JOURNAL OF BONE AND MINERAL RESEARCH Volume 5, Number 7, 1990 Mary Ann Liebert, Inc., Publishers

Prostaglandin EZInhibits Formation of Osteoclastlike Cells in Long-Term Human Marrow Cultures but Is Not a Mediator of the Inhibitory Effects of Transforming Growth Factor ,6 C. CHENU,'.' N. KURIHARA,' G.R. MUNDY,' and G.D. ROODMAN'

ABSTRACT Prostaglandins are important local regulators of bone cell function and have been shown to have multiple effects on osteoclasts. Using a human bone marrow culture system in which multinucleated cells with osteoclast characteristics form, we have recently shown that TGF-P is a potent inhibitor of osteoclastlike cell formation and appears to act at several stages of their development. Because it has been suggested that the effects of TGF-/3 are mediated via a prostaglandin-dependent mechanism, we determined the effects of prostaglandin E, (PGE,) on total and osteoclastlike cell formation (detected by reactivity with the 23c6 monoclonal antibody, which identifies osteoclasts) in human marrow cultures and tested whether prostaglandin synthesis was responsible for the inhibitory effects of TGF-/3 on multinucleated cell formation. These studies show that PGE, is a potent inhibitor of both 23c6-positive and negative multinucleated cell formation in human marrow cultures and that the effects of TGF-P on multinucleated cell formation are not mediated by PGE,

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INTRODUCTION local regulators of bone cell function and have been shown to have multiple effects on osteoclasts. Prostaglandin E, (PGE,) stimulates bone resorption in organ cultures of fetal rat long bones") and in mouse calvariae.'2) Chambers et al. have shown a direct but transient inhibitory effect of PGE, on isolated osteoclasts. ' 3 . 4 ) These workers found that PGE, inhibited the motility of mature rat and human osteolcasts. Using a human bone marrow cultures system in which multinucleated cells (MNC) with osteoclast characterist i c ~ ' ~form ) (multinucleation, appropriate responses to osteotropic factors, contraction in response to calcitonin, formation of resorption lacunae on calcified matrices, and cross-reaction with the 23c6 monoclonal antibody, which

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ROSTAGLANDINS ARE IMPORTANT

identifies osteoclasts), we have recently shown that transforming growth factor fi (TGF-@, which may be an imporis a potent inhibitant regulator of bone tor of multinucleated cell formation and appears to act at multiple stages of their development. TGF-P mainly inhibits the proliferation of the mononuclear precursors and, to a lesser extent, inhibits fusion of these precursors to form multinucleated cells. (I2) TGF-/3 also inhibits osteoclastic bone resorption in fetal rat long bone cultures, probably by inhibiting the proliferation of osteoclast progenitors. ( I 3 ) This effect on bone resorption is independent of prostaglandins. However, Tashjian et al. reported that TGF-P stimulates bone resorption in neonatal mouse calvariae via a prostaglandin-mediated mechanism. ( I 1 ) Further, prostaglandin production has been suggested to be the mediator for the bone-resorbing effects of epidermal

'Research Service and Geriatric Research, Education and Clinical Center of the VA Medical Center and the University of Texas Health Science Center, San Antonio, TX. 2Present address: 53 Rue Bossuet, 69006 Lyon, France,

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growth factor and tumor necrosis factor a in mouse calvariae. ( 1 4 . I S ) Since the role of prostaglandins in osteoclast biology is confusing and the relationship between TGF-/3 and prostaglandins in human osteoclast function unclear, we determined the effects of PGE, on total and osteoclastlike multinucleated cell formation in human bone marrow cultures and tested whether PGE, synthesis was responsible for the inhibitory effects of TGF-/3 on MNC formation. Osteoclastlike cells were identified by reactivity with the 23c6 monoclonal antibody, which preferentially binds to osteoclasts. These studies showed that PGE, is a potent inhibitor of both 23c6-positive and 23c6-negative MNC formation in human marrow cultures and that the effects of TGF-/3 on MNC formation are not mediated by PGE, synthesis. These data further suggest that the effects of PGE, on osteoclast formation differ in rodents and primates.

lated by density gradient centrifugation on Ficoll-Hypaque (Histopaque-1077, Sigma, St. Louis, MO) as previously described.'16) Cells were then cultured in a-MEM containing 20% horse serum (Sterile Systems, Inc., Logan, UT) at lo6 cells per ml in 24-well plates (Linbro, Flow Laboratories, McLean, VA). Cultures were maintained in a humidified atmosphere of 4% CO, and air at 37OC for 3 weeks and fed weekly by removing half the medium and replacing it with an equal volume of fresh medium. Factors were added to the cultures at day 1 and after each medium change (unless otherwise noted in the text). At the end of the culture period, the cells were fixed with 5% glutaraldehyde (Sigma, St. Louis, MO) and stained with either Wright's Giemsa stain or tested for their reactivity with the 23c6 monoclonal antibody (generously provided by Dr. Michael Horton, St. Bartholomew's Hospital, London), which preferentially identifies osteoclasts~'7-19) as previously described. (,O) Cells containing at least three nuclei were scored as multinucleated cells with an inverted microscope. Supernatants from treated and control human bone MATERIALS AND METHODS marrow cultures at 1 , 2 , and 3 weeks were assayed for both PGE, and indomethacin were obtained from Sigma PGE, and PGE, using the commercially available Seragen Chemical Co. (St. Louis, MO). TGF-P from bovine de- radioimmunoassay (Boston, MA). Samples were tested unmineralized bone was generously provided by Dr. Saied diluted in duplicate and expressed as nanomoles per liter Seyedin (Collagen Corp., San Francisco, CA). 1,25-Dihy- using B/Bo. Statistical significance was evaluated by using droxyvitamin D, [ 1,25-(OH),D3] was a gift from Dr. Usko- a two-way analysis of variance for repeated measures. Differences were considered significant for p < 0.05. kovic (Hoffman La Roche, Nutley, NJ). After obtaining informed consent, bone marrow was aspirated from the posterior superior iliac crest of normal donors. Marrow was aspirated into a syringe cointaining RESULTS lo00 u/ml of preservative-free heparin (Sigma, St. Louis, MO) in a minimum essential medium (a-MEM, GIBCO, Figure 1 demonstrates the effects of PGE, on total Grand Island, NY). Marrow mononuclear cells were iso- multinucleated cell formation and on multinucleated cells

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PGE2 FIG. 1. Inhibition of multinucleated cell formation by PGE2. Human bone marrow mononuclear cells were cultured with 1,25-(OH),D3 (lo-* M) for 3 weeks in the presence or absence of 10-6-10-9M prostaglandin E,. The marrow cultures were maintained as described in Materials and Methods. Results are reported as the number of total and 23c6-positive multinucleated cells per culture and represent the mean f SEM of four cultures for a typical experiment.

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PGE, INHIBITS OSTEOCLAST FORMATION reactive with the 23c6 monoclonal antibody (23~6-positive MNC) in cultures stimulated by M 1,25-(OH),D3. In previous studies we have shown that MNC formation is enhanced by 1,25-(OH)2D3.'5~21) Addition of PGE, (10-6-10-8 M) for the entire culture period significantly inhibited the formation of total and 23c6-positive MNC in a dose-dependent manner. PGE, at M almost completely suppressed the formation of MNC stimulated by 1,25(OH),D,. The percentage of 23c6-positive MNC present in marrow cultures treated with (10-6-10-8 M) PGE, was similar to the percentage of 23c6-positive MNC in control cultures. To determine the time course for the effects of PGE, on MNC formation, PGE, (lo-' M) was added only during week 1, 2, or 3 of culture (Fig. 2). Addition of PGE, during only week 1 of culture inhibited both total and 23c6positive MNC formation. Inhibition of MNC formation was also seen when the cultures were exposed to PGE, during only week 2 of culture but was not consistently observed when the cultures were treated with PGE, during the last week.

PGE, (lo+ M) decreased the number of nuclei per MNC in human marrow cultures. MNC formed in cultures treated with 1,25-(OH),D3 contained 11 f 2.6 nuclei per MNC (mean f SD, n = 50) compared to 3 f 0.5 nuclei per MNC in cultures treated with PGE, and 1,25-(OH),D3 @ < 0.005). We then determined whether the inhibitory effects of TGF-@on MNC formation were due to prostaglandin synthesis. As we have previously reported, TGF-@(1 nglml) significantly inhibited MNC formation (Fig. 3). Addition of indomethacin at a concentration of loe6M for the entire 3 weeks of culture decreased MNC formation but did not block the inhibitory effects of TGF-/3 on total or 23c6-positive MNC formation, even though it decreased prostaglandin synthesis. Cultures treated with TGF-/3 alone contained 1.3 0.4 nM PGE, and 0.75 0.3 nM PGE,. Addition of indomethacin M) to these cultures decreased prostaglandin levels to 0.9 f 1 nM PGE, and 0.4 f 0.1 nM PGE,. Similar levels of PGE, and PGE, were present in cultures treated only with 1,25-(OH),D3 alone.

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DISCUSSION

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Treatment of human long-term marrow cultures stimulated with 1,25-(OH),D3 with PGE, for 3 weeks markedly inhibited the formation of both total and 23c6-positive MNC. PGE, appeared to inhibit 23c6-positive and 23c6negative MNC to a similar extent since the percentage of 23c6-positive MNC present in cultures treated with PGE,

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Treatment FIG. 2. Effects of adding prostaglandin E, to human marrow cultures for only week 1, 2, or 3 of culture. Human bone marrow mononuclear cells were cultured with 1,25-(OH),D, M) for 3 weeks as described in Materials and Methods. In selected experiments, lo-' M prostaglandin E, was added for only week 1 (treatment A), week 2 (treatment B), or week 3 (treatment C) of culture. After prostaglandin exposure the media were completely changed, and the cells washed and new growth media added. Nonadherent cells removed by the washes were replaced when the new growth media were added. The results represent the number of total and 23c6-positive multinucleated cells per culture for cultures done in quadruplicate for a typical experiment. * p < 0.05 compared to control cultures treated with 1,25-(OH),D3 for 3 weeks. **p < 0.01 compared to control cultures treated with 1,25(OH),DJ for 3 weeks.

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FIG. 3. Inhibition of multinucleated cell formation by TGF-0 in the presence or absence of indomethacin. Bone marrow mononuclear cells were cultured with 1,25(OH),D, (lo-' M) for 3 weeks. In selected experiments, TGF-/3 (1 ng/ml) was added to the cultures in the presence or absence of M indomethacin. Cultures containing indomethacin are shown by the slashed bars, and cultures lacking indomethacin are shown by the open bars. Results are reported as the mean f SEM for four cultures for a typical experiment. (A) Total MNC; (B) 23c6-positive MNC.

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was similar to that in control cultures. These data demonACKNOWLEDGMENTS strate that PGE, can inhibit the formation of MNC which express the osteoclast phenotype, but this inhibition is not Supported by Research Funds from the Veterans Adspecific for osteoclastlike cells. This conclusion is based on ministration, Grant AM35188 from the National Institutes the observations that the 23c6 monoclonal antibody reacts of Arthritis, Diabetes, and Digestive and Kidney Disease, only with osteoclasts in bone sections, reacts strongly with and Grant CA-40035 from the National Cancer Institute. bone-derived o s t e o c l a s t ~ , (does ~ ~ ) not react with macro- Dr. Roodman is a recipient of a Clinical Investigator phage polykaryons, and does not react strongly with Award from the Veterans Administration Research Sermonocytes, macrophages, ( I B or ) macrophage polykaryons vice. formed in vitro.(20)The nature of the 23c6-negative MNC in long-term marrow cultures is unclear, but they most likely represent cells not in the osteoclast lineage. For exREFERENCES ample, 23c6-positive MNC express calcitonin receptors but 23cGnegative MNC do not (Kurihara et al., manuscript in 1. Klein DC. Raisz LG 1970 Prostaglandins: Stimulation of preparation). bone resorption in tissue culture. Endocrinology 86:1436PGE, inhibited MNC formation most effectively during 1440. the first 2 weeks of culture and did not consistently affect 2. Tashjian AH, Tice JE, Sides K 1977 Biological activities of MNC formation if added during the last week of culture. prostaglandin analogues and metabolites on bone in organ Further, PGE, decreased the number of nuclei per MNC. cultures. Nature 266545447. 3. Chambers TJ, Ali NN 1983 Inhibition of osteoclastic motility The number of nuclei per MNC should reflect the rate of by prostaglandins I,, E,, E, and 6-0x0-E,. J Pathol 139383fusion of the mononuclear precursors for the MNC. Since 397. few if any multinucleated cells are formed during the first 4. Chambers TJ, Fuller K, Athanasou NA 1984 The effect of week of culture and PGE, decreases MNC formation if it prostaglandins I,, E,, E, and dibutyryl cyclic AMP on the ~.~~ data ~ sugis present for only week 1 of c ~ l t u r e , (these cytoplasmic spreading of rat osteoclasts. Br J Exp Pathol65: gest that PGE, inhibits the growth of mononuclear precur557-566. sors for MNC and affects their subsequent fusion. These 5 . Takahashi N, Kukita T, MacDonald BR, Bird A, Mundy data are consistent with the observations that prostaglanGR, McManus LM, Miller M, Boyde A, Jones SJ, Roodman dins are potent inhibitors of granulocyte-macrophage proGD 1989 Osteoclast-like cells form in long-term human bone marrow but not in peripheral blood cultures. J Clin Invest genitor cell (CFU-GM) the probable early precursor for the MNC formed in these ~ u l t u r e s . ( ~ ~ . ~ ~ 83:543-550. ~ 6. Centrella M, Canalis E 1985 Transforming and nontransThe inhibitory effects of TGF-0 on MNC formation forming growth factors are present in medium conditioned were not mediated through prostaglandin synthesis. Addiby fetal rat calvariae. Proc Natl Acad Sci USA 82:7335tion of indomethacin to marrow cultures treated with 7339. TGF-0 did not block the inhibitory effects of TGF-/3 on 7. Seyedin SM, Thompson AY, Bentz H, Rosen DM, McPherMNC formation. These data are in contrast of those of son JM, Conti A, Siege1 NR, Galluppi GR, Piez KA 1986 Tashjian et al.,("' who showed using a mouse calvarial Cartilage-inducing factor A: Apparent identity to transformsystem that the stimulatory effects of TGF-0 on bone reing growth factor-beta. J Biol Chem, 2615693-5695. sorption were mediated via prostaglandin synthesis. How8. Robey PJ, Young MF, Flanders KC, Roche NS, Kondaiah P, Termine JD, Sporn MB, Roberts AB 1987 Osteoblasts ever, these data are consistent with those of Pfeilschifter et synthesize and respond to transforming growth factor-type 0 al.,(lnlwho found in fetal rat long bone assays that the ef(TGF-0) in vitro. J Cell Biol 105:457-463. fects of TGF-0 were not mediated by PGE,. 9. Centrella M, McCarthy TL, Canalis E 1987 Transforming These data suggest that the effects of PGE, on osteogrowth factor p is a bifunctional regulator of replication and clasts and osteoclastlike cell formation may differ among collagen synthesis in osteoclast-enriched cell cultures from species. Ibbotson et aLC2')have demonstrated that PGE, fetal rat bone. J Biol Chem 262:2869-2874. increased MNC formation in feline marrow cultures. Simi- 10. Pfeilschifter J, Mundy GR 1987 Modulation of type b translarly, Akatsu and coworkers'**)demonstrated recently that forming growth factor activity in bone cultures by osteoPGE, also stimulated the formation of osteoclastlike cells tropic hormones. Proc Natl Acad Sci USA 84:2024-2028. in murine bone marrow cultures. However, Roodman et 11. Tashjian AH, Voelkel EF, Lazzaro M, Singer FR, Roberts AB, Derynck R, Winkler ME, Levine L 1985 (Y And 0 human al.(29) could not show any stimulatory effects of PGE, on transforming growth factors stimulate prostaglandin producmultinucleated cell formation in a baboon marrow culture tion and bone resorption in cultured mouse calvaria. Proc system. The basis for the differences between the action of Natl Acad Sci USA 82:4535-4538. PGEl in primate and rodent systems may reflect differences in the cellular composition of rodent and primate 12. Chenu C, Pfeilschifter J, Mundy GR, Roodman GD 1988 Transforming growth factor beta inhibits formation of marrow cultures. Alternatively, the effects of PGE, may osteoclast-like cells in long-term human marrow cultures. be species specific such that PGE, directly inhibits human Proc Natl Acad Sci USA 855683-5687. osteoclast precursors but stimulates murine precursors. 13. Pfeilschifter JP, Seyedin S , Mundy GR 1988 Transforming Further studies are required to distinguish among these growth factor-@ inhibits bone resorption in fetal rat long and other interpretations of these data. bone cultures. J Clin Invest 82:680-685.

PGE,INHIBITS OSTEOCLAST FORMATION 14. Tashjian AH, Levine L 1978 Epidermal growth factor stimulates prostaglandin production and bone resorption in cultured mouse calvariae. Biochem Biophys Res Commun 85: 966-975. 15. Tashjian AH, Voelkel EF, Lazzaro M, Goad D, Bosma T, Levine L 1987 Tumor necrosis factor-a (cachectin) stimulates bone resorption in mouse calvaria via a prostaglandin-mediated mechanism. Endocrinology 120, 2029-2036. 16. Takahashi N, Mundy GR, Roodman GD 1986 Recombinant human gamma interferon inhibits formation of osteoclastlike cells by inhibiting fusion of their precursors. J Immunol 137: 3544-3549. 17. Horton MA, Chambers TJ 1986 Human osteoclast-specific antigens are expressed by osteoclasts in a wide range of nonhuman species. Br J Exp Pathol 67:95-104. 18. Horton MA, Lewis D, McNulty K, Pringle JAS, Chambers TJ 1985 Monoclonal antibodies to osteoclastomas (giant cell bone tumors): Definition of osteoclast-specific antigens. Cancer Res 455663-5669. 19. Davies J, Warwick J , Totty N , Philp R, Helfrich M, Horton M 1989 The osteoclast functional antigen, implicated in the regulation of bone resorption, is biochemically related to the vitronectin receptor. J Cell Biol 109:1817-1826. 20. Kukita T, McManus LM, Miller M, Civin C, Roodman GD 1989 Osteoclast-like cells formed in long-term human bone marrow cultures express a similar surface phenotype as authentic osteoclasts. Lab Invest 60532-538. 21. MacDonald BR, Takahashi N, McManus LM, Mundy GR, Roodman GD 1987 Formation of multinucleated cells which respond to osteotropic hormones in human long-term marrow cultures. Endocrinology 120:2326-2333. 22. Broxmeyer HE, Williams DE, Lu L, Vadhan S , Cooper S , Bicknell DC, Ralph P, Gutterman J, Tricot G 1987 Biomolecules associated with suppression of myelopoiesis in normal conditions and during myeloid leukemia and other related disorders. In: Najman A, Guigonm M et al. (eds) The Inhibitors of Hematopoiesis, John Libby Eurotext, Paris, Vol. 162. pp. 139-149. 23. Pelus LM, Broxmeyer HE, Kurland JI, Moore, MAS 1979 Regulation of macrophage and granulocyte proliferation:

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Specificities of prostaglandin E and lactoferrin. J Exp Med 150277-292. Pelus LMM, Broxmeyer HE, Clarkson BD, Moore MAS 1980 Abnormal responsiveness of colony forming cells from patients with chronic myeloid leukemia to inhibition by prostaglandin E. Cancer Res 40:2512-2515. Kurihara N, Chenu C, Civin C, Roodman GD Identification of committed mononuclear precursors for osteoclast-like cells formed in long term marrow cultures. Endocrinology (in press). Chenu C, Kurihara N, Civin C, Roodman GD 1988 CFUGM are the probable precursors for osteoclasts (abstract). J Bone Min Res 3:128A. Ibbotson KJ, Roodman GD, McManus LM, Mundy GR 1984 Identification and characterization of osteoclast-like cells and their progenitors in cultures of feline marrow mononuclear cells. J Cell Biol 99:471-480. Akatsu T, Takahashi N, Debari K, Morita I, Murota S , Nagata N, Takatani 0, Suda T 1989 Prostaglandins promote osteoclast-like cell formation by a mechanism involving cyclic adenosine 3’,5’-monophosphate in mouse bone marrow cell cultures. J Bone Min Res 4:29-35. Roodman GD, Ibbotson KJ, MacDonald BR, Kuehl TJ, Mundy GR 1985 1,25-Dihydroxyvitamin D, causes formation of multinucleated cells with several osteoclast characteristics in cultures of primate marrow. Proc Natl Acad Sci USA 82: 8213-8217.

Address reprint requests to: G. David Roodman, M.D., Ph.D. Research Service (151) VA Hospital 7400 Merton Minter Blvd. San Antonio, TX 78284

Received for publication May 1, 1989; in revised form December 13, 1989;accepted January 18, 1990.

Prostaglandin E2 inhibits formation of osteoclastlike cells in long-term human marrow cultures but is not a mediator of the inhibitory effects of transforming growth factor beta.

Prostaglandins are important local regulators of bone cell function and have been shown to have multiple effects on osteoclasts. Using a human bone ma...
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