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Positive Multinucleated Cells (positive + multinucleated_cell)
Selected AbstractsBumetanide, the Specific Inhibitor of Na+ -K+ -2Cl, Cotransport, Inhibits 1,,25-Dihydroxyvitamin D3 -Induced Osteoclastogenesis in a Mouse co-culture SystemEXPERIMENTAL PHYSIOLOGY, Issue 5 2003Hyun-A Lee The Na+ -K+ -2Cl, cotransporter (NKCC1) is responsible for ion transport across the secretory and absorptive epithelia, the regulation of cell volume, and possibly the modulation of cell growth and development. It has been reported that a variety of cells, including osteoblasts, contain this cotransporter. In this study, the physiological role of NKCC1 in osteoclastogenesis was exploited in a co-culture system. Bumetanide, a specific inhibitor of NKCC1, reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. In order to investigate the mechanism by which bumetanide inhibits osteoclastogenesis, the mRNA expressions of the receptor activator of nuclear factor (NF)-,B ligand (RANKL) and osteoprotegerin (OPG) were analysed by RT-PCR. Exposure of osteoblastic cells to a medium containing 1 µM bumetanide reduced RANKL mRNA expression induced by 10 nM 1,,25-dihydroxyvitamin D3 (1,,25(OH)2D3, in a dose-dependent manner. In addition, RANKL expression was also analysed with enzyme-linked immunosorbant assay (ELISA) using anti-RANKL antibody. The expression of RANKL was decreased with the increase of bumetanide concentration. In contrast, the expression of OPG mRNA, a novel tumour necrosis factor (TNF) receptor family member was increased in the presence of bumetanide. These results imply that bumetanide inhibits osteoclast differentiation by reducing the RANKL/OPG ratio in osteoblastic cells. However, no significant difference in M-CSF mRNA expression was observed when bumetanide was added. Also, we found that the phosphorylation of c-Jun NH2 -terminal kinase (JNK), which regulates the activity of various transcriptional factors, was reduced by bumetanide treatment. Conclusively, these findings suggest that NKCC1 in osteoblasts has a pivotal role in 1,,25(OH)2D3 -induced osteoclastogenesis partly via the phosphorylation of JNK. [source] Regulation of osteoclastogenesis and RANK expression by TGF-,1JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2001Tao Yan Abstract Transforming growth factor-, (TGF-,) has been shown to both inhibit and to stimulate bone resorption and osteoclastogenesis. This may be due, in part, to differential effects on bone marrow stromal cells that support osteoclastogenesis vs. direct effects on osteoclastic precursor cells. In the present study, we used the murine monocytic cell line, RAW 264.7, to define direct effects of TGF-, on pre-osteoclastic cells. In the presence of macrophage-colony stimulating factor (M-CSF) (20 ng/ml) and receptor activator of NF-,B ligand (RANK-L) (50 ng/ml), TGF-,1 (0.01,5 ng/ml) dose-dependently stimulated (by up to 120-fold) osteoclast formation (assessed by the presence of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells and expression of calcitonin and vitronectin receptors). In addition, TGF-,1 also increased steady state RANK mRNA levels in a time- (by up to 3.5-fold at 48 h) and dose-dependent manner (by up to 2.2-fold at 10 ng/ml). TGF-,1 induction of RANK mRNA levels was present both in undifferentiated RAW cells as well as in cells that had been induced to differentiate into osteoclasts by a 7-day treatment with M-CSF and RANK-L. Using a fluorescence-labeled RANK-L probe, we also demonstrated by flow cytometry that TGF-,1 resulted in a significant increase in the percentage of RANK+ RAW cells (P,<,0.05), as well as an increase in the fluorescence intensity per cell (P,<,0.05), the latter consistent with an increase in RANK protein expression per cell. These data thus indicate that TGF-, directly stimulates osteoclastic differentiation, and this is accompanied by increased RANK mRNA and protein expression. J. Cell. Biochem. 83: 320,325, 2001. © 2001 Wiley-Liss, Inc. [source] Platelet-rich plasma impairs osteoclast generation from human precursors of peripheral bloodJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2010Elisabetta Cenni Abstract Platelet-rich plasma is used to accelerate bone repair for the release of osteogenic growth factors from activated platelets. To date, the effects on osteoclasts have been only scarcely investigated, even though these cells are crucial for bone remodeling. The aim of this research was the evaluation of the effects of thrombin-activated platelets (PRP) on osteoclastogenesis from human blood precursors. We evaluated both the ability to influence osteoclast differentiation induced by the receptor activator of nuclear factor-kappaB ligand (RANKL), and the ability to induce osteoclast differentiation without RANKL. In both assays, the incubation with PRP supernatant at 10% did not significantly affect the formation of tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells that were able to form the F-actin ring. However, when PRP at 25 and 50% was added to the medium without RANKL, the generation of TRACP-positive multinucleated cells was inhibited. PRP, even at 10%, reduced the osteoclast-mediated bone collagen degradation, suggesting inhibition of osteoclast activation. Similarly, after incubation with PRP supernatant, calcitonin receptor mRNA was lower than the untreated samples. In conclusion, PRP at 10% interfered with the complete differentiation process of human osteoclast precursors. At higher concentration it impaired osteoclast formation also at an early stage of differentiation. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:792,797, 2010 [source] Interleukin-27 inhibits human osteoclastogenesis by abrogating RANKL-mediated induction of nuclear factor of activated T cells c1 and suppressing proximal RANK signalingARTHRITIS & RHEUMATISM, Issue 2 2010George D. Kalliolias Objective Interleukin-27 (IL-27) has stimulatory and regulatory immune functions and is expressed in rheumatoid arthritis (RA) synovium. This study was undertaken to investigate the effects of IL-27 on human osteoclastogenesis, to determine whether IL-27 can stimulate or attenuate the osteoclast-mediated bone resorption that is a hallmark of RA. Methods Osteoclasts were generated from blood-derived human CD14+ cells. The effects of IL-27 on osteoclast formation were evaluated by counting the number of tartrate-resistant acid phosphatase,positive multinucleated cells and measuring the expression of osteoclast-related genes. The induction of nuclear factor of activated T cells c1 (NFATc1) and the activation of signaling pathways downstream of RANK were measured by immunoblotting. The expression of key molecules implicated in osteoclastogenesis (NFATc1, RANK, costimulatory receptors, and immunoreceptor tyrosine,based activation motif,harboring adaptor proteins) was measured by real-time reverse transcription,polymerase chain reaction. Murine osteoclast precursors obtained from mouse bone marrow and synovial fluid macrophages derived from RA patients were also tested for their responsiveness to IL-27. Results IL-27 inhibited human osteoclastogenesis, suppressed the induction of NFATc1, down-regulated the expression of RANK and triggering receptor expressed on myeloid cells 2 (TREM-2), and inhibited RANKL-mediated activation of ERK, p38, and NF-,B in osteoclast precursors. Synovial fluid macrophages from RA patients were refractory to the effects of IL-27. In contrast to the findings in humans, IL-27 only moderately suppressed murine osteoclastogenesis, and this was likely attributable to low expression of the IL-27 receptor subunit WSX-1 on murine osteoclast precursors. Conclusion IL-27 inhibits human osteoclastogenesis by a direct mechanism that suppresses the responses of osteoclast precursors to RANKL. These findings suggest that, in addition to its well-known antiinflammatory effects, IL-27 plays a homeostatic role in restraining bone erosion. This homeostatic function is compromised under conditions of chronic inflammation such as in RA synovitis. [source] Development of an ex vivo cellular model of rheumatoid arthritis: Critical role of cd14-positive monocyte/macrophages in the development of pannus tissueARTHRITIS & RHEUMATISM, Issue 9 2007Toshiko Nozaki Objective To establish an ex vivo cellular model of pannus, the aberrant overgrowth of human synovial tissue (ST). Methods Inflammatory cells that infiltrated pannus tissue from patients with rheumatoid arthritis (RA) were collected without enzyme digestion, and designated as ST-derived inflammatory cells. Single-cell suspensions of ST-derived inflammatory cells were cultured in medium alone. Levels of cytokines produced in culture supernatants were measured using enzyme-linked immunosorbent assay kits. ST-derived inflammatory cells were transferred into the joints of immunodeficient mice to explore whether these cells could develop pannus. CD14 and CD2 cells were depleted by negative selection. Results Culture of ST-derived inflammatory cells from 92 of 111 patients with RA resulted in spontaneous reconstruction of inflammatory tissue in vitro within 4 weeks. Ex vivo tissue contained fibroblasts, macrophages, T cells, and tartrate-resistant acid phosphatase,positive multinucleated cells. On calcium phosphate,coated slides, ST-derived inflammatory cell cultures showed numerous resorption pits. ST-derived inflammatory cell cultures continuously produced matrix metalloproteinase 9 and proinflammatory cytokines associated with osteoclastogenesis, such as tumor necrosis factor ,, interleukin-8, and macrophage colony-stimulating factor. More importantly, transferring ST-derived inflammatory cells into the joints of immunodeficient mice resulted in the development of pannus tissue and erosive joint lesions. Both in vitro development and in vivo development of pannus tissue by ST-derived inflammatory cells were inhibited by depleting CD14-positive, but not CD2-positive, cells from ST-derived inflammatory cells. Conclusion These findings suggest that overgrowth of inflammatory cells from human rheumatoid synovium simulates the development of pannus. This may prove informative in the screening of potential antirheumatic drugs. [source] | ||||||||||