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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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The effects of actin cytoskeleton, microtubule network, and myosin II disruption on the morphology of LPS/IFNγ induced MGCs.Day 4 LPS/IFNγ-induced MGCs were incubated with Nocodazole (5 µM), Cytochalasin D (1 µM), Wiscostatin (10 µM), or Blebbistatin (50 µM), for 4 hours. Cells were then stained for F-actin (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-g007: The effects of actin cytoskeleton, microtubule network, and myosin II disruption on the morphology of LPS/IFNγ induced MGCs.Day 4 LPS/IFNγ-induced MGCs were incubated with Nocodazole (5 µM), Cytochalasin D (1 µM), Wiscostatin (10 µM), or Blebbistatin (50 µM), for 4 hours. Cells were then stained for F-actin (green), α-tubulin (red), and DNA (blue). Images shown are representative of three independent experiments. Scale bars represent 50 microns.

Mentions: To disrupt microtubule trafficking of organelles, RANKL+ LPS/IFNγ-induced MGCs were incubated with nocodazole for 3–4 hours after the 96-hour differentiation protocol (Fig. 7). As expected, nocodazole treatment resulted in the depolymerization of the microtubule network, which had a pronounced effect on the cell morphology. The MGCs no longer had the central indentation surrounded by a thicker layer at the periphery (Fig. 7). In fact, they appeared more like the MGCs formed by RANKL and IL-4, albeit with an epitheliod shape and larger size. Furthermore, the nuclei in these cells were no longer limited to the cell periphery and instead, were often found towards the centre of the cell (Fig. 7). These results indicated that the microtubule network played an important role in regulating the shape of these MGCs and the localization of their cytoplasmic contents including nuclei.


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

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

The effects of actin cytoskeleton, microtubule network, and myosin II disruption on the morphology of LPS/IFNγ induced MGCs.Day 4 LPS/IFNγ-induced MGCs were incubated with Nocodazole (5 µM), Cytochalasin D (1 µM), Wiscostatin (10 µM), or Blebbistatin (50 µM), for 4 hours. Cells were then stained for F-actin (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-g007: The effects of actin cytoskeleton, microtubule network, and myosin II disruption on the morphology of LPS/IFNγ induced MGCs.Day 4 LPS/IFNγ-induced MGCs were incubated with Nocodazole (5 µM), Cytochalasin D (1 µM), Wiscostatin (10 µM), or Blebbistatin (50 µM), for 4 hours. Cells were then stained for F-actin (green), α-tubulin (red), and DNA (blue). Images shown are representative of three independent experiments. Scale bars represent 50 microns.
Mentions: To disrupt microtubule trafficking of organelles, RANKL+ LPS/IFNγ-induced MGCs were incubated with nocodazole for 3–4 hours after the 96-hour differentiation protocol (Fig. 7). As expected, nocodazole treatment resulted in the depolymerization of the microtubule network, which had a pronounced effect on the cell morphology. The MGCs no longer had the central indentation surrounded by a thicker layer at the periphery (Fig. 7). In fact, they appeared more like the MGCs formed by RANKL and IL-4, albeit with an epitheliod shape and larger size. Furthermore, the nuclei in these cells were no longer limited to the cell periphery and instead, were often found towards the centre of the cell (Fig. 7). These results indicated that the microtubule network played an important role in regulating the shape of these MGCs and the localization of their cytoplasmic contents including nuclei.

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