Localization of platelet antigens and fibrinogen on osteoclasts

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1 Localization of platelet antigens and fibrinogen on osteoclasts N. A. ATHANASOU*, J. QUINN, A. HERYET and J. O'D. McGEE University of Oxford, Nitffield Department of Pathology, John Radcliffe Hospital, Heading/on, Oxford 0X3 9DU, UK * Author for correspondence Summary The antigenic phenotype of the human osteoclast, which is known to be derived from a circulating mononuclear precursor cell of haemopoietic origin, is controversial. Recent studies have shown that macrophage as well as megakaryocyte/ platelet antigens are expressed by osteoclasts. In this study, we have sought to define, by immunohistochemistry, the nature and possible function of platelet antigens expressed by human osteoclasts in foetal and adult bone specimens. Monoclonal antibodies to platelet glycoprotein Ilia (gpllla) and CD9 antibodies stained osteoclasts in all bone specimens examined. Fibrinogen was also localized to the osteoclast membrane in foetal bone imprints. In addition, we found that CD9 and gpllla antibodies reacted weakly with monocytes in buffy coat smears. Antibodies to factor 8 and glycoproteins Ib and Ilb/IHa did not react with osteoclasts. These results show that osteoclasts, monocytes, macrophages, megakaryocytes and platelets possess common antigens and that fibrinogen is present on the surface of osteoclasts. By analogy with platelets, CD9 and gpllla may play a role in fibrinogen binding by osteoclasts. Possible mechanisms by which platelet antigens and fibrinogen binding could affect osteoclast function are proposed. Key words: osteoclast, platelet, megakaryocyte, fibrinogen, monoclonal antibody, immunohistochemistry. Introduction Osteoclasts are multinucleated cells, which are principally reponsible for bone resorption and remodelling. They are derived from fusion of circulating mononuclear precursor cells of bone marrow origin (Marks, 1983). The nature of the stem cell that gives rise to osteoclast precursors in the bone marrow is uncertain. There is considerable evidence to suggest that osteoclasts are part of the mononuclear phagocyte system and that their precursor cells are divided from the pluripotential haemopoietic stem cell that gives rise to the erythroid, myeloid and megakaryocytoid cell lines in the marrow (Gothlin & Ericsson, 1976; Chambers, 1980). However, there is also evidence from functional (Chambers & Magnus, 1982), transplantation (Loutit & Nisbet, 1982) and immunohistochemical (Horton et al. 1984, 1985a,b) studies that suggests that the osteoclast is derived from a stem cell other than that for peripheral blood cells (Chambers, 1985). Recent immunohistochemical studies of the human osteoclast have shown that macrophage as well as Journal of Cell Science 89, (1988) Printed in Great Britain The Company of Biologists Limited 1988 platelet antigens are present on osteoclasts. Several antimacrophage antibodies, which also react with osteoclasts, stain megakaryocytes and platelets in human foetal bone (Athanasou et al. 1986). In addition, antiplatelet glycoprotein Ilia (gpllla) antibodies have been shown to stain human osteoclasts (Beckstead et al. 1986; Horton, 1986; Athanasou et al. 1986). In order to determine if there are other platelet antigens that are present on osteoclasts, we have used a large panel of platelet monoclonal antibodies of defined specificity to stain osteoclasts in human foetal and adult bone specimens. As gpllla is thought to be the fibrinogen receptor in platelets, we also looked at the ability of osteoclasts to bind fibrinogen. Materials and methods Foetal bone specimens Fresh tissue was obtained from six 12 to 19week human foetuses at (prostaglandininduced) therapeutic terminations of pregnancy. The femora, tibiae and humeri were isolated and placed in Eagles' Minimal Essential Medium (Flow) with 115

2 added benzyl penicillin 100 units ml ' (Glaxo) and streptomycin (loomgml" 1 ) (Glaxo). The bones were cleared of soft tissue and then treated as outlined below. Foetal metaphyseal bone imprints Bones were cut transversely at the midshaft then bisected longitudinally. Metaphyseal bone imprints were made by lightly imprinting the cut surface of the bone onto a Multispot glass slide (Hendley, Essex). The imprints were air dried at room temperature then immediately fixed in cold ( 20 C) acetone for lomin. The fixed slides were stored at 20 C. Ciyostat sections of foetal metaphyseal bone Undecalcified cryostat sections of the metaphysis were produced from bisected foetal long bones that had been snapfrozen and stored in liquid nitrogen. The sections (5ftm) were collected onto polyllysinecoated glass slides, air dried for 24 h, then fixed in acetone for 10 min and air dried at room temperature. Adult bone specimens These consisted of biopsies from three cases of Paget's disease of bone, and tissue from three cases of giant cell tumour of bone. In both cases, the fresh tissue had been snapfrozen and stored in liquid nitrogen prior to cryostat sectioning and immunohistochemistry. Buffy coat preparations Buffy coat smears were prepared by standard techniques (Dacie & Lewis, 1975) from venous blood of patients with normal blood counts. Histological and immunohistochemical procedures Representative sections of foetal bone, giant cell tumour of bone specimens and biopsies of Paget's disease of bone were stained routinely with haematoxylin and eosin (H & E). The naphthol ASB1 techniques for acid phosphatase and alkaline phosphatase were also used to stain both foetal bone sections and imprints (Bancroft & Stevens, 1975). Platelet antigens were located in the bone imprints, tissue sections and buffy coat smears by immunohistochemistry after the application of the monoclonal and polyclonal antibodies listed in Table 1. These included all the monoclonal antibodies in the Platelet panel of the Third International Workshop on Human Leucocyte Differentiation Antigens, Oxford, These antibodies were grouped on the basis of FACS (fluorescence activated cell sorter), biochemical and immunohistological analysis, including serological analysis of mutant platelets lacking plateletspecific glycoproteins. For full details of platelet monoclonal antibodies and the analysis protocol, see Horton & Hogg (1987). In addition, we stained both foetal and adult bone preparations with two antimacrophage antibodies, EBM/ll and Yl/82a, which are also known to stain osteoclasts (Athanasou et al. 1986), and with a polyclonal antibody for fibrinogen (Behring Diagnostics, Hoescht, UK). Immunohistochemical staining of imprints, cryostat sections and buffy coat smears was performed by an indirect immunoperoxidase or alkaline phosphatase antialkaline phosphatase (APAAP) technique as described (Gatter et al. 1984). The monoclonal antibodies were in the form of ascites diluted in hybridoma culture medium to 1:500 and 1:250 (v/v). Negative controls consisted of the substitution of primary antibody by regular culture medium. Results Histology and enzyme histochemistry of tissues examined Abundant multinucleated osteoclasts (2040) were present in H & Estained metaphyseal bone imprints. Their osteoclastic nature has been confirmed by their specific morphological response to calcitonin (Athanasou et al. 1986). Other morphologically identifiable scattered cells in the imprints included erythrocytes, megakaryocytes, platelets, monocytesand polymorphs. Megakaryocytes were easily distinguished from osteoclasts by their generally smaller size and single convoluted polymorphic nucleus. In imprints, some larger mononuclear cells contained a large densely stained, occasionally polymorphic nucleus; these cells stained positively with platelet antibodies and may represent megakaryoblasts or other early megakaryocyte precursors (Williams & Levine, 1982). In metaphyseal bone sections, large numbers of osteoclasts were present on the surface of newly formed bone trabeculae that were being resorbed. The multinucleated cells identified as osteoclasts in both imprints and sections of foetal bone were acid phosphatase positive and alkaline phosphatase negative (not illustrated). Sections of Pagetic bone and giant cell tumour of bone showed the characteristic histological appearance of these conditions and contained abundant osteoclasts and osteoclastlike giant cells, respectively. Immunohistochemistry Localization of platelet antigens and fibrinogen on osteoclasts and monocytes. The results of the immunohistochemical staining of foetal and adult bone preparations with monoclonal antibodies directed against platelet antigens are shown in Table 1. Platelet antibodies of the CD9 group (BU16, FMC56, ALB6) and antigpllla antibodies (VIP12, C17, PL1 and 962C1) stained osteoclasts in all the foetal and adult bone preparations examined (Fig. 1A,B). Fibrinogen was also localized on osteoclasts in foetal bone imprints (Fig. 1C). The other antibodies directed against platelet antigens including those against glycoprotein Ib (gplb) and glycoprotein Ilb/lIIa (gpllb/llla) did not stain osteoclasts (Fig. 1C). In buffy coat smears the CD9 and gpllla antibodies strongly stained platelets; monocytes, but no other white cells, were also weakly stained. In foetal bone imprints, osteoclast staining by both CD9 and gpllla antibodies was largely cytoplasmic with some membrane prominence. Megakaryocytes 116 N. A. Athanasou et al.

3 Table 1. Monoclonal and polyclonal platelet antibodies used in the present study and results of staining Antibody Antigen specificity Source Osteoclast reactivity in foetal and adult bone specimens HPL14 AN51 FMC25 J15 BC5C4 P2 P2S6 HASE2 JC6E6 P112 KB3E6 HPL2 P4 P140 96SL3 1D11D5 V1PL3 1115A5 VlPll 1112B5 111SA1 CLBthrombo/7 FCM D1 V1P12 C17 PL1 962C1 BU16 FMC 56 ALB6 NUTPAN Cl Meg 1 P7 GR21I0 MT32 PS SYB10 SYB11 F Antifibrinogen* EBM/11 Y182a Platelet gplb Platelet gplb Platelet gpix Platelet gpllb/l I la Platelet gpllb/l I la Platelet gpllb/l I la Platelet gpllla and gpll lalike Platelet gpllla and gpll lalike Platelet gpllla and gplllalike Platelet gpllla and gplllalike Platelet CD9 Platelet CD9 Platelet CD9 Platelet CD5like Platelet CD36 Platelet gp 150 Platelet p24 Platelet unclustered (gplblike) Platelet unclustered Platelet unclustered Platelet unclustered Factor8related antigen Fibrinogen Monocyte/macrophage Monocyte/macrophage Hiraiwa McMichael Zola McMichael Ando Brochier Hogg Ando Ando Hogg Ando Hiraiwa Brochier Hogg Knapp Knapp Tetteroo Zola Knapp Lansdorp Ravoet Johnson Zola Bouchier Yokoyama Pilkington Brochier Garrido Rieber Brochier Breard Breard Mason Behring McGee Mason + + +, Strong reaction; +, weak reaction; * Polyclonal antibody., no reaction. and platelets were also strongly stained, as were occasional scattered mononuclear cells. In sections of the foetal bone metaphysis, osteoclasts showed strong cytoplasmic staining (Fig. 2). A few mononuclear cells in the intertrabecular stroma were also stained but mononuclear cells lining the bone trabeculae were almost entirely unstained. Fibrinogen was largely localized to the membrane of osteoclasts in foetal bone imprints (Fig. 1C). Megakaryocytes, platelets and a few scattered mononuclear cells were also positive for fibrinogen. Most background mononuclear cells, however, were negative for fibrinogen. Platelet antigens and fibrinogen on osteoclasts 117

4 B W;. f N. A. Athanasou et al.

5 Fig. 2. Human foetal metaphyseal bone section stained with 962C1 (antigpllla) showing strong diffuse staining of an osteoclast (arrowed) lying against a bone trabecula. Immunoperoxidase; X336. In cryostat sections of Pagetic bone, osteoclasts and a few stromal cells were stained strongly with CD9 and gpllla antibodies (Fig. 3). Osteoclastlike giant cells and scattered mononuclear stromal cells in giant cell tumour of bone also showed strong cytoplasmic staining. Both giant cells and the mononuclear cells that were stained by these antibodies showed no evidence of mitotic activity or nuclear and cellular atypia. In cryostat sections of human foetal and adult bone specimens, it was not possible to localize fibrinogen to any specific cell type due to heavy background staining. Localization of macrophage antigens in osteoclasts, megakaryocytes and platelets. Results of the immunohistochemical staining of the foetal and adult bone preparations with the two antimacrophage antibodies, EBM/11 and Yl/82a, are also shown in Table 1. Both antibodies showed strong cytoplasmic staining of osteoclasts in foetal and Pagetic bone as well as osteoclastlike giant cells in giant cell tumours of bone. In addition, in foetal bone imprints E B M / l l and Yl/82a strongly stained scattered background mononuclear Fig. 1. Human metaphyseal bone imprints stained immunohistochemically by an indirect immunoperoxidase method with: A. C17 (antigpllla) showing strong membrane staining of an osteoclast. Two smaller megakaryocytes (arrowed) and mononuclear cells show diffuse cytoplasmic staining. The nuclei of the megakaryocytes are partially obscured by the histochemical reaction product. X336. B. BU16 (anticd9) showing membrane staining of an osteoclast (top) and diffuse cytoplasmic staining of a megakaryocyte (bottom) and platelets (arrowheads). X336. C. Antifibrinogen showing strong membrane staining of the osteoclast membrane. X336. D. AN51 (antigplb) showing staining of megakaryocytes (arrowed) and platelets but osteoclast (centre) is unstained. X210. cells and megakaryocytes and weakly reacted with platelets (Fig. 4). In buffy coat smears, these antibodies strongly reacted with monocytes and weakly stained platelets. Discussion The antigenic phenotype of the human osteoclast, which is known to be derived from a circulating mononuclear precursor cell of bone marrow origin, is controversial. Horton et al. (1984, 1985«,6) have reported that human osteoclasts do not express macrophage or platelet antigens and argued that this favours the origin of the osteoclast from a stem cell distinct from the pluripotential haemopoietic stem cell. I lowever, there are now several reports of monoclonal antibodies that recognize cell surface antigens on osteoclasts and other myeloid cells, notably macrophages (Nijweide et al. 1985; Oursler et al. 1985; Sminia & Dijkstra, 1986; Athanasou et al. 1986, 1987) and platelets (Becksteade/ al. 1986; Morton et al. 1986; Athanasou et al. 1986). This study has confirmed that osteoclasts express monocyte/macrophage and megakaryocyte/plateletassociated antigens. This finding is consistent with, but not proof of, a common origin or differentiation pathway for monocytes, megakaryocytes and osteoclasts from a single stem cell, i.e. the pluripotential haemopoietic stem cell. Human haemopoietic stem cells have been shown to express glycoprotein Ilia (Kraser et al. 1986). Human blood monocytes and platelets also share cell surface components (Burckhardt et al. 1982; Bai et al. 1984) and, like platelets, monocytes bind fibrin and fibrinogen (Colvin & Dvorak, 1975; Sherman & Lee, 1977; Hogg, 1983). Platelet antigens and fibrinogen on osteoclasts 119

6 Fig. 3. Section of Pagetic bone showing two osteoclasts lying in How ship's lacunae. Both are stained by C17 (antigpllla). Immunoperoxidase; X336. *# Fig. 4. Human metaphyseal bone imprint stained with EBM/ll (antimacrophage) showing diffuse cytoplasmic staining of two osteoclasts and a megakaryocyte (arrowed). Immunoperoxidase; X336. However, immunohistology has also revealed CD9 and gpllla antigens on nonmyeloid tissues; so, the finding that these cell types have common surface antigens does not permit a definite conclusion regarding their origin. The finding of fibrinogen and platelet antigens on osteoclasts is of great interest as it is possible that their presence may signal a common process of differentiation or function by these cell types, gpllla antibodies are known to inhibit platelet aggregation and fibrinogen binding by platelets (Foon & Todd, 1986), whilst CD9 antibodies stimulate platelet aggregation and induce fibrinogen binding (Horton & Hogg, 1987). Analogously, in osteoclasts, the gpllla antigen may similarly influence fibrinogen binding or even form the site of binding of fibrinogen by osteoclasts. In contrast, the 24xlO 3 /W r protein recognized by the CD9 antibodies may have the opposite effect on osteoclast fibrinogen binding. The role of surface fibrinogen on osteoclasts is unknown, but again, by analogy with platelets, it is possible that it is necessary for aggregation or cellular adhesion of mononuclear osteoclast precursors prior to fusion and the formation of multinuclear osteoclasts. Osteoclasts may also require fibrinogen to bind to the bone surface. Another possibility involves plasminogen activator (PA), which is known to be secreted by osteoblasts (Hamilton et al. 1985). The PA/plasmin system has been implicated in mechanisms of connective tissue turnover, remodelling and cell migration (Lack & Rogers, 1958; Beers, 1975; Sherman, 1976), all of which functions are exhibited by the osteoclast 120 N. A. Athanasou et al.

7 (Chambers, 1985). Osteoclasts, like platelets, are highly motile cells that change shape and activity when they are stimulated or inhibited (Chambers & Magnus, 1982; Chambers, 1985). It is possible that PA is one of these stimulatory factors and that it may influence osteoclast fibrinogen binding directly or indirectly through one of the platelet antigens present on osteoclasts. This work was supported by the Arthritis and Rheumatism Council. We thank Dr C. G. Woods and Dr. J. Keeling for supplying the bone specimens, Miss L. Watts for typing the manuscript and Mr G. Richardson for photographic assist References ATHANASOU, N. A., HERYET, A., QUINN, J., GATTER, K. C, MASON, D. Y. & MCGEE, J. O'D. (1986). Osteoclasts contain macrophage and megakaryocyte antigens. J. Path. 150, ATHANASOU, N. A., QUINN, J. & MCGEE, J. O'D. (1987). Leucocyte common antigen is present on osteoclasts. J. Path. 153, BAI, Y., DURBIN, H. & HOGG, N. (1984). Monoclonal antibodies specific for platelet glycoproteins react with human monocytes. Blood 64, BANCROFT, J. D. & STEVENS, A. (1975). Theoiy and Practice of Histological Techniques, pp Edinburgh: Churchill Livingstone. BECKSTEAD, J. H., STENBERG, P. E., MCEVER, R. P., SHUMAN, M. A. & BAINTON, D. F. (1986). Immunochemical localisation of membrane and alpha granule proteins in human megakaryocytes: Applications to plastic embedded bone marrow biopsy specimens. Blood 67, BEERS, W. H. (1975). Follicular plasminogen and plasminogen activator and the effect of plasmin on ovarian follicle wall. Cell 6, BURCKHARDT, J. J., KERR ANDERSON, W. H., KEARNEY, J. F. & COOPER, M. D. (1982). Human blood monocytes and platelets share a cell surface component. Blood 60, CHAMBERS, T. J. (1980). The cellular basis of bone resorption. Clin. Orthop. rel. Res. 151, CHAMBERS, T. J. (1985). The pathobiology of the osteoclast. J. clin. Path. 38, CHAMBERS, T. J. & MAGNUS, C. J. (1982). Calcitonin alters behaviour of isolated osteoclasts. J. Path. 136, COLVIN, R. B. & DVORAK, H. F. (1975). Fibrinogen/fibrin on the surface of macrophages: Detection, distribution, binding requirements and possible role in macrophage adherence phenomena. J. exp. Med. 142, DACIE, J. V. & LEWIS, S. M. (1975). Practical Haematology, pp Edinburgh: Churchill Livingstone. FOON, K. A. & TODD, R. F. (1986). Immunologic classification of leukemia and lymphoma. Blood 68, 131. GATTER, K. C, FAUNI, B. & MASON, D. Y. (1984). The use of monoclonal antibodies in histopathological diagnosis. In Recent Advances in Histopathology, no. 12 (ed. P. P. Anthony & R. N. M. MacSween), pp Edinburgh: Churchill Livingstone. GOTHLIN, G. & ERICSSON, J. L. E. (1976). The osteoclast. Clin. Orthop. 120, HAMILTON, J. A., LINGELBACH, S., PARTRIDGE, N. C. & MARTIN, T. J. (1985). Regulation of plasminogen activator production by boneresorbing hormones in normal and malignant osteoblasts. Endocrinology 116, HOGG, N. (1983). Human monocytes are associated with the formation of fibrin. J. exp. Med. 157, HORTON, M. A. (1986). Expression of platelet glycoprotein Ilia by human osteoclasts. Blood 68, 595. HORTON, M. A. & HOGG, N. (1987). Platelet antigens: new and previously defined clusters. In Leucocyte Typing III (ed. A. McMichael et ai), pp Oxford University Press. HORTON, M. A., LEWIS, D., MCNULTY, K., PRINGLE, J. A. S. & CHAMBERS, T. J. (1985«). Monoclonal antibodies to osteoclastomas (Giant Cell Bone Tumours): Definition of osteoclastspecific cellular antigens. Cancer Res. 45, HORTON, M. A., LEWIS, D., MCNULTY, K., PRINGLE, J. A. S. & CHAMBERS, T. J. (19856). Human fetal osteoclasts fail to express macrophage antigens. Br. J. exp. Path. 66, HORTON, M. A., RIMMER, E. F., LEWIS, D., PRINGLE, J. A. S., FULLER, K. & CHAMBERS, T. J. (1984). Cell surface characterization of the human osteoclast: phenotypic relationship to other bone marrowderived types.j. Path. 141, KRASER, J. K., LEAHY, M. F. & BERRIDGE, M. V. (1986). Expression of antigens of the platelet glycoprotein 11 b/ Ilia complex on human hematopoietic stem cells. Blood 68, LACK, C. W. & ROGERS, H. J. (1958). Action of plasmin on cartilage. Nature, Lond. 182, LOUTIT, J. F. & NISBET, N. W. (1982). The origin of the osteoclast. Iinmunobiology 161, MARKS, S. C. (1983). The origin of osteoclasts. J. oral Path. 12, NUWEIDE, P. J., VRIGHERDHAMMERS, T., MULDER, R. J. P. & BLOK, J. (1985). Cell surface antigens on osteoclasts and related cells in the quail studied with monoclonal antibodies. Histocheniistry 83, OURSLER, M. J., BELL, L. V., CLEVINGER, B. & OSDOBY, P. (1985). Identification of osteoclastspecific monoclonal antibodies. J. Cell Biol. 100, Platelet antigens and fibrinogen on osteoclasts 121

8 SHERMAN, L. A. & LEE, J. (1977). Specific binding of macrophages and osteoclasts in embryonic rat bone. soluble fibrin to macrophages. J. exp. Med. 145, Calc. Tiss. Int. 39, SHERMAN, M. J. (1976). Plasminogen activator in early WILLIAMS, N. & LEVINE, R. F. (1982). The origin, embryogenesis: enzyme production by trophoblast and development and regulation of megakaryocytes. Br. J. parietal endoderm. Cell 9, Haemal. 52, SMINIA, T. & DIJKSTRA, C. D. (1986). The origin of osteoclasts: an immunohistochemical study on (Received 12 August 1987 Accepted 12 October 1987) 122 N. A. Athanasou et al.