ABSTRACT
Application of 'omics' technology, and advances in in vitro methods for studying the growth of Fasciola hepatica, have highlighted the central role of migrating neoblasts in driving forward development and differentiation towards the adult-like form. Neoblast populations present molecular heterogeneity, morphological variation and changes associated with recruitment of these stem cells into their final tissue locations. However, terminal differentiation towards function, has received much less attention than has been the case for the free-living Platyhelminths. An actively replicating neoblast population, comprising cells with heterochromatic nuclei consistent with regulation of gene expression, has been identified in the parenchyma of juvenile Fasciola gigantica migrating in the liver of experimentally infected mice. In some of these cells, early cytoplasmic differentiation towards myocyte function was noted. Neoblasts have also been identified close to, and incorporated in, the subtegumental zone, the gastrodermis and the excretory ducts. In these locations, progressive morphological differentiation towards terminal function has been described. This includes the appearance of specific progenitors of type-1, type-2 and type-3 tegumental cells, the latter possibly contributing to tegumental spine development. 'Cryptic' surface molecular differentiation is postulated to account for recognition and 'docking' of migrating neoblasts with their final site for terminal differentiation.
Subject(s)
Fasciola , Fascioliasis , Liver , Animals , Mice , Fascioliasis/parasitology , Fascioliasis/veterinary , Liver/parasitology , Fasciola/physiology , Cell DifferentiationABSTRACT
The therapeutic goal of increasing bone mass by co-treatment of parathyroid hormone (PTH) and an osteoclast inhibitor has been complicated by the undefined contribution of osteoclasts to the anabolic activity of PTH. To determine whether active osteoclasts are required at the time of PTH administration, we administered a low dose of the transient osteoclast inhibitor salmon calcitonin (sCT) to young rats receiving an anabolic PTH regimen. Co-administration of sCT significantly blunted the anabolic effect of PTH as measured by peripheral quantitative computer tomography (pQCT) and histomorphometry in the femur and tibia, respectively. To determine gene targets of sCT, we carried out quantitative real time PCR and microarray analysis of metaphyseal samples 1.5, 4 and 6.5h after administration of a single injection of PTH, sCT or PTH+sCT. Known targets of PTH action, IL-6, ephrinB2 and RANKL, were not modified by co-administration with sCT. Surprisingly, at all time points, we noted a significant upregulation of sclerostin mRNA by sCT treatment, as well as down-regulation of two other osteocyte gene products, MEPE and DMP1. Immunohistochemistry confirmed that sCT administration increased the percentage of osteocytes expressing sclerostin, suggesting a mechanism by which sCT reduced the anabolic effect of PTH. Neither mRNA for CT receptor (Calcr) nor labeled CT binding could be detected in sclerostin-enriched cells differentiated from primary calvarial osteoblasts. In contrast, osteocytes freshly isolated from calvariae expressed a high level of Calcr mRNA. Furthermore immunohistochemistry revealed co-localization of CT receptor (CTR) and sclerostin in some osteocytes in calvarial sections. Taken together these data indicate that co-treatment with sCT can blunt the anabolic effect of PTH and this may involve direct stimulation of sclerostin production by osteocytes. These data directly implicate calcitonin as a negative regulator of bone formation through a previously unsuspected mechanism.
Subject(s)
Bone Morphogenetic Proteins/genetics , Calcitonin/pharmacology , Genetic Markers/genetics , Osteocytes/metabolism , Parathyroid Hormone/pharmacology , Animals , Cells, Cultured , Computational Biology , Extracellular Matrix Proteins/genetics , Female , Femur/drug effects , Femur/metabolism , Humans , Immunohistochemistry , Interleukin-6/genetics , Mice , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis , Osteocytes/drug effects , Phosphoproteins/genetics , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Tibia/drug effects , Tibia/metabolismABSTRACT
Members of the ephrin and Eph family are local mediators of cell function through largely contact-dependent processes in development and in maturity. Production of ephrinB2 mRNA and protein are increased by PTH and PTHrP in osteoblasts. Both a synthetic peptide antagonist of ephrinB2/EphB4 receptor interaction and recombinant soluble extracellular domain of EphB4 (sEphB4), which is an antagonist of both forward and reverse EphB4 signaling, were able to inhibit mineralization and the expression of several osteoblast genes involved late in osteoblast differentiation. The findings are consistent with ephrinB2/EphB4 signaling within the osteoblast lineage having a paracrine role in osteoblast differentiation, in addition to the proposed role of osteoclast-derived ephrinB2 in coupling of bone formation to resorption. This local regulation might contribute to control of osteoblast differentiation and bone formation at remodeling sites, and perhaps also in modeling.
Subject(s)
Cell Lineage , Ephrin-B2/metabolism , Osteoblasts/cytology , Osteoblasts/metabolism , Receptor, EphB4/metabolism , Signal Transduction , Animals , Cell Communication , Humans , Mice , Osteoclasts/cytology , Osteoclasts/metabolism , Osteogenesis , RatsABSTRACT
Reactive oxygen species such as superoxide are implicated in cardiac hypertrophy, but their contribution to the cardiac complications of insulin resistance is unresolved. We tested the hypothesis that the antioxidant tempol attenuates cardiac hypertrophy in insulin-resistant mice. Mice with cardiac GLUT4 deletion (GLUT4-knockout), superimposed on global GLUT4 suppression (GLUT4-knockdown) were administered tempol for 4 weeks. Age-matched GLUT4-knockdown littermates were used as controls (14 mice/group). GLUT4-knockout mice exhibited marked cardiac hypertrophy: heart to body weight ratio was increased 61+/-7% and expression of the hypertrophic genes beta-myosin heavy chain and B-type natriuretic peptide (BNP) were elevated 5.5+/-0.7- and 6.2+/-1.5-fold relative to control, respectively. Pro-fibrotic pro-collagen III expression was also higher (3.8+/-0.7-fold) in the GLUT4-knockout myocardium (all p<0.001). Both gp91(phox) and Nox1 subunits of NADPH oxidase were also upregulated, 4.9+/-1.2- and 9.3+/-2.8-fold (both p<0.01). Tempol treatment significantly attenuated all of these abnormalities in GLUT4-knockout mice. Heart to body weight ratio was decreased, as was fold expression of beta-myosin heavy chain (to 3.8+/-0.8), BNP (to 2.5+/-0.5), pro-collagen III (to 1.9+/-0.4), gp91(phox) (to 0.9+/-0.3) and Nox1 (to 2.3+/-0.1, all p<0.05 versus untreated GLUT4-knockout mice). In addition, tempol upregulated ventricular expression of both thioredoxin-2 (confirming an antioxidant action) and glycogen synthase kinase-3beta (GSK-3beta). Tempol did not elicit any other significant changes in control mice. Cardiac superoxide generation, however, was not altered by GLUT4-knockout or tempol. In conclusion, tempol treatment reduced morphological and molecular evidence of cardiac hypertrophy in the GLUT4-knockout insulin-resistant mouse in vivo, even at doses insufficient to lower cardiac superoxide. Parallel reductions in pro-collagen III and NADPH oxidase have important implications for our understanding of the molecular basis of cardiac hypertrophy in the setting of insulin resistance. Antioxidants may offer new alternatives in this disorder.
Subject(s)
Antioxidants/pharmacology , Cardiomegaly/drug therapy , Cyclic N-Oxides/pharmacology , Glucose Transporter Type 4/deficiency , Insulin Resistance/genetics , Animals , Female , Glucose Transporter Type 4/genetics , Male , Mice , Mice, Knockout , Spin LabelsABSTRACT
Since parathyroid hormone (PTH) is the only proven anabolic therapy for bone, it becomes the benchmark by which new treatments will be evaluated. The anabolic effect of PTH is dependent upon intermittent administration, but when an elevated PTH level is maintained even for a few hours it initiates processes leading to new osteoclast formation, and the consequent resorption overrides the effects of activating genes that direct bone formation. Identification of PTH-related protein (PTHrP) production by cells early in the osteoblast lineage, and its action through the PTH1R upon more mature osteoblastic cells, together with the observation that PTHrP+/- mice are osteoporotic, all raise the possibility that PTHrP is a crucial paracrine regulator of bone formation. The finding that concurrent treatment with bisphosphonates impairs the anabolic response to PTH, adds to other clues that osteoclast activity is necessary to complement the direct effect that PTH has in promoting differentiation of committed osteoblast precursors. This might involve the generation of a coupling factor from osteoclasts that are transiently activated by receptor activator of nuclear factor-kappaB ligand (RANKL) in response to PTH. New approaches to anabolic therapies may come from the discovery that an activating mutation in the LRP5 gene is responsible for an inherited high bone mass syndrome, and the fact that this can be recapitulated in transgenic mice, whereas inactivating mutations result in severe bone loss. This has focused attention on the Wnt/frizzled/beta-catenin pathway as being important in bone formation, and proof of the concept has been obtained in experimental models.
Subject(s)
Anabolic Agents/therapeutic use , Bone Development/physiology , Bone Diseases/drug therapy , Animals , Humans , Mice , Mice, Knockout , Parathyroid Hormone/physiology , Receptors, Parathyroid Hormone/physiology , Signal TransductionABSTRACT
Summary The ratio of osteoprotegerin [OPG, tumour necrosis factor receptor superfamily, member 11b (TNFRSF11B)] to receptor activator of nuclear factor kappaB ligand [RANKL, tumour necrosis factor (ligand) superfamily, member 11 (TNFSF11)] in bone is critical for the regulation of bone remodelling. Myeloma cells can home to bone, triggering increased RANKL and decreased OPG expression by stromal cells, leading to osteolysis. Whether myeloma cells contribute directly to the pool of RANKL or OPG in bone has been contentious. Here we provide evidence of RANKL expression by reverse transcription polymerase chain reaction and in situ hybridization, demonstrating transcripts encoding both the membrane-bound and secreted forms of RANKL in five human multiple myeloma cell lines (LP-1, NCI-H929, OPM-2, RPMI8226, U266) and myeloma cells purified from bone marrow aspirates of myeloma patients. We demonstrated that RANKL encoding mRNAs are translated to protein by antibody detection of RANKL. In vitro assays showed that myeloma cells induced bone marrow derived mononuclear cells to differentiate into adherent tartrate-resistant acid phosphatase positive multinucleated cells, indicative of the formation of functional osteoclasts. This differentiation could also be achieved with passaged myeloma media alone, implicating secreted products. Finally, we provide evidence that the differentiation observed is at least in part the result of myeloma cell expression of RANKL. We therefore conclude that myeloma cells can directly contribute to the pool of RANKL in bone.
Subject(s)
Carrier Proteins/genetics , Leukocytes, Mononuclear/pathology , Membrane Glycoproteins/genetics , Multiple Myeloma/pathology , Osteoclasts/pathology , RNA, Messenger/analysis , Acid Phosphatase/metabolism , Cell Differentiation , Cell Division , Cell Line, Tumor , Culture Media, Conditioned , Gene Expression , Humans , In Situ Hybridization/methods , Isoenzymes/metabolism , Multiple Myeloma/metabolism , Osteoclasts/immunology , Protein Isoforms/genetics , Proteoglycans , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , Reverse Transcriptase Polymerase Chain Reaction , Syndecans , Tartrate-Resistant Acid Phosphatase , Tumor Cells, CulturedABSTRACT
Osteoclasts form when hematopoietic cells are stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) or tumor necrosis factor-alpha (TNFalpha). Osteoclast precursors derive from M-CSF-dependent proliferating hematopoietic cells but cannot yet be purified from mixed populations. M-CSF stimulation of bone marrow cells results in large numbers of nonadherent, proliferating macrophage precursors. These rapidly form adherent bone marrow macrophages (BMM). BMM and their precursors can be isolated free from mesenchymal and lymphocytic cells. BMM precursors derived from CBA-strain mouse bone marrow, when cocultured with ST2 cells (which express RANKL and M-CSF), formed numerous mononuclear osteoclasts, which resorbed bone and expressed tartrate-resistant acid phosphatase (TRAP) and calcitonin receptors (CTR). Addition of approximately 10 BMM precursors to ST2 cultures resulted in over 80% of these cocultures forming functional osteoclasts, suggesting that they are a highly enriched source of osteoclast progenitors. Supporting this, recombinant RANKL/M-CSF-stimulated BMM precursors formed populations in which all cells expressed TRAP. While only a small proportion of these cells (8.6%) expressed CTR, with transforming growth factor-beta (TGFbeta) present RANKL/M-CSF-stimulated BMM precursors formed almost pure (98.4%) CTR-positive osteoclasts after 7 days. This suggests that TGFbeta stimulated the maturation rate of these cells. Passaged or viably frozen BMM precursors gave rise to BMM that also all formed osteoclasts lineage cells after RANKL/M-CSF stimulation. These data suggest that BMM precursors derived from CBA mice are an expanded pool of osteoclast progenitors. These can be employed to generate osteoclast populations of high purity and in large numbers when stimulated by TGFbeta, which greatly augments the osteoclastogenic effects of RANKL.
Subject(s)
Bone Marrow Cells/cytology , Osteoclasts/cytology , Animals , Bone Marrow Cells/drug effects , Bone Resorption/etiology , Bone Resorption/pathology , Carrier Proteins/pharmacology , Cell Differentiation/drug effects , Cell Line , Coculture Techniques , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Macrophage Colony-Stimulating Factor/pharmacology , Membrane Glycoproteins/pharmacology , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Osteoclasts/drug effects , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , Recombinant Proteins/pharmacology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Transforming growth factor beta (TGF-beta) is abundant in bone and has complex effects on osteolysis, with both positive and negative effects on osteoclast differentiation, suggesting that it acts via more than one mechanism. Osteoclastogenesis is determined primarily by osteoblast (OB) expression of the tumor necrosis factor (TNF)-related molecule receptor activator of NF-kappaB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG), which are increased and decreased, respectively, by osteolytic factors. A RANKL-independent osteoclastogenic mechanism mediated by TNF-alpha has also been shown. Therefore, we investigated TGF-beta effects on osteoclast formation in culture systems in which osteoclastogenic stimulus is dependent on OBs and culture systems where it was provided by exogenously added RANKL or TNF-alpha. Both OPG and TGF-beta inhibited osteoclast formation in hemopoietic cell/OB cocultures, but the kinetics of their action differed. TGF-beta also inhibited osteoclastogenesis in cocultures of cells derived from OPG null (opg-/-) mice. TGF-beta strongly decreased RANKL messenger RNA (mRNA) expression in cultured osteoblasts, and addition of exogenous RANKL to TGFbeta-inhibited cocultures of opg-/- cells partially restored osteoclastogenesis. Combined, these data indicate that the inhibitory actions of TGF-beta were mediated mainly by decreased OB production of RANKL. In contrast, in the absence of OBs, TGF-beta greatly increased osteoclast formation in recombinant RANKL- or TNF-alpha-stimulated cultures of hemopoietic cells or RAW 264.7 macrophage-like cells to levels several-fold greater than attainable by maximal stimulation by RANKL or TNF-alpha. These data suggest that TGF-beta may increase osteoclast formation via action on osteoclast precursors. Therefore, although RANKL (or TNF-alpha) is essential for osteoclast formation, factors such as TGF-beta may powerfully modify these osteoclastogenic stimuli. Such actions may be critical to the control of physiological and pathophysiological osteolysis.
Subject(s)
Carrier Proteins/metabolism , Glycoproteins/metabolism , Membrane Glycoproteins/metabolism , Osteoclasts/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Transforming Growth Factor beta/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Carrier Proteins/genetics , Carrier Proteins/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , Coculture Techniques , Glycoproteins/genetics , Glycoproteins/pharmacology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , Osteoclasts/cytology , Osteoclasts/drug effects , Osteoprotegerin , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Tumor Necrosis Factor , Spleen/cytology , Transforming Growth Factor beta/pharmacology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Bone is a major storage site for TGFbeta superfamily members, including TGFbeta and bone morphogenetic proteins. It is believed that these cytokines are released from bone during bone resorption. Recent studies have shown that both RANKL and macrophage colony-stimulating factor are two essential factors produced by osteoblasts for inducing osteoclast differentiation. In the present study we examined the effects of bone morphogenetic protein-2 on osteoclast differentiation and survival supported by RANKL and/or macrophage colony-stimulating factor. Mouse bone marrow-derived macrophages differentiated into osteoclasts in the presence of RANKL and macrophage colony-stimulating factor. TGFbeta superfamily members such as bone morphogenetic protein-2, TGFbeta, and activin A markedly enhanced osteoclast differentiation induced by RANKL and macrophage colony-stimulating factor, although each cytokine alone failed to induce osteoclast differentiation in the absence of RANKL. Addition of a soluble form of bone morphogenetic protein receptor type IA to the culture markedly inhibited not only osteoclast formation induced by RANKL and bone morphogenetic protein-2, but also the basal osteoclast formation supported by RANKL alone. Either RANKL or macrophage colony-stimulating factor stimulated the survival of purified osteoclasts. Bone morphogenetic protein-2 enhanced the survival of purified osteoclasts supported by RANKL, but not by macrophage colony-stimulating factor. Both bone marrow macrophages and mature osteoclasts expressed bone morphogenetic protein-2 and bone morphogenetic protein receptor type IA mRNAs. An EMSA revealed that RANKL activated nuclear factor-kappaB in purified osteoclasts. Bone morphogenetic protein-2 alone did not activate nuclear factor-kappaB, but rather inhibited the activation of nuclear factor-kappaB induced by RANKL in purified osteoclasts. These findings suggest that bone morphogenetic protein-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and survival. The enhancement of RANKL-induced survival of osteoclasts by bone morphogenetic protein-2 appears unrelated to nuclear factor-kappaB activation.
Subject(s)
Bone Morphogenetic Proteins/pharmacology , Carrier Proteins , Membrane Glycoproteins , Osteoclasts/cytology , Osteoclasts/physiology , Receptors, Tumor Necrosis Factor/physiology , Transforming Growth Factor beta , Animals , Bone Marrow Cells/cytology , Bone Morphogenetic Protein 2 , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Humans , Macrophage Colony-Stimulating Factor/pharmacology , Macrophages/cytology , Male , Mice , RANK Ligand , Receptor Activator of Nuclear Factor-kappa BABSTRACT
To understand the timescales and magnitude of responses that can be expected following catchment and riparian rehabilitation, we forecast changes to selected stream ecosystem attributes following tree planting in a pastoral catchment. All planting scenarios were predicted to lead to decreases in daily maximum water temperature after 15-20 years to levels that would be suitable for sensitive invertebrate species. Cooling and reheating were rapid so that most benefits to water temperature along the mainstem were forecast to accrue from shading all of the stream channel network. All planting scenarios were predicted to increase sediment yields over the status quo over the 25-year timeframe examined, with maximal sediment yield occurring about 15 years after planting due to expected erosion of the streambanks under the developing forest shade. Sediment yield was greatest for full catchment planting over 25 years, although sediment yield would be lowest with this scenario over longer timescales. A macroinvertebrate biotic index was predicted to increase by 25% over 15 years if whole catchment afforestation were implemented, compared to 9% if only the 4th order mainstem were planted with riparian trees. The use of ecological forecasting to predict likely outcomes for a range of scenarios should prove useful for prioritising rehabilitation actions.
Subject(s)
Conservation of Natural Resources , Ecosystem , Environmental Pollution/prevention & control , Trees , Agriculture , Animals , Forecasting , Invertebrates , Population Dynamics , Soil , Temperature , Water MovementsABSTRACT
We have cloned and expressed murine osteoclast inhibitory lectin (mOCIL), a 207-amino acid type II transmembrane C-type lectin. In osteoclast formation assays of primary murine calvarial osteoblasts with bone marrow cells, antisense oligonucleotides for mOCIL increased tartrate-resistant acid phosphatase-positive mononucleate cell formation by 3-5-fold, whereas control oligonucleotides had no effect. The extracellular domain of mOCIL, expressed as a recombinant protein in Escherichia coli, dose-dependently inhibited multinucleate osteoclast formation in murine osteoblast and spleen cell co-cultures as well as in spleen cell cultures treated with RANKL and macrophage colony-stimulating factor. Furthermore, mOCIL acted directly on macrophage/monocyte cells as evidenced by its inhibitory action on adherent spleen cell cultures, which were depleted of stromal and lymphocytic cells. mOCIL completely inhibited osteoclast formation during the proliferative phase of osteoclast formation and resulted in 70% inhibition during the differentiation phase. Osteoblast OCIL mRNA expression was enhanced by parathyroid hormone, calcitriol, interleukin-1alpha and -11, and retinoic acid. In rodent tissues, Northern blotting, in situ hybridization, and immunohistochemistry demonstrated OCIL expression in osteoblasts and chondrocytes as well as in a variety of extraskeletal tissues. The overlapping tissue distribution of OCIL mRNA and protein with that of RANKL strongly suggests an interaction between these molecules in the skeleton and in extraskeletal tissues.
Subject(s)
Cell Division/drug effects , Lectins, C-Type , Lectins/pharmacology , Membrane Proteins/pharmacology , Osteoblasts/metabolism , Osteoclasts/drug effects , Amino Acid Sequence , Animals , Base Sequence , Bone Marrow Cells/metabolism , DNA, Complementary , Humans , Immunohistochemistry , In Situ Hybridization , Lectins/chemistry , Lectins/genetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Molecular Sequence Data , RNA, Messenger/genetics , Rats , Recombinant Proteins/pharmacology , Sequence Homology, Amino AcidABSTRACT
Hydroxyapatite (HA) coatings have been used to improve implant fixation by promoting bone formation around the prosthesis. A macrophage response to HA particulates has been noted around loosened HA-coated prostheses. As biomaterial wear particle-associated macrophages are known to be capable of differentiating into osteoclasts that are capable of bone resorption, we examined whether particulate HA could similarly induce macrophage-osteoclast differentiation. HA-associated macrophages were isolated from granulomas, formed by subcutaneous implantation of HA, and co-cultured with UMR 106 osteoblast-like cells in the presence of 1,25-dihydroxyvitamin D(3) for up to 14 days on glass coverslips and bone slices. HA-associated macrophage-osteoclast differentiation was evidenced by the formation of numerous multinucleated tartrate resistant acid phosphatase (TRAP)-positive cells which formed lacunar resorption pits on bone slices. Polymethylmethacrylate (PMMA) particle-associated macrophages, isolated from subcutaneous PMMA-containing granulomas, caused significantly more osteoclast formation and bone resorption than HA-associated macrophages. These results indicate that macrophages responding to HA particles are capable of osteoclast differentiation. They also suggest that particles derived from uncemented (HA-coated) implants are likely to induce less osteoclast formation and osteolysis than cemented implants.
ABSTRACT
Osteoclast formation in bone is supported by osteoblasts expressing receptor activator of NF-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) expression. Numerous osteotropic factors regulate expression levels of RANKL and the RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts, thereby affecting osteoclast differentiation. However, not only in RANKL widely expressed in soft tissues, but osteoclasts have been noted in extraskeletal lesions. We found that cultured skin fibroblastic cells express RANKL, M-CSF, and OPG messenger (mRNA). Stimulation by 1 alpha,25 dihydroxyvitamin D3 [1,25(OH)2D3] plus dexamethasone (Dex) augmented RANKL and diminished OPG mRNA expression in fibroblastic cells and caused the formation of numerous osteoclasts in cocultures of skin fibroblastic cells with hemopoietic cells or monocytes. The osteoclasts thus formed expressed tartrate-resistant acid phosphatase (TRAP) and calcitonin (CT) receptors and formed resorption pits in cortical bone. Osteoclast formation also was stimulated (in the presence of Dex) by prostaglandin E2 (PGE2), interleukin-11 (IL-11), IL-1, tumor necrosis factor-alpha (TNF-alpha), and parathyroid hormone-related protein (PTHrP), factors which also stimulate osteoclast formation supported by osteoblasts. In addition, granulocyte-macrophage-CSF (GM-CSF), transforming growth factor-beta (TGF-beta), and OPG inhibited osteoclast formation in skin fibroblastic cell-hemopoietic cell cocultures; CT reduced only osteoclast nuclearity. Fibroblastic stromal cells from other tissues (lung, respiratory diaphragm, spleen, and tumor) also supported osteoclast formation. Thus, RANKL-positive fibroblastic cells in extraskeletal tissues can support osteoclastogenesis if osteolytic factors and osteoclast precursors are present. Such mesenchymally derived cells may play a role in pathological osteolysis and may be involved in osteoclast formation in extraskeletal tissues.
Subject(s)
Carrier Proteins/genetics , Fibroblasts/metabolism , Membrane Glycoproteins/genetics , NF-kappa B/metabolism , Osteoclasts/physiology , Stromal Cells/metabolism , Animals , Calcitriol/pharmacology , Cell Differentiation , Cells, Cultured , Coculture Techniques , Dexamethasone/pharmacology , Gene Expression , Glycoproteins/genetics , Ligands , Macrophage Colony-Stimulating Factor/genetics , Mice , Osteoblasts/cytology , Osteoclasts/cytology , Osteoclasts/metabolism , Osteoprotegerin , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Tumor Necrosis Factor , Stromal Cells/drug effectsABSTRACT
OBJECTIVE: To investigate the cellular mechanism of bone destruction in collagen-induced arthritis (CIA). METHODS: After induction of CIA in DA rats, a histologic study of the advanced arthritic lesion was carried out on whole, decalcified joints from the hindpaws of affected animals. To conclusively identify osteoclasts, joint tissue sections were stained for tartrate-resistant acid phosphatase (TRAP) enzyme activity, and calcitonin receptors (CTR) were identified using a specific rabbit polyclonal antibody. The expression of messenger RNA (mRNA) for the osteoclast differentiation factor (also known as receptor activator of nuclear factor kappaB ligand [RANKL]) was investigated using in situ hybridization with a specific riboprobe. RESULTS: TRAP-positive and CTR-positive multinucleated cells were invariably detected in arthritic lesions that were characterized by bone destruction. Osteoclasts were identified at the pannus-bone and pannus-subchondral bone junctions of arthritic joints, where they formed erosive pits in the bone. TRAP-positive multinucleated cells were detected within synovium and at the bone erosive front; however, CTR-positive multinucleated cells were present only at sites adjacent to bone. RANKL mRNA was highly expressed in the synovial cell infiltrate in arthritic joints, as well as by osteoclasts at sites of bone erosion. CONCLUSION: Focal bone erosion in CIA is attributed to cells expressing definitive features of osteoclasts, including CTR. The expression of RANKL by cells within inflamed synovium suggests a mechanism for osteoclast differentiation and activation at sites of bone erosion. Inhibitors of RANKL may represent a novel approach to treatment of bone loss in rheumatoid arthritis.
Subject(s)
Arthritis, Rheumatoid/metabolism , Bone Diseases/metabolism , Carrier Proteins/biosynthesis , Membrane Glycoproteins/biosynthesis , Acid Phosphatase/metabolism , Animals , Arthritis, Rheumatoid/chemically induced , Biomarkers/analysis , Collagen/immunology , Disease Models, Animal , Female , Histocytochemistry , In Situ Hybridization , Isoenzymes/metabolism , RANK Ligand , Rats , Receptors, Calcitonin/biosynthesis , Tartrate-Resistant Acid PhosphataseSubject(s)
Osteoclasts/ultrastructure , Antigens, CD/metabolism , Bone Resorption , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Differentiation , Fusion Regulatory Protein-1 , Humans , In Vitro Techniques , Membrane Glycoproteins/genetics , Osteoclasts/physiology , RANK Ligand , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor Activator of Nuclear Factor-kappa BABSTRACT
Chlamydomonas reinhardtii activates the transcription of the Cyc6 and the Cpx1 genes (encoding cytochrome c(6) and coprogen oxidase) in response to copper deficiency. Mutational analysis of promoter regions of the Cyc6 and Cpx1 genes revealed a four nucleotide sequence, GTAC, which was absolutely essential for copper responsiveness. The Cyc6 promoter contains two copper response elements, each with a functionally important GTAC sequence, whereas the Cpx1 promoter contains only one. This may contribute to the stronger and more tightly regulated expression of the Cyc6 gene. Mutation or deletion of sequences flanking the GTACs implicates additional nucleotides contributing to copper-responsive expression, but none are absolutely essential. Metal ion selectivity of Cpx1 expression is identical to that described previously for Cyc6 and is restricted to the copper deficiency-induced Cpx1 transcript. The Cyc6 and Cpx1 genes are also induced by oxygen deficiency. Reporter gene constructs indicate that the induction occurs at the level of transcription and requires the same GTAC sequence that is critical for copper responsiveness. We suggest that components of the copper-responsive signal transduction pathway are used for some of the changes in gene expression in hypoxic cells.
Subject(s)
Chlamydomonas/genetics , Copper/pharmacology , Coproporphyrinogen Oxidase/genetics , Cytochromes/genetics , Metalloproteins/genetics , Oxygen/pharmacology , Animals , Arylsulfatases/genetics , Arylsulfatases/metabolism , Base Sequence , Cytochromes f , Gene Expression Regulation/drug effects , Genes, Reporter , Molecular Sequence Data , Mutagenesis , Promoter Regions, Genetic , RNA, Messenger/metabolism , Transcriptional ActivationABSTRACT
RANKL (receptor activator of NFkappaB ligand) is a membrane-associated osteoblastic molecule, and along with macrophage-colony-stimulating factor, is crucial for osteoclast formation. RANKL is known to be strongly expressed in osteoblasts and lymphoid tissues. We have sought to determine the skeletal and extraskeletal sites of production of RANKL mRNA and protein using the techniques of in situ hybridization and immunohistochemistry. Expression of RANKL mRNA and protein were determined in the developmental progression of endochondral bone formation in mouse, intramembranous bone formation in a rabbit model (mRNA only), in human giant cell tumors of bone, and at extraskeletal sites in the mouse. RANKL mRNA was expressed in prehypertrophic and hypertrophic chondrocytes at day E15 embryonic mouse long bone, and its expression was maintained at these sites throughout development. In newborn and adult mice, high levels of RANKL mRNA were expressed in mesenchymal cells of the periosteum and in mature osteoblasts, while megakaryocytes within the marrow microenvironment expressed RANKL mRNA from 1 week of age. Immunohistochemical analysis revealed a similar localization pattern of RANKL protein at the sites described. In the intramembranous bone formation model, RANKL mRNA was expressed in mesenchymal cells and in actively synthesizing osteoblasts, but not in flattened lining osteoblasts or late osteocytes. Expression of RANKL mRNA and protein in osteoclasts was variable with those within resorption lacunae showing the strongest signal/staining. Likewise, expression varied in osteoclasts from giant cell tumor of bone with a minority of tartrate-resistant acid phosphatase-positive multinucleated cells having no detectable RANKL mRNA or protein. In extraskeletal tissues, RANKL mRNA and protein were detected in the brain, heart, kidney, skeletal muscle, and skin throughout mouse development, suggesting the possibility of several other functions of the molecule. RANKL was also developmentally regulated, as evidenced by its expression in the intestine, liver, and lung at E15 and newborn mouse but not in the adult.
Subject(s)
Bone and Bones/metabolism , Carrier Proteins/biosynthesis , Membrane Glycoproteins/biosynthesis , RNA, Messenger/metabolism , Amino Acid Sequence , Animals , Bone Development/physiology , Bone Marrow/metabolism , Bone and Bones/embryology , Carrier Proteins/genetics , Female , Giant Cell Tumor of Bone/pathology , Giant Cells/metabolism , Giant Cells/pathology , Humans , Immunohistochemistry , In Situ Hybridization , Male , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Organ Specificity/genetics , Osteoblasts/metabolism , Osteoclasts/cytology , Osteoclasts/metabolism , Pregnancy , RANK Ligand , RNA, Messenger/biosynthesis , Rabbits , Receptor Activator of Nuclear Factor-kappa B , Species SpecificityABSTRACT
Osteoblastic stromal cells are capable of supporting osteoclast formation from hematopoietic precursors in the presence of osteotropic factors such as 1alpha,25(OH)(2)D(3), PTH, and IL-11. Osteoblastic stromal cells produce receptor activator of NF-kappaB ligand (RANKL), a type II membrane protein of the TNF ligand family, in response to these agents. Activated T lymphocytes also produce RANKL; however, the ability of this cell type to support osteoclast formation in vitro is unknown. Human PBMC-derived T cells, extracted using alphaCD3-coated magnetic beads, were cocultured with adherent murine spleen cells in the presence of Con A and a panel of cytokines. In the presence of Con A, bona fide osteoclasts were formed in vitro with activated T cells: IL-1alpha and TGFbeta further enhanced osteoclast numbers. PBMC-derived lymphocytes showed an increase in the mRNA expression of RANKL within 24 h of treatment with the same agents that were used to induce osteoclast formation. In synovial tissue sections with lymphoid infiltrates from RA patients, the expression of RANKL was demonstrated in CD3(+) T cells. The ability of activated T lymphocytes to support osteoclast formation may provide a mechanism for the potentiation of osteoclast formation and bone resorption in disease states such as rheumatoid arthritis.
Subject(s)
Carrier Proteins/genetics , Hematopoietic Stem Cells/cytology , Lymphocyte Activation , Membrane Glycoproteins/genetics , Osteoclasts/cytology , T-Lymphocytes/physiology , Aged , Animals , Animals, Newborn , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/pathology , Carrier Proteins/biosynthesis , Cell Differentiation , Coculture Techniques , Concanavalin A/pharmacology , Female , Gene Expression Regulation , Humans , Interleukin-2/pharmacology , Male , Membrane Glycoproteins/biosynthesis , Mice , Mice, Inbred C57BL , Middle Aged , RANK Ligand , RNA, Messenger/genetics , Receptor Activator of Nuclear Factor-kappa B , Stromal Cells/cytology , Synovial Membrane/immunology , Synovial Membrane/pathology , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Transcription, Genetic/drug effects , Transcription, Genetic/immunology , Transforming Growth Factor beta/pharmacologyABSTRACT
OBJECTIVE: To determine the patient-related costs of care for critically ill patients with severe sepsis or early septic shock. DESIGN: Retrospective, longitudinal, observational study during a 10-month period. SETTING: Adult general intensive care unit (ICU) in a university hospital located in the United Kingdom. PATIENTS: The study population consisted of 213 patients admitted consecutively to the ICU during a 10-month period. Thirty-six patients were identified using standard definitions as having developed sepsis and analyzed by group (according to the day on which sepsis was diagnosed): Group 1 patients were septic at admission to ICU (n = 16); group 2 patients were septic on their second day in the ICU (n = 10); and group 3 patients developed sepsis after their second day in the ICU (n = 10). One hundred and seventy-seven ICU patients without sepsis were used as the comparative group (group 4). INTERVENTIONS: None. MAIN RESULTS: Patient-related costs of care, length of ICU stay, and ICU and hospital mortality rates were compiled. The median daily costs of care for patients in groups 1, 2, and 3 were $930.74 (interquartile range $851.59-$1,263.96); $814.47 ($650.89-$1,123.06), and $1,079.39 ($705.02-$1,295.96), respectively; these were significantly more than the group 4 patient's daily cost of $750.38 ($644.10-$908.55) (p < .01). The median total cost of treating the group 4 patients was $1,666.87 ($979.71-$2,772.03), significantly less than for the patients with sepsis (p < .01). The difference in total costs of care between the sepsis groups was also significant (p < .05), with a group 1 patient costing $3,801.55 ($1,865.28-$11,676.08), a group 2 patient costing $13,089.17 ($5,792.94-$22,235.18), and a group 3 patient costing $17,962.78 ($13,030.83-$28,546.73). Patients in groups 1, 2, and 3 stayed in the ICU for 3.3 days (1.3-11.3), 16.5 days (8.9-22), and 16.1 days (10.9-9), respectively. Significant differences were found among the three groups (p < 0.05), as well as between the patients with sepsis and those without (p < 0.001), whose median length of stay was 1.9 days (0.9-3.6). The ICU mortality rates were 50% each for groups 1 and 2, 60% for group 3, and 20% for group 4. Only one patient with sepsis and 16 patients without sepsis died in the hospital ward, producing overall mortality rates of 56% for group 1 and 29% for group 4. CONCLUSIONS: Patients with severe sepsis or early septic shock had a high mortality rate, spent prolonged periods of time in the ICU, and were significantly more expensive to treat than nonsepsis ICU patients.
Subject(s)
Hospital Costs , Intensive Care Units/economics , Sepsis/economics , APACHE , Adolescent , Adult , Aged , Aged, 80 and over , Analysis of Variance , England/epidemiology , Female , Hospital Mortality , Humans , Length of Stay , Longitudinal Studies , Male , Middle Aged , Retrospective Studies , Sepsis/etiology , Sepsis/mortality , Shock, Septic/economicsABSTRACT
Osteoclasts are the cells responsible for bone resorption, and their number and rate of formation are critical in determining bone mass. To identify and quantify osteoclasts, as well as to study their formation in bone and in osteoclastogenic cultures, osteoclast-specific cell markers are required. Only the calcitonin receptor (CTR) expression unambiguously identifies osteoclasts and distinguishes them from macrophage polykaryons. However, present autoradiographic methods for CTR detection are cumbersome and time consuming. We have developed rabbit polyclonal antibodies specific for the C-terminal intracellular domain of the mouse and rat Cla CTR. These antibodies labeled HEK-293 cells stably transfected with CTR (but not untransfected HEK-293 cells). This labeling is abrogated by preabsorbing the antibodies with the recombinant antigen. The antibodies immunostained primary mouse and rat osteoclasts as well as osteoclasts in sections of mouse bone. Osteoclasts (both mononuclear and multinucleated) formed from mouse bone marrow or spleen cells cocultured with osteoblasts in the presence of 1,25 dihydroxyvitamin D3 and prostaglandin E2 were also specifically immunostained by the CTR antibodies. Cocultures incubated under conditions that did not allow osteoclastogenesis (i.e., omission of mediators or osteoblasts, or culture for less than 4 days) were not immunostained by CTR antibodies. Autoradiographic detection of 125I-labeled salmon calcitonin combined with CTR immunohistochemistry showed that both methods labeled the same cells. A CTR polyclonal antibody and monoclonal antibody F4/80 were used in combination to show immunofluorescence labeling of murine osteoclasts and macrophage populations, respectively, in marrow/osteoblast cocultures. These results indicate that simple and rapid CTR antibody-based methods can be used to identify osteoclasts, and can be used to characterize the antigenic profile of osteoclasts by using double immunofluorescence analysis.