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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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Golgi and stable microtubules are found at the periphery of RANKL+ LPS/IFNγ induced MGCs.(A) Representative images of day 4 MGCs treated with LPS/IFNγ and stained for GM130, a Golgi marker (green), α-tubulin (red), and nuclei (blue). (B) Representative images of day 4 MGCs treated with LPS/IFNγ stained for acetylated α-tubulin (green), α-tubulin (red), and DNA (blue). Images shown are representative of three independent experiments. Scale bars represent 50 microns.
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pone-0104498-g006: Golgi and stable microtubules are found at the periphery of RANKL+ LPS/IFNγ induced MGCs.(A) Representative images of day 4 MGCs treated with LPS/IFNγ and stained for GM130, a Golgi marker (green), α-tubulin (red), and nuclei (blue). (B) Representative images of day 4 MGCs treated with LPS/IFNγ stained for acetylated α-tubulin (green), α-tubulin (red), and DNA (blue). Images shown are representative of three independent experiments. Scale bars represent 50 microns.

Mentions: To better understand how macrophage activation influences MGC formation, we next took a closer look at the nature of MGCs that were modulated by RANKL+ LPS/IFNγ. As described earlier, these were extremely large cells with a thin, compressed cell body. We examined the distribution of organelles in these cells, focusing on Golgi which were detected using antibodies against GM130 [29]. Multiple discrete Golgi were observed predominantly in the thicker, peripheral region of the cell, where the nuclei were located (Fig. 6A). As Golgi frequently localize towards the microtubule organizing centres (MTOCs) which are enriched with stable microtubules, we immunostained cells for acetylated tubulin, a marker of stable microtubules [30]. Acetylated microtubules were prominent encircling the peripheral cytoplasm, with only a few sparse microtubules penetrating the depressed cell interior (Fig. 6B).


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

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

Golgi and stable microtubules are found at the periphery of RANKL+ LPS/IFNγ induced MGCs.(A) Representative images of day 4 MGCs treated with LPS/IFNγ and stained for GM130, a Golgi marker (green), α-tubulin (red), and nuclei (blue). (B) Representative images of day 4 MGCs treated with LPS/IFNγ stained for acetylated α-tubulin (green), α-tubulin (red), and DNA (blue). Images shown are representative of three independent experiments. Scale bars represent 50 microns.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104498-g006: Golgi and stable microtubules are found at the periphery of RANKL+ LPS/IFNγ induced MGCs.(A) Representative images of day 4 MGCs treated with LPS/IFNγ and stained for GM130, a Golgi marker (green), α-tubulin (red), and nuclei (blue). (B) Representative images of day 4 MGCs treated with LPS/IFNγ stained for acetylated α-tubulin (green), α-tubulin (red), and DNA (blue). Images shown are representative of three independent experiments. Scale bars represent 50 microns.
Mentions: To better understand how macrophage activation influences MGC formation, we next took a closer look at the nature of MGCs that were modulated by RANKL+ LPS/IFNγ. As described earlier, these were extremely large cells with a thin, compressed cell body. We examined the distribution of organelles in these cells, focusing on Golgi which were detected using antibodies against GM130 [29]. Multiple discrete Golgi were observed predominantly in the thicker, peripheral region of the cell, where the nuclei were located (Fig. 6A). As Golgi frequently localize towards the microtubule organizing centres (MTOCs) which are enriched with stable microtubules, we immunostained cells for acetylated tubulin, a marker of stable microtubules [30]. Acetylated microtubules were prominent encircling the peripheral cytoplasm, with only a few sparse microtubules penetrating the depressed cell interior (Fig. 6B).

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