T-cell involvement in osteoclast biology: implications for rheumatoid bone erosion

D. O’Gradaigh and J. E. Compston

Bone Research Group, University of Cambridge School of Clinical Medicine, Department of Medicine, Addenbrooke's Hospital, Cambridge CB2 2QQ, UK.

Correspondence to: D. O’Gradaigh, Bone Research Group, University of Cambridge School of Clinical Medicine, Department of Medicine, Addenbrooke's Hospital, Cambridge CB2 2QQ, UK. E-mail: dogradaigh{at}camrheum.fsnet.co.uk


    Introduction
 Top
 Introduction
 Bone remodelling
 T-cell interaction with...
 T-cell activation of osteoclasts...
 Adult periodontal disease: a...
 RANKL expression in RA
 T cells in oestrogen-deficient...
 Interferon-{gamma}
 Other cytokines expressed or...
 Osteopontin
 Therapeutic strategies
 Summary
 References
 
Erosion of periarticular bone is a hallmark of rheumatoid arthritis (RA), resulting in significant joint deformity, pain and disability [1]. Osteoclasts are specialized cells of the macrophage lineage responsible for bone resorption during remodelling. These cells have been demonstrated at the site of erosion in RA [2, 3] and in representative animal models of the disease [4], and are thus believed to be critical in the pathogenesis of joint damage. Developments in research methods have facilitated significant advances in the understanding of osteoclast regulation in conditions characterized by excessive bone resorption, including RA. As the osteoblast-derived signal that regulates osteoclasts [receptor activator of nuclear factor {kappa}B ligand (RANKL); see below] is identical to TRANCE (tumour necrosis factor-related activation-induced cytokine), which was first discovered in studies of T cells [5, 6], the intriguing role of T cells in bone biology has generated a considerable literature, referred to as ‘osteoimmunology’ [7]. In this article we will briefly describe normal bone remodelling and RANKL expression by T cells in the interaction with dendritic cells (DCs). Osteoclast regulation by T-cell expression of RANKL and other cytokines will then be described in the setting of the three clinical conditions most commonly studied—RA and its animal models, osteoporosis, and adult periodontitis. Potential therapeutic strategies for the prevention of bone loss in RA will be highlighted.


    Bone remodelling
 Top
 Introduction
 Bone remodelling
 T-cell interaction with...
 T-cell activation of osteoclasts...
 Adult periodontal disease: a...
 RANKL expression in RA
 T cells in oestrogen-deficient...
 Interferon-{gamma}
 Other cytokines expressed or...
 Osteopontin
 Therapeutic strategies
 Summary
 References
 
Throughout adult life, the skeleton undergoes remodelling in which small packets of bone are resorbed and replaced by new bone. Remodelling is carried out by a ‘bone remodelling compartment’ (in the case of cancellous bone [8]) or a ‘basic multicellular unit’ [9]. These comprise a vascular space in which osteoclasts resorb bone at the leading edge of the structure; osteoblasts follow in their wake, producing osteoid, which subsequently becomes mineralized. This association of osteoblast and osteoclast activity is termed ‘coupling’, while ‘balance’ relates to the relative amounts of bone resorbed and replaced. Ideally, each remodelling event will be balanced, with no net bone loss. In contrast, osteoporosis is the consequence of imbalanced remodelling, where bone resorption exceeds new bone formation.

Bone resorption by osteoclasts requires a signal derived from osteoblasts or from bone lining cells (quiescent osteoblast-like cells on the bone surface). This signal was identified simultaneously in 1998 by two groups [10, 11]. The term RANKL is now preferred to refer to the molecule that has been called TRANCE, ODF (osteoclast differentiation factor) and OPGL (osteoprotegerin ligand) [12]. RANKL is expressed on the surface of osteoblasts, and interacts with its cognate receptor, RANK, on the surface of osteoclasts and their circulating monocytic precursors. Through a sequence of adapter proteins, the tumour necrosis factor (TNF) receptor-associated factors (TRAFs) [13], this signal results in the differentiation of precursors, fusion to form multicellular osteoclasts, and the activation of the osteoclast so formed to resorb bone. Osteoblasts also secrete macrophage colony-stimulating factor (M-CSF), an essential survival factor for osteoclasts, and osteoprotegerin (OPG). OPG acts as a soluble decoy receptor for RANKL, inhibiting the interaction between RANKL and RANK.

As in many other biological systems (e.g. metalloproteinases and their tissue inhibitors), it is the relative amounts of RANKL and OPG that regulate osteoclastic activity [11]. This balance may be affected by local and systemic factors, including mechanical strain, cytokines such as TNF-{alpha} and interleukin (IL)-1, prostaglandin (PG) E2, and hormones (oestradiol, 1,25 dihydroxyvitamin D3, parathyroid hormone).


    T-cell interaction with dendritic cells
 Top
 Introduction
 Bone remodelling
 T-cell interaction with...
 T-cell activation of osteoclasts...
 Adult periodontal disease: a...
 RANKL expression in RA
 T cells in oestrogen-deficient...
 Interferon-{gamma}
 Other cytokines expressed or...
 Osteopontin
 Therapeutic strategies
 Summary
 References
 
CD4+ T cells are the predominant subset that interacts with DCs, the specialized antigen-presenting cells, though CD8+ cells also depend largely on DC activation. CD4+ cells are activated on recognition by the T-cell receptor complex of (foreign) peptide, though antigen presentation normally does not elicit a response without co-stimulatory signalling between CD28 on the T cell and B7.1 (CD80) or B7.2 (CD86) on DCs [14]. CTLA4 is an alternative ligand for CD80 and CD86, having inhibitory effects on T-cell activation [15]. The expression of the co-stimulatory CD80 and CD86 is regulated by interaction between CD40 ligand (CD40L = CD154), expressed on CD4+ T cells, and CD40 on the dendritic cell [16]. B7 expression is also induced by toll-like receptors (TLRs), a group of transmembrane receptors that recognize shared microbial antigens, such as peptidoglycan, lipopolysaccharide and microbial DNA, thus constituting additional signalling of ‘danger’. CD40L, a member of the TNF receptor superfamily, also induces DC maturation and cytokine production and protects DCs from apoptosis.

RANKL, expressed by T cells, was found in the search for other members of the TNF superfamily which promote DC survival [5], and appears to provide a CD40/CD40L-independent pathway of T-cell activation in response to certain viruses [17]. T-cell expression of RANKL is up-regulated following engagement of the T-cell receptor complex, this being enhanced by co-stimulation through CD28 [18]. RANKL expression in vitro (using immobilized anti-CD3 antibody) was maximal after 48 h in culture and was sustained up to 96 h, being 10-fold higher on CD4+ cells compared with the CD8+ subset [18]. Subsequent in vitro studies have also demonstrated RANKL expression by T cells when activated by concanavalin A [19] or phytohaemagglutinin [20, 21]. Wang et al. [22] have shown that intracellular calcium signalling downstream of T-cell receptor activation is critical for RANKL expression, and can be blocked by cyclosporin A. Kong et al. [23] have additionally demonstrated the requirement for protein kinase C and phosphoinositide 3-kinase signalling. Of note, RANKL is cleaved from the surface of the T-cell by a TNF-{alpha}-converting enzyme (ADAM17) [24], resulting in functionally active [23] soluble RANKL. Both forms of RANKL are inhibited by OPG. CD40L signalling increases DC (and B-cell) expression of OPG [25], which then inhibits RANKL activity in a negative feedback loop that limits T-cell/DC activation. DCs from OPG–/– mice were found to present antigen to T cells more efficiently and had increased cytokine expression [25].



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FIG. 1. Potential pathways of osteoclast regulation by T cells in rheumatoid synovium. Synovial tissue (boxed area) is attached to bone (light shading) and overlaps articular cartilage (dark shading). Osteoclasts (OC) are shown on the periosteal bone surface and on subchondral bone. Monocytic precursors (MP) from synovial blood vessels (BV) encounter RANKL expressed by T cells. T cells influence macrophage (M{Phi}) expression of IL-15, IL-1 and TNF-{alpha}, which act directly on osteoclasts and their precursors. Indirect osteoclast-regulating signals include IL-17, TNF-{alpha} and IL-1, which act on periosteal and subchondral osteoblasts (OB) to release RANKL (large open arrows). Fibroblast-like synoviocytes (FLS) produce IL-7, which promotes the release of osteoclastogenic signals from T cells. (RANKL is also produced by FLS). This figure can be viewed in colour as supplementary material at Rheumatology Online.

 
The RANKL–RANK interaction between T cells and DCs may also have an important role in the regulation of certain autoimmune diseases [26]. Using an elegant murine model of CD8+ T-cell mediated diabetes, Green et al. [26, 27] have found that disease can be effectively prevented by CD4+/CD25+ regulatory T cells, whose function and accumulation in the relevant tissue appear to depend on RANKL–RANK signalling.

Very recently, another molecule involved in T-cell interaction with DCs has been shown to also affect bone. DAP12 is an adapter molecule involved in the transduction of activating signals for a range of immune cells, including NK cells, granulocytes, monocyte/macrophages and DCs, and DAP12-deficient mice have impaired TH1 responses (reviewed in [28]). These mice have now been shown to have osteopetrosis, due to an inability of marrow precursors to differentiate to mature, resorptive osteoclasts [29]. Current evidence suggests a model in which DAP12 may modulate responsiveness to certain cytokines, favouring osteoclast formation under normal conditions but promoting the differentiation of precursors to macrophages and the DC lineage in inflammatory conditions [28].


    T-cell activation of osteoclasts in disease
 Top
 Introduction
 Bone remodelling
 T-cell interaction with...
 T-cell activation of osteoclasts...
 Adult periodontal disease: a...
 RANKL expression in RA
 T cells in oestrogen-deficient...
 Interferon-{gamma}
 Other cytokines expressed or...
 Osteopontin
 Therapeutic strategies
 Summary
 References
 
A number of studies have demonstrated T-cell expression of RANKL in vivo at sites of increased bone resorption [23, 3033]. However, whether T cells can direct the formation of an osteoclast independently of other signals in vivo remains debatable. Dendritic cells, tissue macrophages and osteoclasts share a common, circulating precursor cell of the monocyte lineage (CD14+). These cells may differentiate in vitro into mature DCs or macrophages [34] or osteoclasts [35] depending on the cytokine environment. It is therefore plausible that the final differentiation of precursors in vivo is likewise determined by the microenvironment they encounter on leaving the circulation [36]. Data from rheumatoid synovial tissue are noteworthy in this respect—while T cells are predominantly located in the sublining layer and in perivascular areas [37] and are associated with DCs at these sites [38], osteoclasts are only found at the bone–pannus interface (or on subchondral bone) [3, 4]. TGF-ß has been described as a ‘mechanism for coupling a cell to its environment’ [39, 40], in this case the environment being the proximity of bone. Exposure of mononuclear cells to TGF-ß before RANKL increased osteoclast formation, though this effect was dependent upon the presence in cultures of non-adherent mononuclear cells (i.e. lymphocytes) [41]. However, TGF-ß acts primarily as a deactivator of inflammatory macrophages and thus also supports DCs. Indeed, the effects of TGF-ß on osteoclastogenesis are complex (for discussion see [42]), but the importance of both it and RANKL to regulatory T-cell function is intriguing in the context of osteoclastogenesis in an autoimmune disease. Alternative differentiation of precursors into osteoclasts or DCs has been attributed to their expression of toll-like receptors [43]. Takami et al. [43] found that activation of TLR-2, -3, -4 or -9 inhibited the differentiation of precursors to osteoclasts, suggesting that these additional signals of microbial invasion may function to prevent pathogenic invasion into bone. The cytokine environment may also influence the final commitment of precursors. Granulocyte macrophage colony-stimulating factor (GM-CSF) and M-CSF function as a reciprocal pair in the commitment of precursors to the DC or osteoclast lineage, the former reducing the expression of c-Fos, which is required for osteoclast differentiation [44]. Osteoclast formation is therefore most probably a specific response to a combination of signals derived from bone and from adjacent inflammatory cells, including T cells, and not an inevitable consequence of T-cell expression of RANKL alone.

In the following sections, we will review the evidence for osteoclast activation by T-cell-derived RANKL in disease, before considering the role of inhibitory T-cell signals and indirect T-cell-mediated effects.


    Adult periodontal disease: a paradigm of T-cell-mediated bone loss
 Top
 Introduction
 Bone remodelling
 T-cell interaction with...
 T-cell activation of osteoclasts...
 Adult periodontal disease: a...
 RANKL expression in RA
 T cells in oestrogen-deficient...
 Interferon-{gamma}
 Other cytokines expressed or...
 Osteopontin
 Therapeutic strategies
 Summary
 References
 
Much of the understanding of osteoimmunology derives from studies of adult periodontal disease, a common inflammatory T-cell-mediated [45] condition associated with alveolar bone resorption. Numerous Gram-negative bacteria are implicated, of which Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans are the most common. T cells are abundant in the diseased periodontal tissue, with a subtle predominance of proinflammatory (TH1) cytokine profile at the sites of active bone resorption [46]. There is evidence of clonal expansion of T cells specific to the infective pathogen [47]. Various animal models have convincingly demonstrated that CD4+ T cells are activated in a classical antigen-presentation manner [32, 48, 49] with co-stimulation through lipopolysaccharide-induced B7.1 and B7.2 activation of CD28 [49], resulting in expression of RANKL and of inflammatory cytokines [32, 46]. Kawai et al. [49] demonstrated that inhibition of B7 co-stimulation (using CTLA4Ig) was sufficient to prevent alveolar bone loss. However, all aspects of the periodontal disease were suppressed by this treatment, suggesting that it is the T-cell response in total [i.e. including RANKL expression, indirect effects on other cytokines (discussed below), activation of macrophages, etc.] rather than the co-stimulatory signal itself that caused the bone resorption.


    RANKL expression in RA
 Top
 Introduction
 Bone remodelling
 T-cell interaction with...
 T-cell activation of osteoclasts...
 Adult periodontal disease: a...
 RANKL expression in RA
 T cells in oestrogen-deficient...
 Interferon-{gamma}
 Other cytokines expressed or...
 Osteopontin
 Therapeutic strategies
 Summary
 References
 
The precise role of T cells in RA is debated [37, 50, 51], though it is broadly accepted that lymphocytes and macrophages interact in the generation of the cytokines which drive many of the characteristic features of the disease. The cytokine profile reflects an inflammatory response (TH1), though low levels of interferon {gamma} (IFN-{gamma}) have been reported [52]. Various authors have shown expression of RANKL by rheumatoid synovial T cells [23, 30]. However, Kong et al. [23] found that synovial T cells from all osteoarthritis samples they examined also expressed RANKL. The study by Gravallese et al. [30] demonstrated RANKL expression only on T cells (extracted from RA synovium) after sequential exposure to immobilized anti-CD3 and expansion in IL-2, a procedure which is known to induce RANKL expression irrespective of the T-cell origin. Nonetheless, Kotake et al. [20] has demonstrated RANKL (protein) expression by CD3+/4+ cells in rheumatoid synovium by dual immunohistochemical labelling, while Horwood et al. [19] used a combination of in situ hybridization and immunohistochemistry to show expression of RANKL mRNA by CD3+ T cells. Curiously, both of these studies failed to detect significant expression of RANKL by synovial fibroblasts, which others have shown to be a further source of this cytokine [30, 5355]. In the studies that demonstrate RANKL expression by T cells, it has been assumed that it is these cells rather than the fibroblast-like synoviocytes or osteoblasts that are the critical source of RANKL in the generation of osteoclasts in RA. Perhaps the most compelling argument that this might be the case comes from the proximity of perivascular T cells to the blood vessels from which the continuous supply of pluripotent precursors emerges.

While a number of potential antigens have been proposed in the pathogenesis of RA, the evidence that T-cell activation is antigen-driven remains elusive [50]. Recently, Brennan et al. [56] have suggested that T cells activated by a combination of cytokines are similar in their T-cell/monocyte interactions to rheumatoid synovial T cells (specifically in the expression of TNF-{alpha}). We have found that such cytokine-activated T cells also up-regulate RANKL expression, significantly increasing osteoclastogenesis in co-culture with CD14+ precursors [57]. These T cells expressed significantly less IFN-{gamma} than anti-CD3 or phorbol-myristate acetate (PMA)/ionomycin-activated T cells. This is of particular interest, as IFN-{gamma} has been identified as a critical inhibitor of excessive osteoclast formation by the immune system [58] (see later).


    T cells in oestrogen-deficient osteoporosis
 Top
 Introduction
 Bone remodelling
 T-cell interaction with...
 T-cell activation of osteoclasts...
 Adult periodontal disease: a...
 RANKL expression in RA
 T cells in oestrogen-deficient...
 Interferon-{gamma}
 Other cytokines expressed or...
 Osteopontin
 Therapeutic strategies
 Summary
 References
 
A remarkable discovery was that nude mice (athymic, i.e. lacking T cells) did not develop osteoporosis following ovariectomy, but that bone loss did occur following adoptive transfer of T cells from an ovariectomized normal mouse [59]. Nude mice have an essentially normal bone phenotype, indicating that physiological bone modelling and remodelling are not T-cell-dependent. Following ovariectomy, the number of bone marrow T cells expressing TNF-{alpha} is increased, significantly augmenting M-CSF- and RANKL-dependent osteoclastogenesis [59, 60]. However, in vitro osteoclastogenesis from bone marrow of these mice did not occur in the absence of M-CSF and RANKL (i.e. the normal physiological signalling). These findings indicate that complete abrogation of bone loss in the absence of T cells cannot be attributed to a mechanism that can only amplify the normal remodelling process. While replication of these studies has not been reported, the available data imply that T-cell expression of TNF-{alpha} is critically involved in initiating the excessive (RANKL-mediated) remodelling which characterizes oestrogen-deficiency osteoporosis. Potential mechanisms for TNF-{alpha}-initiated remodelling include apoptosis of bone lining cells, which exposes additional remodelling sites, or alterations in putative initiating signals such as nitric oxide or osteopontin expression by osteocytes. The finding that high-dose anti-TNF therapy can halt (and even reverse) radiographic progression in RA [61] suggests that TNF-{alpha} may well have a similar role in rheumatoid bone erosion, in addition to its effects on inflammation.


    Interferon-{gamma}
 Top
 Introduction
 Bone remodelling
 T-cell interaction with...
 T-cell activation of osteoclasts...
 Adult periodontal disease: a...
 RANKL expression in RA
 T cells in oestrogen-deficient...
 Interferon-{gamma}
 Other cytokines expressed or...
 Osteopontin
 Therapeutic strategies
 Summary
 References
 
Given that RANKL expression by T cells is an almost universal consequence of antigen presentation, a regulatory mechanism which prevents excessive osteoclast formation is likely to exist. One such inhibitory signal was identified by Takayanagi et al. [58]. Building on the observation of increased bone loss in collagen-induced arthritis in IFN-{gamma} receptor (IFNGR)-deficient mice [62, 63], Takayanagi and colleagues demonstrated increased bone loss in a T-cell-dependent model of endotoxin-induced bone resorption in similarly IFNGR–/– mice. Co-culture of splenic T cells activated by anti-CD3 antibodies with bone marrow-derived precursors strongly inhibited osteoclastogenesis, an effect that was cancelled in cultures with osteoclast precursors from the IFNGR1-deficient mice [58]. In contrast, Kong et al. [23] had found that the activation of T cells by anti-CD3 stimulated osteoclastogenesis in co-culture with similar precursors. Important differences between the methods described by Kong et al. and by Takayanagi et al. were the duration of T-cell culture (4 days in the former case, 1 day in the latter) and the additional use of anti-CD28 antibody [18] by Kong et al. As IFN-{gamma} is rapidly up-regulated on T-cell activation (peak within 24 h) whilst expression of RANKL under similar activating conditions is maximal at 48 h [18], it is likely that the timing of the addition of T cells to co-cultures is a critical determinant of the net effect of RANKL and IFN-{gamma} on osteoclastogenesis in vitro. More importantly, these studies suggest that it is the balance of these signals in an inflammatory tissue adjacent to bone that most influences bone resorption. Further insight into this agonist–antagonist dynamic was recently provided by Huang et al. [64]. Pretreatment of osteoclast precursors (splenocytes or the macrophage cell line RAW264.7) with RANKL rendered them resistant to the inhibitory effects of IFN-{gamma}, highlighting the importance of early exposure to RANKL-expressing T cells (or other cells), mentioned above. We have observed a similar effect in the interaction between TNF-{alpha} and RANKL [65], and the effect of the initial cytokine during exposure to multiple signals has also been noted in macrophages [66, 67]. That the effects of IFN-{gamma} are complex is also suggested by a study of adult periodontal disease, in which mice unable to express IFN-{gamma} had less bone loss than wild-type controls [32]. This has been attributed to reduced induction in the IFN-{gamma}–/– animals of the key cytokines TNF-{alpha} and IL-1, which are known to increase bone resorption both directly and indirectly.


    Other cytokines expressed or regulated by T cells
 Top
 Introduction
 Bone remodelling
 T-cell interaction with...
 T-cell activation of osteoclasts...
 Adult periodontal disease: a...
 RANKL expression in RA
 T cells in oestrogen-deficient...
 Interferon-{gamma}
 Other cytokines expressed or...
 Osteopontin
 Therapeutic strategies
 Summary
 References
 
Interactions between lymphocytes and monocytes produced soluble factors with osteoclast-activating activity [68], subsequently identified as TNF-{alpha} and -ß [69] and IL-1ß [70]. TNF-{alpha} and IL-1 act on osteoblastic cells to induce RANKL expression and both cytokines increase the resorptive activity of osteoclasts independently of stromal cells [71]. Some authors have reported RANKL-independent osteoclastogenesis in the presence of TNF-{alpha} [7274], though others have suggested that simultaneous [75] or prior [76] exposure to RANKL is necessary. This debate has been reviewed recently in the context of rheumatoid bone erosion [77]. We have noted that TNF-{alpha}-mediated, RANKL-independent osteoclastogenesis from CD14+ precursors is T-cell-dependent and is blocked by adding the IL-1 receptor antagonist [65].

T-cell expression of osteoclastogenic cytokines in response to IL-1 and TNF-{alpha} is mediated by stromal cell expression of IL-7 [78]. IL-7 is produced by cultured RA synovial fibroblasts in response to TNF-{alpha} and IL-1 [79]. In a positive feedback loop, IL-7 stimulates expression of TNF-{alpha} by synovial monocytes and by T cells [80].

IL-15, expressed by synovial fibroblasts and monocytes [79], is a potent activator of TNF-{alpha} expression by CD4+ T cells [81] and dose-dependently increased the number of osteoclasts formed in stromal cell-depleted rat marrow cultures (under osteoclastogenic conditions) [82]. This effect was not mediated by TNF-{alpha} induction and appeared to be a direct effect on early osteoclast precursors independent of T cells.

IL-15 also stimulates production of IL-17 (in a cyclosporin-inhibited signalling pathway) [83]. IL-17 is exclusively expressed by activated memory T cells (CD4+CD45RO+) [84] and is found in significantly greater amounts in RA synovial tissue [85, 86] and in the synovial fluid of RA joints compared with osteoarthritic, traumatic or gouty joint effusions [86]. Increased osteoclastogenesis in response to IL-17 requires osteoblast expression of RANKL. IL-17 also increases the expression of IL-6, TNF-{alpha} and IL-1 [87], which interact in the pathogenesis of joint erosion, as the inhibition of joint erosion was greater with combined blockade of TNF-{alpha}, IL-1 and IL-17 than with any of these treatments alone [88].

IL-18 levels are increased in RA synovial tissues compared with osteoarthritis [89], particularly in active disease. Paradoxically, IL-18 expression by osteoblasts [90] induces T-cell expression of GM-CSF, which inhibits osteoclast formation [44, 91].

IL-4 gene therapy completely inhibits the expression of IL-17 by CD4+ cells, thus preventing bone erosion [92]. IL-4 has also been shown to reduce the expression of RANKL by T cells activated by anti-CD3 antibody [18]. Interestingly, in in vitro osteoclastogenesis experiments, IL-4 (and IL-13) induced osteoclast formation in mixed peripheral blood mononuclear cell cultures but significantly suppressed osteoclastogenesis in cultures from which lymphocytes had been depleted [93]. The contrast between these in vitro data and the results of the in vivo gene therapy studies highlight the complexity of cytokine and cell–cell interactions, particularly between T cells and osteoclast precursors, which are difficult to replicate in vitro.


    Osteopontin
 Top
 Introduction
 Bone remodelling
 T-cell interaction with...
 T-cell activation of osteoclasts...
 Adult periodontal disease: a...
 RANKL expression in RA
 T cells in oestrogen-deficient...
 Interferon-{gamma}
 Other cytokines expressed or...
 Osteopontin
 Therapeutic strategies
 Summary
 References
 
Osteopontin (OPN) is a bone matrix protein [94] promoting the adhesion and spreading of osteoclasts on the bone surface [95]. OPN is identical to Eta-1 which is expressed by T cells [96] and is chemotactic to T cells, stimulating their expression of proinflammatory (TH1) cytokines [97]. In RA and osteoarthritis synovial tissue, OPN expression was almost exclusively limited to fibroblasts [98]. A number of studies [99102] have indicated that matrix OPN (probably expressed by osteocytes [103]) is not required for osteoclast formation or activity per se but is critical for osteoclastic resorption in pathological states. A recent study of collagen antibody-induced arthritis in the OPN-deficient mouse found significant reductions in all aspects of joint pathology in the absence of OPN [104]. In particular, a biochemical marker of bone resorption (deoxypyridinoline) was not increased in the OPN-deficient mice [104]. OPN binds to {alpha}vß3 integrin, which has recently been studied as a therapeutic target in RA [105] (reviewed in [106]).


    Therapeutic strategies
 Top
 Introduction
 Bone remodelling
 T-cell interaction with...
 T-cell activation of osteoclasts...
 Adult periodontal disease: a...
 RANKL expression in RA
 T cells in oestrogen-deficient...
 Interferon-{gamma}
 Other cytokines expressed or...
 Osteopontin
 Therapeutic strategies
 Summary
 References
 
Notwithstanding the data described above demonstrating RANKL-independent osteoclastogenesis under particular experimental conditions, several lines of evidence indicate that the final common pathway leading to the generation of osteoclasts in RA and its related animal models depends on RANKL signalling. Perhaps the most compelling evidence comes from studies of a serum transfer model of arthritis in RANKL knockout mice [107]. By histological and microcomputed tomography, bone erosion was dramatically reduced in the knockout animals. Interestingly, in this model, cartilage erosion was not prevented. The serum transfer approach bypasses the requirement for T cells, and this study therefore does not exclude the possibility that RANKL-independent, T-cell-mediated pathways are involved in rheumatoid erosion [21, 78]. Furthermore, RANKL knockout animals have an osteopetrotic phenotype, rendering the bone more resistant to any potential RANKL-independent mechanisms. However, TNF-{alpha} mediated joint destruction was inhibited by the RANKL inhibitor osteoprotegerin [108], again implying a final, RANKL-mediated pathway for osteoclast formation and activity. In an adjuvant-induced arthritis model, treatment with OPG at the onset of disease resulted in only minimal loss of trabecular and cortical bone, associated with a dramatic reduction in osteoclast numbers [23]. Furthermore, there was a striking reduction in cartilage damage [23]. Reductions in erosive damage were also obtained with OPG treatment of collagen-induced arthritis [109]. In the most relevant study with respect to therapeutic strategies, treatment with OPG was compared with pamidronate (a potent aminobisphosphonate), a combination of both agents, or the anti-TNF-{alpha} drug infliximab [108]. As anticipated, clinical features of inflammation were only suppressed in the infliximab-treated animals. However, radiographic and histological assessments of joint erosion demonstrated similar efficacy with either OPG or pamidronate (56 and 53% reduction respectively compared with controls), and a significantly greater response when both agents were used in combination (81% reduction). While these data do not prove the presence of RANKL-independent pathways, they do suggest that OPG cannot adequately inhibit the multiple converging osteoclastogenic pathways in inflammatory bone erosion, particularly in view of the potent synergy between TNF-{alpha} and RANKL [74]. In addition, there may be reduced bioavailability of OPG at particular sites that is unable to inhibit overwhelming levels of RANKL at those sites. A similar effect has been observed in the inhibition of endosteal but not of periosteal osteoclasts with OPG [110], and while OPG expression has been demonstrated in periodontal tissues [111], treatment with OPG-Fc (a fusion protein with functional RANKL inhibitory effects) was required to reduce bone loss significantly in an animal model of periodontal disease [33].


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TABLE 1. Effects of T cell-related cytokines on osteoclastic bone resorption

 
Novel therapeutic agents with potential advantages over OPG have been reported recently in abstract form. These include a cyclized peptidomimetic that inhibits TNF-{alpha} action and blocks RANKL-induced osteoclastogenesis in vitro [112], with higher affinity for the soluble form of RANKL (therefore potentially offering some selectivity towards pathological bone resorption). Recombinant soluble frizzled-related protein has also been shown to inhibit both RANKL-dependent and independent osteoclastogenesis in vitro [113]. In contrast, a non-peptide molecule has been developed that selectively inhibits RANKL activation of NF{kappa}B [114]. A vaccination strategy resulted in the formation of polyclonal antibodies to RANKL [115]. As this approach would require immunoglobulin-producing cells at the site of excess RANKL production, would compete with the endogenous decoy receptor OPG, and would be irreversible even if the resorptive disease was controlled, its value in RA may be limited.

As described above, combined biological therapies directed at both the erosive (IL-17, IL-1) and inflammatory (IL-1 and TNF-{alpha}) components in an ex vivo model of RA has shown promise [88], and gene transfer of IL-4 has also been effective in reducing bone damage [92].

It is unclear whether T-cell derived RANKL acts in RA only during cell–cell contact with monocytic osteoclast precursors, or whether soluble RANKL cleaved from the cell surface is also relevant. If so, inhibitors of the TNF-{alpha} converting enzyme (ADAM17) [24], already under investigation as inhibitors of metalloproteinases, might be of value in preventing bone erosion [116].

Cyclosporin has been demonstrated in clinical studies to significantly delay radiographic progression [117], an effect previously attributed to its general disease-modifying effects. However, cyclosporin interferes with calcineurin signalling, which is known to be required for RANKL expression [22] and activity [118]. This drug might therefore have quite specific effects on prevention of bone erosion. Conversely, cyclosporin has also been associated with osteopenia [119]. Other anti-T-cell therapies have been investigated, though effects on radiographic progression were not reported in most of these relatively short-term studies (reviewed in [50]). Recently, CTLA4Ig (an inhibitor of CD80/CD86 co-stimulation which may also have inhibitory effects on T cells through B7 signalling [120]) has entered clinical trials in RA [121] and may be of greater efficacy in the inhibition of T-cell RANKL induction, given the results of this treatment in animal models of periodontal disease [49].


    Summary
 Top
 Introduction
 Bone remodelling
 T-cell interaction with...
 T-cell activation of osteoclasts...
 Adult periodontal disease: a...
 RANKL expression in RA
 T cells in oestrogen-deficient...
 Interferon-{gamma}
 Other cytokines expressed or...
 Osteopontin
 Therapeutic strategies
 Summary
 References
 
T cells, particularly CD4+ cells expressing the TH1 cytokine profile, express RANKL in response to antigen presentation (or cytokine activation). RANKL is required for T-cell interaction with DCs and may be involved in regulatory T-cell accumulation in the suppression of some autoimmune diseases. Osteoclast formation and activation require RANKL and M-CSF, but may additionally require other signals from the bone microenvironment to drive monocytic pluripotent precursors towards the osteoclast lineage in vivo. T-cell involvement in osteoclastogenesis in RA is likely to be the combined result of RANKL expression, monocyte/T-cell interactions generating other osteoclastogenic cytokines, such as IL-17, IL-1, TNF-{alpha} and TGF-ß, and possibly requires additional signals from the bone microenvironment. Inhibition of RANKL with OPG significantly reduces bone erosion in various relevant disease models. Inhibition of osteoclast activity at the bone surface (with bisphosphonate) may have additional benefit, particularly where high RANKL concentrations or synergy with TNF-{alpha} overwhelm OPG. The in vivo relevance of experimentally observed RANKL-independent mechanisms of osteoclastogenesis is unclear.


    Acknowledgments
 
DO’G is supported by the Arthritis Research Campaign (Clinical Research Fellowship O0157) and by a Sackler Foundation Scholarship (University of Cambridge). We are grateful to Professor J. S. H. Gaston (University of Cambridge) and Professor T. J. Chambers (St George's Hospital Medical School) for critical reading of the manuscript.

The authors have declared no conflicts of interest.


    References
 Top
 Introduction
 Bone remodelling
 T-cell interaction with...
 T-cell activation of osteoclasts...
 Adult periodontal disease: a...
 RANKL expression in RA
 T cells in oestrogen-deficient...
 Interferon-{gamma}
 Other cytokines expressed or...
 Osteopontin
 Therapeutic strategies
 Summary
 References
 

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Submitted 12 March 2003; Accepted 21 May 2003