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In Vitro Investigation of the Roles of the Proinflammatory Cytokines Tumor Necrosis Factor-α and Interleukin-1 in Murine Osteoclastogenesis

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The TNF Superfamily

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1155))

Abstract

Whereas the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of NF-кB ligand (RANKL) are essential and sufficient for osteoclastogenesis, a number of other cytokines including two proinflammatory cytokines, tumor necrosis factor-α (TNF-α), and interleukin-1 (IL-1), can exert profound effects on the osteoclastogenic process. However, the precise mode of action of TNF-α and IL-1 in osteoclastogenesis remains controversial. While some groups demonstrated that these two cytokines can promote murine osteoclastogenesis in vitro in the presence of M-CSF only, we and others showed that TNF-α-/IL-1-mediated osteoclastogenesis requires permissive levels of RANKL. This chapter describes the method that we have used to investigate the effects of TNF-α and IL-1 on osteoclast formation in in vitro osteoclastogenesis assays using primary murine bone marrow macrophages (BMMs). Detailed experimental conditions are provided and critical points are discussed to help the reader use the method to independently evaluate the roles of TNF-α and IL-1 in osteoclastogenesis in vitro. Moreover, this method can be used to further elucidate the signaling mechanisms by which these two cytokines act in concert with RANKL or with each other to modulate osteoclastogenesis.

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Abbreviations

BMM:

Bone marrow macrophage

FBS:

Fetal bovine serum

IACUC:

Institutional Animal Care and Use Committee

IL-1:

Interleukin-1

LPS:

Lipopolysaccharide

M-CSF:

Macrophage/monocyte-colony stimulating factor

NFATc1:

Nuclear factor of activated T cells, cytoplasmic 1

OPG:

Osteoprotegerin

OSHA:

Occupational Safety and Health Administration

PBS:

Phosphate-buffered saline

PI:

Principal investigator

RANK:

Receptor activator of nuclear factor-кB

RANKL:

Receptor-activator of nuclear factor-кB ligand

TNF-α:

Tumor necrosis factor-α

TRAF:

TNF receptor-associated factor

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Acknowledgments

This work is supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health grant AR47830 (to X. F.) NIAMS, National Institutes of Health Graduate Research Supplement to AR47830 (to J. J.), and by a Within Our Reach innovative basic research grant from the Research and Education Foundation of American College of Rheumatology (to X. F.).

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Authors and Affiliations

  1. Department of Pathology, University of Alabama at Birmingham, Shelby Building, Room 870, 1825 University Blvd, Birmingham, AL, 35294, USA

    Joel Jules

  2. Department of Pathology, University of Alabama at Birmingham, Volker Hall G019E, 1670 University Blvd, Birmingham, AL, 35294, USA

    Xu Feng

Authors
  1. Joel Jules
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  2. Xu Feng
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Corresponding author

Correspondence to Xu Feng .

Editor information

Editors and Affiliations

  1. INSERM Unité 1138, Paris, France

    Jagadeesh Bayry

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Jules, J., Feng, X. (2014). In Vitro Investigation of the Roles of the Proinflammatory Cytokines Tumor Necrosis Factor-α and Interleukin-1 in Murine Osteoclastogenesis. In: Bayry, J. (eds) The TNF Superfamily. Methods in Molecular Biology, vol 1155. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0669-7_10

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  • DOI: https://doi.org/10.1007/978-1-4939-0669-7_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0668-0

  • Online ISBN: 978-1-4939-0669-7

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