Abstract
We describe a method to visualize the migration of osteoclast precursors within intact murine bone marrow in real time using intravital multiphoton microscopy. Conventionally, cell migration has been evaluated using in vitro systems, such as transmigration assays. Although these methods are convenient for quantification and are highly reproducible, these in vitro assay systems may not accurately reflect in vivo cellular behavior. In addition to in vitro analyses, recent technological progress in two-photon excitation-based laser microscopy has enabled the visualization of dynamic cell behavior deep inside intact living organs. Combining this imaging method with in vitro chemoattraction analyses, we have revealed that sphingosine-1-phosphate (S1P), a lipid mediator enriched in blood, bidirectionally controls the trafficking of osteoclast precursors between the circulation and bone marrow cavities via G protein-coupled receptors (GPCRs).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Harada S, Rodan GA (2003) Control of osteoblast function and regulation of bone mass. Nature 423:349–355
Teitelbaum SL, Ross FP (2003) Genetic regulation of osteoclast development and function. Nat Rev Genet 4:638–649
Henriksen K et al (2009) Local communication on and within bone controls bone remodeling. Bone 44:1026–1033
Yu X et al (2003) Stromal cell-derived factor-1 (SDF-1) recruits osteoclast precursors by inducing chemotaxis, matrix metalloproteinase-9 (MMP-9) acrivity, and collagen transmigration. J Bone Miner Res 18:1404–1418
Wright LM et al (2005) Stromal cell-derived factor-1 binding to its chemokine receptor CXCR4 on precursor cells promotes the chemotactic recruitment, development and survival of human osteoclasts. Bone 36:840–853
Koizumi K et al (2009) Role of CX3CL1/fractalkine in osteoclast differentiation and bone resorption. J Immunol 183:7825–7831
Kim MS, Day CJ, Morrison NA (2005) MCP-1 is induced by receptor activator of nuclear factor-kB ligand, promotes human osteoclast fusion, and rescues granulocyte macrophage colony-stimulating fctor suppression of osteoclast formation. J Biol Chem 280:16163–16169
Choi SJ et al (2000) Macrophage inflammatory protein 1-α is a potential osteoclast stimulatory factor in multiple myeloma. Blood 96:671–675
Lean JM et al (2002) CCL9/MIP-1 gamma and its receptor CCR1 are the major chemokine ligand/receptor species expressed by osteoclasts. J Cell Biochem 87:386–393
Ha J et al (2010) CXC chemokine ligand 2 induced by receptor activator of NF-κB ligand enhances osteoclastogenesis. J Immunol 184:4717–4724
Kwak HB et al (2008) Reciprocal cross-talk between RANKL and interferon-gamma-inducible protein 10 is responsible for bone-erosive experimental arthritis. Arthritis Rheum 58:1332–1342
Binder NB et al (2009) Estrogen-dependent and C-C chemokine receptor-2-dependent pathways determine osteoclast behavior in osteoporosis. Nat Med 15:417–424
Ishii M et al (2009) Sphingosine-1-phospate mobilizes osteoclast precursors and regulates bone homeostasis. Nature 458:524–528
Ishii M et al (2010) Chemorepulsion by blood S1P regulates osteoclast precursor mobilization and bone remodeling in vivo. J Exp Med 207:2793–2798
Cahalan MD et al (2002) Two-photon tissue imaging: seeing the immune system in a fresh light. Nat Rev Immunol 2:872–880
Germain RN et al (2006) Dynamic imaging of the immune system: progress, pitfalls and promise. Nat Rev Immunol 6:497–507
Wang BG, Konig K, Halbhuber KJ (2010) Two-photon microscopy of deep intravital tissues and its merits in clinical research. J Microsc 238:1–20
Germain RN et al (2008) Making friends in out-of-the-way places: how cells of the immune system get together and how they conduct their business as revealed by intravital imaging. Immunol Rev 221:163–181
Jung S et al (2000) Analysis of fractalkine receptro CX3CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol 20:4106–4114
Burnett SH et al (2004) Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene. J Leukoc Biol 75:612–623
Rivera J, Proia RL, Olivera A (2008) The alliance of sphingosine-1-phosphate and its receptors in immunity. Nat Rev Immunol 8:753–763
Matloubian M et al (2004) Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 6972:355–360
Wer SH et al (2005) Sphingosine 1-phospate type 1 receptor agonism inhibits transendothelial migration of medullary T cells to lymphatic sinuses. Nat Immunol 12:1228–1235
Osaka M et al (2002) Enhancement of sphingosine 1-phosphate-induced migration of vascular endothelial cells and smooth muscle cells by an EDG-5 antagonist. Biochem Biophys Res Commun 299:483–487
Allende M et al (2003) G-protein-coupled receptor S1P1 acts within endothelial cells to regulate vascular maturation. Blood 102: 3665–3667
Ferron M, Vacher J (2005) Targeted expression of Cre recombinase in macrophages and osteoclasts in transgenic mice. Genesis 41: 138–145
Kono M et al (2007) Deafness and stria vascularis defects in S1P2 receptor-null mice. J Biol Chem 282:10690–10696
Tomimori Y et al (2009) Evaluation of pharmaceuticals with a novel 50-hour animal model of bone loss. J Bone Miner Res 24:1194–1205
Liu Y et al (2000) Edg-1, the G-protein-coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation. J Clin Invest 106:951–961
Acknowledgment
This work was supported by Grants-in-Aid for Encouragement of Young Scientists (A) (22689030), for Scientific Research on Innovative Areas (22113007) and by a Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program) from the Ministry of Education, Science, Sports and Culture of Japan, by a Grant-in-Aid for Research on Allergic Disease and Immunology (H21-010) from the Ministry of Health, Labor and Welfare of Japan, and by Grants from the International Human Frontier Science Program (CDA-00059/2009 and RGY-0077/2011).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Ishii, T., Kawamura, S., Nishiyama, I., Kikuta, J., Ishii, M. (2012). Use of Intravital Microscopy and In Vitro Chemotaxis Assays to Study the Roles of Sphingosine-1-Phosphate in Bone Homeostasis. In: Pébay, A., Turksen, K. (eds) Sphingosine-1-Phosphate. Methods in Molecular Biology, vol 874. Humana Press. https://doi.org/10.1007/978-1-61779-800-9_10
Download citation
DOI: https://doi.org/10.1007/978-1-61779-800-9_10
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-799-6
Online ISBN: 978-1-61779-800-9
eBook Packages: Springer Protocols