Figure 5

Interleukin (IL)-17 and IL-32 synergistically induce osteoclastogenesis. (A) Tartrate-resistant acid phosphatase (TRAP) staining for identification of osteoclasts. Osteoclast precursors were cultured in the presence of IL-17 (0.1 ng/ml) or/and IL-32 (5 ng/ml) with macrophage colony-stimulating factor (M-CSF; 25 ng/ml). The receptor activator of the nuclear factor kappa-B ligand (RANKL; 30 ng/ml)-treated group was the positive control. The medium and stimulus were changed every 3 days. After 15 days, the cells were stained for TRAP activity. (B) Numbers of multinucleated TRAP-positive cells per well. TRAP-positive cells containing two or more nuclei were scored as osteoclasts. TRAP-positive cells were counted three times by blind scoring. *P < 0.05 (compared with M-CSF+IL-32+IL-17), **P < 0.01 (compared with M-CSF+IL-32+IL-17). (C) The mRNAs of TRAP, Cathepsin K, calcitonin receptor (CTR) and matrix metallopeptidase 9 (MMP9), as osteoclast markers were quantified by real-time PCR. *P < 0.05 (compared with M-CSF+IL-32+IL-17), **P < 0.01 (compared with M-CSF+IL-32)+IL-17), ***P < 0.001 (compared with M-CSF+IL-32+IL-17). (D) Formation (left) and percent area (right) of resorption pits by osteoclasts on dentine discs. Cell culture was performed as described with dentine discs in 96-well plates. The osteoclast precursors were cultured in the presence of IL-17 (1 ng/ml) or IL-32 (5 ng/ml) with M-CSF (25 ng/ml) and RANKL (30 ng/ml). At day 21, the cells were removed from dentine. Resorption area was evaluated by light microscopy and measured using the image analysis software. (A), (B) and (D) are representative of two or three experiments with similar results. (C) Means ± SD of more than three separate experiments.