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The receptor activator of nuclear factor (NF)-B ligand (RANKL) is a new chemotactic factor for human monocytes¹

VÉRONIQUE BREUIL^*,, HEIDY SCHMID-ANTOMARCHI^*, ANNIE SCHMID-ALLIANA^*, ROGER REZZONICO^*, LIANA EULLER-ZIEGLER and BERNARD ROSSI^*,2

^* INSERM Unit 364, IFR 50 Faculté de Médecine Pasteur, Nice, Cedex, France; and
Rheumatology Department, L’Archet Hospital, Nice, France

²Correspondence: Unit INSERM 364, IFR 50 Faculté de Médecine Pasteur, Avenue de Valombrose 06107, Nice, Cedex, France. E-mail: rossi{at}unice.fr doi: 10.1096/fj.02-1188fje

SPECIFIC AIMS

The receptor activator of nuclear factor-B ligand (RANKL) plays a critical role in bone metabolism, especially in promoting monocyte-derived osteoclast precursor maturation. RANKL exists as a membrane-anchored or a soluble form (sRANKL). Starting from the observations that RANKL is expressed at the surface of inflamed vascular endothelium, inflammation induces bone resorption, and RANKL is involved in the recruitment of leucocytes during lymph node organogenesis, we posed the hypothesis that sRANKL may have a chemoattractive effect on monocytes from which osteoclasts are derived.

PRINCIPAL FINDINGS

1. sRANKL induces the migration of the monocytic cell line MonoMac-6
Boyden chamber experiments revealed that sRANKL started to exhibit a chemoattractive effect on MonoMac-6 cells after 8 h, increasing up to 24 h of incubation (Fig. 1 ). This kinetic profile was different from the time-course observed for the MonoMac-6 cells migration in response to the reference chemokine for monocyte MCP-1, which culminated at 8 h (Fig. 1) . The migration of MonoMac-6 cells in response to sRANKL was dose-dependent, and a half-maximal effect was at 0.6 nM (20 ng/ml). 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 ).

View larger version (17K): [in this window] [in a new window]   Figure 1. Time-course of MonoMac-6 (MM6) cell migration across fibronectin-coated filters in response to monocyte-chemoattractant protein-1 (MCP-1) and sRANKL. [3H]-Methyl thymidine-labeled MonoMac-6 cells were loaded in the upper well of the chemotaxis chamber. The lower well contained serum-free medium (open bars), serum-free medium supplemented with 5 nM MCP-1 (shaded bars), or sRANKL (solid bars) at 100 ng/ml. At the indicated time, the amount of migrated cells was evaluated by liquid scintillation ß-counts. The control group (open bars) is referred to as 100% migration and corresponds to 1% migration of total loaded cells at 8 h, 1.7% at 12 h, and 6.2% at 24 h, respectively. The results are expressed as mean ± SEM of triplicate measurements and are representative of three separate experiments.

2. sRANKL also induces the migration of freshly isolated human peripheral blood mononuclear cells (PBMC) or CD14⁺-purified PBMC
To test whether RANKL could also attract normal cells, freshly isolated human total PBMC or CD14⁺-purified PBMC were exposed to a gradient of sRANKL in migration chambers. 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.

3. sRANKL is a true chemotactic agent toward monocytic cells
sRANKL-induced migration of MonoMac-6 cells was abolished when sRANKL was added 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 sRANKL-induced chemotaxis experiments in the presence of a threefold excess of osteoprotegerin (OPG), the soluble decoy receptor for RANKL. Under these conditions, addition of OPG along with RANKL in the lower reservoir abrogated the chemoattractive effect of sRANKL on MonoMac-6 cells, as well as on freshly isolated PBMC (Fig. 2 ).

View larger version (23K): [in this window] [in a new window]   Figure 2. Inhibition by OPG of sRANKL-induced chemotaxis on MonoMac-6 (MM6) cells (A) and freshly isolated human PBMC (B). A) [3H]-Methyl thymidine-labeled MonoMac-6 cells were loaded in the upper wells of the chemotaxis chamber. The lower well contained serum-free medium (open bars), serum-free medium supplemented with 100 ng/ml sRANKL (shaded bars), or serum-free medium supplemented with 100 ng/ml sRANKL and 300 ng/ml OPG (solid bars). After 24 h, migrated cells were evaluated by measuring [3H]-methyl thymidine incorporation by scintillation spectroscopy as described above. Control group is referred as 100% migration and represents 4% migration of input cells. Results are expressed as the mean ± SEM of triplicate measurements and are representative of three separate experiments. B) Freshly isolated human PBMC were loaded in the upper well of the chemotaxis chamber. The lower well contained serum-free medium (open bars), serum-free medium supplemented with 100 ng/ml sRANKL (shaded bars), or serum-free medium supplemented with 100 ng/ml sRANKL and 300 ng/ml OPG (solid bars). After a 90-min incubation of chambers at 37°C in air with 5% CO2, filters were fixed in phosphate-buffered saline–formaldehyde 1%, and filters’ lower side were stained for 30 s with hematoxyline. Five fields were counted on each filter (x100 magnification). Control group is referred to as 100% migration, corresponding to a median of 65 cells per field counted (five fields per filter were counted). Results are expressed as the mean ± SD of triplicate measurements and are representative of three separate experiments.

Given that sRANKL-induced chemotaxis increases up to 24 h, we wanted to rule out the possibility that the observed chemotactic effect of sRANKL was a result of the secretion of a monocyte-attracting chemokine. RNase protection assay experiments revealed that none of the transcripts encoding for MCP-1, regulated on activation, normal T expressed and secreted (RANTES), macrophage-inflammatory protein-1 (MIP-1), MIP-1ß, or interleukin (IL)-8 was significantly modified, compared with the level of glyceraldehyde 3-phosphate dehydrogenase, when cells were exposed to sRANKL for the indicated time-points.

4. The effect of sRANKL on monocytes migration 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. sRANKL exerted an effect that was additive to that induced by a maximal concentration (5 nM) of MCP-1.

CONCLUSIONS AND SIGNIFICANCE

Since its recent discovery, RANKL has been shown to play a role in various functions in bone metabolism and immune system, but to date, no chemotactic activity has been ascribed to RANKL. Our results provide evidence that sRANKL can act as a potent chemotactic factor for human monocytes (Fig. 3 ).

View larger version (90K): [in this window] [in a new window]   Figure 3. Chemotactic effect of sRANKL, the cleaved form of membrane-bound RANKL (mRANKL), on monocytes.

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 first used the MonoMac-6 cell line, recognized as a suitable model for studying monocyte migration. The ability of human recombinant sRANKL to induce the migration of MonoMac-6 cells was measured through an acellular fibronectin-coated filter, which presents the advantage, compared with transendothelial migration, to avoid the possible sRANKL-induced secretion of chemokine(s) by the endothelial monolayer, which may interfere with the test. sRANKL promoted the migration of MonoMac-6 cells in a dose-dependent manner with a half-maximal effect at 0.6 nM (20 ng/ml), according to the affinity of sRANKL for its receptor. 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. Similar results were obtained with normal PBMC or CD14⁺-purified cells, strengthening the physiological relevance of the chemoattractive properties of sRANKL toward monocyte.

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. As sRANKL-induced chemotaxis increased up to 24 h, we verified that this effect was not indirectly a result of the secretion of chemokine(s) induced by sRANKL. Although one has to consider that secretion of chemokines by monocyte in the upper well could unlikely trigger their migration, in fact, none of the chemokines known to induce monocyte migration (RANTES, interferon-inducible protein 10, MIP-1, MIP-1ß, MCP-1, IL-8) was increased in the presence of sRANKL, as assessed by RNase protection assay. 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 (sRANKL) derived post-translationally by cleavage by tumor necrosis factor -converting enzyme. It is interesting that 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. 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 versus soluble status. This is reminiscent of the situation observed for fractalkine, a membrane-associated chemokine, which is mainly involved in the cell–cell adhesion processes in its membrane form, whereas the soluble form of fractalkine, resulting from a proteolytic cleavage, exhibits chemotactic properties toward natural killer cells and CD8 lymphocytes.

It is now well established that inflammation induces bone resorption, as observed in inflammatory chronic diseases such as rheumatoid arthritis and ankylosing spondylitis. A number of recent discoveries have improved our knowledge of the interactions on these cellular systems, in which the RANKL/RANK/OPG system plays a major role. Indeed, the RANKL network is strongly implicated in the normal physiology of differentiation and function of bone and immune cells and in the pathophysiology of inflammatory chronic diseases, such as rheumatoid arthritis, characterized by local and systemic bone loss. However, important questions remain to be answered before fully understanding the relationship between bone and immune cells.

In conclusion, our data, by providing evidence that sRANKL behaves as a true chemoattractant for monocytes, shed a new light on the complex mechanisms that link bone metabolism and immunity.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1188fje; doi: 10.1096/fj.02-1188fje

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