Atypical multinucleated cells form in long-term marrow cultures from patients with Paget's disease.

Although Paget's disease is the most flagrant example of a primary osteoclast disorder, little is known of osteoclast biology in this disease. In this report we have studied the formation of cells with the osteoclast phenotype in long-term cultures of marrow mononuclear cells derived from patients with Paget's disease, and compared these with similar cells formed in long-term marrow cultures from normal individuals, and with osteoclasts present in pagetic bone. Osteoclasts formed in pagetic marrow cultures resembled osteoclasts present in pagetic bone, but were distinctly different from osteoclasts formed in normal marrow cultures. Osteoclast formation was 10-20-fold greater in pagetic marrow cultures than in normal cultures. The multinucleated cells formed in cultures of pagetic marrow were much larger in size, were hyperresponsive to 1,25(OH)2 vitamin D, had more nuclei per cell, had increased levels of tartrate-resistant acid phosphatase activity and had ultrastructural features which were not seen in multinucleated cells formed from normal marrow mononuclear cells. These pagetic marrow-derived multinucleated cells formed large resorption lacunae on calcified matrices and cross-reacted with monoclonal antibodies which preferentially bind to osteoclasts. The multinucleated cells formed from marrow obtained from uninvolved sites in Paget's patients also displayed these abnormal features.

Interleukin 6. A potential autocrine/paracrine factor in Paget's disease of bone.

Pagetic osteoclasts are greatly increased in number and size and have increased numbers of nuclei per cell compared to normal osteoclasts. The mechanisms responsible for enhanced osteoclast formation in Paget's disease are unknown. We have used our recently described model system for pagetic osteoclast formation to evaluate culture media conditioned by these atypical multinucleated cells (MNC) to determine if pagetic osteoclasts produce an autocrine or paracrine factor that enhances osteoclast formation. Conditioned media from long-term bone marrow cultures from patients with Paget's disease stimulated osteoclast-like MNC formation in normal marrow cultures. At least part of this activity could be ascribed to interleukin 6 (IL-6). In contrast, conditioned media from normal marrow cultures contained lower levels of IL-6 and did not stimulate formation of osteoclast-like MNC. 7 of 8 bone marrow plasma samples taken from involved bones and 18 of 27 peripheral blood serum samples from Paget's patients had high levels of IL-6. Normal marrow plasma and peripheral blood serum had no or very low levels of IL-6. These results suggest that IL-6 produced by marrow and/or bone cells in patients with Paget's disease may be an autocrine/paracrine factor for pagetic osteoclasts.

Intracellular multiplication of Legionella pneumophila in HL-60 cells functionally differentiated in response to 22-oxacalcitriol.

The agent 1,25-dihydroxyvitamin D3 (D3) induces the differentiation of HL-60 human leukemia cells into functional monocyte-like cells that can support the intracellular multiplication of Legionella pneumophila. 22-Oxacalcitriol (OCT), a synthetic analogue of D3, exhibits greater differentiation-inducing activity than D3 in WEHI-3 mouse leukemia cells and has been suggested to be clinically more useful because of its lower hypercalcemic activity. The abilities of OCT and D3 to induce the functional differentiation of human leukemia HL-60 cells have now been investigated. OCT induced the differentiation of HL-60 cells into monocyte-like cells to a similar extent as D3. Thus, both OCT and D3 increased (1) the surface expression of CD11b, CD11c, CD14, and CD35; (2) nonspecific esterase staining; and (3) phagocytic activity toward fluorescent beads. HL-60 cells differentiated in response to OCT also supported the intracellular multiplication of L. pneumophila. Activation of both OCT- and D3-treated HL-60 cells with human recombinant interferon-gamma (IFN-gamma) for 24 h before infection markedly inhibited L. pneumophila multiplication. IFN-gamma activation enhanced superoxide anion generation by D3-treated HL-60 cells but not by OCT-treated HL-60 cells, suggesting that the inhibition of L. pneumophila multiplication in IFN-gamma-activated cells is independent of superoxide generation. Finally, D3, but not OCT, markedly stimulated the formation of osteoclast-like multinucleated cells from mouse bone marrow cells, consistent with the lower hypercalcemic activity of OCT.

Clinical features and distribution of malignant melanoma and pigmented nevi on the soles of the feet in Japan.

This study, based on 842 cases of malignant melanoma from 108 medical schools and medical centers in Japan (from 1983 to 1985), presents information regarding the incidence, sex and age distribution, anatomical location, and clinical features of malignant melanoma in Japan and compares it with data from the United States. In the white population, the epidemiologic evidence indicates that exposure to sunlight may be an important causal factor of the disease. On the other hand, in more heavily pigmented races, such as Negroids and Orientals, many of the malignant melanomas occur on the skin of the unexposed soles of the foot. In our survey, 241 out of 833 cases of melanoma (28.9%) in Japanese patients involved the skin of the soles. Whites tend to have a wide distribution of cutaneous melanoma over the whole body surface, showing the greatest aggregation of lesions on the head, neck, and trunk, with small number on the soles of the feet. This comparative study revealed that the anatomical distribution and clinical types of malignant melanoma in the United States and Japan were different. The racial difference--the highest frequency or proportion of melanoma on the skin of the soles in the pigmented races, and the lowest ratio of this anatomical area in the least-pigmented races--remains to be investigated.

Percutaneous absorption of betamethasone 17-benzoate measured by radioimmunoassay.

Percutaneous absorption was studied in patients following topical application of betametahsone 17-benzoate cream and gel with occlusion by means of a sensitive and specific radioimmunoassay method. Concentrations of betamethasone 17-benzoate in plasma were between 0.3 and 5 ng/ml, indicating approximately 0.05 to 0.3% of the steroid applied to the skin was detected in plasma. Plasma betamethasone 17-benzoate levels increased in proportion to the amount of the steroid applied to the skin. High correlation between plasma betamethasone 17-benzoate levels and percent inhibition of plasma cortisol was also observed. Approximately 3 ng/ml levels of betamethasone 17-benzoate in plasma induced 90% inhibition of plasma cortisol. The data suggest that betamethasone 17-benzoate in gel base was more readily absorbed than in cream base.

Thermochemotherapy for malignant melanoma: combination therapy of ACNU and hyperthermia in mice.

B16 melanoma (mouse melanoma) and C24 melanoma (human malignant melanoma) transplanted in mice were treated by a combined therapy of ACNU [1-(4-amino-2-methyl - 5 - pyrimidinyl) - methyl - 3 - (2 - chlorethyl) - 3 - nitrosourea hydrochloride] (10 mg/kg) and hyperthermia (43 degrees C, 30 min). In both types of melanoma, a marked synergistic effect of the combined therapy was noted. Particularly in C24 melanoma a reduction in the size of tumor was observed. Histopathologic findings revealed a strong degeneration such as destruction of the tumor structure and vacuolization of nuclei.

Thermochemotherapy for malignant melanoma: overcoming heterogeneity in drug sensitivity.

Endo B (melanotic) and W (amelanotic) human malignant melanomas originated from the same tumor, both known to be heterogeneous in drug sensitivity to ACNU [( 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitroso- urea hydrochloride]), were treated experimentally with a combination therapy of ACNU and hyperthermia in mice. Whereas Endo W melanoma has no sensitivity, Endo B melanoma is sensitive to ACNU alone. However, in both types of melanomas, a marked synergistic effect of the combination therapy was noted. Histologically, marked degeneration of both tumor cells was detected. These results strongly suggest that thermochemotherapy may overcome the tumor heterogeneity in drug sensitivity.

12-O-tetradecanoylphorbol-13-acetate inhibits osteoclast-like cell differentiation in rat bone marrow cultures by inducing macrophage polykaryons.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) at 10(-9) M stimulated the formation of osteoclast-like multinucleated cells (MNCs) in the presence of 1 alpha,25-dihydroxyvitamin D3 in rat bone marrow cultures. However, at 10(-7) M, it clearly inhibited 1 alpha,25-dihydroxyvitamin D3-dependent osteoclast-like MNC formation at 6 days of culture. In cultures treated with 10(-7) M TPA, numerous MNCs that lack the marker enzyme tartrate-resistant acid phosphatase (TRAP) were formed. These TRAP-negative MNCs had neither receptors for calcitonin nor dentine-resorbing activity. The reactivity of the cells against antirat macrophage antibodies was completely different from that of authentic osteoclasts. These data suggest that TRAP-negative MNCs formed in the presence of 10(-7) M TPA are macrophage polykaryons. Time-course studies showed that 10(-7) M TPA stimulated osteoclast-like MNC formation at 4 days of culture, but these osteoclast-like MNCs were converted to TRAP-negative MNCs. Furthermore, 1-(5-isoquinolinyl-sulfonyl)2-methylpiperazine (H-7), a protein kinase-C inhibitor, inhibited osteoclast-like MNC formation in a dose-dependent fashion. These results suggest that activation of protein kinase-C may play a role in osteoclast differentiation.

Regulation of osteoclastogenesis by antisense oligodeoxynucleotides specific to zinc finger nuclear transcription factors Egr-1 and WT1 in rat bone marrow culture system.

Differentiation of osteoclasts is defined by the transcription factors expressed in response to bone microenvironments. In this work, we examined the effects of an expressional blockage of Egr-1 and/or WT1 on the differentiation of osteoclasts using specific antisense oligodeoxynucleotides (ODN). In a culture system forming preosteoclast-like cells (POC) from rat bone marrow cells depleted of marrow stromal cells, POC formation was markedly stimulated by the addition of Egr-1 antisense ODN compared to that in cultures in which sense ODN was added, whereas Egr-1 antisense ODN inhibited the formation of macrophage-like cells. The formation of multinucleated osteoclast-like cells was also stimulated by the addition of Egr-1 antisense ODN in whole bone marrow cultures. In contrast, WT1 antisense ODN did not affect POC formation induced by the treatment with Egr-1 antisense ODN; however, WT1 antisense ODN dramatically suppressed the formation of osteoclast-like multinucleated cells induced by the blockage of Egr-1 expression using Egr-1 antisense ODN. These data suggest that Egr-1 acts as the suppressor, not as the inducer, in osteoclastogenesis. The findings also suggested that WT1 could be involved in the multinucleation step of osteoclastogenesis, at least when Egr-1 expression was blocked.

In vitro differentiation of the murine macrophage cell line BDM-1 into osteoclast-like cells.

Osteoclasts are derived from hematopoietic stem cells, but details about their precursor are still obscure. We present here a mouse macrophage cell line, BDM-1 cells, that showed a high potential to differentiate into osteoclast-like multinucleate cells (MNCs) when cocultured with primary osteoblasts for 14 days in the presence of 10(-8) M 1 alpha,25-dihydroxyvitamin D3. These MNCs had tartrate-resistant acid phosphatase (TRAP) activity and strong ability to resorb dentine. In this culture system, 10(-10)-10(-8) M 12-O-tetradecanoylphorbol-13-acetate stimulated the formation of TRAP-positive MNCs, whereas salmon calcitonin inhibited it. Time-course effect studies showed that 12-O-tetradecanoylphorbol-13-acetate had an effect on the late phase of osteoclast differentiation but not on precursor proliferation. By immunocytochemical staining, all BDM-1 cells expressed Mac-1, Mac-2, and MOMA-2 antigens, and a large number of them expressed F4/80 antigen, but the rest of them were negative for this antigen. To select subclones able to differentiate into TRAP-positive MNCs, we sought to isolate several subclones from BDM-1 cells by mean of different specificity for F4/80 antigen expression. TRAP-positive MNCs were not generated from F4/80-positive subclones, but were obtained from subclones containing F4/80-negative cells. These results suggest that an F4/80-negative macrophage subpopulation is responsible for the differentiation of this cell line into osteoclasts.

Involvement of lymphocyte function-associated antigen-1 and intercellular adhesion molecule-1 in osteoclastogenesis: a possible role in direct interaction between osteoclast precursors.

In our search for molecules involved in the process of osteoclast differentiation, we examined the surface phenotypes of the preosteoclast-like cells and osteoclast-like multinucleated cells (MNCs) formed in bone marrow cultures, using monoclonal antibodies recognizing different antigen molecules expressed on hematopoietic cells. Among these cell surface antigens, lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) were highly expressed on mononuclear cells in the cultures for forming preosteoclast-like mononuclear cells. The double detection of these two antigen molecules with osteoclast-specific antigen and with calcitonin receptor, using a fluorescence-activated cell sorter or autoradiography technique, revealed that LFA-1 and ICAM-1 were expressed on the preosteoclasts. The expression of ICAM-1 was detected on both preosteoclasts and osteoclast-like MNCs, whereas the expression of LFA-1 was restricted to preosteoclasts. We designed a peptide with the sequence of the binding site of ICAM-1 against the ligand LFA-1. In the whole bone marrow culture system for forming osteoclast-like MNCs, a significant inhibition of MNC formation was observed by the addition of this peptide. These results strongly suggest the involvement of an LFA-1/ICAM-1-interaction in osteoclastogenesis.

Inhibitory effects of the bone-derived growth factors osteoinductive factor and transforming growth factor-beta on isolated osteoclasts.

Demineralized bone matrix contains a number of growth factors for osteoblast-like cells. Two of these, the novel glycoprotein osteoinductive factor (OIF) and transforming growth factor-beta (TGF beta), act together to cause ectopic bone formation in vivo. Since OIF, like TGF beta, is likely released from bone when the matrix is resorbed, we examined the effects of homogeneous OIF and TGF beta on osteoclast function. Osteoclast function was tested in isolated avian osteoclasts and was measured in terms of tartrate-resistant acid phosphatase (TRAP) activity, oxygen-derived free radical production, and formation of characteristic resorption lacunae on slices of sperm whale dentine. OIF (50-100 ng/ml) inhibited the capacity of these osteoclasts to form lacunae whether assessed by the number of excavations per slice or by the total area resorbed. OIF (10-100 ng/ml) or TGF beta (10-20 ng/ml) caused a decrease in TRAP activity as well as a reduction in oxygen-derived free radical generation detected by nitroblue tetrazolium staining. TGF beta had no effect on the resorption capacity of isolated osteoclasts in concentrations that inhibited TRAP activity and nitroblue tetrazolium staining. These results suggest that growth regulatory factors, such as OIF and TGF beta, released during the resorption of bone may be endogenous inhibitors of continued osteoclastic activity. This cessation of osteoclast activity may be an essential preliminary step to the new bone formation that occurs at resorption sites during bone remodeling.

Trypsinized osteoclast-like multinucleated cells formed in rat bone marrow cultures efficiently form resorption lacunae on dentine.

Rat bone marrow cultures containing 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] formed multinucleated cells (MNCs) that had many characteristics of osteoclasts. These MNCs, which have a tartrate-resistant acid phosphatase (TRAP) activity, could be classified into two morphological types: one type had smooth cellular margins (smooth-margined MNCs) and the other type had irregular spike-like margins (stellate MNCs). When bone marrow cells depleted of authentic osteoclasts were seeded and cultured on dentine slices, only low numbers of resorption lacunae could be detected. However, when preformed MNCs were detached by trypsinization and replated on dentine slices, numerous resorption lacunae were observed by scanning electron microscopy on these slices. Formation of lacunae occurred reproducibly during the five to ten days of culture. We also examined the effect of retinoic acid on TRAP-positive MNC formation in this bone marrow culture system. Although RA inhibited total TRAP-positive MNC formation, it increased the ratio of stellate MNCs to smooth-margined MNC, suggesting that RA may have the ability to regulate the formation of active osteoclasts.

Tumor necrosis factor-alpha cooperates with receptor activator of nuclear factor kappaB ligand in generation of osteoclasts in stromal cell-depleted rat bone marrow cell culture.

A member of the tumor necrosis factor (TNF) family, receptor activator of nuclear factor kappaB ligand (RANKL; also known as ODF, OPGL, and TRANCE), plays critical roles in osteoclast differentiation and activation in the presence of macrophage colony-stimulating factor (M-CSF). Recently, TNF-alpha has also been shown to induce the formation of multinucleated osteoclast-like cells (MNCs) in the presence of M-CSF from mouse macrophages. We demonstrated that mononuclear preosteoclast-like cells (POCs) were formed in the presence of conditioned medium of osteoblastic cells in a rat bone marrow culture depleted of stromal cells. Using this culture system, in this study we examined whether TNF-alpha affects differentiation into POCs from hematopoietic progenitor cells. Human TNF-alpha (hTNF-alpha) markedly stimulated the formation of POCs. Moreover, a concentration as low as 0.005 ng/mL of hTNF-alpha increased the level of mRNA for calcitonin receptor (CTR) and cathepsin-K of POCs. The POCs induced by hTNF-alpha formed MNCs, which showed dentine-resorbing activity after coculture with primary osteoblasts. Stimulation was observed after 24 h of treatment with hTNF-alpha only on day 1 or day 2 of the culture. After 24 h of hTNF-alpha treatment, expression of the receptor activator of nuclear factor kappaB (RANK) mRNA was markedly increased. The addition of soluble RANKL (sRANKL) to the preformed POCs efficiently induced MNCs. Interestingly, treatment of bone marrow cells with hTNF-alpha and sRANKL synergistically augmented the formation of MNCs. This formation was abolished by the addition of human osteoprotegerin (hOPG). These results suggest that cooperation of TNF-alpha and RANKL is important for osteoclastogenesis.

Osteoclast differentiation antigen, distinct from receptor activator of nuclear factor kappa B, is involved in osteoclastogenesis under calcitonin-regulated conditions.

Although calcitonin has been clinically utilized as a primary treatment for several metabolic bone diseases, its inhibitory effects against osteoclastic function diminish after several days owing to the calcitonin 'escape phenomenon'. We have previously found a unique cell-surface antigen (Kat1-antigen) expressed on rat osteoclasts. Here we show evidence that, in the presence of calcitonin, the Kat1-antigen is involved in osteoclastogenesis. Treatment of bone marrow cultures for forming osteoclast-like cells with anti-Kat1-antigen monoclonal antibody (mAb Kat1) provoked a marked stimulation of osteoclast-like cell formation only in the presence of calcitonin but not in its absence. Osteoclastogenesis stimulated by the receptor activator of nuclear factor kappa B (NF-kappaB) ligand/osteoclast differentiation factor was further augmented by mAb Kat1 in the presence of calcitonin. Furthermore, even in the presence of the osteoprotegerin/osteoclast inhibitory factor, mAb Kat1 induced osteoclast-like cell formation. Our current data suggest that the Kat1-antigen is a molecule that is distinct from receptor activator of NF-kappaB. The presence of the unique Kat1-antigen on cells in the osteoclast lineage appears to contribute to the fine regulation of osteoclastogenesis in vivo. Expression of this cell-surface molecule in cells in the osteoclast lineage may partly explain the mechanism responsible for the escape phenomenon.

Osteoclast differentiation antigen.

Osteoclasts are the primary cells which perform bone resorption. The origin of these multinucleated giant cells is the haematopoietic stem cells. The differentiation pathway of the osteoclasts has so far been well studied and the cell-lineage of these bone resorbing cells is considered to be close but not identical to the monocytes/macrophages. Owing to the development of in vitro culture systems for evaluating osteoclast differentiation, it has been elucidated that various cytokines are involved in the differentiation of the osteoclasts. However, there is still ambiguity concerning the molecular mechanism of the differentiation of the osteoclasts. One approach for clarifying the molecular mechanism is to find unique antigen molecules involved in the process of osteoclast differentiation. In this review article, we introduce such immunological studies concerning osteoclast differentiation. We also refer to our recent establishment of a panel of monoclonal antibodies recognizing rat osteoclasts. One of the monoclonal antibodies recognizes cell surface antigen (Kat1-antigen) expressed on cells in osteoclast-lineage and not on monocytes/macrophages. Cross-linking of the cell surface antigen using this monoclonal antibody showed that the Kat1-antigen is the unique cell surface molecule involved in the regulation of the affinity of the calcitonin receptor and also involved in the modulation of bone resorption. In this review article, we overview, the current issues which should be elucidated for understanding the differentiation and activation of the osteoclasts. We further emphasize the utility of the immunological approach for solving these current target issues.

Behavior of melanocytes in reticulate acropigmentation of Kitamura.

Four patients with reticulate acropigmentation of Kitamura (RAPK) had reticulate pigmentation that was slightly depressed below the skin surface. Histologic findings revealed epidermal atrophy, elongation of the rete ridges with large amounts of melanin, and an increased number of dopa-positive basal melanocytes. Electron microscopic findings showed many melanosome complexes within the keratinocytes, dendrites filled with numerous melanosomes, and some melanosome complexes within the melanocytes. It is concluded that the hyperpigmentation of RAPK is due to increased number of active melanocytes and to increased transfer of melanosomes to surrounding keratinocytes.

Systemic plasmacytosis. A syndrome of peculiar multiple skin eruptions, generalized lymphadenopathy, and polyclonal hypergammaglobulinemia.

A description is given of two patients with peculiar multiple skin eruptions, asymptomatic generalized lymphadenopathy, and polyclonal hypergammaglobulinemia. Both patients were admitted to our hospital for further evaluation of an increased erythrocyte sedimentation rate and hypergammaglobulinemia discovered during routine medical examinations. Despite various investigations, the underlying disease causing the hypergammaglobulinemia was not found. Histologic examination disclosed dense perivascular infiltration of plasma cells in the dermis. In the lymph nodes, considerable plasma cell infiltration was found from the cortex to the medulla. These plasma cells were mature and showed no cellular atypism. The association of peculiar multiple skin eruptions, lymphadenopathy, and polyclonal hypergammaglobulinemia, which we have called "systemic plasmacytosis," signifies a new syndrome that can be differentiated from diseases reported previously.

Pretibial epidermolysis bullosa. Successful therapy with a skin graft.

A patient with pretibial epidermolysis bullosa was successfully treated with a skin graft. Ultrastructural examination revealed a decreased number of and rudimentary anchoring fibrils (AFs) in the pretibial area in contrast to normal AFs in the grafted skin obtained from a nonpredilection site. Our results indicate the importance of AFs in the pathogenesis of pretibial epidermolysis bullosa.

Ultrastructural study of tissue reaction of mice against Sporothrix schenckii infection.

In the present study, in order to clarify the defense mechanisms against S. schenckii infections, we examined the tissue reactions of mice against the pathogen over a period of time by both light and electron microscopy. The histological features were, at an early stage, a mixed cell granuloma consisting of polymorphonuclear leukocytes (PMNs) and macrophages, and, later on, enlargement and vacuolation of the macrophages at the periphery. In electron microscopy, blastospores had been phagocytized by the PMNs and macrophages, and no extracellular blastospores were seen. PMNs taking up blastospores were phagocytized by other PMNs or by macrophages. After 3 or 4 months, the phagosomes of the macrophages had grown in size, and contained a number of blastospores. During the experiment, the ultrastructure of the blastospores was well preserved, and their viability was considered high. Of the defense mechanisms against S. schenckii infection, PMN phagocytosis is of great importance, but the fact that proliferation of this organism was observed within the macrophages, suggested that the macrophages were not able to destroy the organisms, but rather were responsible for the disease becoming chronic.