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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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Biochemical characterization of the osteoclast-likeness of the various MGCs.(A) Cell lysates were taken at indicated time points during the differentiation protocol and analyzed for MMP-9 by Western blotting. 1* indicates lysates from RAW 264.7 cells prior to LPS/IFNγ or IL-4 addition. Numbers below MMP-9 blot represent densitometry readings for MMP-9 expression with 1.0 being day 2, RANKL-treated cells. (B) Cell lysates were taken from day 2 – day 4 during the differentiation protocol and analyzed for Cathepsin K expression. The last lane is the cell lysate from day 4 untreated RAW 264.7 cells. (C) Same as (B) but blots were analyzed for Arginase and iNOS (arrow). GAPDH was used as a loading control. Blots are representative of three independent experiments.
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pone-0104498-g005: Biochemical characterization of the osteoclast-likeness of the various MGCs.(A) Cell lysates were taken at indicated time points during the differentiation protocol and analyzed for MMP-9 by Western blotting. 1* indicates lysates from RAW 264.7 cells prior to LPS/IFNγ or IL-4 addition. Numbers below MMP-9 blot represent densitometry readings for MMP-9 expression with 1.0 being day 2, RANKL-treated cells. (B) Cell lysates were taken from day 2 – day 4 during the differentiation protocol and analyzed for Cathepsin K expression. The last lane is the cell lysate from day 4 untreated RAW 264.7 cells. (C) Same as (B) but blots were analyzed for Arginase and iNOS (arrow). GAPDH was used as a loading control. Blots are representative of three independent experiments.

Mentions: Other means to characterize osteoclast activity include probing for characteristic osteoclast protein biomarkers. First, we probed for MMP-9, a protease secreted by functional OCs [13], [14] (Fig. 5A). RANKL-treated cells showed a gradual increase in MMP-9 expression, showing a ∼2-fold increase between day 2 and day 4. RANKL+ LPS/IFNγ-treated cells also showed a similar trend, although the MMP-9 levels were more significantly enhanced at days 3 and 4 (Fig. 5A). In these cells, there was a ∼10-fold increase in MMP-9 expression between day 2 and day 4. In contrast, RANKL+ IL-4-treated cells showed an initial pronounced increase in MMP-9 expression between days 1 and 2 which then persisted throughout the time window of analysis (Fig. 5A). Thus, based on MMP-9 expression, RANKL and RANKL+ LPS/IFNγ showed similar MMP-9 temporal trends, albeit of different magnitudes.


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

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

Biochemical characterization of the osteoclast-likeness of the various MGCs.(A) Cell lysates were taken at indicated time points during the differentiation protocol and analyzed for MMP-9 by Western blotting. 1* indicates lysates from RAW 264.7 cells prior to LPS/IFNγ or IL-4 addition. Numbers below MMP-9 blot represent densitometry readings for MMP-9 expression with 1.0 being day 2, RANKL-treated cells. (B) Cell lysates were taken from day 2 – day 4 during the differentiation protocol and analyzed for Cathepsin K expression. The last lane is the cell lysate from day 4 untreated RAW 264.7 cells. (C) Same as (B) but blots were analyzed for Arginase and iNOS (arrow). GAPDH was used as a loading control. Blots are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104498-g005: Biochemical characterization of the osteoclast-likeness of the various MGCs.(A) Cell lysates were taken at indicated time points during the differentiation protocol and analyzed for MMP-9 by Western blotting. 1* indicates lysates from RAW 264.7 cells prior to LPS/IFNγ or IL-4 addition. Numbers below MMP-9 blot represent densitometry readings for MMP-9 expression with 1.0 being day 2, RANKL-treated cells. (B) Cell lysates were taken from day 2 – day 4 during the differentiation protocol and analyzed for Cathepsin K expression. The last lane is the cell lysate from day 4 untreated RAW 264.7 cells. (C) Same as (B) but blots were analyzed for Arginase and iNOS (arrow). GAPDH was used as a loading control. Blots are representative of three independent experiments.
Mentions: Other means to characterize osteoclast activity include probing for characteristic osteoclast protein biomarkers. First, we probed for MMP-9, a protease secreted by functional OCs [13], [14] (Fig. 5A). RANKL-treated cells showed a gradual increase in MMP-9 expression, showing a ∼2-fold increase between day 2 and day 4. RANKL+ LPS/IFNγ-treated cells also showed a similar trend, although the MMP-9 levels were more significantly enhanced at days 3 and 4 (Fig. 5A). In these cells, there was a ∼10-fold increase in MMP-9 expression between day 2 and day 4. In contrast, RANKL+ IL-4-treated cells showed an initial pronounced increase in MMP-9 expression between days 1 and 2 which then persisted throughout the time window of analysis (Fig. 5A). Thus, based on MMP-9 expression, RANKL and RANKL+ LPS/IFNγ showed similar MMP-9 temporal trends, albeit of different magnitudes.

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