Proc. Nati. Acad. Sci. USA Vol. 82, pp. 8213-8217, December 1985 Medical Sciences 1,25-Dihydroxyvitamin D3 causes formation of multinucleated cells with several osteoclast characteristics in cultures of primate marrow (acid phosphatase) G. D. ROODMAN*, K. J. IBBOTSON, B. R. MACDONALD, T. J. KUEHLt, AND G. R. MUNDY *Research Service, Audie Murphy Veterans Administration Hospital, and Departments of Medicine, University of Texas Health Science Center, tSouthwest Foundation for Biomedical Research, San Antonio, TX Communicated by H. F. DeLuca, July 25, 1985 ABSTRACT 1,25-Dihydroxyvitamin D3 induces cells de- rived from mononuclear phagocytes such as HL-60 and U937 to differentiate to macrophage-like cells and causes alveolar macrophages to form multinucleated cells in vitro. Since the osteoclast is thought to be derived from early marrow cells of the macrophage lineage, we cultured normal primate marrow mononuclear cells in medium containing 1,25-dihydroxy- vitamin D3. We found that large multinucleated cells (3-30 nuclei per cell) formed after 2-3 weeks of culture. Addition of 1,25-dihydroxyvitamin D3 to these cultures markedly stimu- lated multinucleated cell formation. Other vitamin D ana- logues, parathyroid hormone, prostaglandin E2, and calcitonin failed to stimulate multinucleated cell formation. However, calcitonin inhibited the stimulation of multinucleated cell formation by 1,25-dihydroxyvitamin D3. The multinucleated cells that formed in these cultures contained a tartrate-resistant acid phosphatase activity, a marker enzyme for osteoclasts. This activity was increased by 1,25-dihydroxyvitamin D3 and parathyroid hormone, and calcitonin inhibited the increase in acid phosphatase activity stimulated by these compounds. Ultrastructurally, the multinucleated cells had several features similar to those of osteoclasts. These data suggest that the multinucleated cells in our culture have several characteristics of osteoclasts and that the potent bone-resorbing activity of 1,25-dihydroxyvitamin D3 in vivo and in vitro may be mediated in part by stimulation of marrow mononuclear cells to form osteoclasts. The osteoclast is thought to be derived from immature marrow cells of the monocyte/macrophage lineage (1-3). However, the osteoclast has been difficult to study directly because of its relative inaccessibility. Therefore investigators have used other cells of the monocyte/macrophage series, such as peritoneal macrophages (4), alveolar macrophages (5), HL-60 cells (6), and U937 cells (7), as models for osteoclasts or their precursors. Recently, it has been shown that the most potent biologically active metabolite of vitamin D, la,25-dihydroxyvitamin D3, causes fusion of alveolar macrophages (5) and differentiation of mouse leukemic cells and HL-60 and U937 human leukemic cells in culture into macrophages (6-8). Since the normal osteoclast precursor is believed to be present in the marrow mononuclear cell population, we cultured normal primate marrow mononu- clear cells with 1,25-dihydroxyvitamin D3. We utilized the long-term culture system of Testa et al. (9), which they and we have used to examine formation of multinucleated cells with biologic and morphologic characteristics of osteoclasts in cultures of normal feline marrow (9, 10). We found that in the presence of 1,25-dihydroxyvitamin D3, a subpopulation of primate marrow mononuclear cells formed multinucleated cells with some of the characteristics of osteoclasts. MATERIALS AND METHODS Collection and Processing of Baboon Marrow Cells. Bone marrow was aspirated from the sternum or posterior iliac crest of adult baboons anesthetized with ketamine (10 mg/kg). Marrow was collected in syringes containing mini- mal essential medium alpha (a-MEM, GIBCO) supplemented with 5% (vol/vol) fetal bovine serum (Sterile Systems, Logan, UT) and preservative-free heparin (100 units/ml; Sigma). Marrow mononuclear cells were harvested from the interface of Ficoll/Hypaque density gradients (11) that had been centrifuged at 400 x g for 30 min at 12TC. The marrow mononuclear cells were washed twice with medium and then cultured in 24-well plates (Linbro) at 106 cells per ml, in a-MEM with 20o horse serum. In some experiments marrow mononuclear cells were incubated for 1 hr in a-MEM con- taining 20% fetal calf serum in 35-mm plastic tissue culture dishes, and the nonadherent cells were collected. The non- adherent cells were cultured in a similar fashion as the unfractionated marrow mononuclear cells. All cultures were maintained in a humidified 4% CO2 atmosphere at 370C. In selected experiments, various concentrations of osteotropic hormones were added at the initiation of the cultures and with each feeding. Cultures were fed weekly by removing half of the medium and nonadherent cells and replacing it with an equal volume of fresh medium. No attempt was made to recover the nonadherent cells. After culture, cells were fixed with 5% (vol/vol) glutaraldehyde (Sigma) and then stained with Wright stain. Cells were examined with an inverted phase-contrast microscope; those cells containing more than three nuclei were counted as multinucleated. Assay of Acid Phosphatase Activity. Marrow cells were cultured as described above. After the completion of culture, cells were fixed and stained with an acid phosphatase kit (Sigma) in the presence of 0.67 M sodium tartrate. Cells were scored for the red, acid phosphatase stain as follows: 0, no staining; 1, <25% of the cytoplasm was stained; 2, 25-50% of the cytoplasm was stained; 3, >50% of the cytoplasm was intensely stained. The acid phosphatase acid score was determined per cell and the mean + SEM score was deter- mined. Ultrastructural Studies. Cells were fixed in their culture flasks with 1.5% glutaraldehyde/2% formaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 hr, washed in buffer, dehydrated, and embedded as a monolayer in Polybed (Polyscience, Warrington, PA). Sections parallel to the culture dish or perpendicular to it were obtained with a diamond knife and examined on a JEOL CS-100 electron microscope after staining with uranyl acetate and lead citrate. 8213 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 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Proc. Nati. Acad. Sci. USAVol. 82, pp. 8213-8217, December 1985Medical Sciences
1,25-Dihydroxyvitamin D3 causes formation of multinucleatedcells with several osteoclast characteristics in cultures ofprimate marrow
(acid phosphatase)
G. D. ROODMAN*, K. J. IBBOTSON, B. R. MACDONALD, T. J. KUEHLt, AND G. R. MUNDY*Research Service, Audie Murphy Veterans Administration Hospital, and Departments of Medicine, University of Texas Health Science Center, tSouthwestFoundation for Biomedical Research, San Antonio, TX
Communicated by H. F. DeLuca, July 25, 1985
ABSTRACT 1,25-Dihydroxyvitamin D3 induces cells de-rived from mononuclear phagocytes such as HL-60 and U937to differentiate to macrophage-like cells and causes alveolarmacrophages to form multinucleated cells in vitro. Since theosteoclast is thought to be derived from early marrow cells ofthe macrophage lineage, we cultured normal primate marrowmononuclear cells in medium containing 1,25-dihydroxy-vitamin D3. We found that large multinucleated cells (3-30nuclei per cell) formed after 2-3 weeks of culture. Addition of1,25-dihydroxyvitamin D3 to these cultures markedly stimu-lated multinucleated cell formation. Other vitamin D ana-logues, parathyroid hormone, prostaglandin E2, and calcitoninfailed to stimulate multinucleated cell formation. However,calcitonin inhibited the stimulation of multinucleated cellformation by 1,25-dihydroxyvitamin D3. The multinucleatedcells that formed in these cultures contained a tartrate-resistantacid phosphatase activity, a marker enzyme for osteoclasts.This activity was increased by 1,25-dihydroxyvitamin D3 andparathyroid hormone, and calcitonin inhibited the increase inacid phosphatase activity stimulated by these compounds.Ultrastructurally, the multinucleated cells had several featuressimilar to those of osteoclasts. These data suggest that themultinucleated cells in our culture have several characteristicsof osteoclasts and that the potent bone-resorbing activity of1,25-dihydroxyvitamin D3 in vivo and in vitro may be mediatedin part by stimulation of marrow mononuclear cells to formosteoclasts.
The osteoclast is thought to be derived from immaturemarrow cells of the monocyte/macrophage lineage (1-3).However, the osteoclast has been difficult to study directlybecause of its relative inaccessibility. Therefore investigatorshave used other cells of the monocyte/macrophage series,such as peritoneal macrophages (4), alveolar macrophages(5), HL-60 cells (6), and U937 cells (7), as models forosteoclasts or their precursors. Recently, it has been shownthat the most potent biologically active metabolite of vitaminD, la,25-dihydroxyvitamin D3, causes fusion of alveolarmacrophages (5) and differentiation of mouse leukemic cellsand HL-60 and U937 human leukemic cells in culture intomacrophages (6-8). Since the normal osteoclast precursor isbelieved to be present in the marrow mononuclear cellpopulation, we cultured normal primate marrow mononu-clear cells with 1,25-dihydroxyvitamin D3. We utilized thelong-term culture system of Testa et al. (9), which they andwe have used to examine formation of multinucleated cellswith biologic and morphologic characteristics of osteoclastsin cultures of normal feline marrow (9, 10). We found that inthe presence of 1,25-dihydroxyvitamin D3, a subpopulation
of primate marrow mononuclear cells formed multinucleatedcells with some of the characteristics of osteoclasts.
MATERIALS AND METHODSCollection and Processing of Baboon Marrow Cells. Bone
marrow was aspirated from the sternum or posterior iliaccrest of adult baboons anesthetized with ketamine (10mg/kg). Marrow was collected in syringes containing mini-mal essential medium alpha (a-MEM, GIBCO) supplementedwith 5% (vol/vol) fetal bovine serum (Sterile Systems,Logan, UT) and preservative-free heparin (100 units/ml;Sigma). Marrow mononuclear cells were harvested from theinterface of Ficoll/Hypaque density gradients (11) that hadbeen centrifuged at 400 x g for 30 min at 12TC. The marrowmononuclear cells were washed twice with medium and thencultured in 24-well plates (Linbro) at 106 cells per ml, ina-MEM with 20o horse serum. In some experiments marrowmononuclear cells were incubated for 1 hr in a-MEM con-taining 20% fetal calf serum in 35-mm plastic tissue culturedishes, and the nonadherent cells were collected. The non-adherent cells were cultured in a similar fashion as theunfractionated marrow mononuclear cells. All cultures weremaintained in a humidified 4% CO2 atmosphere at 370C. Inselected experiments, various concentrations of osteotropichormones were added at the initiation ofthe cultures and witheach feeding. Cultures were fed weekly by removing half ofthe medium and nonadherent cells and replacing it with anequal volume of fresh medium. No attempt was made torecover the nonadherent cells. After culture, cells were fixedwith 5% (vol/vol) glutaraldehyde (Sigma) and then stainedwith Wright stain. Cells were examined with an invertedphase-contrast microscope; those cells containing more thanthree nuclei were counted as multinucleated.Assay of Acid Phosphatase Activity. Marrow cells were
cultured as described above. After the completion of culture,cells were fixed and stained with an acid phosphatase kit(Sigma) in the presence of 0.67M sodium tartrate. Cells werescored for the red, acid phosphatase stain as follows: 0, nostaining; 1, <25% of the cytoplasm was stained; 2, 25-50%of the cytoplasm was stained; 3, >50% of the cytoplasm wasintensely stained. The acid phosphatase acid score wasdetermined per cell and the mean + SEM score was deter-mined.
Ultrastructural Studies. Cells were fixed in their cultureflasks with 1.5% glutaraldehyde/2% formaldehyde in 0.1 Mcacodylate buffer (pH 7.4) for 2 hr, washed in buffer,dehydrated, and embedded as a monolayer in Polybed(Polyscience, Warrington, PA). Sections parallel to theculture dish or perpendicular to it were obtained with adiamond knife and examined on a JEOL CS-100 electronmicroscope after staining with uranyl acetate and lead citrate.
8213
The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Adult baboon bone marrow mononuclear cells were cultured (106cells per ml; 0.5 ml per culture) in minimal essential medium alphaplus 20% horse serum, in the presence or absence of the variouscompounds as indicated. After 3 weeks, cultures were fixed withglutaraldehyde and stained with Wright stain. Cells containing threeor more nuclei per cell were scored. Results are presented as themean ± SEM for quadruplicate determinations except where noted.*Mean of duplicate determinations.tNot done.
Statistical Methods. The mean ± SEM was calculated forall results. All results were compared by using a pairedStudent's t test except for acid phosphatase scores, whichwere compared by using a mixed-model analysis of variance.Results were considered significantly different for P < 0.05.
RESULTS
Variable numbers ofmultinucleated cells formed in long-termbaboon marrow cultures in the absence of osteotropic hor-mones (Tables 1-4). Addition of 10 nM 1,25-dihydroxyvita-min D3 significantly stimulated multinucleated cell formation(Table 1). Concentrations of 1,25-dihydroxyvitamin D3 <1nM failed to significantly stimulate multinucleated cell for-mation (Table 2). Concentrations of 1,25-dihydroxyvitaminD3 :100 nM decreased. multinucleated cell formation (datanot shown). In contrast to 1,25-dihydroxyvitamin D3, para-thyroid hormone (10-400 ng/ml), calcitonin (10-200 ng/ml),and prostaglandin E2 (10 ,M-1 nM) failed to consistentlystimulate multinucleated cell formation (Table 1).The multinucleated cells that formed (Fig. 1) had 3-30
nuclei per cell and each nucleus had one prominentnucleolus. The peripheral cytoplasm was clear (clear zone).The cells stained positively for nonspecific esterase andtartrate-resistant acid phosphatase.
Multinucleated cell formation was maximal between 2 and4 weeks of culture (Table 3). No multinucleated cells formedbefore 13 days in culture. Addition of 10 nM 1,25-dihydroxyvitamin D3 increased the number but not the rateof formation of multinucleated cells.
Table 2. Multinucleated cell formation in cultures treated withvarious concentrations of 1,25-dihydroxyvitamin D3
Multinucleated cellsper culture
Concentration, nM Exp. 1 Exp. 2
0 0 55 610 46±24 134±51 2 ± 1 99 ± 60.10 0 71 + 8
Adult baboon marrow mononuclear cells were cultured and scoredfor multinucleated cell formation as described in Materials andMethods and the legend to Table 1. Various concentrations of1,25-dihydroxyvitamin D3 were added at the start of culture. Resultsare presented as the mean ± SEM for triplicate determinations.
FIG. 1. Multinucleated cells formed in long-term cultures ofbaboon marrow in the presence of 1,25-dihydroxyvitamin D3. After3 weeks in culture, the cells were fixed with glutaraldehyde andstained for acid phosphatase. (x570.)
Addition of 10 nM 25-hydroxy- or 24,25-dihydroxyvitaminD3 or of 10 nM vitamin D3 did not increase multinucleated cellformation above control values (Table 4). Concentrations ofany of the vitamin D analogues .100 nM inhibited multinu-cleated cell formation.Although calcitonin did not affect multinucleated cell
formation by itself (Tables 1 and 4), addition of calcitonin tomarrow cultures containing 1,25-dihydroxyvitamin D3 con-sistently inhibited multinucleated cell formation. For exam-ple, few if any multinucleated cells formed in the absence of1,25-dihydroxyvitamin D3 (Table 4). Addition of 1,25-dihydroxyvitamin D3 increased the number ofmultinucleatedcells to 96 per culture. Addition of calcitonin to these1,25-dihydroxyvitamin D3-stimulated cultures decreasedmultinucleated cell formation to 54 per culture. For fourindependent experiments in which calcitonin and 1,25-dihy-droxyvitamin D3 were added simultaneously, multinucleatedcell formation was decreased to 47 + 9% (mean ± SEM) ofvalues obtained when only 1,25-dihydroxyvitamin D3 wasadded.The tartrate-resistant acid phosphatase activity present in
the cells was increased by 1,25-dihydroxyvitamin D3 in fourof six experiments (Table 5). In the two experiments in which1,25-dihydroxyvitamin D3 did not increase acid phosphataseactivity, it increased multinucleated cell formation onlymodestly (40-60%). However, parathyroid hormone, al-though unable to increase multinucleated cell formation, did
Table 3. Time course of multinucleated cell formationMultinucleated cells per culture
Control 1,25-(OH)2D3Days in culture Exp. 1 Exp. 2 Exp. 1 Exp. 2
Adult baboon marrow mononuclear cells were cultured in theabsence (Control) or presence [1,25-(OH)2D3] of 10 nM 1,25-dihydroxyvitamin D3. Results are presented as the mean ± SEM forquadruplicate determinations (Exp. 1) or the average of duplicatedeterminations (Exp. 2). Similar patterns of multinucleated cellformation were seen in four other independent experiments done induplicate. ND, not done.
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Table 4. Effects of vitamin D analogues and calcitonin onmultinucleated cell formation in long-term baboonmarrow cultures
Baboon marrow mononuclear cells were cultured in the absence orpresence of vitamin D analogues (10 nM) or calcitonin (100 ng/ml).Values represent means of duplicate determinations.
increase acid phosphatase activity (Table 5). Althoughcalcitonin did not decrease acid phosphatase activity byitself, it consistently inhibited the stimulation of acid phos-phatase activity induced by 1,25-dihydroxyvitamin D3 orparathyroid hormone. Multinucleated cells in control cul-tures had low levels of acid phosphatase staining (Table 5).Only rare mononuclear cells contained detectable tartrate-resistant acid phosphatase activity. Preformed multinucle-ated cells responded to osteotropic hormones within 48 hr ofexposure to parathyroid hormone, calcitonin, or 1,25-dihydroxyvitamin D3 (Table 5, Exp. 3).On semi-thin sections, the multinucleated cells showed a
characteristic peripheral distribution of their nuclei. Onsections parallel to the culture dishes, the nuclei werearranged as a circle at the periphery of the cells (not shown);when sectioned perpendicularly to the culture dish, the nucleiwere also at the periphery of the cells but only at the poleopposed to the site of attachment to the plastic (Fig. 2). Thearea ofattachment to the plastic dish was devoid oforganellesand was composed of dense filamentous material (Fig. 2).The cytoplasm contained numerous mitochondria, some ofwhich were pleomorphic; an abundant rough endoplasmicreticulum; large Golgi complex areas; and numerouslysosomes and residual bodies containing myelinic figures.Free ribosomes were relatively scarce, and the nuclei werehighly indented with abundant euchromatin. Although ruffledborders were not observed at the site of attachment to theculture dish, intense ruffling of the membrane facing the
culture medium was observed (Fig. 2). A very frequent andcharacteristic feature of these cells was the presence of acanalicular system with membrane ruffling extending deeplyinto the cytoplasm (Fig. 2).We have found that the precursor for the osteoclast-like
cells in feline marrow cultures was a nonadherent immaturemonocyte (10). To determine whether a similar cell type is theprecursor for the osteoclast-like cells formed in primatemarrow, we depleted baboon marrow mononuclear cells ofcells adherent to plastic tissue culture dishes prior to long-term marrow culture. Removal of adherent cells prior toculture increased multinucleated cell formation 2-fold: un-fractionated marrow mononuclear cells formed 110 ± 5multinucleated cells per culture in the presence of 1,25-dihydroxyvitamin D3 and nonadherent marrow cells formed220 ± 33 multinucleated cells per culture (n = 4).
DISCUSSIONLong-term primate marrow cultures formed multinucleatedcells with several of the characteristics of osteoclasts. Thecells were multinucleated; had several ultrastructural fea-tures similar to those of osteoclasts not lying adjacent to anendosteal bone surface (12); responded to the osteotropichormones 1,25-dihydroxyvitamin D3, parathyroid hormone,and calcitonin; and contained a tartrate-resistant acid phos-phatase, a marker enzyme for osteoclasts (13-15). The majorprecursor for these cells is a nonadherent marrow mononu-clear cell.
Variable numbers of multinucleated cells formed in theabsence of osteotropic hormones, whereas 1,25-dihydroxy-vitamin D3 stimulated the formation and acid phosphataseactivity ofthe baboon marrow multinucleated cells. The largevariation in multinucleated cell formation in these studiesmost probably reflects differences in marrow samples fromdifferent individuals as well as the variable amount ofperipheral blood admixed during marrow aspiration. Periph-eral blood mononuclear cells did not form multinucleatedcells under these culture conditions (data not shown). Similarvariability in other hemopoietic progenitor cell concentra-tions present in different baboon marrow samples has beenreported (16). Other analogues ofvitamin D3 did not affect theformation of these osteoclast-like cells. These data suggestthat both the precursor for baboon marrow multinucleatedcells and the osteoclast-like cells themselves can respond to1,25-dihydroxyvitamin D3. Other studies have shown that1,25-dihydroxyvitamin D3 stimulates precursors to form
Table 5. Tartrate-resistant acid phosphatase activity in baboon marrow multinucleated cellstreated with osteotropic hormones
In Exps. 1 and 2, baboon marrow mononuclear cells were cultured in the presence or absence of1,25-dihydroxyvitamin D3 (10 nM), calcitonin (100 ng/ml) or parathyroid hormone (200 ng/ml) for 3weeks as described in Materials and Methods. In Exp. 3, bone marrow was initially grown in thepresence of 1,25-dihydroxyvitamin D3 for 3 weeks; the vitamin then was removed and the cells wereincubated for 48 hr in medium and then for 48 hr in the absence or presence of osteotropic hormones.Treatment of cultures with calcitonin alone did not affect acid phosphatase activity.*Acid phosphatase activity was scored 0-3 (see Materials and Methods). Results represent the mean± SEM for triplicate cultures, with at least 20 cells scored per culture. Results were compared usinga mixed-model analysis of variance. ND, not done.tP < 0.05 compared to control.tP < 0.05 compared to 1,25-dihydroxyvitamin D3.§P < 0.05 compared to parathyroid hormone.
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FIG. 2. (a) Perpendicular section of a multinucleated cell, area facing the culture medium. The nuclei (N) are deeply indented and are alllocated at the periphery ofthe cell. Numerous microvilli are present (thick arrows) along the plasma membrane in contact with the culture medium(at top). The double arrow shows the canalicular system extending deep into the cytoplasm of the cell. Golgi complexes (G) are found in thecytoplasm but not in a perinuclear organization. Mitochondria are numerous and some are pleomorphic (curved arrow). (x6400.) (b) View ofa highly developed canalicular system. (x9600.) (c) Higher magnification (x20,000) of the cytoplasm and zone of attachment of a multinucleatedcell to the culture dish (at bottom). The plasma membrane (thick arrows) is closely attached to the plastic culture dish. The attachment zone(AZ) is devoid of organelles and consists of filamentous material. The cytoplasm is characterized by the presence of numerous mitochondriaand secondary lysosomes (L), often containing myelinic figures. A canalicular system is seen at the top (double arrow). G, Golgi complex.
authentic osteoclasts. Tinkler et al. (17) showed that admin-istration of 1,25-dihydroxyvitamin D3 in vivo increasedosteoclast formation in mice. Hefley and Stern (18) showedthat 1,25-dihydroxyvitamin D3 increased osteoclast forma-tion at least 2-fold in cultured rat fetal long bones. Although1,25-dihydroxyvitamin D3 also promotes fusion of mousealveolar macrophages (5) and peripheral blood monocytes(19), the multinucleated cells formed from these macrophageshave not been shown to respond to parathyroid hormone orcalcitonin or to have ultrastructural characteristics of osteo-clasts.
In our studies, 1-10 nM 1,25-dihydroxyvitamin D3 wasrequired for optimal multinucleated cell formation. Similarconcentrations of 1,25-dihydroxyvitamin D3 have been re-quired by others (5) to elicit fusion of cells derived from adultanimals. Only 1% of 1,25-dihydroxyvitamin D3 is unbound inour cultures (data not shown), so the concentration of1,25-dihydroxyvitamin D3 actually available to the cells maybe on the order of 10-100 pM. The time required for theeffects of 1,25-dihydroxyvitamin D3 on multinucleated cellformation was much longer than the time required to detectthe effects of 1,25-dihydroxyvitamin D3 on bone in organculture (18). These differences may reflect the fact that thecompound may be stimulating fusion of already differentiatedosteoclast progenitors in bone. In marrow cultured in theabsence of bone, granulocyte/macrophage progenitors may
first need to differentiate to early macrophages which, underthe influence of 1,25-dihydroxyvitamin D3, further differen-tiate to osteoclast progenitors. These osteoclast progenitorsthen fuse to form multinucleated cells. We have found inpreliminary studies that treatment of our cultures withcolony-stimulating factor, the hormone required formacrophage differentiation from committed progenitor cells,followed by 1,25-dihydroxyvitamin D3 markedly increasesmultinucleated cell formation in our cultures (data notshown). The time course ofmultinucleated cell formation wasunchanged. Colony-stimulating factor alone did not consis-tently increase multinucleated cell formation. These data (notshown) suggest that 1,25-dihydroxyvitamin D3 may have adual role in bone cells: (i) inducing differentiation ofosteoclast progenitor cells and (ii) stimulating preformedosteoclasts to resorb bone.
Several studies have shown that the osteoclast precursor ispresent in the marrow. Walker (20) showed that bone marrowtransplantation cured osteopetrosis in lethally irradiatedosteopetrotic mice. Coccia et al. (21) successfully treated apatient with malignant osteopetrosis with bone marrowtransplantation and showed that the osteoclasts that formedwere of donor origin. Ko and Bernard (22) demonstrated thatbone marrow mononuclear cells formed osteoclasts inosteoclast-free bone in vitro. We previously have reported(10) that the precursor for osteoclast-like cells in feline
marrow cultures is a nonadherent immature marrowmonocyte, and Burger et al. (23) have shown that theprecursor for the osteoclasts formed in mouse marrowcultures is a nonadherent immature monocyte.
Parathyroid hormone did not increase multinucleated cellformation but did increase acid phosphatase activity inmultinucleated cells in baboon marrow cultures. This resultwas unexpected because treatment with parathyroid hor-mone increased osteoclast-cell formation in feline marrowcultures (10) and increased osteoclast numbers in animals (24,25). Chambers and Dunn (26) and Rodan and Martin (27) havesuggested that parathyroid hormone exerts its effects onosteoclasts indirectly through intermediate cells. These datasuggest that baboon marrow cultures lack the accessory cellsrequired for parathyroid hormone to stimulate multinucle-ated cell formation but that once formed the multinucleatedcells can respond to the hormone.
Calcitonin inhibited both the 1,25-dihydroxyvitamin D3stimulation of multinucleated cell formation and the 1,25-dihydroxyvitamin D3 and parathyroid hormone stimulation ofacid phosphatase activity in baboon marrow multinucleatedcells. These data further support the osteoclast-like nature ofthe multinucleated cells formed in baboon marrow cultures.Hefley and Stem (18) have shown that calcitonin inhibitsosteoclast formation in fetal rat long bone cultures by 40%o.Similarly, Chambers and Moore (28) have shown thatcalcitonin inhibits the motility of isolated mature osteoclasts.
Ultrastructurally, the multinucleated cells have some fea-tures of osteoclasts but were also different from typicalosteoclasts in a number of respects. The high number ofmitochondria, some pleomorphic; a zone of attachment thatlacks organelles; and the existence of deep membraneinvaginations that create a canalicular system are character-istic of osteoclasts. The peripheral localization of the nuclei,the scarcity of free polysomes, the high number of secondarylysosomes, and the disposition of the Golgi stacks in a Golgicomplex area rather than in a perinuclear fashion werefeatures differing from those of osteoclasts. Clearly, thatthese cells are grown on plastic rather than on their naturalbone substratum might prevent the full development of thiscanalicular system into a ruffled-border. Further work will berequired to differentiate these cells from other giant cells, ifindeed these and osteoclasts are two different cell types.
In summary, baboon marrow cultures form multinucleatedcells with some osteoclast-like characteristics. The datapresented here suggest that the potent bone-resorbing activ-ity of 1,25-dihydroxyvitamin D3 may be mediated, in part, bystimulating the formation of osteoclasts and increasing acidhydrolase activity in these cells. Such culture systems shouldbe a useful model for further defining the growth require-ments and hormonal responsiveness of osteoclast precur-sors.
The expert assistance of Lynn Neff and Joye Laderer is gratefullyacknowledged. We thank Dr. R. Baron for his help in performing andinterpreting the ultrastructural studies.
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