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Tumor necrosis factor-alpha inhibits osteoclastogenesis by inducing osteoblasts to release granulocyte-macrophage colony-stimulating factor
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Abstracts / Bone 44 (2009) S253–S338
however the process by which it forms is still not fully understood and little is known about the mechanisms that lead to the dissolution of the ruffled border. To study the dynamics of ruffled border formation, osteoclasts were treated with pharmacological inhibitors of resorption. Mature rabbit osteoclasts were seeded on to dentine and treated with either calcitonin, a peptide hormone which disrupts osteoclast sealing zones, or cytochalasin D, a fungal toxin and potent inhibitor of actin polymerisation. The osteoclasts were then allowed to recover in medium without inhibitors for various lengths of time prior to fixation. Confocal microscopy was used to analyse the effects of these agents on actin ring formation and transmission electron microscopy was used to analyse changes in membrane distribution and vacuolar content at the ultrastructural level. Treating osteoclasts with either calcitonin or cytochalasin D completely disrupted and inhibited actin ring and ruffled border formation, but in a reversible manner; the osteoclasts were able to repolarise and reform their ruffled border within 6 h after the end of treatment. In the osteoclasts treated with cytochalasin D there also seemed to be a complete loss of the functional secretory domain. Disappearance of the ruffled border and functional secretory domain following treatment with cytochalasin D confirms that the actin cytoskeleton plays an important role in the formation of the ruffled border. Treatment with both agents caused a large increase in the number of vesicles within osteoclasts, leading to the conclusion that the ruffled border membrane becomes internalised in vesicles and vacuoles within the osteoclast. Interestingly, the size and number of vesicles seen after calcitonin treatment was larger than seen after cytochalasin D treatment. Our current work is focusing on the further identification of these vacuoles and secretory vesicles in ruffled border dynamics using immuno-EM techniques. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.618
P193 Tumor necrosis factor-alpha inhibits osteoclastogenesis by inducing osteoblasts to release granulocyte-macrophage colony-stimulating factor E. Atanga⁎, S. Dolder, W. Hofstetter Bone Biology Group, Department of Clinical Research, University of Bern, Bern, Switzerland Introduction: The cytokine Tumor Necrosis Factor-alpha (TNFα) mediates bone loss in inflammatory diseases, stimulating osteoclast development and activity. Previously, we demonstrated that TNFα inhibits the development of osteoclasts in vitro via an osteoblastmediated mechanism. Gene array analysis suggested GranulocyteMacrophage Colony-Stimulating Factor (GM-CSF) to be a candidate molecule. Within this study, the role of the cytokine in TNFα dependent inhibition of osteoclastogenesis was characterized. Methods: Osteoclast development was investigated either in cocultures of murine osteoblasts (C57Bl/6J mice) and bone marrow cells (BMC) or in cultures of BMC. GM-CSF was assessed at transcript and protein levels by RT-PCR and ELISA in murine osteoblasts treated with TNFα (1–30 ng/ml) and 10 nM dihydroxy-cholecalciferol (1,25(OH)2VitD3). Results: Expression of GM-CSF transcripts was induced in cells treated with TNFα within 24 h. Treatment with 1,25(OH)2D3/TNFα led to a further 15-fold stimulation. The protein accumulated in the supernatant (CM) during the course of 72 h, reaching concentrations of 25 pg/ml in CM from osteoblasts treated with TNFα (1 ng/ml) and 150 pg/ml in CM from cells treated with TNFα/1,25(OH)2D3. No GM-CSF was detected in CM from control cells. In co-cultures of GM-CSF-/-
osteoblasts and wt BMC, the inhibitory activity of TNFα (1 ng/ml) on osteoclast development was abolished whereas in co-cultures with wt osteoblasts, the number of osteoclasts was reduced by 80%. However, CM from GM-CSF-/- and wt osteoblasts reduced osteoclast numbers to 50% and 10%, respectively. Preincubation of wt CM with αGM-CSF Ab restored numbers of osteoclasts to approx. 50% again. Conclusion: GM-CSF, which is released by osteoblasts in response to TNFα/1,25(OH)2D3, mediates part of the TNFα-induced inhibition of osteoclast formation in vitro. In co-cultures with GMCSF-/-osteoblasts, TNFα does not affect osteoclast number. On the other hand, CM from GM-CSF-/-osteoblasts inhibits osteoclastogenesis. The finding of TNFα not only acting as a stimulator but also as an inhibitor of osteoclastogenesis and bone resorption opens new possibilities for the interactions of inflammatory processes and bone metabolism. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.619
P194 Expression of inclusion body-associated proteins in Paget's disease of bone E.A. Azzama,*, D.I. Scotta, J.S. Gregorya, A.D. Mckinnonb, M.H. Helfricha a School of Medicine and Dentistry b Histology-EM Facility, University of Aberdeen, Aberdeen, UK Paget's disease of bone (PDB) is a late-onset disease characterized by focal increases in bone remodelling. A pathognomic feature of PDB is the presence of cytoplasmic and nuclear inclusion bodies in osteoclasts. The aim of this study was to assess whether pagetic inclusions may be similar to inclusion bodies in neurodegenerative diseases by studying key proteins expressed in such structures: VCP, ubiquitin, p62, phosphorylated tau, beta amyloid and the 20S proteasomal subunit alpha and beta. We used decalcified, wax embedded bone biopsies of 4 patients with PDB. Presence of osteoclast inclusions in these biopsies was confirmed by transmission electron microscopy. As controls we used biopsies from patients with osteoarthritis (n = 2,) osteoporosis (n = 1)renal osteodystrophy (n = 1) and prostate carcinoma (n = 1). Brain tissue from a patient with Alzheimer's disease and osteoclastoma tissue served as additional positive controls for inclusion bodies and nonpagetic osteoclasts respectively. Sections of 5 μm thickness were used and stained using a slide staining robot (Bond Max) and using manufacturer's detection kits. For each antibody staining conditions were optimised using the positive control tissues and in many cases antigen retrieval was required for best staining results. Stained sections were analyzed by light microscopy and numbers of osteoclasts and staining intensity assessed semi-quantitatively taking into consideration whether staining was seen in the nucleus or the cytoplasm or both. For VCP staining a quantitative method, using Volocity software, was used to analyse whether staining intensity differed between osteoclasts in PDB and controls. We found that PDB osteoclasts showed a higher level of VCP protein per cell than non-pagetic osteoclasts (P = 0.001). We also found a significant difference in the number of stained osteoclasts between PDB and non-paget controls for p62, ubiquitin, and the proteasomal beta subunit (P = 012; P = 0.016 and P = 0.016 resp.). Nuclear staining was frequently seen in osteoclasts in both PDB and controls, with levels of nuclear staining higher in PDB. In addition, ubiquitin and p62 were seen in cytoplasmic aggregates in PDB osteoclasts. No staining was seen for beta amyloid and phosphorylated tau in any of the bone sections. Our results indicate that VCP, ubiquitin, p62, proteasomal alpha and beta subunits are overexpressed in osteoclasts in patients with PDB and
Abstracts / Bone 44 (2009) S253–S338
however the process by which it forms is still not fully understood and little is known about the mechanisms that lead to the dissolution of the ruffled border. To study the dynamics of ruffled border formation, osteoclasts were treated with pharmacological inhibitors of resorption. Mature rabbit osteoclasts were seeded on to dentine and treated with either calcitonin, a peptide hormone which disrupts osteoclast sealing zones, or cytochalasin D, a fungal toxin and potent inhibitor of actin polymerisation. The osteoclasts were then allowed to recover in medium without inhibitors for various lengths of time prior to fixation. Confocal microscopy was used to analyse the effects of these agents on actin ring formation and transmission electron microscopy was used to analyse changes in membrane distribution and vacuolar content at the ultrastructural level. Treating osteoclasts with either calcitonin or cytochalasin D completely disrupted and inhibited actin ring and ruffled border formation, but in a reversible manner; the osteoclasts were able to repolarise and reform their ruffled border within 6 h after the end of treatment. In the osteoclasts treated with cytochalasin D there also seemed to be a complete loss of the functional secretory domain. Disappearance of the ruffled border and functional secretory domain following treatment with cytochalasin D confirms that the actin cytoskeleton plays an important role in the formation of the ruffled border. Treatment with both agents caused a large increase in the number of vesicles within osteoclasts, leading to the conclusion that the ruffled border membrane becomes internalised in vesicles and vacuoles within the osteoclast. Interestingly, the size and number of vesicles seen after calcitonin treatment was larger than seen after cytochalasin D treatment. Our current work is focusing on the further identification of these vacuoles and secretory vesicles in ruffled border dynamics using immuno-EM techniques. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.618
P193 Tumor necrosis factor-alpha inhibits osteoclastogenesis by inducing osteoblasts to release granulocyte-macrophage colony-stimulating factor E. Atanga⁎, S. Dolder, W. Hofstetter Bone Biology Group, Department of Clinical Research, University of Bern, Bern, Switzerland Introduction: The cytokine Tumor Necrosis Factor-alpha (TNFα) mediates bone loss in inflammatory diseases, stimulating osteoclast development and activity. Previously, we demonstrated that TNFα inhibits the development of osteoclasts in vitro via an osteoblastmediated mechanism. Gene array analysis suggested GranulocyteMacrophage Colony-Stimulating Factor (GM-CSF) to be a candidate molecule. Within this study, the role of the cytokine in TNFα dependent inhibition of osteoclastogenesis was characterized. Methods: Osteoclast development was investigated either in cocultures of murine osteoblasts (C57Bl/6J mice) and bone marrow cells (BMC) or in cultures of BMC. GM-CSF was assessed at transcript and protein levels by RT-PCR and ELISA in murine osteoblasts treated with TNFα (1–30 ng/ml) and 10 nM dihydroxy-cholecalciferol (1,25(OH)2VitD3). Results: Expression of GM-CSF transcripts was induced in cells treated with TNFα within 24 h. Treatment with 1,25(OH)2D3/TNFα led to a further 15-fold stimulation. The protein accumulated in the supernatant (CM) during the course of 72 h, reaching concentrations of 25 pg/ml in CM from osteoblasts treated with TNFα (1 ng/ml) and 150 pg/ml in CM from cells treated with TNFα/1,25(OH)2D3. No GM-CSF was detected in CM from control cells. In co-cultures of GM-CSF-/-
osteoblasts and wt BMC, the inhibitory activity of TNFα (1 ng/ml) on osteoclast development was abolished whereas in co-cultures with wt osteoblasts, the number of osteoclasts was reduced by 80%. However, CM from GM-CSF-/- and wt osteoblasts reduced osteoclast numbers to 50% and 10%, respectively. Preincubation of wt CM with αGM-CSF Ab restored numbers of osteoclasts to approx. 50% again. Conclusion: GM-CSF, which is released by osteoblasts in response to TNFα/1,25(OH)2D3, mediates part of the TNFα-induced inhibition of osteoclast formation in vitro. In co-cultures with GMCSF-/-osteoblasts, TNFα does not affect osteoclast number. On the other hand, CM from GM-CSF-/-osteoblasts inhibits osteoclastogenesis. The finding of TNFα not only acting as a stimulator but also as an inhibitor of osteoclastogenesis and bone resorption opens new possibilities for the interactions of inflammatory processes and bone metabolism. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.619
P194 Expression of inclusion body-associated proteins in Paget's disease of bone E.A. Azzama,*, D.I. Scotta, J.S. Gregorya, A.D. Mckinnonb, M.H. Helfricha a School of Medicine and Dentistry b Histology-EM Facility, University of Aberdeen, Aberdeen, UK Paget's disease of bone (PDB) is a late-onset disease characterized by focal increases in bone remodelling. A pathognomic feature of PDB is the presence of cytoplasmic and nuclear inclusion bodies in osteoclasts. The aim of this study was to assess whether pagetic inclusions may be similar to inclusion bodies in neurodegenerative diseases by studying key proteins expressed in such structures: VCP, ubiquitin, p62, phosphorylated tau, beta amyloid and the 20S proteasomal subunit alpha and beta. We used decalcified, wax embedded bone biopsies of 4 patients with PDB. Presence of osteoclast inclusions in these biopsies was confirmed by transmission electron microscopy. As controls we used biopsies from patients with osteoarthritis (n = 2,) osteoporosis (n = 1)renal osteodystrophy (n = 1) and prostate carcinoma (n = 1). Brain tissue from a patient with Alzheimer's disease and osteoclastoma tissue served as additional positive controls for inclusion bodies and nonpagetic osteoclasts respectively. Sections of 5 μm thickness were used and stained using a slide staining robot (Bond Max) and using manufacturer's detection kits. For each antibody staining conditions were optimised using the positive control tissues and in many cases antigen retrieval was required for best staining results. Stained sections were analyzed by light microscopy and numbers of osteoclasts and staining intensity assessed semi-quantitatively taking into consideration whether staining was seen in the nucleus or the cytoplasm or both. For VCP staining a quantitative method, using Volocity software, was used to analyse whether staining intensity differed between osteoclasts in PDB and controls. We found that PDB osteoclasts showed a higher level of VCP protein per cell than non-pagetic osteoclasts (P = 0.001). We also found a significant difference in the number of stained osteoclasts between PDB and non-paget controls for p62, ubiquitin, and the proteasomal beta subunit (P = 012; P = 0.016 and P = 0.016 resp.). Nuclear staining was frequently seen in osteoclasts in both PDB and controls, with levels of nuclear staining higher in PDB. In addition, ubiquitin and p62 were seen in cytoplasmic aggregates in PDB osteoclasts. No staining was seen for beta amyloid and phosphorylated tau in any of the bone sections. Our results indicate that VCP, ubiquitin, p62, proteasomal alpha and beta subunits are overexpressed in osteoclasts in patients with PDB and