Characterization of a monoclonal antibody (DA1) which reacts with bone matrix and bone cell...
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46 Abstracts from the Bone and Tooth Society Meetrng Both cysteine proteinase and collagenase inhibitors (Z- Phe-Ala-CHN2:Delaisse et al., 1980; E-64:Delaisse et al., 1984; Cl-1:Delaisse et al., 1985) have been shown to in- hibit bone resorption as measured by the release of Ca or hydroxyproline from cultured embryonrc mouse bones. W e have tested the effects of Z-Phe-Ala-CHN2, E-64, and Cl-l on the resorption of sperm whale dentine (SWD) by me- chanically isolated chick osteoclasts (0~s). Ocs were cul- tured in MEM without serum in the presence or absence of 12.5 FM Z-Phe-Ala-CHN2, 40 or 60 PM E-64, or 40 or 100 FM Cl-l for 12 or 3 days. In addition, Ocs were cultured in MEM (without serum) with or without added 12.5 PM Z-Phe-Ala-CHN2 on anorganic oyster shell. Specimens were examined (1) after neutral red staining of live cells for counting Ocs, (2) when stained with toluidine blue, after fixatron and cell removal, for counting and characterrsrng resorption pits (RP), and (3) by SEM for stereopair re- cording of RP. The depths, areas, and volumes of RP were measured by stereophotogrammetry. The numbers and sizes of the RP on SWD were significantly reduced by Z-Phe-Ala-CHN2 and E-64, Z-Phe-AlaCHN2 having the greater effect. For a given plan area, the volumes and the depths of RPs were smaller in these test compared with control SWD specimens. However, Cl-l did not have this inhibitory effect on the numbers or sizes of RP on SWD. and Z-Phe-Ala-CHN2 did not significantly reduce resorp- tion of the (noncollagenous, calcitic) oyster shell by the ocs CHARACTERIZATION OF A MONOCLONAL ANTIBODY (DAl) WHICH REACTS WITH BONE MATRIX AND BONE CELL CYTOPLASM D.H. Al-FadhI,’ R M. Sharrard,’ L.J. Partridge,* D.R. Burton,’ J.A. Gallagher,3 C.E. Sheard,4 L. Bobrow. J.A.S. Pringle,6 and R.G.G. Russell’ ‘Department of Human Metabolism & Ciinical Biochemistry, University of Sheffield Medical School, Sheffield SlO 2RX, 2Department of Biochemistry, University of Sheffield, Sheffield SIO 2TN, 3Department of Anatomy, University of Liverpool, Liverpool L69 3BX. 4Department of Anatomy & Embryology, University College, London, 5Department of Hlstopathology, University College, London, and 6Deparfment of Morbid Anatomy, Institute of Orthopaedics, RNOH, Stanmore, UK. Monoclonal antibody DA1 is one of a panel of antibodies raised against cultured human bone cells. Immunocyto- chemistry of cultured human bone cells showed the an- tigen to be in the extracellular matrix and, where the cells were made permeable by fixation before staining, also in the cytoplasm Fluorescence-actrvated cell sorting showed cultured human bone cells to be posrtlve for DA1 after fixation. lmmunodot labeling showed the DA1 antigen in cytoplasmic fractions of the cells but not in detergent extracts of the membrane fraction. Thus the DA1 antigen appears to be a cytoplasmlc species that is secreted into the matrix but is not detectable in the cell membrane. In cryostat sections of adult human bone, osteoblasts and osteocytes were positive. DA1 also recognized human chondrocytes. However. in a plastic-embedded section of fetal human femur, DA1 recognized a minor subpopulation of cells distributed throughout the epiphyseal region. Ex- tensrve testing of soft tissue sections with DA1 gave marnly negative results. In sectlons of some human osteosar- coma, there was intense staining of the extracellular ma- trix. In a sample of giant cell tumour, the giant cells were negative but some stromal cells were positive. In an immu- noblot of an SDS-polyacrylamide gel of proteins from cul- tured human bone cells, DA1 reacted strongly with a band of molecular weight approximately 35,000 and with a band at about 50,000. We are presently involved in further char- acterization of the antigen by immunoprecrpitation and two-dimensional gel blotting. We thus hope to elucidate the nature of the DA1 antigen and its role in the matrix of bone. Supported by YCRC, British Council and Action Re- search. HORMONAL INDUCTION OF OSTEOCLASTIC PHENOTYPE FROM HAEMOPOIETIC PRECURSORS IN VITRO K. Fuller and T.J. Chambers Department of Histopathology, St George’s Hospital MedIcal School, London SW1 7 ORE, UK Mammalian spleen cells, peripheral blood mononuclear cells, or neonatal and adult mammalian bone marrow (after depletion of preexisting osteoclasts) were incubated in the presence or absence of agents known to stimulate osteoclastic bone resorption. Osteoclast phenotype was identified as the ability to excavate bone, a function that seems specific for osteoclasts. Osteoclasts were undetec- table in the first week of incubation but were regularly and progressively induced to appear in succeeding weeks, but only in the presence of hormonal stimulation, in spleen and marrow cultures. No osteoclastic differentiation was ob- served using blood mononuclear cells. These results, in particular those for spleen cells, cannot be explained merely on the basis of improved survival or stimulation of osteoclasts already present. Rather, they demonstrate hormone-dependent induction of osteoclastic differentia- tion from cells not previously demonstrating osteoclastic function. This experimental system should enable rnvesti- gation of osteoclast differentiation and lineage and of the hormonal mechanisms involved in regulation of osteo- elastic production and may enable production of osteo- clasts In vitro In numbers sufficient to assist analysis of the mechantsms and regulation of osteoclastic bone resorp- tion. BONE MEMBRANE AND THE SODIUM-POTASSIUM PUMP C.L. Haycox,’ W.J. Heideger,’ and K.W. Beach ‘Departmenf of Chemical Engineering and “Department of Surgery, University of Washington, Seattle, WA 98195, USA. The concentration of calcium In the circulation is controlled in part by its transport to and from bone, the body’s prin- cipal reservoir for both calcium and phosphate. An elec- trochemical potential difference has been demonstrated across the bone membrane, that layer of cells lining bone surfaces as the barrier between bone and blood. Active transport of one or more ionic species is implied by this potential, and the control of body calcium is linked to that transport system. We have developed a technique for the separate analysis of Ionic accumulation in the fluid phase
Transcript of Characterization of a monoclonal antibody (DA1) which reacts with bone matrix and bone cell...
46 Abstracts from the Bone and Tooth Society Meetrng
Both cysteine proteinase and collagenase inhibitors (Z- Phe-Ala-CHN2:Delaisse et al., 1980; E-64:Delaisse et al., 1984; Cl-1:Delaisse et al., 1985) have been shown to in- hibit bone resorption as measured by the release of Ca or hydroxyproline from cultured embryonrc mouse bones. We have tested the effects of Z-Phe-Ala-CHN2, E-64, and Cl-l on the resorption of sperm whale dentine (SWD) by me- chanically isolated chick osteoclasts (0~s). Ocs were cul- tured in MEM without serum in the presence or absence of 12.5 FM Z-Phe-Ala-CHN2, 40 or 60 PM E-64, or 40 or 100 FM Cl-l for 12 or 3 days. In addition, Ocs were cultured in MEM (without serum) with or without added 12.5 PM Z-Phe-Ala-CHN2 on anorganic oyster shell. Specimens were examined (1) after neutral red staining of live cells for counting Ocs, (2) when stained with toluidine blue, after fixatron and cell removal, for counting and characterrsrng resorption pits (RP), and (3) by SEM for stereopair re- cording of RP. The depths, areas, and volumes of RP were measured by stereophotogrammetry. The numbers and sizes of the RP on SWD were significantly reduced by Z-Phe-Ala-CHN2 and E-64, Z-Phe-AlaCHN2 having the greater effect. For a given plan area, the volumes and the depths of RPs were smaller in these test compared with control SWD specimens. However, Cl-l did not have this inhibitory effect on the numbers or sizes of RP on SWD. and Z-Phe-Ala-CHN2 did not significantly reduce resorp- tion of the (noncollagenous, calcitic) oyster shell by the ocs
CHARACTERIZATION OF A MONOCLONAL ANTIBODY (DAl) WHICH REACTS WITH BONE MATRIX AND BONE CELL CYTOPLASM
D.H. Al-FadhI,’ R M. Sharrard,’ L.J. Partridge,* D.R. Burton,’ J.A. Gallagher,3 C.E. Sheard,4 L. Bobrow. J.A.S. Pringle,6 and R.G.G. Russell’
‘Department of Human Metabolism & Ciinical Biochemistry, University of Sheffield Medical School, Sheffield SlO 2RX, 2Department of Biochemistry, University of Sheffield, Sheffield SIO 2TN, 3Department of Anatomy, University of Liverpool, Liverpool L69 3BX. 4Department of Anatomy & Embryology, University College, London, 5Department of Hlstopathology, University College, London, and 6Deparfment of Morbid Anatomy, Institute of Orthopaedics, RNOH, Stanmore, UK.
Monoclonal antibody DA1 is one of a panel of antibodies raised against cultured human bone cells. Immunocyto- chemistry of cultured human bone cells showed the an- tigen to be in the extracellular matrix and, where the cells were made permeable by fixation before staining, also in the cytoplasm Fluorescence-actrvated cell sorting showed cultured human bone cells to be posrtlve for DA1 after fixation. lmmunodot labeling showed the DA1 antigen in cytoplasmic fractions of the cells but not in detergent extracts of the membrane fraction. Thus the DA1 antigen appears to be a cytoplasmlc species that is secreted into the matrix but is not detectable in the cell membrane. In cryostat sections of adult human bone, osteoblasts and osteocytes were positive. DA1 also recognized human chondrocytes. However. in a plastic-embedded section of fetal human femur, DA1 recognized a minor subpopulation of cells distributed throughout the epiphyseal region. Ex- tensrve testing of soft tissue sections with DA1 gave marnly negative results. In sectlons of some human osteosar-
coma, there was intense staining of the extracellular ma- trix. In a sample of giant cell tumour, the giant cells were negative but some stromal cells were positive. In an immu- noblot of an SDS-polyacrylamide gel of proteins from cul- tured human bone cells, DA1 reacted strongly with a band of molecular weight approximately 35,000 and with a band at about 50,000. We are presently involved in further char- acterization of the antigen by immunoprecrpitation and two-dimensional gel blotting. We thus hope to elucidate the nature of the DA1 antigen and its role in the matrix of bone. Supported by YCRC, British Council and Action Re- search.
HORMONAL INDUCTION OF OSTEOCLASTIC PHENOTYPE FROM HAEMOPOIETIC PRECURSORS IN VITRO
K. Fuller and T.J. Chambers
Department of Histopathology, St George’s Hospital MedIcal School, London SW1 7 ORE, UK
Mammalian spleen cells, peripheral blood mononuclear cells, or neonatal and adult mammalian bone marrow (after depletion of preexisting osteoclasts) were incubated in the presence or absence of agents known to stimulate osteoclastic bone resorption. Osteoclast phenotype was identified as the ability to excavate bone, a function that seems specific for osteoclasts. Osteoclasts were undetec- table in the first week of incubation but were regularly and progressively induced to appear in succeeding weeks, but only in the presence of hormonal stimulation, in spleen and marrow cultures. No osteoclastic differentiation was ob- served using blood mononuclear cells. These results, in particular those for spleen cells, cannot be explained merely on the basis of improved survival or stimulation of osteoclasts already present. Rather, they demonstrate hormone-dependent induction of osteoclastic differentia- tion from cells not previously demonstrating osteoclastic function. This experimental system should enable rnvesti- gation of osteoclast differentiation and lineage and of the hormonal mechanisms involved in regulation of osteo- elastic production and may enable production of osteo- clasts In vitro In numbers sufficient to assist analysis of the mechantsms and regulation of osteoclastic bone resorp- tion.
BONE MEMBRANE AND THE SODIUM-POTASSIUM PUMP
C.L. Haycox,’ W.J. Heideger,’ and K.W. Beach
‘Departmenf of Chemical Engineering and “Department of Surgery, University of Washington, Seattle, WA 98195, USA.
The concentration of calcium In the circulation is controlled in part by its transport to and from bone, the body’s prin- cipal reservoir for both calcium and phosphate. An elec- trochemical potential difference has been demonstrated across the bone membrane, that layer of cells lining bone surfaces as the barrier between bone and blood. Active transport of one or more ionic species is implied by this potential, and the control of body calcium is linked to that transport system. We have developed a technique for the separate analysis of Ionic accumulation in the fluid phase
