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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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Functional assays of generated MGCs.Day 4 MGCs were assessed on their ability to undergo phagocytosis of IgG-opsonized sheep red blood cells (IgG-sRBCs) and on their ability to produce TRAP. (A) Representative images of cells after phagocytosis assay (see Methods). IgG-sRBCs are in red, F-actin in green, and DNA in blue. (B) Quantification of internalized IgG-sRBCs per 100 MGCs (mean ± standard deviation). Quantification was done on three independent experiments, with between 100–150 MGCs counted per experiment. * indicates p<0.001 (ANOVA). (C) Representative images of day 4 MGCs stained for TRAP. Top panel: RAW 264.7 macrophages on glass substrate. Bottom panel: RAW 264.7 macrophages on Osteo Assay Surface. Scale bars represent 50 microns.
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pone-0104498-g003: Functional assays of generated MGCs.Day 4 MGCs were assessed on their ability to undergo phagocytosis of IgG-opsonized sheep red blood cells (IgG-sRBCs) and on their ability to produce TRAP. (A) Representative images of cells after phagocytosis assay (see Methods). IgG-sRBCs are in red, F-actin in green, and DNA in blue. (B) Quantification of internalized IgG-sRBCs per 100 MGCs (mean ± standard deviation). Quantification was done on three independent experiments, with between 100–150 MGCs counted per experiment. * indicates p<0.001 (ANOVA). (C) Representative images of day 4 MGCs stained for TRAP. Top panel: RAW 264.7 macrophages on glass substrate. Bottom panel: RAW 264.7 macrophages on Osteo Assay Surface. Scale bars represent 50 microns.

Mentions: A key component of macrophage function is their ability to carry out phagocytosis [21]. One type of phagocytosis employed by these cells is through Fcgamma (Fcγ) receptors, which engage IgG-opsonized targets (or IgG-sensitized red blood cells). Osteoclasts characteristically lose their phagocytic ability throughout differentiation, which is attributed to reduced Fcγ receptor display [22]. We thus determined the ability of the various induced MGCs to internalize IgG-opsonized sheep red blood cells (IgG-sRBCs). Of the three different MGC types, only RANKL+ IL-4-treated MGCs showed significant ability to internalize IgG-sRBCs (Fig. 3A,B; p<0.0001). While there were some RANKL− and RANKL+ LPS/IFNγ-treated cells that did have internalized IgG-sRBCs, the frequency was very rare (∼5–10 internalized IgG-sRBCs per 100 MGCs) (Fig. 3A,B). These observations suggested that RANKL-treatment alone and RANKL+ LPS/IFNγ-treatment either decreased Fcγ receptor expression post macrophage fusion, or significantly decreased internalization post fusion, and IL-4 treatment either prevented this loss, or activated their expression.


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

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

Functional assays of generated MGCs.Day 4 MGCs were assessed on their ability to undergo phagocytosis of IgG-opsonized sheep red blood cells (IgG-sRBCs) and on their ability to produce TRAP. (A) Representative images of cells after phagocytosis assay (see Methods). IgG-sRBCs are in red, F-actin in green, and DNA in blue. (B) Quantification of internalized IgG-sRBCs per 100 MGCs (mean ± standard deviation). Quantification was done on three independent experiments, with between 100–150 MGCs counted per experiment. * indicates p<0.001 (ANOVA). (C) Representative images of day 4 MGCs stained for TRAP. Top panel: RAW 264.7 macrophages on glass substrate. Bottom panel: RAW 264.7 macrophages on Osteo Assay Surface. Scale bars represent 50 microns.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104498-g003: Functional assays of generated MGCs.Day 4 MGCs were assessed on their ability to undergo phagocytosis of IgG-opsonized sheep red blood cells (IgG-sRBCs) and on their ability to produce TRAP. (A) Representative images of cells after phagocytosis assay (see Methods). IgG-sRBCs are in red, F-actin in green, and DNA in blue. (B) Quantification of internalized IgG-sRBCs per 100 MGCs (mean ± standard deviation). Quantification was done on three independent experiments, with between 100–150 MGCs counted per experiment. * indicates p<0.001 (ANOVA). (C) Representative images of day 4 MGCs stained for TRAP. Top panel: RAW 264.7 macrophages on glass substrate. Bottom panel: RAW 264.7 macrophages on Osteo Assay Surface. Scale bars represent 50 microns.
Mentions: A key component of macrophage function is their ability to carry out phagocytosis [21]. One type of phagocytosis employed by these cells is through Fcgamma (Fcγ) receptors, which engage IgG-opsonized targets (or IgG-sensitized red blood cells). Osteoclasts characteristically lose their phagocytic ability throughout differentiation, which is attributed to reduced Fcγ receptor display [22]. We thus determined the ability of the various induced MGCs to internalize IgG-opsonized sheep red blood cells (IgG-sRBCs). Of the three different MGC types, only RANKL+ IL-4-treated MGCs showed significant ability to internalize IgG-sRBCs (Fig. 3A,B; p<0.0001). While there were some RANKL− and RANKL+ LPS/IFNγ-treated cells that did have internalized IgG-sRBCs, the frequency was very rare (∼5–10 internalized IgG-sRBCs per 100 MGCs) (Fig. 3A,B). These observations suggested that RANKL-treatment alone and RANKL+ LPS/IFNγ-treatment either decreased Fcγ receptor expression post macrophage fusion, or significantly decreased internalization post fusion, and IL-4 treatment either prevented this loss, or activated their 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