The unexpected links between bone and the immune system




Anne TETI, PhD


Nadia RUCCI, MD
Department of Experimental
Medicine, University of L’Aquila
L’Aquila, ITALY

The unexpected links between bone and the immune system

by A. Teti and N. Rucci ,Italy

Bone is a tissue of central importance, maintaining several relationships with other organs. Among these, the immune system, with which it shares molecular pathways, transcription factors, and several cytokines responsible for bone and immune cell regulation. A paradigm of this crosstalk comes from the studies of Hiroshi Takayanagi on the mechanisms underlying the development of rheumatoid arthritis, demonstrating the central role of a subset of T lymphocytes in the induction of exaggerated osteoclast activity, thus leading to erosion in the affected joints. RANKL/RANK (receptor activator of nuclear factor–kappaB [ligand]) is an important pathway shared by bone and the immune system. This pathway is essential for both osteoclastogenesis and lymphocyte differentiation, so that diseases due to inactivating mutations of RANKL or RANK, such as osteopetrosis, result in immunological defects in addition to altered bone phenotype. This review focuses on the description of the principal molecules/pathways shared with the immune system, which under both physiological and pathological conditions, regulate bone remodeling by acting on osteoclast formation and activity. We propose that the evidence available today strongly points to the osteoclast as a cell with immunological properties, in addition to its role in bone resorption.

Medicographia. 2010;32:341-348 (see French abstract on page 348)

The perception of bone as a static organ has changed dramatically over the past several years. The literature has clearly shown that bone is a tissue of central importance and that, in addition to its role in locomotion and in the regulation of calcium and phosphate homeostasis, bone actively maintains multiple relationships with other organs.

Recent observations have evidenced crucial crosstalk between bone and the immune system, thus leading to the launch of a new interdisciplinary field, osteoimmunology.1 Indeed, several cytokines, molecular pathways, and transcription factors are shared by the immune and skeletal systems. Moreover, immune cells, like bone cells, arise from hematopoietic stem cells (HSCs) found in the bone marrow, which is physically as well as functionally associated with bone tissue. Interestingly, cell differentiation from HSCs has been shown to be subject to a fine regulation by the osteoblasts, which form the HSC niche.2 Kollet et al have consistently found that, once subjected to specific stressful stimuli, activated osteoclasts degrade endosteal components, thus promoting the mobilization of hematopoietic progenitors.3

Studies on autoimmune diseases, such as rheumatoid arthritis, performed by Hiroshi Takayanagi, have provided a pivotal contribution in development of the field of osteoimmunology, with identification of a subset of a T cell population that produces high quantities of interleukin (IL)-17, a pro-osteoclastogenic cytokine that increases osteoclast differentiation by direct and indirect mechanisms, thus leading to bone destruction.1 Conversely, animal models lacking molecules pivotal for the regulation of the immune system frequently show an abnormal osteoclast phenotype.1

Based on this evidence, we believe that a more extensive investigation of the mechanisms underlying the bone-immune interplay could allow the identification of new strategies for the management of immune system and bone disorders. In this review, we summarize the recent findings that have contributed to consolidation of the field of osteoimmunology, with particular focus on the close relationship between the osteoclasts and the immune cells.

The bone remodeling process

It is well known that bone tissue is in dynamic flux, continually renewed lifelong by a physiological process termed bone remodeling.4,5 This process is mandatory for the replacement of immature bone with mechanocompetent bone, as well as for repair of fractures and for proper calcium balance. Indeed, it has been estimated that at least 10% of bone is renewed per year.

Bone remodeling follows the activation-resorption-formation (ARF) sequence (Figure 1). The first step, called the activation phase, starts with stimulation of the lining cells, quiescent osteoblasts, which, in response to appropriate stimuli, increase their own surface expression of receptor activator of the nuclear factor-kappaB (NF-κB) ligand (RANKL), which in turn interacts with its receptor RANK (receptor activator of NF-κB), expressed by preosteoclasts. RANKL/RANK interaction triggers preosteoclast fusion and differentiation to multinucleated osteoclasts. Once differentiated, osteoclasts polarize, adhere to the bone surface, and dissolve bone (resorption phase), then they undergo apoptosis, which is a physiological process, required to prevent excessive bone resorption.

After this resorptive process, there is an intermediate phase preceding bone formation, called a reversal phase, during which some macrophage-like uncharacterized mononuclear cells are observed at the site of remodeling, whose function consists of removal of debris produced during matrix degradation.

The final step, bone formation, is triggered by several growth factors stored in the bone matrix and released after its degradation, including bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and transforming growth factor–β (TGFβ), which are likely to be responsible for recruitment of osteoblasts in the resorbed area. Once recruited, osteoblasts produce new bone matrix, initially not mineralized (osteoid), and then they promote its mineralization, thus completing the bone remodeling process.

Under physiological conditions, the coupling of bone formation with previous resorption occurs faithfully. In contrast, an imbalance between the resorption and formation reflects improper bone remodeling, which in turn affects the bone mass, eventually leading to a pathological condition.

Osteoblast regulation of osteoclastogenesis

Although the principal function of the osteoblasts is to synthesize bone matrix proteins and to promote the process of mineralization, a crucial role of osteoblasts in osteoclast biology has been clearly demonstrated by the release of key molecules that regulate osteoclastogenesis and bone resorption. Osteoclasts are multinucleated cells that arise from the monocyte/ macrophage cell line.6 Starting from multipotent HSCs, transcription factor PU.1, along with the macrophage-colony stimulating factor (M-CSF), allows the commitment toward a common progenitor for macrophages and osteoclasts (Figure 2). In particular, PU.1 positively regulates the M-CSF receptor, c-Fms, while M-CSF stimulates proliferation of osteoclast precursors and upregulates RANK expression. With the expression of c-Fms and RANK receptors, the precursors become fully committed to osteoclast lineage.7 The main source of RANKL in bone is the osteoblast, which expresses RANKL on its membrane surface, thus inducing osteoclast differentiation by interacting with the RANK receptor expressed by the osteoclast precursors. Therefore, triggering of the RANKL/RANK pathway requires a cell-cell contact (Figure 3, page 344). However, lower quantities of soluble RANKL are also released after enzymatic cleavage of the surface molecule by metalloproteinase (MMP)-14. Another key molecule produced by osteoblasts that interfere with the RANKL/RANK pathway is osteoprotegerin (OPG), a decoy receptor for RANKL8 with an osteoprotective role. Indeed, OPG is a secreted protein sharing the same structure of the extracellular domain of RANK so that it binds RANKL, preventing its interaction with RANK and subsequent inhibition of osteoclastogenesis.

Figure 1
Figure 1. The bone remodeling process.

Bone remodeling starts with activation of the lining cells, which increase surface expression of RANKL. RANKL interacts with its receptor RANK, thus triggering osteoclast differentiation (Activation phase). Osteoclasts resorb bone (Resorption phase), thus allowing the release of factors usually stored in the bone matrix (BMPs, TGFβ, FGFs) that
recruit osteoblasts in the resorbed area. Once recruited, osteoblasts produce the new bone matrix and promote its mineralization (Formation phase), thus completing the bone remodeling process
Abbreviations: BMPs, bone morphogenetic proteins; FGFs, fibroblast growth factors; Pre-OCLs, preosteoclasts; OCL, osteoclast; OBLs, osteoblasts; TGFβ, transforming growth factor–β

Figure 2
Figure 2. Schematic representation of osteoclastogenesis.

The transcription factor PU.1, together with M-CSF (macrophage-colony stimulating factor), allow the commitment of hematopoietic stem cells (HSCs) to a common progenitor for macrophages and osteoclasts (CFU-M). RANK expression on pre-osteoclast surface and its interaction with RANKL trigger cellcell fusion and the formation of osteoclasts (OCL).
Mature osteoclasts can resorb bone.

RANKL/RANK signaling

RANKL is a type II membrane protein belonging to the TNF superfamily, while its receptor RANK is a type I membrane protein. Osteotropic hormones and factors such as 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], parathyroid hormone (PTH), prostaglandin E2 (PGE2), and IL-11 upregulate the expression of RANKL in osteoblast/stromal cell plasma membrane. As previously mentioned, RANKL interacts with its receptor RANK, located on the preosteoclast surface, which in turn activates signaling by recruiting adaptor molecules belonging to the TNF-receptor–associated factors (TRAF) family (Figure 3). Indeed, the RANK cytoplasmic tail contains three binding sites for TRAF69 and this interaction is mandatory for osteoclast differentiation, since TRAF6 knockout mice develop osteopetrosis.10 Binding of TRAF6 to RANK induces trimerization of TRAF6, leading to activation of nuclear factor–kappaB (NFkappaB) and of mitogen-activated protein kinases (MAPKs).11

Figure 3
Figure 3. RANKL/RANK pathway activation.

RANKL expressed on the membrane surface of the osteoblast (OBL) interacts with RANK, expressed by preosteoclasts (preOCL). This interaction recruits TRAF6 that
activates NF-κB and c-Fos, the latter dimerizing with c-Jun and forming the AP-1 complex. Finally, NFATc1, AP-1, PU.1, and MITF cooperate to induce transcription of genes involved in osteoclast differentiation.

NF-κB includes a family of dimeric transcription factors, which reside in the cytoplasmunder nonstimulated conditions. However, they enter the nucleus upon cell stimulation by various factors, including RANKL. NF-κB is central to the osteoclastogenic process since the double knockout of the p52 and p50 subunits leads to blockade of osteoclast formation.12

Another transcription factor crucial for osteoclast differentiation is activator protein 1 (AP-1) complex, which consists of c-Fos, c-Jun, and ATF proteins. In particular, c-Fos is specifically induced by RANK and is critical for osteoclastogenesis, since c-Fos knockout mice develop osteopetrosis due to the lack of osteoclasts.13

NF-κB upregulates the expression of another key molecule for osteoclast differentiation, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) transcription factor.14,15 This initial induction requires the interaction of NF-κB with NFATc2, which is recruited to the NFATc1 promoter independently of RANKL stimulation.16 The essential role of NFATc1 in osteoclastogenesis was demonstrated by evidence that NFATc1- deficient embryonic stem cells did not differentiate into osteoclasts, while the ectopic expression of NFATc1 induced osteoclast differentiation also in the absence of RANKL.17

Chromatin immunoprecipitation experiments revealed that NFATc1 is recruited to the NFATc1 promoter region 24 hours after RANKL stimulation, and this occupancy persists during the terminal differentiation of osteoclasts, thus indicating a mechanism of autoamplification.18

In cooperation with AP-1, PU.1, NF-κB, and microphthalmia-associated transcription factor (MITF), NFATc1 regulates the transcription of several target genes involved in osteoclast differentiation and function (Figure 3). These include cathepsin K, calcitonin receptor, tartrate-resistant acid phosphatase (TRAcP),17,19 β3 integrin, osteoclastassociated receptor (OSCAR),7 and dendritic cell–specific transmembrane protein (DC-STAMP), the latter involved in osteoclast fusion.

RANKL/RANK signaling is shared by bone and the immune system

When we talk about the role of RANKL/ RANK in the immune system, we need to point out that RANKL, also known as TNF-related activationinduced cytokine (TRANCE) according to the nomenclature of the immune system, and its receptor RANK, were first identified as molecules expressed by T cells and dendritic cells, respectively, and their physical interaction increased the ability of dendritic cells to stimulate naive T cell proliferation as well as dendritic cell survival.

Therefore, the RANKL/RANK pathway was “born” in an immunologic context. At the same time, bone researchers identified the so called osteoclast differentiation factor (ODF), expressed by the osteoblasts, which increased osteoclast formation,20 and OPG, an osteoblast-derived secreted member of the TNF receptor family, which, in contrast, inhibited osteoclast development and bone resorption acting as a decoy receptor. The molecule able to interact with OPG, named OPG-ligand (OPGL),21 was then identified. Finally, bone researchers and immunologists joined in the conclusion that RANKL/TRANCE, ODF, and OPGL are the same molecule, and that RANKL-expressing T cells can also activate osteoclasts, thus mimicking the effect of osteoblasts. Based on this evidence it is not surprising to find bone loss in patients with disorders characterized by abnormal activation of the immune system, such as rheumatoid arthritis or other chronic inflammatory diseases.

Immunological role of the RANKL/RANK pathway

As far as the role of RANKL in the immune system is concerned, it has been demonstrated that, in addition to bone phenotype, due to the lack of osteoclasts, RANKL-deficient mice show a defect in the development of secondary lymphoid tissue.22 However, these mice do not present a severe immunodeficiency, likely due to the fact that lack of RANKL in T cells is compensated by CD40.23 RANKL also seems to be important for mammary development24 and has been found to be involved in inflammatory bowel diseases by stimulating dendritic cells.25

Figure 4
Figure 4. Ig-like receptors and regulation of osteoclastogenesis.

Interaction of the Immunoglobulin-like receptor (Ig-like receptor) expressed on the pre-osteoclast (OCL) surface, with its ligand, induces phosphorylation of DAP12 or FcRγ , with subsequent activation of calcium signaling. Calcium (Ca2+) promotes activation of both c-Fos and NFATc1 through CAMKIV/CREB and calcineurin, respectively.

Recent evidence identified a role for RANKL as a chemokine that can attract RANK-expressing tumor cells and osteoclasts,26,27 thus pointing to a role of this factor in tumor- induced bony metastases.

Finally, a recent study (2009) identified an unexpected role of RANKL/RANK in the central nervous system, showing that this pathway was involved in thermoregulation and central fever response in inflammation.28

RANKL/RANK–linked diseases

The versatility of the RANKL/RANK axis mirrors the complexity of the diseases in which this pathway is lacking. Among them, osteopetrosis is a rare genetic disorder characterized by sclerosis of the skeleton due to reduced or complete lack of osteoclast function and, as a consequence, impairment of bone resorption.29 This disease is clinically very heterogeneous, ranging from severe to asymptomatic. Autosomal recessive osteopetrosis (ARO) is the most severe form of osteopetrosis, usually diagnosed within the first year of life and in patients with a resultant life expectancy of 3 to 4 years. Similar clusters of patients with ARO harbor mutations in the genes encoding for RANKL and RANK.30,31 In contrast with all the other forms characterized by a normal or increased number of osteoclasts that are unable to resorb bone, obviously this is an osteoclast- poor ARO form.

Importantly, beside bone phenotype, there are also immunological defects, such as hypogammaglobulinemia due to impairment in immunoglobulin-secreting B cells. This is in line with evidence showing the importance of RANKL/RANK in the immune system, which should be taken into account in the management of this form of ARO. Indeed, it has been demonstrated that two ARO patients harboring RANK mutations exhibited impaired fever during pneumonia.28

Ig-like receptors and osteoclast regulation

Beside the well-known RANKL/RANK pathway, osteoblasts can regulate osteoclast differentiation by interacting with immunoglobulin (Ig)-like receptors, such as OSCAR, whose ligand has not yet been clearly identified. These receptors are associated with immunoreceptor tyrosine-based activation motif (ITAM)-harboring adaptor molecules DAP12 and Fcreceptor common gamma subunit (FcRγ). The role of the latter molecules in osteoclast regulation has been underlined by evidence that mice deficient in both DAP12 and FcRγ have an osteopetrotic phenotype.32 Phosphorylation of the ITAM sequence in DAP12 or FcRγ, resulting after RANK activation, allows the recruitment of splenocyte tyrosine kinase (SYK) and resultant activation of phospholipase C gamma (PLCγ), which in turn triggers calcium signaling. Calcium signaling promotes osteoclastogenesis by activating calcium/calmodulin-dependent protein kinase type IV (CAMKIV), which concurs to c-Fos and calcineurin activation, both cooperating to potentiate NFATc1 autoamplification (Figure 4).1 Among the molecules that have a dual role in the regulation of immune cells and osteoclasts, a recent study33 identified the transcription factor B lymphocyte-induced maturation protein–1 (Blimp1). This is a transcriptional repressor involved in the differentiation of B lymphocytes toward plasma cells by direct repression of the transcription factors Pax5, Bcl, and Myc.34 Nishikawa and colleagues demonstrated that Blimp1 stimulates osteoclastogenesis by repressing the transcription factors IFN regulatory factor-8 (IRF-8) and v-Maf musculo-aponeurotic fibrosarcoma oncogene family, protein B (MafB), both negatively affecting osteoclastogenesis.35,36

Inflammatory cytokines and osteoclastogenesis

Among the cells of the immune system, T lymphocytes play a crucial role in the regulation of osteoclastogenesis, which is indeed the result of a balance between positive and negative factors produced by T cells. As far as the RANKL/ RANK pathway is concerned, it has been demonstrated that activated T cells express RANKL on their surface, thus activating osteoclastogenesis by cell–cell contact.37 Activated T cells also produce IL-10, IL-12, and IL-18, which, in contrast, negatively affect osteoclastogenesis.38 As described below, the CD4+ T helper (TH)-cell subset TH1 and TH2 produce interferon gamma (IFN-γ), which suppresses RANKL signaling by degrading TRAF6, and IL-4, another cytokine with an anti-osteoclastogenic role.

Other cells of the immune system, such as the macrophages, contribute to osteoclast differentiation and function by producing IL-1, IL-6, and TNF-α.20,39 Moreover, a recent study40 showed that lipopolysaccharides (LPS) upregulated the expression of membrane RANKL in human blood neutrophils. LPS-activated neutrophils were then able to stimulate osteoclastogenesis and bone resorption in co-cultures with osteoclast precursors.

Finally, a recent report from Rifas and Weitzmann41 described the identification of a new T cell cytokine, called secreted osteoclastogenic factor of activated T cells (SOFAT), which induces both osteoblastic IL-6 production and osteoclast formation in the absence of osteoblasts or RANKL, and was insensitive to the effects of the RANKL inhibitor OPG.

Immune diseases and osteoclast activation

_ Rheumatoid arthritis
One of the milestones that was pivotal in defining the new field of osteoimmunology came from research by Takayanagi et al on rheumatoid arthritis.1 This is an autoimmune disease characterized by inflammation of synovial joints, with CD4+ T-lymphocyte infiltration and synovial cell proliferation, leading to severe bone destruction mediated by osteoclasts.42 The clinical feature of bone loss is not restricted to the affected joints, since systemic osteoporosis can also occur.43 Although the increased inflammatory cytokine levels present in affected joints may contribute to enhanced osteoclastogenesis, the mechanism of systemic osteoporosis associated with arthritis remains unclear.

Recent reports have highlighted the crucial role of osteoclasts in the development of this disease. Indeed, it has been demonstrated that osteoclast-deficient mice were protected from bone erosion in arthritis models, despite the presence of inflammation.44,45 Moreover, high RANKL expression has been detected specifically in the synovium of rheumatoid arthritis patients.46 In rheumatoid arthritis affected joints, different cell types can be found, including macrophages, fibroblasts, dendritic cells, plasma cells, and CD4+ T helper cells. Takayanagi1 demonstrated that in the latter cell type there is a subpopulation, defined as osteoclastogenesis TH cells (THO cells), which, at variance with CD4+ TH 1 cells, does not produce the anticlastogenic cytokines IFN-γ and IL-4, but secretes high quantities of IL-17. This cytokine, in turn, triggers RANKL expression by synovial fibroblasts. IL-17 also stimulates local inflammation, thus inducing macrophages to secrete proinflammatory cytokines such as TNF-α, IL-1, and IL-6. These cytokines in turn activate osteoclastogenesis, directly as well as by stimulating RANKL expression by synovial fibroblasts. Finally, it has been shown that THO cells themselves express RANKL, thus activating osteoclastogenesis by direct induction of precursor differentiation.

_ Psoriatic arthritis
This is a disease characterized by musculoskeletal inflammation, and several studies have reported the crucial role of TNF in its pathogenesis. Elevated levels of TNF have been found in the sera, synovial fluid, and synovial membranes of psoriatic patients.47 A marked reduction in inflammation and progressive joint damage was consistently observed in patients treated with anti-TNF drugs, which is not only due to their ability to reduce inflammation, but also to reduce osteoclast activation, since it is well known that TNF promotes osteoclast formation. On the other hand, recent reports showed that TNF can affect bone formation by inducing Dickkopf-related protein 1 (DKK-1) to impair bone-forming osteoblast development via inhibition of Wnt signaling.

Is the osteoclast an immune cell?

Based on the aforementioned evidence, it has been hypothesized that osteoclasts are cells that belong to the immune system.48 This raises the question as to why there is a need for an immune cell to resorb bone.49 Chambers50 had previously proposed that the bone matrix is recognized by osteoclasts as a peculiar “foreign body.” In fact, as described in the above (see the bone remodeling process), during the resting condition the bone matrix is covered by a layer of osteoblasts, or lining cells (Figure 1), which segregates the bone matrix from the interstitial fluid, thus probably preventing recognition by the immune system. An external stimulus, such as an inflammatory response, or exposure to PTH/parathyroid hormone– related protein (PTHrP), could trigger osteoblast retraction, so that the mineralized bone matrix can be exposed and recognized as a “foreign body” by immune cells, which have all the requirements to induce osteoclast formation and bone resorption.

Under physiological conditions, this process, once activated, must be switched off and, in fact, there are several paracrine and autocrine mechanisms that negatively regulate osteoclast activity. Consequently, osteoblasts are recalled in the previously resorbed site where they refill the lacunae with newformed bone matrix and again segregate the bone surface from the interstice so that “foreign bone” is no longer exposed to the immune system. If the negative regulation of osteoclast activity fails, this process proceeds longer than necessary, thus resulting in excess bone resorption, with pathological consequences.

Conclusions

It is now clear that bone is a tissue of central importance, therefore, when we study the molecular mechanisms underlying bone remodeling and bone pathological events, we cannot ignore its multiple interactions with other tissues. The discipline of osteoimmunology has shown that osteoclasts and immune cells share a common origin. These two types of cells arise from the HSCs in the bone marrow, another organ closely related to bone. Immunology has also clarified the involvement of bone cells in the development of diseases initially classified in an immunological context, and has identified the central role of some cytokines, known to be produced by immune cells, in the regulation of bone cells. Furthermore, recent advances suggest the potential involvement of osteoclasts and osteoblasts in the regulation of HSCs directed to an immunological commitment.We believe that these findings should encourage immunologists and bone researchers to continue investigating this field, all the more so as better understanding of the relationships between bone and immune cells could help identify new strategies for the management of patients suffering from bone diseases. _

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Keywords: osteoimmunology; bone tissue; immune system; hematopoietic stem cell; osteoclast; cytokine; rheumatoid arthritis; bone remodeling