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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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Related in: MedlinePlus

Microtubule-organizing centre movement during the differentiation of RANKL+ LPS/IFNγ-treated cells.Representative images of cells treated with RANKL and LPS/IFNγ from early Day 3 of the differentiation protocol until the end of experimentation (day 4). Cells were stained with tubulin (red) and DNA (DAPI). Images shown are representative of three independent experiments. Scale bars represent 50 microns.
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pone-0104498-g008: Microtubule-organizing centre movement during the differentiation of RANKL+ LPS/IFNγ-treated cells.Representative images of cells treated with RANKL and LPS/IFNγ from early Day 3 of the differentiation protocol until the end of experimentation (day 4). Cells were stained with tubulin (red) and DNA (DAPI). Images shown are representative of three independent experiments. Scale bars represent 50 microns.

Mentions: Since the microtubule network seemed to play a role in the overall morphology of RANKL+ LPS/IFNγ-mediated MGCs, we looked at the microtubule organizing centre (MTOC) in these cells. In mononuclear cells, the movements of the MTOC and nucleus are closely coupled [34]. Furthermore, MTOCs are closely associated with the Golgi apparatus [35], [36]. We had observed in earlier experiments that α-tubulin staining of the various MGCs showed bright, punctate staining for MTOCs (data not shown). These were clearly visible and had microtubule strands emitting from them. Therefore, we decided to take fixed images of RANKL+ LPS/IFNγ-treated cells from early day 3 up to day 4 (Fig. 8). During our differentiation protocol, macrophage fusion began during late day 2 to early day 3. The nuclei in these cells were centralized, with MTOCs scattered between and/or around them (Fig. 8). At day 3, the MTOCs were found progressively further from the central core and closer to the cell periphery. A more noticeable movement of the nuclei towards the cell periphery occurred from late day 3 onwards (Fig. 8). Interestingly, the microtubule staining pattern followed a similar trend (Fig. 8). Up until late day 3, the staining profile was fairly even throughout the cell. Afterwards, a less dense microtubule staining was seen at the cell centre, again correlating the microtubule distribution with nuclear positioning.


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

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

Microtubule-organizing centre movement during the differentiation of RANKL+ LPS/IFNγ-treated cells.Representative images of cells treated with RANKL and LPS/IFNγ from early Day 3 of the differentiation protocol until the end of experimentation (day 4). Cells were stained with tubulin (red) and DNA (DAPI). 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-g008: Microtubule-organizing centre movement during the differentiation of RANKL+ LPS/IFNγ-treated cells.Representative images of cells treated with RANKL and LPS/IFNγ from early Day 3 of the differentiation protocol until the end of experimentation (day 4). Cells were stained with tubulin (red) and DNA (DAPI). Images shown are representative of three independent experiments. Scale bars represent 50 microns.
Mentions: Since the microtubule network seemed to play a role in the overall morphology of RANKL+ LPS/IFNγ-mediated MGCs, we looked at the microtubule organizing centre (MTOC) in these cells. In mononuclear cells, the movements of the MTOC and nucleus are closely coupled [34]. Furthermore, MTOCs are closely associated with the Golgi apparatus [35], [36]. We had observed in earlier experiments that α-tubulin staining of the various MGCs showed bright, punctate staining for MTOCs (data not shown). These were clearly visible and had microtubule strands emitting from them. Therefore, we decided to take fixed images of RANKL+ LPS/IFNγ-treated cells from early day 3 up to day 4 (Fig. 8). During our differentiation protocol, macrophage fusion began during late day 2 to early day 3. The nuclei in these cells were centralized, with MTOCs scattered between and/or around them (Fig. 8). At day 3, the MTOCs were found progressively further from the central core and closer to the cell periphery. A more noticeable movement of the nuclei towards the cell periphery occurred from late day 3 onwards (Fig. 8). Interestingly, the microtubule staining pattern followed a similar trend (Fig. 8). Up until late day 3, the staining profile was fairly even throughout the cell. Afterwards, a less dense microtubule staining was seen at the cell centre, again correlating the microtubule distribution with nuclear positioning.

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