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 circulatingmononuclear precursor cell of haemopoietic ori-gin, is controversial. Recent studies have shownthat macrophage as well as megakaryocyte/ plate-let antigens are expressed by osteoclasts. In thisstudy, we have sought to define, by immunohisto-chemistry, the nature and possible function ofplatelet antigens expressed by human osteoclastsin foetal and adult bone specimens. Monoclonalantibodies to platelet glycoprotein Ilia (gpllla)and CD9 antibodies stained osteoclasts in all bonespecimens examined. Fibrinogen was also local-ized to the osteoclast membrane in foetal boneimprints. In addition, we found that CD9 and

gpllla antibodies reacted weakly with monocytesin buffy coat smears. Antibodies to factor 8 andglycoproteins Ib and Ilb/IHa did not react withosteoclasts. These results show that osteoclasts,monocytes, macrophages, megakaryocytes andplatelets possess common antigens and that fi-brinogen is present on the surface of osteoclasts.By analogy with platelets, CD9 and gpllla mayplay a role in fibrinogen binding by osteoclasts.Possible mechanisms by which platelet antigensand fibrinogen binding could affect osteoclastfunction are proposed.

Key words: osteoclast, platelet, megakaryocyte, fibrinogen,monoclonal antibody, immunohistochemistry.

Introduction

Osteoclasts are multinucleated cells, which are princi-pally reponsible for bone resorption and remodelling.They are derived from fusion of circulating mono-nuclear precursor cells of bone marrow origin (Marks,1983). The nature of the stem cell that gives rise toosteoclast precursors in the bone marrow is uncertain.There is considerable evidence to suggest that osteo-clasts are part of the mononuclear phagocyte systemand that their precursor cells are divided from thepluripotential haemopoietic stem cell that gives rise tothe erythroid, myeloid and megakaryocytoid cell linesin the marrow (Gothlin & Ericsson, 1976; Chambers,1980). However, there is also evidence from functional(Chambers & Magnus, 1982), transplantation (Loutit& Nisbet, 1982) and immunohistochemical (Horton etal. 1984, 1985a,b) studies that suggests that the osteo-clast is derived from a stem cell other than that forperipheral blood cells (Chambers, 1985).

Recent immunohistochemical studies of the humanosteoclast have shown that macrophage as well asJournal of Cell Science 89, 115-122 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

platelet antigens are present on osteoclasts. Severalanti-macrophage antibodies, which also react withosteoclasts, stain megakaryocytes and platelets in hu-man foetal bone (Athanasou et al. 1986). In addition,anti-platelet glycoprotein Ilia (gpllla) antibodies havebeen shown to stain human osteoclasts (Beckstead et al.1986; Horton, 1986; Athanasou et al. 1986). In order todetermine if there are other platelet antigens that arepresent on osteoclasts, we have used a large panel ofplatelet monoclonal antibodies of defined specificity tostain osteoclasts in human foetal and adult bonespecimens. As gpllla is thought to be the fibrinogenreceptor in platelets, we also looked at the ability ofosteoclasts to bind fibrinogen.

Materials and methods

Foetal bone specimensFresh tissue was obtained from six 12- to 19-week humanfoetuses at (prostaglandin-induced) therapeutic terminationsof pregnancy. The femora, tibiae and humeri were isolatedand placed in Eagles' Minimal Essential Medium (Flow) with

115

added benzyl penicillin 100 units ml ' (Glaxo) and strepto-mycin (lOOmgml"1) (Glaxo). The bones were cleared of softtissue and then treated as outlined below.

Foetal metaphyseal bone imprintsBones were cut transversely at the midshaft then bisectedlongitudinally. Metaphyseal bone imprints were made bylightly imprinting the cut surface of the bone onto a Multi-spot glass slide (Hendley, Essex). The imprints were airdried 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 boneUndecalcified cryostat sections of the metaphysis were pro-duced from bisected foetal long bones that had been snap-frozen and stored in liquid nitrogen. The sections (5ftm)were collected onto poly-L-lysine-coated glass slides, air driedfor 24 h, then fixed in acetone for 10 min and air dried at roomtemperature.

Adult bone specimensThese consisted of biopsies from three cases of Paget's diseaseof bone, and tissue from three cases of giant cell tumour ofbone. In both cases, the fresh tissue had been snap-frozenand stored in liquid nitrogen prior to cryostat sectioning andimmunohistochemistry.

Buffy coat preparationsBuffy coat smears were prepared by standard techniques(Dacie & Lewis, 1975) from venous blood of patients withnormal blood counts.

Histological and immunohistochemical proceduresRepresentative sections of foetal bone, giant cell tumour ofbone specimens and biopsies of Paget's disease of bone werestained routinely with haematoxylin and eosin (H & E). Thenaphthol AS-B1 techniques for acid phosphatase and alkalinephosphatase were also used to stain both foetal bone sectionsand imprints (Bancroft & Stevens, 1975).

Platelet antigens were located in the bone imprints, tissuesections and buffy coat smears by immunohistochemistryafter the application of the monoclonal and polyclonalantibodies listed in Table 1. These included all the mono-clonal antibodies in the Platelet panel of the Third Inter-national Workshop on Human Leucocyte DifferentiationAntigens, Oxford, 1986. These antibodies were grouped onthe basis of FACS (fluorescence activated cell sorter), bio-chemical and immunohistological analysis, including sero-logical analysis of mutant platelets lacking platelet-specificglycoproteins. For full details of platelet monoclonal anti-bodies and the analysis protocol, see Horton & Hogg (1987).In addition, we stained both foetal and adult bone prep-arations with two anti-macrophage antibodies, EBM/ll andYl/82a, which are also known to stain osteoclasts (Athanasouet al. 1986), and with a polyclonal antibody for fibrinogen(Behring Diagnostics, Hoescht, UK).

Immunohistochemical staining of imprints, cryostat sec-tions and buffy coat smears was performed by an indirectimmunoperoxidase or alkaline phosphatase anti-alkalinephosphatase (APAAP) technique as described (Gatter et al.

1984). The monoclonal antibodies were in the form of ascitesdiluted in hybridoma culture medium to 1:500 and 1:250(v/v). Negative controls consisted of the substitution ofprimary antibody by regular culture medium.

Results

Histology and enzyme histochemistry of tissuesexamined

Abundant multinucleated osteoclasts (20-40) werepresent in H & E-stained metaphyseal bone imprints.Their osteoclastic nature has been confirmed by theirspecific morphological response to calcitonin (Athana-sou et al. 1986). Other morphologically identifiablescattered cells in the imprints included erythrocytes,megakaryocytes, platelets, monocytesand polymorphs.Megakaryocytes were easily distinguished from osteo-clasts by their generally smaller size and single con-voluted polymorphic nucleus. In imprints, some largermononuclear cells contained a large densely stained,occasionally polymorphic nucleus; these cells stainedpositively with platelet antibodies and may representmegakaryoblasts or other early megakaryocyte precur-sors (Williams & Levine, 1982). In metaphyseal bonesections, large numbers of osteoclasts were present onthe surface of newly formed bone trabeculae that werebeing resorbed. The multinucleated cells identified asosteoclasts in both imprints and sections of foetal bonewere acid phosphatase positive and alkaline phospha-tase negative (not illustrated).

Sections of Pagetic bone and giant cell tumour ofbone showed the characteristic histological appearanceof these conditions and contained abundant osteoclastsand osteoclast-like giant cells, respectively.

Immunohistochemistry

Localization of platelet antigens and fibrinogen onosteoclasts and monocytes. The results of the immuno-histochemical staining of foetal and adult bone prep-arations with monoclonal antibodies directed againstplatelet antigens are shown in Table 1. Platelet anti-bodies of the CD9 group (BU-16, FMC56, ALB6) andanti-gpllla antibodies (VI-P12, C17, PL1 and 96-2C1)stained osteoclasts in all the foetal and adult bonepreparations examined (Fig. 1A,B). Fibrinogen wasalso localized on osteoclasts in foetal bone imprints(Fig. 1C). The other antibodies directed againstplatelet antigens including those against glycoproteinIb (gplb) and glycoprotein I lb / l I Ia (gpl lb/ l l la) didnot stain osteoclasts (Fig. 1C). In buffy coat smears theCD9 and gpllla antibodies strongly stained platelets;monocytes, but no other white cells, were also weaklystained.

In foetal bone imprints, osteoclast staining by bothCD9 and gpllla antibodies was largely cytoplasmicwith 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

Antibody

HPL14AN51FMC25J15BC5-C4P2P2S6HAS-E2JC6-E6P112KB3-E6HPL2P4P14096-SL31D1-1D5V1-PL3111-5A5Vl-Pll111-2B5111-SA1CLB-thrombo/7FCM 24111-3D1V1-P12

C17

PL1

96-2C1

BU-16FMC 56ALB6NU-TPANCl Meg 1P7GR21I0M-T32

PSSYB10SYB11F8.86.3Anti-fibrinogen*EBM/11Y182a

+ + , Strong reaction; +, weak reaction;* Polyclonal antibody.

Antigenspecificity

Platelet gplbPlatelet gplbPlatelet gpIXPlatelet gp l lb / l l l aPlatelet gpl lb/ l I laPlatelet gp l lb / l l l aPlatelet gp l lb / l l l aPlatelet gp l lb / l l l aPlatelet gp l lb / l l l aPlatelet gp l lb / l l l aPlatelet gp l lb / l l l aPlatelet gpl lb/ l I laPlatelet gp l lb / l l l aPlatelet gp l lb / l l l aPlatelet gp l lb / l l l aPlatelet gp l lb / l l l aPlatelet gp l lb / l l l aPlatelet gp l lb / l l l aPlatelet gp l lb / l l l aPlatelet gp l lb / l l l aPlatelet gp l lb / l l l aPlatelet gpl lb/ l I laPlatelet gp l lb / l l l aPlatelet gp l lb / l l l aPlatelet gpllla and

gpll la-likePlatelet gpllla and

gpll la-likePlatelet gpllla and

gpllla-likePlatelet gpllla and

gpllla-likePlatelet CD9Platelet CD9Platelet CD9Platelet CD5-likePlatelet CD36Platelet gp 150Platelet p24Platelet unclustered

(gplb-like)Platelet unclusteredPlatelet unclusteredPlatelet unclusteredFactor-8-related antigenFibrinogenMonocyte/macrophageMonocyte/macrophage

—, no reaction.

Source

HiraiwaMcMichaelZolaMcMichaelAndoBrochierHoggAndoAndoHoggAndoHiraiwaBrochierHoggVilellaVilellaKnappVilellaKnappVilellaVilellaTetterooZolaVilellaKnapp

Lansdorp

Ravoet

Vilella

JohnsonZolaBouchierYokoyamaPilkingtonBrochierGarridoRieber

BrochierBreardBreardMasonBehringMcGeeMason

Osteoclast reactivityin foetal and adult

bone specimens

—-----------------------

+ +

+ +

+ +

+ +

+++-----

----

+ ++ ++ +

and platelets were also strongly stained, as were oc-casional scattered mononuclear cells. In sections of thefoetal bone metaphysis, osteoclasts showed strongcytoplasmic staining (Fig. 2). A few mononuclear cellsin the intertrabecular stroma were also stained butmononuclear cells lining the bone trabeculae were

almost entirely unstained. Fibrinogen was largely local-ized to the membrane of osteoclasts in foetal boneimprints (Fig. 1C). Megakaryocytes, platelets and afew scattered mononuclear cells were also positive forfibrinogen. Most background mononuclear cells, how-ever, were negative for fibrinogen.

Platelet antigens and fibrinogen on osteoclasts 117

B

W;

. f1

118 N. A. Athanasou et al.

In cryostat sections of Pagetic bone, osteoclasts and afew stromal cells were stained strongly with CD9 andgpllla antibodies (Fig. 3). Osteoclast-like giant cellsand scattered mononuclear stromal cells in giant celltumour of bone also showed strong cytoplasmic stain-ing. Both giant cells and the mononuclear cells thatwere stained by these antibodies showed no evidence ofmitotic activity or nuclear and cellular atypia. Incryostat sections of human foetal and adult bonespecimens, it was not possible to localize fibrinogen toany specific cell type due to heavy background staining.

Localization of macrophage antigens in osteoclasts,megakaryocytes and platelets. Results of the immuno-histochemical staining of the foetal and adult bonepreparations with the two anti-macrophage antibodies,EBM/11 and Yl/82a, are also shown in Table 1. Bothantibodies showed strong cytoplasmic staining of osteo-clasts in foetal and Pagetic bone as well as osteoclast-like giant cells in giant cell tumours of bone. Inaddition, in foetal bone imprints EBM/l l and Yl/82astrongly stained scattered background mononuclear

Fig. 1. Human metaphyseal bone imprints stainedimmunohistochemically by an indirect immunoperoxidasemethod with: A. C17 (anti-gpllla) showing strongmembrane staining of an osteoclast. Two smallermegakaryocytes (arrowed) and mononuclear cells showdiffuse cytoplasmic staining. The nuclei of themegakaryocytes are partially obscured by the histochemicalreaction product. X336. B. BU-16 (anti-CD9) showingmembrane staining of an osteoclast (top) and diffusecytoplasmic staining of a megakaryocyte (bottom) andplatelets (arrowheads). X336. C. Anti-fibrinogen showingstrong membrane staining of the osteoclast membrane.X336. D. AN51 (anti-gplb) showing staining ofmegakaryocytes (arrowed) and platelets but osteoclast(centre) is unstained. X210.

Fig. 2. Human foetal metaphyseal bonesection stained with 96-2C1 (anti-gpllla)showing strong diffuse staining of anosteoclast (arrowed) lying against a bonetrabecula. Immunoperoxidase; X336.

cells and megakaryocytes and weakly reacted withplatelets (Fig. 4). In buffy coat smears, these anti-bodies strongly reacted with monocytes and weaklystained platelets.

Discussion

The antigenic phenotype of the human osteoclast,which is known to be derived from a circulatingmononuclear precursor cell of bone marrow origin, iscontroversial. Horton et al. (1984, 1985«,6) havereported that human osteoclasts do not express macro-phage or platelet antigens and argued that this favoursthe origin of the osteoclast from a stem cell distinctfrom the pluripotential haemopoietic stem cell. I low-ever, there are now several reports of monoclonalantibodies that recognize cell surface antigens onosteoclasts and other myeloid cells, notably macro-phages (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 expressmonocyte/macrophage and megakaryocyte/platelet-associated antigens. This finding is consistent with, butnot proof of, a common origin or differentiationpathway for monocytes, megakaryocytes and osteo-clasts from a single stem cell, i.e. the pluripotentialhaemopoietic stem cell. Human haemopoietic stemcells have been shown to express glycoprotein Ilia(Kraser et al. 1986). Human blood monocytes andplatelets also share cell surface components (Burck-hardt 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

Fig. 3. Section of Pagetic boneshowing two osteoclasts lying inHow ship's lacunae. Both arestained by C17 (anti-gpllla).Immunoperoxidase; X336.

• * #

However, immunohistology has also revealed CD9 andgpllla antigens on non-myeloid tissues; so, the findingthat these cell types have common surface antigensdoes not permit a definite conclusion regarding theirorigin.

The finding of fibrinogen and platelet antigens onosteoclasts is of great interest as it is possible that theirpresence may signal a common process of differen-tiation or function by these cell types, gpllla anti-bodies are known to inhibit platelet aggregation andfibrinogen binding by platelets (Foon & Todd, 1986),whilst CD9 antibodies stimulate platelet aggregationand induce fibrinogen binding (Horton & Hogg, 1987).Analogously, in osteoclasts, the gpllla antigen maysimilarly influence fibrinogen binding or even form thesite of binding of fibrinogen by osteoclasts. In contrast,

Fig. 4. Human metaphysealbone imprint stained withEBM/ll (anti-macrophage)showing diffuse cytoplasmicstaining of two osteoclasts and amegakaryocyte (arrowed).Immunoperoxidase; X336.

the 24xlO3/Wr protein recognized by the CD9 anti-bodies may have the opposite effect on osteoclastfibrinogen binding.

The role of surface fibrinogen on osteoclasts isunknown, but again, by analogy with platelets, it ispossible that it is necessary for aggregation or cellularadhesion of mononuclear osteoclast precursors prior tofusion and the formation of multinuclear osteoclasts.Osteoclasts may also require fibrinogen to bind to thebone surface. Another possibility involves plasminogenactivator (PA), which is known to be secreted byosteoblasts (Hamilton et al. 1985). The PA/plasminsystem has been implicated in mechanisms of connec-tive 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.

(Chambers, 1985). Osteoclasts, like platelets, arehighly motile cells that change shape and activity whenthey are stimulated or inhibited (Chambers & Magnus,1982; Chambers, 1985). It is possible that PA is one ofthese stimulatory factors and that it may influenceosteoclast fibrinogen binding directly or indirectlythrough one of the platelet antigens present on osteo-clasts.

This work was supported by the Arthritis and RheumatismCouncil. We thank Dr C. G. Woods and Dr. J. Keeling forsupplying the bone specimens, Miss L. Watts for typing themanuscript 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 megakaryocyteantigens. J . Path. 150, 239-246.

ATHANASOU, N. A., QUINN, J. & MCGEE, J. O'D. (1987).

Leucocyte common antigen is present on osteoclasts. J.Path. 153, 121-126.

BAI, Y., DURBIN, H. & HOGG, N. (1984). Monoclonal

antibodies specific for platelet glycoproteins react withhuman monocytes. Blood 64, 139—146.

BANCROFT, J. D. & STEVENS, A. (1975). Theoiy and

Practice of Histological Techniques, pp. 258-294.Edinburgh: Churchill Livingstone.

BECKSTEAD, J. H., STENBERG, P. E., MCEVER, R. P.,

SHUMAN, M. A. & BAINTON, D. F. (1986).

Immunochemical localisation of membrane and alphagranule proteins in human megakaryocytes: Applicationsto plastic embedded bone marrow biopsy specimens.Blood 67, 285-293.

BEERS, W. H. (1975). Follicular plasminogen andplasminogen activator and the effect of plasmin onovarian follicle wall. Cell 6, 379-386.

BURCKHARDT, J. J., KERR ANDERSON, W. H., KEARNEY, J.F. & COOPER, M. D. (1982). Human blood monocytesand platelets share a cell surface component. Blood 60,767-771.

CHAMBERS, T. J. (1980). The cellular basis of boneresorption. Clin. Orthop. rel. Res. 151, 283-293.

CHAMBERS, T. J. (1985). The pathobiology of theosteoclast. J . clin. Path. 38, 241-252.

CHAMBERS, T. J. & MAGNUS, C. J. (1982). Calcitonin

alters behaviour of isolated osteoclasts. J. Path. 136,27-39.

COLVIN, R. B. & DVORAK, H. F. (1975). Fibrinogen/fibrin

on the surface of macrophages: Detection, distribution,binding requirements and possible role in macrophageadherence phenomena. J. exp. Med. 142, 1377-1390.

DACIE, J. V. & LEWIS, S. M. (1975). Practical

Haematology, pp. 77-78. Edinburgh: ChurchillLivingstone.

FOON, K. A. & TODD, R. F. (1986). Immunologic

classification of leukemia and lymphoma. Blood 68,1-31.

GATTER, K. C , FAUNI, B. & MASON, D. Y. (1984). The

use of monoclonal antibodies in histopathologicaldiagnosis. In Recent Advances in Histopathology, no. 12(ed. P. P. Anthony & R. N. M. MacSween), pp. 35-67.Edinburgh: Churchill Livingstone.

GOTHLIN, G. & ERICSSON, J. L. E. (1976). The osteoclast.

Clin. Orthop. 120, 201-231.HAMILTON, J. A., LINGELBACH, S., PARTRIDGE, N. C. &

MARTIN, T. J. (1985). Regulation of plasminogenactivator production by bone-resorbing hormones innormal and malignant osteoblasts. Endocrinology 116,2186-2191.

HOGG, N. (1983). Human monocytes are associated withthe formation of fibrin. J. exp. Med. 157, 473-485.

HORTON, M. A. (1986). Expression of platelet glycoproteinIlia by human osteoclasts. Blood 68, 595.

HORTON, M. A. & HOGG, N. (1987). Platelet antigens: newand previously defined clusters. In Leucocyte Typing III(ed. A. McMichael et ai), pp. 733-746. OxfordUniversity Press.

HORTON, M. A., LEWIS, D., MCNULTY, K., PRINGLE, J.

A. S. & CHAMBERS, T. J. (1985«). Monoclonalantibodies to osteoclastomas (Giant Cell BoneTumours): Definition of osteoclast-specific cellularantigens. Cancer Res. 45, 5663-5669.

HORTON, M. A., LEWIS, D., MCNULTY, K., PRINGLE, J.

A. S. & CHAMBERS, T. J. (19856). Human fetalosteoclasts fail to express macrophage antigens. Br. J.exp. Path. 66, 103-108.

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 marrow-derivedtypes .J. Path. 141, 281-294.

KRASER, J. K., LEAHY, M. F. & BERRIDGE, M. V. (1986).

Expression of antigens of the platelet glycoprotein11 b / Ilia complex on human hematopoietic stem cells.Blood 68, 762-769.

LACK, C. W. & ROGERS, H. J. (1958). Action of plasminon cartilage. Nature, Lond. 182, 948-949.

LOUTIT, J. F. & NISBET, N. W. (1982). The origin of the

osteoclast. Iinmunobiology 161, 193-203.MARKS, S. C. (1983). The origin of osteoclasts. J . oral

Path. 12, 226-256.NUWEIDE, P. J., VRIGHERD-HAMMERS, T., MULDER, R. J.

P. & BLOK, J. (1985). Cell surface antigens on osteoclastsand related cells in the quail studied with monoclonalantibodies. Histocheniistry 83, 315-324.

OURSLER, M. J., BELL, L. V., CLEVINGER, B. & OSDOBY,

P. (1985). Identification of osteoclast-specific monoclonalantibodies. J . Cell Biol. 100, 1592-1600.

Platelet antigens and fibrinogen on osteoclasts 121

SHERMAN, L. A. & LEE, J. (1977). Specific binding of macrophages and osteoclasts in embryonic rat bone.soluble fibrin to macrophages. J . exp. Med. 145, 76-85. Calc. Tiss. Int. 39, 263-266.

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, 231-240. Haemal. 52, 173-180.

SMINIA, T. & DIJKSTRA, C. D. (1986). The origin ofosteoclasts: an immunohistochemical study on (Received 12 August 1987 - Accepted 12 October 1987)

122 N. A. Athanasou et al.