT3 is associated with a reduction of the suppressor of cytokine signaling 3 (SOCS3) [81]. Furthermore, in the collagen-induced arthritis model of RA, the expression of dominant-negative STAT3 or SOCS3 results in amelioration of arthritis and a reduction of IL-6 and TNF-a within the joints [81]. Although the mechanism for the suppression of these cytokines was not defined

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in these experiments, another study demonstrated that apoptosis is induced in RA and osteoarthritis synovial fibroblasts by expression of a dominant-negative STAT3, in the absence of an additional apoptotic signal [82]. Together, these observations suggest that STAT3 is important in protecting against apoptosis in the rheumatoid joint, and that it may be an excellent therapeutic target. Sentrin-1/SUMO-1 Sentrin-1/SUMO-1, which may modulate Fas signaling, may be important in the rheumatoid joint. SUMO-1 is a small, ubiquitin-like protein, which shares great similarity to human ubiquitin [83]. In contrast to ubiquitin that prepares molecules for degradation, SUMO-1 modifies target molecules by the process of ‘‘sumolation,’’ which regulates their stability and localization, and their interaction with other protein, thus affecting the signal transduction. The overexpression of SUMO-1 provides protection against anti-Fas – and TNF-a –induced cell death [83]. Compared with the negligible expression of SUMO-1 mRNA in normal or osteoarthritis synovial tissues, SUMO-1 mRNA is highly expressed in rheumatoid synovial tissues, predominantly in the lining layer and at sites of cartilage invasion [68]. In addition, synovial fibroblasts from RA tissues maintained the high expression of SUMO-1 over the 60-day period following implantation into the SCID mice. Therefore, although the potential mechanism has not been characterized, these observations suggest that the expression of SUMO-1 in the rheumatoid joint may contribute to apoptosis resistance by synovial fibroblasts [68]. Synoviolin/Hrd1 Synoviolin/Hrd1 was recently cloned by screening the proteins from adherent rheumatoid synovial cells using antibodies generated by immunization of mice with the cells pooled from several patients with RA [84]. Analysis of the protein structure revealed that synoviolin/Hrd1 belongs to the family of E3 ubiquitin ligases. Synoviolin/Hrd1 was highly expressed in RA synovial tissues, and its expression correlated with the development of collagen-induced arthritis. Furthermore, 30% of synoviolin/Hrd1 –overexpressing mice develop spontaneous arthritis, with no other abnormality throughout their life. Conversely, Synoviolin/ Hrd1+/
mice, which exhibit a markedly reduced expression of synoviolin/Hrd1, are resistant to the induction of collagen-induced arthritis and demonstrate increased apoptosis in the joints. Furthermore, inhibition of synoviolin/Hrd1 expression by small, interfering RNA suppresses the growth of RA synovial fibroblasts, and sensitizes them to apoptosis induced by disruption of endoplasmic reticulum function. These observations further support the notion that resistance to apoptosis contributes to the pathogenesis of RA. Bcl-2 family members Proapoptotic (Bax, Bak, Bid) and antiapoptotic (Bcl-2, Bcl-xL, A1, and Mcl-1) Bcl-2 family members are critical in regulating survival by regulating mitochon-

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drial integrity. The current authors’ data demonstrated that Bcl-2 is highly expressed in RA synovial tissue, compared with osteoarthritis, particularly in CD68
synovial fibroblasts [25]. In vitro, synovial fibroblasts infected by an adenovirus expressing a Bcl-2 ribozyme result in the reduction of Bcl-2, the loss of mitochondria transmembrane potential, and apoptotic cell death [25]. Furthermore, it has been demonstrated that exogenous IL-15 enhances the expression of Bcl-2 and Bcl-xL mRNA in rheumatoid synovial fibroblasts expressing functional IL-15 receptor [85]. In addition, the suppression of endogenously produced IL-15 results in apoptosis of RA synovial fibroblasts. In support of these findings, Bcl-2 is present at the sites of early erosions and correlates with the erosion and inflammation scores in the adjuvant-induced arthritis model of RA [8]. These observations suggest that the enhanced expression of antiapoptotic Bcl-2 family members may be important in protecting synovial fibroblasts from cell death in the rheumatoid joint. p53 The expression of the tumor suppressor gene product p53 is up-regulated in lining cells within the rheumatoid joint [86]. Inactivating somatic mutations of p53 have been identified in rheumatoid synovial fibroblasts [12]. Because p53 is important in apoptosis, these mutations may contribute to the resistance of these synovial fibroblasts to apoptosis and promote their pathogenicity [87,88]. A recent study, using microdissected RA synovial tissue sections, observed p53 mutations located mainly in the synovial lining rather than in the sublining [11]. The regions with greater amounts of IL-6 mRNA had more p53 mutations, suggesting that the p53 mutations might also contribute to the increased IL-6 [11]. The sequences of the p53 mutations suggest that they were induced by oxidative stress within the rheumatoid joint, which further suggests that they were induced by and contributed to the chronic inflammation [11]. In summary, oxidative stress may result in p53 mutations that may protect against the induction of apoptosis and promote inflammation. Further supporting the relevance of p53, DBA/l, p53
/
mice develop more severe collagen-induced arthritis and express more IL-6 compared with DBA/l wild-type mice. In support of the potential of p53 as a therapeutic target, the intra-articular injection of an adenoviral vector expressing wild-type p53 induced synovial apoptosis and reduced inflammation, without affecting cartilage metabolism [89]. Osteoclasts Osteoclasts are bone-resorbing cells derived from monocyte precursors. Within the rheumatoid joint, the interaction of RANK on the osteoclasts with RANKL on the surface of activated T-cell or synovial fibroblasts results in the activation of NF-kB, which enables osteoclasts to mature and become activated, capable of resorbing bone. In contrast, the soluble decoy receptor for RANKL, osteoprotegerin (OPG), blocks the interaction between RANK and RANKL, thus

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preventing the osteoclast differentiation. The enhanced activity of osteoclasts contributes to the pathologic bone loss and the erosions observed in patients with RA (reviewed in reference [90]). TNF-a does not seem to be a major factor in the osteoclastogenesis that is part of normal bone remodeling; however, within the inflammatory joint, TNF-a, in the presence of RANKL, promotes the differentiation and activation of osteoclasts. In TNF-a transgenic mice, which are a model of RA, systemic TNF-a increases the number of circulating osteoclast precursors [91]. In addition, the inhibition of TNF-a in patients with RA protects against joint destruction, even if inflammation is not suppressed. The NF-kB activation that occurs following the addition of TNF-a is essential for the survival of osteoclasts. When the NF-kB activation is inhibited by the ectopic expression of a super-repressor IkBa, TNF-a results in osteoclast apoptosis, supporting the importance of NF-kB activation in osteoclast survival [92]. Even in the absence of TNF-a, the inhibition of NF-kB activation using decoy oligodeoynucleotides induces osteoclast apoptosis [93]. Because RANK – RANKL interactions are important for osteoclast survival, interruption of these interactions may also promote apoptosis. In an experimental model of arthritis, administration of OPG, the decoy RANKL, at the onset of disease prevented bone and cartilage destruction, but not inflammation [94,95]. Because osteoclast survival depends on RANKL, this effect may have been mediated by osteoclast depletion through induction of apoptosis [94,95] These observations suggest that TNF-a is a critical mediator of osteoclast differentiation and activation, and that interruption of RANK – RANKL interactions or the inhibition of NF-kB activation may be effective strategies to induce osteoclast apoptosis, protecting chronically inflamed joints against erosion and destruction.

Future directions in targeting apoptosis in the treatment of patients with rheumatoid arthritis As mentioned previously, RA is characterized by the paucity of apoptosis in the inflamed joint. The persistent activation of the NF-kB, PI3K/Akt-1, and STAT3 pathways, and the resulting increased expression of antiapoptotic molecules (see Table 2), may contribute to the resistance to apoptosis of cells in the joint, especially macrophages, synovial fibroblasts, neutrophils, and T lymphocytes. In addition, currently used medications, although not specifically designed to promote apoptosis, may function in part through the induction of apoptosis (see Table 1). These observations support the potential role of specifically targeting the induction of apoptosis as a therapeutic approach for RA. Although the activation of T lymphocytes might contribute to the initiation of RA, to date, therapies that target T cells have not been successful. This lack of effect may be because the inflammation in the joints is already chronic at the time of diagnosis or because the correct targets for T-cell modulation have not been identified. Nonetheless, the persistent activation of synovial macrophages, the

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Table 3 Potential targets for the induction of apoptosis in patients with rheumatoid arthritis Cell type

Molecular target

Synovial T cells

Bcl-xL Bcl-2 FLIP TRAIL NF-kB FLIP A1 Mcl-1 Mcl-1 A1 FLIP Akt-1 STAT3 Sentrin/SUMO-1 Synoviolin/Hrd1 Bcl-2 p53 NF-kB RANKL OPG

Synovial macrophages

Neutrophils Synovial fibroblasts

Osteoclasts

abnormal proliferation of synovial fibroblasts, and the accumulation of neutrophils and osteoclasts, partly because of the failure of elimination of these cells by apoptosis within the joint, seems to be important in the pathogenesis of established RA.

Summary A better understanding of the mechanisms contributing to the resistance of synovial macrophages and fibroblasts to apoptosis will not only provide better insights into the mechanisms contributing to the perpetuation of RA but will also help identify targets (Table 3) for the development of novel, more effective, and long-lasting therapies for the treatment of patients with RA. To avoid toxicity, such as the induction of apoptosis of critical organs such as the liver or brain, the mechanisms by which these molecules are targeted and therapy delivered must be carefully selected, using the insights obtained from studies characterizing the mechanisms that promote chronic inflammation.

Acknowledgments The authors thank Yingyu Ma for help with the preparation of the figure presented in the manuscript.

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