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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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Resorption ability of generated MGCs.RAW 264.7 macrophages were plated and differentiated on Osteo Assay Surface plates. At the end of the assay, the MGCs were assessed on their ability to resorb the substrate throughout the protocol. (A) Two representative images of resorption pits by the various treated MGCs. (B) Quantification of resorption area (mean ± standard deviation of 3 independent assays) as determined by percentage resorbed versus total area. * indicates p<0.001, ** indicates p<0.01, and *** indicates p<0.05 (ANOVA).
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pone-0104498-g004: Resorption ability of generated MGCs.RAW 264.7 macrophages were plated and differentiated on Osteo Assay Surface plates. At the end of the assay, the MGCs were assessed on their ability to resorb the substrate throughout the protocol. (A) Two representative images of resorption pits by the various treated MGCs. (B) Quantification of resorption area (mean ± standard deviation of 3 independent assays) as determined by percentage resorbed versus total area. * indicates p<0.001, ** indicates p<0.01, and *** indicates p<0.05 (ANOVA).

Mentions: In order to identify whether the MGCs generated on non-glass substrates had bone resorption capabilities, RAW 264.7 cells were plated and differentiated on Osteo surfaces. Results showed that all three MGC-types were able to resorb bone (Fig. 4). RANKL+ LPS/IFNγ-treated MGCs showed the greatest resorption ability, likely due to their size difference, followed by RANKL+ IL-4-treated cells, and finally RANKL-alone cells. This data, coupled with TRAP analysis of the MGCs suggest that while the various MGC may have different morphologies, they do share similar functional characteristics as OCs.


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

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

Resorption ability of generated MGCs.RAW 264.7 macrophages were plated and differentiated on Osteo Assay Surface plates. At the end of the assay, the MGCs were assessed on their ability to resorb the substrate throughout the protocol. (A) Two representative images of resorption pits by the various treated MGCs. (B) Quantification of resorption area (mean ± standard deviation of 3 independent assays) as determined by percentage resorbed versus total area. * indicates p<0.001, ** indicates p<0.01, and *** indicates p<0.05 (ANOVA).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104498-g004: Resorption ability of generated MGCs.RAW 264.7 macrophages were plated and differentiated on Osteo Assay Surface plates. At the end of the assay, the MGCs were assessed on their ability to resorb the substrate throughout the protocol. (A) Two representative images of resorption pits by the various treated MGCs. (B) Quantification of resorption area (mean ± standard deviation of 3 independent assays) as determined by percentage resorbed versus total area. * indicates p<0.001, ** indicates p<0.01, and *** indicates p<0.05 (ANOVA).
Mentions: In order to identify whether the MGCs generated on non-glass substrates had bone resorption capabilities, RAW 264.7 cells were plated and differentiated on Osteo surfaces. Results showed that all three MGC-types were able to resorb bone (Fig. 4). RANKL+ LPS/IFNγ-treated MGCs showed the greatest resorption ability, likely due to their size difference, followed by RANKL+ IL-4-treated cells, and finally RANKL-alone cells. This data, coupled with TRAP analysis of the MGCs suggest that while the various MGC may have different morphologies, they do share similar functional characteristics as OCs.

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