P bone remodeling with the initial pathologic event being. Cell Biology of Paget s Disease

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1 JOURNAL OF BONE AND MINERAL RESEARCH Volume 14, Supplement 2, 1999 Blackwell Science, Inc. Q 1999 American Society for Bone and Mineral Research Cell Biology of Paget s Disease SAKAMURI V. REDDY, CHEIKH MENAA, FREDERICK R. SINGER: ANNE DEMULDER? and G. DAVID ROODMAN *4 ABSTRACT Paget s disease is characterized by markedly increased osteoclast formation and bone resorption followed by excessive new bone formation. Osteoclasts in Paget s disease are increased both in number and size, contain paramyxoviral-like nuclear inclusions, and can have up to 100 nuclei per cell. Marrow culture studies have identified several abnormalities in osteoclast formation in Paget s disease. Osteoclast-like multinucleated cells formed more rapidly in marrow cultures from patients with Paget s disease, produced increased levels of interleukin-6 0, and expressed high levels of IL-6 receptors compared to normals. IE6 levels were also increased in bone marrow and peripheral blood of patients with Paget s disease. In addition, osteoclast precursors from patients with Paget s disease are hyperresponsive to 1,25dihydroxyvitamin D3 (la(oh),d3) and calcitonin. The increased sensitivity of osteoclast precursors to 1,25(OH),D3 is mediated through the vitamin D receptor OR), since 24-hydroxylase activity is also up-regulated at concentrations of l,%(oh),d3 that are one log less than that needed to induce 24-hydroxylase activity in osteoclast precursors from normals. However, VDR numbers and affinity for l,e(oh),d3 do not differ in osteoclast precursors from Paget s patients compared to those from normals. Synergistic interactions between cytokines such as IL-6 and l,s(oh),d3 also cannot explain the enhanced sensitivity of osteoclast precursors from patients with Paget s disease to 1,25(OH),D3. Interestingly, coculture studies of osteoclast precursors and cells from the marrow microenvironment of patients with Paget s disease and normals have demonstrated that the marrow microenvironment is more osteoclastogenic than normal. Thus, studies of the cell biology of osteoclasts in Paget s disease have demonstrated an increased rate of osteoclast formation and abnormalities in both osteoclast precursors and the marrow microenvironment. Enhanced IL-6 praduction by osteoclasts in Paget s disease may further amplify the increased osteoclast formation already ongoing in the pagetic lesion, and may explain the increased bone turnover at uninvolved sites distant from the pagetic lesion. (J Bone Miner Res 1999,14, suppl. 2:M) INTRODUCTION AGET S DISEASE is characterized by grossly distorted P bone remodeling with the initial pathologic event being increased bone resorption.( * ) The greatly increased bone resorption is then followed by abundant new bone formation. The bone is rapidly laid down and is of poor quality. However, the primary cellular abnormality in Paget s disease is in the osteoclast rather than in the osteoblast. Osteoclasts from patients with Paget s disease have several distinct abnormalities that distinguish them from normal. In bone biopsy specimens of bone lesions from patients with Paget s disease, osteoclasts are markedly increased in number and size with the osteoclasts containing up to 100 nuclei per These osteoclasts appear to induce increased bone resorption, although the amount of resorption quantitated per osteoclast nuclei may actually be lower than that seen with normal o~teoclasts.(~) Ultrastructural studies of osteoclastic lesions from patients with Paget s disease have shown that they contain Department of MedicineMematology, University of Texas Health Science Center, San Antonio, Texas, U.S.A. 2John Wayne Cancer Center, Santa Monica, California, U.S.A. Brugmann Hospital, Brussels, Belgium. 4Audie Murphy Veterans Administration Hospital, San Antonio, Texas, U.S.A. 3

2 4 REDDY ET AL. paramyxoviral-like nuclear and cytoplasmic inclusion^,(^.^) and immunocytochemical studies have shown crossreactivity of these inclusions against measles virus nucleocapsid antibodies and respiratory syncytial virus nucleocap- sid antibodies, as well as other related paramyxo~iruses.(*.~) In situ hybridization studies on bone biopsy specimens from patients with Paget s disease have identified both measles virus transcripts and canine distemper virus transcripts in osteoclasts from patients with Paget s disease.( * ) Interestingly, other cells in the bone, in addition to osteoclasts, also express transcripts from measles virus or canine distemper virus (CDV) in these studies. In situ polymerase chain reaction studies have recently reported that 15 out of 15 bone biopsy specimens from patients with Paget s disease express canine distemper virus nucleocapsid messenger RNA, ) while osteoclasts from patients with osteoarthritis show no detectable viral sequences. However, the basis for the increased osteoclast formation and osteoclast activity in patients with Paget s disease has not been clearly defined. MATERIALS AND METHODS We have applied bone marrow culture techniques using bone marrow samples from bones affected with Paget s disease to try to unravel the underlying pathobiology of the osteoclasts in Paget s disease. In these studies, bone marrow was obtained from affected bones from patients with Paget s disease, then cultured in the presence of 1,25- dihydroxyvitamin D, (1,25(OH),D,) to induce osteoclastlike cell formation.( ) Multinucleated cells (MNC) that formed in these cultures expressed an osteoclast phenotype, that is, they reacted strongly with the 23c6 monoclonal antibody that identifies the osteoclast vitronectin re~eptor, ~) expressed tartrate-resistant acid phosphatase (TRAP) and calcitonin receptors, and formed resorption lacunae on calcified matrices.( 3) The osteoclasts formed in these cultures were markedly increased in number and size and had increased nuclear number, with up to 100 nuclei per cell. These MNC also expressed paramyxoviral antigens for measles virus and respiratory syncytial virus,(15) and shared ultrastructural features of osteoclasts isolated from patients with Paget s disease.(16) We have used these marrow culture techniques to examine osteoclasts and osteoclast precursors in Paget s patients. IL-6 and Paget s disease RESULTS An interesting feature of Paget s disease is that bones not clinically involved with the disease show increased bone remodeling.(17) Although several authors have suggested that the increased bone remodeling in bones not clinically involved with Paget s disease may be due to secondary hyperparathyr~idism,( ~~ ) increased parathyroid hormone (FTH) levels have not been consistently found in patients with Paget s disease, and the levels of PTH in Paget s patients in whom elevated PTH levels have been detected are not markedly increased. Therefore, we proposed that pagetic osteoclasts or other cells present in the bone lesions in patients with Paget s disease secreted a stimulatory factor that enhanced osteoclast formation and activity both within the lesion as well as at sites distant from the lesion. To test this hypothesis we obtained bone marrow from involved bones from patients with Paget s disease and cultured it with 1,25(OH),D, for 3 weeks to induce osteoclast formation. At the end of the culture period, the media was removed, serum-free media was added, and the cultures continued for 24 h. This Paget s conditioned media was then used as the only stimulatory factor in normal bone marrow cultures to induce osteoclast formation. Interestingly, when conditioned media from marrow cultures from patients with Paget s disease were used to stimulate osteoclast formation in normal marrow cultures, osteoclast formation was increased in a dose-dependent fashion between 5% and 25% v/v of the Paget s conditioned media. Higher concentrations were toxic to the cultures. In contrast, conditioned media obtained from marrow cultures from normals did not stimulate osteoclast formation under these conditions. We then wanted to identify the factor(s) present in the conditioned media from marrow cultures from patients with Paget s disease that was stimulating osteoclast formation in normal marrow cultures. Therefore, we added neutralizing antibodies to cytokines that were known to induce osteoclast formation, including interleukin-1 (IL-l), interleukin-6 (IL-6), transforming growth factor alpha (TGF-a), and granulocyte macrophage colony stimulating factor (GM-CSF) to normal marrow cultures stimulated with the Paget s conditioned media. ) We found that a neutralizing antibody to IL-6, but not to IL-1, tumor necrosis factor (TNF), or GM-CSF, completely blocked the osteoclast stimulatory activity present in the Paget s conditioned media. Bone marrow cultures contain mixed cell populations and it was unclear if stromal cells, macrophages, or the osteoclast-like cells formed in these marrow cultures from Paget s disease were the primary source for the IL-6 that was being produced. Therefore, we performed in situ hybridization studies to identify the cell types that expressed high levels of messenger RNA for IL-6. We found that the osteoclast-like cells were producing prodigious amounts of IL-6 messenger RNA. We then measured IL-6 levels in the conditioned media from both normal marrow cultures and marrow cultures from patients with Paget s disease and found that the patients with Paget s disease had significantly elevated IL-6 levels in this conditioned media compared to normals.(* ) However, it was unclear if these results were due simply to an in vitro phenomenon or reflective of what was occurring in patients with Paget s disease. Therefore, we obtained bone marrow aspirates and peripheral blood samples from patients with Paget s disease. The cells were separated immediately from the supernatants of the marrow aspirates and the peripheral blood samples after collection, and the supernatants from the marrow and the plasma from the peripheral blood were then tested by enzyme-linked immunosorbent assay (ELISA) for levels of IL-6. We found that

3 OSTEOCLASTS IN PAGET S DISEASE 5 IL-6 levels were markedly increased in the peripheral blood in 17 out of 21 patients with Paget s disease and in 9 out of 10 marrow samples from patients with Paget s disease compared to normals.(20) In fact, IL-6 levels up to 3 ng per ml were detectable in marrow supernatants from patients with Paget s disease, while IL-6 was essentially undetectable in both peripheral blood or marrow samples from normals. Consistent with our findings of IL-6 production by osteoclasts from Paget s patients are the findings of Hoyland and coworkers.(21) They performed in situ hybridization studies of bone biopsy specimens from patients with Paget s disease and patients with osteoarthritis or normal bone. These workers found that IL-6 nuclear factor, as well as IL-6 and IL-6 receptor messenger RNA, were markedly increased in osteoclasts from patients with Paget s disease compared to controls. They proposed that a paramyxoviral infection induces up-regulation of IL-6 and IL-6 receptor in osteoclasts from patients with Paget s disease, in that this IL-6 then acted in a paracrine fashion to stimulate osteoclast formation from osteoclast precursors. We have previously shown that IL-6 could stimulate normal osteoclast formation.( ) Consistent with this model for the up-regulation of IL-6 by paramyxovirus infection of osteoclasts are our preliminary findings in which, as a model for osteoclast precursors, we have infected normal peripheral blood monocytes with measles virus and shown that monocytes infected with measles virus express high levels of IL-6 and do not express to any significant degree other inflammatory cytokines such as IL-1 or TNF. Similarly, Mee et al.(lo) have shown that CDV infection of canine marrow cultures induces IL-6. Thus, IL-6 appears to be an autocrine/paracrine factor that may play an important role in amplifying the osteoclast formation in patients with Paget s disease, and may be responsible for the increased bone remodeling seen in bones not clinically involved with Paget s disease at sites distant from the pagetic lesion. Osteoclast precursors from Paget s patients are hyperresponsive to 1,25(OH),D, In our initial studies of bone marrow cultures from patients with Paget s disease, we found that osteoclast formation was markedly increased in these marrow cultures compared to those from normals, and osteoclast formation was increased as much as 20- to 100-fold above that seen in normal marrow cultures.( 3) Interestingly, the rate of osteoclast formation was also increased such that maximum osteoclast formation occurred within 2 weeks of initiation of these marrow cultures compared with 3 weeks in normal marrow cultures. Furthermore, we found that osteoclast formation in marrow cultures from patients with Paget s disease occurred with concentrations of 1,25(OH),D, that was at least one log less than that required for osteoclast formation in normal marrow cultures. Significant levels of osteoclast formation could be detected at concentrations at lo- M of 1,25(OH),D,, a concentration where no osteoclast formation occurred in normal marrow cultures. These data suggested that possibly there were intrinsic abnormalities in osteoclast precursors from patients with Paget s dis- ease compared to normal osteoclast precursors. Alternatively, these abnormalities in osteoclast formation and 1,25(OH),D, sensitivity could reflect intrinsic differences in the marrow microenvironment in Paget s disease, in particular, marrow stromal cells. To further determine if there were intrinsic abnormalities in osteoclast precursors from patients with Paget s disease or if abnormalities existed in the marrow microenvironment in pagetic lesions, we examined osteoclast precursors from Paget s patients and normals and compared their relative sensitivity to 1,25(OH),D, and to the marrow microenvironment. We had shown previously that the earliest identifiable osteoclast precursor is the granulocyte macrophage colony forming cell.(23) This cell then gives rise to an early osteoclast precursor that is bipotent and can form either monocyte-macrophages or differentiate further to form committed precursors for the osteoclasts. These committed precursors are unipotent and can only form osteoclasts. We isolated highly purified populations of both early and the committed osteoclast precursors and examined the capacity of highly purified populations of granulocytemacrophage colony-forming unit (CFU-GM) derived cells to form osteoclasts with varying concentrations of 1,25(OH),D,. Demulder and coworkers( ) demonstrated that early osteoclast precursors from patients with Paget s disease could form osteoclast-like cells in vitro with concentrations of 1,25(OH),D, that are at least one log less than that required for osteoclast formation by normal early osteoclast precursors. To confirm that these early osteoclast precursors from patients with Paget s disease were hyperresponsive to 1,25(OH),D,, 24-hydroxylase gene expression (the first gene product that is induced when 1,25(OH),D3 binds to VDR) was measured by reverse transcriptase polymerase chain reaction (RT-PCR). We found that 24-hydroxylase mrna expression could be detected in early precursors from patients with Paget s disease at concentrations of 1,25(OH),D, that are at least one to two logs less than that required for 24-hydroxylase expression by normal precursors. Increased sensitivity to 1,25(OH),D, could result from increased numbers of VDRs being expressed in early osteoclast precursors from patients with Paget s disease or an increased affinity of the VDR for 1,25(OH),D,. Therefore, we used RT-PCR to quantitate VDR expression in purified early osteoclast precursors from Paget s patients and normals. We found that the relative amount of VDR expressed by early precursors from Paget s patients was similar to that found with normals. Since all of these studies were done with RT-PCR, we then wanted to determine if VDR protein were being expressed in osteoclast-like cells formed in marrow cultures as well as by early osteoclast precursors and the more differentiated osteoclast precursors. We demonstrated by Western blot that osteoclast-like cells formed in vitro expressed VDR, and that both early and late osteoclast precursors expressed VDR. Interestingly, the amount of VDR expressed by osteoclast precursors decreases with osteoclast precursor differentiation. In collaboration with Dr. Barsony at the National Institutes of Health, Bethesda, MD, U.S.A., we measured the

4 6 REDDY ET AL. relative affinity of VDR for 1,25(OH),D, in osteoclast precursors from Paget s patients and normals using a fluoresceinated derivative of l,z(oh),d, and confocal microscopy.(25) We found that the relative affinity of VDR in early osteoclast precursors from Paget s patients was increased compared to normals. These data suggest that the increased sensitivity of osteoclast precursors from patients with Paget s disease was not due to increased numbers of VDRs but to increased affinity of VDR for 1,25(OH),D3, and that increased 1,25(OH),D, responsiveness was an intrinsic property of the osteoclast precursor. An alternative possibility that might explain the increased sensitivity of early osteoclast precursors from Paget s patients to 1,25(OH),D, was that cytokines made by these cells or by osteoclasts or stromal cells in the marrow microenvironment could enhance the sensitivity of these cells to 1,25(OH),D,. Since osteoclasts from patients with Paget s disease express IL-6, we tested if low levels of IL-6 could enhance the sensitivity of normal osteoclast precursors to 1,25(OH),D,. When normal bone marrow was cultured with varying concentrations of 1,25(OH),D, and 10 pg/ml of IL-6, the 1,25(OH),D3 dose-response curve for osteoclast formation was unchanged. Taken together, these data demonstrate that in patients with Paget s disease, enhanced sensitivity of osteoclast precursors to 1,25(OH),D3 is an intrinsic property of these cells. The enhanced sensitivity to 1,25(OH),D, is not due to interactions between other cytokines, nor is it due to abnormal numbers of VDR. The basis for the enhanced affinity of l,z(oh),d, for VDR in osteoclast precursors from patients with Paget s disease remains to be determined. Mee and coworkers have demonstrated that CDV infection of canine marrow cells increased their sensitivity to 1,25(OH),D,.( 0) Possibly, paramyxoviral infection of osteoclast precursors from Paget s patients may in part be responsible for their enhanced responsiveness to 1,25(OH),D,. Effects of the marrow microenvironment on osteoclast formation in patients with Paget s disease Another possible mechanism for increased osteoclast formation in patients with Paget s disease was that the marrow microenvironment may be abnormal. This seemed possible, since Paget s disease is a highly localized disease and patients do not develop new lesions throughout the course of their disease. We quantitated early osteoclast precursors (CFU-GM-derived cells) in bone marrow aspirates from involved bones from patients with Paget s disease or normals to determine if early osteoclast precursor numbers were increased. Demulder and coworkers( ) demonstrated that CFU-GM cells were significantly increased in marrow aspirates from patients with Paget s disease compared to normal. However, when these early osteoclast precursors were separated from marrow stromal cells using a monoclonal antibody against CD34 (an antigen that is expressed on hematopoietic precursors and not on stromal cells(26)) the number of osteoclast precursors present in normal marrow and marrow from patients with Paget s disease was very similar. Therefore, studies were performed using marrow stromal cells from normals and from patients with Pag- et s disease and cocultured with CD34-positive cells from normals or patients with Paget s disease, to determine if the stromal cells could enhance the growth of early osteoclast precursors. When normal CD3Cpositive cells were cocultured with normal stromal cells, the expected number of CFU-GM-derived colonies formed. However, when normal CD34-positive cells were cultured with stromal cells from patients with Paget s disease, there was a significant enhancement of CFU-GM growth; approximately 2-fold greater than the expected value. Similarly, when CD34- positive cells from Paget s patients were cocultured with stromal cells from Paget s patients, there was a 2.5-fold increase CFU-GM colony formation above the expected values. Interestingly, when CD34-positive cells from Paget s patients were cocultured with normal stromal cells, there again was enhanced growth of CFU-GM, to levels that were 2-fold above that expected. These data suggest that the marrow microenvironment from affected bones of patients with Paget s disease could enhance the growth of early osteoclast precursors, and that the early osteoclast precursors in patients with Paget s disease was hyperresponsive to the marrow microenvironment. To further dissect the abnormalities present in the marrow microenvironment from patients with Paget s disease compared to normal, marrow stromal cell lines were produced from normal marrow and marrow from patients with Paget s disease. Cell lines were produced by isolating CD34-negative cells, and then infecting these cells with a recombinant adenovirus which contains the SV40 large T antigen.( ) Both of these immortalized cell lines could support osteoclast formation, although the marrow stromal cell line from Paget s patients was more osteoclastogenic. These marrow stromal cell lines produced IL-6 and IL-1, which could enhance osteoclast formation. However, levels of RANK ligand mrna, a newly described stimulator of osteoclast formation which may be the common mediator for the effects of other osteoclastogenic factors on osteoclast formation,( ) was significantly elevated in the stromal cell line derived from patients with Paget s disease compared to that from normals. The enhanced expression of RANK ligand by stromal cells from patients with Paget s disease may explain in part the enhanced capacity of stromal cells from patients with Paget s disease to induce increased levels of osteoclast formation. DISCUSSION There are several abnormalities in osteoclast precursors as well as the marrow microenvironment in patients with Paget s disease (Fig. 1). Early osteoclast precursors are hyperresponsive to 1,25(OH),D3 and the marrow microenvironment. These cells appear to show enhanced growth even with the normal marrow microenvironment. In addition, these early osteoclast precursors contain measles virus nucleocapsid transcripts. The committed osteoclast precursors also contain measles virus nucleocapsid transcripts and appear to form osteoclasts at an increased rate compared to normal osteoclast precursors. The mature osteoclast formed in Paget s patients also contains measles virus tran-

5 OSTEOCLASTS IN PAGET S DISEASE 7 Model for Osteoclast Formation Macrophages Monocytes Hyper-responsive to: Micrmnvironment Abnormal Morphology CFU-Blast CFU-GM Early OCL Precursors Late OCL Precursors FIG. 1. Abnormalities in osteoclast precursors as well as the marrow microenvironment in patients with Paget s disease. scripts or canine distemper virus transcripts and produces prodigious amounts of IL-6, which can further amplify the increased osteoclast formation and activity in patients with Paget s disease. The marrow microenvironment also appears to be abnormal and has an enhanced capacity to induce osteoclast formation compared with the normal marrow microenvironment. The marrow stromal cells from patients with Paget s disease appear to demonstrate elevated levels of RANK ligand expression compared with normal marrow stromal cells. Thus, although the pathophysiology responsible for the increased osteoclast formation and activity in patients with Paget s disease is still beginning to be clarified, interesting and potentially important differences between osteoclast precursors from normals and patients with Paget s disease have been detected. ACKNOWLEDGMENTS This work was supported by Research Funds from the Veterans Administration and National Institutes of Health NIAMS Grants AR and AR 41336, NIADDK Grant AR 36125, and NCI Grant CA REFERENCES 1. Hosking DJ 1981 Paget s disease of bone. Br Med J 283:68& Yates AJ 1988 Paget s disease of bone. Baillieres Clin Endocrinol Metab Krane S 1986 Paget s disease of bone. Calcif Tissue Int 38: Rebel A, Basle M, Pouplard A, Kouyoumdjian S, Filmon R, LePatezour A 1980 Viral antigens in osteoclasts from Paget s disease of bone. Lancet Kanis JA 1991 Pathophysiology and Treatment of Paget s Dis- ease of Bone. Carolina Academic Press, Martin Dunitz, London, U.K. 6. Harvey L, Gray T, Beneton MNC, Douglas DL, Kanis JA, Russell RGG 1982 Ultrastructural features of the osteoclasts from Paget s disease of bone in relation to a viral aetiology. J Clin Pathol Rebel A, Malkani K, Basle M, Bregeon CH 1976 Osteoclast ultrastructure in Paget s disease. Calcif Tissue Res 24k Mills BG, Singer FR, Weiner LP, Suffin SC, Stabile E, Holst P 1984 Evidence for both respiratory syncytial virus and measles virus antigens in the osteoclasts of patients with Paget s disease of bone. Clin Orthop 183: Basle MF, Russell WC, Goswami KA, Rebel A, Giraudon P, Wild R, Filmon R 1985 Paramyxovirus antigens in osteoclasts from Paget s bone tissue detected by monoclonal antibodies. J Gen Virol66: Gordon MT, Mee AP, Anderson DC, Sharpe PT 1995 Canine bone marrow cultures infected with canine distemper virus: An in vitro model of Paget s disease. Bone Basle MF, Fournier JG, Rozenblatt S, Rebel A, Bouteille M 1986 Measles virus RNA detected in Paget s disease bone tissue by in situ hybridization. J Gen Virol Mee AP, Dixon JA, Hoyland JA, Davies M, Selby PL, Mawer EB 1998 Detection of canine distemper virus in 100% of Paget s disease samples by in situ-reverse transcriptase-polymerase chain reaction. Bone B Kukita A, Chenu C, McManus LM, Mundy GR, Roodman GD 1990 Atypical multinucleated cells form in long-term marrow cultures from patients with Paget s disease. J Clin Invest 85:128& Horton MA, Lewis D, McNulty K, Pringle JAS, Chambers TJ 1985 Monoclonal antibodies to osteoclastomas (giant cell bone tumors): Definition of osteoclast-specific cellular antigens. Cancer Res Mills BG, Frausto A, Singer FR, Ohsaki Y, Demulder A, Roodman GD 1994 Multinucleated cells formed in vitro from Paget s bone marrow express viral antigens. Bone Roodman GD, Ohsaki Y, Miller MM, Demulder A, Hosking D, Singer F, McManus L 1998 Pagetic osteoclasts formed in vitro: Absence of paracrystalline inclusions. J Submicrosc Cyto1 Pathol30:

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