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.