The FASEB Journal express article10.1096/fj.02-1188fje. Published online July 18, 2003.
The receptor activator of nuclear factor (NF)κB ligand (RANKL) is a new chemotactic factor for human monocytes Véronique Breuil,*,† Heidy Schmid-Antomarchi,* Annie Schmid-Alliana,* Roger Rezzonico,* Liana Euller-Ziegler,† and Bernard Rossi* *INSERM Unit 364, IFR 50 Faculté de Médecine Pasteur, Avenue de Valombrose, 06107 Nice Cedex 2, France. †Rheumatology Department, L’Archet Hospital, Route St Antoine de Ginestière, 06200 Nice, France Corresponding author: Bernard Rossi, Unit INSERM 364, IFR 50 Faculté de Médecine Pasteur, Avenue de Valombrose, 06107 Nice Cedex, France. E-mail:
[email protected] ABSTRACT Bone resorption is regulated by the immune system, where receptor activator of nuclear factor (NF)κB ligand (RANKL), a new member of the tumor-necrosis factor family, may contribute to pathological conditions. Due to the role of RANKL in the maturation of monocyte-derived osteoclasts, we hypothesized that RANKL could exert chemotactic properties toward monocytic cells. Our results demonstrate that RANKL induces the migration of MonoMac-6 monocytic cells as well as human freshly isolated total peripheral blood mononuclear cells (PBMC) and CD14+ purified PBMC. RANKL induces the migration of MonoMac-6 cells in a dose-dependent manner and with an efficacy similar to MCP-1. After an 8-h incubation, the soluble form of RANKL (sRANKL) started to exhibit a chemoattractive effect on MonoMac-6 cells, with an increased effect observed up to 24 h. RANKL elicits an additive chemotactic effect to MCP-1. Furthermore, addition of the RANKL decoy receptor osteoprotegerin in the lower well or RANKL in the upper well abrogates the RANKL-induced migration of MonoMac-6 cells, hallmarking a true specific activity. RNase protection assay experiments indicate that exposure of MonoMac-6 cells to RANKL had no significant effect on the expression of a variety of chemokines, known to attract monocytes. This study provides evidence that RANKL behaves as a chemotactic factor for monocytic cells, emphazing the cross-talk between bone and immune systems. Key words: migration • CD14+ • MonoMac6 cell line • osteoprotegerin
T
he transmembrane receptor activator of nuclear factor (NF)κB ligand (RANKL), also called TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine) or OPGL (osteoprotegerin ligand), is a member of the TNF family, that plays a critical role in osteoclast formation and activation (1–3). RANKL is expressed at the surface of T and B lymphocytes, dendritic cells, osteoblasts, and bone marrow stromal cells (4–6). RANKL interacts with RANK, a cell surface receptor member of the TNF receptor family, expressed by monocyte-derived osteoclast precursors, T cells, B cells, and dendritic cells (4–6). Like TNF-α, RANKL is synthesized as a membrane-anchored precursor and is cleaved by a metalloproteasedisintegrin TNF-α convertase (TACE) to generate the soluble form of RANKL (sRANKL) (1, 7). In T cells, RANKL expression is induced by T cell receptor engagement, suggesting that
activated T cells may influence bone metabolism through the RANKL/RANK pathway and contribute to pathological conditions (6, 8). Indeed, in rheumatoid arthritis, characterized by an infiltration of activated T cells and monocytes, RANKL is expressed in T cells and fibroblastlike cells in the synovium, and treatment by osteoprotegerin (OPG), the decoy receptor for RANKL, prevents bone erosions in a model of adjuvant arthritis (9). RANKL and RANK null mice present a severe osteopetrosis, with a complete lack of osteoclasts due to the inability of monocytic precursors to maturate, whereas maturation of monocytes and macrophages is not affected (10, 11). Furthermore, RANKL null mice exhibit a complete lack of lymph nodes, suggesting a possible role of RANKL in the homing of lymphocyte populations, according to the expression of RANKL/RANK in B and T lymphocytes (3, 12). Consistent with its role in osteoclastogenesis, expression of RANKL is greatly increased by inflammatory factors known to stimulate bone resorption, such as interleukin (IL)-1β, TNF-α, IL-11, and PGE 2 (13). To reach the sites of bone resorption from bone marrow or peripheral blood, osteoclast precursors need to migrate through the vascular endothelium. However, to date, the mechanisms by which osteoclast progenitors are attracted to the bone remodeling site remains to be elucidated (14, 15). In this regard, recent studies are of particular interest: (i) RANKL is expressed on microvascular endothelial cells in response to IL-1α and TNF-α, especially on capillaries in the vicinity of resorbing osteoclasts in active bone remodeling regions (16); (ii) the adhesion of osteoclast precursors to endothelial cells requires IL-1β or TNF-α stimulation (17). Based on these findings, we hypothesized that recruitment of osteoclasts at the bone remodeling sites could be mediated by sRANKL-induced monocyte chemoattraction. We show here that sRANKL, besides its role in osteoclast maturation, exhibits chemotactic properties toward human monocytes. MATERIALS AND METHODS Cell culture MonoMac-6 cells (DSM ACC124), originally established from a patient with monoblastic leukemia, were obtained from the German Collection of Microorganisms (Braunschweig, Germany) (18). These cells were grown in suspension in RPMI 1640 medium (Gibco, CergyPontoise, France) supplemented with 10% heat-inactivated fetal calf serum (Gibco), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), nonessential amino acids (Gibco), sodium pyruvate (1 mM, Gibco), insulin (9 µg/ml, Sigma, St. Louis, MO). Cells were diluted every 3–6 days using a split ratio 1:3. Isolation of peripheral blood mononuclear cells (PBMC) and CD14+ monocytes PBMC were obtained from buffy coats of blood from normal donors. Ten milliliters of blood were collected by standard venipuncture into heparinized tubes. Each tube was diluted with an equal volume of phosphate-buffered saline (PBS) and 10 ml of blood preparation overlaid onto 15 ml of Ficoll-Paque (Eurobio, Les Ulis, France). Tubes were centrifuged (700g, 15 min, 4°C); the buffy coat interface was removed, added to 10 ml of PBS, and then centrifuged (300g, 10 min, room temperature); and the pellet resuspended in α-MEM + 0.1% BSA (Gibco). The number of PBMC was determined using a hematocytometer.
For CD14+ experiments, positive selection was performed by adding MACS colloidal superparamagnetic microbeads (Miltenyi Biotec, Paris, France) conjugated with monoclonal anti-human CD14 antibodies to freshly prepared PBMC preparation, according to the manufacturer’s instructions. In brief, cells and microbeads were incubated for 15 min at 4°C. The cells were then washed with MACS buffer, resuspended, and loaded onto the top of the separation column. The eluent containing CD14– cells was withdrawn, and trapped CD14+ PBMC were eluted and resuspended in MEMα medium + 0.1% BSA. The purity of the CD14+ cells was determined by flow cytometry analysis at 93.6%, corresponding to 4.3% of the total PBMC. Products Human rMCP-1 was purchased from R&D Systems (Abigton, UK). Fibronectin was obtained from Sigma (Saint Quentin Fallavier, France). Recombinant soluble human RANKL (sRANKL) and OPG were kindly provided by Dr. C. Dunstan (Amgen, Thousand Oaks, CA). Chemotaxis assays As described previously, chemotactic responses of MonoMac-6 cells were evaluated using 24well chemotaxis chambers and polyethylene terephtalate (PET) inserts with 8-µm pores (BectonDickinson, San Jose, CA) coated with 6.5 µg/ml fibronectin on both sides (19). MonoMac-6 cells, incubated for 16 h with [3H]-methyl thymidine (ICN, 2.5 µCi/ml), were placed in the upper well (106 cells/100 µl/ well), and monocyte chemotactic protein-1 (MCP-1), OPG, or sRANKL, at various concentrations, was added to the lower well. After incubation at 37°C in 5% CO2 atmosphere, migrated cells were collected at different time points in the lower well and evaluated by measuring [3H]-methyl thymidine incorporation by scintillation spectroscopy. Each experiment was performed in triplicate. Alernatively, migration was evaluated, as described previously by Barleon et al. (20). Briefly, 50 µl of PBMC (1.5 × 106/ml) were seeded in the upper wells. sRANKL (100 ng/ml) in α-MEM + 0.1% BSA was added to the lower wells of chemotaxis chamber (5-µm pore size polycarbonate filter, without coating) (Costar, Corning, NY). After a 90-min incubation of chambers at 37°C in air with 5% CO2, filters were removed and cells remaining in the upper well were discarded. Filters were fixed in PBS-formaldehyde 1%, and the filters’ lower sides were stained for 30 s with hematoxylin. Five fields were counted on each filter (×100 magnification), and each experiment was performed in triplicate. The same procedure of the chemotaxis assay for human total PBMC was then used for CD14+ cells. RNase extraction and RNase protection assay (RPA) analysis MonoMac-6 cells were starved overnight in RPMI containing 0.1% BSA and seeded (7 × 106 cells/condition in 1 ml) into the upper well of six-well chemotaxis chambers and PET inserts with 8-µm pores (Becton-Dickinson) coated with 6.5 µg/ml fibronectin on both sides. Serumfree RPMI medium or serum-free RPMI medium supplemented with 100 ng/ml sRANKL was added to the lower well. After incubation at 37°C in 5% CO2 atmosphere, at the indicated time points, the cells of the upper well were isolated and total RNA was extracted by lysing MonoMac-6 cells with TRIzol reagent (Life Technologies, Cergy Pontoise, France) according to
the manufacturer’s instructions. The level of expression of RANTES, IP-10, MIP-1α, MIP-1β, MCP-1, IL-8, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analyzed by RiboQuant RPA, using the hck-5 multiprobe template set from Becton-Dickinson. In brief, riboprobes were 32P-labeled and hybridized overnight in solution with 10 µg of the RNA samples. The hybridized RNA was digested with RNase and the remaining “RNase-protected” probes were purified, resolved on denaturating polyacrylamide gels, and imaged by autoradiography according to the RiboQuant protocol. RESULTS sRANKL induces migration of the monocytic cell line MonoMac-6 in a dose-dependent manner Before testing the ability of sRANKL to induce the migration of MonoMac-6 cells, we first confirmed that MonoMac-6 cells did express the RANK receptor by reverse transcriptasepolymerase chain reaction (RT-PCR) (data not shown). To determine the optimal conditions of migration, MonoMac-6 cells were exposed for various periods of time to a gradient of human recombinant sRANKL. We found that sRANKL started to exhibit a chemoattractive effect on MonoMac-6 cells after 8 h, with an increasing effect observed for up to 24 h of incubation (Fig. 1). This kinetic profile was different from the time course observed for the MonoMac-6 cell migration in response to the reference chemokine for monocyteMCP-1, which culminated at 8 h (Fig. 1). The migration of MonoMac-6 cells in response to sRANKL was dose-dependent, with a halfmaximal effect at 0.6 nM (20 ng/ml) (Fig. 2). At 24 h, the sRANKL-induced chemotaxis was 1.73-fold that of the basal value (+73% ± 10%), corresponding to a slightly higher efficacy than MCP-1 (+36% ± 2%) (Fig. 1). Chemoattractive properties of sRANKL are confirmed on freshly isolated PBMC and CD14+ purified PBMC To test whether RANKL could also attract normal cells, we exposed freshly isolated human total PBMC or CD14+ purified PBMC to a gradient of sRANKL. As shown in Table 1, sRANKL, at 100 ng/ml, increased by 1.4-fold the migration of PBMC as well as CD14+ purified monocytes, close to the value observed with MCP-1. CD14+ cells migrated at a lesser extent than PBMC, both in basal, MCP-1, or RANKL conditions, likely because CD14-coated beads attached to monocytes hindered their migration. sRANKL is a true chemotactic agent toward monocytic cells As shown in Figure 3, the migration of MonoMac-6 cells was abolished when sRANKL was added both in the upper and lower reservoir, indicating that the sRANKL effect did correspond to chemotaxis and not to chemokinesis. To analyze the sRANKL specificity of monocyte chemoattraction, we performed sRANKLinduced chemotaxis experiments in the presence of threefold excess of OPG, the soluble decoy receptor for RANKL. Under these conditions, addition of OPG abrogated the chemoattractive
effect of sRANKL on MonoMac-6 cells (Fig. 4A), as well as on freshly isolated PBMC (Fig. 4B). Given that sRANKL-induced chemotaxis increased up to 24 h, we wanted to rule out the possibility that the observed chemotactic effect of sRANKL was due to the secretion of a monocyte-attracting chemokine. To this end, we measured, by RNase protection assay, the level of transcripts encoding for a panel of chemokines known to efficiently attract monocytes. As shown in Figure 5 and Table 2, none of the transcripts encoding for MCP-1, RANTES, MIP-1α, MIP-1β, or IL-8 were significantly modified, compared with the level of GAPDH, when cells were exposed to sRANKL for the indicated times. The effect of sRANKL is additive to the chemoattraction induced by MCP-1 To gain information on the means by which sRANKL mediated its chemotactic action, we measured the migration of MonoMac-6 cells in the presence of sRANKL alone or in combination with MCP-1. As shown in Figure 6, sRANKL exerted an effect that was additive to that induced by a maximal concentration (5 nM) of MCP-1. DISCUSSION Since its recent discovery, RANKL has been shown to play a role in various functions in bone metabolism and the immune system (1–6). Although other members of the TNF family are known to exert chemotactic effects toward monocytes, to date, no chemotactic activity has been ascribed to RANKL (21). Our results provide evidence that sRANKL can act as a potent chemotactic factor for human monocytes, which may be of importance to better understand interactions between bone and immune system. To test the hypothesis that sRANKL might intervene not only as a maturating factor of osteoclast precursors, but also in the recruitment process of circulating monocytes at the bone remodeling site, we used initially the MonoMac-6 cell line, which has proven to be a suitable model for studying the effect of chemokines on cells of the monocytic lineage (19). The ability of human recombinant sRANKL to induce the migration of [3H]-thymidine-labeled MonoMac-6 cells was measured through an acellular fibronectin-coated filter, as described previously (19). These experimental conditions present the advantage, compared with a transmigration system through an endothelial monolayer, of avoiding the possible sRANKL-induced secretion of chemokine(s) by endothelial cells, which may interfere with the test. sRANKL promoted the migration of MonoMac-6 cells in a dose-dependent manner, with a halfmaximal effect at 0.6 nM (20 ng/ml), according to the affinity of sRANKL for its receptor (3). Note that, under optimal conditions (i.e., 0.6 nM and 24 h migration), sRANKL-induced MonoMac-6 migration was as efficient as MCP-1, the reference chemokine for monocyte migration (22). Similar results were obtained with normal PBMC or CD14+ purified cells, strengthening the physiological relevance of the chemoattractive properties that sRANKL exerts toward cells of the monocyte lineage. The effect of sRANKL was totally inhibited by the addition of its decoy receptor, OPG, reinforcing the idea that sRANKL-induced monocyte chemoattraction is specific. Because sRANKL-induced chemotaxis increases up to 24 h, we verified that this effect was not indirectly
due to the secretion of chemokine(s) induced by sRANKL. However, sRANKL-induced secretion of chemokines by monocytes in the upper reservoir can very unlikely trigger migration toward the lower reservoir. In fact, none of the chemokines known to induce monocyte migration (RANTES, IP-10, MIP-1α, MIP-1β, MCP-1, IL-8) was increased in the presence of sRANKL, as assessed by RNase protection assay, ruling out the indirect implication of other chemokines (23). These data support the hypothesis that sRANKL is able, per se, to induce monocyte chemotaxis. Furthermore, the additive effect that sRANKL exerted over the MCP-1-induced migration of MonoMac-6 cells suggests that sRANKL mediates its action through its own signaling pathways. RANKL exists in two biologically active forms: a cellular membrane-bound form (mRANKL) and a soluble form derived posttranslationally by cleavage by TACE (1, 7). Interestingly, the morphology of osteoclasts maturated in the presence of sRANKL slightly differs from that obtained by culturing PBMC in contact with stromal cells expressing mRANKL (24). Moreover, sRANKL induces a persistent expression of CD11c and CD18 integrin that has been shown to be crucial for MCP-1-induced monocyte migration, whereas mRANKL expressed by stromal cells fails to do so (24, 25). The fact that only sRANKL might behave as a chemoattractant for osteoclast precursors points to the attractive hypothesis that RANKL could exert distinct effects depending on its membrane vs. soluble status. This is reminiscent of the situation observed for fractalkine, a membrane-associated chemokine, which, in its membrane form, is mainly involved in cell-cell adhesion processes (26), whereas the soluble form of fractalkine, resulting from a proteolytic cleavage, exhibits chemotactic properties toward NK cells and CD8 lymphocytes (27, 28). It is now well established that inflammation induces bone resorption, as observed in inflammatory chronic diseases such as rheumatoid arthritis and ankylosing spondylitis (13, 29– 31). Several recent discoveries have improved our knowledge of the interactions on these cellular systems, in which the RANKL/RANK/OPG system plays a major role (32–34). Indeed, the RANKL network is strongly implicated in the normal physiology of differentiation and function of both bone and immune cells and in the pathophysiology of inflammatory chronic diseases, such as rheumatoid arthritis, characterized by local and systemic bone loss (13, 29–33). However, important questions remain to be answered before the intricacies of the relationship between bone and immune cells can be fully understood (35). In conclusion, our data, by providing evidence that sRANKL behaves as a true chemoattractant for monocytes, shed new light on the complex mechanisms that link bone metabolism and immunity. ACKNOWLEDGMENTS We thank the Institut National de la Santé et de la Recherche Médicale (INSERM) for its financial support and Dr. Pascal Staccini for the statistical analysis. REFERENCES 1.
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Table 1 sRANKL is a chemotactic factor for freshly isolated human PBMC and CD14+ purified PBMCa Experiment PBMC - 1 PBMC - 2 PBMC - 3 PBMC - 4 CD14+ - 1 a
Control Median 95% CI 65 60–68.25 68 66–69.98 68 66–73.63 69 65.75–71.25 54 52–58
RANKL (100 ng/ml) Median 95% CI 82 76.12–87.25 98 92.5–101 97 92.1–102.6 96 92.4–100.3 71 68.50–77.00
P
