Proc. Natl. Acad. Sci. USAVol. 80, pp. 5907-5911, October 1983Cell Biology

Induction of monocytic differentiation and bone resorption by1,25-dihydroxyvitamin D3*

(differentiation antigens/osteoclast/esterase/lysozyme)

Zvi BAR-SHAVITt, STEVEN L. TEITELBAUMt, PIETER REITSMAt, ANN HALLt, LYLE E. PEGGt, JOANN TRIAL§,AND ARNOLD J. KAHN:tDepartments of Pathology and Medicine, The Jewish Hospital of St. Louis, tDivision of Cell Biology, Washington University School of Dental Medicine, and§Department of Genetics, Washington University Medical School, St. Louis, Missouri 63110

Communicated by Donald C. Shreffler, June 30, 1983

ABSTRACT 1,25-Dihydroxyvitamin D3 [1,25(0H)2D3] stim-ulates bone resorption in man and other vertebrates, in part, byincreasing the number of osteoclasts, the principal resorbing cellsof bone. Because osteoclasts are very likely derived from a mem-ber(s) of the mononuclear phagocyte family, we determined if1,25(OH)2D3 promotes maturation of these cells by studying itseffects on the human promyelocytic leukemia cell line HL-60. Ofthe vitamin D3 metabolites tested, only 1,25(OH)2D3, at 10-10 to10-7 M, induces the differentiation of HL60 into mono- and mul-tinucleated macrophage-like cells. Phenotypic change is evidentwithin 24 hr and reaches a plateau between 72 and 96 hr of in-cubation. The changes are metabolite-specific and include (i) ad-herence to substrate, (ii) acquisition of the morphological featuresof mature monocytes, (iii) a 4- to 6-fold enhancement in lysozymesynthesis and secretion, (iv) increase in the fraction of a-naphthylacetate esterase-positive cells from approximately 2% to 100% ofthe population, and (v) the acquisition of several monocyte-asso-ciated cell surface antigens. More importantly, treated HL-60 cellsacquire the capacity to bind and degrade bone matrix, two of theessential, functional characteristics of osteoclasts and related bone-resorbing cells. These results, considered together with the re-ported action of 1,25(OH)2D3 on nontransformed mononuclear cells,are consistent with the view that vitamin D3 enhances bone re-sorption and osteoclastogenesis in vivo by promoting the differ-entiation of precursor cells.

Of the several circulating factors known to affect bone resorp-tion, one of the most potent is 1,25-dihydroxyvitamin D3[1,25(OH)2D3]. This compound, when administered in picomo-lar to nanomolar concentrations, markedly stimulates resorp-tive activity (1) and promotes a readily measurable increase inthe number of osteoclasts, the principal resorbing cells of bone(2). It is generally assumed that the appearance of increasednumbers of osteoclasts is responsible for enhanced bone re-sorption. However, the mechanism(s) by which 1,25(OH)2D3alters the size of the osteoclast population, and therefore re-sorptive activity, is presently unknown.

Osteoclasts originate by fusion of circulating mononuclearprecursor cells and almost certainly represent one of the end-stage cells of mononuclear phagocyte differentiation (3, 4). Likeosteoclasts, other mature, nonproliferative members of thisfamily-e.g., monocytes and macrophages (Mos)-possess thecapacity to attach to and degrade bone matrix (5, 6), and there-fore they serve as useful models with which to study the mech-anisms of bone resorption in tissue culture. In a previous study,we showed that Mos isolated from vitamin D-deficient animalsexhibit, in vitro, the same bone resorptive dysfunction char-acteristically observed in the intact, calciferol-deprived animal

(7). This dysfunction is not corrected by the direct addition of1,25(OH)2D3 to M4 cultures, but administration of the me-tabolite to vitamin D-depleted animals for several days prior tocell isolation restores bone resorptive capability to normal. Thisfinding is in keeping with the observed lag period between theintroduction of 1,25(OH)2D3 and the increase in the size of theosteoclast population (2). It is also consistent with the hypoth-esis that the steroid exerts its effect on the mononuclear phago-cyte system, at least in part, by promoting cell maturation.

In recent years, a number of tumor cell lines with monocyteand myeloid characteristics have been isolated and conditionedfor growth in tissue culture (8). These lines vary in their degreeof maturation, and several of them (the murine line M-1 andthe human lines U937 and HL-60) have been shown to respondto chemical inducers by differentiating into cells with more de-finitive MO-like features (9-14). We have found that those celllines that are inherently more mature bind and degrade bonein a fashion indistinguishable from that of differentiated Mos,whereas the immature cell lines lack these properties (15). Inthe present study, we used the human promyelocytic cell lineHL-60 to test the hypothesis that 1,25(OH)2D3 enhances boneresorption and osteoclastogenesis by stimulating maturation ofcells of the mononuclear phagocyte family. Our data show thatthis vitamin D3 metabolite elicits monocyte differentiation andmultinucleation in HL-60 and that this transformation is ac-companied by a marked increase in the ability of these cells tobind and degrade bone matrix, the two essential functionalcharacteristics of bone-resorbing cells.

MATERIALS AND METHODSCells. MO-like cell line J774.2 was obtained from the Amer-

ican Type Culture Collection, and the human promyelocyticleukemia HL-60 from E. Huberman (Argonne National Lab-oratory). Cells are grown in RPMI 1640 medium containing 20%fetal bovine serum, glutamine, and nonessential amino acids.For the bone degradation assay, cells are plated in flat-bottomwells (16-mm diameter) at 2.5 X 105 cells per well.

Vitamin D Treatment. Vitamin D metabolites [1,25(OH)2D3,25(OH)D3, and 24,25(OH)2D3; Hoffmann-La Roche] are dis-solved in ethanol and added to the cells in the concentrationsindicated. Control cultures are treated with the appropriateamount of the carrier (ethanol), which never exceeds 0.1%.

Bone Binding Assay. J774.2, a naturally adherent cell line,and HL-60 rendered adherent by exposure to 1,25(OH)2D3 are

Abbreviations: 1,25(OH)2D3, 1,25-dihydroxyvitamin D3 (other vitaminD metabolites are abbreviated similarly); MO, macrophage; Me2SO, di-methyl sulfoxide.* This work was presented in part at the meetings of the American So-ciety for Bone and Mineral Research (15) and the American Society ofCell Biology (30).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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detached from culture dishes as follows: Monolayers are rinsedin a-Mops [Eagle's minimal essential medium buffered to pH7.4 with 3-(N-morpholino)propanesulfonic acid] and incubatedfor 20 min at 40C in phosphate-buffered saline without Ca2"and Mg2" followed by gentle scraping. The cells are then washedand suspended in a-Mops. Nonadherent HL-60 cells are re-covered by gentle pipetting of untreated cultures.

Cells are radiolabeled by incubating them for 1 hr at 370Cin a solution containing Na51CrO4 (250-500 mCi/mg of Cr;Amersham; 1 Ci = 3.7 X 1010 Bq) in a-Mops (1 puCi per 106cells). The cells are then washed and aliquoted onto the strippedendocranial surface of freshly dissected 7-day-old rat pup cal-varia and the cells are allowed to attach for 60 min at 370C. Thenonadherent cells are then removed by sequential rinsing inphosphate-buffered saline and the calvaria with attached ra-dioactive cells are subjected to gamma counting.

Bone Degradation. Bone degradation is determined fromthe cell-mediated release of isotope from in vivo 'Ca-labeleddevitalized bone particles as described (6). The cell lines areplated at 2.5 x 105 cells per well and the bone particles (di-ameter 25-43 tkm) are added at 115 ug per well. The assay isperformed in RPMI 1640 medium containing 20% fetal bovineserum for 22 hr.

Esterase. Cytochemical assays for a-naphthyl acetate ester-ase and for naphthol AS-D chloroacetate esterase are per-formed with commercially available kits (Sigma nos. 90-Al and90-C2). Results are expressed as percent reactive cells.

Lysozyme. HL-60 cells are seeded in 24-well plates at 2 X105 cells per ml, 1 ml per well. Cells are incubated with1,25(OH)2D3 or with ethanol carrier for the time and concen-tration indicated. Lysozyme is measured by using the methodof Litwack (16). The standard used is egg white lysozyme (Sigma).

Analysis of Cell Surface Antigens by Immunofluorescence.Cells are harvested from culture wells by vigorous pipettingand are washed several times at 4°C with phosphate-buffered

saline plus 0.1% bovine serum albumin, 0.2% sodium azide,and dextran sulfate at 0.25 ug/ml. The same solution is usedfor antibody dilutions and further washings. Primary antibody(25 ,ul) is added to 104 cells, which are allowed to react at 40Cfor 30 min. After thorough washing, fluorescein isothiocyanate-conjugated F(ab')2 fragment of rabbit anti-mouse Ig (diluted1:10, Cappel Laboratories, Cochranville, PA) is added and al-lowed to react. After further washing, 2% (wt/vol) paraformal-dehyde with 0.005% Evan's blue stain (17) is added and the cellsare examined with a Zeiss fluorescence microscope.An alternative approach involves a protocol designed to block

nonspecific Fc receptor binding of the primary antibody. Hu-man IgG is added at 0.1% to phosphate-buffered saline, andthis solution is used for all washing and antibody dilutions. Thesecondary antibody is a fluorescein isothiocyanate-conjugatedF(ab')2 sheep anti-mouse IgG with no crossreactivity to humanIgG (Cappel Laboratories). Results with this protocol are nodifferent than those obtained with the previous one (data notshown).Immunofluorescence Flow Cytometry. Cells are harvested

and treated, as for immunofluorescence microscopy, in plastictubes (2 x 106 cells per tube). Cell fluorescence is analyzed byan EPICS V flow cytometer (Coulter).

RESULTSInduction and Characterization of Differentiation in HL-60

Cells. HL-60 cells exhibit conspicuous morphological changeswithin 24 hr of exposure to 1,25(OH)2D3 at concentrations aslow as 10-10 M. During this initial period, the cells begin toattach to the substrate, spread, lose cytoplasmic basophilia, anddisplay a decrease in nucleus-to-cytoplasm ratio. Moreover, thenuclei assume the reniform appearance typical of monocytes.These morphological changes occur progressively in culture andreach a plateau after approximately 72 hr of exposure to1,25(OH)2D3 (compare the treated and control cells in Fig. 1).

-7 .: ;i X

.:w .*-.M..

j4

j

FIG. 1. Micrographs of HL-60 cells: Control (Left) or treated with 10-8 M 1,25(OH)2D3 for 48 hr (Right). Note the decrease in nucleus-to-cytoplasm ratio and the appearance of monocyte-like reniform nuclei under the influence of 1,25(OH)2D3. (x90.)

Proc. Natl. Acad. Sci. USA, 80 (1983)

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Proc. Natl. Acad. Sci. USA 80 (1983) 5909

Other tested vitamin D3 metabolites, including 25(OH)D3 and24,25(OH)2D3, do not induce morphological transformation inHL-60 even when used at concentrations as high as 10-6 M.

Treatment with 1,25(OH)2D3 sharply curtails proliferation ofHL-60 cells, with the first measurable decrements occurringbetween 24 and 48 hr of incubation {Fig. 2). This cessation ofmitotic activity is accompanied by the attachment of an in-creasing fraction of the population to the substrate; by 72-96hr of culture (Fig. 2) 40-50% of the treated population is at-tached. In-contrast, no attachment is detectable in control cells.Mos synthesize and secrete copious amounts of lysozyme in

vitro (18), and 1,25(OH)2D3-treated HL-60 cells exhibit muchthe same characteristic. Treated cells show about a 2-fold in-crease in secreted enzyme by 36 hr of incubation and a 4- to6-fold increase over controls by 96 hr (not.shown). The amountof enzyme produced varies as a function of metabolite con-centration, with the midpoint-in the dose-response curve oc-curring at 5 X 10-.8 M and a;plateau at 10-7 M (Fig. 3). Ap-proximately 90% of the lysozyme produced by: treated cells issecreted into the culture medium (not shown).

Monocytes and granulocytes may be distinguished, at leastin part, by differences in esterase substrate specificity; mono-cytes contain an enzyme, evidently not present in granulocytes,capable of degrading a-naphthyl acetate, whereas granulocytesare uniquely able (relative to monocytes) to hydrolyze naphtholAS-D chloroacetate (19). Untreated HL-60 cells exhibit littleenzyme activity of either type-but rapidly acquire the ability todegrade a-naphthyl acetate upon exposure to 1,25(OH)2D3 (Fig.4). By 24 hr, approximately 50% of the nonadherent and 80%of the adherent treated cells are naphthyl.acetate esterase pos-itive; by 48-96 hr, essentiallyall treated cells exhibit such stain-ing. Moreover, the dose-response relationship of a-naphthylacetate es.terase activity in response to 1,25(OH)2D3 indicatessensitivity to the vitamin/hormone at physiological concentra-tions. Specifically, 72-hr exposure of HL-60 cells to 1,25(OH)2D3yields 30%.reactive cells at 10-10 M, 78% reactive cells at 10-9M, and 100% reactivecells at 10-8 and 10-7 M. The established-monocytic inducer phorbol 12-tetradecanoate 13-acetate at 5 x10-9 M has a similar effect on the-appearance of the esterase.

In contrast to a-naphthyl acetate esterase, AS-D chloroac-etate esterase activity diminishes to less than control levels in1,25(OH)2D3-treated cells (Fig.. 4B). On the other hand, ex-posure of cells to the granulocyte inducer Me2SO elicits an =4-fold enhancement of the latter enzyme (Fig. 4B). These find-ings are consistent with the view that 1,25(OH)2D3 promotesmonocyte but not granulocyte differentiation in HL-60.

*0ko

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0 1,000

;, 8000

& 600

E 4000>). 200

0 10-11 10-1010-9 10-8 10-7 10-6 10-5

1,25(OH)2D3, M

FIG. 3. Lysozyme synthesis by HL-60 cells -as a function of 1,25-(OH)2D3 concentration. o, Lysozyme per culture; 9, lysozyme per cell.The apparent continued increase in enzyme synthesis per cell at highermetabolite concentrations is due to a diminution in cell number. Thedata represent the mean of two experiments. Samples were collectedafter 72 hr of incubation.

Cell Surface Antigens in Untreated and Induced HL-60 Cells.Additional evidence supporting the monocytic nature of vita-min D3-induced HL-60 cells comes from .examining cell-sur-face antigens with monoclonal antibodies prepared against hu-man monocytes and other leukocytes. As can be seen in Table1, HL-60 cells, whether control or treated with 1,25(OH)2D3or Me2SO, contain antigens recognized by antibodies MMA (20)and GAP 8.3 (21), which is consistent with the broad reactivityof these reagents. A different pattern of staining is apparentwith those antibodies that recognize antigens more restrictedin distribution. For example, both forms of treatment lead toacquisition of HLA-D/DR, as assessed by L243 (22). Similarly,untreated cells are Mac-120 negative (23), whereas inductionleads to 81% positively in vitamin D3-treated cells and 24% pos-itivelyin Me2SO-stimulated cells. The fact many of the lattercells are recognized by a reportedly monocyte-specific anti-body suggests that induction by Me2SO leads to incompletecommitment to the granulocyte pathway. On the other hand,the observation that 63D3 recognizes a sizeable proportion ofboth 1,25(OH)2D3 and Me2SO-treated cells is consistent withthe original-observation that this antibody reacts with bothgranulocytes and monocytes, although much more avidly withthe latter than the former (24). This difference in avidity is clearlyevident in fluorescence analysis of Me2SO- and 1,25(OH)2D3-

a1)C.)a).)

Cua)

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12-

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0 24 48 72 96Time, hr

'FIG. 2. Growth -and attachment to plastic of 1,25(OH)AD-treated,(., A) and untreated ()-HL-60 cells. *, Total cells; A, adherent cells.Notice that both the curtailment of mitotic activity and the appearance-of adherent cells occur between 24 and 48 hr of incubation. Each pointrepresents the mean of six replicates.

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A. a-Naphthylacetateesterase

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B. Naphthol AS-Dchloroacetateesterase

0

.-

-24 48 72 96Time, hr

FIG. 4. a-Naphthyl acetate (A) and naphthol-AS-D chloroacetate(B) esterase activities in HL60 cells (cytochemical method). Note thatboth the plastic-adherent (A) and nonadherent (A) fractions of the1;25(OH)2D3 (10-8 M-treated HL-60 cells acquire the monocytic marker(A), but not the granulocytic marker (B). Phorbol ester (-) (a knownmonocyte inducer in HL-60) elicits the same differential effect. Di-methyl sulfoxide (Me2SO) (o), which is believed to induce granulocyticdifferentiation in HL-60 cells, enhances the granulocytic marker (B)but not the monocytic (A). -Untreated cells (e) exhibit little of eitheresterase.

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5910 Cell Biology: Bar-Shavit et al. Proc. Natl. Acad. Sci. USA 80 (1983)

Table 1. Analysis of HL-60 surface antigensby immunofluorescence

Reactive cells, %Con- 1,25-

Antibody Specificity trol (OH)2D3 Me2SOMMA Myelomonocytic (20) 100* 100* 87*GAP 8.3 Leukocytic (21) 100 98 96L243 D/DR (22) 0 16 29Mac-120 MO (HLD-DR-) (23) 8 81* 2463D3 Monocytes (24) 1 93* 71JE9t C3 0 21 0T2C5* C4 0 0 0

All antibodies are to human cells or proteins. The HL-60 cells weretreated with the indicated agents and fluorescence was measured. C,complement.* Very bright fluorescence.t Binds to purified human C3 (J. Bednarczyk and J. D. Capra, personalcommunication).

* Binds to purified human C4 (J. Bednarczyk and J. D. Capra, personalcommunication).

treated cells (Fig. 5). Finally, treatment with 1,25(OH)2D3 leadsto the appearance, of an antigen that reacts with J1E9 (anti-hu-man C3) but not T2C5 (anti-C4). This observation not only un-derscores the specificity of the assay but also raises the inter-esting possibility that the steroid stimulates not only the synthesisof C3 but also the expression of C3b receptors. Indeed, vitaminD3-treated but not control cells form rosettes with human com-plement-coated sheep erythrocytes (data not shown).

Bone-Binding and Resorption by Induced HL-60 Cells. Theappearance of some of the characteristics of mature monocytesin 1,25(OH)2D3-treated HL-60 cells is paralleled by a markedenhancement in the ability of this line to bind and resorb bonematrix. For example, the plastic-adherent fraction of treatedcells binds to calvarial surfaces at a rate (not shown) and to alevel (Fig. 6A) comparable to those observed in the "mature"cell line J774.2. On the other hand, untreated HL-60 cells andthe plastic (substrate) nonadherent fraction of 1,25(OH)2D3-treated cells attach poorly to bone. Similarly, 1,25(OH)2D3-treated adherent cells resorb bone matrix to about the sameextent as J774.2, whereas control and nonadherent treated HL-60 cells remain poor resorbers (Fig. 6B). Finally, in contrast tothe properties observed in vitamin D3-treated adherent cells,HL-60 cells induced with Me2SO bind poorly to plastic and bone

340320300

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131211109876543

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1

50 100 150 200 250Fluorescence intensity, channel no.

FIG. 5. Flow cytometry analysis of 63D3 binding to HL-60 cells. *,Control treated with ethanol; o, treated with 1,25(OH)2D3 (10 M, 72hr); A, treated with Me2SO (1.75%, vol/vol, 3 days and 6 days).

A30

00

10. 1

0J774.2 Untreated Nonad- Adher-

HL-60 herent ent

1,25(OH)2D3-treated HL-60

B14

01

J774.2 Untreated Nonad- Adher-HL-60 herent ent

1,25(OH)2D3-treated HL-60

FIG. 6. Bone binding (A) and degradation (B) by HL-60 and J774.2cells. 1,25(OH)2D3 treatment was 48 hr, 10-8 M.

and do not exhibit enhanced resorptive activity (data not shown).Multinucleation in 1,25(OH)2D3-Treated HL-60 Cells. One

of the features that distinguishes osteoclasts from other boneand bone marrow cells is multinucleation. It is of interest,therefore, that 1,25(OH)2D3, an osteoclastogenic agent, elicitsmultinucleation in HL-60 cells. Specifically, after 48 hr of ex-posure, approximately 20% of the nuclei in the substrate-adher-ent fraction and approximately 10% of the nuclei in the non-adherent fraction are in multinucleated cells; by 96 hr, ap-proximately 50% of the nuclei in the adherent fraction are inmultinucleated cells (Fig. 7). On the other hand, there are al-most no multinucleated cells in control populations.

DISCUSSIONIt has been known for many years that 1,25(OH)2D3 plays a ma-jor role in regulating bone resorption, but the mechanisms by

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FIG. 7. Kinetics of multinucleation in HL-60 cells in response to1,25(0H)2D3. HL,60 cells were concentrated by cytospin centrifuga-tion, applied to slides, fixed, and stained. Two hundred cells in tripli-cate slides were scored. *, Control treated with ethanol; A, plastic-ad-herent cells treated with 1,25(OH)2D3 (10-8 M); *, nonadherent cellstreated with 1,25(OH)2D3 (10-8 M).

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Proc. Natl. Acad. Sci. USA 80 (1983) 5911

which the steroid exerts this effect remain poorly understood.Recently, we made the observation that the bone resorptivedefect in Mos isolated from vitamin D-deficient animals couldbe corrected by in vivo, but not in vitro, administration of1,25(OH)2D3 (7). This finding suggested to us that vitamin D3does affect members of the mononuclear phagocyte family (in-cluding osteoclasts) but that the principal site of action is onimmature rather than fully differentiated cells. This view wassupported by the publications of Suda and his collaborators,showing that 1,25(OH)2D3 elicits monocyte differentiation in amouse myeloid tumor cell line (MI) in vitro (13) and demon-strating that the introduction of this compound or la(OH)D3significantly increases the life span of nude or syngeneic micerendered leukemic by the injection of MI cells (25). The lattertherapeutic effect presumably occurs because the vitamin D3metabolites induce maturation and cessation of proliferation ofthe injected cells. Similarly, Suda and colleagues have also re-ported that 1,25(OH)2D3 induces the differentiation of HL-60cells in vitro, concluding that this compound promotes matura-tion along a granulocyte pathway (26, 27). The latter finding is,of course, at variance with the results of the present report andprobably reflects the relative nonspecificity of the criteria usedby these authors to establish cell phenotype (Fc receptors,phagocytosis). We note, however, that three independent ob-jective criteria were used to assess the nature of HL-60 dif-ferentiation in the current study: (i) the acquisition of a-naph-thyl acetate esterase activity, (ii) the synthesis and secretion oflysozyme, and (iii) the appearance of several monocyte-asso-ciated antigenic determinants (recognized by 63D3, Mac-120,and JIE9) on the cell surface. By all three criteria, 1,25(OH)2D3-treated HL-60 cells are monocytic.Of course, more than the response of tumor cells is required

to make a persuasive argument that vitamin D3 is an importantregulator of monocyte/osteoclast differentiation in vivo. To thisend, we call attention to the following additional observationsregarding the action of 1,25(OH)2D3 on nontransformed cells:(i) suppression of MO colony formation by mouse bone marrowcells (CFU-GM) in vitro (unpublished observations), (ii) cor-rection of MO function in vitamin D3-deficient mice (28), and(iii) correction of bone-binding and resorptive deficiencies inperipheral blood monocytes of a patient with malignant osteo-petrosis [assessed, in vitro, after injection of the vitamin intothe patient (unpublished observations)]. It is important to notethat the proliferation-inhibiting and differentiation-inducingeffects of 1,25(OH)2D3 on marrow cells were obtained at phys-iological concentrations of the vitamin D3 metabolite. It seemsto us that the data from the present study considered togetherwith the earlier observations on vitamin D-deficient animalsand the acute administration of the vitamin, make a compellingargument that 1,25(OH)2D3 promotes the differentiation ofmononuclear phagocytes. Because the latter cells are the prob-able precursors of osteoclasts (3, 4), it follows that such actionmight also result in osteoclastogenesis and, as a further con-sequence, increased resorptive activity. In this regard, it is ofinterest that 1,25(OH)2D3 does elicit multinucleation in HL-60cells (Fig. 7), one of the events associated with the establish-ment of the osteoclast phenotype. Finally, it should be notedthat the action of the steroid on differentiation is apparently notrestricted to the mononuclear phagocyte family; alteration inthe proliferation and differentiation of gut mucosal cells in re-sponse to the vitamin has also been reported (29). Thus,1,25(OH)2D3 should, perhaps, be viewed in a more global con-text, i.e., as a general promoter of cell maturation.

Note Added in Proof. McCarthy et al. (31) have recently affirmed that1,25(OH)2D3 induces monocyte differentiation in HL-60 and reporteda similar response in normal human marrow cells.

We thank Drs. John Bednarczyk and J. Donald Capra of the Uni-versity of Texas Health Science Center at Dallas for the gift of anti-bodies against human complement components; Dr. Howard Raff ofthe University of Washington, Seattle, for the gift of Mac-120; Dr.Milan Uskokovic of Hoffmann-LaRoche for the gift of vitamin D me-tabolites; and Dr. E. Hubermann of Argonne National Laboratories forHL-60. Hybridomas secreting MMA, GAP 8.3, and 63D3 were ob-tained from the American Type Culture Collection. This work was sup-ported by National Institutes of Health Grants DE05413 and DE-04629.Z.B.-S. is a recipient of an Arthritis Foundation Investigator Award.P.R. is a Fellow of the Netherlands Organization for the Advancementof Pure Research (Z.W.O.).

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Cell Biology: Bar-Shavit et al.

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