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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 multinucleated giant cells generated by RANKL, RANKL+ LPS/IFNγ, and by RANKL+ IL-4.(A) Schematic of protocol used to generate the various multinucleated giant cells (MGCs). RAW 264.7 cells were plated on 6-well plates, with or without glass coverslips (160 000–180 000 cells/well). Cells were plated in AMEM with 25 ng/ml RANKL. The next day (day 1; 24 hrs later), LPS and IFNγ (0.1 µg/ml and 100 U/ml, respectively) or IL-4 (10 U/ml) were added. On day 2 (48 hrs later), the media was changed, and RANKL, LPS, IFNγ, or IL-4 were added. Experiments were terminated on day 4. (B) Representative images of day 4 MGCs, analyzed by immunofluorescence microscopy for DNA (blue) and F-actin (green; top panel) and α-tubulin (green; bottom panel). Scale bars represent 50 microns. (C) Quantification of cell size (area) of the various MGCs (mean ± standard deviation). MGCs were generated four independent times and between 40–50 MGCs were measured at each time point. * indicates p<0.0001 (ANOVA). (D) Quantification of fusion index of the MGCs generated in (C). * indicates p<0.01, ** indicates p<0.0005 (ANOVA).
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pone-0104498-g001: Characterization of the multinucleated giant cells generated by RANKL, RANKL+ LPS/IFNγ, and by RANKL+ IL-4.(A) Schematic of protocol used to generate the various multinucleated giant cells (MGCs). RAW 264.7 cells were plated on 6-well plates, with or without glass coverslips (160 000–180 000 cells/well). Cells were plated in AMEM with 25 ng/ml RANKL. The next day (day 1; 24 hrs later), LPS and IFNγ (0.1 µg/ml and 100 U/ml, respectively) or IL-4 (10 U/ml) were added. On day 2 (48 hrs later), the media was changed, and RANKL, LPS, IFNγ, or IL-4 were added. Experiments were terminated on day 4. (B) Representative images of day 4 MGCs, analyzed by immunofluorescence microscopy for DNA (blue) and F-actin (green; top panel) and α-tubulin (green; bottom panel). Scale bars represent 50 microns. (C) Quantification of cell size (area) of the various MGCs (mean ± standard deviation). MGCs were generated four independent times and between 40–50 MGCs were measured at each time point. * indicates p<0.0001 (ANOVA). (D) Quantification of fusion index of the MGCs generated in (C). * indicates p<0.01, ** indicates p<0.0005 (ANOVA).

Mentions: Previous studies of osteoclastogenesis and multinucleated giant cell formation have used various protocols resulting in sometimes conflicting results. Protocol variations include the amount, timing, and duration of LPS, IFNγ, or IL-4 treatment, the presence or absence of RANKL in the media, the precursor cell line, the substrate on which the cells are initially plated, and the assessments used to determine osteoclast-likeness. In order to maintain consistency, the following protocol was used (Fig. 1A). In brief, RAW 264.7 murine macrophages were initially plated on 6-well plates (with or without glass coverslips) or on Corning Osteo Assay Surface (24-well) Plates (Corning; Corning, NY) in AMEM media containing 25 ng/ml RANKL. Following 24 hours (24 hours post-plating; Day 1), either LPS and IFNγ (0.1 µg/ml and 100 U/ml, respectively) or IL-4 (10 U/ml) was added. The next day (48 hours post-plating; Day 2), the media was replaced with fresh media containing RANKL and LPS/IFNγ or IL-4. On Day 4 (96 hours post-plating), the experiment was terminated (or drugs were added for a specific time and then the experiment was terminated). The quantity of LPS, IFNγ and IL-4 added were based on what is commonly used to activate macrophages [15], [20]. At the end of the experiment, the generated MGCs were assessed, via microscopy and protein analysis, for osteoclast-likeness (TRAP staining, phagocytic ability, and cathepsin K and MMP-9 expression).


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 multinucleated giant cells generated by RANKL, RANKL+ LPS/IFNγ, and by RANKL+ IL-4.(A) Schematic of protocol used to generate the various multinucleated giant cells (MGCs). RAW 264.7 cells were plated on 6-well plates, with or without glass coverslips (160 000–180 000 cells/well). Cells were plated in AMEM with 25 ng/ml RANKL. The next day (day 1; 24 hrs later), LPS and IFNγ (0.1 µg/ml and 100 U/ml, respectively) or IL-4 (10 U/ml) were added. On day 2 (48 hrs later), the media was changed, and RANKL, LPS, IFNγ, or IL-4 were added. Experiments were terminated on day 4. (B) Representative images of day 4 MGCs, analyzed by immunofluorescence microscopy for DNA (blue) and F-actin (green; top panel) and α-tubulin (green; bottom panel). Scale bars represent 50 microns. (C) Quantification of cell size (area) of the various MGCs (mean ± standard deviation). MGCs were generated four independent times and between 40–50 MGCs were measured at each time point. * indicates p<0.0001 (ANOVA). (D) Quantification of fusion index of the MGCs generated in (C). * indicates p<0.01, ** indicates p<0.0005 (ANOVA).
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pone-0104498-g001: Characterization of the multinucleated giant cells generated by RANKL, RANKL+ LPS/IFNγ, and by RANKL+ IL-4.(A) Schematic of protocol used to generate the various multinucleated giant cells (MGCs). RAW 264.7 cells were plated on 6-well plates, with or without glass coverslips (160 000–180 000 cells/well). Cells were plated in AMEM with 25 ng/ml RANKL. The next day (day 1; 24 hrs later), LPS and IFNγ (0.1 µg/ml and 100 U/ml, respectively) or IL-4 (10 U/ml) were added. On day 2 (48 hrs later), the media was changed, and RANKL, LPS, IFNγ, or IL-4 were added. Experiments were terminated on day 4. (B) Representative images of day 4 MGCs, analyzed by immunofluorescence microscopy for DNA (blue) and F-actin (green; top panel) and α-tubulin (green; bottom panel). Scale bars represent 50 microns. (C) Quantification of cell size (area) of the various MGCs (mean ± standard deviation). MGCs were generated four independent times and between 40–50 MGCs were measured at each time point. * indicates p<0.0001 (ANOVA). (D) Quantification of fusion index of the MGCs generated in (C). * indicates p<0.01, ** indicates p<0.0005 (ANOVA).
Mentions: Previous studies of osteoclastogenesis and multinucleated giant cell formation have used various protocols resulting in sometimes conflicting results. Protocol variations include the amount, timing, and duration of LPS, IFNγ, or IL-4 treatment, the presence or absence of RANKL in the media, the precursor cell line, the substrate on which the cells are initially plated, and the assessments used to determine osteoclast-likeness. In order to maintain consistency, the following protocol was used (Fig. 1A). In brief, RAW 264.7 murine macrophages were initially plated on 6-well plates (with or without glass coverslips) or on Corning Osteo Assay Surface (24-well) Plates (Corning; Corning, NY) in AMEM media containing 25 ng/ml RANKL. Following 24 hours (24 hours post-plating; Day 1), either LPS and IFNγ (0.1 µg/ml and 100 U/ml, respectively) or IL-4 (10 U/ml) was added. The next day (48 hours post-plating; Day 2), the media was replaced with fresh media containing RANKL and LPS/IFNγ or IL-4. On Day 4 (96 hours post-plating), the experiment was terminated (or drugs were added for a specific time and then the experiment was terminated). The quantity of LPS, IFNγ and IL-4 added were based on what is commonly used to activate macrophages [15], [20]. At the end of the experiment, the generated MGCs were assessed, via microscopy and protein analysis, for osteoclast-likeness (TRAP staining, phagocytic ability, and cathepsin K and MMP-9 expression).

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