Impaired differentiation of osteoclasts in TREM-2-deficient individuals

Abstract

TREM-2 is an immunoglobulin-like cell surface receptor associated with DAP12/KARAP that activates monocyte-derived dendritic cells (DCs) in vitro. Recently, it has been shown that genetic defects of human DAP12/KARAP and TREM-2 result in a rare syndrome characterized by bone cysts and presenile dementia called Nasu-Hakola disease. This observation suggests that TREM-2 may function in myeloid cells other than DCs, most probably osteoclasts (OCs) and microglial cells, which are involved in bone modeling and brain function. Consistent with this prediction, here we show that OC differentiation is dramatically arrested in TREM-2-deficient patients, resulting in large aggregates of immature OCs that exhibit impaired bone resorptive activity. These results demonstrate a critical role for TREM-2 in the differentiation of mononuclear myeloid precursors into functional multinucleated OCs.

Figure 1.

Monocytic precursors from normal donors express TREM-2 at early times of culture with M-CSF or GM-CSF and IL-4. TREM-2 is expressed within 48–72 h of culture with M-CSF or GM-CSF and IL-4, reaching a maximum level at days 3–4. It is worth noting that IL-4 alone is sufficient to induce TREM-2 expression, consistent with a possible role of Th2 cytokines in regulation of TREM-2.

Figure 2.

Patient A exhibits a selective lack of TREM-2 expression in monocytes cultured with M-CSF or GM-CSF and IL-4. (a) TREM-2 and CD16 expression in monocytes cultured for 48 or 72 h in the presence of M-CSF from normal or TREM-2–deficient patients. (b) TREM-2 and CD1b expression on control, TREM-2–deficient, and DAP12-deficient monocytes cultured for 4 d in GM-CSF and IL-4. (c) TREM-1 expression on freshly isolated monocytes from normal, TREM-2–deficient, and DAP12-deficient individuals. (d) Expression of TREM-2 and DAP12 proteins in normal and TREM-2–deficient monocyte-derived DCs at day 6 of culture assessed by immunoblot. Similar results were obtained with monocytes derived from patient B (not depicted).

Figure 3.

Arrest of differentiation, defect of actin polymerization, reduced levels of vitronectin receptor (VNR), and lack expression of calcitonin receptor (CTR) in OCs from TREM-2–deficient patients. (a–d) Images of OC cultures derived from normal (a and c) and TREM-2–deficient individuals (b and d). Images were taken on a Nikon phase contrast microscope with a 10DL objective. In c and d, cells were stained for intracellular TRAP. Upon stimulation with M-CSF, RANKL, and mAb 3.8B1, TREM-2–deficient monocytic precursors do not fuse, but form large aggregates of cells intensely positive for the TRAP reaction. Multinucleated cells with up to three nuclei were rarely detected in the OC cultures from TREM-2–deficient patients (red arrow). (e–h) Actin cytoskeleton of OCs from normal (e and g) and TREM-2–deficient individuals (f and h) generated in glass chamber slides, stained on day 10 of culture with Phalloidin-Oregon Green and analyzed by confocal microscopy (×20). (i) Content of F actin/cell was determined in control and TREM-2–deficient cells by flow cytometry 48 h after stimulation of monocytes with M-CSF, RANKL, and the 3.8B1 mAb. At this early time point immature OCs could still be recovered from culture wells. (j) Three-fold dilutions of cDNAs obtained from control OC cultures, control monocytes, or TREM-2–deficient OC cultures were amplified by RT-PCR with oligonucleotide pairs specific for vitronectin receptor (VNR), calcitonin receptor (CTR), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PCR products were separated by agarose gel electrophoresis and visualized with ethidium bromide. Molecular weight markers are indicated.

Figure 4.

Impaired resorptive function of TREM-2–deficient immature OCs. Resorptive function of normal and TREM-2–deficient OCs on dentin slices (a–f). Monocytic precursors from healthy individuals (a and b) and TREM-2–deficient patients (c–f) were cultured on dentin slices. At day 10, dentin slices were stained with hematoxylin and inspected by light microscopy before (a, c, and e) and after (b, d, and f) cell removal. Resorption pits appear as dark spots stained with hematoxylin after cell removal. Normal monocytes differentiated into multinucleated OCs (a) that generated resorption pits (b). In general, TREM-2–deficient mononuclear immature OCs (c) did not excavate resorptive pits (d). Only a few pits were detected (f) that corresponded in number to the few multinucleated cells generated (e).

Figure 5.

A subset of TREM-2–deficient DCs expresses the macro-phage marker CD16. Immature DCs from normal (top panels) and TREM-2–deficient individuals (bottom panels) were generated by culturing peripheral blood monocytes with GM-CSF and IL-4. At day 4, DCs were analyzed for expression of CD1a, CD16, CD83, and CD86. A population of CD1a⁻ CD16⁺ macrophage-like cells was detected in the cultures of TREM-2–deficient patient A but not in control cultures. Identical results were obtained from DC cultures of patient B (not depicted). DC activation was monitored 24 h after stimulation with LPS. CD86 and CD83 were up-regulated on LPS-activated DCs in both TREM-2–deficient and control DC.