NLRP3 inflammasome plays a critical role in the pathogenesis of hydroxyapatite-associated arthropathy.

The proinflammatory and catabolic cytokine IL-1ß has been implicated in the pathogenesis of osteoarthritis (OA) by mediating synovial inflammation and cartilage degeneration. Although synovial macrophages are suggested to be the source of IL-1ß, the mechanism remains unclear. Ectopic deposition of hydroxyapatite (HA) crystals in joints is closely associated with OA and other arthropathies, but the precise role of HA in arthritis pathogenesis has not been clearly demonstrated. Here we show that HA crystals of a particular size and shape can stimulate robust secretion of proinflammatory cytokines IL-1ß and IL-18 from murine macrophages in a NLRP3 inflammasome-dependent manner. HA-induced inflammasome activation is dependent on potassium efflux, generation of reactive oxygen species (ROS), and lysosomal damage, but independent of cell death. Mice lacking the inflammasome components are protected against HA-induced neutrophilic inflammation in the air-pouch model of synovitis, and they show decreased joint pathology accompanying spontaneous HA deposition in the ank-deficient mouse model of arthritis. Moreover, calcium crystal positive synovial fluids from some OA patients exhibited inflammasome-stimulatory activity in vitro. These results demonstrate that the NLRP3 inflammasome mediates the pathological effect of HA crystals in vitro and in vivo and suggest a critical role for the inflammasome in the pathogenesis of OA.

Important roles of PI3Kgamma in osteoclastogenesis and bone homeostasis.

G protein-coupled receptor-regulated PI3Kgamma is abundantly expressed in myeloid cells and has been implicated as a promising drug target to treat various inflammatory diseases. However, its role in bone homeostasis has not been investigated, despite the fact that osteoclasts are derived from myeloid lineage. We therefore carried out thorough bone phenotypic characterization of a PI3Kgamma-deficient mouse line and found that PI3Kgamma-deficient mice had high bone mass. Our analyses further revealed that PI3Kgamma deficiency did not affect bone formation because no significant changes in osteoblast number and bone formation rate were observed. Instead, the lack of PI3Kgamma was associated with decreased bone resorption, as evidenced by decreased osteoclast number in vivo and impaired osteoclast formation in vitro. The decreased osteoclast formation was accompanied by down-regulated expression of osteoclastogenic genes, compromised chemokine receptor signaling, and an increase in apoptosis during osteoclast differentiation. Together, these data suggest that PI3Kgamma regulates bone homeostasis by modulating osteoclastogenesis. Our study also suggests that inhibition of PI3Kgamma, which is being considered as a potential therapeutic strategy for treating chronic inflammatory disorders, may result in an increase in bone mass.

Macrophage fusion: the making of osteoclasts and giant cells.

The fusion of cells is a fundamental biological event that is essential for a variety of developmental and homeostatic processes. Fusion is required for the formation of multinucleated osteoclasts and giant cells, although the mechanisms that govern these processes are poorly understood. A new study now reveals an unexpected role for the receptor, dendritic cell-specific transmembrane protein (DC-STAMP), in this process. The potential mechanism by which DC-STAMP governs fusion and the implications of this finding will be discussed.

IL-1 receptor-associated kinase M is a central regulator of osteoclast differentiation and activation.

Osteoporosis is a serious problem worldwide; it is characterized by bone fractures in response to relatively mild trauma. Osteoclasts originate from the fusion of macrophages and they play a central role in bone development and remodeling via the resorption of bone. Therefore, osteoclasts are important mediators of bone loss that leads, for example, to osteoporosis. Interleukin (IL)-1 receptor (IL-1R)-associated kinase M (IRAK-M) is only expressed in cells of the myeloid lineage and it inhibits signaling downstream of IL-1R and Toll-like receptors (TLRs). However, it lacks a functional catalytic site and, thus, cannot function as a kinase. IRAK-M associates with, and prevents the dissociation of, IRAK-IRAK-4-TNF receptor-associated factor 6 from the TLR signaling complex, with resultant disruption of downstream signaling. Thus, IRAK-M acts as a dominant negative IRAK. We show here that mice that lack IRAK-M develop severe osteoporosis, which is associated with the accelerated differentiation of osteoclasts, an increase in the half-life of osteoclasts, and their activation. Ligation of IL-1R or TLRs results in hyperactivation of NF-kappaB and mitogen-activated protein kinase signaling pathways, which are essential for osteoclast differentiation. Thus, IRAK-M is a key regulator of the bone loss that is due to osteoclastic resorption of bone.

Role of MKP-1 in osteoclasts and bone homeostasis.

Bone mass is maintained through the complementary activities of osteoblasts and osteoclasts; yet differentiation of either osteoblasts and osteoclasts engages the mitogen-activated protein kinase (MAPK) pathway. The MAPKs are negatively regulated by a family of dual-specificity phosphatases known as the MAPK phosphatases (MKPs). MKP-1 is a stress-responsive MKP that inactivates the MAPKs and plays a central role in macrophages; however, whether MKP-1 plays a role in the maintenance of bone mass has yet to be investigated. We show here, using a genetic approach, that mkp-1(-/-) female mice exhibited slightly reduced bone mass. We found that mkp-1(+/+) and mkp-1(-/-) mice had equivalent levels of bone loss after ovariectomy despite mkp-1(-/-) mice having fewer osteoclasts, suggesting that mkp-1(-/-) osteoclasts are hyperactive. Indeed, deletion of MKP1 led to a profound activation of osteoclasts in vivo in response to local lipopolysaccharide (LPS) injection. These results suggest a role for MKP-1 in osteoclasts, which originate from the fusion of macrophages. In support of these observations, receptor activator for nuclear factor-kappaB ligand induced the expression for MKP-1, and osteoclasts derived from mkp-1(-/-) mice had increased resorptive activity. Finally, receptor activator of nuclear factor-kappaB ligand-induced p38 MAPK and c-Jun NH2-terminal kinase activities were enhanced in osteoclasts derived from mkp-1(-/-) mice. Taken together, these results show that MKP-1 plays a role in the maintenance of bone mass and does so by negatively regulating MAPK-dependent osteoclast signaling.

Macrophage fusion: are somatic and cancer cells possible partners?

Macrophages are present in all tissues and can fuse with themselves to differentiate into multinucleate osteoclasts or giant cells that play a central role in osteoporosis and chronic inflammatory diseases, respectively. Yet, the mechanism by which they fuse remains uncharacterized. The macrophage fusion receptor (MFR) and its ligand CD47 might mediate homotypic fusion of macrophages and allow for their recognition as 'self' before fusion. Although a novel process and controversial idea, macrophages might exploit a similar mechanism for fusion with somatic cells or tumor cells, with resultant organ repair or metastasis, respectively. Hence, macrophages might be the 'double-edged swords' of tissues.

Deletion of mitogen-activated protein kinase phosphatase 1 modifies the response to mechanical bone marrow ablation in a mouse model.

The maintenance of bone mass results from a delicate balance between bone formation by osteoblasts and bone resorption by osteoclasts. Understanding these processes is essential for the development of effective treatments for skeletal diseases. Mechanical bone marrow ablation provides a unique animal model to study bone repair and the roles of specific genes in this process. Ablation of marrow induces the formation of intramembranous bone in the medullary cavity, which is subsequently resorbed by osteoclasts. We used this model to ask whether mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP1) affects the bone formed in response to marrow ablation. MKP1 is a negative regulator of MAPK signaling, which is essential for a wide variety of cellular mechanisms, including those critical for osteoblast and osteoclast function. At 10 d after mechanical bone marrow ablation, the femurs of male mkp1(+/+) and mkp1(-/-) mice were compared with those of unoperated baseline mice by using radiography, peripheral quantitative computed tomography, and microcomputed tomography. Both genotypes developed increased bone mass after marrow ablation, but the increase was more pronounced in mkp1(-/-) mice compared with mkp1(+/+) mice. These results indicate that MKP1 affects the bone formed in response to marrow ablation and suggest encouraging possibilities for the use of inhibitors of MKP1 to modulate bone repair.

Macrophage fusion: molecular mechanisms.

Macrophages are the most versatile, plastic, and mobile cells in the animal kingdom. They are present in all tissues and might even define a true " body-wide" network that maintains health and ensures the repair of tissues and organs. In specific and rare instances, macrophages fuse to form multinucleate osteoclasts and giant cells in bone and in chronic inflammatory reactions, respectively. While macrophages lose most of their plasticity and mobility after they become multinucleate, at the same time they acquire the capacity to resorb calcified tissues, such as bone, and foreign bodies, such as pathogens and implants, and they mediate the replacement of the resorbed tissue by new tissue. There is evidence to suggest that macrophages might also fuse with somatic cells to repair tissues and with tumor cells to trigger the metastatic process. The molecular machinery of macrophage fusion remains poorly characterized, but it is likely to be shared by all fusing macrophages.

Methods to fuse macrophages in vitro.

Macrophages are mononucleate cells that fuse in rare and specific instances to form osteoclasts in bone or giant cells in chronic inflammatory conditions. Because of the central role these cells play in bone metabolism and in inflammation, respectively, methods to study their formation in vitro are described.

Cell-cell fusion.

Cell-cell fusion is a highly regulated and dramatic cellular event that is required for development and homeostasis. Fusion may also play a role in the development of cancer and in tissue repair by stem cells. While virus-cell fusion and the fusion of intracellular membranes have been the subject of intense investigation during the past decade, cell-cell fusion remains poorly understood. Given the importance of this cell-biological phenomenon, a number of investigators have begun analyses of the molecular mechanisms that mediate the specialized fusion events of a variety of cell types and species. We discuss recent genetic and biochemical studies that are beginning to yield exciting insights into the fusion mechanisms of Saccharomyces cerevisiae mating pairs, Caenorhabditis elegans epithelial cells and gametes, Drosophila melanogaster and mammalian myoblasts, and mammalian macrophages.

[Macrophage fusion: are somatic and cancer cells possible partners?]. / La fusion des macrophages: partenaires des cellules somatiques et cancéreuses ?

The fusion of cells is a fundamental biological event that plays a central role in a variety of developmental and homeostatic processes. Macrophages are present in all tissues and can initiate interaction and fusion. The putative macrophage-fusion machinery is still poorly understood, but some of its components have been identified. Macrophages recognize each other as << self >> in order to fuse but some essential questions remain: do macrophages fuse with somatic cells to repair tissues and organs? Do macrophages fuse with tumor cells to trigger metastasis? Agnès Vignery discusses these novel and challenging ideas in this review.

Kcnn4 is a regulator of macrophage multinucleation in bone homeostasis and inflammatory disease.

Macrophages can fuse to form osteoclasts in bone or multinucleate giant cells (MGCs) as part of the immune response. We use a systems genetics approach in rat macrophages to unravel their genetic determinants of multinucleation and investigate their role in both bone homeostasis and inflammatory disease. We identify a trans-regulated gene network associated with macrophage multinucleation and Kcnn4 as being the most significantly trans-regulated gene in the network and induced at the onset of fusion. Kcnn4 is required for osteoclast and MGC formation in rodents and humans. Genetic deletion of Kcnn4 reduces macrophage multinucleation through modulation of Ca(2+) signaling, increases bone mass, and improves clinical outcome in arthritis. Pharmacological blockade of Kcnn4 reduces experimental glomerulonephritis. Our data implicate Kcnn4 in macrophage multinucleation, identifying it as a potential therapeutic target for inhibition of bone resorption and chronic inflammation.

Additive Effects of Mechanical Marrow Ablation and PTH Treatment on de Novo Bone Formation in Mature Adult Rats.

Mechanical ablation of bone marrow in young rats induces rapid but transient bone growth, which can be enhanced and maintained for three weeks by the administration of parathyroid hormone (PTH). Additionally, marrow ablation, followed by PTH treatment for three months leads to increased cortical thickness. In this study, we sought to determine whether PTH enhances bone formation after marrow ablation in aged rats. Aged rats underwent unilateral femoral marrow ablation and treatment with PTH or vehicle for four weeks. Both femurs from each rat were analyzed by X-ray and pQCT, then analyzed either by microCT, histology or biomechanical testing. Marrow ablation alone induced transient bone formation of low abundance that persisted over four weeks, while marrow ablation followed by PTH induced bone formation of high abundance that also persisted over four weeks. Our data confirms that the osteo-inducive effect of marrow ablation and the additive effect of marrow ablation, followed by PTH, occurs in aged rats. Our observations open new avenues of investigations in the field of tissue regeneration. Local marrow ablation, in conjunction with an anabolic agent, might provide a new platform for rapid site-directed bone growth in areas of high bone loss, such as in the hip and wrist, which are subject to fracture.

Dramatic increase in cortical thickness induced by femoral marrow ablation followed by a 3-month treatment with PTH in rats.

We previously reported that following mechanical ablation of the marrow from the midshaft of rat femurs, there is a rapid and abundant but transient growth of bone, and this growth is enhanced and maintained over a 3-week period by the bone anabolic hormone parathyroid hormone (PTH). Here, we asked whether further treatment with PTH or bisphosphonates can extend the half-life of the new bone formed in lieu of marrow. We subjected the left femur of rats to mechanical marrow ablation and treated the animals 5 days a week with PTH for 3 weeks (or with vehicle as a control) to replace the marrow by bone. Some rats were euthanized and used as positive controls or treated with vehicle, PTH, or the bisphosphonate alendronate for a further 9 weeks. We subjected both femurs from each rat to soft X-ray, peripheral quantitative computed tomography (pQCT), micro-computed tomography (microCT), dynamic histomorphometry analysis, and biomechanical testing. We also determined the concentrations of serum osteocalcin to confirm the efficacy of PTH. Treatment with PTH for 3 months dramatically enhanced endosteal and periosteal bone formation, leading to a 30% increase in cortical thickness. In contrast, alendronate protected the bone that had formed in the femoral marrow cavity after marrow ablation and 3 weeks of treatment with PTH but failed to promote endosteal bone growth or to improve the biomechanical properties of ablated femurs. We further asked whether calcium-phosphate cements could potentiate the formation of bone after marrow ablation. Marrow cavities from ablated femurs were filled with one of two calcium-phosphate cements, and rats were treated with PTH or PBS for 84 days. Both cements helped to protect the new bone formed after ablation. To some extent, they promoted the formation of bone after ablation, even in the absence of any anabolic hormone. Our data therefore expand the role of PTH in bone engineering and open new avenues of investigation to the field of regenerative medicine and tissue engineering. Local bone marrow aspiration in conjunction with an anabolic agent, a bisphosphonate, or a calcium-phosphate cement might provide a new platform for rapid preferential site-directed bone growth in areas of high bone loss.

Replacement of bone marrow by bone in rat femurs: the bone bioreactor.

During development and repair of bone, two distinct yet complementary mechanisms, intramembranous and endochondral, mediate new bone formation via osteoblasts. Because mechanical bone marrow ablation leads to the rapid and transient formation of new bone in the marrow cavity, we postulated that parathyroid hormone (PTH), which is a bone anabolic hormone, enhances the formation of new bone that forms after marrow ablation. We subjected the left femur of rats to mechanical marrow ablation, or sham operation, and injected the animals daily with PTH or vehicle for 1, 2, or 3 weeks in a first experiment, then with PTH, parathyroid hormone-related peptide (PTHrP), or vehicle for 3 weeks in a second experiment. We subjected both femurs from each rat to soft X-ray, peripheral quantitative computed tomography, computed tomography on a microscale, and histological analysis, and determined the concentration of serum osteocalcin. In addition, in the second experiment, we determined the serum concentration of calcium, tartrate-resistant acid phosphatase (TRAP), and receptor activator of NF-kappaB ligand (RANKL) at 3 weeks, and subjected femurs to biomechanical testing. Following treatment with PTH or PTHrP for 3 weeks, bone filled the marrow cavity of the shafts whose marrow had been ablated. PTH increased trabecular density in the right femur, but failed to induce bone formation in the medullary region of the right unoperated femoral shafts. The newly formed bone endowed left femoral shafts with improved biomechanical properties when compared to those of right femurs and left femurs from control, sham-operated, and vehicle-treated rats. PTHrP, like PTH, increased serum osteocalcin, but neither increased serum calcium, TRAP, or RANKL at 3 weeks. Our results reveal that the newly formed bone that follows marrow ablation is responsive to PTH, expand the role of PTH in bone, and might open new avenues of investigations to the field of regenerative medicine and tissue engineering. Local bone marrow removal in conjunction with pharmacologic intervention with an anabolic agent might provide a technique for rapid preferential site-directed bone growth in areas of high bone loss.

CD200 and its receptor, CD200R, modulate bone mass via the differentiation of osteoclasts.

Fusion of macrophages is an essential step in the differentiation of osteoclasts, which play a central role in the development and remodeling of bone. Osteoclasts are important mediators of bone loss, which leads, for example, to osteoporosis. Macrophage fusion receptor/signal regulatory protein alpha (MFR/SIRPalpha) and its ligand CD47, which are members of the Ig superfamily (IgSF), have been implicated in the fusion of macrophages. We show that CD200, which is not expressed in cells that belong to the myeloid lineage, is strongly expressed in macrophages at the onset of fusion. By contrast, the CD200 receptor (CD200R), which, like CD200, belongs to the IgSF, is expressed only in cells that belong to the myeloid lineage, including osteoclasts, and in CD4+ T cells. Osteoclasts from CD200-/- mice differentiated at a reduced rate. Activation of the NF-kappaB and MAP kinase signaling pathways downstream of RANK, a receptor that plays a central role in the differentiation of osteoclasts, was depressed in these cells. A soluble recombinant protein that included the extracellular domain of CD200 rescued the fusion of CD200-/- macrophages and their activation downstream of RANK. Conversely, addition of a soluble recombinant protein that included the extracellular domain of CD200R or short-hairpin RNA-mediated silencing of the expression of CD200R prevented fusion. Thus CD200 engagement of the CD200R at the initiation of macrophage fusion regulated further differentiation to osteoclasts. Consistent with in vitro observations, CD200-/- mice contained fewer osteoclasts and accumulated more bone than CD200+/+ mice. The CD200-CD200R axis is therefore a putative regulator of bone mass, via the formation of osteoclasts.

The intracellular domain of CD44 promotes the fusion of macrophages.

Macrophages seed all tissues in which they have the ability, in specific and rare instances, to fuse with themselves and to differentiate into osteoclasts in bone or into giant cells in chronic inflammatory reactions. Although these cells play a central role in osteoporosis and in foreign body rejection, respectively, the molecular mechanism used by macrophages to fuse remains poorly understood. Macrophages might also fuse with somatic and tumor cells to promote tissue repair and metastasis, respectively. We reported that CD44 expression is highly induced in macrophages at the onset of fusion in which it plays a role. We report now that the intracellular domain of CD44 (CD44ICD) is cleaved in macrophages undergoing fusion and that presenilin inhibitors prevent the release of CD44ICD and fusion. We also show that CD44ICD promotes the fusion of tissue macrophages and bone marrow-derived macrophages. Finally, we report that CD44ICD is localized in the nucleus of macrophages in which it promotes the activation of NF-kappaB. These observations open avenues to study the role of CD44ICD in blood cells and tumors.