Localization of 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, gpllla antibodies reacted weakly with which is known to be derived from a circulating in buffy coat smears. Antibodies to factor 8 and mononuclear precursor cell of haemopoietic ori- glycoproteins Ib and Ilb/IHa did not react with gin, is controversial. Recent studies have shown osteoclasts. These results show that osteoclasts, that as well as / plate- monocytes, , and let antigens are expressed by osteoclasts. In this possess common antigens and that fi- study, we have sought to define, by immunohisto- brinogen is present on the surface of osteoclasts. chemistry, the nature and possible function of By analogy with platelets, CD9 and gpllla may platelet antigens expressed by human osteoclasts play a role in fibrinogen binding by osteoclasts. in foetal and adult specimens. Monoclonal Possible mechanisms by which platelet antigens antibodies to platelet glycoprotein Ilia (gpllla) and fibrinogen binding could affect osteoclast and CD9 antibodies stained osteoclasts in all bone function are proposed. specimens examined. Fibrinogen was also local- ized to the osteoclast membrane in foetal bone Key words: osteoclast, platelet, megakaryocyte, fibrinogen, imprints. In addition, we found that CD9 and monoclonal antibody, immunohistochemistry.

Introduction platelet antigens are present on osteoclasts. Several anti-macrophage antibodies, which also react with Osteoclasts are multinucleated cells, which are princi- osteoclasts, stain megakaryocytes and platelets in hu- pally reponsible for and remodelling. man foetal bone (Athanasou et al. 1986). In addition, They are derived from fusion of circulating mono- anti-platelet glycoprotein Ilia (gpllla) antibodies have nuclear precursor cells of origin (Marks, been shown to stain human osteoclasts (Beckstead et al. 1983). The nature of the stem cell that gives rise to 1986; Horton, 1986; Athanasou et al. 1986). In order to osteoclast precursors in the bone marrow is uncertain. determine if there are other platelet antigens that are There is considerable evidence to suggest that osteo- present on osteoclasts, we have used a large panel of clasts are part of the mononuclear system platelet monoclonal antibodies of defined specificity to and that their precursor cells are divided from the stain osteoclasts in human foetal and adult bone pluripotential haemopoietic stem cell that gives rise to specimens. As gpllla is thought to be the fibrinogen the erythroid, myeloid and megakaryocytoid cell lines receptor in platelets, we also looked at the ability of in the marrow (Gothlin & Ericsson, 1976; Chambers, osteoclasts to bind fibrinogen. 1980). However, there is also evidence from functional (Chambers & Magnus, 1982), transplantation (Loutit & Nisbet, 1982) and immunohistochemical (Horton et Materials and methods al. 1984, 1985a,b) studies that suggests that the osteo- Foetal bone specimens clast is derived from a stem cell other than that for peripheral cells (Chambers, 1985). Fresh tissue was obtained from six 12- to 19-week human foetuses at (prostaglandin-induced) therapeutic terminations Recent immunohistochemical studies of the human of pregnancy. The femora, tibiae and humeri were isolated osteoclast have shown that macrophage as well as and placed in Eagles' Minimal Essential Medium (Flow) with Journal of Cell Science 89, 115-122 (1988) Printed in Great Britain © The Company of Biologists Limited 1988 115 added benzyl penicillin 100 units ml ' (Glaxo) and strepto- 1984). The monoclonal antibodies were in the form of ascites mycin (lOOmgml"1) (Glaxo). The were cleared of soft diluted in hybridoma culture medium to 1:500 and 1:250 tissue and then treated as outlined below. (v/v). Negative controls consisted of the substitution of primary antibody by regular culture medium. Foetal metaphyseal bone imprints Bones were cut transversely at the midshaft then bisected longitudinally. Metaphyseal bone imprints were made by Results lightly imprinting the cut surface of the bone onto a Multi- spot glass slide (Hendley, Essex). The imprints were air Histology and enzyme histochemistry of tissues dried at room temperature then immediately fixed in cold examined (—20°C) acetone for lOmin. The fixed slides were stored at Abundant multinucleated osteoclasts (20-40) were -20°C. present in H & E-stained metaphyseal bone imprints. Their osteoclastic nature has been confirmed by their Ciyostat sections of foetal metaphyseal bone specific morphological response to (Athana- Undecalcified cryostat sections of the were pro- sou et al. 1986). Other morphologically identifiable duced from bisected foetal long bones that had been snap- frozen and stored in liquid nitrogen. The sections (5ftm) scattered cells in the imprints included erythrocytes, were collected onto poly-L-lysine-coated glass slides, air dried megakaryocytes, platelets, monocytesand polymorphs. for 24 h, then fixed in acetone for 10 min and air dried at room Megakaryocytes were easily distinguished from osteo- temperature. clasts by their generally smaller size and single con- voluted polymorphic nucleus. In imprints, some larger Adult bone specimens mononuclear cells contained a large densely stained, These consisted of biopsies from three cases of Paget's disease occasionally polymorphic nucleus; these cells stained of bone, and tissue from three cases of tumour of positively with platelet antibodies and may represent bone. In both cases, the fresh tissue had been snap-frozen or other early megakaryocyte precur- and stored in liquid nitrogen prior to cryostat sectioning and sors (Williams & Levine, 1982). In metaphyseal bone immunohistochemistry. sections, large numbers of osteoclasts were present on the surface of newly formed bone trabeculae that were Buffy coat preparations being resorbed. The multinucleated cells identified as Buffy coat smears were prepared by standard techniques osteoclasts in both imprints and sections of foetal bone (Dacie & Lewis, 1975) from venous blood of patients with were positive and alkaline phospha- normal blood counts. tase negative (not illustrated). Histological and immunohistochemical procedures Sections of Pagetic bone and giant cell tumour of Representative sections of foetal bone, giant cell tumour of bone showed the characteristic histological appearance bone specimens and biopsies of Paget's disease of bone were of these conditions and contained abundant osteoclasts stained routinely with haematoxylin and eosin (H & E). The and osteoclast-like giant cells, respectively. naphthol AS-B1 techniques for acid phosphatase and alkaline phosphatase were also used to stain both foetal bone sections Immunohistochemistry and imprints (Bancroft & Stevens, 1975). Localization of platelet antigens and fibrinogen on Platelet antigens were located in the bone imprints, tissue osteoclasts and monocytes. The results of the immuno- sections and buffy coat smears by immunohistochemistry histochemical staining of foetal and adult bone prep- after the application of the monoclonal and polyclonal arations with monoclonal antibodies directed against antibodies listed in Table 1. These included all the mono- clonal antibodies in the Platelet panel of the Third Inter- platelet antigens are shown in Table 1. Platelet anti- national Workshop on Human Leucocyte Differentiation bodies of the CD9 group (BU-16, FMC56, ALB6) and Antigens, Oxford, 1986. These antibodies were grouped on anti-gpllla antibodies (VI-P12, C17, PL1 and 96-2C1) the basis of FACS (fluorescence activated cell sorter), bio- stained osteoclasts in all the foetal and adult bone chemical and immunohistological analysis, including sero- preparations examined (Fig. 1A,B). Fibrinogen was logical analysis of mutant platelets lacking platelet-specific also localized on osteoclasts in foetal bone imprints glycoproteins. For full details of platelet monoclonal anti- (Fig. 1C). The other antibodies directed against bodies and the analysis protocol, see Horton & Hogg (1987). platelet antigens including those against glycoprotein In addition, we stained both foetal and adult bone prep- Ib (gplb) and glycoprotein Ilb/lIIa (gpllb/llla) did arations with two anti-macrophage antibodies, EBM/ll and not stain osteoclasts (Fig. 1C). In buffy coat smears the Yl/82a, which are also known to stain osteoclasts (Athanasou CD9 and gpllla antibodies strongly stained platelets; et al. 1986), and with a polyclonal antibody for fibrinogen (Behring Diagnostics, Hoescht, UK). monocytes, but no other white cells, were also weakly Immunohistochemical staining of imprints, cryostat sec- stained. tions and buffy coat smears was performed by an indirect In foetal bone imprints, osteoclast staining by both immunoperoxidase or alkaline phosphatase anti-alkaline CD9 and gpllla antibodies was largely cytoplasmic phosphatase (APAAP) technique as described (Gatter et al. with some membrane prominence. Megakaryocytes

116 N. A. Athanasou et al. Table 1. Monoclonal and polyclonal platelet antibodies used in the present study and results of staining

Osteoclast reactivity Antigen in foetal and adult Antibody specificity Source bone specimens

HPL14 Platelet gplb Hiraiwa — AN51 Platelet gplb McMichael - FMC25 Platelet gpIX Zola - J15 Platelet gpllb/llla McMichael - BC5-C4 Platelet gpllb/l I la Ando - P2 Platelet gpllb/llla Brochier - P2S6 Platelet gpllb/llla Hogg - HAS-E2 Platelet gpllb/llla Ando - JC6-E6 Platelet gpllb/llla Ando - P112 Platelet gpllb/llla Hogg - KB3-E6 Platelet gpllb/llla Ando - HPL2 Platelet gpllb/l I la Hiraiwa - P4 Platelet gpllb/llla Brochier - P140 Platelet gpllb/llla Hogg - 96-SL3 Platelet gpllb/llla Vilella - 1D1-1D5 Platelet gpllb/llla Vilella - V1-PL3 Platelet gpllb/llla Knapp - 111-5A5 Platelet gpllb/llla Vilella - Vl-Pll Platelet gpllb/llla Knapp - 111-2B5 Platelet gpllb/llla Vilella - 111-SA1 Platelet gpllb/llla Vilella - CLB-thrombo/7 Platelet gpllb/l I la Tetteroo - FCM 24 Platelet gpllb/llla Zola - 111-3D1 Platelet gpllb/llla Vilella - V1-P12 Platelet gpllla and Knapp + + gpll la-like C17 Platelet gpllla and Lansdorp + + gpll la-like PL1 Platelet gpllla and Ravoet + + gpllla-like 96-2C1 Platelet gpllla and Vilella + + gpllla-like BU-16 Platelet CD9 Johnson + FMC 56 Platelet CD9 Zola + ALB6 Platelet CD9 Bouchier + NU-TPAN Platelet CD5-like Yokoyama - Cl Meg 1 Platelet CD36 Pilkington - P7 Platelet gp 150 Brochier - GR21I0 Platelet p24 Garrido - M-T32 Platelet unclustered Rieber - (gplb-like) PS Platelet unclustered Brochier - SYB10 Platelet unclustered Breard - SYB11 Platelet unclustered Breard - F8.86.3 Factor-8-related antigen Mason - Anti-fibrinogen* Fibrinogen Behring + + EBM/11 /macrophage McGee + + Y182a Monocyte/macrophage Mason + +

+ + , Strong reaction; +, weak reaction; —, no reaction. * Polyclonal antibody.

and platelets were also strongly stained, as were oc- almost entirely unstained. Fibrinogen was largely local- casional scattered mononuclear cells. In sections of the ized to the membrane of osteoclasts in foetal bone foetal bone metaphysis, osteoclasts showed strong imprints (Fig. 1C). Megakaryocytes, platelets and a cytoplasmic staining (Fig. 2). A few mononuclear cells few scattered mononuclear cells were also positive for in the intertrabecular stroma were also stained but fibrinogen. Most background mononuclear cells, how- mononuclear cells lining the bone trabeculae were ever, were negative for fibrinogen.

Platelet antigens and fibrinogen on osteoclasts 117 B

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. f1

118 N. A. Athanasou et al. Fig. 2. Human foetal metaphyseal bone section stained with 96-2C1 (anti-gpllla) showing strong diffuse staining of an osteoclast (arrowed) lying against a bone . Immunoperoxidase; X336.

In cryostat sections of Pagetic bone, osteoclasts and a cells and megakaryocytes and weakly reacted with few stromal cells were stained strongly with CD9 and platelets (Fig. 4). In buffy coat smears, these anti- gpllla antibodies (Fig. 3). Osteoclast-like giant cells bodies strongly reacted with monocytes and weakly and scattered mononuclear stromal cells in giant cell stained platelets. tumour of bone also showed strong cytoplasmic stain- ing. Both giant cells and the mononuclear cells that Discussion were stained by these antibodies showed no evidence of mitotic activity or nuclear and cellular atypia. In The antigenic phenotype of the human osteoclast, cryostat sections of human foetal and adult bone which is known to be derived from a circulating specimens, it was not possible to localize fibrinogen to mononuclear precursor cell of bone marrow origin, is any specific cell type due to heavy background staining. controversial. Horton et al. (1984, 1985«,6) have Localization of macrophage antigens in osteoclasts, reported that human osteoclasts do not express macro- megakaryocytes and platelets. Results of the immuno- phage or platelet antigens and argued that this favours histochemical staining of the foetal and adult bone the origin of the osteoclast from a stem cell distinct preparations with the two anti-macrophage antibodies, from the pluripotential haemopoietic stem cell. I low- EBM/11 and Yl/82a, are also shown in Table 1. Both ever, there are now several reports of monoclonal antibodies showed strong cytoplasmic staining of osteo- antibodies that recognize cell surface antigens on clasts in foetal and Pagetic bone as well as osteoclast- osteoclasts and other myeloid cells, notably macro- like giant cells in giant cell tumours of bone. In phages (Nijweide et al. 1985; Oursler et al. 1985; addition, in foetal bone imprints EBM/ll and Yl/82a Sminia & Dijkstra, 1986; Athanasou et al. 1986, 1987) strongly stained scattered background mononuclear and platelets (Becksteade/ al. 1986; Morton et al. 1986; Fig. 1. Human metaphyseal bone imprints stained Athanasou et al. 1986). immunohistochemically by an indirect immunoperoxidase This study has confirmed that osteoclasts express method with: A. C17 (anti-gpllla) showing strong monocyte/macrophage and megakaryocyte/platelet- membrane staining of an osteoclast. Two smaller associated antigens. This finding is consistent with, but megakaryocytes (arrowed) and mononuclear cells show not proof of, a common origin or differentiation diffuse cytoplasmic staining. The nuclei of the pathway for monocytes, megakaryocytes and osteo- megakaryocytes are partially obscured by the histochemical clasts from a single stem cell, i.e. the pluripotential reaction product. X336. B. BU-16 (anti-CD9) showing haemopoietic stem cell. Human haemopoietic stem membrane staining of an osteoclast (top) and diffuse cells have been shown to express glycoprotein Ilia cytoplasmic staining of a megakaryocyte (bottom) and platelets (arrowheads). X336. C. Anti-fibrinogen showing (Kraser et al. 1986). Human blood monocytes and strong membrane staining of the osteoclast membrane. platelets also share cell surface components (Burck- X336. D. AN51 (anti-gplb) showing staining of hardt et al. 1982; Bai et al. 1984) and, like platelets, megakaryocytes (arrowed) and platelets but osteoclast monocytes bind fibrin and fibrinogen (Colvin & (centre) is unstained. X210. Dvorak, 1975; Sherman & Lee, 1977; Hogg, 1983).

Platelet antigens and fibrinogen on osteoclasts 119 Fig. 3. Section of Pagetic bone showing two osteoclasts lying in How ship's lacunae. Both are stained by C17 (anti-gpllla). Immunoperoxidase; X336.

• *#

Fig. 4. Human metaphyseal bone imprint stained with EBM/ll (anti-macrophage) showing diffuse cytoplasmic staining of two osteoclasts and a megakaryocyte (arrowed). Immunoperoxidase; X336.

3 However, immunohistology has also revealed CD9 and the 24xlO /Wr protein recognized by the CD9 anti- gpllla antigens on non-myeloid tissues; so, the finding bodies may have the opposite effect on osteoclast that these cell types have common surface antigens fibrinogen binding. does not permit a definite conclusion regarding their The role of surface fibrinogen on osteoclasts is origin. unknown, but again, by analogy with platelets, it is The finding of fibrinogen and platelet antigens on possible that it is necessary for aggregation or cellular osteoclasts is of great interest as it is possible that their adhesion of mononuclear osteoclast precursors prior to presence may signal a common process of differen- fusion and the formation of multinuclear osteoclasts. tiation or function by these cell types, gpllla anti- Osteoclasts may also require fibrinogen to bind to the bodies are known to inhibit platelet aggregation and bone surface. Another possibility involves plasminogen fibrinogen binding by platelets (Foon & Todd, 1986), activator (PA), which is known to be secreted by whilst CD9 antibodies stimulate platelet aggregation (Hamilton et al. 1985). The PA/plasmin and induce fibrinogen binding (Horton & Hogg, 1987). system has been implicated in mechanisms of connec- Analogously, in osteoclasts, the gpllla antigen may tive tissue turnover, remodelling and similarly influence fibrinogen binding or even form the (Lack & Rogers, 1958; Beers, 1975; Sherman, 1976), site of binding of fibrinogen by osteoclasts. In contrast, all of which functions are exhibited by the osteoclast

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122 N. A. Athanasou et al.