Multinucleation and preservation of nucleolar integrity of macrophages

Rat bone marrow-derived macrophages formed multinucleated giant cells spontaneously when cultured in slide glass chambers or when induced with the polyanion acetyl lignin. Nuclei in such cells tended to cluster in distinct rings. DNA fragmentation appeared to occur in multinucleated cells, as detected by 3' end-labeling. Southern blot analyses, using probes specific for nucleolar and non-nucleolar genes, indicated that chromatin DNA was fragmented whereas nucleolar DNA was relatively intact. Autoradiography revealed preservation, in multinucleated cells, of nucleoli into which radiolabeled uridine was incorporated. Multinucleated macrophages appeared to eventually fragment. Preserved integrity of nucleoli seems to be a feature of macrophage multinucleation, a process which apparently culminates in cell death.     

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.     

Differentiation of monocytes into multinucleated giant bone-resorbing cells: two-step differentiation induced by nurse-like cells and cytokines

Bone resorption in the joints is the characteristic finding in patients with rheumatoid arthritis (RA). Osteoclast-like cells are present in the synovial tissues and invade the bone of patients with RA. The characteristics of these cells are not completely known. In the work reported here, we generated these cells from peripheral-blood monocytes from healthy individuals. The monocytes were co-cultured with nurse-like cells from synovial tissues of patients with RA (RA-NLCs). Within 5 weeks of culture, the monocytes were activated and differentiated into mononuclear cells positive for CD14 and tartrate-resistant acid phosphatase (TRAP). These mononuclear cells then differentiated into multinucleated giant bone-resorbing cells after stimulation with IL-3, IL-5, IL-7, and/or granulocyte-macrophage-colony-stimulating factor. TRAP-positive cells with similar characteristics were found in synovial fluid from patients with RA. These results indicate that multinucleated giant bone-resorbing cells are generated from monocytes in two steps: first, RA-NLCs induce monocytes to differentiate into TRAP-positive mononuclear cells, which are then induced by cytokines to differentiate into multinucleated giant bone-resorbing cells.     

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.     

Fine needle aspiration cytology of fibroadenoma with multinucleated stromal giant cells. A review of cases in a six-year period

OBJECTIVE: To describe the fine needle aspiration cytology findings of fibroadenoma with multinucleated stromal giant cells, with histologic correlation. STUDY DESIGN: The author reviewed the cytologic findings of two cases of fibroadenoma with multinucleated stromal giant cells from the file of Pamela Youde Nethersole Eastern Hospital in a six-year period from 1995 to the end of 2000. The diagnosis was confirmed by histologic examination of the lumpectomy specimens. RESULTS: The two cases had similar cytologic findings. The direct smears contained cohesive clusters of bland-looking ductal cells arranged in a "staghorn" pattern. Numerous naked nuclei were also seen in the background. Also, there were occasional multinucleated giant cells in isolation. These giant cells contained 5-10 randomly arranged, round to oval nuclei, fine chromatin and sometimes distinct nucleoli. The cytoplasm was abundant and pale staining, and the cell border was ill defined. Associated epithelioid histiocytes and foamy macrophages were not seen. Histologic examination of the lumpectomy specimens showed architectural features of fibroadenoma with pericanalicular and intracanalicular patterns. In addition, scattered multinucleated giant cells with focal degenerative change were noted in the tumor stroma. Their stromal nature was confirmed by immunohistochemical study. CONCLUSION: Multinucleated stromal giant cells are rarely identified in fine needle aspiration biopsies of fibroadenoma. Recognition of this peculiar finding may help to avoid misdiagnosis of other, more sinister conditions, such as phyllodes tumor and metaplastic carcinoma.     

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.     

Computerized video time-lapse (CVTL) analysis of the fate of giant cells produced by X-irradiating EJ30 human bladder carcinoma cells

This study was designed to examine the viability and proliferation of uninucleated and multinucleated giant cells formed after 6 Gy X irradiation. The pedigrees of 102 individual EJ30 giant cells present 5 days after irradiation were analyzed from time-lapse movies captured over 6.3 days from 100 fields (100x). Pedigree analysis enabled us to study the proliferation of giant cells. The average starting size (area) of the giant cells (14500 +/- 9100 microm(2)) was approximately 25 times larger than the normal-sized cells observed after irradiation (560 +/- 200 microm(2)). From a total of 76 pedigrees of uninucleated giant cells, 42 had giant cells that either died or were arrested, while 34 divided at least once and produced progeny that divided again (five three times and three four times) before the progeny died or were arrested. Twenty-four pedigrees contained progeny that were lost from observation after dividing at least once. While most progeny continued to have giant cell morphology, two uninucleated giant cells ultimately produced progeny that contained two normal-sized cells. From a total of 26 multinucleated giant cells, only three divided. Two divided only once, but one produced progeny that divided two times. In all, 37 out of 102 giant cells divided at least once; eight of these divided four or five times with two of these pedigrees ultimately producing two normal-sized daughter cells. These results suggest that a small fraction of giant cells might be potentially clonogenic.     

Beating of multinucleated giant myocardial cells in culture

Cultured mouse myocardial cells grown as cell sheets in Petri dishes fused together and formed multinucleated giant cells on treatment with HVJ (Sendai virus). The giant cells had well organized myofibrils and beat spontaneously and rhythmically. The spontaneous beating activity of the giant cells changed in response to changes of the external potassium and calcium concentrations and on addition of ouabain in the same way as the beating of cultured myocardial cells not treated with HVJ. When a microelectrode was inserted into giant cells that exhibited spontaneous beating, action potentials were easily recorded.     

Nuclear fusion in multinucleated giant cells during the sexual development of Dictyostelium discoideum

Macrocyst development in Dictyostelium discoideum, is generally considered a sexual phase. This development is initiated by the formation of a giant cell, the result of the fusion of two different mating type haploid cells, such as NC4 and HM1. The giant cell engulfs unfused surrounding cells to develop into a macrocyst. Therefore, if the macrocyst is a sexual structure, the giant cell must be a diploid zygote. However, under certain conditions, a very large multinucleated giant cell containing several dozens of nuclei is formed, followed by normal development into a macrocyst. In such a multinucleated giant cell, it was found that only two nuclei fuse together to produce a diploid zygote and all others disappear at the early stage of development. The diploid nucleus undergoes meiosis and subsequently subdivides into a number of haploid progeny cells later released from the macrocyst to initiate new life cycles.     

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.     

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.     

Functional and biochemical characterization of osteoclast-like cells derived from giant cell tumours of bone.

Cells harvested from human giant cell tumours of bone were characterized on the basis of morphological features, proliferative capacity, total(AP) and tartrate resistant acid phosphatase (TRAP) activity, and hormonal response. Culture were formed by mononucleated and multinucleated cells. Mononucleated cells showed fibroblastic morphology, whereas multinucleated cells showed osteoclastic phenotype. We conclude that in these cultures mature osteoclasts and their mononuclear precursors are present.     

Breast carcinoma with tumor giant cells. Report of a case with fine needle aspiration cytology

A 31-year-old woman presented with a cystic mass in the left breast. At fine needle aspiration (FNA), the mass felt gritty, and a firm mass remained after drainage of the cyst. Cytologic examination of the aspirate showed mononucleated malignant cells and an array of bizarre malignant multinucleated giant cells. A diagnosis of carcinoma of breast with malignant giant cells was made. Subsequent histologic study of the lesion showed a central cystic cavity lined by bizarre tumor giant cells. Immunocytochemistry and lectin cytochemistry confirmed the epithelial nature of the malignant giant cells. The entities that may yield giant cells on FNA of breast masses are discussed.     

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.     

Demonstration of bone resorbing cells in elasmobranchs: Comparison with osteoclasts

We have previously shown that Elasmobranchs-characterized by a partially calcified cartilaginous endoskeleton-presented a bony vertebral arch containing osteoblasts, osteocytes and resorbing cells. The aim of this study is to test the ability of Elasmobranchs to resorb bone tissue. The subcutaneous implantation in dogfish (Scyliorhinus canicula) of devitalized mineral-containing bone particles, obtained from a bony fish (the eel, Anguilla anguilla) resulted, after 21 2 months, in the formation of mononucleated as well as multinucleated cells around and between the bone fragments. By light microscopy, the multinucleated giant cells presented the general aspect of osteoclastic cells whereas, by transmission electron microscopy they never showed ruffled borders which are considered as the typical features of osteoclasts. Except for this character, the mononucleated and multinucleated cells exhibited the typical ultrastructural aspects leading us to say that these cells are involved in the resorption of the bone fragments. This study shows that Elasmobranchs are able to resorb implanted bone.     

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.     

Kinetics and characteristics of murine macrophage-lymphocyte interaction in presence of PHA: appearance of multinucleated giant cells in vitro

The appearance of cellular associations between macrophages and lymphocytes--which we have denominated macrophage-lymphocyte rosettes--and their kinetic formation in the presence of phytohemagglutinin (PHA) have been studied in B10 A (4R) mice. The greatest number of macrophage-lymphocyte rosettes was found from 6 to 12 hours after incubation with PHA. During this time, 42.38 +/- 10.70 of the total number of macrophages had lymphocytes attached to their membranes. This percentage decreased to 17.33 +/- 2.07% after 24 hours. The activation of macrophages after PHA treatment was tested by the phagocytic capacity of these cells. This activity increased significantly 24 hours after incubation. In our assay, an increase in the appearance of multinucleated giant cells when compared to controls was also observed. When the macrophages were lymphocyte depleted, the appearance of the multinucleated giant cells was significantly lower. The kinetics for these formations are also discussed.     

Pneumatosis cystoides gastrica associated with adenocarcinoma of the stomach

This paper reports the finding of benign foreign-body-type multinucleated giant cells and clumps of adenocarcinoma cells in gastric brushing cytology specimens of a case confirmed by radiology and histology as pneumatosis cystoides gastrica associated with an ulcerated carcinoma. Pneumatosis cystoides gastrica should be included in the differential diagnosis of benign multinucleated giant cells found in gastric cytology smears.     

Cutting edge: microRNA regulation of macrophage fusion into multinucleated giant cells

Cellular fusion of macrophages into multinucleated giant cells is a distinguishing feature of the granulomatous response to inflammation, infection, and foreign bodies (Kawai and Akira. 2011. Immunity 34: 637-650). We observed a marked increase in fusion of macrophages genetically deficient in Dicer, an enzyme required for canonical microRNA (miRNA) biogenesis. Gene expression profiling of miRNA-deficient macrophages revealed an upregulation of the IL-4-responsive fusion protein Tm7sf4, and analyses identified miR-7a-1 as a negative regulator of macrophage fusion, functioning by directly targeting Tm7sf4 mRNA. miR-7a-1 is itself an IL-4-responsive gene in macrophages, suggesting feedback control of cellular fusion. Collectively, these data indicate that miR-7a-1 functions to regulate IL-4-directed multinucleated giant cell formation.     

Identification of osteoclasts and their differentiation from mononuclear phagocytes by enzyme histochemistry

A double staining method is presented which allows the enzyme histochemical differentiation between osteoclasts (mono- and multinucleated forms) and mononuclear phagocytes (macrophages, multinucleated inflammatory giant cells). Osteoclasts are characterized by a strong acid phosphatase activity whereas macrophages and inflammatory giant cells show a variable non-specific esterase activity. The described method may be useful in studying the osteoclast origin and the extraosseous distribution of these cells.