TNF-α in combination with TGF-β induces preosteoclasts at a low concentration of M-CSF.

<p>(A) Induction of TRAP⁺ mononuclear cells in the presence of various concentrations of TNF-α or TGF-β in the presence of M-CSF (3 ng/ml). Each value represents the mean ± SEM of three cultures. (B) Representative photomicrograph of TRAP⁺ mononuclear cells induced by M-CSF (3 ng/ml), TNF-α (20 ng/ml), and TGF-β (1 ng/ml) (top); or TRAP⁺ MNCs induced from preosteoclasts by RANKL (30 ng/ml) as illustrated in (C) (middle); or TRAP⁺ mononuclear cells induced by RANKL (3 ng/ml) and M-CSF (10 ng/ml) (bottom). Bar = 50 µm. (C) Illustration of sequence of culture conditions. (D) RT-PCR analysis of mRNA expression of osteoclast-specific genes and genes encoding cell surface proteins in cultures of BMM, preosteoclasts (pre-OC), and MNCs. (E) Relative expression levels of <i>ctsk, calcr, tnfrs11a</i> (RANK), <i>nfatc,</i> and <i>c-fos</i> were normalized with β<i>-actin</i>. (F) Real-time RT-PCR analysis of expression levels of <i>ctsk, tm7sf4</i> (DC-STAMP), and <i>csf-1r</i> (c-fms). Expression levels were normalized with <i>gapdh</i>. NABMCs were cultured for 3 days in the presence of various concentrations of TNF-α, TGF-β, M-CSF, and RANKL. To induce TRAP⁺ MNCs, RANKL was added to preosteoclasts induced by treatment with TNF-α, TGF-β, and M-CSF, and cultures were continued for 2 days. At the end of culture, TRAP-staining was performed and TRAP⁺ mononuclear cells were counted. Total RNA from cells was prepared and analyzed by semi-quantitative RT-PCR or real-time RT-PCR using specific primers as in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047930#s2" target="_blank">Materials and Methods</a>.</i> Each value represents the mean ± SEM (n = 3). Statistical significance was determined by Student’s <i>t</i> test; *P<0.05, **P<0.01 compared with BMM. Experiments were performed three times using three different rats, and similar results were obtained.</p

Analysis of expression of genes encoding cell surface proteins in BMM, preosteoclasts, and MNCs.

<p>(A) Relative mRNA expression levels of <i>emr1</i> (F4/80), <i>itgam</i> (CD11b), <i>CCR1, CCR2,</i> and <i>CCR5</i> in cultures of BMM, preosteoclasts (pre-OC) and MNCs. (B) Immunofluorescence images of preosteoclasts stained for Kat1 antigen (red) and CD11b/c (green). (C) Percentages of Kat1<b>⁻</b>CD11b/c⁺, Kat1⁺CD11b/c⁺ or Kat1⁺CD11b/c<b>⁻</b> cells among cells induced by M-CSF in combination with TNF-α and/or TGF-β. NABMCs were cultured for 3 days in the presence of various combinations of TNF-α, TGF-β, and M-CSF to induce formation of BMM or preosteoclasts. After formation of preosteoclasts, RANKL was added to induce formation of MNCs. Total RNA from cells was prepared and analyzed by real-time RT-PCR using specific primers as in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047930#s2" target="_blank">Materials and Methods</a></i>. Expression levels were normalized with <i>gapdh</i> (A). The cells were stained for Kat1 and CD11b and analyzed by confocal microscopy using LSM5Pascal software. The photographs show typical cultures. (B). Each value represents the mean ± SEM (n = 3, A) or (cell counts from ten-microscope fields, C). Statistical significance was determined by Student’s <i>t</i> test; *P<0.05, **P<0.01 compared with BMM (A) or TGF-β alone (C). Bar = 20 µm. The experiments were performed three times, and similar results were obtained.</p

Kat1⁺ cells represent a late differentiation stage of preosteoclasts.

<p>(A) Photographs of cells that were Kat1<b>⁻</b>c-fms⁺, Kat1⁺c-fms⁺, or Kat1⁺c-fms<b>⁻</b> in time-course cultures. Cells were stained for Kat1 and c-fms, and analyzed by confocal microscopy with LSM5Pascal software. Bar = 50 µm. (B) Distribution of cells from cultures shown in (A). Each value represents the mean ± SEM of ten fields in a typical culture. Statistical significance was determined by Student’s <i>t</i> test; *P<0.05, **P<0.01 compared with 48 hours. (C) Formation of TRAP⁺ MNCs from enriched populations of Kat1⁺ or Kat1⁻ cells in preosteoclasts cultures. NABMCs were cultured with TNF-α, TGF-β, and M-CSF for 2.5 days. Kat1⁺ cells and Kat1⁻ cells in preosteoclasts cultures were isolated by panning with mAb Kat1 as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047930#s2" target="_blank">Materials and Methods</a></i>, and cultured for 2 days in the presence of RANKL (30 ng/ml) and M-CSF (3 ng/ml). Statistical significance was determined by Student’s <i>t</i> test. **P<0.01 compared with Kat-1⁻ cells. We have repeated this experiments and similar results were obtained.</p

Chemotaxis of preosteoclasts toward chemokines and effect of chemokines on multinucleation of preosteoclasts.

<p>Chemotaxis of preosteoclasts toward chemokine MIP-1α (A) and MCP-1 (B). (C) Effect of MIP-1α or MCP-1 on the formation of TRAP⁺ MNCs from preosteoclasts in the presence of RANKL and M-CSF. NABMCs were cultured in the presence of TNF-α, TGF-β, and M-CSF. The cells were detached and cultured in a transwell. Preosteoclasts were added to the upper chamber of the transwell, and various concentrations of MIP-1α or MCP-1 were added to the lower chamber (A, B). After 4 hours of culture, migration of cells to the lower side of the filter was analyzed. Preosteoclasts induced by TNF-α, TGF-β, and M-CSF were then cultured in the presence of RANKL, M-CSF, and various concentrations of MIP-1α or MCP-1 for 2 days (C). TRAP⁺ cells having three or four, or more than five nuclei were counted as MNCs. Each value represents the mean ± SEM of three cultures. Significance was determined by Student’s <i>t</i>-test; *P<0.05 compared to cultures without chemokines.</p

List of primers used for RT-PCR and real-time RT-PCR.

<p>List of primers used for RT-PCR and real-time RT-PCR.</p

TNF-α and TGF-β specifically increase subpopulations of Kat1⁺c-fms⁺ and Kat1⁺c-fms⁻ preosteoclasts.

<p>(A) Confocal microscopic analysis of Kat1⁺, c-fms⁺, or Kat1⁺c-fms⁺ cells in cultures induced by M-CSF and various concentrations of TNF-α, and TGF-β. Bar = 50 µm. (B) Percentages of Kat1<b>⁻</b>c-fms⁺, Kat1⁺c-fms⁺ or Kat1⁺c-fms<b>⁻</b> cells among cells induced by M-CSF and various concentrations of TNF-α and TGF-β. NABMCs were cultured for 2.5 days. Cells were stained for Kat1 and c-fms, and analyzed by confocal microscopy with LSM5Pascal software. The photographs show typical cultures. Each value represents the mean ± SEM of ten fields in a typical culture. Statistical significance was determined by Student’s <i>t</i> test; **P<0.01 compared with TNF-α or TGF-β alone. We have repeated these experiments and similar results were obtained.</p

Treatment with TNF-α and TGF-β specifically induces the formation of Kat1⁺ preosteoclasts.

<p>(A) Immunostaining image of Kat1 antigen (red) in rat bone marrow cultures. Arrows: multinucleated osteoclasts. Arrow heads: osteoclast precursors. Bar = 50 µm. Rat bone marrow cells were cultured in the presence of 1,25(OH)₂D₃ for 6 days, and were then stained with Kat1 mAb. (B) Immunofluorescence image of stained Kat1 antigen (green) in rat mandible. Living osteoclasts were stained <i>in situ</i> by the direct injection of the Kat1 mAb; rats were perfused with 4% paraformaldehyde followed by dissection of mandible, and sections were prepared as in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047930#s2" target="_blank">Materials and Method</a>.</i> Tissue sections were stained with FITC-conjugated anti-mouse IgM and observed by fluorescence microscopy. Arrow heads: osteoclasts. Right panel; phase-contrast view of same field as left panel. B: bone. BM: bone marrow. Bar = 50 µm. NABMC were cultured in the presence of various concentrations of M-CSF, TNF-α, and TGF-β for 2 days (C, D). (C) Cells were stained for Kat1 and analyzed by confocal microscopy. Immunofluorescence images of Kat1 antigen (red) and nuclei (blue) in cells induced by M-CSF alone (left), or TNF-α (20 ng/ml), TGF-β (1 ng/ml), and M-CSF (right). Bar = 50 µm. (D) FACS analysis with Kat1 mAb (red) and control IgM (blue). Cells were induced by M-CSF alone (left), or TNF-α (20 ng/ml), TGF-β (1 ng/ml), and M-CSF (right).</p

Analysis of differentiation potentials of osteoclast precursor cells induced by various conditions.

<p>(A) Time course of formation of TRAP⁺ MNCs from mononuclear cells induced by M-CSF in combination with TNF-α and TGF-β separately or together. (B) Effect of hydroxyurea (HU) on RANKL-induced formation of TRAP⁺ MNCs from preosteoclasts. NABMCs were cultured with TNF-α, TGF-β, and M-CSF for 2 days, and were then cultured further for 1–3 days (A) in the presence of RANKL (30 ng/ml) and M-CSF (3 ng/ml), or (B) in the presence or absence of HU (100 µM). Each value represents the mean ± SEM of three cultures. Statistical significance was determined by Student’s <i>t</i> test; *P<0.05, **P<0.01.</p