Related Experiment Video
Updated: Apr 26, 2026
A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation
Published on: March 15, 2018
Modulation of osteoclastogenesis with macrophage M1- and M2-inducing stimuli
Sujeeve Jeganathan1, Cara Fiorino2, Urja Naik2
1Ontario 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.
Insights
Inflammatory stimuli alter macrophage differentiation into multinucleated giant cells. Lipopolysaccharide/interferon gamma induced Langhans cells, while interleukin-4 produced foreign body giant cells with osteoclast features.
Area of Science:
- Immunology
- Cell Biology
- Histology
Background:
- Macrophages are crucial immune cells derived from monocytes, playing roles in innate and adaptive immunity.
- Macrophages differentiate into osteoclasts, multinucleated cells responsible for bone resorption, under specific conditions (RANKL and M-CSF).
- Macrophage polarization by inflammatory stimuli is known, but its effect on multinucleated giant cell formation is less understood.
Purpose of the Study:
- To investigate how inflammatory stimuli influence the differentiation of macrophages into distinct types of multinucleated giant cells.
- To characterize the morphology and function of multinucleated giant cells generated under different inflammatory conditions.
- To explore the modulation of osteoclastogenesis by inflammatory stimuli.
Main Methods:
- Macrophage differentiation into multinucleated giant cells using receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF).
- Treatment with inflammatory stimuli: lipopolysaccharide (LPS)/interferon gamma (IFNγ) and interleukin-4 (IL-4).
- Microscopy (bright field, epifluorescence, scanning electron), functional assays, and western blotting for osteoclast markers.
Main Results:
- RANKL alone induced osteoclasts.
- RANKL combined with LPS/IFNγ generated Langhans-type giant cells.
- RANKL combined with IL-4 produced giant cells with foreign body giant cell morphology and osteoclast-like characteristics.
- Characterization of multinucleated giant cell formation and morphology.
Conclusions:
- Inflammatory stimuli significantly alter macrophage differentiation into specific multinucleated giant cell types.
- LPS/IFNγ and IL-4 induce distinct multinucleated giant cell phenotypes, diverging from typical osteoclasts.
- This study provides insights into the plasticity of macrophage differentiation and its impact on osteoclastogenesis and immune responses.
