J Quinn

Human Osteoclast Formation from Blood Monocytes, Peritoneal Macrophages, and Bone Marrow Cells

Abstract. Mononuclear precursors of the human osteoclast have been identified in both bone marrow and the circulation in man, but osteoclast membership of the mononuclear phagocyte system (MPS) and its precise cellular ontogeny remain controversial. We isolated human hematopoietic marrow cells, blood monocytes, and peritoneal macrophages and incubated each of these cell populations with UMR106 osteoblast-like cells on glass coverslips and dentine slices in both the presence and absence of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3), macrophage-colony stimulating factor (M-CSF), and dexamethasone. Cells isolated from peripheral blood and peritoneal dialysis fluid were positive only for monocyte/macrophage markers (CD11a, CD11b, CD14, and HLA-DR) and negative for osteoclast markers [tartrate-resistant acid phosphatase (TRAP), vitronectin reception (VNR), and calcitonin (CT) receptors and did not form resorption pits on dentine slices after 24 hours in culture. Similarly marrow cells did not form resorption pits on dentine slices after 24 hours in culture. However, after 14 days in co-culture with UMR106 cells, in the presence of 1,25(OH)2D3 and M-CSF, numerous TRAP, CT receptor, and VNR-positive multinucleated cells capable of extensive lacunar resorption were formed in co-cultures of all these preparations. The presence of 1,25 (OH)2D3, M-CSF, and UMR106 were absolute requirements for osteoclast differentiation. It is concluded that precursor cells capable of osteoclast differentiation are present in the marrow compartment, the monocyte fraction of peripheral blood, and in the macrophage compartment of extraskeletal tissues and that these cells are capable of differentiating into mature functional osteoclasts. These findings argue in favor of osteoclast membership of the human MPS.

Biomaterial particle phagocytosis by bone-resorbing osteoclasts.

Abundant implant-derived biomaterial wear particles are generated in aseptic loosening and are deposited in periprosthetic tissues in which they are phagocytosed by mononuclear and multinucleated macrophage-like cells. It has been stated that the multinucleated cells which contain wear particles are not bone-resorbing osteoclasts. To investigate the validity of this claim we isolated human osteoclasts from giant-cell tumours of bone and rat osteoclasts from long bones. These were cultured on glass coverslips and on cortical bone slices in the presence of particles of latex, PMMA and titanium. Osteoclast phagocytosis of these particle types was shown by light microscopy, energy-dispersive X-ray analysis and SEM. Giant cells containing phagocytosed particles were seen to be associated with the formation of resorption lacunae. Osteoclasts containing particles were also calcitonin-receptor-positive and showed an inhibitory response to calcitonin. Our findings demonstrate that osteoclasts are capable of phagocytosing particles of a wide range of size, including particles of polymeric and metallic biomaterials found in periprosthetic tissues, and that after particle phagocytosis, they remain fully functional, hormone-responsive, bone-resorbing cells.

The origin and nature of stromal osteoclast-like multinucleated giant cells in breast carcinoma: implications for tumour osteolysis and macrophage biology.

The origin and nature of osteoclast-like multinucleated giant cells (OMGCs), in extraskeletal neoplasms, is uncertain. The ultrastructure, antigenic phenotype and function of OMGCsm in a breast carcinoma were studied in order to clarify the relationship between OMGCs, osteoclasts and other cells of the mononuclear phagocyte system (MPS). OMGCs resorbed cortical bone in a manner similar to osteoclasts. However, unlike osteoclasts, OMGCs did not possess a ruffled border or clear zone, and expressed HLA-DR and Fc receptors and CD14, CD16, CD18 and CD11 (p150,95) antigens. In addition, OMGCs failed to respond morphologically to calcitonin and were directly stimulated by parathyroid hormone (PTH) to increase bone resorption. These findings suggest that OMGCs are a specific type of macrophage polykaryon distinct from both osteoclasts and other types of inflammatory polykaryon. Occasional smaller (20-25 microns) macrophage-like cells were also associated with resorption pits. Bone resorption by OMGCs isolated from the breast indicates that a cell of the MPS can be transplanted to a new tissue location and perform a highly specialised function appropriate to an MPS cell of that tissue (i.e. the osteoclast). PTH stimulation of bone resorption by OMGCs suggests that PTH or a PTH-like protein, may be involved in the bone resorption and consequent hypercalcaemia associated with metastatic breast cancer.

New sites of cellular vitronectin receptor immunoreactivity detected with osteoclast-reacting monoclonal antibodies 13C2 and 23C6.

The immunohistochemical profile of osteoclast-reacting monoclonal antibodies, 13C2 and 23C6, known to detect the alpha-chain of the vitronectin receptor, is described. Both antibodies reacted with several cell types apart from osteoclasts including megakaryocytes, smooth muscle cells, endothelial cells, thyroid follicular epithelium, renal glomeruli and tubular epithelium, myoepithelial and epithelial cells in the breast and prostate, and both cytotrophoblast and syncytiotrophoblast. In addition, macrophage polykaryons, synovial lining cells, a small number of mononuclear cells in buffy coats, and a few macrophage-like cells in the stroma of various tissues were also stained. The epitopes recognized by these antibodies are thus not osteoclast-specific and are present on other cells of the mononuclear phagocyte system. The implications of these results for osteoclast ontogeny, the nature of the antigens described and the question of osteoclast-specific antibodies are discussed.

Osteoclasts are capable of particle phagocytosis and bone resorption.

Osteoclasts are multinucleated cells specialized for the function of lacunar bone resorption. Although they are known to be capable of phagocytosis of inert particles, it is not known whether this abolishes their ability to respond to hormones or to form resorption lacunae. Human and rat osteoclasts were isolated from giant cell tumours of bone and rat long bones, respectively, and cultured on coverslips and cortical bone slices, both in the presence and in the absence of particles of latex (1 μm diameter) and polymethylmethacrylate (PMMA) (<50 μm). By light microscopy, it was evident that osteoclasts which had phagocytosed both latex and PMMA particles remained responsive to calcitonin. Osteoclast phagocytosis of particles was also evident on scanning electron microscopy, where it could also be seen that these cells were associated with the formation of resorption lacunae. These findings underline the fact that the osteoclast is a true member of the mononuclear phagocyte system and that phagocytosis does not abrogate either its hormonal response to calcitonin or its highly specialized function of bone resorption. That osteoclasts which have phagocytosed biomaterial particles such as PMMA are still able to carry out lacunar bone resorption is of interest in clinical conditions such as aseptic loosening, where a heavy foreign body particle load is often associated with extensive bone resorption.

Human tumour-associated macrophages differentiate into osteoclastic bone-resorbing cells

Macrophages are commonly found within osteolytic secondary carcinomas in bone, but the manner in which these cells contribute to malignant bone resorption is uncertain. Macrophages isolated from primary breast carcinomas were co‐cultured for up to 21 days with UMR 106 rat osteoblast‐like cells on bone slices and glass coverslips in the presence and absence of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] and human macrophage colony stimulating factor (M‐CSF). Cell cultures were then assessed for the presence of phenotypic markers of macrophage and osteoclast differentiation. Isolated cells were negative for osteoclast markers including tartrate‐resistant acid phosphatase (TRAP), vitronectin receptor (VNR), and the ability to carry our lacunar bone resorption, but were positive for CD11b and CD14, macrophage markers which are not present on osteoclasts. In 21‐day co‐cultures of breast carcinoma tumour‐associated macrophages (TAMs) and UMR 106 cells, incubated in the presence of 1,25(OH)2D3 and M‐CSF, numerous TRAP‐ and VNR‐positive multinucleated cells capable of extensive lacunar resorption were formed. Contact with UMR 106 cells and the presence of 1,25(OH)2D3 and M‐CSF were absolute requirements for differentiation of human breast carcinoma TAMs into mature functional osteoclasts. TAM–osteoclast differentiation may represent an important cellular mechanism of osteolysis in metastatic skeletal carcinomas.

Origin of marrow stromal cells and haemopoietic chimaerism following bone marrow transplantation determined by in situ hybridisation.

The origin and cell lineage of stromal cells in the bone marrow is uncertain. Whether a common stem cell exists for both haemopoietic and stromal cells or whether these cell lines arise from distinct stem cells is unknown. Using in situ hybridisation for detection of the Y chromosome, we have examined histological sections of bone marrow from seven patients who received marrow transplants from HLA-matched donors of the opposite sex. Stromal cells (adipocytes, fibroblasts, endothelial cells, osteoblasts and osteocytes) were identified in these recipients as being of host origin. This result is consistent with the concept of a distinct origin and separate cell lineage for cells of the haemopoietic and stromal systems. It also shows that engraftment of marrow stromal cell precursors does not occur and that host stromal cells survive conditioning regimens for marrow transplantation. With the exception of one case, with a markedly hypocellular marrow, mixed chimaerism was seen in haemopoietic cells, indicating that this is not a rare event after marrow transplantation.

Bone resorption by macrophage polykaryons of giant cell tumour of tendon sheath

The antigenic phenotype, ultrastructure and bone resorbing ability of mononuclear and multinucleated giant cells of four giant cell tumour of tendon sheath (GCTTS) lesions was assessed. Both the giant cells and the mononuclear cells exhibited the antigenic phenotype of cells of the monocyte/macrophage lineage. The giant cells, unlike osteoclasts, did not respond morphologically to calcitonin and showed ultrastructural and immunophenotypic features of macrophage polykaryons. However, like osteoclasts, the giant cells showed direct evidence of resorption pit formation on bone slices. This indicates that the GCTTS is composed of cells of histiocytic differentiation with the giant and mononuclear cell components expressing a similar antigenic phenotype. Bone resorption by macrophage polykaryons shows that this is not a unique defining characteristic of osteoclasts. Qualitative differences in the degree and pattern of bone resorption by macrophage polykaryons distinguish it from that of osteoclasts and may underlie the clinical behaviour of osteolytic lesions.

Extracellular matrix receptor and platelet antigens on osteoclasts and foreign body giant cells

SummaryOsteoclasts (OCs) and other cells of the mononuclear phagocyte system possess receptors for adhesive proteins present in the extracellular matrix. The antigenic phenotype of OCs and foreign body giant cells (FBGCs) was investigated for the presence of several integrin molecules and other largely platelet-associated antigens involved in cell adhesion reactions. Both OCs and FBGCs expressed the α-chains of the vitronectin receptor (CD51) and of the VLA-2 (CDw49b) and VLA-4 (CDw49d) molecules as well as their respective β-chains, gpIIIa (CD61) and CD29. OCs and FBGCs also expressed CD9 and CD55 (DAF-Decay Accelerating Factor) and strongly reacted with antibodies directed against fibrinogen, fibronectin and vitronectin; the latter are ligands for several of the above matrix protein receptors. The data suggest that cell-cell and cell-matrix interactions involving adhesive proteins may be important in OC and FBGC function.