JOURNAL OF BONE AND MINERAL RESEARCH Volume 6, Number 10, 1991 Mary Ann Lieberl, Inc., Publishers
Inhibition of Bone Resorption by a Monoclonal Antibody That Reacts with a 150 kD Membrane Protein in Chicken Osteoclasts TEUVO A. HENTUNEN, PAIVI T. LAKKAKORPI, T I M 0 RAUTIALA, and H . KALERVO VAANANEN
ABSTRACT Bone resorption is a multistep process that includes the maturation of osteoclast precursors, the special attachment of fully differentiated osteoclasts to mineralized bone surface, and the dissolution of inorganic mineral, as well as the breakdown of organic matrix. We have produced a large panel of monoclonal antibodies directed against chicken osteoclasts to obtain specific probes for studying the function of osteoclasts. One of our antibodies, K20, inhibited bone resorption of isolated osteoclasts almost completely. Several pieces of evidence suggested that the antigen detected by this antibody was located in the plasma membrane of the osteoclast. In western blot analysis K20 antibody specifically recognized a 150 kD protein in the medullary bone microsome fraction under reducing and nonreducing conditions. In addition to osteoclasts and some bone and bone marrow mononuclear cells, a positive immunoreaction was seen in the kidney tubules. These data suggest that monoclonal antibody K20 reacts with an osteoclast surface antigen that i s functionally important in bone resorption.
INTRODUCTION
could be useful for developing specific antibodies against these special membrane domains. The membrane of the osteoclast that faces the bone surface consists of the clear ONOCLONAL ANTIBODIES are powerful tools in the studies on the characterization of functional cell sur- zone and the ruffled border areas. The former mediates face determinants and the differentiation of cells. Using the attachment of the cell to the bone matrix, and it is osteoclast-reactive monoclonal antibodies it has been characterized by the specific organization of' cytoskeletal shown that vitronectin receptor in the membrane of osteo- proteins."+'O) The latter membrane region forms the actual clasts mediates the attachment of osteoclasts to bone.('.*] bone resorption site on the bone surface. The resorption lacunae have been shown to be acid by Also, the fact that Arg-Gly-Asp peptides inhibit osteoclasts cultured on bone slices supports the idea that inte- acridine orange accumulation experiments'""2) and direct grins play an important role in the attachment of osteoclasts micropuncture measurement^.''^.'^^ Omeprazole [S-methto the bone m a t r i ~ . ' ~Another .~) functional antigen in the oxy-24 [4-methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulfimembrane of osteoclasts identified and characterized by a ny1)-lH-bentzimidazole], a drug activated only in acid monoclonal antibody is 121F antigen.(5.6)This antigen is (pK, of 4.0)to form reactive sulfonamides and then atrelated to the manganese form of superoxide dismutase,(61 tacks accessible SH groups, has also been shown to inhibit bone r e ~ o r p t i o n . ( This ' ~ ~ ~finding ~~ further indicates the and it appears to be involved in bone r e ~ o r p t i o n . ' ~ ) To understand osteoclastic bone resorption it is impor- importance of acid compartments in resorption. In the ruftant to understand the different membrane structures of fled borders of osteoclasts there is an electrogenic vacuothe osteoclast. Resorbing osteoclasts show three distinct lar-type H+-ATPase that is responsible for pumping pro- ~ ~acid ) pH favor the cell membrane domains. Monoclonal antibody techniques tons into resorption l a c ~ n a . ( ' ~The
M
Department of Anatomy, University of Oulu, Finland.
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solubilization of the bone mineral, and it also seems to be important for osteoclastic lysosomal enzymes capable of collagen degradation. ( ' L ' ~ ) The plasma membrane domain facing away from bone (basolateral membrane) is rich in N ~ + , K + - A T P ~and S ~Ca2'-ATPase.(z4.25) '~~) Our aim is to study the differentiation and function of osteoclasts using specific monoclonal antibodies. In the previous study we demonstrated that osteoclasts and few mononuclear cells in bone, bone marrow, and blood share common antigens, suggesting the existence of osteoclast precursors also outside the bone.'*6)In the present study we describe how we screened by in vitro resorption assay a panel of monoclonal antibodies and found that one of the antibodies almost completely inhibited bone resorption. This K20 antibody was further characterized by immunohistochemistry and immunoblotting.
MATERIALS AND METHODS Antibodies The production of monoclonal antibodies used in this study has been described earlier. ( 2 6 ) They were raised against isolated chicken osteoclasts and selected primarily according to their reactivity with intact chicken osteoclasts.
In vitro resorption assay In vitro culture system for osteoclasts on bone slices was similar, as described earlier.('0~27-29) Osteoclasts were obtained from 4-6 days old white Leghorn chicks. Tibiae and femora were dissected out, placed in phosphate-buffered saline (PBS), and cleaned of any adherent soft connective tissue. Epiphyses of the bones were cut away and metaphyses were then cut longitudinally into two pieces. Bone marrow was removed, and metaphyseal area was scraped with a scalpel blade to release cells into Dulbecco's modified Eagle's medium (DMEM) buffered with 20 mM HEPES and containing 10% heat-inactivated fetal calf serum (FCS), pH 7.4 (GIBCO Laboratories, Grand Island, NY). Every step was performed at 4°C. Larger bone fragments were then allowed to settle for 5 minutes, and the supernatant was collected and centrifuged for 10 minutes at 200 x g. The cell pellet containing osteoclasts and other bone and bone marrow cells was suspended in the same medium as before (400 p1 cell suspension per chick). Bone slices (0.7 cm x 0.7 cm x 150 pm) were prepared from bovine cortical femur with a low-speed, water-cooled diamond saw (Gillings-Hamco; Hamco Machines, Inc., Rochester, NY). Slices were cleaned by ultrasonication for 30 s in distilled water before use. Approximately 40 pl chicken bone cell suspension was pipetted onto a slice in the multidish with 24 wells (Nunc a/s, Roskilde,
FIG. 1. (A) Chicken osteoclasts cultured for 2 days on the bovine bone slices and stained with toluidine blue ( x 422). (B) Resorption lacunae are also clearly seen by light microscopy after removing the cells ( x 219). (C) Nonresorbed bone ( x 273).
INHIBITION OF OSTEOCLASTIC BONE RESORPTION
Y, 1
1
FIG. 2. Effect of different monoclonal antibodies on bone resorption in vitro. Resorption activity is expressed as a ratio of the number of excavations to the number of multinucleated cells. The average number of multinucleated cells per bone slice is 85. Values are means f standard error of the mean (SEM) of 9-54 bone slices. Data are collected from 10 different assays. Significance of difference: * * p < 0.001; **p < 0.01; * p < 0.05.
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Denmark). After I h incubation 200 pl medium was added to each well. Cells were allowed to attach to the bone for 5 h at 37°C in a humidified atmosphere with 5% CO,. Nonadherent cells were removed by washing the bone slices twice with PBS. To each well was added 500 p1 DMEM10% FCS with or without a monoclonal antibody. Monoclonal antibodies were diluted either 1 :4 (culture medium of hybridomas) or 150 (ascitic fluid). In the controls there was the same amount of NS-1 culture medium or PBS instead of a monoclonal antibody. The cultures were kept in the incubator for an additional 2 days. At the end of the culture the medium was sucked away from the wells and the bone slices were fixed with 2% paraformaldehyde in PBS for 20 minutes at room temperature. Bone slices were washed first with PBS containing 0.15 M glycine and then with PBS. Cells on the bone slices were stained with 1 % toluidine blue in 1% sodium borate for 2 The number of multinucleated cells was counted by light microscope (Leitz Aristoplan, Wetzlar, Germany), after which the cells were removed from the bone slices by ultrasonication for 10 s in distilled water. Resorption pits were quantified by light microscope with phase-contrast optics. Resortion activity was expressed as a ratio of the number of
FIG. 3. Immunofluorescence staining of chicken bone and bone marrow cells with K20 antibody. (A) Intact osteoclasts and some mononuclear cells in suspension ( x 547) and (C) in frozen section of medullary bone ( x 190) are heavily stained. (B and D) Phase-contrast pictures of the same areas ( x 547 and x 190).
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resorption pits to the number of multinucleated cells on the bone slice.
photometer (UV-300; Shimadzu Corp., Tokyo, Japan). Acridine orange fluorescence was measured with excitation and emission wavelengths of 492 and 547 nm, respectively, as previously described.(20)The final dilution of K20 asIrnrnunohistochernistry citic fluid was either 1:lOOO or 150. Three different conIntact bone cells, bone marrow cells, and peritoneal trols were used: 10 nM bafilomycin (positive control for macrophages from white Leghorn chickens were isolated inhibition), PBS, or ascitic fluid of antibody K5 diluted as and stained in suspension as previously described.lz6) K20 (negative controls). Pieces from tissues were frozen immediately in liquid nitrogen after removing from animals. Sections (8 p ) were RESULTS cut using a cryostat microtome (Cryo-cut microtome) at -20°C. Unspecific binding in frozen sections was blocked Monoclonal antibodies used in this study were raised by incubating sections in Tris-buffered saline (TBS), pH against isolated chicken osteoclasts.(26)According to their 7.4. containing 5% bovine serum albumin (BSA) for 1 h at staining patterns antibodies recognized different epitopes room temperature. BSA was then replaced by primary in the osteoclast membrane. antibody (ascitic fluid, 1:100) diluted with TBS, pH 7.4, Bone cells on the bovine bone slices were stained with containing 0.5% BSA and 0.2% Triton X-100. Sections 1 Vo toluidine blue, and multinucleated cells were counted were incubated for 1 h at room temperature. Unbound by normal light microscope with phase-contrast optics antibody was washed twice with PBS for 15 minutes. Sec(Fig. 1A). After removing the cells from the bone slices by ondary antibody was rhodamine-conjugated rabbit antiultrasonication, resorption pits were clearly visible for mouse immunoglobulin (DAKOPATTS a/s, Glostrup, counting (Fig. 1B). Excavations were deep and sharply deDenmark) diluted 1: 100. Negative control sections were infined after a 2 day culture period. The K20 cell culture mecubated in the same way but omitting the primary antidium inhibited bone resorption up to 89% of control. body. Sections were incubated and washed as before. SecThree other antibodies (K27, K35, and K41) inhibited bone tions were examined by a light microscope (Leitz Aristoresorption clearly but not to the same extent as K20 plan, Wetzlar, Germany) equipped with appropriate filters and phase-contrast optics. Fluorescent and phase-contrast (Fig. 2). In immunohistochemistry the staining of osteoclasts images were photographed on Agfa Pan 400 film. with antibody K20 was strong, suggesting a high level of the antigen (Fig. 3). K20-labeled osteoclasts derived either Electrophoresis and irnrnunoblotting from 4- to 6-day-old chickens or egg-laying hens with the same extent. The antigen seemed to be distributed evenly Bone microsomes were prepared from medullary bone over the osteoclast. That antibody K20 stained intact of regularly laying hens as previously described.120)This osteoclasts when incubations were done in suspensions microsome fraction primarily consisted of osteoclast memover ice suggested that the epitope was located in the outer brane vesicles. A 12 pg sample of isolated bone microsurface of the osteoclast (Fig. 3A). Table 1 shows the imsomes was first fractioned in 9% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under REACTIVITY OF K20 nonreducing or reducing conditions,(J0)and the proteins TABLE1. IMMUNOHISTOLOGIC WITH DIFFERENT CHICKEN ANTIBODY were then transferred onto 0.45 pm pore size nitrocellulose CELLSAND TISSUES membrane (Schleicher & Schuell, Dassel, Germany) as previously described.(J” Nonspecific binding was blocked by Tissue and cells Antibody K2Oa incubating the membrane in TBS, pH 7.4, containing 3% BSA overnight at 4°C. The membrane was cut into strips Bone and incubated with the primary monoclonal antibody K20 Osteoclasts ++ (ascitic fluid) diluted with 0.5% BSA and 0.1 Vo Tween 20 Mononuclear cellsb ++ in TBS, pH 7.4 (1:100), for 1 h at room temperature. Bone marrow mononuclear cellsb + Strips were then washed three times with 0.5% BSA and Peritoneal macrophages 0.1% Tween 20 in TBS, pH 7.4, for 10 minutes at room Liver temperature. Bound antibody was detected by incubating Kidney + the strips in affinity-purified goat antimouse IgG horseProventriculus radish peroxidase conjugate (Bio-Rad Laboratories, RichSuprarenal glands mond, CA) at 1:3OOO dilution for 1 h at room temperature. Intestine The strips were washed in the same way as mentioned earlier, and the antigen-antibody-conjugates were visualStriated muscle ized with diaminobenzidine (DAB) (Sigma Chemical ComBrain pany, St. Louis, MO). Lung Aorta Spleen Proton transport measurement Proton transport by isolated bone cell membrane vesicles was assayed in a dual-beam, dual-wavelength spectro-
a + +, Strong reaction; +, weak reaction; -, no reaction. bFew, thus far unidentified, mononuclear cells.
INHIBITION OF OSTEOCLASTIC BONE RESORPTION munologic reactivity of antibody K20 with different chicken cells and tissues. In addition to osteoclasts and some bone- and bone marrow-derived mononuclear cells, positive immunoreaction was also seen in the frozen sections of kidney tubules. In kidney the apical membrane of a few cells, probably intercalated cells, were positively stained with antibody K20 (Fig. 4A and B). Peritoneal macrophages, liver, proventriculus, suprarenal glands, intestine, striated muscle, brain, lung, aorta, and spleen were not stained with K20 (Table I). The molecular weight of the antigen detected with antibody K20 was approximiately 150 kD under both reducing and nonreducing conditions when analyzed by immunoblotting (Fig. 5 ) . The sample giving strongest reaction in Western blot analysis was bone microsome fraction prepared from the medullary bone of regularly laying hens. The enrichment of the antigen in the bone cell membrane vesicle fraction further suggested that it was located in the plasma membrane of the osteoclast. Antibody K20 did not have any significant effect on proton transport by isolated bone cell membrane vesicles, although in some experiments a slight inhibition was observed (Fig. 6). Figure 6 also shows that the acidification of the bone cell-derived microsomes is ATP driven and that the addition of the proton-potassium uncoupler nigericin (Nig) rapidly dissipates the pH gradient. Moreover, 10 nM bafilomycin, a specific inhibitor of vacuolar-type H +-ATPase, almost completely blocked proton transport, demonstrating the existence of an active proton pump in our membrane vesicle preparations.
DISCUSSION We previously produced a panel of monoclonal antibodies against isolated and partially purified osteoclasts from chicken medullary bone.(z6)The bone resorption assay using freshly isolated mixed bone cell preparation on bone or dentine slices is today a widely used system for studying the effect of different agents on O S ~ ~ O C ~ ~Using S ~
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this assay system we found that the monoclonal antibody K20 almost completely inhibited the resorption activity of cultured osteoclasts. The effect of antibody K20 was obviously specific, since several other monoclonals with approximately the same IgG concentration and belonging to the same IgG class(26)were not able to inhibit bone resorption in this assay system. It is unlikely that antibodies directed against intracellular domains could inhibit bone resorption in our resorption assay system, although it is known that osteoclasts have a high phagocytotic capacity. Immunofluorescence stainings of intact bone cells at 4°C indicated that antibody K20 detected an epitope in the plasma membrane of the osteoclast. Western blot analysis showed that the membrane antigen in question was about I50 kD protein. The extensive screening of several other tissues by immunofluorescence staining revealed specific binding of K20 antibody also in kidney. The staining pattern in kidney was of special interest, since it fitted well into the distribution of intercalated cells in the kidney tubules. This suggested us to test whether K20 has any effect on proton transport of isolated osteoclast membrane vesicles, since osteoclasts and intercalated cells are known to have the same type of proton pump in their apical membanes.(I720) The result was that K20 antibody did not have any significant effect on proton transport. However, it must be kept in mind that the orientation of plasma membrane vesicles capable of proton transport must be inside-out, that is, reversed to in situ situation. Our results also suggested that K20 did not have, either, any significant effect on Ca”-stimulated ATPase activity of isolated bone microsonies (data not shown). Two research groups have reported to have monoclonal antibodies that inhibited osteoclastic bone resorption in vitro. Chambers et aI.(’)screened a panel of human osteoclastoma-reactive monoclonal antibodies and found that some of them inhibited bone resorption. Later those antibodies were shown to recognize vitronectin receptor (VNR) in osteoclasts.(2~321 The molecular size of VNR in osteoS clasts . ~ ~and ~ ~a monkey ~ ~ - ~ kidney ~ ) cell line (Vero) has been re-
FIG. 4. (A and B) Frozen sections of chicken kidney recognized by K20 antibody and visualized with immunofluorescence staining ( x 133 and x 266). In kidney tubules, the apical membranes of certain cells, probably intercalated cells, are stained. (C) Control staining without the primary antibody ( x 167).
HENTUNEN ET AL. ported to be 140 kD (for the a chain) and 85 kD (for the /3 chain) under nonreducing conditions. Reducing conditions resulted in a 120/100 kD dimer and a low-molecular-mass A B C peptide of 30 kD size.(2)Recently, Oursler and Osdoby(s' described a monoclonal antibody (121F) that detected a kD 150 kD protein in the plasma membrane of the osteoclastt6)and also inhibited bone resorption.'') Under nonre205 ducing conditions the molecular mass of the antigen was reported to be above 200 kD.(331This 121F antigen has 116 97 been suggested to be related to the manganese form of superoxide dismutase.'6) It is not known how this enzyme 66 is associated with bone resorption, but it seems that oxygen-derived free radicals, particularly superoxide anion, 45 are involved in the formation and activation of osteoc l a s t ~ . ' The ~ ~ ) 121F antigen was expressed either in osteoclasts or bone marrow-derived giant cells, not in the bone marrow mononuclear cells. These results suggest that our K20 antibody detects some other protein than VNR or 121F antigen. FIG. 5. Western blot analysis of chicken bone microTo summarize, we have produced a monoclonal antisomes using monoclonal antibody K20 revealed a single protein band of about 150 kD under nonreducing (B) or body that detects a functionally important 150 kD antigen reducing conditions (C). The control lane stained without in the plasma membrane of osteoclasts. Further characterization of this interesting antigen is now in process. the primary antibody (A).
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ACKNOWLEDGMENTS This study was supported by the grants from Sigrid Juselius Foundation and Paul0 Foundation. We thank Satu Koljonen and Marja Paloniemi for their technical assistance and Eero Oja for preparing photographs. Presented in part at Tenth International conference on Calcium Regulating Hormones and Eleventh Annual Meeting of the American Society for Bone and Mineral Research, Joint Meeting, Montreal, Quebec, Canada, September 914, 1989.
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FIG. 6. Effects of 10 nM bafilomycin and monoclonal antibodies K5 and K20 on H+ transport of isolated bone microsomes. The final dilution of antibodies in the assay was 150.
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Teuvo Hentunen Department of Anatomy University of Oulu Kajaanintie 52A SF-90220 Oulu, Finland Received for publication November 14, 1990; in revised form February 7, 1991; accepted March 11, 1991.