The differentiation of monocytes into macrophages,epithelioid cells,and multinucleated giant cells in subcutaneous granulomas

The morphological changes occurring in monocytes during their differentiation into macrophages, epithelioid cells, Langhans-type giant cells, and foreign-body-type giant cells were investigated in foreign-body granulomas induced by subcutaneous implantation of pieces of Melinex plastic. Analysis based on Adams's (1974) criteria for discrimination between the several types of cell of the monocyte line, showed that each type has a characteristic type of granule. Primary and secondary granules, numerous in the Golgi area of monocytes were generally found close to the cell membrane and decreased in number in maturing macrophages. This was accompanied by an increase in the number of microtubules. Mature macrophages show numerous characteristic macrophage granules, which are round (average diameter: 280 nm) and have a halo between the limiting membrane and granular matrix. Mature epithelioid cells have characteristic epithelioid cell granules, and multinucleated giant cells a heterogenous population of granules. Fusing macrophages generally have their Golgi areas facing each other, and also show a reduced thickness of the cell coat. The morphology of the multinucleated giant cell is closely related to the number of nuclei present. In Langhans-type giant cells, which generally have two to ten nuclei, a giant centrosphere with numerous aggregated centrioles is found. In transition forms between Langhans-type and foreign-body-type giant cells, which generally contain 10--30 nuclei, the centrioles show less aggregation. In the foreign-body-type giant cells, which generally have more than 30 nuclei, centrioles are virtually absent and never aggregated. These differences between the Langhans-type giant cells, the foreign-body-type giant cells, and the transition forms, support our previous finding that Langhans-type giant cells are the precursors of foreign-body-type giant cells.     

Three-dimensional study of the cytoskeleton in macrophages and multinucleate giant cells by quick-freezing and deep-etching method

The three-dimensional ultrastructure of multinucleate giant cells in subcutaneous granulomas was compared with those of peritoneal macrophages using a quick-freezing and deep-etching method. Subcutaneous granulomas were induced by implanting plastic coverslips in the dorsal subcutaneous tissue of rats. The quick-freezing and deep-etching replicas were prepared from the cells attached to the coverslips. Dense networks of actin filaments were distributed along all peripheral aspects (beneath the plasma membrane, and on free and coverslip-attached surfaces) of the multinucleate giant cells. On the coverslip-attached surface, numerous clathrin-coated pits and vesicles occurred between the actin filaments. In these cells, intermediate filaments, but not actin filaments, were the predominant cytoskeletal components in perinuclear regions and were attached to the cell nucleus, mitochondria and other vesicular cell organelles. A similar distribution of cytoskeletal components was observed in the mononuclear macrophages of the granulomas and the peritoneal macrophages. These results show that the cytoskeletal organization varies in different regions of the cytoplasm of multinucleate giant cells, while the characteristic cytoskeletal arrangement, resembling that of mononuclear macrophages, is maintained.     

Genetic backgrounds and redox conditions influence morphological characteristics and cell differentiation of osteoclasts in mice

Osteoclasts (OCLs) are multinucleated giant cells and are formed by the fusion of mononuclear progenitors of monocyte/macrophage lineage. It is known that macrophages derived from different genetic backgrounds exhibit quite distinct characteristics of immune responses. However, it is unknown whether OCLs from different genetic backgrounds show distinct characteristics. In this study, we showed that bone-marrow macrophages (BMMs) derived from C57BL/6, BALB/c and ddY mice exhibited considerably distinct morphological characteristics and cell differentiation into OCLs. The differentiation of BMMs into OCLs was comparatively quicker in the C57BL/6 and ddY mice, while that of BALB/c mice was rather slow. Morphologically, ddY OCLs showed a giant cell with a round shape, C57BL/6 OCLs were of a moderate size with many protrusions and BALB/c OCLs had the smallest size with fewer nuclei. The intracellular signaling of differentiation and expression levels of marker proteins of OCLs were different in the respective strains. Treatment of BMMs from the three different strains with the reducing agent N-acetylcysteine (NAC) or with the oxidation agent hydrogen peroxide (H₂O₂) induced changes in the shape and sizes of the cells and caused distinct patterns of cell differentiation and survival. Thus, genetic backgrounds and redox conditions regulate the morphological characteristics and cell differentiation of OCLs.     

Differentiation of granuloma cells (epithelioid cells and multinucleated giant cells): a morphometric analysis. Investigations using the model of experimental autoimmune (anti-TBM) tubulo-interstitial nephritis

Morphometric analysis disclosed distinct differences between blood monocytes, tissue monocytes (i.e. immature macrophages), epithelioid cells and multinucleated giant cells as well as phagocytic macrophages (i.e. mature macrophages) in the granuloma model of autoimmune (anti-TBM) tubulo-interstitial nephritis. The numerical density of lysosomes decreased slightly in tissue monocytes compared with blood monocytes but showed a pronounced increase during the formation of epithelioid cells. The lysosomal compartments of epithelioid cells and multinucleated giant cells resembled each other very closely, but the giant cells obviously produced additional lysosomes of small diameter (80-120 nm). Phagocytic macrophages displayed a total numerical density of lysosomes similar to that of tissue monocytes but the mean diameter of the lysosomes was markedly greater. Thus the volume density of lysosomes was highest in phagocytic macrophages. The blood monocytes exhibited the smallest lysosomal compartment. In tissue monocytes, epithelioid cells, and multinucleated giant cells the volume densities of the lysosomes were greater than in blood monocytes and remained relatively constant because the increase in numerical density was counterbalanced by a decrease in mean granule diameter. We found only minor differences in mitochondrial volume densities among the five cell populations. The shape of the mitochondria, however, changed steadily from short rotational ellipsoids in the blood monocytes to rather elongated and slender bodies in the multinucleated giant cells. The results suggest that epithelioid cells and multinucleated giant cells are active cells which may contribute by their specific performances, to the immunologic microenvironment of the granuloma.     

Three-dimensional study of the cytoskeleton in macrophages and multinucleate giant cells by quick-freezing and deep-etching method.

The three-dimensional ultrastructure of multinucleate giant cells in subcutaneous granulomas was compared with those of peritoneal macrophages using a quick-freezing and deep-etching method. Subcutaneous granulomas were induced by implanting plastic coverslips in the dorsal subcutaneous tissue of rats. The quick-freezing and deep-etching replicas were prepared from the cells attached to the coverslips. Dense networks of actin filaments were distributed along all peripheral aspects (beneath the plasma membrane, and on free and coverslip-attached surfaces) of the multinucleate giant cells. On the coverslip-attached surface, numerous clathrin-coated pits and vesicles occurred between the actin filaments. In these cells, intermediate filaments, but not actin filaments, were the predominant cytoskeletal components in perinuclear regions and were attached to the cell nucleus, mitochondria and other vesicular cell organelles. A similar distribution of cytoskeletal components was observed in the mononuclear macrophages of the granulomas and the peritoneal macrophages. These results show that the cytoskeletal organization varies in different regions of the cytoplasm of multinucleate giant cells, while the characteristic cytoskeletal arrangement, resembling that of mononuclear macrophages, is maintained.     

Induction of multinucleated giant cell formation from human blood-derived monocytes by phorbol myristate acetate in in vitro culture

Multinucleated giant cells (MGC) of mononuclear phagocyte origin occur in different tissues in various inflammatory states and pathological conditions. Although MGC are believed to be derived from monocyte-derived macrophages by fusion, their mechanism of formation is not known. In this study, we investigated the role of PMA, a protein kinase C activator, in the induction and formation of MGC from blood monocyte-derived macrophages in in vitro culture. The addition of PMA (1 x 10(-9) to 8 x 10(-8) M) to 3-wk-old cultures of blood-derived monocytes induces cell fusion with a 30% to 80% fusion rate. Moreover, IFN-gamma-treated blood-derived monocyte cultures showed an additional enhancement of fusion rate with the addition of PMA. 1(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a protein kinase inhibitor, inhibited the formation of macrophage-derived giant cells when added before phorbol ester and IFN-gamma. These findings suggest that protein kinase C may play an important role in the formation of macrophage-derived MGC.     

In vitro fusion of newt macrophages

Spontaneous formation of multinucleate giant cells is often observed in in vitro cultures of peritoneal adherent macrophages from the newts, Notophthalmus viridescens and Taricha granulosa (urodele amphibians). The frequency of such giant cells in these cultures is increased by the addition of phorbol myristic acetate at the initiation of the cultures. This high frequency of multinucleate cells permitted us to evaluate whether multinucleate giant cells arise by cell fusion and/or by repeated nuclear division without cytokinesis. Cell fusion is readily detectable by scanning electron microscopy. To determine whether nuclear division without cytokinesis also occurs, some cultures were treated with colchicine to arrest mitotic figures; others were pulsed with tritiated thymidine to detect DNA synthesis. Mitotic figures were not seen in acridine orange-stained samples. In monolayers that were processed for autoradiography, only a few nuclei were marked with tritium. These observations suggest that nuclear division does not contribute significantly, if at all, to the formation of multinucleate giant cells from cultured newt peritoneal macrophages.     

Comparative study on peroxidatic activity in inflammatory cells on cutaneous and peritoneal implants

Summary Inflammatory reactions were evoked by simultaneous implantation of pieces of Melinex plastic in the subcutaneous tissues of the dorsum and in the peritoneal cavity of rats. The cellular composition of the Melinex-adherent cells and their peroxidatic (PO) activity were investigated in relation to the duration of implantation. Several striking differences were found between the subcutaneous and peritoneal implants. On the 7th and 14th days, multinucleated giant cells were abundantly present on the subcutaneous implants, whereas they were relatively rare on the peritoneal implants. The subcutaneous implants bore no mast cells and only a few eosinophilic granulocytes, but both types of cell were observed frequently on the peritoneal implants.Macrophages and multinucleated giant cells on the subcutaneous implants show PO activity only in the granules or are PO negative. On the peritoneal implants three types of macrophages can be distinguished: exudate macrophages which have PO activity restricted to granules or are PO-negative; macrophages with PO activity in granules and both the rough endoplasmic reticulum (RER) and nuclear envelope; and resident macrophages with PO activity only in the RER and nuclear envelope. In addition, two types of multinucleated giant cells are found, one with and the other without PO activity in the RER and nuclear envelope. Multinucleated giant cells with PO activity in the RER and nuclear envelope as well as exudate macrophages with PO activity in the RER and nuclear envelope were mainly found 32 h and 3 days after implantation of the Melinex in the peritoneal cavity. These findings are discussed in the light of current knowledge of the PO activity in macrophages and multinucleated giant cells. It is concluded that the appearance of PO activity in the RER and nuclear envelope of exudate macrophages and multinucleated giant cells is in all probability a transient phenomenon, and that there is no objective evidence to support the opinion that exudate macrophages with PO activity in the RER and nuclear envelope are transitional cells between exudate and resident macrophages.     

The differentiation of monocytes into macrophages,epithelioid cells,and multinucleated giant cells in subcutaneous granulomas

Summary The peroxidatic (PO) activity of monocytes differentiating into macrophages, epithelioid cells, and multinucleated giant cells in subcutaneous granulomas was investigated with three different media for the demonstration of PO activity. Irrespective of the stage of differentiation, these cells did not show PO activity in the rough endoplasmic reticulum (RER) or nuclear envelope. In addition, it was found that the morphologically characteristic types of granule of the various cells of the monocyte line (the primary granules and secondary granules of monocytes, the macrophage granules, and the epithelioid cell granules), all have distinct cytochemical characteristics.Monocytes lose their primary and secondary granules during differentiation into mature macrophages. Simultaneously, the granules of both types become elongated and the secondary granules lose their halo. In contrast to monocytes, mature macrophages may contain a few microperoxisomes. During the differentiation of macrophages into epithelioid cells or multinucleated giant cells there is an increase in the number of microperoxisomes.     

PCC4azal teratocarcinoma stem cell differentiation in culture: III. Cell-to-cell communication properties

The cell-to-cell communication properties of the PCC4azal embryonal carcinoma cells and their differentiated endoderm-like cells and giant cells were characterized by ionic coupling, metabolic coupling, and transfer of an injected fluorescein dye. All PCC4azal cell types communicate well with one another: stem cells with stem cells, endoderm-like cells with endoderm-like cells, and giant cells with giant cells. In addition, the stem cells communicate well with giant cells as assayed by metabolic coupling. The ability of the undifferentiated teratocarcinoma cells to metabolically couple with a variety of heterologous cell types was examined. The stem cells were always efficient donors but very poor recipients in almost all combinations with cells of fibroblast or epithelial morphology that were derived from several different species. In contrast, these same heterologous cell types were both efficient donors and efficient recipients with each other.     

The origin of chicken hematopoietic colonies as assayed in semisolid agar.

This investigation was undertaken to determine whether primitive stem cells and/or fully differentiated macrophages were the source of in vitro colonies derived from hematopoietic tissues. The chicken colony-forming cell (CFC) present in uncultured yolk sac was a nonadherent, presumably undifferentiated cell. The efficiency of colony formation in this case was approximately 0.08%. In contrast to uncultured yolk sac, the CFC present in one-week old yolk sac cultures was evidently a macrophage. Yolk sac cultures, which consisted of greater than 99% macrophages, produced colonies with an efficiency of 1-5% while cultures derived from peritoneal macrophages produced colonies with an efficiency of 10%. Silica selectively destroyed macrophages and reduced the colony forming efficiency of cells derived from yolk sac cultures.     

Freeze-fracture features of epithelioid cells,multinucleated giant cells,and phagocytic macrophages

The freeze-fracture morphology of epithelioid cells, multinucleated giant cells (Langhans' type), and phagocytic macrophages was investigated. The intensely folded and interdigitating surface membranes of epithelioid cells and multinucleated giant cells displayed no specialized areas of cell contact. The size of the intramembranous particles (IMP) and the fact that the area density of IMPs was higher in the cytoplasmic (P) faces than in the external (E) faces of the cell membranes agreed with observations in other eukaryotic cells. The area densities of the IMPs suggest lower transport rates of molecules across the cell membranes of granuloma cells than of certain epithelial cells. Small pits were detected in the surface membranes of the granuloma cells but an extrusion of granules was not observed. The cytoplasmic granules displayed very different sizes and shapes ranging from spherical to rod-shaped. The latter type of granules (probably primary lysosomes) dominated in multinucleated giant cells. The granule membranes were studded with IMPs whose area densities increased with the granule size. Multilamellar bodies with smooth (lipid) fracture faces were found only in phagocytic macrophages. The nuclear pores of the granuloma cells were distributed over the entire surfaces of the nuclei and displayed moderate clustering. The values of the area densities of the nuclear pores were in keeping with the values observed in mammalian and human epithelial or mesenchymal cells, indicating similar exchange rates of molecules between the nucleoplasm and the cytoplasm in these different cell types. In a single phagocytic macrophage the E-face of the inner membrane of the nuclear envelope displayed a network of fine filaments whose nature is at present unknown.     

In vitro stimulation of immune functions by lipids derived from macrophages exposed to bacterial peptidoglycan.

It has previously been established that the processing of Bacillus subtilis cell walls by the macrophage-like cell line RAW264 leads to the formation of peptidoglycolipids containing glycopeptides of bacterial origin. In view of the immunologic activities associated with lipophilic muramyl peptide derivatives, lipid extracts derived from macrophages exposed to bacterial cell walls were assayed for mitogenicity. The crude lipid extract derived from RAW264 cells exposed to bacterial cell walls gave a strong mitogenic response when delivered as liposomes to murine splenocytes, whereas lipids derived from macrophages not exposed to bacterial cell walls did not give a mitogenic response. Fractionation of the RAW264 lipids into neutral lipids, glycolipids, and phospholipids demonstrated that mitogenicity was associated primarily with the phospholipid fraction and thus correlated with the presence of the aforementioned peptidoglycolipids. Mitogenicity was specific to the lipids derived from macrophages exposed to bacterial cell walls and was not observed in lipids derived from macrophages that had either been nonspecifically activated by exposure to Con A or had been allowed to phagocytize latex beads. The mitogenic response was found to be dependent on the presence of macrophages in the splenocyte population and to, at least in part, involve macrophage activation.     

Polarized distribution of Na+,K+-ATPase in giant cells elicited in vivo and in vitro

Giant cell formation was analyzed to determine whether it results in the high level of Na+,K+-ATPase expression that characterizes multinucleated cells such as osteoclasts. Giant cells and fusing alveolar macrophages were subjected to morphological, immunological, and biochemical studies. Both subunits of the Na+,K+-ATPase were found to be present on the plasma membrane of giant cells. Their localization was restricted to the non-adherent domain of the cell surface. Dynamic studies of giant cell differentiation demonstrated that on culture and/or multinucleation, an increase in sodium pump alpha-subunit synthesis occurred and led to a high level of expression of Na pumps. Conversely, the adherent plasma membrane of giant cells was enriched in a lysosomal membrane antigen. This study demonstrates that culture and/or multinucleation induces a significant increase in the expression of sodium pumps. The polarized distribution of these pumps and of a lysosomal component suggests that fusing macrophages undergo biochemical and morphological alterations which prepare them for a new and specialized function in chronic inflammatory reactions. Giant cells may offer a suitable model system to study the differentiation of other related multinucleated cells, such as osteoclasts.     

TRANSFORMATION OF MONOCYTES IN TISSUE CULTURE INTO MACROPHAGES, EPITHELIOID CELLS, AND MULTINUCLEATED GIANT CELLS : An Electron Microscope Study

The sequential transformation of chicken monocytes into macrophages, epithelioid cells, and multinucleated giant cells in vitro was studied by electron microscopy after fixation and embedment in situ. The following changes occur. In the nucleus, margination of chromatin, evident in monocytes, decreases in later forms. Nucleoli become more complex and nuclear pores increase in number. In cytoplasm, a progressive increase in volume of the ectoplasm and endoplasm occurs in culture. Lysosomes increase in number and size prior to phagocytosis. During phagocytosis (most active from 1 to 3 days of culture) lysosome depletion occurs. Lysosomes are present in greatest number and show maximal structural variation in the epithelioid and young giant cells. Aging giant cells lose lysosomes. All stages possess variably large quantities of rough-surfaced endoplasmic reticulum and free ribosomes. The Golgi apparatus, small in monocytes, increases in size and complexity. Massive accumulations of lysosomes within the Golgi apparatus of macrophages and epithelioid cells suggest that lysosomes originate there. In giant cells, multiple Golgi regions occur, often ringing the nuclei. Monocytes and macrophages have few mitochondria. Mitochondria of epithelioid cells are larger, more numerous, and may have discontinuous outer membranes. Mitochondria are most numerous in giant cells where they increase with age and become polymorphous. Cytoplasmic filaments are approximately 50 to 60 A in diameter and of indeterminate length. They occur both singly and in bundles which touch cytoplasmic vesicles and mitochondria. Few filaments occur in monocytes and macrophages. A large increase in the number of filaments occurs in epithelioid cells, where filaments (90 to 100 A) surround the cytocentrum as a distinctive annular bundle often branching into the cytoplasm. The greatest concentration of filaments occurs in aged giant cells. Pseudopodia are always present. They are short and filiform in monocytes and giant cells, and broad, with abundant micropinocytotic vesicles, in macrophages and epithelioid cells. At every stage, the cell membrane contains dense cuplike structures. These may represent the membranous residue of lysosomes which have discharged to the outside, analogous to merocrine secretion. Contiguous epithelioid cells display elaborate cytoplasmic interdigitation. In places, the plasma membranes break down and epithelioid cells fuse to form giant cells.     

Multinuclear giant cell formation is enhanced by down-regulation of Wnt signaling in gastric cancer cell line, AGS

AGS cells, which were derived from malignant gastric adenocarcinoma tissue, lack E-cadherin-mediated cell adhesion but have a high level of nuclear beta-catenin, which suggests altered Wnt signal. In addition, approximately 5% of AGS cells form multinuclear giant cells in the routine culture conditions, while taxol treatment causes most AGS cells to become giant cells. The observation of reduced nuclear beta-catenin levels in giant cells induced by taxol treatment prompted us to investigate the relationship between Wnt signaling and giant cell formation. After overnight serum starvation, the shape of AGS cells became flattened, and this morphological change was accompanied by decrease in Myc expression and an increase in the giant cell population. Lithium chloride treatment, which inhibits GSK3beta activity, reversed these serum starvation effects, which suggests an inverse relationship between Wnt signaling and giant cell formation. Furthermore, the down-regulation of Wnt signaling caused by the over-expression of ICAT, E-cadherin, and Axin enhanced giant cell formation. Therefore, down-regulation of Wnt signaling may be related to giant cell formation, which is considered to be a survival mechanism against induced cell death.     

Second site escape of a T20-dependent HIV-1 variant by a single amino acid change in the CD4 binding region of the envelope glycoprotein

Background

Many novel studies and therapies are possible with the use of human embryonic stem cells (hES cells) and their differentiated cell progeny. The hES cell derived CD34 hematopoietic stem cells can be potentially used for many gene therapy applications. Here we evaluated the capacity of hES cell derived CD34 cells to give rise to normal macrophages as a first step towards using these cells in viral infection studies and in developing novel stem cell based gene therapy strategies for AIDS.

Results

Undifferentiated normal and lentiviral vector transduced hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34 hematopoietic progenitor cells. The differentiated CD34 cells isolated from cystic bodies were further cultured in cytokine media to derive macrophages. Phenotypic and functional analyses were carried out to compare these with that of fetal liver CD34 cell derived macrophages. As assessed by FACS analysis, the hES-CD34 cell derived macrophages displayed characteristic cell surface markers CD14, CD4, CCR5, CXCR4, and HLA-DR suggesting a normal phenotype. Tests evaluating phagocytosis, upregulation of the costimulatory molecule B7.1, and cytokine secretion in response to LPS stimulation showed that these macrophages are also functionally normal. When infected with HIV-1, the differentiated macrophages supported productive viral infection. Lentiviral vector transduced hES cells expressing the transgene GFP were evaluated similarly like above. The transgenic hES cells also gave rise to macrophages with normal phenotypic and functional characteristics indicating no vector mediated adverse effects during differentiation.

Conclusion

Phenotypically normal and functionally competent macrophages could be derived from hES-CD34 cells. Since these cells are susceptible to HIV-1 infection, they provide a uniform source of macrophages for viral infection studies. Based on these results, it is also now feasible to transduce hES-CD34 cells with anti-HIV genes such as inhibitory siRNAs and test their antiviral efficacy in down stream differentiated cells such as macrophages which are among the primary cells that need to be protected against HIV-1 infection. Thus, the potential utility of hES derived CD34 hematopoietic cells for HIV-1 gene therapy can be evaluated.     

S-100-immunoreactive giant macrophages in lymphoid tissues of the guinea pig

Giant cells containing S-100 protein of the lymphoid tissues in the guinea pig were studied by immunohistochemistry using S-100 antiserum. S-100-immunoreactive giant cells were dendritic in shape, contained one or two irregular-shaped, euchromatic nuclei, phagosomes of various diameter, numerous mitochondria and microfilaments in the perikaryon, and extended cell processes free of cell organelles. These cells predominantly lined the superficial cortex facing the subcapsular sinus, were less numerously scattered in the medulla of lymph nodes and located at the marginal zone of the spleen. They also stained with S-100 alpha monoclonal antiserum and showed active phagocytosis for aldehyde-fixed red cells or colloidal carbon in the popliteal lymph node and spleen. S-100-immunoreactive giant cells also appeared in the corticomedullary zone of the thymus and in the interfollicular area of the Peyer's patches of the gut. Small sinus macrophages, which exhibited active phagocytosis for colloidal carbon but were less active for red cells in the popliteal lymph node and spleen, were not stained with S-100 antiserum. These findings indicate that S-100-immunoreactive giant cells of the lymph node and spleen are a subpopulation of macrophages different from S-100-negative cells of the small type.     

Modulation of Osteoclastogenesis with Macrophage M1- and M2-Inducing Stimuli

Macrophages are generated through the differentiation of monocytes in tissues and they have important functions in innate and adaptive immunity. In addition to their roles as phagocytes, macrophages can be further differentiated, in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), into osteoclasts (multinucleated giant cells that are responsible for bone resorption). In this work, we set out to characterize whether various inflammatory stimuli, known to induce macrophage polarization, can alter the type of multinucleated giant cell obtained from RANKL differentiation. Following a four-day differentiation protocol, along with lipopolysaccharide (LPS)/interferon gamma (IFNγ) as one stimulus, and interleukin-4 (IL-4) as the other, three types of multinucleated cells were generated. Using various microscopy techniques (bright field, epifluorescence and scanning electron), functional assays, and western blotting for osteoclast markers, we found that, as expected, RANKL treatment alone resulted in osteoclasts, whereas the addition of LPS/IFNγ to RANKL pre-treated macrophages generated Langhans-type giant cells, while IL-4 led to giant cells resembling foreign body giant cells with osteoclast-like characteristics. Finally, to gain insight into the modulation of osteoclastogenesis, we characterized the formation and morphology of RANKL and LPS/IFNγ-induced multinucleated giant cells.