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Modulation of osteoclastogenesis with macrophage M1- and M2-inducing stimuli.

Jeganathan S, Fiorino C, Naik U, Sun HS, Harrison RE - PLoS ONE (2014)

Bottom Line: Following a four-day differentiation protocol, along with lipopolysaccharide (LPS)/interferon gamma (IFNγ) as one stimulus, and interleukin-4 (IL-4) as the other, three types of multinucleated cells were generated.Using various microscopy techniques (bright field, epifluorescence and scanning electron), functional assays, and western blotting for osteoclast markers, we found that, as expected, RANKL treatment alone resulted in osteoclasts, whereas the addition of LPS/IFNγ to RANKL pre-treated macrophages generated Langhans-type giant cells, while IL-4 led to giant cells resembling foreign body giant cells with osteoclast-like characteristics.Finally, to gain insight into the modulation of osteoclastogenesis, we characterized the formation and morphology of RANKL and LPS/IFNγ-induced multinucleated giant cells.

View Article: PubMed Central - PubMed

Affiliation: Ontario Cancer Institute and Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

ABSTRACT
Macrophages are generated through the differentiation of monocytes in tissues and they have important functions in innate and adaptive immunity. In addition to their roles as phagocytes, macrophages can be further differentiated, in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), into osteoclasts (multinucleated giant cells that are responsible for bone resorption). In this work, we set out to characterize whether various inflammatory stimuli, known to induce macrophage polarization, can alter the type of multinucleated giant cell obtained from RANKL differentiation. Following a four-day differentiation protocol, along with lipopolysaccharide (LPS)/interferon gamma (IFNγ) as one stimulus, and interleukin-4 (IL-4) as the other, three types of multinucleated cells were generated. Using various microscopy techniques (bright field, epifluorescence and scanning electron), functional assays, and western blotting for osteoclast markers, we found that, as expected, RANKL treatment alone resulted in osteoclasts, whereas the addition of LPS/IFNγ to RANKL pre-treated macrophages generated Langhans-type giant cells, while IL-4 led to giant cells resembling foreign body giant cells with osteoclast-like characteristics. Finally, to gain insight into the modulation of osteoclastogenesis, we characterized the formation and morphology of RANKL and LPS/IFNγ-induced multinucleated giant cells.

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Characterization of the ultrastructural differences of the various MGCs.Representative scanning electron microscopy images of the generated day 4 MGCs. Scale bars represent 50(middle-top and middle-bottom, respectively).
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pone-0104498-g002: Characterization of the ultrastructural differences of the various MGCs.Representative scanning electron microscopy images of the generated day 4 MGCs. Scale bars represent 50(middle-top and middle-bottom, respectively).

Mentions: To further analyze the morphology of the various generated MGCs at an ultrastructural level, scanning electron microscope images were taken (Fig. 2). Once again, RANKL-treated MGCs exhibited no defined morphology, and heterogenous shapes and cellular protrusions were observed. However, RANKL+ LPS/IFNγ-treated MGCs had a characteristic indentation throughout most of the cell body that was surrounded by a visibly thicker border area (Fig. 2). In some of these cells, there were upward protrusions in the indentation. RANKL+ IL-4-treated MGCs showed a consistent oval/circular morphology and, as the actin and microtubule staining suggested, the centre of the cell was visibly thicker than the periphery (Fig. 2).


Modulation of osteoclastogenesis with macrophage M1- and M2-inducing stimuli.

Jeganathan S, Fiorino C, Naik U, Sun HS, Harrison RE - PLoS ONE (2014)

Characterization of the ultrastructural differences of the various MGCs.Representative scanning electron microscopy images of the generated day 4 MGCs. Scale bars represent 50(middle-top and middle-bottom, respectively).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125219&req=5

pone-0104498-g002: Characterization of the ultrastructural differences of the various MGCs.Representative scanning electron microscopy images of the generated day 4 MGCs. Scale bars represent 50(middle-top and middle-bottom, respectively).
Mentions: To further analyze the morphology of the various generated MGCs at an ultrastructural level, scanning electron microscope images were taken (Fig. 2). Once again, RANKL-treated MGCs exhibited no defined morphology, and heterogenous shapes and cellular protrusions were observed. However, RANKL+ LPS/IFNγ-treated MGCs had a characteristic indentation throughout most of the cell body that was surrounded by a visibly thicker border area (Fig. 2). In some of these cells, there were upward protrusions in the indentation. RANKL+ IL-4-treated MGCs showed a consistent oval/circular morphology and, as the actin and microtubule staining suggested, the centre of the cell was visibly thicker than the periphery (Fig. 2).

Bottom Line: Following a four-day differentiation protocol, along with lipopolysaccharide (LPS)/interferon gamma (IFNγ) as one stimulus, and interleukin-4 (IL-4) as the other, three types of multinucleated cells were generated.Using various microscopy techniques (bright field, epifluorescence and scanning electron), functional assays, and western blotting for osteoclast markers, we found that, as expected, RANKL treatment alone resulted in osteoclasts, whereas the addition of LPS/IFNγ to RANKL pre-treated macrophages generated Langhans-type giant cells, while IL-4 led to giant cells resembling foreign body giant cells with osteoclast-like characteristics.Finally, to gain insight into the modulation of osteoclastogenesis, we characterized the formation and morphology of RANKL and LPS/IFNγ-induced multinucleated giant cells.

View Article: PubMed Central - PubMed

Affiliation: Ontario Cancer Institute and Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

ABSTRACT
Macrophages are generated through the differentiation of monocytes in tissues and they have important functions in innate and adaptive immunity. In addition to their roles as phagocytes, macrophages can be further differentiated, in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), into osteoclasts (multinucleated giant cells that are responsible for bone resorption). In this work, we set out to characterize whether various inflammatory stimuli, known to induce macrophage polarization, can alter the type of multinucleated giant cell obtained from RANKL differentiation. Following a four-day differentiation protocol, along with lipopolysaccharide (LPS)/interferon gamma (IFNγ) as one stimulus, and interleukin-4 (IL-4) as the other, three types of multinucleated cells were generated. Using various microscopy techniques (bright field, epifluorescence and scanning electron), functional assays, and western blotting for osteoclast markers, we found that, as expected, RANKL treatment alone resulted in osteoclasts, whereas the addition of LPS/IFNγ to RANKL pre-treated macrophages generated Langhans-type giant cells, while IL-4 led to giant cells resembling foreign body giant cells with osteoclast-like characteristics. Finally, to gain insight into the modulation of osteoclastogenesis, we characterized the formation and morphology of RANKL and LPS/IFNγ-induced multinucleated giant cells.

Show MeSH
Related in: MedlinePlus