|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on August 8, 2002; DOI 10.1182/blood-2002-04-1121.
NEOPLASIA
From the Hematology Departments of Internal Medicine
and Biomedical Science, Experimental Medicine, and Pathology,
University of Parma, Parma, Italy; and INSERM U463,
Insitut de Biologie, Nantes, France.
The biologic mechanisms involved in the pathogenesis of multiple
myeloma (MM) bone disease are not completely understood. Recent
evidence suggests that T cells may regulate bone resorption through the cross-talk between the critical osteoclastogenetic factor,
receptor activator of nuclear factor- Multiple myeloma (MM) is a plasma cell malignancy
localized in the bone marrow (BM) and characterized by the high
capacity to induce bone destruction. Almost all patients with MM show
early osteolytic lesions that result from an increased bone resorption due to the stimulation of osteoclast recruitment and activity in close
contact with myeloma cells.1 However, the biologic mechanisms involved in the pathogenesis of MM-induced bone disease are
still unclear.
In recent years, the osteoprotegerin ligand, namely, receptor
activator of nuclear factor- Growing evidence suggests that T lymphocytes may regulate bone
resorption and maintain bone homeostasis through the cross-talk between
RANKL and interferon Reagents
Cells and culture conditions
Human cell lines.
Human myeloma cell line (HMCL) XG-6 was established in Dr Bataille's
laboratory (INSERM U463, Nantes, France) and cultured as previously
described.13 HMCL U266 and osteosarcoma cell line Saos-2
were obtained from the American Type Culture Collection (Rockville,
MD); HMCL RPMI-8226, OPM-2, and human B-cell leukemia cell line REH
were purchased from DSM (Brunswick, Germany). Epstein-Barr virus-positive (EBV+) cells, established from healthy
subjects, were a kind gift from Dr P. Sansoni (University of Parma,
Italy). For some experiments, human cell lines XG-6, RPMI-8226, U266,
REH, and EBV+ cells (5 × 106 cells) were
incubated in the presence or absence of IL-6 (20 ng/mL) in 5 mL RPMI
1640 medium supplemented with 2% heat-inactivated fetal bovine serum
(FBS; Gibco Invitrogen, Milan, Italy), penicillin (50 U/mL),
streptomycin (50 µg/mL), and glutamine (2 mM) for 24 hours.
Cell cultures.
CD3+ cells, T-lymphocyte subsets CD4+ and
CD8+, and CD19+ B cells were purified using an
immunomagnetic method (MACS Miltenyi, Bergisch Gladbach,
Germany) from peripheral blood (PB) and BM mononuclear cells
(MNCs) obtained from healthy subjects and patients with MM, acute
B-cell leukemia, and B lymphoma with BM involvement. T-cell
activation was performed incubating 2 × 106 MNCs or
CD3+ cells in 6-well plates precoated with anti-CD3 (0.1 µg/well) plus CD28 (5 µg/well) mAb for 2 to 4 days. U266 or
RPMI-8226 (5 × 106 cells) were cocultured with
activated T cells (2 × 106) in a transwell system
(0.45-µM pore size; Falcon, Becton Dickinson, Oxford, United
Kingdom) for 24 to 48 hours in 5 mL complete medium in the presence or
absence of anti-human IL-6 mAb (clone 6708.111; R & D Systems; 10 µg/mL) or anti-IL-7 polyclonal antibody (Peprotech, London, United
Kingdom; 0.03 µg/mL) or anti-IgG. At the end of the coculture period
mRNA and cellular proteins were extracted from T lymphocytes. Fresh MM
cells were purified from patients at diagnosis or relapse using an
anti-CD138 mAb as previously described.14 Purified MM
cells (5 × 106) were cocultured with autologous purified
CD3+ T lymphocytes (2 × 106) in the
above-mentioned experimental conditions.
Osteoclastogenesis
Patients We studied 8 women and 7 men (mean age, 62 years; range, 53-73 years) with newly diagnosed MM (stages I-III) and 20 sex- and age-matched controls. BM and PB samples were obtained after informed consent was given. Approval was obtained from the Institutional Review Board of the University of Parma for these studies. Informed consent was provided according to the Declaration of Helsinki. BM CD3+ T lymphocytes were purified from patients at diagnosis or relapse and from controls. Control lymphocytes were obtained from diagnostic BM samples for nonneoplastic diseases without any skeletal involvement. Only cell populations with a purity of more than 95% were tested.RNA isolation and RT-PCR amplification For reverse transcription-polymerase chain reaction (RT-PCR) analysis, total cellular RNA was extracted from cells using Trizol reagent (Gibco). Then, 1 µg RNA was reverse-transcribed with 400 U Moloney murine leukemia virus reverse transcriptase (Gibco) according to the manufacturer's protocol. cDNAs were amplified by PCR with the following specific primer pairs. RANKL: sense: 5'-AGCACATCAGAGCAGAGAAAGC-3', antisense: 5'-CAGTAAGGAGGGGTTGGAGACC-3'; IL-7: sense: 5'-TTTTATTCCGTGCTGCTCGC-3', antisense: 5'-GCCCTAATCCGTTTTGACCA-3'; and 2-microglobulin: sense:
5'-CTCGCGCTACTCTCTTCTCTTTCTGG-3', antisense:
5'-GCTTACATGTCTCGATCCCACTTAA-3'. PCRs were performed in a thermal
cycler (MiniCycler MyResearch, Watertown, MA) for 30 cycles (annealing temperature: 60°C, 66°C, 63°C for RANKL, IL-7,
and 2-microglobulin, respectively). Then, 10 µL of
each amplified reaction was electrophoresed through a 2% agarose gel, stained with ethidium bromide (1 µ/mL) in 1 times TBE buffer (0.1 M
Tris [tris(hydroxymethyl)aminomethane], 90 mM boric acid, 1 mM EDTA [ethylenediaminetetraacetic acid], pH 8.4) and
visualized under UV light. Product size (464 base pair (bp) for RANKL,
429 bp for IL-7, and 334 bp for 2-microglobulin) was
established by comigration with a 100-bp ladder marker (Gibco).
Pictures of the electrophoresed cDNAs were recorded with a digital DC
120 Kodak camera and quantified by ID Image Analysis Software (Kodak Digital Science-Eastman Kodak, Rochester, NY). To analyze the effects of the experimental conditions independently of variability due
to the amount of cDNA amplified, the signal of specific cDNAs was
normalized to the respective signal of 2-microglobulin
which expression was found to be comparable under all the conditions used. To confirm the proper identity, PCR products of the appropriate size were directly sequenced.
Western blot analysis Western blot analysis for RANKL expression was performed as previously described.4 T lymphocytes alone or in transwell cocultures with HMCLs or fresh MM cells were resuspended in 100 µL lysis buffer (10 mM Tris-HCL, pH 7.6, 150 mM NaCl, 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride [PMSF], 2 mg/mL aprotinin [Sigma, St Louis, MO], and 1% Triton X-100). COS-7 cells lipofected with plasmid expression vector pCEP4, pCEP4-murine RANKL (Amgen, Thousand Oaks, CA), were used as positive controls. Protein levels were determined using a standard procedure (Uptima, Interchim, France). After 40 minutes on ice, lysates were cleared by centrifugation at 12 000g for 30 minutes at 4°C. Proteins (70 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% polyacrylamide gels and transferred onto polyvinylidene difluoride membrane. After blocking, membranes were incubated overnight with a monoclonal anti-RANKL antibody (1.5 µg/mL) obtained from R & D Systems. After washing, membranes were incubated with a horseradish peroxidase (HRP)-conjugated goat antimouse antibody (1:10 000; Becton Dickinson) at room temperature for 30 minutes. Blots were then developed using the Supersignal West Dura Extended Duration Substrate detection system (Pierce, Rockford, IL).Immunoreactive bands were visualized by a 5-minute exposure (Kodak X-OMAT). The intensity of each band was quantified by ID Image Analysis Software. To detect soluble RANKL (sRANKL), immunoprecipitation of conditioned media was performed using RANK-Fc (R & D Systems). Briefly, 1 mL supernatant was incubated with 0.5 µg RANK-Fc for 1 hour at 4°C; then, 20 µL protein G PLUS-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) was added and incubated with mixing for 2 hours at 4°C. Pellets were collected by centrifugation and washed 4 times with RIPA buffer (Tris-HCL 100 mM, NaCl 150 mM, EDTA 1 mM, sodium deoxycholate 1%, SDS 0.1%, Triton X-100 1%). After the final wash, the immunoprecipitated material was recovered by boiling in sample loading buffer and separated by electrophoresis. Gel was stained with a silver stain plus kit (Bio-Rad Laboratories, Milan, Italy) to detect the presence of sRANKL. In addition, an immunoblot analysis was performed using the above-mentioned procedure. ELISA The amount of sRANKL, IFN- , IL-6, IL-1, TNF- , IL-7, and
IL-11 in conditioned media, BM plasma, and PB serum was determined by
commercially available enzyme-linked immunosorbent assay (ELISA) kits.
The assay kit for RANKL was from Biomedica (Vienna, Austria). IFN-,
IL-6, IL-1, and TNF assay kits were obtained from Endogen. IL-7 and
IL-11 kits were purchased from R & D Systems. The sensitivity range of
ELISA tests was 0.4 to 50 pmol/L (8-1000 pg/mL) for RANKL, 25.6 to 1000 pg/mL for IFN-, 10.24 to 400 pg/mL for IL-6, 10.2 to 400 pg/mL for
IL-1, 15.6 to 1000 pg/mL for TNF-, 0.156 to 20 pg/mL for IL-7, and
15.6 to 1000 pg/mL for IL-11. The intra-assay and interassay
coefficients of variation (CV) were of 5% and 7% for RANKL;
2% and 6% for IFN-; less than 10% for IL-6, IL-1, and TNF;
3.3% and 7.8% for IL-7; and 2.4% and 6.6% for IL-11. ELISA for
sRANKL is an enzyme immunoassay designed to determine soluble
uncomplexed human RANKL. sRANKL binds to the precoated recombinant OPG
and forms a sandwich with the detection antibody. The detection
antibody is a rabbit antihuman soluble RANKL that is specific for RANKL
with a negligible cross-reactivity (< 1%) to human TNF-related
apoptosis-inducing ligand (TRAIL), as certified by the producer.
Statistical analysis was performed using analysis of variance (ANOVA) for repeated measurements, followed by a Tukey-Kramer test. P < .05 was considered significant. Results are expressed as the mean ± SE. Fluorescence-activated cell sorting analysis Fluorescein isothiocyanate (FITC)-conjugated and phycoerythrin (PE)-conjugated mAbs or CyChrome-conjugated mAbs recognizing CD138, CD126, CD3, CD4, CD8, CD25, or HLA-DR (human leukocyte antigen-DR) and the negative controls of IgG1 or IgG2a isotype of irrelevant specificity were purchased from Becton Dickinson. Purified cells were resuspended in phosphate-buffered saline (PBS) containing 1% fetal calf serum (FCS), 1% human serum, 10% mouse serum, and 0.01% sodium azide, then stained for 20 minutes at 4°C with combinations of saturating amounts of fluorochrome-conjugated mAbs. After staining, cells were washed extensively and analyzed. Flow cytometry analysis was performed using a fluorescence-activated flow cytometer (FACScan, Becton Dickinson) as previously reported.17
Effect of HMCLs and fresh MM cells on RANKL production We have previously demonstrated that HMCLs and fresh MM cells purified from several patients with MM do not express RANKL mRNA and protein.4As expected, T-cell activation with anti-CD3 plus anti-CD28 mAbs
induces RANKL mRNA in both CD4+ and CD8+
lymphocytes as shown by RT-PCR analysis (Figure
1A); consistently, RANKL protein was
induced in CD3+-activated T lymphocytes, with a maximal
effect observed after 4 days of activation (Figure 1B).
To investigate the potential effect of HMCLs on RANKL expression in activated T lymphocytes, a series of cocultures was performed in a transwell system. We found that HMCL U266 further increased RANKL mRNA expression in PB-activated T lymphocytes (Figure 1C) after 24 hours of coculture. Consistently, RANKL protein expression was stimulated by U266 and RPMI-8266 after 48 hours of coculture (Figure 1D). The stimulatory effect was more pronounced in CD8+ cells rather than CD4+ T lymphocytes as shown in Figure 1E, where a typical experiment with U266 is presented. The addition of anti-IL-6 mAb was able to reduce RANKL induction by HMCL RPMI-8226 (Figure 1F) as well as by U266 cells (data not shown). Consistently, IL-6 concentration was significantly higher in the supernatant of cocultures with RPMI-8226 or U266 (1.80 ± 0.03 ng/mL and 1.20 ± 0.02 ng/mL, respectively, versus 0.63 ± 0.01 ng/mL activated T lymphocytes; P < .001). Fresh purified MM, similarly to HMCLs, stimulated RANKL expression by autologous T lymphocytes obtained from MM patients in a transwell system. Densitometric analysis showed a 3.8-fold increase of RANKL in T lymphocytes cocultured with myeloma cells (Figure 1G). The concentrations of sRANKL were significantly increased in
supernatants of activated T lymphocytes cocultured with RPMI-8226 or
U266 myeloma cells in comparison with control (Figure
2A). This effect was observed in both
CD8+ and CD4+ T cells (Figure 2B). Similarly,
fresh purified MM cells stimulated RANKL secretion (Figure 2C) by
autologous T lymphocytes; sRANKL levels were undetectable in
supernatants of MM cell cultures.
To confirm the presence of sRANKL in conditioned medium of CD3+ cells and in the cocultures with HMCL, an immunoprecipitation with RANK-Fc was performed. sRANKL was identified as a band with a molecular weight of about 26 kDa (Figure 2D). Western blot analysis confirmed that HMCL U266 stimulates sRANKL production by activated T lymphocytes (Figure 2E). In contrast to the stimulatory effect on sRANKL production, a slight
inhibitory effect was observed on TNF- Effect of HMCLs and fresh MM cells on IFN- secretion
by activated CD3+ T cells (mean percent of inhibition ± SD versus control: 69% ± 3%; P < .001; Figure
3A). Similarly fresh MM cells inhibited IFN- secretion by both activated and nonactivated autologous T
lymphocytes in a transwell coculture system (mean percent of inhibition ± SD:  43% ± 7%; P < .001 and
28 ± 6%; P < .01, respectively; Figure
3B,C).
T-cell-mediated osteoclastogenesis induced by HMCL: effect of OPG To demonstrate that RANKL overexpression in T lymphocytes is able to induce osteoclast formation, we assessed human CD34+-derived osteoclastogenesis in a stromal cell-free condition. In this system we observed that conditioned medium of CD3- plus CD28-activated T lymphocytes cocultured with U266 significantly increased the formation of multinucleated TRAP+ cells after 10 days of culture (56% ± 6%; P < .01) in comparison with conditioned medium of activated T lymphocytes cultured alone. A significant inhibition on osteoclast formation was observed in the presence of rhOPG at 100 ng/mL (188 ± 11 versus 300 ± 8; P < .05 ) and 1 µg/mL (162 ± 10 versus 300 ± 8; P < .05). The presence of a neutralizing antibody anti-IL-7 inhibited the osteoclastogenesis induced by conditioned medium of cocultures (176 ± 16 versus 300 ± 8; P < .05; Figure 4).
Similar results were obtained using human PB MNCs at high density to generate osteoclasts (data not shown). Role of IL-7 in RANKL overexpression by T cells in cocultures and IL-7 expression and production by HMCLs and MM patients It has been recently demonstrated that IL-7 stimulates RANKL expression in T lymphocytes18; therefore, we hypothesized an involvement of this cytokine in the induction of RANKL in T cells by HMCLs. The addition of a neutralizing anti-IL-7 polyclonal antibody reduced RANKL overexpression induced by an HMCL (RPMI-8226) in activated T lymphocytes, whereas an irrelevant anti-IgG polyclonal antibody did not produce any effect (Figure 5A).
Moreover, to support our hypothesis we investigated whether myeloma cells were able to express or produce IL-7. We found that several HMCLs (RPMI-8226, OPM-2, XG-6, U266) and fresh purified CD138+ MM cells expressed IL-7 mRNA, whereas B-cell leukemia REH as well as PB MNCs and purified BM CD19+ B lymphocytes obtained from healthy donors and from patients with B-cell lymphoblastic leukemia did not (Figure 5B). However, IL-7 mRNA was detected in a portion of healthy donors as well as in some patients with infectious disease (data not shown). IL-7 was undetectable in conditioned medium of MNCs or normal B cells or the B-cell leukemic cell line REH. A slight stimulatory effect on IL-7 mRNA expression in HMCLs stimulated by IL-6 was found (Figure 5C, top). In addition IL-7 was significantly up-regulated when HMCLs were cultured in the presence of IL-6 (Figure 5C). In contrast, IL-6 did not induce IL-7 production in normal B cells and REH (Figure 5C) as well as in EBV+ cells and in B cells obtained from patients with acute lymphoblastic leukemia or B lymphoma with BM involvement (data not shown). All cells were previously evaluated for CD126 expression by flow cytometry. When we tested IL-7 levels in vivo we found that IL-7 serum levels were significantly higher in MM patients in comparison to healthy subjects (median, 12.15 pg/mL; range, 2.41-29.5 pg/mL versus 1.91 pg/mL; range, 0-3.43 pg/mL; P < .05). Similarly, IL-7 levels in BM plasma were significantly increased in MM patients in comparison with healthy subjects (median, 8.67 pg/mL; range, 2.68-36.8 pg/mL versus 0.40 pg/mL; range, 0-0.46 pg/mL; P < .05; Figure 5D). RANKL expression by T lymphocytes from MM patients Flow cytometry analysis demonstrated a significantly increased number of activated T lymphocytes bearing activation markers indicated by the presence of CD25, HLA-DR, and CD38 (data not shown). To address our in vitro results toward a clinical perspective we evaluated RANKL expression in CD3+ cells purified from 11 BM samples from 15 MM patients previously studied for skeletal involvement. Using RT-PCR we found that CD3+ cells obtained from BM of MM patients with extensive osteolysis and pathologic fractures (5 patients) expressed RANKL mRNA, whereas MM patients without osteolysis (6 patients) or control subjects did not (Figure 6B).
The present study shows that myeloma cells are able to induce an
up-regulation of RANKL and a down-regulation of IFN- The biologic mechanisms involved in the pathogenesis of MM bone disease are still not completely understood.1 Recently, in agreement with other authors,5 we have demonstrated that myeloma cells produce an imbalance in the OPG/RANKL expression in the BM environment.4 We show that myeloma cells are also able to affect RANKL expression in T lymphocytes. Using a coculture system, we demonstrate that HMCLs stimulate RANKL production and secretion by activated CD3+ cells. In addition, we show that RANKL overexpression is mediated by the release of a soluble factor because the stimulatory effect was observed in a transwell system. Among the molecules that could be responsible for the stimulation of RANKL, we focused our attention on IL-7. The role of IL-7 in the regulation of T homeostasis and activation is well established19; more recently, it has been postulated that IL-7 might be involved in osteoclast activation because IL-7 stimulates RANKL production by T cells.18 We found that HMCLs and fresh MM cells express IL-7 mRNA in contrast to normal or leukemic B cells and that IL-7 secretion was up-regulated in the presence of IL-6. This stimulatory effect of IL-6 on IL-7 expression and secretion seems to be specific for myeloma cells because we did not find any stimulatory effect of IL-6 on BM normal B cells as well as in EBV+ cells or in B leukemia cells. The role of IL-7 on RANKL stimulation in T lymphocytes by myeloma cells was confirmed by the inhibitory effect exerted by an antibody anti-IL-7 in the cocultures; furthermore, we found that IL-7 neutralization inhibited MM-induced osteoclastogenesis. An inhibitory effect on RANKL in the cocultures was also observed in the presence of anti-IL-6 mAb. It is likely that IL-6 is indirectly involved in the mechanism underlying RANKL stimulation by HMCLs because no evidence indicates that IL-6 stimulates RANKL in T lymphocytes or in other cell systems.4 On the other hand, activated T cells secrete IL-6 and we found that IL-6 levels were significantly higher in the supernatant of the cocultures. An increase of IL-6 production by T lymphocytes was also demonstrated in MM patients.20 In addition, our data indicate that IL-6 stimulates HMCLs to produce IL-7, which in turn stimulates RANKL in T lymphocytes.18 The potential involvement of IL-7 in the pathophysiology of MM is supported by the in vivo finding of higher IL-7 levels in PB serum and BM plasma of MM patients than in healthy subjects. Our in vitro model of osteoclastogenesis widely used by
others8,15,18 supports the possibility that the effect of
myeloma cells on RANKL production and secretion by activated T cells is involved in MM-induced osteoclast formation. In fact, we observed that
supernatants of CD3+ cells cocultured with HMCLs increased
osteoclast formation inhibited by rhOPG. However, OPG did not
completely blunt the stimulatory effect on RANKL production by
conditioned medium of cocultures. The OPG inability to completely block
the osteoclastogenetic activity of T cells, also reported by
others,8 suggests that RANKL-independent mechanisms are
also involved in this effect. Besides RANKL, other cytokines are
secreted by activated T cells,21-23 either
osteoclastogenetic such as TNF- On the other hand, it is unlikely that IL-7 produced by HMCLs could
affect IFN- Our in vitro model was supported by the in vivo finding of an increased number of activated T lymphocytes in the BM of MM patients. This observation confirms previous studies showing the presence of an activated T-cell subpopulation34 with a CD8 clonal expansion35-37 in peripheral blood of MM patients. In addition, the stimulatory effect on RANKL production by T cells was confirmed by the finding of RANKL mRNA expression in BM CD3+ T cells purified from MM patients with massive osteolysis (> 3 osteolytic lesions) in comparison with patients without osteolysis. Moreover, this observation should be confirmed in a larger cohort of patients. In line with our observation, activated CD3+ cells were found by others to be positive for RANKL expression in bone biopsies of MM patients.5 Our results suggest that in MM the RANKL overexpression by T lymphocytes is involved in osteoclast activation, as has been observed in other pathophysiologic conditions, such as rheumatoid arthritis,11,38 periodontal infection,39 osteoporosis,40 and hypercalcemia in adult T-cell leukemia.12 It is likely that myeloma cells induce osteoclastic cell activation
through the induction of OPG/RANKL imbalance in BM stromal cells as has
been recently demonstrated.4,5 The present study indicates
that myeloma cells up-regulate RANKL and down-regulate IFN- In conclusion, on the basis of these data and previous reports we can
now propose a new physiopathologic mechanism (Figure 7) underlying MM bone destruction that
highlights the critical role of the RANKL system.
Submitted April 19, 2002; accepted June 22, 2002.
Prepublished online as Blood First Edition Paper, August 8, 2002; DOI 10.1182/blood-2002-04-1121.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Nicola Giuliani, Hematology Department of Internal Medicine and Biomedical Science, University of Parma, via Gramsci 14, 43100 Parma, Italy; e-mail: nicola.giuliani{at}unipr.it.
1. Bataille R, Manolagas SC, Berenson JR. Pathogenesis and management of bone lesions in multiple myeloma. Hematol Oncol Clin North Am. 1997;11:349-361[CrossRef][Medline] [Order article via Infotrieve]. 2. Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 1998;93:165-176[CrossRef][Medline] [Order article via Infotrieve].
3.
Khosla S.
Minireview: the OPG/RANKL/RANK system.
Endocrinology.
2001;142:5050-5055 4. Giuliani N, Bataille R, Mancini C, Lazzaretti M, Barille S. Myeloma cells induce imbalance in the osteoprotegerin/osteoprotegerin ligand system in the human bone marrow environment. Blood. 2001;98:3527-3533.
5.
Pearse RN, Sordillo EM, Yaccoby S, et al.
Multiple myeloma disrupts the TRANCE/osteoprotegerin cytokine axis to trigger bone destruction and promote tumor progression.
Proc Natl Acad Sci U S A.
2001;98:11581-11586
6.
Sezer O, Heider U, Jakob C, Eucker J, Possinger K.
Human bone marrow myeloma cells express RANKL.
J Clin Oncol.
2002;20:353-354 7. Altamirano CV, Ma HJ, Parker KM, et al. RANKL is expressed in malignant multiple myeloma cell lines [abstract]. Blood. 2000;96:360a. 8. Weitzmann MN, Cenci S, Rifas L, Haug J, Dipersio J, Pacifici R. T cell activation induces human osteoclast formation via receptor activator of nuclear factor kappaB ligand-dependent and -independent mechanisms. J Bone Miner Res. 2001;16:328-337[CrossRef][Medline] [Order article via Infotrieve].
9.
Kim N, Odgren PR, Kim DK, Marks SC Jr, Choi Y.
Diverse roles of the tumor necrosis factor family member TRANCE in skeletal physiology revealed by TRANCE deficiency and partial rescue by a lymphocyte-expressed TRANCE transgene.
Proc Natl Acad Sci U S A.
2000;97:10905-10910 10. Takayanagi H, Ogasawara K, Hida S, et al. T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma. Nature. 2000;408:600-605[CrossRef][Medline] [Order article via Infotrieve]. 11. 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;402:304-309[CrossRef][Medline] [Order article via Infotrieve]. 12. Nosaka K, Miyamoto T, Sakai T, Mitsuya H, Suda T, Matsuoka M. Mechanism of hypercalcemia in adult T-cell leukemia: overexpression of receptor activator of nuclear factor kappaB ligand on adult T-cell leukemia cells. Blood. 2002;99:634-640.
13.
Zhang XG, Gaillard JP, Robillard N, et al.
Reproducible obtaining of human myeloma cell lines as a model for tumor stem cell study in human multiple myeloma.
Blood.
1994;83:3654-3663
14.
Barille S, Akhoundi C, Collette M, et al.
Metalloproteinases in multiple myeloma: production of matrix metalloproteinase-9 (MMP-9), activation of proMMP-2, and induction of MMP-1 by myeloma cells.
Blood.
1997;90:1649-1655
15.
Matayoshi A, Brown C, DiPersio JF, et al.
Human blood-mobilized hematopoietic precursors differentiate into osteoclasts in the absence of stromal cells.
Proc Natl Acad Sci U S A.
1996;93:10785-10790 16. Faust J, Lacey DL, Hunt P, et al. Osteoclast markers accumulate on cells developing from human peripheral blood mononuclear precursors. J Cell Biochem. 1999;72:67-80[CrossRef][Medline] [Order article via Infotrieve].
17.
Fagnoni FF, Vescovini R, Passeri G, et al.
Shortage of circulating naive CD8(+) T cells provides new insights on immunodeficiency in aging.
Blood.
2000;95:2860-2868 18. Weitzmann MN, Cenci S, Rifas L, Brown C, Pacifici R. Interleukin-7 stimulates osteoclast formation by up-regulating the T-cell production of soluble osteoclastogenic cytokines. Blood. 2000;96:1873-1878. 19. Fry TJ, Mackall CL. Interleukin-7: master regulator of peripheral T-cell homeostasis? Trends Immunol. 2001;22:564-571[CrossRef][Medline] [Order article via Infotrieve]. 20. Lapena P, Prieto A, Garcia-Suarez J, et al. Increased production of interleukin-6 by T lymphocytes from patients with multiple myeloma. Exp Hematol. 1996;24:26-30[Medline] [Order article via Infotrieve]. 21. Andersson U, Sander B, Andersson J, Moller G. Concomitant production of different lymphokines in activated T cells. Eur J Immunol. 1988;18:2081-2084[Medline] [Order article via Infotrieve].
22.
Wang R, Zhang L, Zhang X, et al.
Differential regulation of the expression of CD95 ligand, receptor activator of nuclear factor-kappa B ligand (RANKL), TNF-related apoptosis-inducing ligand (TRAIL), and TNF-alpha during T cell activation.
J Immunol.
2001;166:1983-1990
23.
Josien R, Wong BR, Li HL, Steinman RM, Choi Y.
TRANCE, a TNF family member, is differentially expressed on T cell subsets and induces cytokine production in dendritic cells.
J Immunol.
1999;162:2562-2568
24.
Feldmann M, Londei M, Haworth C.
T cells and lymphokines.
Br Med Bull.
1989;45:361-370 25. Dinarello CA. Interleukin-18, a proinflammatory cytokine. Eur Cytokine Netw. 2000;11:483-486[Medline] [Order article via Infotrieve].
26.
Chen NJ, Huang MW, Hsieh SL.
Enhanced secretion of IFN-gamma by activated Th1 cells occurs via reverse signaling through TNF-related activation-induced cytokine.
J Immunol.
2001;166:270-276
27.
Portier M, Zhang XG, Caron E, Lu ZY, Bataille R, Klein B.
28. Frassanito MA, Cusmai A, Dammacco F. Deregulated cytokine network and defective Th1 immune response in multiple myeloma. Clin Exp Immunol. 2001;125:190-197[CrossRef][Medline] [Order article via Infotrieve]. 29. Cook G, Campbell JD, Carr CE, Boyd KS, Franklin IM. Transforming growth factor beta from multiple myeloma cells inhibits proliferation and IL-2 responsiveness in T lymphocytes. J Leukoc Biol. 1999;66:981-988[Abstract]. 30. Yamamoto N, Zou JP, Li XF, et al. Regulatory mechanisms for production of IFN-gamma and TNF by antitumor T cells or macrophages in the tumor-bearing state. J Immunol. 1995;154:2281-2290[Abstract].
31.
Holter W, Kalthoff FS, Pickl WF, et al.
Transforming growth factor-beta inhibits IL-4 and IFN-gamma production by stimulated human T cells.
Int Immunol.
1994;6:469-475 32. Bonig H, Banning U, Hannen M, et al. Transforming growth factor-beta1 suppresses interleukin-15-mediated interferon-gamma production in human T lymphocytes. Scand J Immunol. 1999;50:612-618[CrossRef][Medline] [Order article via Infotrieve]. 33. Borger P, Kauffman HF, Postma DS, Vellenga E. IL-7 differentially modulates the expression of IFN-gamma and IL-4 in activated human T lymphocytes by transcriptional and post-transcriptional mechanisms. J Immunol. 1996;156:1333-1338[Abstract].
34.
Dianzani U, Pileri A, Boccadoro M, et al.
Activated idiotype-reactive cells in suppressor/cytotoxic subpopulations of monoclonal gammopathies: correlation with diagnosis and disease status.
Blood.
1988;72:1064-1068
35.
Moss P, Gillespie G, Frodsham P, Bell J, Reyburn H.
Clonal populations of CD4+ and CD8+ T cells in patients with multiple myeloma and paraproteinemia.
Blood.
1996;87:3297-3306
36.
Sze DM, Giesajtis G, Brown RD, et al.
Clonal cytotoxic T cells are expanded in myeloma and reside in the CD8(+)CD57(+)CD28( 37. Raitakari M, Brown RD, Sze D, et al. T-cell expansions in patients with multiple myeloma have a phenotype of cytotoxic T cells. Br J Haematol. 2000;110:203-209[CrossRef][Medline] [Order article via Infotrieve]. 38. Kotake S, Udagawa N, Hakoda M, et al. Activated human T cells directly induce osteoclastogenesis from human monocytes: possible role of T cells in bone destruction in rheumatoid arthritis patients. Arthritis Rheum. 2001;44:1003-1012[CrossRef][Medline] [Order article via Infotrieve]. 39. Teng YT, Nguyen H, Gao X, et al. Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection. J Clin Invest. 2000;106:59-67.
40.
Roggia C, Gao Y, Cenci S, et al.
Up-regulation of TNF-producing T cells in the bone marrow: a key mechanism by which estrogen deficiency induces bone loss in vivo.
Proc Nat Acad Sci U S A.
2001;98:13960-13965
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  M. F. Faienza, G. Brunetti, S. Colucci, L. Piacente, M. Ciccarelli, L. Giordani, G. C. Del Vecchio, M. D'Amore, L. Albanese, L. Cavallo, et al. Osteoclastogenesis in Children with 21-Hydroxylase Deficiency on Long-Term Glucocorticoid Therapy: The Role of Receptor Activator of Nuclear Factor-{kappa}B Ligand/Osteoprotegerin Imbalance J. Clin. Endocrinol. Metab., July 1, 2009; 94(7): 2269 - 2276. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. E. Kearns, S. Khosla, and P. J. Kostenuik Receptor Activator of Nuclear Factor {kappa}B Ligand and Osteoprotegerin Regulation of Bone Remodeling in Health and Disease Endocr. Rev., April 1, 2008; 29(2): 155 - 192. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Vega, N. M. Maalouf, and K. Sakhaee The Role of Receptor Activator of Nuclear Factor-{kappa}B (RANK)/RANK Ligand/Osteoprotegerin: Clinical Implications J. Clin. Endocrinol. Metab., December 1, 2007; 92(12): 4514 - 4521. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Weinstock-Guttman, J. Hong, R. Santos, M. Tamano-Blanco, D. Badgett, K. Patrick, M. Baier, J. Feichter, E. Gallagher, N. Garg, et al. Interferon-{beta} modulates bone-associated cytokines and osteoclast precursor activity in multiple sclerosis patients Multiple Sclerosis, September 1, 2006; 12(5): 541 - 550. [Abstract] [PDF]
|
|
|
|
 |
|
 N. Giuliani, F. Morandi, S. Tagliaferri, S. Colla, S. Bonomini, G. Sammarelli, and V. Rizzoli Interleukin-3 (IL-3) is overexpressed by T lymphocytes in multiple myeloma patients Blood, January 15, 2006; 107(2): 841 - 842. [Full Text] [PDF]
|
|
|
|
|
|
N. Giuliani, S. Colla, F. Morandi, M. Lazzaretti, R. Sala, S. Bonomini, M. Grano, S. Colucci, M. Svaldi, and V. Rizzoli Myeloma cells block RUNX2/CBFA1 activity in human bone marrow osteoblast progenitors and inhibit osteoblast formation and differentiation Blood, October 1, 2005; 106(7): 2472 - 2483. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Shibata, M. Abe, K. Hiura, J. Wilde, K. Moriyama, T. Sano, K.-i. Kitazoe, T. Hashimoto, S. Ozaki, S. Wakatsuki, et al. Malignant B-Lymphoid Cells with Bone Lesions Express Receptor Activator of Nuclear Factor-{kappa}B Ligand and Vascular Endothelial Growth Factor to Enhance Osteoclastogenesis Clin. Cancer Res., September 1, 2005; 11(17): 6109 - 6115. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Terpos and M.-A. Dimopoulos Myeloma bone disease: pathophysiology and management Ann. Onc., August 1, 2005; 16(8): 1223 - 1231. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Colucci, G. Brunetti, R. Rizzi, A. Zonno, G. Mori, G. Colaianni, D. Del Prete, R. Faccio, A. Liso, S. Capalbo, et al. T cells support osteoclastogenesis in an in vitro model derived from human multiple myeloma bone disease: the role of the OPG/TRAIL interaction Blood, December 1, 2004; 104(12): 3722 - 3730. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Meyer, L. C. Hofbauer, A. Neubauer, M. Schoppet, C. M. Lu, R. C. Walker, B. Barlogie, and J. Shaughnessy Jr. DKK1 in Multiple Myeloma N. Engl. J. Med., April 1, 2004; 350(14): 1464 - 1466. [Full Text] [PDF]
|
|
|
|
 |
|
 S. Yaccoby, M. J. Wezeman, A. Henderson, M. Cottler-Fox, Q. Yi, B. Barlogie, and J. Epstein Cancer and the Microenvironment: Myeloma-Osteoclast Interactions as a Model Cancer Res., March 15, 2004; 64(6): 2016 - 2023. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Giuliani, S. Colla, V. Rizzoli, S. Barille-Nion, and R. Bataille Do Human Myeloma Cells Directly Produce the Receptor Activator of Nuclear Factor {kappa}B Ligand (RANKL) or Induce RANKL in the Bone Marrow Microenvironment? Cancer Res., January 15, 2004; 64(2): 772 - 773. [Full Text] [PDF]
|
|
|
|
|
|
U. Heider, I. Zavrski, C. Jakob, C. Fleissner, O. Sezer, A. C. W. Zannettino, B. Pan, A. N. Farrugia, G. J. Atkins, and L. B. To Identification and Clinical Relevance of Receptor Activator of Nuclear Factor {kappa}B Ligand Expression of Myeloma Cells Cancer Res., January 15, 2004; 64(2): 773 - 775. [Full Text] [PDF]
|
|
|
|
|
|
O. Sezer, U. Heider, I. Zavrski, C. A. Kuhne, and L. C. Hofbauer RANK ligand and osteoprotegerin in myeloma bone disease Blood, March 15, 2003; 101(6): 2094 - 2098. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
B ligand (RANKL) in T lymphocytes: a potential role in
multiple myeloma bone disease
Top
Abstract
Introduction
B ligand (RANKL), and interferon 
