D.R.Haynes,T.N.Crotti,M.Loric,G.I.Bain 1,G.J.Atkins andd.m.findlay 1

Transcription

1 Rheumatology 2001;40:623±630 Osteoprotegerin and receptor activator of nuclear factor kappab ligand RANKL) regulate osteoclast formation by cells in the human rheumatoid arthritic joint D.R.Haynes,T.N.Crotti,M.Loric,G.I.Bain 1,G.J.Atkins andd.m.findlay 1 Department of Pathology and 1 Department of Orthopaedics and Trauma, The University of Adelaide and The Royal Adelaide Hospital, Adelaide 5000, SA, Australia Abstract Objective. This study investigated the involvement of the recently identi ed regulators of osteoclast formation RANKL wreceptor activator of nuclear factor kappab RANK) ligand, osteoclast differentiation factor, TRANCE, osteoprotegerin ligandx and its natural inhibitor, osteoprotegerin OPG), in the bone erosion of rheumatoid arthritis RA). Methods. mrna was extracted from cells isolated from the pannus and synovial membrane regions of joints of 11 RA patients. Semiquantitative reverse transcription±polymerase chain reaction was carried out, and the isolated cells were also cultured to determine their ability to form osteoclasts. Results. mrnas encoding RANKL, RANK, OPG and macrophage-colony stimulating factor were expressed by cells isolated from RA joints. In addition, mrna encoding for tumour necrosis factor apoptosis-inducing ligand and the osteoclast markers tartrate-resistant acid phosphatase and calcitonin receptor were also often expressed. Osteoclasts capable of forming resorption lacunae were generated from cells in the RA joints. At 50 nguml, recombinant OPG completely inhibited the resorptive activity of these cells. There was a signi cant correlation between the ratio of RANKL mrna to OPG mrna and the number of resorption pits produced P = 0.028). Conclusion. These data suggest that RANKL is an essential factor for osteoclast formation by cells in the rheumatic joint and that OPG may prevent the bone erosion seen in RA joints. KEY WORDS: Bone, Rheumatoid arthritis, Joint damage, Cytokines, Osteoclasts, Osteoprotegerin. Progressive joint destruction is a hallmark of rheumatoid arthritis RA). The in ammatory changes in RA are associated with the breakdown of both soft tissue and bone in the rheumatoid joint. The bone erosion can be localized to the in amed joint w1x as well as being generalized, secondary osteoporosis often being associated with RA w2x. Despite the widespread occurrence of RA, we still have an incomplete understanding of the processes of this chronic systemic disease. Most studies have investigated the in ammation that occurs in the soft tissues; however, recent advances in our understanding of bone metabolism allow us now to better investigate the mechanisms of bone loss in RA. A cell surface molecule, receptor activator of nuclear factor kappab ligand RANKL; also called osteoclast Submitted 18 May 2000; revised version accepted 11 December Correspondence to: D. R. Haynes. differentiation factor, TRANCE and osteoprotegerin ligand) and its receptor, receptor activator of nuclear factor kappab RANK) have been shown recently to be key factors stimulating osteoclast formation w3, 4x. It has been shown that the binding of RANKL to RANK on the surface of osteoclast precursors promotes the differentiation of these cells to mature osteoclasts. It is now clear that, together with macrophage-colony stimulating factor M-CSF), RANKL is required for osteoclast formation. The soluble tumour necrosis factor TNF) receptor-like molecule osteoprotegerin OPG) is a natural inhibitor of RANKL w5x. OPG binds to RANKL and prevents its ligation to RANK. The importance of these molecules in regulating bone metabolism is demonstrated by transgenic and gene knockout studies in mice w6x. The relative levels of RANKL and OPG are likely to be important in determining whether osteoclasts will form w7x. As these factors control physiological osteoclast formation, it is reasonable 623 ß 2001 British Society for Rheumatology

2 624 D. R. Haynes et al. to propose that they may also be key regulators of pathological bone resorption, such as in RA. It has been reported previously that, under certain conditions, human osteoclasts can be derived from cells present in or near the tissues of arthritic joints w8, 9x. More recently, it has been shown that RANKL is expressed within the rheumatoid joint and that synoviocytes and activated T cells are implicated in its production w10±13x. Moreover, in a mouse model of RA, administration of OPG prevented the bony erosions that often accompany joint in ammation in RA w12x. In the present work, we sought to test the concept that the production of RANKL by cells within the human RA pannus and synovial membrane leads to osteoclastic bone resorption. Cells isolated from the synovial membrane and at the bone±pannus interface were used in this study. We found a positive association between RANKL expression and bone resorption in vitro, and that resorption was completely prevented by exogenous OPG. surgery by the surgeon co-author GIB); the pannus tissue was considered to be that part of the synovial membrane that had in ltrated the bone and cartilage. The tissue classi ed as synovial membrane was capsular synovial tissue separate from bone±cartilage in ltrate. Histology was carried out routinely on samples of these tissues and examples are shown in Fig. 1. Generally, the samples were similar and contained large numbers of in ltrating mononuclear cells. The only major difference was that the pannus tissues did not have a layer of cells lining the surface of the synovial membrane. On removal, the tissue samples were placed immediately into Hanks' balanced salt solution HBSS; Gibco BRL, Life Technologies, Melbourne, Australia) then digested at 378C in calcium- and magnesium-free HBSS solution Gibco BRL, Life Technologies) containing 1 mguml Materials and methods Chemicals Human recombinant RANKL and OPG were gifts from Amgen Thousand Oaks, CA, USA). Patient tissue samples and cell isolation Tissue samples were taken at surgery from patients who had been diagnosed as suffering from RA, which they had had for 6±25 yr. All patients showed advanced erosion of the bone on X-ray, and had disease that was active but not burnt out, as seen in the late stages of the disease. Details of the samples taken from the patients and of the medication at the time of surgery are shown in Table 1. The tissue samples were classi ed as corresponding to either the pannus region adjacent to the bone where erosion was occurring, or to the synovial membrane not adjacent to the bone. In all cases the classi cation of the tissue was made at the time of TABLE 1. Details of samples from patient tissues Patient sample Age Sex Disease duration yr) Medications Site Region 1 58 M 17 Gold, prednisolone Wrist Pannus 2 71 F 19 NSAID Knee Pannus 3a 37 M 15 NSAID Elbow Pannus 3b Elbow Membrane 4 48 F 20 Gold Wrist Pannus 5 52 F 30 Methotrexate Wrist Membrane 6 44 F 6 Herbal therapies Wrist Pannus only 7a 51 M 17 Methotrexate Foot Membrane 7b Foot Pannus 8 57 F 25 NSAID Wrist Pannus 9 52 F 22 Methotrexate Wrist Membrane F 9 Methotrexate Elbow Membrane F 15 Gold, prednisolone Wrist Pannus NSAID, non-steroidal anti-in ammatory drug. FIG. 1. a) Typical section of synovial membrane. This consisted of a membrane lining layer of cells and a sublining layer of connective tissue of variable thickness in ltrated by a large number of mononuclear cells. b) Pannus tissue was very similar but lacked the layer of cells that lined the membrane of the synovium. Both sections were formalin- xed, paraf n-embedded and stained with haematoxylin. Magni cation 3100.

3 Factors regulating bone loss in RA joints 625 collagenase Sigma, Castle Hill, Australia) and 1 mguml dispase Sigma). After 60 min, 0.5 mguml trypsin in HBSS solution Sigma) was added, and the tissue was incubated for a further 30 min. Cells were separated from undigested connective tissue with a 70-mm cell sieve Falcon, Becton Dickinson Labware, Bedford, MA, USA) and the cell suspension was washed once in HBSS. Cells were suspended in RPMI 1640 medium at a concentration of cellsuml. Cells were isolated from a total of 11 patients with RA, and tissue was taken from two separate sites in two patients. The yield of cells varied depending on the size and the cell density of the tissue sample. An average of cells from each milligram of wet weight of tissue was obtained. mrna was extracted from cells for analysis by reverse transcription±polymerase chain reaction RT-PCR). Suf cient cells for the studies of osteoclast generation, as described below, were obtained from eight of the patients. Isolation of human bone-derived cells Human bone cells were derived as outgrowths from trabecular bone fragments obtained from patients undergoing primary hip replacement, as described previously w14x. Culture of osteoclasts and osteoclast precursors Cells isolated from the rheumatoid tissues were tested for osteoclast formation anduor activity, as described previously in detail in studies in which the differentiation of monocytes to osteoclasts was promoted by coculture with rodent osteoblastic cells as stromal cells w15x. Brie y, where human bone cells were used as a stromal population, 13-mm diameter sterile glass coverslips or mm thick discs of dentine were seeded with human bone-derived cells 24 h before addition of cells isolated from the rheumatoid tissues. Rheumatoid cells w coverslips) or dentine)x were added and after 1 h the non-adherent cells were removed by washing. The individual coverslips and pairs of dentine slices were placed in 16-mm diameter wells with 1 ml of amem medium containing M 1a,25 OH) 2 D 3 vitamin D 3 ), M dexamethasone Fauldings, Adelaide, Australia) and 25 nguml recombinant human M-CSF a kind gift from the Genetics Institute, Cambridge, MA, USA). Medium was replenished every 3 days throughout the experiment and all experiments were carried out in duplicate for each rheumatoid sample. Tartrate-resistant acid phosphatase TRAP) After 1 or 14 days of culture, cells staining positive for TRAP were quantitated using a commercial staining kit Sigma). Identi cation of resorption pit formation To assess the extent of bone resorption by cells in the cocultures, dentine discs were examined for resorption lacunae on day 14, as described previously w14x. Preparation of total RNA and RT-PCR analysis The total cell population isolated from pannus or synovial membrane, as described above, was lysed by the addition of Trizol reagent Life Technologies, Gaithersburg, MD, USA) and total RNA was prepared according to the manufacturer's instructions. cdna was synthesized using an AMV RT cdna kit Promega, Madison, WI, USA). cdna was ampli ed by PCR in a thermal cycler Eppendorf, Hamburg, Germany). Each ampli cation mixture contained 1 ml of the cdna sample or water control, 0.2 mm dntps and 1 U of Platinum Taq DNA polymerase Life Technologies), 100 ng each of 59 and 39 primers, 1.5 mm MgCl 2, 2 ml 10 3 reaction buffer, and sterile diethyl pyrocarbonate H 2 O. Twenty-two cycles of PCR were performed for glyceraldehyde-3-phosphate dehydrogenase GAPDH) and 30±34 cycles for the other primer pairs. Primer sequences and predicted PCR product sizes have been published previously w15, 16x. Ampli cation products were resolved by electrophoresis on a 2% wuv agarose gel and post-stained with SYBR Gold catalogue no. S-11494; Molecular Probes, Eugene, OR, USA). The intensity of the PCR products was quanti ed using a Molecular Imager Fx uorescent scanner and Quant-1 software Bio-Rad, Hercules, CA, USA). Preliminary experiments were performed to ensure that the number of PCR cycles was within the exponential phase of the ampli cation curve. This allowed semiquantitative comparisons to be made between the levels of expression of the various RNA species in the samples, as described previously w15, 16x. Results Expression of regulators of osteoclast formation in rheumatoid tissues RT-PCR) Figure 2 shows PCR products corresponding to mrna for RANKL, OPG and M-CSF, as determined by RT-PCR, in cells digested from rheumatoid tissues sampled from 11 patients. PCR products corresponding to each of the mrna species could be ampli ed from nearly all tissue samples. We also observed that TRAIL, a TNF-a-related molecule that has been shown to bind to and antagonize the inhibitory actions of OPG w17, 18x, was expressed in all the samples tested. mrna encoding for the osteoclast markers TRAP and calcitonin receptor CTR) were also expressed in many of the rheumatoid tissues. TRAP was expressed in all but two samples. In contrast, CTR was expressed in all the pannus tissue samples but only one of the synovial membrane samples. Generation of osteoclasts from rheumatoid tissues The population of cells digested from RA tissues that were adherent to glass coverslips contained many cells with the appearance of osteoclasts after culture for 24 h Fig. 3a). These osteoclast-like cells were large and TRAP-positive and contained many nuclei. This suggested that osteoclast-like cells were resident in the

4 626 D. R. Haynes et al. FIG. 2. PCR products corresponding to RANKL, OPG, RANK, M-CSF, TRAIL, TRAP and CTR mrna detected by semiquantitative RT-PCR for the 13 samples of RA tissue described in Table 1. Expression is shown relative to the housekeeping gene GAPDH. arthritic tissues. Many more osteoclastic cells were seen after 14 days of culture, at which time large multinucleated TRAP-staining cells were seen regularly amongst cells cultured either alone Fig. 3b) or in the presence of human osteoblast-like cells. Addition of exogenous OPG did not markedly affect the number of TRAP-positive cells that formed from alone Fig. 3c) or cultured with human bone-derived cells. When were cultured alone on dentine slices, large numbers of resorption lacunae were usually seen by day 14 Fig. 3d). Slightly more resorption pits were observed when were cultured with human bone-derived cells Fig. 3e). Treatment with 50 nguml OPG completely inhibited the formation of resorption pits in cultures of alone Fig. 3f ) and in cocultures of with human bone-derived cells. Using inverted-phase microscopy, resorption pits could be observed during the culture period, and no pits were seen before day 7. The numbers of resorption pits and TRAP-positive cells seen at day 14 are compared in Table 2. Culturing the in the presence of human bone-derived cells resulted in a slight increase in the mean numbers of resorption pits and cells expressing TRAP compared with cells incubated alone. Although OPG treatment resulted in complete inhibition of pit formation, there was no reduction in the number of cells expressing TRAP, as stated above. There is growing experimental evidence w7x that i) RANKL and OPG mrna levels are good surrogates for the expression of the corresponding proteins, and ii) that osteoclastic resorption is dependent upon the effective concentration of RANKL, which is in turn determined by the local concentration of OPG. We therefore compared the ratio of RANKL mrna to OPG mrna, as measured by RT-PCR, with the numbers of resorption pits produced by cells digested from the RA tissues cultured alone Fig. 4). There was a strong positive correlation between these values wspearman rank non-parametric) correlation 0.762; P = 0.028x, consistent with a role for RANKL in promoting osteoclastic bone resorption in human RA tissues. A similar comparison of the numbers of resorption pits with the ratio of TRAIL mrna to OPG mrna was carried out but no signi cant correlation

5 Factors regulating bone loss in RA joints 627 FIG. 3. a) Multinucleated cells expressing TRAP dark-staining cells) were present in the RA tissues and were seen after 24 h of culture of alone. Magni cation b) Many more multinucleated cells expressing TRAP were seen after 14 days, when were cultured alone. Magni cation 350. c) Treatment of these cells with 50 nguml OPG did not markedly reduce the numbers of cells expressing TRAP at day 14. Magni cation 350. d) usually formed many resorption pits in dentine slices after 14 days when cultured alone. Magni cation e) Slightly more resorption pits were seen when the RA cells were cultured with human bone-derived cells. Magni cation f ) Treatment with 50 nguml OPG for 14 days completely inhibited the formation of resorption pits in cultures of alone. Magni cation was noted P > 0.05), indicating that TRAIL may have less of a role than OPG and RANKL in regulating osteoclast formation in this pathology. Inhibition of osteoclast formation by OPG Cells isolated from RA tissues were cultured on dentine slices in the presence of 50 nguml OPG to determine whether RANKL expressed by the cells was essential for the formation of osteoclasts. As shown pictorially in Fig. 3, OPG completely inhibited the ability of rheumatoid cells to form resorption lacunae Table 2). This inhibition occurred in the presence or absence of added human bone cells. Interestingly, the numbers of cells expressing TRAP was only slightly reduced by OPG treatment. Discussion This study has shown that bone-resorbing osteoclasts can be generated in culture from cells present within the

6 628 D. R. Haynes et al. TABLE 2. TRAP expression and resorption pit formation during 14 days of culture of cells isolated from RA tissue; the effects of coculture with human bone-derived cells NHB) and treatment with 50 nguml OPG are shown +NHB alone +NHB +OPG alone Pits per dentine slice 88.8 " 28.0 a 45.1 " " " 0.0 TRAP + cells per " " " " 35 a Mean " S.E.M. for n=8. FIG. 4. Association of the ratio of RANKL mrna to OPG mrna with the number of resorption pits produced by cells digested from the same tissue samples. The data points were derived from the experiments described in Fig. 2 and Table 2. The individual samples are numbered as described in Table 1. The regression analysis was carried out using the SAS version 6.21 software package, and signi cance was determined from the resulting r value. RA joint. The data indicate that these tissues contain both mature osteoclasts and their precursors, as well as producing factor s) essential for osteoclast formation. Our results show that RANKL mrna is expressed in cells within the RA tissues, and the ratio of RANKL mrna to the mrna of its inhibitor, OPG, expressed in the RA tissues signi cantly correlated with the formation of functional osteoclasts. These results are supported by recent reports showing the expression of RANKL and RANK in rheumatoid tissues in humans w11, 13x and in animal models w12, 19x. Our results differ from a previous report w8x which found that additional rat osteoblast-like cells were required for RA synovial macrophage and blood monocytes to produce osteoclastic resorption. The reason for the difference may be that we mainly used cells isolated from tissue adjacent to bone, corresponding to the invading pannus, whereas the previous study used synovial macrophages and blood monocytes from the RA patients. More studies would need to be carried out to determine if indeed there are functional differences in osteoclast formation in these different regions of synovial tissues. It is signi cant to note the relationship between the ratio of RANKL mrna to OPG mrna and osteoclast formation. These data suggest a correlation between the mrna levels of RANKL and OPG and the corresponding protein levels. In addition, our ndings support the concept that the relative levels of RANKL and OPG is a key factor in determining bone loss in these tissues. Although we found abundant expression of M-CSF mrna in cells digested from RA tissues, it is clear that M-CSF, an essential cofactor for the induction of osteoclast differentiation by RANKL, is limiting in human osteoclastogenesis in culture w4x. As in other studies w8, 13x, M-CSF was included in all our cell cultures as it allowed us to investigate the activities of RANKL independently of a requirement for M-CSF. The number of multinucleated TRAP-positive cells that developed from cells isolated from the rheumatoid tissues was not markedly reduced by OPG treatment, while OPG treatment totally prevented resorption pit formation, as reported previously w13x. This may indicate that osteoclast precursors in the rheumatoid tissues have differentiated further towards becoming mature osteoclasts than the less differentiated cells in the peripheral monocyte population, as TRAP expression is markedly inhibited by OPG in culture systems using peripheral blood monocytes w3, 4x. While development of the rheumatoid cells into multinucleated TRAPpositive cells may not be affected by inhibiting RANKL with OPG, RANKL does appear to be required to stimulate and maintain osteoclastic bone resorption in these tissues. This concept is further supported by the observation that TRAP mrna was expressed by cells isolated from most of the patient tissue samples, but strong expression did not always correlate with the formation of resorption pits by cells isolated from these tissues. CTR expression may be a more appropriate indicator of osteoclast differentiation than TRAP expression in this pathology. CTR was expressed in all the pannus samples but in only one of the synovial membrane samples. This is consistent with a recent report, using in situ hybridization, showing that CTR mrna is not expressed in the synovial membrane but is expressed in the tissue±bone interface in RA w11x. CTR was also consistently expressed in rheumatoid tissues from which osteoclasts readily formed, further supporting the concept that CTR expression is associated with the later stages of osteoclast differentiation. RANKL has been shown to be required for osteoclast formation, and is normally provided by osteoblast-like cells in bone w3, 4x. Recent reports indicate that stromal support may be provided by either synovial broblasts w11, 13x or lymphocytes present in the rheumatoid tissues w10, 12x. Horwood et al. w10x showed that CD3 + T cells from the human rheumatoid joint express RANKL and can promote osteoclast formation from rodent spleen cell precursors. Kong et al. w12x demonstrated that, in addition to the production of RANKL by lymphocytes, the inhibition of RANKL by OPG treatment in vivo

7 Factors regulating bone loss in RA joints 629 reduced both bone and cartilage destruction in a model of adjuvant arthritis in rats. Our study strongly supports these ndings and suggests that OPG may be similarly ef cacious in human RA. Other mediators known to be produced by in ammatory cells in the soft tissues of the RA joint may promote bone loss indirectly by inducing the expression of RANKL. The in ammatory mediators interleukin IL) -1b and TNF-a have been shown to stimulate RANKL mrna expression w20x, and prostaglandin E 2 is reported to enhance the stimulation of osteoclast formation by RANKL w21x. IL-17 is also present in synovial uids in RA and has similarly been reported to stimulate osteoclastogenesis w22x. In addition, in ammatory chemokines produced in the in amed joint may also contribute to osteoclast formation directly w23x or by attracting monocyte osteoclast precursors w8x. It might be signi cant that TRAIL mrna was consistently expressed by cells isolated from RA tissues. TRAIL is a TNF-a-related molecule that induces apoptosis; however, it has also been shown to bind OPG and is able to suppress the inhibitory action of OPG in osteoclast formation w17, 18x. Its expression in the RA joint may therefore represent an additional pro-osteoclastogenic in uence. A recent report has demonstrated that TRAIL may suppress lymphocyte proliferation and have anti-in ammatory activity in an animal model of arthritis w24x. This is the rst report of the presence of TRAIL expression in tissues of the human RA joint. Further studies will be needed in humans to determine whether TRAIL produced in the RA joint is involved in regulating both bone loss and in ammation in this pathology. It is possible that bone lysis and the cytokines that cause it also contribute to the progression of in ammation in RA. In the rodent model of adjuvant arthritis, treatment with OPG reduced both cartilage and bone destruction but did not reduce in ammation w12x. This has yet to be demonstrated for human RA. However, in the light of our results here, the separate consideration of in ammation and bone treatment may result in speci c targeted treatments for these two components of human RA. Bone loss is a major cause of the loss of function of the human RA joint. Therefore, inhibiting this bone loss would be of great assistance to patients with RA. Alone, or in combination with anti-in ammatory therapies, treatments that inhibit osteoclast formation may help maintain joint function. There is now good evidence that OPG is effective in treating the bone loss seen in an animal model of this disease w12x. Together with the results presented here, this raises the exciting possibility that OPG, or a structural mimetic of OPG, might be useful in maintaining joint function for sufferers of RA. Acknowledgements The authors thank Mr Dale Caville for photographic assistance and Professor Barrie Vernon-Roberts for helpful advice. This work was supported by the National Health and Medical Research Council of Australia and the University of Adelaide Faculty of Medicine. References 1. Gravellese EM, Harada Y, Wang JT, Gorn AH, Thornhill TS, Goldring SR. Identi cation of cell types responsible for bone resorption in rheumatoid arthritis and juvenile rheumatoid arthritis. Am J Pathol 1998;152:943± Gough A, Sambrook P, Devlin J et al. Osteoclastic activation is the principal mechanism leading to secondary osteoporosis in rheumatoid arthritis. J Rheumatol 1998; 25:1282±9. 3. Yasuda H, Shima N, Nakagawa N et al. Osteoclast differentiation factor is a ligand for osteoprotegerinuosteoclastogenesis-inhibitory factor and is identical to TRANCEuRANKL. Proc Natl Acad Sci USA 1998; 95:3597± Lacey DL, Timms E, Tan HL et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998;9:165± Simonet WS, Lacey DL, Dunstan CR et al. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 1997;89:309± Bucay N, Sarosi I, Dunstan CR et al. Osteoprotegerinde cient mice develop early onset osteoporosis and arterial calci cation. Genes Dev 1998;19:1260±8. 7. Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Boyle WJ, Riggs BL. The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption. Bone Miner Res 2000;15:2± Fujikawa Y, Sabokbar A, Neale SD, Athanasou NA. Human osteoclast formation and bone resorption by monocytes and synovial macrophages in rheumatoid arthritis. Ann Rheum Dis 1996;55:816± Toritsuka Y, Nakamura N, Lee SB et al. Osteoclastogenesis in iliac bone marrow of patients with rheumatoid arthritis. J Rheumatol 1997;24:1690± Horwood NJ, Kartsogiannis V, Quinn JM, Romas E, Martin TJ, Gillespie MT. Activated T lymphocytes support osteoclast formation in vitro. Biochem Biophys Res Commun 1999;264:144± Gravallese EM, Manning C, Tsay A et al. Synovial tissue in rheumatoid arthritis is a source of osteoclast differentiation factor. Arthritis Rheum 2000;43:250± Kong YY, Feige U, Sarosi I et al. Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 1999; 402:304± Takayanagi H, Iizuka H, Juji T et al. Involvement of receptor activator of nuclear factor kappab ligandu osteoclast differentiation factor in osteoclastogenesis from synoviocytes in rheumatoid arthritis. Arthritis Rheum 2000;43:259± Haynes DR, Hay SJ, Rogers SD, Otha S, Howie DW, Graves SE. Regulation of bone cells by particle-activated mononuclear phagocytes. J Bone Joint Surg Br 1997; 79B:988± Haynes DR, Atkins GJ, Loric M, Crotti TN, Geary SM, Findlay DM. Bidirectional signaling between stromal and hemopoietic cells regulates interleukin-1 expression during human osteoclast formation. Bone 1999;25:269±78.

8 630 D. R. Haynes et al. 16. Atkins GJ, Haynes DR, Geary SM, Loric M, Crotti TN, Findlay DM. Coordinated cytokine expression by stromal and hematopoietic cells during human osteoclast formation. Bone 2000;26:653± Emery JG, McDonnell P, Burke MB et al. Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J Biol Chem 1998;273:14363± Wong BR, Josien R, Choi Y. TRANCE is a TNF family member that regulates dendritic cell and osteoclast function. Leukoc Biol 1999;65:715± Romas E, Bakharevski O, Hards DK et al. Expression of osteoclast differentiation factor at sites of bone erosion in collagen-induced arthritis. Arthritis Rheum 2000; 43:821± Hofbauer LC, Lacey DL, Dunstan CR, Spelsberg TC, Riggs BL, Khosla S. Interleukin-1beta and tumor necrosis factor-alpha, but not interleukin-6, stimulate osteoprotegerin ligand gene expression in human osteoblastic cells. Bone 1999;25:255± Wani MR, Fuller K, Kim NS, Choi Y, Chambers T. Prostaglandin E2 cooperates with TRANCE in osteoclast induction from hemopoietic precursors: Synergistic activation of differentiation, cell spreading and fusion. Endocrinology 1999;140:1927± Kotake S, Udagawa N, Takahashi N et al. IL-17 in synovial uids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis. J Clin Invest 1999;103:1345± Zheng MH, Fan Y, Smith A, Wysocki S, Papadimitriou JM, Wood DJ. Gene expression of monocyte chemoattractant protein-1 in giant cell tumors of bone osteoclastoma: possible involvement in CD68+ macrophage-like cell migration. J Cell Biochem 1998; 70:121± Song K, Chen Y, Goke R et al. Tumor necrosis factorrelated apoptosis-inducing ligand TRAIL) is an inhibitor of autoimmune in ammation and cell cycle progression. J Exp Med 2000;19:1095±104.