Macrophage Polykaryons Research Articles

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.

Osteotropic factor responsiveness of highly purified populations of early and late precursors for human multinucleated cells expressing the osteoclast phenotype

Recently we have adapted human long-term bone marrow cultures to form multinucleated cells (MNC) that express the osteoclast phenotype and used semisolid culture techniques to identify early (bipotent) and late (unipotent) mononuclear precursors for these MNC. The early precursor can form both osteoclast-like MNC and macrophage polykaryons; the late precursor forms only osteoclast-like MNC. In this study we examined the effects of osteotropic hormones and cytokines of MNC formation from highly purified populations of these early or late mononuclear precursor cells. MNC expressing the osteoclast phenotype were identified by their cross-reactivity with the 23c6 monoclonal antibody, which preferentially identifies osteoclasts. 1,25-(OH)2D3 (10(-8) M), IL-1 beta (10 u/ml), and IL-6 (100 pg/ml) stimulated formation of 23c6-positive MNC from highly purified populations of early or late precursor cells. In contrast, PTH (50 ng/ml) did not act directly on late precursor cells but only stimulated 23c6-positive MNC formation from early precursors. These results show that (1) 1,25-(OH)2D3, IL-1 beta, and IL-6 can stimulate 23c6-positive MNC formation from a highly enriched population of early and late precursors, and (2) PTH does not act on late precursors but may act indirectly on the late precursors.

Immunophenotypic differences between osteoclasts and macrophage polykaryons: immunohistological distinction and implications for osteoclast ontogeny and function.

The antigenic phenotype of human fetal osteoclasts was compared with that of human tissue macrophages and macrophage polykaryons in foreign body lesions using a large number of monoclonal antibodies directed against myeloid (granulocyte/mononuclear phagocyte) antigens. Osteoclasts expressed a restricted range of macrophage-associated antigens including CD13, CD15A, CD44, CD45, CD54, (ICAM-1), CD71 (transferrin receptor), and CD68. These antigens were also present on macrophages and macrophage polykaryons both of which also strongly expressed CD11a,b,c, CD18, (LFA family), CD14, CD31, CD36, CD37, CD39 and CD43 antigens. There was also weak and occasional expression of CD16 (FcRIII), CD25 (interleukin 2 receptor), CD32 (FcRII), CD35 (C3b receptor) and HLA-DR by macrophage polykaryons. The presence of some macrophage associated antigens on osteoclasts is consistent with their originating from cells of the mononuclear phagocyte system. The numerous differences in antigenic phenotype between osteoclasts and macrophage polykaryons, however, suggest that their pathways of development and differentiation are not identical. The differences discerned in antigenic phenotype should also permit distinction between these polykaryons (and possibly their mononuclear precursors) in normal and diseased tissues.

Sequential Expression of Phenotype Markers for Osteoclasts during Differentiation of Precursors for Multinucleated Cells Formed in Long Term Human Marrow Cultures*

Long-term human marrow cultures form multi-nucleated cells (MNC) which express the osteoclast phenotype. Mononuclear precursors for these MNC can be identified and highly enriched. We tested early (bipotent) and late (unipotent) precursors of these MNC for expression of several osteoclast differentiation markers: 1) the osteoclast vitronectin receptor, identified by the 23c6 monoclonal antibody, 2) the vacuolar-type proton pump, identified by the E11 monoclonal antibody, and 3) the calcitonin (CT) receptor, by autoradiography with 125I-labeled salmon calcitonin. We wished to determine if the proton pump was expressed in cells in long term marrow cultures and if its expression correlated with expression of the CT receptor and the vitronectin receptor. About 30% of early precursor cells reacted with the 23c6 monoclonal antibody, but none expressed CT receptors or showed amplified proton pump expression. The CT receptor and amplified proton pump expression were detected first on the late precursor, a stage in which every cell reacted strongly with the 23c6 monoclonal antibody. Over 80% of MNC formed from these late precursors expressed abundant CT receptors, and all MNC expressed the vitronectin receptor and showed amplified proton pump expression. In contrast, macrophage polykaryons and mononuclear macrophages formed in vitro failed to express the osteoclast vitronectin and CT receptors and expressed the proton pump at levels indistinguishable from those of surrounding cells.

Macrophage polykaryons are capable of bone resorption

Mouse myeloid leukemic M1 cells are induced to differentiate into macrophage-like cells by a differentiation-inducing factor (D-factor) and various agents. IFN-γ alone did not induced differentiation of clone T22-3 of M1 cells but inhibited their differentiation by D-factor. That is, IFN-γ at 4 U/ml inhibited 50% of phagocytic activity of T22-3 cells induced by 7 × 10−11 M D-factor. In addition, it inhibited the induction of lysozyme activity and morphological differentiation of these cells by D-factor. IFN-γ also inhibited dexamethasone-induced differentiation of T22-3 cells. Previously interferon-α/β was shown not to induce differentiation of M1 cells itself, but to enhance induction of their differentiation by D-factor. The present study showed that IF-α/gb and IFN-γ had opposite effects on induction of differentiation of T22-3 cells by D-factor. The effect of IFN-γ on the differentiation of M1 cells varied with the clone of M1 cells used: IFN-γ inhibited D-factor-induced differentiation of cells of clones T22-3 and S2, but induced differentiation of cells of clones B24 and S1.

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 α-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.

P21. Resorption of bone by foreign body macrophage polykaryons

Osteoclasts are large multinucleated giant cells that contains between 3 and 100 nuclei per cell, but usually contain 10–20 nuclei per cell and are highly motile. They form by fusion of mononuclear precursors and become adherent to bone. Osteoclasts are polarized cells that have a basolateral domain that doesn't face bone and a resorptive surface that forms a characteristic sealing zone and F-actin ring at sites of bone contact. When osteoclasts are plated on bone surfaces, a characteristic “resorption pit” is formed below the cell within the sealing zone. These resorption lacunae are never seen in the absence of osteoclasts and are not produced by macrophages or macrophage polykaryons. The capability to efficiently excavate bone is a unique function of osteoclasts and requires many specialized systems as well as exquisite regulation to maintain healthy bone. Bone remodeling is an essential process, which creates the marrow space utilized for hematopoietic stem cell differentiation, shapes and sculpts the bones during growth, enables tooth eruption, is critical for maintaining bone quality and strength, and is part of the system generating calcium homeostasis. Excessive bone remodeling is a feature of a number of pathological states, such as rheumatoid arthritis, hypercalcemia of malignancy, Paget's disease, and osteoporosis, while defective bone remodeling is seen in osteopetrosis. Without remodeling, the skeleton would eventually collapse. Humans remodel their skeleton at different rates depending upon the bone location and the number of additional factors, including mechanical forces, autocrine and paracrine hormone status, and immunological influences.

4. Bone resorption by macrophage polykaryons derived from a giant cell tumour of tendon sheath

4. Bone resorption by macrophage polykaryons derived from a giant cell tumour of tendon sheath

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.

9. Resorption of bone by tumour-associated macrophage polykaryons

9. Resorption of bone by tumour-associated macrophage polykaryons

Depolarization of macrophage polykaryons in the absence of external sodium induces a cyclic stimulation of a calcium-activated potassium conductance

Macrophage polykaryons associated with the foreign body granuloma display several electrophysiological properties when studied with intracellular microelectrodes. One of the most evident properties is the slow hyperpolarization (2–5 s long, 10–60 mV amplitude), due to transient openings of Ca 2+-dependent K + channels, that is similar to those observed in macrophages. How this oscillation of membrane potential is triggered is not well known and the only way to repeatedly activate it under experimental control is through the intracellular injection of Ca 2+. Although this technique is important for understanding the properties of the K + channels, no information has been obtained about the way Ca 2+ levels are raised and controlled in the cytosol. Slow hyperpolarizations can also be triggered by electrical stimulation, but reproducibility is low with cells bathed in physiological solutions. We then decided to investigate the effect of depolarization on the electrophysiological properties of macrophage polykaryons exposed to bathing solutions of several ionic compositions. We show in this paper that cell membrane depolarization induced by a long current pulse can trigger several patterns of membrane potential changes and that, in the absence of extracellular Na +, repetitive oscillations of decaying amplitudes are observed in almost all the cells. They are very similar to the slow hyperpolarizations, are dependent on the presence of extracellular Ca 2+, and are blocked by quinine and D-600. Whole-cell patch clamp recording under voltage clamp conditions showed an outward current that oscillates and that also exhibits decaying amplitudes. The data presented here indicate that these oscillations are a consequence of the cyclic opening of the Ca 2+-activated K + channels and support the hypothesis that favors the participation of Ca 2+ channels and of the Ca 2+/Na + exchange system in their triggering. These two mechanisms are not enough to explain either why the K + channels close or why the membrane potential returns to the original level at the end of each cycle. The possibility of using these oscillations as a model to study the slow hyperpolarization is discussed.

Depolarization of macrophage polykaryons in the absence of external sodium induces a cyclic stimulation of a calcium-activated potassium conductance

Depolarization of macrophage polykaryons in the absence of external sodium induces a cyclic stimulation of a calcium-activated potassium conductance

Leucocyte common antigen is present on osteoclasts.

Using a large panel of monoclonal antibodies which recognize the leucocyte common antigen (LCA), the presence of LCA on osteoclasts in both fetal and adult human bone specimens has been determined by immunohistochemistry. LCA is evident on the surface of fetal human osteoclasts in bone imprints and cryostat sections. LCA was also found on osteoclasts in specimens of fixed, decalcified osteoarthritic bone. The intensity and pattern of osteoclast reactivity were similar to those of foreign-body type macrophage polykaryons in inflammatory lesions. These results favour derivation of osteoclasts and their precursors from the multipotential haemopoietic stem cell which produces peripheral blood leucocytes and argues against their origin from a separate stem cell.

Electrophysiology of phagocytic membranes: intracellular K + activity and K + equilibrium potential in macrophage polykaryons

The role of K + as current carrier during the slow membrane hyperpolarizations (SH) elicited by iontophoretic Ca 2+ injections into macrophage polykaryons is studied. The intracellular K + activity ( a K) and the K + equilibrium potential ( E K) are measured using ion-sensitive microelectrodes. The mean value of a K is 84 ± 5 mM in a culture medium containing 5.3 mM K +, but increases to 100 ± 8 mM when the extracellular K + concentration is raised to 30.3 mM. Under the same conditions the values of E K obtained from the Nernst equation are −81 ± 2 mV and −40 ± 2 mV, respectively. The reversal potentials ( E R) of the SH are calculated from changes observed in transmembrane potential and input resistance, according to an equivalent model based only on passive ionic fluxes. The mean E R values obtained are −74 ± 8 mV in the presence of low K + concentration and −37 ± 3 mV for the high K + medium. These values are significantly smaller than the estimated E K for the corresponding situations. Evidence for the existence of an electrogenic (Na + + K +)-ATPase activity is also presented. The evidence indicates that an increase in the membrane potassium permeability can account for about 90% of the total permeability change occurring during the SH.

Electrophysiology of phagocytic membranes. Role of divalent cations in membrane hyperpolarizations of macrophage polykaryons

The electrophysiological properties of the membrane of mouse peritoneal macrophage polykaryons are studied. Slow hyperpolarizations can be elicited by iontophoretic injections of either Ca 2+ or Sr 2+ into the cytoplasm. The effect of both cations is identical, since: (1) it is invariably triggered by the cation injection, (2) the amplitude is dependent on the K + gradient, (3) quinine blocks reversibly the response to both cation injections. Mg 2+, Ba 2+ and Mn 2+ did not elicit responses when injected into the cytoplasm. Ca 2+ induced slow hyperpolarizations were reversibly blocked by the addition of Ba 2+ to the external saline, but were not affected by the presence of external tetraethylammonium chloride. Cells maintained in saline containing high concentrations of Ca 2+, Sr 2+ or Mn 2+ exhibited sustained hyperpolarizations. Quinine blocked the hyperpolarization induced by high Ca 2+ or Sr 2+, but was ineffective for the case of Mn 2+. Cells hyperpolarized by external Mn 2+ frequently exhibited nonlinear, voltage-current characteristics. Similar patterns could also be observed in a small fraction (less than 10%) of the cells in control conditions. Current-induced shifts between two stable membrane potentials were seen either in high Ca 2+ or normal medium. The great variability of the responses described for this phagocytic membrane is discussed. The evidence supports the assumption that Ca 2+ and Sr 2+ can induce transient or persistent hyperpolarized states by activating a potassium permeability. External Mn 2+ may act in part by reducing impalement-related current leakage from the phagocytic membrane.

Giant cell formation in rabbit long-term bone marrow cultures: immunological and functional studies.

A method for the long-term culture of rabbit newborn bone marrow has been developed. It is characterized by the rapid appearance of an adherent, adipocyte-containing stromal layer, proliferation of mature myeloid cells, and the formation of numerous, large multinucleate giant cells. By the combined use of morphological, immunological, and functional criteria these giant cells have been characterized as macrophage polykaryons and not osteoclastic giant cells. We conclude that long-term bone marrow culture in the rabbit favors the proliferation, maturation, and fusion of macrophage, but not osteoclast, precursors--new experimental models will have to be developed to enable the developmental biology of osteoclasts to be studied in the rabbit.

Phenotypic Characterization of Gamma Interferon-Induced Human Monocyte Polykaryons

Abstract Multinucleated giant cells of mononuclear phagocyte origin (monocyte or macrophage polykaryons [MPs] ) are seen in numerous different normal and pathologic states. We have previously shown that gamma interferon (IFN-gamma) induces fusion of uninuclear monocytes (UMs) to form MPs. This study was designed to characterize these IFN-gamma-induced MPs. Control and IFN-gamma-treated UMs and MPs did not have peroxidase activity, but they stained intensely for nonspecific esterase and acid phosphatase. The esterase of UMs and MPs was abolished by fluoride, but the acid phosphatase of UMs and MPs was only minimally decreased by tartrate. The phagocytosis of polystyrene spheres and glutaraldehyde- fixed erythrocytes by MPs was moderately depressed as compared with control or treated UMs, whereas the phagocytosis of IgG-coated erythrocytes was markedly depressed. Populations of control monocytes produced less H2O2 in response to 200 nmol/L of phorbol myristate acetate than did IFN-gamma-treated monocytes (37 +/- 7 v 199 +/- 29 nmol/h per milligram of cell protein). However, when examined microscopically, individual MPs had less ability to reduce NBT (18% +/- 5% positive for MP, 91% +/- 3% for treated UMs, and 67% +/- 3% for control UMs). The surface membrane antigens Leu M3, OKM1 (C3bi receptor), DU-HL60′3, DU-HL60′4, TE5, and V1 were not expressed or were expressed poorly in MPs; they were expressed normally in control and treated UMs. However, HLA-DR expression was increased in treated UMs and MPs. The binding of the lectins RCA, Con A, WGA, DBA, UEA, and PNA was equivalent in all cells. Thus, MPs formed by fusion of UMs in vitro after culture with IFN-gamma differ in several features from UMs.

Phenotypic characterization of gamma interferon-induced human monocyte polykaryons

Multinucleated giant cells of mononuclear phagocyte origin (monocyte or macrophage polykaryons [MPs] ) are seen in numerous different normal and pathologic states. We have previously shown that gamma interferon (IFN-gamma) induces fusion of uninuclear monocytes (UMs) to form MPs. This study was designed to characterize these IFN-gamma-induced MPs. Control and IFN-gamma-treated UMs and MPs did not have peroxidase activity, but they stained intensely for nonspecific esterase and acid phosphatase. The esterase of UMs and MPs was abolished by fluoride, but the acid phosphatase of UMs and MPs was only minimally decreased by tartrate. The phagocytosis of polystyrene spheres and glutaraldehyde-fixed erythrocytes by MPs was moderately depressed as compared with control or treated UMs, whereas the phagocytosis of IgG-coated erythrocytes was markedly depressed. Populations of control monocytes produced less H2O2 in response to 200 nmol/L of phorbol myristate acetate than did IFN-gamma-treated monocytes (37 +/- 7 v 199 +/- 29 nmol/h per milligram of cell protein). However, when examined microscopically, individual MPs had less ability to reduce NBT (18% +/- 5% positive for MP, 91% +/- 3% for treated UMs, and 67% +/- 3% for control UMs). The surface membrane antigens Leu M3, OKM1 (C3bi receptor), DU-HL60-3, DU-HL60-4, TE5, and V1 were not expressed or were expressed poorly in MPs; they were expressed normally in control and treated UMs. However, HLA-DR expression was increased in treated UMs and MPs. The binding of the lectins RCA, Con A, WGA, DBA, UEA, and PNA was equivalent in all cells. Thus, MPs formed by fusion of UMs in vitro after culture with IFN-gamma differ in several features from UMs.

A functional and morphological study of cells adjacent to ectopic bone implants in rats

Subcutaneous implantation of bone chips into normal and osteopetrotic (ia) rats results in the formation of multinucleate giant cells (MNGCs) adjacent to the bone surface. In this study the resorptive and morphological characteristics of the cells surrounding these implants were assessed to determine if the bone-resorbing defects seen in ia animals would be mimicked, thus giving validity to the use of this system as a model for the study of osteoclastic lineage and function. Direct measurement of in vivo bone resorption was achieved through the use of (45)Ca-labelled bone-chip pairs that were primarily osteoid-exposed and freeze-thawed (FT), primarily mineral-exposed and bleached (B), or primarily mineral-exposed and collagenase-treated (CT). Comparison of the (45)Ca content of implanted chips to that of controls indicated the total (45)Ca release during a two-week implantation period. There was no significant difference in the amount of label released between normal and ia animals. Both normal and ia rats showed 23% greater total (45)Ca release from mineral- versus osteoid-exposed matrix. Cellular events occurring on the bony substrate were evaluated by light and electron microscopy. At 3 days, bone chips were surrounded primarily by mononuclear cells. By 14 days, MNGCs were present at the bone surface in both ia and normal animals. In mineral-exposed implants, 40-50% of the bone surface was covered by MNGCs as compared to 20% of the osteoid-exposed surface. These MNGCs possessed occasional clear zones, but did not exhibit ruffled borders; therefore, they could not be classified as osteoclasts. Thus, the defects seen in ia mutants were not reproduced in this implant system. The (45)Ca release that occurred was probably due to the action of mononuclear phagocytes and macrophage polykaryons rather than to true osteoclastic bone resorption.

Recombinant human gamma-interferon induces human monocyte polykaryon formation.

Monocyte or macrophage polykaryons (MP) are seen in different tissues in various inflammatory states and in normal bone (osteoclasts). The factors controlling the formation and the function of MP are not completely understood. This study was designed to evaluate the effects of the lymphokine gamma-interferon (IFN-gamma) on human monocyte function in vitro. Purified recombinant IFN-gamma [20-200 units/ml (0.1-1.0 nM)] caused the appearance of MP in cultures of normal human monocytes cultured in 10% unheated autologous serum. The MP were noted by as early as 36 hr of culture with fusion indices of 40%-60% and up to 160 nuclei per cell. The effect was seen with both recombinant IFN-gamma and natural IFN-gamma produced by Staphylococcal enterotoxin A-stimulated lymphocytes, but IFN-alpha (leukocyte-derived and recombinant) and IFN-beta did not induce MP formation. The activity of the IFN-gamma was destroyed by heating at 56 degrees C for 4 hr, incubating at pH 2 for 3 hr, or incubating with antibody against IFN-gamma. Populations of monocytes incubated 3 days with 100 units of IFN-gamma per ml (0.5 nM) had enhanced capacity to produce H2O2 in response to phorbol 12-myristate 13-acetate and increased content of acid phosphatase and plasminogen activator. As determined by autoradiography, the MP did not incorporate [3H]dThd into their nuclei. Thus, the IFN-gamma appears to induce MP formation by a process of monocyte fusion, and to "activate" monocytes, as judged by various parameters.