Lymphocytes and the Dap12 Adaptor Are Key Regulators of Osteoclast Activation Associated with Gonadal Failure

Bone resorption by osteoclasts is necessary to maintain bone homeostasis. Osteoclast differentiation from hematopoietic progenitors and their activation depend on M-CSF and RANKL, but also requires co-stimulatory signals acting through receptors associated with DAP12 and FcRγ adaptors. Dap12 mutant mice (KΔ75) are osteopetrotic due to inactive osteoclasts but, surprisingly, these mice are more sensitive than WT mice to bone loss following an ovariectomy. Because estrogen withdrawal is known to disturb bone mass, at least in part, through lymphocyte interaction, we looked at the role of mature lymphocytes on osteoclastogenesis and bone mass in the absence of functional DAP12. Lymphocytes were found to stimulate an early osteoclast differentiation response from Dap12-deficient progenitors in vitro. In vivo, Rag1-/- mice lacking mature lymphocytes did not exhibit any bone phenotype, but lost their bone mass after ovariectomy like KΔ75 mice. KΔ75;Rag1-/- double mutant female mice exhibited a more severe osteopetrosis than Dap12-deficient animals but lost their bone mass after ovariectomy, like single mutants. These results suggest that both DAP12 and mature lymphocytes act synergistically to maintain bone mass under physiological conditions, while playing similar but not synergistic co-stimulatory roles in protecting bone loss after gonadal failure. Thus, our data support a role for lymphocytes during osteoclast differentiation and suggest that they may function as accessory cells when regular osteoclast function is compromised

Ostéoimmunologie : une vision globale et intégrée du tissu squelettique et du système immunitaire

L’interface entre le système immunitaire et la physiologie osseuse a conduit à l’émergence de l’ostéo-immunologie, nouveau champ de recherche qui ouvre de nouvelles perspectives dans la compréhension et le traitement des pathologies inflammatoires ostéo-articulaires. De nombreuses molécules et voies de signalisation, décrites initialement dans les cellules du système immunitaire, agissent également dans les ostéoclastes, cellules ayant pour rôle le maintien de l’homéostasie osseuse en assurant le remodelage du tissu osseux. Ainsi, la différenciation ostéoclastique requiert deux voies de signalisation « immunologiques » : la voie RANK/RANKL (receptor activator of NF-κB ligand) et celle des protéines adaptatrices à domaine ITAM (immunoreceptor tyrosine based activation motif), DAP12 (DNAX-activating protein) et FcRγ (récepteur du fragment Fc des immunoglobulines). Mais la façon dont les deux voies s’associent pour induire le facteur de transcription clé de l’ostéoclastogenèse NFATc1 (nuclear factor of activated T-cells), restait à élucider. Une étude réalisée par le groupe de H. Takayanagi vient de permettre d’identifier les protéines tyrosine kinases Bruton et Tec, qui forment un complexe protéique capable d’intégrer ces deux voies de signalisation et, ainsi, permettent l’expression des gènes nécessaires à la différenciation ostéoclastique

DAP12 overexpression induces osteopenia and impaired early hematopoiesis.

ITAM-bearing transmembrane signaling adaptors such as DAP12 and FcRγ are important players in bone homeostasis, but their precise role and functions are still unknown. It has been shown that osteoclast differentiation results from the integration of the RANK and of the DAP12 and FcRγ signaling pathways. DAP12-deficient mice suffer from a mild osteopetrosis and culture of their bone marrow cells in the presence of M-CSF and RANKL, fails to give rise to multinucleated osteoclasts. Here, we report that mice overexpressing human DAP12 have an osteopenic bone phenotype due to an increased number of osteoclasts on the surface of trabecular and cortical bone. This enhanced number of osteoclasts is associated with an increased number of proliferating myeloid progenitors in Tg-hDAP12 mice. It is concomitant with an arrest of B cell development at the Pre-Pro B/Pre B stage in the bone marrow of Tg-hDAP12 mice and important decrease of follicular and marginal B cells in the spleen of these animals. Our data show that the overexpression of DAP12 results in both increased osteoclastogenesis and impaired hematopoiesis underlining the relationship between bone homeostasis and hematopoiesis

Immature denditric cell transdifferentiation into osteoclasts: a novel pathway sustained by the rheumatoid arthritis microenvironment

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Genome-wide expression analyses establish dendritic cells as a new osteoclast precursor able to generate bone-resorbing cells more efficiently than monocytes

Dendritic cells (DCs), mononuclear cells that initiate immune responses, and osteoclasts (OCs), multinucleated bone-resorbing cells, are hematopoietic cells derived from monocytic precursor cells. Using in vitro generated dendritic cells, we previously showed that human and murine DCs could transdifferentiate into resorbing osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). In this study we globally compared by transcriptomic profiling this new osteoclast differentiation pathway from DCs with the canonical differentiation pathway from monocytes. DNA chip data revealed that starting from two very distinct cell types, treatment with M-CSF and RANKL generated two highly similar types of osteoclast. In particular, DC-derived osteoclasts expressed all the characteristic marker genes of monocyte-derived osteoclasts. Two major molecular events could be observed during osteoclastogenesis: downregulation of a large set of monocyte or DC specific markers, together with upregulation of characteristic osteoclast marker genes. Most interestingly, our transcriptomic data showed a closer molecular profile between DCs and OCs than between monocytes and OCs. Our data establish DCs as a new osteoclast precursor able to generate OCs more efficiently than monocytes. © 2010 American Society for Bone and Mineral Research

Proliferation of spleen and bone marrow osteoclast precursors.

<p>Spleen cells of three 3-month-old WT and Tg-hDAP12 mice were seeded in triplicate in wells of 96-well plates at 78×10³ cells/well. At each time point, cells were incubated with BrdU during 3 hours. Incorporated BrdU was measured at 370 nm with a 492 nm reference wavelenght, using a colorimetric immunoassay. White circles: WT cells, black squares: Tg-hDAP12 cells. Results are means ± SE. <i>p</i><0,001 (***);<i>p</i><0,01 (**).</p

RANKL dose-dependent response of spleen and bone marrow osteoclast precursors.

<p>Bone marrow and spleen cells of three 3-month-old WT and Tg-hDAP12 mice were seeded in triplicate in wells of 96-well plates. 12,5×10³ cells/well for bone marrow cells and 78×10³per well for spleen cells. Bone marrow and spleen cells were grown in presence of M-CSF and increasing concentrations of RANKL either 20, 30, 50 and 100 ng/ml for bone marrow cells (A) or 20, 30, 40, 50 and 100 ng/ml for spleen cells (B). After 4 days in culture for bone marrow cells or 6 days in culture for spleen cells, TRAP-positive osteoclasts with ≥3 nuclei were counted. White bars:WT cells; Grey bars:Tg-hDAP12 cells. Results are expressed as mean number of osteoclasts with nuclei ≥3 present on the total surface of each of the three wells ± SE. In (B), ***indicated <i>p</i><0.001.</p

Characterization of monocytic precursors in the spleen of 3-month-old Tg-hDAP12 female

<p><b>mice.</b> (A) FACS analyses of myeloid B22O⁻CD11b⁺ cells in the spleen of Tg-hDAP12 mice by comparison with WT mice (four WT and four Tg-hDAP12 animals). The total number of B220⁻CD11b⁺ cells is expressed as a percentage of total nucleated splenocytes. Results are means ± SE. >0.1. (B) One representative FACS dot plot showing the myeloid B220⁻CD11b⁺ population in a black oval and the B220+CD11b–lymphoid population (dashed black oval). (C) Left, Number of CFU-GM colonies obtained after seeding of 2×10⁵ WT or Tg-hDAP12 spleen cells of 3-month-old female mice in Methocult 3534® containing SCF, IL-3 and IL-6 (see Materials and Methods). Results of one representative out of two experiments. Results are means of the number of colonies in the three wells ± SE. *: <i>p</i><0.01. Right, Photographs of one typical colony obtained after 7 days in culture (left picture) and of colony-derived cells after Wright-Giemsa staining. (right picture). The cells have the typical morphology of macrophages. (D) number of CFU-M colonies counted 7 days after seeding WT or Tg-hDAP12 bone marrow or spleen cells in methocult supplemented with 30 ng/ml M-CSF. One experiment performed. Grey bars: Tg-hDAP12 mice; white bars: WT mice.</p

Osteoclastogenesis from bone marrow and spleen cells cultures from 3-month-old Tg-hDAP12 and WT female mice.

<p>(A) Osteoclastogenesis from mononuclear bone marrow cells cultured for five days in the presence of M-CSF and RANKL; cells were seeded at 12×10³ cells/well in triplicate in 96-well plates; Scale bar 200 µm. (B and C) Osteoclastogenesis from mononuclear spleen cells cultured in the presence of the two cytokines. Cells were seeded at 78×10³ cells/well in triplicate in 96-well plates (B) Kinetic studies of osteoclastogenesis over 8 days. Scale bar 100 µm. Cells were fixed in paraformaldehyde and stained for TRAP activity at each indicated time point of the kinetic. Arrows show osteoclast precursors on day 2 (D2) in cultures of WT and Tg-hDAP12 splenocytes. On day 4 (D4) of WT cultures, the images show two different microscopic fields, with osteoclasts formed in foci of proliferating precursors (image on the left). On day 6 (D6) of Tg-hDAP12 cultures, the images show also two different microscopic fields with dead osteoclasts (*in the image on the right). On day 8 (D8) of Tg-hDAP12 cultures the stars (*) also show lysed osteoclasts. One experiment is shown, representative of six independent experiments. (C) Quantifications of TRAP-positive multinucleated cells present in spleen cell cultures at each time point of the kinetic were done on two experiments. Results are mean ± SE.</p

Osteoblastogenesis and osteoclastogenic activities of osteoblasts in bone marrow cell cultures from 3-month-old Tg-hDAP12 female mice.

<p>Osteoblasts were classically obtained after treatment of bone marrow cells with ß sodium-glycerophosphate (10 mM) and ascorbic acid (50 µg/ml) for 7 days. Total RNA was extracted at different times of culture: day 3, day 7 and day 14. (A) Gene expression of osteoblast-associated markers: alkaline phosphatase (ALP), osteocalcin (OCN), measured by RT-PCR at the indicated times of culture. Gene expression was normalized to the L32, house-keeping gene values. Results are means of two independent experiments ± SE. (B) Mineralized colonies obtained from osteoblasts generated from bone marrow. Colonies doubly-stained with alkaline phosphatase and von Kossa appear as black dots. Results are plotted as the mean number of nodules ±SE of three wells for 3-month-old WT and Tg-hDAP12 female mice and were representative of three independent experiments. (C) Expression of human DAP12 transgene in 7-day-old bone marrow-derived osteoblasts using RT-PCR. 1: WT osteoblasts; 2: Tg-hDAP12 osteoblasts. 3: hDAP12 expression in human monocyte-derived osteoclasts used as control; 4: RT-PCR without template. White stars indicate the 373 bp PCR fragment corresponding to hDAP12. One experiment is shown, representative of three independent experiments. Grey bars: Tg-hDAP12 mice; white bars: WT mice.</p