JOURNAL OF BONE AND MINERAL RESEARCH Volume 6, Number 2, 1991 Mary Ann Liebert, Inc., Publishers

on the Effects of Transforming Growth Factor Regulation of Osteoclastic Development and Function GARY HATTERSLEY and TIMOTHY J . CHAMBERS

ABSTRACT Transforming growth factor (TGF) PI is a multifunctional cytokine with powerful effects on osteoblastic cells. Its role in the regulation of osteoclast generation and function, however, is unclear. It has been reported both to stimulate and to inhibit resorption in organ culture and to inhibit multinuclear cell formation in bone marrow cultures. We tested the effects of TGF-6, on bone resorption by osteoclasts isolated from neonatal rat long bones. We found potent stimulation of osteoclastic bone resorption, mediated by osteoblastic cells, with an ECs, of 10 pg/ml, considerably lower than that of well-documented osteotropic hormones. Stimulation was not mediated by Swiss mouse 3T3 cells, a nonosteoblastic cell line. TGF-0, strongly inhibited the generation of calcitonin receptor (CTR)-positive cells in mouse bone marrow cultures, but as for isolated osteoclasts, bone resorption per CTR-positive cell was increased. The inhibition of CTR-positive cell formation was associated with suppression of maturation of other bone marrow derivatives and may be related more to the known ability of TGF-PI to suppress the proliferation of primitive hematopoietic cells than to a specific role of TGF-PI in osteoclast generation.

INTRODUCTION

0 (TGF-0) modulates the growth and differentiation of a wide variety of cell types.“’ It may play an important role in bone physiology: it is produced in bone,(’ 3, by bone cells,(4‘) and bone is the largest tissue source.(b71It has potent but often discrepant effects on osteoblastic proliferation, differentiation, matrix synthesis, and bone formation, both in vivo and in vitr0.1~s.8-13) In contrast to the extensive literature on the modulation of osteoblastic function, there is little information on the effects of TGF-0, on osteoclastic bone resorption. What there is is contradictory but suggests powerful effects: organ cultures of neonatal mouse calvariae show stimulation of resorption,(’4)but fetal rat long bones show the opposite.(”’ It has also been reported that TGF-8, inhibits the formation of multinucleate cells in liquid bone marrow cultures. ( I 6 ’ The organ culture experiments are difficult to interpret in terms of osteoclastic regulation, not only because they

T

RANSFORMING GROWTH FACTOR

are contradictory but because osteoclast regulation is often indirect in such systems: the different results may then be due to differences in the cellular constitutions of the two organ culture systems. An analysis of the mechanisms by which TGF-0, regulates bone resorption in organ culture would be of particular interest since osteoblastic cells, which exert a major influence on osteoclastic function,“7‘’91 not only respond to TGF-0, but synthesize and TGF-0, is released from resorbing bone(2o’: TGF-0, may thus be involved in the regulation of osteoclasts by osteoblasts. Regulation of bone resorption in vivo is generally associated not only with changes in the activity of osteoclasts already present, but by changes in osteoclast number. The effects of TGF-0, on the generation of multinucleate cells has been described.‘L6’However, two phenotypically distinct types of multinuclear cells are generated in bone marrow cultures: multinucleate macrophages and osteoclasts.‘”~’*) Multinuclear cell numbers are not a reliable guide to osteoclast generation since not all multinuclear cells are osteoclasts and not all osteoclasts are multinu-

Department of Histopathology, St. George’s Hospital Medical School, London, UK.

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multiwell dish containing bone slices. After 15 minutes sedimentation the bone slices were removed, washed in medium 199, and incubated overnight in a Nunclon multiwell tissue culture plate (GIBCO) in the presence or absence of bovine parathyroid hormone (PTH; Dr. Zanelli, Hampstead, UK) or human TGF-@, (Calbiochem, San Diego) in incubation medium (MEM + FBS). Bone resorption was assessed as described later. Similar experiments were performed using osteoclast suspensions obtained after a prolonged sedimentation period (35 minutes). This modification produces cultures containing increased numbers of osteoblastic cells'26)and restores osteoclastic responsiveness t o PTH. To further MATERIALS AND METHODS test the indirect responsiveness of osteoclasts to TGF-@,, HEPES-buffered medium 199 (Flow Laboratories, Ir- osteoclasts obtained after a short sedimentation time were vine, UK) was used for cell isolated and sedimentation. cocultured with either a cloned, osteoblastlike UMR-106 Hanks-buffered Eagle's minimum essential medium cells (Dr. T.J. Martin, Melbourne, Australia) or Swiss (MEM; GIBCO, Paisley, UK) was used for subsequent in- mouse embryo 3T3 cells (Flow; 5 x lo4 cells per ml) on cubation, supplemented with 100 IU/ml of benzylpenicillin bone slices in the presence or absence of TGF-0,. Bone re(GIBCO), 100 &ml of streptomycin (GIBCO), 2 mM glu- sorption was assessed after incubation as described subsetamine (GIBCO), and 10% heat-inactivated fetal bovine quently. serum (FBS; GIBCO). Incubations were performed at 37°C in a humidified 5% CO, atmosphere. Bone marrow cell cultures Slices of bovine cortical bone were employed as subCBA/ca mice (6-8 weeks old) were killed by cervical disstrates for osteoclastic resorption. Bone slices (2 x 2 x 0.1 mm) were prepared from adult bovine femora using a location. Femora and tibiae were aseptically removed and low-speed saw (Buehler, Lake Bluff, IL) as previously de- dissected free of adherent tissue. The bone ends were cut scribed,lZs)cleaned by ultrasonication, washed, and stored and the marrow cavity flushed out with medium 199 by slowly injecting medium at one end of the bone using a dry at room temperature. sterile 25 gauge needle. The bone marrow cells were washed twice and suspended ( 2 x lo6 cells per ml) in Isolation of osteoclastsfrom neonatal rat long bones M hydrocortiMEM supplemented with 10% FBS and Osteoclasts were disaggregated, as previously described, sone (Sigma, Poole, UK). Cultures were supplemented frm neonatal rat long bones.("' Male Wistar rats from the with hydrocortisone to improve hematopoiesis and reduce St. George's Medical School colony were decapitated variability between batches of FBS.I*') This suspension within 48 h of birth. The long bones were removed and was placed (200 pl/ml) in the wells of multiwell plates, dissected free of adherent tissue. Bones were then cut each well of which contained a 6 mrn Thermanox coverslip across their epiphyses, curetted in medium 199, and the (Lux, Flow) and a slice of bovine cortical bone. The culcurettings vigorously agitated with a plastic pipette. Large tures were incubated in the presence of TGF-@, or vehicle. fragments were allowed to sediment for 10 s. The cell sus- 1,25-Dihydroxyvitamin D, [ 1,25-(OH),D,; Roche, Welwyn pension was then transferred to a 200 x 200 mm Sterilin Garden City, UK] or vehicle were added from day 7. Culclear; osteoclast generation measured as multinuclear cells can diverge even in direction from osteoclast generation,Iz3') as judged by generation of CTR-positive cells and resorptive function. We therefore elected to test the effect of TGF-P, on osteoclasts isolated from neonatal rat long bone and to test the effects of TGF-@, on osteoclast generation, as measured by the development of resorptive function and calcitonin receptors, in an attempt to identify the role of TGF@,in the regulation of osteoclastic bone resorption.

TABLE l . EFFECTOF TGF-0, AND P T H ON BONERESORPTION BY ISOLATEDOSTEOCLASTS~

oc OC OC OC OC OC

+ PTH, + TGF-PI,

40ng/ml lOOOpg/ml + TGF-@,, 100pg/ml + TGF-P,, 10pg/ml + TGF-PI, 1 pg/ml

Plan area of bone resorbed per bone slice (Fm' x lo-=, mean f SEM)

No. pits per bone slice (mean + SEM)

9.6 f 2.8 8.7 f 1.7 9.7 f 0.7 11.1 f 2.1 10.5 f 1.3 10.6 f 1.9

12.5 f 2.0 11.4 f 1.1 10.8 f 2.0 9.5 f 1.4 9.7 f 0.8 10.2 f 0.9

aOsteoclasts were sedimented onto bone slices for 15 minutes. After incubation overnight the bone resorption was quantified in the scanning electron microscope. Each figure is derived from 12 bone slices in two experiments.

167

REGULATION OF OSTEOCLASTS BY TGF-PI tures were fed every 3 days by replacing 100 yl culture medium with fresh medium and hormone or vehicle. No attempt was made to replace nonadherent cells. After incubation bone slices were prepared for scanning electron microscopy and the coverslips for 'zSI-labeled salmon calcitonin ([1z51]sCT)autoradiography, as described later. To test the effect of TGF-0, on the production of monocytes and polymorphonuclear leukocytes, bone marrow cells isolated as described subsequently were cultured in 25 cm' tissue culture flasks (2 x lo6 ml; Falcon) in 10 ml growth medium in the presence of TGF-PI or vehicle and 1,25-(OH),D, or vehicle at day 7. Cultures were fed weekly by removing all of the growth medium and replacing with fresh medium and hormones or vehicle. The nonadherent cells removed during feeding were counted using a hemocytometer. Cytocentrifuge preparations (Cytospin, Shandon, Cheshire, UK) were prepared to allow assessment of morphology after staining (May-Grunwald Giemsa) by standard hematologic criteria. ( z 8 1

Measurement o$ bone resorption Cells were removed from the bone slices by immersion in 10% sodium hypochlorite (BDH, Poole, Dorset, UK) for 10 minutes. The bone slices were then dehydrated in ethanol, sputter coated with gold, and examined in a Cambridge S90 scanning electron microscope (Cambridge Instruments, Cambridge, UK). The extent of bone resorption was measured blind by examining the whole surface of each bone slice for resorption lacunae. Resorption by bone marrow cells was quantified using a 1 cm' grid at x200 screen magnification. Resorption by isolated osteoclasts was quantified by measuring the number of osteoclastic excavations and the plan area of bone resorbed, as previously described.

/''sIJsCT autoradiography Salmon calcitonin (sCT; Sandoz Ltd, Basel, Switzerland) was iodinated using a modification of the chloroamine-T method,"" as previously de~cribed."~'labeled sCT (1 nM) was incubated with cultures in HEPES-buffered medium 199 with 0.1% bovine serum albumin (Sigma) for 1 h at 22°C. Nonspecific binding was assessed by incubating some cultures with labeled sCT and excess (300 nM) unlabeled sCT. After incubation the cells were fixed by immersing the coverslips in formalin for 30 s before extensive washing in water. The coverslips were coated with K5 nuclear emulsion (Ilford, UK), developed after 14 days at 4°C and counterstained with Mayer's hemat oxylin. Calcitonin-receptor (CTR)-positive cells were scored as those that demonstrated sufficient grain density to outline the cell clearly. The number of CTR-positive cells present in 10 random fields per coverslip at x 100 magnification was counted. Differences between groups were analyzed by either Student's [-test or a Bonferroni correction using analysis of variance, where significance was determined at 95% by Gabriel's test.

RESULTS In osteoclastic populations obtained after a short (15 minute) sedimentation time (which minimizes contamination by nonosteoclastic cells and consequent indirect hormonal responses) we found that neither TGF-P, nor PTH showed any effect on bone resorption (Table 1). We have previously found that more prolonged sedimentation of bone cells is associated with the adhesion of greater numbers of nonosteoclastic, predominantly alkaline phospha-

1

0.5

FIG. 1. The effect of TGF-0, on bone resorption by osteoclasts obtained after prolonged sedimentation of bone cells onto bone slices. Each point (mean f standard error of mean) is derived from 12 bone slices in two experiments. *Test SEM number of excavations per bone slice in control bone slices, 7.4 f 1.3; versus control significant at 95%. Mean mean plan area resorbed per bone slice, 10.1 1.7 x lo-, pmz.

*

*

HATTERSLEY AND CHAMBERS

168

Since TGF-P, stimulates bone resorption by mature tase-positive bone cells onto the bone slices and with restoration of the hormone responsiveness of intact bone.1z61 osteoclasts in the presence of osteoblastic cells, it seemed Under these circumstances TGF-0, strongly stimulated possible that the enhanced bone resorption in bone marosteoclastic bone resorption in the range 10-lo00 pg/ml row cultures may have been due not to increased osteoclast (Fig. 1) to a level similar to that seen in the presence of generation, but to TGF-P,-induced stimulation of osteoclasts by osteoblastic cells, which are known to be present PTH.Iz6) Similar experiments were performed in which osteoclas- in such bone marrow cultures. We therefore used CTR tic populations obtained after a short sedimentation were positivity, a marker specific for osteoclasts among bone incubated with or without added UMR-106 cells. Without marrow cells, which correlates with osteoclast formaUMR-106 cells bone resorption was unresponsive to PTH; tion,I3’) to assess the number of cells of osteoclastic in the presence of UMR-106 cells PTH stimulated bone re- phenotype formed in the presence of TGF-0,. The number sorption and TGF-@, induced significant stimulation of of such cells was considerably reduced by TGF-6, at 100 bone resorption at 100 and lo00 pg/ml (Fig. 2). When 3T3 and lo00 pg/ml (Fig. 4) without change in the number of cells were substituted for UMR cells there was no stimula- nuclei per CTR-positive cell (Table 2). Enumeration of the tion of bone resorption in the presence of TGF-P, (lo00 number of mature hematopoietic progeny formed by the pg/ml; plan area of bone resorbed, n = 6 in pmz x lo-,? * bone marrow cultures showed that the generation of macSEM per bone slice: 3T3 + osteoclasts, 76.9 25.6; 3T3 rophages and granulocytes was also markedly reduced by + osteoclasts + TGF-PI, 61.9 f 17.4). The results suggest TGF-0, (Fig. 5 ) . that TGF-P, is a potent stimulator of bone resorption and that it acts, like other agents that stimulate bone resorpDISCUSSION tion, through the intermediary of cells of osteoblastic phenotype. We found that osteoclasts isolated from neonatal rat We have found that incubation of mouse bone marrow cells in the presence of 1 ,25-(OH),D3 for 7-14 days is asso- long bone and incubated on bone slices showed n o differciated with the generation of CTR-positive cells and re- ence in bone resorption in the presence or absence of TGFsorptive function in the TGF-0, (100 and 10 0,.When the culture conditions were altered such that pg/ml) increased the extent of resorption of bone slices in- greater number of nonosteoclastic bone cells were allowed cubated with the bone marrow cells in the presence of 1,25- to adhere to the bone slices, TGF-P, stimulated bone re(OH),D,, although the cytokine (lo00 pg/ml) did not itself sorption in the range 10-lo00 pg/ml. This compares with induce resorptive function in the absence of 1,25-(OH),D, stimulation seen in the range 200-2000 pg/ml previously and is consistent with the (Fig. 3). Furthermore, in a separate experiment, 10 and described in organ 100 pg/ml of TGF-6, failed to induce resorption (control greater sensitivity of the bone slice assay compared to 1.2 + 0.9; 10 pg/ml of TGF-PI, 0.7 f 1.0; 100 pg/ml of organ culture systems observed using other hormones, to TGF-PI, 2.3 f 1.7; results derived from six cultures on which the bone slice assay shows responsiveness within or dose to the physiologic range.(2s.26.331 With 50% of maxiday 14 and expressed as mm2/cm’, mean f SEM).

*

*

20 -

* T

*

ii O UMR c

oc + PTH

oc +

Oc

+

TGFR UMR 1000pg/ml + PTH

OC

UMR

+

OC

UMR

+

TGFR TGFB TGFR 1000pg/ml lOOpg/ml 1Opglml

FIG. 2. The effect of TGF-0, on bone resorption by osteoclasts and by osteoclasts cocultured with UMR-106 cells. Each point derived from 12 bone slices in two experiments. *Test versus control significant at 95vo. Mean f SEM number of excavations per bone slice in control bone slices, 7.4 + 1.3; mean plan area resorbed per bone slice, 10.1 f 1.7 x pmz.

169

REGULATION OF OSTEOCLASTS BY TGF-(3,

1 70 60 -

50

-

40 -

30 20 10 -

'T(;FpI 1000pglml Control

0-

7

21

1'4

Incubation time (days)

FIG. 3. The effect of TGF-P, on bone resorption by cultures of mouse bone marrow cells. Results expressed as mean f standard error of mean. Each point derived from 12 bone slices in two experiments. *Test versus 1,25-(OH),D,l significant at 95%.

I400

-

1200 N

I -

1000 -

U

a

G.-

800

-

600

-

400

-

VI

5

?

g

/n'

T

8 ?

.-EE .-"

-0

2

200 -

0I I

7

14

I

21

Incubation time (davs)

FIG. 4. The effect of TGF-P, on CTR-positive cell number in cultures of mouse bone marrow cells. Results expressed as mean f standard error of mean. Each point derived from 12 cultures (two experiments). *Test versus 1,25-(OH)zD1 significant at 95%.

ma1 stimulation occurring at 10 pg/ml, TGF-(3, exceeds IL-1 (ECso 50 ng/ml), TNF-a (2 ng/ml), TNF-/3 (4ng/ml), PTH (50 pg/ml), and 1,25-(OH),D1 (1 ng/ml) in pot e n ~ y . " ~ . ~ ~This - ' ~ 'extreme sensitivity of bone resorption to the presence of TGF-(3, and the magnitude of the response suggest that TGF-(3, may play a major role as a stimulator of osteoclastic bone resorption.

Similarly, osteoclastic cells responded to TGF-PI if osteoblastic cells (UMR-106) were added to the cultures. These results suggest that TGF-(3,. like many other boneresorbing agents (see Ref. 19), has no direct action on osteoclasts but stimulates resorption through a primary interaction with cells of the osteoblastic lineage. Paradoxically, we found that TGF-(3, was essentially in-

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TABLE 2. PROPORTION OF CTR-POSITIVE CELLS CONTAINING ONE,Two TO FIVE,AND SIXOR MORENUCLEI IN CULTURES INCUBATED FOR 7 OR 14 DAYSWITH ~ , ~ ~ - ( O H ) > D J ~ % CTR-positive cells that contained

Incubation conditions

(+ SEM)

Duration of incubation (days) after addition of 1,2S-(OH)ZD,

I Nucleus

7 14 7 14 7 14

78*9 7 0 + 3 92* 4 69*8 71 * 9 66 + 10

2-5 Nuclei 2 0 * 20* 8 * 22* 26 21

* *

8 3 4 5 12 8

6 Nuclei or more 2*2 10 f 6 0 9 + 6 3*4 11 3

*

aEach result is the mean of six cultures. None of the points for cultures incubated with TGF-0, differed significantly from those incubated without TGF-0,.

60

50

T

T

10

** T

*

I

0

FIG. 5. Effect of TGF-0, (lo00 pg/ml) on the number of nonadherent cells in cultures of mouse bone marrow cells. Cytocentrifuge preparations were also prepared and cell types categorized to distinguish between mature macrophages (solid), mature neutrophils (granulocytes, dotted), and other mature hematopoietic progeny (open), such as megakaryocytes and mast cells, including primate cells of indeterminate morphology. * p > 0.05 by Student's 1-test.

hibitory for osteoclast generation from mouse bone marrow cells. By itself it did not induce bone resorptive function or CTR-positive cells to differentiate. However, it reduced the number of CTR-positive cells generated in the presence of 1,25-(OH)*DJ to approximately control levels at 1 ng/mi of TGF-0,. Despite the reduced numbers of CTR-positive cells TGF-P, (10-100 pg/ml) was associated with an increase in the extent of resorption of the surface

bone slices incubated in the bone marrow cultures. It seems that fewer osteoclasts are formed in the presence of TGF-P,, but those formed are stimulated by the cytokine to increased bone resorption. TGF-0, is known to directly inhibit the proliferation of bone marrow cells, in which it has a role in the regulation of cell cycle status of multipotential hematopoietic precursors.136.37) Accordingly we found that the number of mac-

171

REGULATION OF OSTEOCLASTS BY TGF-6, rophages and granulocytes produced in our bone marrow cultures was reduced to a similar extent to CTR-positive cells by TGF-0,. This suggests that the reduced number of CTR-positive cells generated in the presence of TGF-@, may have been a consequence of this general inhibition of hematopoiesis and may not represent a specific inhibition by TGF-0, of osteoclast generation. Many cell types produce TGF-@,,and almost all cell types have TGF-@,receptors (see Ref. 1); TGF-@,is essentially a local hormone that regulates cell interactions in many, often unrelated systems. The role of TGF-0, in the regulation of proliferation of multipotent hematopoietic precursors is unlikely to be part of the mechanism by which bone resorption is regulated: it is difficult to see how TCF-@, could effectively regulate osteoclast formation through inhibition of proliferation of multipotent hematopoietic cells. Such an action would disrupt the production of other hematopoietic progeny, and alterations in hematopoiesis related to other progeny would disrupt the regulation of osteoclast formation. It also seems theoretically unlikely that TGF-0, in the same microenvironment both stimulates the function of mature cells but inhibits differentiation of function. We have not excluded the possibility that, in keeping with its effect on bone resorption by mature osteoclasts, TGF-0, may even cause a relative increase in osteoclastic differentiation, since such an effect would be masked in the liquid marrow culture system through reduction, by TGF-PI, of the size of the pool of hematopoietic cells available for osteoclastic differentiation, an effect likely to be a tissue culture artefact caused by disruption of the compartmentalization of hematopoietic populations that exists in vivo.‘38’ This question needs to be addressed through an assessment of the effect of TGF-@, on osteoclast formation from committed hematopoietic precursors (CFU-C). Although the generation of CTR-positive cells was impaired by TGF-p,, resorption per CTR-positive cell was increased. Since osteoblastic cells are a component of bone marrow cultures,‘.’9)this may represent an osteoblast-mediated stimulation of resorption by bone marrow-derived osteoclasts. Our results may explain the discrepancy between the effect of TGF-@, on bone resorption in organ culture systems f 14.15).. resorption was increased in mouse calvariae but inhibited on prolonged culture of rat long bones. This might be expected if resorption in the latter, which contains a developing bone marrow cavity, were more dependent upon recruitment of osteoclasts from hematopoietic precursors. In keeping with this, TGF-P, inhibition of resorption in long bones was more marked in the later half of prolonged cultures and resembled the effects of hydroxyurea, a potent inhibitor of DNA synthesis.(ls’ Our results suggest that TGF-@, is essentially a stimulator of osteoclastic bone resorption. If so, the inhibition of resorption observed in prolonged incubation of rat long bones may be explicable as due to an effect on hematopoietic cells that is not primarily relevant, as discussed earlier, to the regulation of osteoclastic bone resorption.

ADDENDUM Since this manuscript was submitted, a paper by Shinar and Rodan (Endocrinology 1990 126:3153-3158) has been published that uses TRAP-positive multinuclear cell (MNC) numbers, which the authors find to correlate with CTR-positive cells, to quantify osteoclast formation. Like us they found a biphasic response to TGF-P for TRAPpositive MNC production in the presence of 1,25-(OH),D,. Increased TRAP-positive MNC production seemed dependent upon but not attributable to PG production.

ACKNOWLEDGMENTS This work was supported by the Medical Research Council. We are grateful to Vincent Ang for assistance with the iodination of calcitonin and to Valerie Emmons for typing the manuscript.

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Address reprint requests to: T.J . Chambers Department of Histopathology St. George’s Hospital Medical School Cranmer Terrace London S WI 7 ORE UK Received for publication June 18, 1990; in revised form August 17, 1990; accepted October 1, 1990.