Figure 1.
LL-37 induces monocyte differentiation.
Negatively isolated monocytes at the concentration of 1×106/mL were incubated in RPMI 1460 medium with 10% FBS in the presence or absence of LL-37. A. Cell morphology was shown by phase contrast microscopy (magnification 200×). B. Monocytes were incubated in the presence or absence of 5 µM LL-37 for 6 days. Cells were harvested and counted by hemocytometer. Data (mean ± SE) was from four independent experiments performed. ** p<0.01 vs control. C–D. Monocytes treated with 5 µM LL-37 for 14 days (C) or 63 days (D) were fixed, permeablized, stained with DAPI, and observed by fluorescent microscopy. Multinucleated giant cell formation was found in 63-day LL-37-treated monocytes. Images represent one of four independent experiments.
Figure 2.
LL-37-differentiated monocytes are a novel type of bone forming cells (monoosteophils).
Monocytes were incubated in the presence of M-CSF/RANKL (both at 25 ng/mL, A, E) or 5 µM LL-37 (B,C,D,F,G,H) on BioCoat™ Osteologic™ Discs in 5% CO2 atmosphere. After incubation for 3 weeks, cells were removed with bleach and observed by phase contrast microscopy (magnification ×200, A, B) or scanning electron microscopy (SEM) (C, D). Pit formation was shown on the disc incubated with M-CSF/RANKL-differentiated monocytes (A). Refractile specks were shown on the disc incubated with LL-37-differentiated monocytes (B). SEM showed the refractile specks are built-up granules with a shallow absorbed zone (C, D). After incubation for 7 weeks, pit formation and osteoclast are shown on the disc incubated with M-CSF/RANKL-differentiated monocytes by using SEM (E), and built-up structures and cells are shown on the disc of LL-37-differentiated monocytes (F–H). Monocytes at concentration of 1×106/mL were incubated with 5 µM LL-37 or M-CSF/RANKL (both at 25 ng/mL). I. Morphology was evaluated by phase contrast microscopy (magnification 200×). J. After incubation for 6 days, LL-37- and M-CSF/RANKL-differentiated monocytes were harvested, stained with antibodies, and analyzed by flow cytometry. K. Cytokine levels in the supernatant of LL-37- and M-CSF/RANKL-differentiated monocytes were analyzed. In the LPS treated experiment, 6-day LL-37- and M-CSF/RANKL-differentiated monocytes were collected, resuspended at the concentration of 1×106/mL, and incubated with 100 ng/mL LPS for 18 h and cytokine levels were detected in the supernatant (mean ± SE, n = 3). Data represent three independent experiments performed. M+R: M-CSF+RANKL. ** p<0.01, *** p<0.001 in comparison with M-CSF/RANKL-differentiated monocytes; ## p<0.01 in comparison with M-CSF/RANKL-differentiated monocytes treated with LPS for 18 h.
Figure 3.
Bone formation of monoosteophils in vivo.
NOD/SCID mice were implanted subcutaneously with hydroxyapatite/tricalcium phosphate only (A,E, I), or with 5 µM LL-37 (B, F, J), or with 5×106 monocytes (C,G,K), or 5 µM LL-37 plus 5×106 monocytes (D,H,L). Implants were harvested 7 weeks later and sections stained with haematoxylin and eosin (A–D), masson trichrome (E–H), and anti-human BSP II antibody (I–L) (100×).
Figure 4.
Comparison of monoosteophils with monocyte-derived macrophages.
Negatively isolated monocytes at the concentration of 1×106/mL were incubated in the presence or absence of 100 ng/mL LPS, 5 µM LL-37, 10 ng/mL GM-CSF, or 50 ng/mL M-CSF for 6 days. A. Cell morphology was shown by phase contrast microscopy (magnification 200×). B. 6-day medium-, LPS-, LL-37-, GM-CSF-, or M-CSF-differentiated monocytes at the concentration of 1× 106/mL were mixed with fluorescent labeled latex beads at a multiplicity ratio of 1∶500, incubated at 37°C for 1 h, and analyzed by flow cytometry to show phagocytosis (MFI, mean fluorescence intensity). C. After monocytes were incubated with medium, LPS, LL-37, GM-CSF, and M-CSF for 6 days, cytokine/chemokine levels in cultured supernatant were analyzed (pg/mL, mean ± SE). D. 6-day medium-, LPS-, LL-37-, GM-CSF-, or M-CSF-differentiated monocytes were harvested, stained with antibodies, and analyzed by flow cytometry. Data shown were from three or four independent experiments.
Figure 5.
Monoosteophils are distinct from dendritic cells.
Negatively isolated monocytes at the concentration of 1× 106/mL were incubated in the presence or absence of 5 µM LL-37, 1,000 U/mL GM-CSF/500 U/mL IL-4, GM-CSF/IFNα (both at 1,000 U/mL). A. After incubation for 6 day, LL-37-differentiated monocytes were harvested, stained with antibodies, and analyzed by flow cytometry. B. Morphology of medium-, LL-37-, GM-CSF/IL-4-, and GM-CSF/IFNα-differentiated monocytes in the presence or absence of LPS were shown by phase contrast microscopy (magnification 200×). Images shown were from one of four independent experiments.
Figure 6.
Characteristics of monoosteophils.
Monocytes were incubated in the presence of 5 µM LL-37 for 6 days. A. cells were fixed, permeabilized, stained with phalloidin-Alexa 594 and DAPI, and observed by using fluorenscence microscope. B. cells were harvested, stained with antibodies, and analyzed by using flow cytometry to show surface staining of proteins. C. Cells were harvested, fixed and permeabilized, stained with antibodies, and analyzed by using flow cytometry to show intracellular staining of proteins. Data shown were from one of four independent experiments.