Peripheral giant cell granuloma: Evidence for osteoclastic differentiation Franc0 Bonetti, IUD, Giuseppe Pelosi, MD. Guido Martignoni, MD, Aldo Mombello, Giuseppe Zamboni, MD, Maurizio Pea, MD, Aldo Scarpa, MD, and Marco Chilosi, M Chem Sci, Verona, Italy


INSTITUTO DI ANATOMIA PATOLOGICA, UNIVERSITX DI VERONA Nine cases of peripheral giant cell granuloma of the oral cavity have been immunohistochemically analyzed to assess the nature of the giant cells. Giant cells were unreactive when tested with antibodies recognizing myelomonocytic and macrophage markers (lysozyme, MAC 387, HAM 58) but showed strong immunoreactivity with MBl, an antibody reactive with osteoclasts. It is concluded that giant cells characterizing giant cell granuloma exhibit a phenotype distinct from other giant cells found in sites of chronic inflammation and may be true osteoclasts. (ORAL SURC ORAL MED ORAL PATHOL 1990;70:471-5)

Peripheral giant cell granuloma (PGCG), is a relatively common lesion of the oral cavity (from 0.4% to 1.9% of oral surgical pathology material) and appears as a localized tumorlike enlargement of the gingiva.’ It occurs in all age groups but most frequently in young to middle-aged adults; women are affected twice as often as men.2g3 The maxilla and mandible are affected with equal frequency, the premolar/molar region being the area most often involved.4 A soft to firm mass forms in the gingiva, may push the teeth aside, and may erode the mandible. Microscopically, the lesion arises from, or is at least attached to, the periodontal ligament or the mucoperiosteum and consistsof a massof multinucleated giant cells in an active vascular stoma with plump spindle-shaped cells; occasionally, small amounts of neoformed bone are evident. Despite ultrastructural studies, the true nature of the giant cells in PGCG remains debatable.5Previous studies have shown that multinucleated giant cells present in sites of chronic inflammation exhibit features of mononuclear phagocytic differentation6 Flanagan and coworkers7 have provided evidence of the osteoclastic nature of giant cell granulomas of the jaws with functional and immunohistochemical studies of fresh-frozen tissues. Recently, we have demonstrated that MBl, a

Supported by the Associazione Italiana per la Ricerca sul Cancro, Milano, and Minister0 Pubblica Istruzione (60%), Roma. 7/14/19990

recently developed monoclonal antibody (mAb) reacting, on paraffin-embedded material, with mature B lymphocytes and a minority of T lymphocytes and monocytes,8 strongly reacts with fetal osteoclasts. Multinucleated cells of giant cell tumor of bone (so called osteoclastoma) and osteoclast-like cells found in rare casesof breast carcinoma are also reactive with MB1 ,9,lo In this report we demonstrate that the giant cells in PGCG exhibit an identical immunophenotype to that expressed by osteoclasts, whereas they are negative for antibodies that react with macrophages. Recognition of MB 1 immunoreactivity can be useful for osteoclast identification and can be obtained on retrospective and archival, conventionally processed material. MATERIAL


Nine formalin-fixed and paraffin-embedded gingival samples with the histologic diagnosis of PGCG were retrieved from the files of the Department of Pathology, University of Verona. Histochemical and immunohistochemical methods have been previously described in detail. *1Source and dilution of antibodies are reported in Table I. Briefly, 4 pm sectionsof theselesions were analyzed immunohistochemically with polyclonal and monoclonal (mAb) antibodies, which react, on paraffinembedded material, with monocytes and macrophages-lysozyme (polyclonal),12 HAM 56 (mAb),13 MAC 387 (mAb)14-and with MBI, a recently developed mAb (Biotest, Dreieich, West Germany) which reacts, on conventionally processedmaterial, 471



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Fig. 1. Paraffin sections of giant cell granuloma. A large number of osteoclast-like giant cells are evident in a vascular stroma rich with mononuclear cells. (Hematoxylin-eosin stain.)

Table I. Antibodies used: Sources and dilutions Antibody




Lysozyme HAM 56 MAC 387 MB1

DAK0 Enzo DAK0 Biotest

l/200 l/1000 l/40 l/l

Polyclonal Monoclonal Monoclonal Monoclonal

not only with a minority of B and T lymphocytes, but also with osteoclasts.8 Dewaxed sections were incubated in a moist chamber with primary antibody (60 minutes for lysozyme, HAM 56, MAC 387 using diluted antibodies; overnight with undiluted antibody for MBl); then (for mAbs) with biotinylated polyvalent sheepantimouse immunoglobulin (1 hour) and preformed avidin-biotinylated horseradish peroxidase complex (Amersham, Amersham, U.K.) or (for polyclonals) with PAP solution (1:200, DAKO, Copenhagen, Denmark) for 30 minutes. The final reaction product was produced by incubation with hydrogen peroxide 33 ‘-diaminobenzidine (Sigma Chemical Co., St. Louis, MO.) and Hz02 in TRIS buffer aspreviouslydescribed.l 5 The speciticity of reaction was verified by replacing the primary antibody with phosphate-bufferedsalinesolution and with normal rabbit polyclonal serum or with mouse monoclonalserum of comparabledilution.

RESULTS Histology

Microscopically, the lesions exhibited the characteristic features of PGCG. A large number of giant cells were evident in a vascular stroma of collagen fibers and plump spindle-shaped cells (Fig. 1). The multinucleated giant cells exhibited an abundant eosinophilic cytoplasm. The arrangement of the nuclei, filling the cells except for a zone of cytoplasm around the periphery, was similar to those of osteoclasts. The matrix consisted of spindle-shaped cells and a variable amount of collagen fibers with numerous capillary vessels.Giant cells were unevenly distributed. Newly formed bone tissue was observed in three of nine cases.All lesions were separated from the covering epithelium, normal or with acanthosis, by a narrow zone of fibrous tissue. lmmunohistochemistry

Immunohistochemical results are summarized in Table II. Positive staining was graded from + (weak) to +++ (strong), depending on the degree of immunoprecipitate density. The osteoclast-like giant cells were negative for monocyte-macrophage markers lysozyme, HAM 56, and MAC 387. On the other hand, they were clearly immunostained by MBl, with a diffuse granular product confined to the cytoplasm (Fig. 2). Spindle-shaped plump cells present in the

Peripheral giant cell granuloma

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Fig. 2. Same case as Fig. 1 stained with mAb MBl, immunoreactive with osteoclasts. Giant cells are strongly positive, whereas only rare mononuclear cells (possibly precursors) are stained.

lesions were invariably devoid of specific staining for MBl. A sparse population of monocytes and macrophages, morphologically distinguishable from osteoclast-like cells, was also present and variably immunostained by lysozyme, HAM 56, and MAC 387 (Fig. 3). - - DISCUSSION

PGCG is a lesion of unknown etiology, even if it is often associated with local irritation. At present it is generally agreed that it is a reactive, nonneoplastic lesion formed by granuloma-like tissue dominated by multinucleated giant cells.2*4 Polykarions of PGCG are often morphologically indistinguishable from other types of multinucleated giant cells, as those that form by fusion of mononuclear phagocytes at sites of chronic inflammation t6-18and immunophenotypic analysis can provide usef;l markers for detecting and characterizing giant cells with osteoclastic features. Giant cells with osteoclast morphology from the nine lesions examined in this study presented diffuse granular MB1 immunoreactivity, strong and confined to cytoplasm, which supports the characterization of these MBl-positive cells as osteoclasts.10 MB1 mAb recognizes three bands of 200,110, and 100 kd, when characterized by the immunoblot procedure.8At the moment the presenceof all of these

II. Immunoreactivity of osteoclast-like giant cells of giant cell granuloma


Case1 Case Case Case Case Case Case Case Case

2 3 4 5 6 7 8 9




+ ++ + ++ +t tt t+ ++ t



MAC 387






Myelomonocyte and macrophage markers (lysozyme, HAM 56, MAC 387) were absent in giant cells but present in scattered mononuclear cells in the stroma. MB1 (mAb reactive with osteoclasts)immunoreactivity was present with variable intensity in the giant cells of all casesand in occasional mononuclear cells.

molecules in osteoclastsof PGCG and their functional significance remain unknown. The negative immunoreaction of the same giant cells of PGCG for monocyte/macrophage markers, such as MAC 387, HAM 56, and lysozyme, allows for the distinction of these from other multinucleated giant cells of mononuclear phagocytic differentiation, the latter of which are a common feature in granulo-

matous reactions.6 There is controversy whether osteoclasts are de-

474 Bonetti et al.


Fig. 3. Samecaseas Fig. 1 stainedwith mAb MAC 387, immunoreactive with myelomonocytic antigen. Giant cc:lls are negative, whereas scattered mononuclear cells are positive.

rived by fusion of mononuclear macrophagesor have a lineage distinct from macrophages. Although osteoclasts are undoubtedly derived from a bone marrow stem ce11,19 different authors have provided evidence in functional, phenotypic, and transplantation studies that they constitute a cell line that is separate from the lineage of conventional blood cells.20,*I Other authors, however, have provided evidence of the presenceof common markers in both osteoclasts and hematopoietic cells.22-25 When all these data are taken together, it is concluded that both macrophages and osteoclasts share a common stem cell; however, osteoclasts acquire in their process of differentiation markers (such as MB1 immunoreactivity) that allow their clear distinction from macrophages. The activation and/or recruitment mechanism of osteoclastsin PGCG is unknown yet may be related to an abnormal proliferation and/or accumulation of osteoclasts,normally involved in resorbing deciduous teeth and alveolar bone during tooth eruption, because this lesion develops where such teeth have been replaced.26T 27 Further studies of the distribution of cells of the osteoclast lineage in such lesions, together with biochemical investigations on the factors inducing their local recruitment and/or activation, are still neededto understand the forming mechanism and, more generally, the functional significance of osteoclaststhat occur in this lesion.

We thank Mrs. Alessandra Molin for her skillful technical assistance. REFERENCES Cawson RA, Eveson JW. Oral pathology and diagonsis. London: William Heinemann Medical Books, 1987:10.5-10.6. 2. Giansanti JS, Waldron CA. Peripheral giant cell granuloma. Review of 720 cases.J Oral Sure 1969:27:787-91. 3. Lucas RB. Pathology of tumoursof the oral tissues. London: Churchill Livingstone, 1984:268-71. 4. Rosai J. Ackerman’s surgical pathology. St. Louis: The CV Mosby Company, 1989:191-2. 5. Sapp JP. Ultrastructure and histogenesis of peripheral giant cell reparative granuloma of the jaw. Cancer 1972;30:1119-29. 6. Papadimitrou JM, Van Bruggen I. Evidence that multinucleate giant cells are examples of mononuclear phagocytic differentiation. J Path01 1986;148:149-57. I. Flanagan AM, Tinkler SMB, Horton MA, Williams DM, Chambers TJ. The multinucleate cells in giant cell granulomas of the iaw are osteoclasts.Cancer 1988;62:1139-45. 8. Poppema S, Hollema H, Visser L, Vos H. Monoclonal antibodies (MTl.MTZ.MBl.MB2.MB3) reactive with leukocyte subsets in paraffin embedded material. Am J Path01 1987;127:418-29. 9. Chilosi M, Bonetti F, Menestrina F, Lestani M. Breast carcinoma with stromal multinucleated giant cells [Letter]. J Path01 1987;152:55-6. 10. Chilosi M, Gilioli E, Lestani M, Menestrina F, Fiore-Donati L. Immunohistochemical characterization of osteoclasts and osteoclast-like cells with monoclonal antibody MB1 on paraffin-embedded tissues. J Path01 1988;156:251-4. 11. Bonetti F, Chilosi M, Menstrina F, et al. Immunohistological analysis of Rosai-Dorfman histiocytosis: a diseaseof S- lOO+ CDl-histiocytes. Virchows Arch [B] 1987;411:129-35. 12. Mason DY, Taylor CR. The distribution of muramidase (lysozyme) in human tissues. J Clin Path01 1975;28:124-32. 13. Gown AM, Tsukada T, Ross R. Human atherosclerosis II. Immunocytochemical analysis of the cellular composition of 1.

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Volume 70 Number 4 human atheroscleroticlesions. Am J Pathol1986;125:191-207. 14. Brandtzaeg P, JonesDB, Flavell DJ, Fagerhol MK. MAC 387 antibody and detection of formalin-resistant myelomonocytic Ll antigen. J Clin Pathol 1988;41:963-70. 15. Chilosi M, Bonetti F, Iannucci A. Carcinogenic 3,3’-diaminobenzidine (DAB) [Letter]. Am J Clin Path01 1981;75:638. 16. Papadimitriou JM, Walters MN-I. Macrophage polykarya. CRC Crit Rev Toxic01 1979:6:21l-55. 17. Papadimitriou JM, Walters’MN-I. Macrophage polykarya. CRC Crit Rev Toxic01 1979;6:21l-55. 18. Chambers TJ. Multinucleate giant cells. J Path01 1978; 126:125-48. 19. Ash P, Loutit JF, Townsend MS. Osteoclasts derived from haematopoietic stem cells. Nature 1980;283:669-70. 20. Horton MA, Pringle JAS, Chambers TJ. Identification of osteoclasts with monoclonal antibodies. N Engl J Med 1985; 312:923-4. 21. Horton MA, Lewis D, McNulty K, Pringle JAS. Monoclonal antibodies to osteoclastomas(giant cell bone tumors). definition of osteoclast-specific antigens. Cancer Res 1985;45:5639. 22. Horton MA, Rimmer EF, Lewis D, Pringle JAS, Fuller K, Chambers TJ. Cell surface characterization of the human os-

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teoclast: phenotypic relationship to other bone marrow-derived cell types. J Path01 1984;144:281-94. Athanasou NA, Heryet A, Quinn J, Gatter KC, Mason DY, McGee JO. Osteoclasts contain macrophage and megakaryocyte antigens. J Path01 1986;150:239-46. Athanasou NA, Quinn J, McGee JO. Leucocyte common antigen is present on osteoclasts.J Path01 1987;153:121-6. Regezi JA, Zarbo RJ, Lloyd RV. Muramidase, alpha-l antitrypsin, alpha-l antichymotrypsin, and S-100 protein immunoreactivity in giant cell lesions. Cancer 1987;59:64-8. Markus SC Jr, Cahill DR. The ultrastructure of alveolar bone during tooth eruption. Am J Anat 1986;177:427-34. Markus SC Jr, Grolman M-L. Tartrate-reistant acid phosphatase in mononuclear and multinuclear cell during the bone resorption of tooth eruption. J Histochem Cytochem 1987; 35:1227-30.

Reprint requests to: Dr. Franc0 Bonetti lstituto di Anatomia Patologica Policlinico di B. Roma 37234 Verona, Italy

Peripheral giant cell granuloma: evidence for osteoclastic differentiation.

Nine cases of peripheral giant cell granuloma of the oral cavity have been immunohistochemically analyzed to assess the nature of the giant cells. Gia...
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