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Effect of calcitonin and PTH on bone cell acid phosphatase
157
Abstracts from the Bone and Tooth Society
substrata. Resorption was still occurring in SWD seeded with marrow or bone-derived cell fractions at 6 weeks, and pits of all sizes were being produced. However, it was concluded that the marrow fraction was a better and longer source of resorbing cells than the bone fraction, probably because of the presence of osteoclast precursors, and that trypsinization should be avoided for maintenance of the multinucleate osteoclast population. Reference Boyde et al., Proc Roy Microse Sot 18, 357, 1983.
EFFECT OF CALCITONIN ACID PHOSPHATASE
AND PTH ON BONE CELL
I.P. Braidman, L.A. Burby, W.R. Robertson*, D.C. Anderson
and
Departments of Medicine and Chemical Pathology*, University of Manchester Medical School, Clinical Sciences Building, Hope Hospital, Eccles Old Road, Salford M6 8HD We have measured responses of individual bone cells in culture by microdensitometry of cytochemical reaction product for marker enzymes in bone. Thus we have found that low concentrations of PTH (O-10 pg/ml) stimulate acid phosphatase in cells of the osteoclast lineage (Ocl) only in the presence of osteoblasts (Ob). We have now examined the effects of calcitonin in this system and its interac?ion with PTH. Bone cells were released from fetal rat calvariae by enzymic digestion and separated into Ocl and Ob by equilibrium density centrifugation. Cells, grown on Thermanox cover discs, were reacted cytochemically for acid phosphatase, a known marker of osteoclasts in intact calvariae, and reaction product within individual cells was measured by microdensitometry. Culture of Ocl with 0.5-lOO/pg/ml salmon calcitonin for 16 h produced a significant decrease in enzyme activity, with maximal effects at IO/pg/ml concentration (a reduction from geometric mean absorbance of 0.063 in controls to 0.033 in hormone treated cells, P
OSTEOBLASTIC CELLS INDUCE CHEMOTAXIS IN OSTEOCLASTS
sibitity is that osteoblastic cells produce factors, at sites appropriate for bone resorption, that are chemotactic for osteoclasts. If such factors are produced, it is likely that their production is governed by local morphogenetic and structural stimuli and augmented by systemic stimulators of bone resorption. We attempted to detect such factors by incubating osteoclasts and osteoblastic cells on different halves of the same culture dish, beneath a coverslip, in the presence or absence of PTH. Cultures were fixed after incubation, and the direction of migration of osteoclasts was deduced from pseudopodial orientation, with which migration was found to correspond in time-lapse observations. The ratio of osteoclasts localized toward, compared to away from, osteoblasts, in the presence and absence of PTH, was 4.2 + 1.2 and 1.1 + 0.2 respectively. This response was unaffected by the simultaneous presence of indomethacin. These results suggest that osteoblastic cells produce diffusible effecters of osteoclastic localization.
CONTROL OF BONE RESORPTION BY RECOMBINANT IMMUNE CELL PRODUCTS: INTERLEUKIN 1, INTERLEUKIN 2, AND INTERFERON GAMMA M. Gowen*
and G.R. Mundy
Department of Medicine, University of Texas Health Science Center at San Antonio, TX 78284 USA. * Present address: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, UK The identity of osteoclast activating factor(s) (OAF) has been the subject of much research and speculation since the activity was first described in 1972. However, the term is commonly used in a way that would suggest it is a specific and distinct protein. We showed previously that monocytes are capable of producing an OAF with physicochemical characteristics very similar to those of interleukin I (IL-l) (Nature 306, 378-380, 1983). We now demonstrate that purified recombinant murine IL-l is a potent and powerful stimulator of bone resorption in the mouse calvarial system, active over a concentration range of 1-300/pM. IL-l-stimulated bone resorption was inhibited by salmon calcitonin, but unaffected by the cyclooxygenase inhibitor indomethatin. IL-I to stimulated bone resorption was inhibited by interferon gamma (100 U/ml). IL-2 was found to have no effect on bone resorption, either alone or in the presence of IL-l. Histology of the bones cultured with IL-l showed increased numbers of osteoclasts, markedly decreased amounts of mineralized bone matrix and osteoid in sections of calvariae, and increased numbers of periosteal and marrow cells. It has been recognized for many years that cells of the immune system play a role in the control of bone turnover. We have now described immune cell products capable of both stimulating and inhibiting bone resorption, the first stage of bone remodeling.
B.M. Thomson and T.J. Chambers Department of Histopathology, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE The development of the shape and structure of bones is associated with intricate and dynamic patterns of osteoelastic localization and activity. Nothing is known of the mechanism by which this localization is effected. One pos-
EFFECT OF HUMAN CALCITONIN GENE-RELATED PEPTIDE ON HUMAN BONE-DERIVED CELLS IN CULTURE A. Crawford, D.B. Evans, H. Skjodt, J.N. Beresford*, I. MacIntyre,+ and R.G.G. Russell Department of Human Metabolism and Clinical
Abstracts from the Bone and Tooth Society
substrata. Resorption was still occurring in SWD seeded with marrow or bone-derived cell fractions at 6 weeks, and pits of all sizes were being produced. However, it was concluded that the marrow fraction was a better and longer source of resorbing cells than the bone fraction, probably because of the presence of osteoclast precursors, and that trypsinization should be avoided for maintenance of the multinucleate osteoclast population. Reference Boyde et al., Proc Roy Microse Sot 18, 357, 1983.
EFFECT OF CALCITONIN ACID PHOSPHATASE
AND PTH ON BONE CELL
I.P. Braidman, L.A. Burby, W.R. Robertson*, D.C. Anderson
and
Departments of Medicine and Chemical Pathology*, University of Manchester Medical School, Clinical Sciences Building, Hope Hospital, Eccles Old Road, Salford M6 8HD We have measured responses of individual bone cells in culture by microdensitometry of cytochemical reaction product for marker enzymes in bone. Thus we have found that low concentrations of PTH (O-10 pg/ml) stimulate acid phosphatase in cells of the osteoclast lineage (Ocl) only in the presence of osteoblasts (Ob). We have now examined the effects of calcitonin in this system and its interac?ion with PTH. Bone cells were released from fetal rat calvariae by enzymic digestion and separated into Ocl and Ob by equilibrium density centrifugation. Cells, grown on Thermanox cover discs, were reacted cytochemically for acid phosphatase, a known marker of osteoclasts in intact calvariae, and reaction product within individual cells was measured by microdensitometry. Culture of Ocl with 0.5-lOO/pg/ml salmon calcitonin for 16 h produced a significant decrease in enzyme activity, with maximal effects at IO/pg/ml concentration (a reduction from geometric mean absorbance of 0.063 in controls to 0.033 in hormone treated cells, P
OSTEOBLASTIC CELLS INDUCE CHEMOTAXIS IN OSTEOCLASTS
sibitity is that osteoblastic cells produce factors, at sites appropriate for bone resorption, that are chemotactic for osteoclasts. If such factors are produced, it is likely that their production is governed by local morphogenetic and structural stimuli and augmented by systemic stimulators of bone resorption. We attempted to detect such factors by incubating osteoclasts and osteoblastic cells on different halves of the same culture dish, beneath a coverslip, in the presence or absence of PTH. Cultures were fixed after incubation, and the direction of migration of osteoclasts was deduced from pseudopodial orientation, with which migration was found to correspond in time-lapse observations. The ratio of osteoclasts localized toward, compared to away from, osteoblasts, in the presence and absence of PTH, was 4.2 + 1.2 and 1.1 + 0.2 respectively. This response was unaffected by the simultaneous presence of indomethacin. These results suggest that osteoblastic cells produce diffusible effecters of osteoclastic localization.
CONTROL OF BONE RESORPTION BY RECOMBINANT IMMUNE CELL PRODUCTS: INTERLEUKIN 1, INTERLEUKIN 2, AND INTERFERON GAMMA M. Gowen*
and G.R. Mundy
Department of Medicine, University of Texas Health Science Center at San Antonio, TX 78284 USA. * Present address: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, UK The identity of osteoclast activating factor(s) (OAF) has been the subject of much research and speculation since the activity was first described in 1972. However, the term is commonly used in a way that would suggest it is a specific and distinct protein. We showed previously that monocytes are capable of producing an OAF with physicochemical characteristics very similar to those of interleukin I (IL-l) (Nature 306, 378-380, 1983). We now demonstrate that purified recombinant murine IL-l is a potent and powerful stimulator of bone resorption in the mouse calvarial system, active over a concentration range of 1-300/pM. IL-l-stimulated bone resorption was inhibited by salmon calcitonin, but unaffected by the cyclooxygenase inhibitor indomethatin. IL-I to stimulated bone resorption was inhibited by interferon gamma (100 U/ml). IL-2 was found to have no effect on bone resorption, either alone or in the presence of IL-l. Histology of the bones cultured with IL-l showed increased numbers of osteoclasts, markedly decreased amounts of mineralized bone matrix and osteoid in sections of calvariae, and increased numbers of periosteal and marrow cells. It has been recognized for many years that cells of the immune system play a role in the control of bone turnover. We have now described immune cell products capable of both stimulating and inhibiting bone resorption, the first stage of bone remodeling.
B.M. Thomson and T.J. Chambers Department of Histopathology, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE The development of the shape and structure of bones is associated with intricate and dynamic patterns of osteoelastic localization and activity. Nothing is known of the mechanism by which this localization is effected. One pos-
EFFECT OF HUMAN CALCITONIN GENE-RELATED PEPTIDE ON HUMAN BONE-DERIVED CELLS IN CULTURE A. Crawford, D.B. Evans, H. Skjodt, J.N. Beresford*, I. MacIntyre,+ and R.G.G. Russell Department of Human Metabolism and Clinical