Calcif Tissue Int (1991) 49:317-320

Calcified Tissue International 9 1991 Springer-Verlag New York Inc.

Production and Characterization of New Monoclonal Antibodies to Human Osteoclasts P. N. Nelson, 1 J. A. S. Pringle, 2 and T. J. Chambers 1 1Department of Histopathology, St. George's Hospital Medical School, Tooting, London SWl7 ORE U.K., U.K., and 2Department of Morbid Anatomy, Institute of Orthopaedics, Royal National Orthopaedic Hospital, Stanmore, Middlesex, U.K. Received July 2, 1990, and in revised form December 18, 1990

Summary. Two monoclonal antibodies raised against human osteoclastoma were found to show antiosteoclastic activity on frozen sections of tumor. Immunoreactivity was localized on the membrane surface. These antibodies exhibited no activity against tissue macrophages and human visceral tissue except kidney, where they stained tubules but not glomeruli. In addition, no activity was observed against rabbit or rat osteoclasts, suggesting that they might react with unique epitopes on human osteoclasts.

Table 1. lsotype characterization of McAbs Hybridoma

Resultant designated clone/McAb

Mouse isotype

PNG211D PNG312G

211D 312G

~/lk ~/3k

Materials and Methods Key words: Osteoclasts - Monoclonal antibodies.

The osteoclast (OC)serves as the major effector cell of bone resorption [1] and is involved in skeletal morphogenesis and remodeling and calcium homeostasis [2]. Its origin is controversial [3], although evidence suggests that it is bone marrow derived [4]. However, the exact nature of its mononuclear precursor cell requires clarification as does its relationship with other hemopoietic cells, particularly macrophages. Phenotypic characterization of OCs in species such as chicken [5] and quail [6] has been difficult as only a limited number of reagents are available which identify cell surface specific markers. Similarly against human OCs, only two antibodies (23C6 and 13C2) have been fully characterized [7] which also cross-react with OCs of other mammalian species. Clearly, more reagents are needed that recognize novel antigenic determinants on human OCs that may prove useful in lineage and functional studies and clinical diagnosis. The hybridoma technique [8] offers the potential to provide an unlimited supply of monoclonal antibody (McAb) which invariably recognizes a single antigenic determinant. Thus, McAbs have proved useful probes for elucidating the antigenic phenotype of ceils [9]. Criteria for producing McAbs include good hybridization and screening techniques plus the utilization of a suitable immunogen. Osteoclastomas (OCL), giant cell tumors of bone, provide a rich source of functionally normal, non-neoplastic OCs [10, 11] that could be used as immunogen. In this study we report on the production and characterization of two McAbs (211D and 312G) derived by hybridizing spleen cells of OCL immunized mice with plasmacytoma cells. These McAbs showed specificity for membrane determinants on OCs present in frozen sections of OCL and human fetal bone. In addition, these antibodies showed no reactivity with rabbit or rat OCs, suggesting that they recognized unique epitopes on human osteoclasts.

Offprint requests to: P. N. Nelson

OCL tumor was confirmed pathologically using standard diagnostic criteria [12]. OCs were also isolated from human fetal long bone (obtained from 18-week therapeutic abortion), rabbit, and rat long bone. A range of normal adult tissue was obtained from surgical specimens and postmortems; these included blood, spleen, tonsil, lung, liver, kidney, and skin. Tissue to be used for frozen section immunohistology was snap-frozen in OCT embedding medium and stored in liquid nitrogen. Cryostat sections (5-8 ~m) were then prepared, or alternatively, tissue imprints were made by lightly dabbing material onto 4 • 1 cm multispot glass microscope slides. Frozen sections and tissue imprints were air dried after preparation, fixed in acetone, dried, and stored at - 70~ OCL was also fixed in buffered 10% formalin, paraffin-embedded and dewaxed using standard procedures [13]. Peripheral blood and osteosarcoma cells (UMR 106.06 and Saos-2) were prepared as cytospins. For immunoperoxidase staining, tissue sections were thawed and blocked in 0.05 M Tris buffered saline (TBS) pH 7.6 containing 3% bovine serum albumen. Sections were then incubated with primary antibody (100 OJ hybridoma supernatant) and optimaly diluted horse radish peroxidase conjugated polyvalent anti-mouse immunoglobulin (Dakopatts, High Wycombe, Bucks, UK) for 30 minutes respectively in a moist chamber at room temperature. Between incubations, sections were washed in TBS and excess medium was removed. Sections were then developed for 8 minutes in TBS containing diaminobenzidine hydrochloride as chromogen (0.5 mg/ml, Sigma, Poole, Dorset, UK), 0.05% hydrogen peroxide, and 0.1% sodium azide. After rinsing in TBS, sections were counterstained with Mayer's hematoxylin, dehydrated in graded alcohols, and mounted. To develop McAbs, female BALB/c mice were immunized intraperitoneally (i.p.) with OCL cell suspension (in saline) derived from cryopreserved tumor. Mice were boosted by repetitive inoculation every second week until test-bleeds showed high levels of antibody. A final boost (i.p.) was given 3 days prior to fusion whereupon spleen cells from immunized mice were fused with X63.Ag8 plasmacytoma cells using the method of Galfre et al. [14]. Fused cells were plated out into 96-well Linbro plates (Flow, Rickmansworth, Herts., UK) and selected in RPMI-1640 medium containing hypoxanthine, aminopterin, and thymidine, supplemented with 20% fetal calf serum, penicillin/streptomycin (Sigma). On day 20, hybridomas were screened for anti-OC activity using OCL tumor imprints. Selected hybridomas were cloned by limiting dilution, and the immunoglobulin subclass was determined by hemagglutination using a polyclonai isotype kit (Serotec, Kidlington, Oxford, UK), For characterizing McAb epitopes, frozen sections of OCL were

318

P.N. Nelson et al.: Monoclonal Antibodies to Human Osteoclasts

Fig. 1. Frozen section of OCL incubated with (a) 211D, (b) 312G. LM x 250.

initially treated with the following enzymes in sodium phosphate buffer pH 7.0: pronase, 50 ixg/ml (Koch-Light, Haverhill, Suffolk, UK); protease, 50 ixg/ml (Sigma); N-glycosidase F, 1 U/ml (Boehringer, Lewes, Sussex, UK); O-glycosidase 10 mU/ml (Boehringer). Results Of 111 hybridomas screened for antibody activity against t i s s u e i m p r i n t s of O C L , t w o t e r m e d P N G 2 l l D and PNG312G show specificity for OC. These hybridomas were cloned, designated 211D and 312G, and isotyped 3,1k and ~/3k, respectively (Table I). Tissue culture supernatants of 211D and 312G reacted with membrane determinants of multiculeated OCs (Fig. 1), although staining was not localized at the bone-apposed surface (Fig. 2). The epitopes recognized by 211D and 312G were not effected by glycosides, but were abrogated by proteases (Table 2). The reactivity of these antibodies was further evaluated on frozen sections of fetal OC and other cells from human and nonhuman species and compared with anti-OC McAbs (23C6 and 13C2) and antimacrophage McAb, EBM11 (Table 3). Antibody reactivity was graded strong, moderate, or weak in comparison to positive control supernatant W6/32 (anti-HLA, Dakopatts) and negative control (X63.Ag8 culture supernatant). Both 211D and 312G exhibited moderate staining of all multinucleated cells (OC) and some mononuclear cells from fetal bone. H o w e v e r , neither antibody showed activity on formalin-fixed OCL sections. Staining of serial frozen sections of OCL with 211D, 312G, 23C6, and 13C2 revealed 100% co-localization on osteoclasts. In contrast to 23C6 and 13C2, McAbs 211D and 312G showed no reactivity with rabbit OC. The latter McAbs also failed to react with Saos-2 and U M R osteosarcoma cell lines and showed no activity against tissue macrophages identified by McAb E B M l l . Of tissues examined, 211D and 312G were found to cross-react soley with kidney tubules (Fig. 3), whereas 23C6 and 13C2 showed activity against both tubules

Fig. 2. Section displaying lack of staining (using 211D) at boneopposed surface (arrow) of OC. LM x 250. and glomeruli. In particular, 211D and 312G stained epithelial cells of both proximal and distal convoluted tubules. Discussion Phenotypic characterization of the osteoclast has been lim-

P. N. Nelson et al.: Monoclonal Antibodies to Human Osteoclasts

319

Table 2. Effect of proteases and glycosidases on McAb reactivity Treated OCL

McAb

Untreated OCL (control)

Pronase

Protease

N-Glycosidase

O-Glycosidase

2lID 312G 23C6 13C2

+ + + +

-

-

+ + + +

+ + + +

+ indicates positive staining of osteoclasts, - no staining observed

Table 3. Reactivity of McAbs with osteoclasts and other cells in human and nonhuman species Tissue (frozen section) Human osteoclasts (osteoclastoma/fetal bone) Osteosarcomas (saos-2/UMR 1 0 6 . 0 6 ) Blood Skin Breast Tonsil Lung Liver Kidney Rabbit osteoclast Rat osteoclast OCL (formalin fixed)

McAbs 211D

312G

23C6

13C2

++

++

+++

+

. . . . . . . ++ (tubules) . -

. . . . . . .

. . . . . . . + + (tubules) -

.

.

. . . . . . . ++ (glomerulii & tubules) + .

EBMll

++ _}_ _} a .~b q_ q_ _{ a

+++a q- q- ..b a q_ q_ q_a

++ (glomeruli & tubules) +

+++a

-

Human tissue: results graded + (weak), + + (moderate), + + + (strong) compared with positive control W6/32 (anti-HLA) or -ve compared with supernatant from X63.Ag8 plasmacytoma cells a Mononuclear phagocytes; bconnective tissue

ited due to the sparsity of suitable reagents. In this study we describe the development of two McAbs which appear to recognize novel epitopes on human osteoclasts. Previous studies [7, 11] have shown that osteoclasts from OCL are antigenically and functionally normal. The data from this investigation appears to be consistent with this observation as 211D and 312G showed no discrimination between OC from osteoclastoma or fetal bone. Presumably the difference in staining intensity exhibited by these antibodies compared with 23C6 and 13C2 reflected differences in functional affinity for target epitopes. Evidently the latter were not effected by glycosidases which cleaved N- or O-linked carbohydrate moieties. H o w e v e r , proteases markedly reduced the binding of all four McAbs suggesting that antigenic determinants were protein in nature. 211D and 312G also showed no activity against osteoblast-like cells as substantiated by their lack of reactivity with human and rat osteosarcomas. The epitopes recognized by these antibodies were not expressed on rabbit or rat osteoclasts. This pattern of reactivity was in contrast to 23C6 and 13C2 which detected cross-reactive epitopes on human and rabbit osteoclasts considered functionally important for regulation [15]. Because 23C6, 13C2 and 2 l I D , 312G co-localize on osteoclast membrane, the different reactivity profiles of these McAb sets suggest that they recognize disparate epitopes. The epitopes recognized by 211D and 312G were not expressed on tissue macrophages. This result provides further evidence for dissimilarity in phenotype between OC and

macrophages and their precursors. Unfortunately, both M c A b s failed to r eact against formalin-fixed paraffinembedded sections of osteoclastoma, indicating that their respective epitopes were denatured by this fixation process. Similarly, Western-blot analysis [16] of211D and 312G failed to reveal distinct bands on a plasma preparation of O C L (data not shown) suggesting that target epitopes were conformational. The lack of reactivity of 211D and 312G with other human tissues followed a pattern similar to 23C6 and 13C2, although against kidney the latter group reacted with both tubules and glomeruli. In contrast, 211D and 312G stained epithelial cells of proximal and distal convoluted tubules only. The reactivity of these McAbs may be due to the presence of crossreactive determinants of possible antibody multispecificity [17, 18]. It has been established that calcitonin receptors are expressed on osteoclasts [19] and exist in the kidney [20]. In particular, calcitoniu receptors in the latter tissue appear to be found in the distal convoluted tubules and cortical and upper medullary regions of the ascending loop of Henle [21]. Hence, the reactivity profile of 211D and 312G for both proximal and distal c o n v o l u t e d tubules suggests that these McAbs are unlikely to recognize the calcitonin receptor. In summary, the antibodies p r o d u c e d in this study showed specificity for human osteoclasts. The difference in reactivity exhibited by these McAbs compared with 23C6 and 13C2 suggests that they recognize novel membrane antigenic determinants.

320

P. N. Nelson et al.: Monoclonal Antibodies to Human Osteoclasts

7.

8. 9.

10.

11. 12. 13.

14. 15.

Fig. 3. Frozen section of human kidney incubated with 211D (312G shows a similar staining pattern). LM x 250.

References

16.

17. 18.

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Cell surface antigens on osteoclasts and related cells in the quail studied with monoclonal antibodies. Histochemistry 83:315-324 Horton MA, Lewis D, McNulty K, Pringle JAS, Chambers TJ (1985) Monoclonal antibodies to osteoclastomas (Giant cell bone tumors): definition of osteoclast-specific cellular antigens. Cancer Res 45:5663-5669 Kohler G, Milstein C (1975) Continuous culture of fused cells secreting specific antibody. Nature 47:329 Stein H, Bonk A, Tolksdorf G, Lennert K, Rodt H, Gerdes J (1980) Immunohistologic analysis of the organization of normal lymphoid tissue and non-Hodgkin's lymphoma. J Histochem Cytochem 28:746 Horton MA, Rimmer EF, Lewis D, Pringle JAS, Fuller K, Chambers TJ (1984) Cell surface characterization of the human osteoclast: phenotypic relationship to other bone marrowderived cell types. J Pathol 144:281-294 Chambers TJ, Fuller K, McSheehy PMJ, Pringle JAS (1985) The effects of calcium-regulating hormones on bone resorption by isolated human osteoclastoma cells. J Pathol 145:297-305 Jaffe HL, Lichtenstein L, Portis RB (1940) Giant cell tumor of bone: its pathological appearance, grading, supposed variants, and treatment. Arch Pathol 30:993-1031 Chilosi M, Gilioli E, Lestani M, Menestrina F, Fiore-Donati L (1988) Immunohistochemical characterization of osteoclasts and osteoclast-like cells with monoclonal antibody MB 1 on paraffin-embedded tissues. J Pathol 156;251-254 Galfre G, Howe SC, Milstein C, Butcher GW, Howard JC (1977) Antibodies to major bistocompatibility antigens produced by hybrid cells. Nature 266:550 Horton MA, Chambers TJ (1986) Human osteoclast-specific antigens are expressed by osteoclasts in a wide range of nonhuman species. Br J Exp Pathol 67:95-104 Burnette W (1981) "Western Blotting." Electrophoretic transfer of proteins from dodecyl sulphate-polyacrylamide gels to unmodified nitrocellulose and radioiodinated protein A. Anal biochem 112:195-203 Gosh S, Campbell AM (1986) Multispecific monoclonal antibodies. Immun Today 7:217-222. Nelson PN, Fletcher SM, de Lange GG, van Leeuwen AM, Goodall M, Jefferis R (1990) Evaluation of monoclonal antibodies with putative specificity for human IgG allotypes. Vox Sang 59:190-197 Nicholson GC, Moseley JM, Sexton PM, Mendelsohn FAO, Martin TJ (1986) Abundant calcitonin receptors in isolated rat osteoclasts. J Clin Invest 78:335-360 Marx S J, Woodard C, Aurbach GD (1972) Calcitonin receptors of kidney and bone. Science 178:99%1001 Chabardes D, Imbert-Teboul M, Montegut M, Clique A, Morel F (1976) Distribution of calcitonin-sensitive adenylate cyclase activity along the rabbit kidney tubule. Proc Natl Acad Sci USA 73:3608-3612