33.1

Receptor activator of nuclear factor-κB ligand, receptor activator of nuclear factor-κB, and osteoprotegerin

Receptor activator of nuclear factor-κB (RANK) ligand (RANKL) is tumor necrosis factor (TNF) superfamily ligand 11 (TNFSF11) and together with its RANK and osteoprotegerin (OPG) (TNFRSF11A and TNFRSF11B, respectively) forms the critical paracrine system, which regulates osteoclast formation .

The discovery of RANKL, which has potent activity to increase the formation of osteoclasts from precursor cells and the bone-resorbing activity and life span of mature osteoclasts, defined a new paradigm for how a variety of cells in the bone marrow microenvironment regulate bone resorption . RANKL directs the terminal differentiation of osteoclast precursor cells. In addition, it stimulates and maintains the resorptive activity of mature cells. Most importantly, this activity of RANKL is reproduced in vitro in the absence of bone marrow stromal cells .

RANKL-deficient mice have significant osteopetrosis and no osteoclasts, but a normal number of monocyte/macrophages . They also fail to erupt teeth, which is a common finding in all causes of developmental osteopetrosis. Marrow stromal cells, hypertrophic chondrocytes, osteoblasts, and osteocytes produce RANKL . In addition, the production of RANKL by B-lymphocytes is important for regulating the enhanced bone resorption and decreased bone mass that occurs after ovariectomy in mice , while T-lymphocyte-generated RANKL may mediate bone loss in patients with HIV infection . Targeted gene deletion of RANKL in the osteocytes and maturing osteoblasts of mice demonstrated the critical nature of RANKL expression by these cells for the maintenance of osteoclastogenesis after fetal development . Recently, it was found that membrane-bound RANKL can bind RANK on vesicles, which are secreted by maturing osteoclasts, and reverse signal into osteoblasts to regulate their activity .

RANKL exists either in a cell membrane-bound or soluble form . Loss of soluble RANKL increased bone mass and decreased osteoclast number in adult, but not in growing mice. However, deletion of soluble RANKL had no effect on the bone loss that occurs after ovariectomy . Many well-known resorption stimulators, including cytokines and hormones, exert their primary osteoclastogenic activity by inducing RANKL expression in mesenchymal lineage cells . Conversely, the shedding of membrane-bound RANKL is a mechanism for inhibiting osteoblast-mediated osteoclast formation by removing RANKL from the cell surface of osteoblasts . This process depends on expression of matrix metalloproteinase (MMP) 14 , since mice that lacked this enzyme have increased osteoclast number.

OPG is a novel, secreted inhibitor of osteoclast formation that acts as a decoy receptor for RANKL . It was initially identified as a soluble factor, having inhibitory effects on osteoclastogenesis in vitro , and an inducer of osteopetrosis when it was transgenically overexpressed in mice . In marrow, it is produced by a variety of cells including stromal cells, B-lymphocytes, and dendritic cells . Besides RANKL, OPG also binds the TNF-like ligand TRAIL (TNF-related apoptosis inducing ligand) . Mice that lack OPG have severe osteoporosis, an increased number of osteoclasts and arterial calcification . The latter finding highlights a potential genetic link between osteoporosis and vascular calcification . Overexpression of OPG in transgenic mice caused osteopetrosis, decreased osteoclast numbers, and extramedullary hematopoiesis . Serum levels of OPG increase with age in humans and are directly correlated with whole body bone mass . Conversely, serum titers of OPG antibodies are inversely correlated with bone mass . Genetic inactivation of OPG is a cause of juvenile Paget’s disease , while decreases in OPG are associated with glucocorticoid-induced osteoporosis in animal and in vitro models .

The biologically active receptor for RANKL is RANK. It was first identified on dendritic cells , but it is also present on osteoclast precursors and mature osteoclasts . RANK expression at the RNA level is detected in a variety of cell types and tissues . RANK-deficient mice phenocopy the defective osteoclast development of RANKL-deficient mice, confirming the exclusive specificity of RANKL for RANK . In humans, gain-of-function mutations in RANK are associated with familial expansile osteolysis, expansible skeletal hyperphosphatasia, and juvenile Paget’s disease, all of which are diseases that are caused by excessive formation and activity of osteoclasts .

While osteoclast-like cells can form in vitro in the absence of RANK or TNF receptor associated factor (TRAF) 6 signaling when exposed to a cocktail of cytokines and growth factors , the significance of this in vitro finding is controversal since osteoclasts are generally absent in RANK-deficient animals . In most instances, cytokines and growth factors other than RANKL, which are produced at sites of inflammation or physiologically during bone turnover, act as cofactors that enhance or modulate the response of osteoclasts and their precursors to RANKL–RANK stimulation . However, it was demonstrated that in the absence of the p100 precursor of NF-κB, TNFα was a potent stimulator of osteoclastogenesis . In addition, in inflammatory arthritis RANKL-independent osteoclastogenesis in vivo has been observed .

33.2

Colony-stimulating factor-1

In addition to RANKL, colony-stimulating factor (CSF)-1 (also known as macrophage CSF) is critical for normal osteoclast formation. This cytokine was originally identified by its ability to regulate macrophage formation . However, it was subsequently shown that a spontaneous mouse mutant (the op / op mouse) with a phenotype of absent osteoclasts and defective macrophage/monocyte formation was deficient in CSF-1 . Injection of CSF-1 into op / op mice corrected the defect in osteoclast formation and bone resorption , as did the expression of CSF-1 protein specifically in osteoblastic cells .

Stimulators of bone resorption can increase the production of CSF-1 in bone , and multiple transcripts of CSF-1 are produced by alternative splicing resulting in either a soluble or membrane-bound form of the protein . In vivo treatment with CSF-1 increased osteoclast number and bone resorption as well as the rate of fracture repair . Expression of the membrane-bound form of CSF-1 is regulated by stimulators of resorption and facilitates the differentiation of precursor cells into mature osteoclasts . However, the deletion of just the soluble form of CSF-1 in mice prevented the bone loss that occurs with estrogen deficiency . This result demonstrates the critical importance of soluble CSF-1 in mediating bone loss in this animal model of postmenopausal osteoporosis.

CSF-1 has multiple effects on osteoclast precursors. It stimulates their replication and differentiation , and it regulates their motility . In mature osteoclasts, CSF-1 augments RANKL-induced resorptive activity . Environmental factors appear to modulate the response of osteoclast precursor cells to CSF-1 since it was found that there were differences in in vitro responses depending on whether cells were cultured on plastic or bone .

CSF-1 receptor (CSF-1R, also known as c-Fms), which binds CSF-1 to activate intracellular signaling, is a tyrosine kinase . Signaling through CSF-1R involves the immunoreceptor tyrosine-based activation motif–containing protein, DAP12, and β-catenin .

The role of CSF-1 in regulating osteoclast apoptosis has also been examined. Addition of CSF-1 to mature osteoclast cultures prolongs their survival . This response may be important for the development of the osteopetrotic phenotype in op / op mice, since transgenic expression of B-cell CLL/lymphoma 2 (Bcl-2), which blocks apoptosis in myeloid cells, partially reversed the defects in osteoclast and macrophage development in these animals . The effects of CSF-1 on osteoclasts has been linked to the activation of a Na/HCO cotransporter . CSF-1 is also a potent stimulator of RANK expression in osteoclast precursor cells and is critical for expanding the osteoclast precursor pool size . Strategies are being developed to inhibit CSF-1 signaling as a mechanism to treat disease .

Most recently, interleukin (IL)-34 was found to be an additional ligand for CSF-1R . Like CSF-1, it can be added with RANKL to in vitro cultures to stimulate osteoclastogenesis . Furthermore, injection of IL-34 into mice increased the proportion of CD11b-positive cells, which contain osteoclast precursors, and decreased trabecular bone mass . Production of IL-34 may be responsible for the spontaneous rescue of the osteoclast phenotype that occurs in op/op mice with age and may also explain why the CSF-1R deficient mouse has a more severe phenotype than the op/op mouse . Differences in the ability of CSF-1R and IL-34 to polarize macrophages into M1 and M2 subsets have been identified .

33.3

Additional colony stimulating factors

Like CSF-1, the CSFs, granulocyte macrophage CSF (GM-CSF) and IL-3, affect osteoclast differentiation . Both have complex actions that are dependent on the lineage stage of the myeloid precursor cells with which they interact. IL-3 is produced by osteoblasts . It has multiple effects on in vitro osteoclastogenesis , which have been associated with both stimulatory and inhibitory responses, depending on the cells that are examined and the culture conditions . One mechanism by which IL-3 inhibits osteoclastogenesis is through the regulation of c-Fos and Id protein expression . Another is through differential regulation of soluble and membrane-bound RANKL expression in osteoblastic lineage cells . IL-3 is also reported to inhibit osteoblast differentiation in multiple myeloma and to enhance mesenchymal stem cell differentiation into osteoblasts .

In early multipotential myeloid precursors, GM-CSF inhibits RANKL-mediated osteoclastogenesis and enhances the number of osteoclast precursor cells . It does this by directing the common myeloid precursor cell toward the dendritic cell lineage . One mechanism for this effect is increased shedding of CSF-1R through upregulation of “a disintegrin and metalloproteinase 17” (ADAM17), which is also known as TNFα converting enzyme . Interestingly, GM-CSF-expanded dendritic lineage cells can still maintain their capacity to differentiate into osteoclasts and may be a source of osteoclast progenitors in inflammatory conditions .

GM-CSF also inhibits the expression of monocyte chemotactic protein 1 [MCP-1, C-C motif chemokine ligand (CCL)2] by osteoclast precursor cells . MCP-1 is a chemokine involved in osteoclast motility. Both GM-CSF and IL-3 inhibit the expression of TNF receptors on myeloid precursor cells . However, in prefusion osteoclast precursors, which are myeloid cells that have been stimulated with RANKL and CSF-1 for 3 days to a point where they will shortly (within 6 hours) fuse into osteoclasts, treatment with GM-CSF+CSF-1 enhanced osteoclastogenesis and mimicked the response to RANKL+CSF-1 . This increased osteoclastogenesis was mediated by upregulation of the cell fusion protein, dendritic cells-specific transmembrane protein in the prefusion osteoclasts . It has also been shown that if multipotential myeloid precursor cells are cultured sequentially with GM-CSF and then with CSF-1+RANKL, they form osteoclasts and, in some instances, act as dendritic cells, which present antigen to T-lymphocytes and initiate the adaptive immune response . IL-3 and GM-CSF may also support osteoclast differentiation by stimulating CSF-1 production .

At relatively high doses granulocyte-colony stimulating factor (G-CSF) decreased bone mass in rodents when injected systemically . This response appeared to result from increased osteoclast formation, decreased osteoblast function, and increased osteoblast apoptosis. Similar effects are seen in humans . G-CSF also mobilized the migration of hematopoietic precursor cells from the bone marrow into the circulation and increased the number of circulating osteoclast precursor cells , which may be related to its ability to increase osteoclast resorptive activity.

In mice, overexpression of G-CSF inhibited the ability of osteoblasts to respond to bone morphogenetic protein . Short-term treatment of mice with G-CSF decreased endosteal and trabecular osteoblasts by increasing their apoptosis and inhibiting osteoblast precursor cell differentiation . Mice overexpressing G-CSF had increased bone resorption, which, in contrast to wild type mice, was not increased with ovariectomy . Targeted deletion of ADAM17 in mice using Cre/Lox technology and the Sox9 promoter to target Cre recombinase to chondroprogenitor cells, produced a phenotype of enhanced G-CSF production, osteoporosis and extramedullary hematopoiesis . These results suggest that the production of ADAM17 on osteoblasts and/or chondroblasts regulates the expression of G-CSF.

33.4

Interleukin-1

There are two separate IL-1 genes, IL-1α and IL-1β, which have identical activities . IL-1 is a potent peptide stimulator of bone resorption . It directly affects RANKL-primed osteoclasts to resorb by a mechanism dependent on microphthalmia transcription factor . It also has indirect resorptive actions through its ability to stimulate RANKL production . In addition, both RANKL- and 1,25-dihydroxyvitamin D ₃ -stimulated osteoclast formation in vitro was mediated, in part, by their effects on IL-1 production . IL-1 also increases prostaglandin synthesis in bone , which may enhance its resorptive activity since prostaglandins are potent resorption stimuli . Direct stimulation of osteoclastogenesis by IL-1 in mixed murine stromal and hematopoietic cell cultures is dependent on RANKL but not TNFα expression in the stromal/osteoblastic cells .

In mouse models, IL-1 appears to be involved in normal growth plate development and bone turnover . It also may be essential for the systemic bone loss that is seen in some inflammatory conditions due to high TNF production . In addition, it mediates some of the effects of estrogen withdrawal on bone loss in both mice and humans . In humans, levels of IL-1have been negatively correlated to measurements of bone mass .

IL-1 is produced in bone , and its activity is present in bone marrow serum . One source of bone cell-derived IL-1 is osteoclast precursor cells, which produce IL-1 when they interact with bone matrix . There is also a natural inhibitor of IL-1, IL-1 receptor antagonist, which is an analog of IL-1 that binds, but does not activate the biologically important type I IL-1 receptors .

There are two known receptors for IL-1: type I and type II . All known biologic responses to IL-1 appear to be mediated exclusively through the type I receptor . IL-1 receptor type I requires interaction with a second protein, IL-1 receptor accessory protein, to generate postreceptor signals . Signaling through type I receptors involves the activation of specific TRAFs and NF-κB . IL-1 receptor type II is a decoy receptor that prevents activation of type I receptors . One report found a decrease in the bone mass of mice that were deficient in the bioactive type I IL-1 receptor . However, this has not been our experience .

Expression of myeloid differentiation factor 88 but not Toll/IL-1 receptor domain-containing adapter inducing interferon-beta (TRIF) was necessary for IL-1 to stimulate RANKL production in osteoblasts and to prolong the survival of osteoclasts . Survival of osteoclasts by treatment with IL-1 required PI3-kinase/AKT and extracellular signal-regulated kinase (ERK) .

The effects of IL-1 on bone in inflammatory states, such as rheumatoid arthritis, are multiple and mediated by both direct and indirect mechanisms. IL-1 stimulates bone resorption and inhibits bone formation through its effects on osteoclasts and osteoblasts, respectively . In inflammatory conditions, it is directly involved in the production of a relatively unique population of very active osteoclasts . In addition, it stimulates the production of a variety of secondary factors in the bone microenvironment including prostaglandins and GM-CSF, which have complex effects on bone cells themselves . IL-1 also has been reported both to inhibit and to stimulate the production of OPG in various osteoblastic cell models in vitro . IL-1’s effects on osteoclast precursor cell migration are mediated by its ability to stimulate production of the chemokine CX3CL1 .

IL-1 induces the differentiation of mesenchymal stem cells toward osteoblasts via the noncanonical Wnt-5a/Ror2 pathway . However, it also inhibits osteoblast migration and the ability of mesenchymal stem cells to promote tissue regeneration . IL-1 also is involved in the mechanisms of mechanosensing in bone . Osteocytes regulate osteoclastogenesis by producing a variety of factors including RANKL . Treatment of the MLO-Y4 osteocyte-like cell line with IL-1 enhanced RANKL and decreased OPG production, which was reversed by mechanically loading the cells .

33.5

Tumor necrosis factor

Like IL-1, TNF represents a family of two related polypeptides (α and β) that are the products of separate genes . TNFα and TNFβ have similar biologic activities and are both potent stimulators of bone resorption .

In vivo administration of TNFα was shown to increase the serum calcium of mice and to stimulate new osteoclast formation and bone resorption . Like IL-1, TNF also enhances the formation of osteoclast-like cells in bone marrow culture through its ability to increase RANKL production . However, RANKL is not the only cytokine that TNF stimulates in bone, and many of these enhance the response to RANKL. For example, TNF stimulates osteoclast formation in mixed stromal cell/osteoclast precursor cell cultures by a mechanism that was partially dependent on the production of IL-1 . In addition, TNF-induced osteolysis was found to be dependent on CSF-1 production .

TNF can also directly stimulate osteoclast formation in vitro, independent of RANK, since it occurs in cells from RANK-deficient mice . However, the significance of this in vitro finding is controversial. In vivo administration of TNF to RANK-deficient mice caused only an occasional osteoclast to form . In addition, RANK-deficient mice that also overexpressed TNF had severe osteopetrosis and no osteoclasts . It was also demonstrated that TNF can stimulate osteoclastogenesis in mice that are deficient in the p100 precursor protein of NF-κB, which is a critical signaling molecule in RANKL-mediated stimulation of osteoclastogenesis and bone resorption . These results demonstrate that even though both TNF and RANKL are members of the same cytokine superfamily and share multiple overlapping signaling pathways, there are crucial differences between their downstream signaling molecules in osteoclast precursor cells. It has also been shown that in cherubism, a disease with elevated production of TNF due to a gain-of-function mutation in the SRC homology 3 domain binding protein 2 (Sh3bp2) gene, bone resorption can occur through the action of cells that are not classic osteoclasts . There may also be a role for sclerostin, a Wnt signaling inhibitor, in the ability of TNF to induce inflammatory arthritis since deletion or inhibition of sclerostin exacerbated the inflammatory joint destruction seen in TNF transgenic mice .

As with IL-1, TNF binds to two cell surface receptors, TNF receptor 1 or p55, and TNF receptor 2 or p75 . In contrast to IL-1, both receptors transmit biologic responses. However, the principal effects on bone cells appear to be mediated through TNF receptor 1 . Mice deficient in both TNF receptor 1 and TNF receptor 2 have been produced . These animals appear healthy and are not reported to have an abnormal bone phenotype.

TNF can stimulate the expression of the CSF-1R in osteoclast precursor cells and, through this mechanism, increase their number . It also enhances RANK signaling, which activates osteoclasts and their precursor cells and enhances the expression of the costimulatory molecule, paired Ig-like receptor A, which enhances nuclear factor of activated T cells cytoplasmic 1 (NFATc1) activation . Recently, it was found that RANKL enhances TNF-induced osteoclastogenesis independent of effects on TRAF6 by degrading TRAF3 in osteoclast precursor cells . Mice that overexpress TNF have an increased number of CD11b high osteoclast precursor cells in their spleen and blood . The enhancement of osteoclastogenesis that occurs with TNF signaling involves multiple pathways, including phosphoinositide 3-kinase/AKT (PI3K/Akt)-mediated Blimp1 expression and recombination signal binding protein for immunoglobulin kappa J region-mediated regulation of miR-182 .

TNF has biphasic effects on bone formation and osteoblast function, which may be dose and time related . At lower doses, it stimulated the differentiation of mesenchymal precursor cells into osteoblasts , while at higher concentrations it inhibited osteoblast function and bone formation . It was reported to promote fracture repair by enhancing the recruitment of precursor cells to the osteoblastic lineage . The inhibitory effects of TNF on osteoblasts appear to be direct and mediated by downregulation of the critical transcription factor genes, RUNX2 and osterix . TNF has many actions on osteoblasts including inhibiting type 1 collagen and osteocalcin synthesis , which are essential for differentiated osteoblast function. It also stimulated osteoblast apoptosis , suppressed production of insulin-like growth factor (IGF)-1 and downregulated EphB4 signaling . Some of the inhibitory effects of TNF on osteoblast differentiation are mediated by activated transcription factor 3 through a pathway involving c-Jun N-terminal kinase (JNK) .

Most recently, patients with inflammatory diseases who were treated with anti-TNF therapy were found to have increased bone mineral density . However, in a study of patients with inflammatory bowel disease who were treated with anti-TNF therapy, the incidence of fractures was not affected even though bone mass increased . This result suggests that circulating TNF, which is produced by localized inflammatory pathology, has systemic effects on bone mass but not necessarily on bone strength. Production of TNF on B-lymphocytes may also contribute to the osteoclastogenesis produced in periodontitis . Finally, a polymorphism in the TNF-α gene has been associated with the risk of developing osteoporosis in women .

33.6

Additional tumor necrosis factor superfamily members

33.7

Interleukin-6

IL-6, like IL-1 and TNF, has a wide variety of activities on immune cell function and on the replication and differentiation of a number of cell types . Osteoblastic cells (both rodent and human) produce IL-6 and IL-6 receptors . Another source of IL-6 in the bone microenvironment is bone marrow stromal cells, which can produce IL-6 after they are stimulated with IL-1 and TNF . The receptor for IL-6 is composed of two parts: a specific IL-6 binding protein (IL-6 receptor), which can be either membrane-bound or soluble, and gp130, an activator protein that is common to a number of cytokine receptors . Soluble IL-6 receptor binds IL-6, and this complex can then activate cells that contain the gp130 signal peptide . The shedding of IL-6 receptor from osteoblasts is stimulated by IL-1 and TNFα .

The ability of IL-6 to affect bone resorption in vitro is variable and depends on the assay system that is used as both stimulatory and inhibitory effects have been observed . It appears that a major effect of IL-6 is to regulate the differentiation of osteoclast progenitor cells into mature osteoclasts . IL-6 also directly stimulates both RANKL and OPG mRNA production in bone and enhances production of prostaglandins . In addition, one publication suggested that IL-6 can stimulate osteoclastogenesis in vitro by a RANKL-independent mechanism . In contrast, two other publications found IL-6 to directly inhibit RANKL signaling in osteoclast precursor cells and decrease osteoclast formation .

Examination of IL-6-deficient mice at 8 months of age demonstrated that bone mass was increased, as was tartrate-resistant acid phosphatase (TRAP)-positive osteoclast number and alkaline phosphatase activity in osteoblasts. However, curiously, cathepsin K-positive osteoclasts were decreased in IL-6-deficient mice suggesting that loss of IL-6 inhibited osteoclast maturation and in this way enhanced bone mass. Some of this discrepancy may be related to differences in the responses of cortical and trabecular bone to membrane-bound and soluble IL-6 receptor . There was also enhanced apoptosis of osteoclasts in IL-6-deficient mice . Deletion of IL-6 in mice promoted fracture healing , and IL-6 appears to influence the bone loss that is seen in mice on a high fat diet .

IL-6 has a variety of effects on mesenchymal cells. It promotes osteogenic differentiation of bone marrow mesenchymal cells but inhibits more mature osteoblast differentiation through the activation of the Janus kinase/signal transduction and activators of transcription (JAK/STAT), Src homology region 2 domain-containing phosphatase-2/dual specificity mitogen-activated protein kinase kinase 2 (SHP2/MEK2) and SHP2/AKT pathways . It also inhibits osteogenesis in glucocorticoid-treated mice . Osterix, a key transcription factor in early osteoblast differentiation, appears to inhibit IL-6 production since mice heterozygous for osterix deficiency had increased IL-6 production . In contrast, bone morphogenetic protein, which enhances osteoblast differentiation, was shown in vitro to enhance IL-6 production in osteoblasts .

IL-6 may mediate some of the bone pathology that is seen with aging and in a variety of clinical syndromes including Paget’s disease , hypercalcemia of malignancy , fibrous dysplasia , giant cell tumors of bone inflammatory states mediated by TNF or RANKL and Gorham–Stout disease . There has been conflicting data about the role of IL-6 in PTH-mediated responses in bone as some investigators have found it critical while others have not . Inhibition of the IL-6 receptor blocked osteoclastogenesis in vitro and in vivo .

In mice, increased IL-6 expression augmented the effects of p62/sequestosome-1 mutations (which is linked to the development of Paget’s disease in humans) but did not fully reproduce the Pagetic phenotype, suggesting that additional mechanisms are involved . It was also shown in mice that measles virus nuclear capsid protein, which is also implicated as driving the development of Paget’s disease, increased IL-6 expression .

IL-6 is a mediator of inflammation, and IL-6 inhibition is now used therapeutically to treat inflammatory diseases, such as rheumatoid arthritis . Recently, anti-IL-6 therapy was shown to reduce systemic bone loss and osteoclast precursor cell number in a mouse model of rheumatoid arthritis . Furthermore, in humans with rheumatoid arthritis, serum levels of IL-6 inversely correlated with bone mass and directly correlated with disease activity .

33.8

Additional interleukin-6 family members

IL-6 is a member of a group of cytokines that share the gp130 activator protein in their receptor complex . Each family member utilizes unique ligand receptors to generate specific binding. Signal transduction through these receptors utilizes the JAK/STAT pathway .

33.9

Interleukin-7

IL-7 is a cytokine that has diverse effects on the hematopoietic and immunologic systems and is best known for its nonredundant role in supporting B- and T-lymphopoiesis. Studies have demonstrated that IL-7 also plays an important role in the regulation of bone homeostasis . However, the precise nature of how IL-7 affects osteoclasts and osteoblasts is controversial, since it has a variety of actions in different target cells. Systemic administration of IL-7 upregulated osteoclast formation from human peripheral blood cells by increasing osteoclastogenic cytokine production in T-lymphocytes . Furthermore, mice with global overexpression of IL-7 had a phenotype of decreased bone mass with increased osteoclast and no change in osteoblast number . Significantly, IL-7 did not induce bone resorption and bone loss in T-cell-deficient nude mice in vivo . In addition, treatment of mice with a neutralizing anti-IL-7 antibody inhibited ovariectomy-induced proliferation of early T-cell precursors in the thymus, demonstrating that ovariectomy upregulates T-cell development through IL-7. This latter effect may be a mechanism by which IL-7 regulates ovariectomy-induced bone loss . However, the interpretation of results from in vivo IL-7 treatment studies is complicated by secondary effects of IL-7, which result from the production of bone-resorbing cytokines by T-cells in response to activation by IL-7 .

In contrast with previously reported studies , we found differential effects of IL-7 on osteoclastogenesis . IL-7 inhibited osteoclast formation in murine bone marrow cells that were cultured for 5 days with CSF-1 and RANKL . Furthermore, IL-7-deficient mice had markedly increased osteoclast number and decreased trabecular bone mass compared to wild type controls . In addition, we found that trabecular bone loss after ovariectomy was similar in wild type and IL-7-deficient mice . Other investigators have found that IL-7 could stimulate osteoclastogenesis by a mechanism that was independent of RANKL but dependent on STAT5, and IL-7 was found to decrease bone mass and increase RANKL through a mechanism dependent on cFos/c-Jun .

IL-7 mRNA levels in bone increase with ovariectomy and this effect may be linked to alterations in osteoblast function with estrogen withdrawal . Treatment of newborn murine calvaria cultures with IL-7 inhibited bone formation, as did the injection of IL-7 above the calvaria of mice in vivo . When IL-7 was overexpressed locally by osteoblasts, trabecular bone mass was increased compared with wild type mice . Furthermore, targeted overexpression of IL-7 in IL-7-deficient mice rescued the osteoporotic bone phenotype of the IL-7-deficient mice . These studies indicated that the actions of IL-7 on bone cells are dependent on whether IL-7 is delivered systemically or locally. Production of IL-7 by osteoblasts appeared critical for normal B-lymphopoiesis and was dependent on mechanistic target of rapamycin complex 1 signaling in osterix-expressing cells . In sepsis, B-lymphopoiesis decreases through a mechanism in which osteoblastic cells are depleted as presumably is IL-7 production by these cells . The induction of this cytokines in osteoblasts is mediated by G _s α-dependent signaling. Osteoclast mediated bone resorption can influence B-lymphopoiesis through effects on local IL-7 production in the bone marrow .

33.10

Interleukin-8 and other chemokines

Recruitment and homing of myeloid cells often occurs under the direction of chemokines and their receptors. This family of relatively small proteins induces interactions through cognate G-protein-coupled receptors to initiate cytoskeletal rearrangement, adhesion, and directional migration . Chemokines can be divided into four branches, depending on the spacing and sequence motifs of their first cysteine (C) residues. These are CXC, CC, C, and CX ₃ C, where X is any other amino acid . The majority of chemokine receptor interactions occur through the CC and CXC chemokines, which are referred to as major, while C and CX ₃ C chemokines are referred to as minor.

33.11

Interleukin-10

IL-10 is produced by activated T- and B-lymphocytes . It is a direct inhibitor of osteoclastogenesis and osteoblastogenesis , which are effects that are associated with increased tyrosine phosphorylation of multiple proteins in osteoclast precursor cells . The direct effects of IL-10 on RANKL-stimulated osteoclastogenesis include decreases in NFATc1 expression, reduced translocation of this transcription factor into the nucleus and suppressed c-Fos and c-Jun expression . Administration of IL-10 may have utility as a mechanism to control wear-induced osteolysis and the alveolar bone loss of periodontal disease . In dental follicle cells, which function to regulate tooth eruption, in vitro treatment with IL-10 inhibited RANKL production and enhance OPG . Hence, there appears to also be an indirect effect of IL-10 on osteoclastogenesis that is mediated by its ability to regulate RANKL and OPG production.

Treatment of bone marrow cell cultures with IL-10 suppressed the production of osteoblastic proteins and prevented the onset of mineralization . IL-10 also inhibited osteoclast formation in bone marrow cultures without affecting macrophage formation or the resorptive activity of mature osteoclasts . This effect appears to involve the production of novel phosphotyrosine proteins in osteoclast precursor cells . IL-10 also stimulates a novel inducible nitric oxide synthase . IL-10-deficient mice have decreased alveolar bone and decreased indices of osteoblast differentiation . A polymorphism in the IL-10 gene has been linked to postmenopausal osteoporosis .

4-1BB is an inducible T-cell costimulatory molecule, which interacts with 4-1BB ligand. In vitro treatment of RANKL-stimulated osteoclast precursor cells with 4-1BB ligand enhanced IL-10 production. In addition, expression of IL-10 was greater in RANKL-stimulated wild type osteoclast precursor cell cultures than in cultured cells from 41-BB-deficient mice . These results imply that some effects of IL-10 on osteoclasts may be mediated through the interactions of 4-1BB with 4-1BB ligand.

33.12

Interleukin-12, interleukin-23, interleukin-27, and interleukin-35

IL-12 is a cytokine that is produced by myeloid and other cell types. It induces T _H 1 differentiation in T-lymphocytes and the subsequent expression of INF γ . Most authors have found that IL-12 has an inhibitory effect on osteoclastogenesis. However, the mechanisms by which this effect occurs in vitro are controversial. Some authors demonstrated direct inhibitory effects of IL-12 on RANKL-stimulated osteoclastogenesis in purified primary osteoclast precursors and RAW 264.7 cells . This was associated with the inhibition of NFATc1 expression in the osteoclast precursor cells. Interestingly, the inhibitory effects of IL-12 on osteoclastogenesis were absent in cells that were pretreated with RANKL . In contrast, others found that the inhibitory effects of IL-12 on osteoclastogenesis are indirect. One group demonstrated that the inhibitory effects of IL-12 are mediated by T-lymphocytes and do not involve production of INF γ . A second group disputes this result and found inhibition of osteoclastogenesis by IL-12 in cells from T-lymphocyte depleted cultures and cells from T-lymphocyte-deficient nude mice . The latter authors also demonstrated that antibody neutralization of INF γ blocked some of the inhibitory effect of IL-12 on RANKL-stimulated osteoclast formation. In contrast to the majority of reports that IL-12 is an inhibitor of osteoclastogenesis, one group demonstrated that IL-12 induced RANKL expression in human periodontal ligament cells .

The effects of IL-12 on TNFα-induced osteoclastogenesis have been examined in vivo . It was found that osteoclastogenesis, which was stimulated by the injection of TNFα over the calvaria of mice, was decreased when the mice were also treated with IL-12. Furthermore, this effect was not altered by antibody neutralization of T-lymphocytes in the mice. Induction of Fas by TNFα and FasL by IL12 in bone was critical for this response

IL-23 is an IL-12-related cytokine composed of one subunit of p40, which it shares with IL-12, and one subunit of p19, which is unique . It is critical for the differentiation of the T _H 17 subset of T-lymphocytes along with TNFβ (TGFβ) and IL-6 . IL-23 appears most important for expanding the population of T _H 17 T-lymphocytes. This subset of T-lymphocytes, which produces RANKL, has a high osteoclastogenic potential, which is mediated by their production of IL-17 . In an LPS-induced model of inflammatory bone destruction, it was found that there was markedly less bone loss in mice that were deficient either in IL-17 or IL-23 . Hence, the production of both is involved in the bone loss in this model. IL-23 induces RANKL expression in CD4 T-lymphocytes and RANK expression in osteoclast precursor cells . However, the actions of IL-23 on bone in vivo are controversial. IL-23-deficient mice have decreased bone mass in one report but increased bone mass in another . In some studies, IL-23 inhibited osteoclastogenesis through actions that were mediated by CD4 T-lymphocytes . In another study, IL-23 stimulated osteoclastogenesis in mixed osteoblast–osteoclast precursor cultures . One study identified upregulation of leukotriene B4 as a mechanism by which IL-23 regulated osteoclasts .

Curiously, individual neutralization of either IL-17 or IL-23 was found to be more efficacious than their combination as an inhibitor of ovariectomy-induced bone loss . IL-23 has also been identified as a critical mediator of the actions of ankylosing spondylitis on bone .

Another IL-12-related cytokine, IL-27, was found to have inhibitory effects on osteoclastogenesis in murine bone marrow cultures that were mediated by T-lymphocytes . However, the direct inhibitory effects of IL-27 on RANKL-stimulated osteoclastogenesis were identified as being mediated by inhibition of c-Fos . Recent data have implicated INF γ as a mediator of IL-27’s inhibitory effect on osteoclastogenesis , and IL-27 can also inhibit RANKL expression in CD4 T-lymphocytes . In osteoblasts, IL-27 inhibited apoptosis through a mechanism dependent on induction of early growth response-2 gene . Bone cells can be a source of IL-27 during inflammation .

IL-35, the most recently discovered IL-12 family member, is a direct inhibitor of osteoclastogenesis .

33.13

Interleukin-15

IL-15, like IL-7, is a member of the IL-2 superfamily and shares many activities with IL-2 including the ability to stimulate lymphocytes. It has been shown to enhance osteoclast progenitor cell number in culture . Its receptor is composed of a unique IL-15 receptor α and the β and γ chains of the IL-2 receptor . Deletion of IL-15 receptor α in mice produced a phenotype of decreased bone mineralization . IL-15 production by T-lymphocytes has been linked to the increased osteoclastogenesis and bone destruction seen in rheumatoid arthritis . In animal models of inflammatory bowel disease and staphylococcus aureus sepsis, lack of IL-15 or treatment with an IL-15 inhibitor reduced bone loss and the severity of the disease . In vitro, IL-15 treatment of mixed murine bone marrow and osteoblast cocultures demonstrated a role of natural killer (NK) cells in the ability of IL-15 to induce apoptosis in osteoblasts . Polymorphisms of the IL-15 gene have also been linked to variations in bone mineral density in women . In rheumatoid arthritis, IL-15 is reported to promote osteoclastogenesis via a pathway that is dependent on phospholipase D1 .

33.14

Interleukin-17 and interleukin-25

IL-17 is a family of related cytokines, which are unique and contain at least six members (A–F) IL-17E is also called IL-25 . These cytokines are central for the development of the adaptive immune response and the products of a subset of CD4 T-lymphocytes with a unique cytokine expression profile, termed T _H 17. This contrasts with the more established T-lymphocyte cytokine-expressing subsets T _H 1 and T _H 2.

IL-17A was initially identified as a stimulator of osteoclastogenesis in mixed cultures of mouse hematopoietic cells and osteoblasts . This enhanced resorptive activity was mediated through the increased production of prostaglandin and RANKL . The direct effects of IL-17A on the differentiation of osteoclast precursor cells is controversial with some investigators finding stimulatory effects and others finding it to be inhibitory . One report found that low levels of IL-17 regulated osteoclast precursor autophagy . In another report, IL-17 stimulated rheumatoid synoviocytes to produce RANKL only with 1,25 dihydroxyvitamin D ₃ and prostaglandin E ₂ . The production of IL-17A in rheumatoid arthritis appears to be involved in the production of activated osteoclasts and bone destruction in involved joints . Effects of IL-17 on osteoclastogenesis and bone resorption are enhanced by TNFα, which is also produced in the inflamed joints of patients with rheumatoid arthritis . Inhibition of IL-17A in an antigen-induced arthritis model reduced the joint and bone destruction that is typically seen and decreased the production of RANKL, IL-1 β, and TNFα in the involved lesions . Multiple reports have now implicated IL-17 as a critical mediator of the bone loss that occurs in animal models after estrogen withdrawal . One of these suggested that studies of IL-17 neutralization be initiated to determine its role as a potential therapy to reverse postmenopausal bone loss in humans and to enhance bone regeneration after fracture . Data also implicate T-cell IL-17 production in the ability of PTH to stimulate bone resorption .

The effects of IL-17 on bone formation are complex. It stimulates mesenchymal cell proliferation and the expression of genes associated with early stages of osteoblast differentiation , an effect that may be mediated by the production of IL-17 by γδ T-cells . However, it inhibits mature osteoblast differentiation in vitro and the reparative response to a calvarial critical size defect in vivo , possibly through effects on Wnt signaling . Paradoxically, one study found that it augmented the osteogenic response to BMP-2 .

33.15

Interleukin-18, interleukin-33, and interleukin-37

IL-18 is similar to IL-1 in its structure and a member of the IL-1 superfamily . IL-18 synergizes with IL-12 to induce INF γ production , and its levels are increased at sites of inflammation such as rheumatoid arthritis . Osteoblastic cells express IL-18, and its production is induced by treatment with endothelin-1 . IL-18 inhibits osteoclast formation through a variety of mechanisms. These include its ability to stimulate GM-CSF , which is produced by T-cells in response to IL-18 treatment . It also stimulates INF γ production in vivo in bone , and its inhibitory effects on osteoclastogenesis and bone resorption are enhanced by cotreatment with IL-12 . IL-18 has been shown to indirectly stimulate osteoclastogenesis through its effects on T-lymphocytes . Finally, IL-18 is reported to increase production of OPG . In IL-18 overexpressing transgenic mice, osteoclasts were decreased, although, curiously, so was bone mass. These results indicate that there also may be effects of IL-18 on bone growth . In confirmation of this hypothesis, it was demonstrated that PTH treatment of osteoblasts stimulated IL-18 production. In addition, the anabolic effect of intermittent PTH treatment on trabecular bone mass in IL-18-deficient mice was reduced . IL-18 is also a mitogen for osteoblastic cells in vitro .

IL-18 binding protein (IL-18BP) is an antagonist of IL-18 action with antiinflammatory actions. Treatment of ovariectomized mice with IL1-BP prevented bone loss. It was also found that osteoporotic women had decreased IL-18BP levels and increased the amounts of serum IL-18 .

IL-33 is another member of the IL-1 family that has primarily been studied for its effects on T-lymphocytes . Its specific receptor is the orphan IL-1 receptor ST2 (also called IL-1R-like-1) . IL-33 is expressed by osteoblasts and production in these cells is stimulated by PTH and OSM . Its effects on bone cells are varied. One report found it to stimulate osteoclastogenesis , while multiple others have found it to be inhibitory ( Table 33.1 ) . In addition, there is a report that it had no effects on bone remodeling although these same authors suggested that it may be involved in the development of osteonecrosis of the femoral head caused by inadequate blood supply . The activation of HIF1α in osteoblasts resulted in increased IL-33 production . Osteoclastogenesis was decreased in transgenic mice that overexpress IL-33 in osteoblasts, . Mice deficient in IL-33 or ST2 had decreased bone mass . Interestingly, loss of ST2 in mice prevented ovariectomy-induced bone loss in the maxilla but not in the femur .

IL-37 is also an IL-1 family member with osteoclast inhibitory activity . Serum levels of IL-37 are increased in patients with rheumatoid arthritis and ankylosing spondylitis and correlate with disease activity .

33.16

Interferons

Interferons (INF) γ is a type II interferon with a wide variety of biologic activities. In vitro, INF γ has generally been found to have inhibitory actions on bone resorption . These appear to be direct and are mediated by its effects on osteoclast progenitor cells. INF γ inhibits the ability of 1,25-dihydroxyvitamin D ₃ , PTH and IL-1 to stimulate the formation of osteoclast-like cells in cultures of human bone marrow . INF γ also inhibits RANK signaling by accelerating the degradation of TRAF6 through the activation of the ubiquitin/proteasome system , by inhibiting NFATc1 expression and by activating the NF-κB and JNK pathways . Curiously, it is reported to not inhibit resorption in mature osteoclasts . However, INF γ is also reported to have stimulatory effects on resorption through its ability to increase RANKL and TNF-α production in T-lymphocytes and through its ability to enhance the fusion of preosteoclasts . It also appears to mediate the ability of γδ T-lymphocytes and IL-27 to inhibit osteoclastogenesis and resorptive activity .

In osteoblasts INF γ is an inhibitor of proliferation and has variable effects on differentiation .

The effects of INF γ on bone in vivo are also variable, as both inhibitory and stimulatory effects have been reported. In mice with collagen-induced arthritis, loss of the INF γ receptor (INF γR) leads to increased bone destruction . Similarly, in mice that are injected over their calvaria with bacterial endotoxin, which activates toll-like receptors (TLRs), loss of INF γR resulted in an enhanced resorptive response . This result is consistent with more recent findings demonstrating that the inhibitory effects of INF γ on osteoclastogenesis are enhanced by activation of TLRs . The ability of M1 macrophages and Foxp3+CD8 T-cells to inhibit osteoclastogenesis was dependent on their production of INF γ . Finally, in mice that underwent ovariectomy to induce estrogen withdrawal, the administration of INF γ enhanced bone mass and prevented the development of the bone loss that is otherwise seen in this condition .

In contrast, intraperitoneal injection of INF γ for 8 days in rats induced osteopenia . In patients who have osteopetrosis, because they produce defective osteoclasts, administration of INF γ stimulated bone resorption and appeared to partially reverse the disease . The latter effects are possibly due to the ability of INF γ to stimulate osteoclast superoxide synthesis , osteoclast formation in vivo , or a generalized immune response .

Type I interferons (INF α and INF β) are typically produced in response to invading pathogens . Mice deficient in the INF α/β receptor component interferon alpha and beta receptor subunit 1 (IFNAR1) have reduced trabecular bone mass and an increased number of osteoclasts . RANKL induces INF β in osteoclasts, and INF β, in turn, inhibits RANKL-mediated osteoclastogenesis by decreasing c-fos expression and inducing the production of miR-155 . Osteocytes are a source of INF α . INF β has also been shown to inhibit bone resorption in vitro although its mechanism of action is not as well studied as that of INF γ and α . In vivo, INF α had no effect on bone turnover .

33.17

Additional cytokines

IL-4 and IL-13 are members of a group of locally acting factors that have been termed “inhibitory cytokines.” The effects of IL-4 and IL-13 seem related and appear to affect both osteoblasts and osteoclasts. Transgenic mice that overexpress IL-4 had an osteoporotic phenotype . This effect may result from both an inhibition of osteoclast formation and activity and an inhibition of bone formation . IL-13 and IL-4 inhibited IL-1-stimulated bone resorption by decreasing the production of prostaglandins and the activity of cyclooxygenase-2 . The direct inhibitory effects of IL-4 on osteoclast precursor cell maturation are more potent than that of IL-13 and involve effects on STAT6, NF-κB, peroxisome proliferator-activated receptor γ1, mitogen-activated protein kinase signaling, Ca ⁺⁺ signaling, NFATc1, and c-Fos . IL-4 along with GM-CSF induces multipotential myeloid cell differentiation toward the dendritic cell lineage and away from the osteoclast lineage .

IL-13 and IL-4 induce cell migration (chemotaxis) in osteoblastic cells , and they regulate the ability of osteoblasts and vascular endothelial cells to control OPG and RANKL production .

IL-32 is a cytokine that is involved in innate and adoptive immunity. It is produced by T-lymphocytes, NK cells and epithelial cells and has six different splice variants . IL-32 stimulated the formation of multinuclear cells that were TRAP- and vitronectin receptor-positive but did not resorb. In addition, it inhibited resorption that was stimulated by RANKL . IL-32 and IL-17 can reciprocally stimulate each other in inflamed synovium and influence osteoclastic resorption . In osteoblasts IL-32γ stimulated bone formation through a mechanism dependent on the miR, miR-29a .

Macrophage migration inhibitory factor (MIF) was initially identified as an activity in conditioned medium from activated T-lymphocytes that inhibited macrophage migration in capillary tube assays . Once purified and cloned , it became available for functional studies and was shown to have a variety of activities. In addition to T-lymphocytes, it is produced by pituitary cells and activated macrophages. MIF is a direct inhibitor of osteoclastogenesis in vitro through its ability to activate Lyn tyrosine kinase .

In vivo, MIF’s effects are complex. Mice that overexpress MIF globally have high turnover osteoporosis while, curiously, mice deficient in MIF have low turnover osteoporosis with decreased serum indices of bone resorption and bone formation . MIF-deficient mice are also reported to not lose bone mass or increase osteoblast or osteoclast number in bone with ovariectomy . Hence, MIF may be another mediator of the effects that estrogen withdrawal has on bone. Estrogen downregulates MIF expression in activated macrophages . Hence, a similar response may occur in bone or bone marrow and mediate some of the effects that ovariectomy has on bone mass. MIF also mediates the homing of osteoclast precursor cells to osteolytic sites .

MIF is made by osteoblasts , and its production in these cells was upregulated by a variety of factors including TGF-β, FGF-2, IGF-II, and fetal calf serum . In vitro, MIF increased MMP9 and MMP13 expression in osteoblasts and inhibited RANKL-stimulated osteoclastogenesis by decreasing the fusion of precursors, possibly through its ability to inhibit the migration of these cells .

Deletion of CD74, a putative MIF receptor, in mice produced a phenotype of enhanced osteoclastogenesis and decreased bone mass . In human studies a polymorphism in the MIF gene has been linked to osteoporosis , and it may mediate some of the pathology of ankylosing spondylosis .