Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro

Summary

This protocol includes dissection of murine long bones and bone marrow isolation, to generate bone marrow macrophages and produce osteoclasts using macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL) or tumor necrosis factor alpha (TNF-α) and their arrest by anti-c-fms antibody, M-CSF receptor.

Abstract

Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by osteoclasts in response to different stimuli. Osteoclast precursors differentiate into multinuclear osteoclasts in response to macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL). Under pathologic conditions, the cytokine profile is different and involves a mixture of inflammatory cytokines. Tumor necrosis factor alpha (TNF-α) is one of the most important cytokines as it is found in large amounts in areas involved with inflammatory osteolysis. The purpose of this protocol is to provide a method by which murine bone marrow is isolated to generate osteoclasts through induction with M-CSF and either RANKL or TNF-α which will be subsequently inhibited by increasing doses of anti-c-fms antibody, the receptor for M-CSF. This experiment highlights the therapeutic value of anti-c-fms antibody in diseases of inflammatory bone resorption.

Introduction

Osteoclasts are highly specialized cells, and they differentiate from hematopoietic stem cells through the fusion of multiple osteoclast precursors. They are essential for healthy bone remodeling and contribute to pathologic bone resorption associated with inflammatory osteolytic diseases such as rheumatoid arthritis and periodontal disease¹.

Osteoclastogenesis and osteoclast function are mediated by two key factors; macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Both M-CSF and RANKL are important for osteoclast differentiation². A....

Protocol

All animal procedures and animal care were performed according to Tohoku University rules and regulations.

1. Murine Hindlimb Dissection and Handling

1. Before dissection, fill a plate with approximately 50 mL of α-MEM depending on the size of the container and place it on ice. This will serve as a collection container until the dissection of all bones is complete. For this and subsequent experiments, use α-MEM containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin G,.......

Discussion

In this study, we investigated the effect of anti-c-fms antibody on RANKL-induced osteoclast formation, TNF-α-induced osteoclast formation and M-CSF-induced osteoclast precursor proliferation. We found that the effective amount of anti-c-fms antibody for the inhibition of osteoclastogenesis among RANKL-induced osteoclast formation, TNF-α induced osteoclast formation and M-CSF-induced proliferation of osteoclast precursors is different.

RANKL mediates osteoclastogenesis in the presenc.......

Materials

Name	Company	Catalog Number	Comments
Anti-c-Fms antibody			AFS98, a rat monoclonal, antimurine, c-Fms antibody (IgG2a)
TNF-α			Recombinant murine TNF-α prepared in our laboratory. TNF-α cDNA fragment cloned by RT-PCR and cloned into a pGEX-6P-I (Amersham Biosciences, Piscataway, NJ) to generate a GST-fusion protein. GST-TNF-α was expressed in Escherichia coli BL21 cells cells (Stratagene, La Jolla, CA). The cells were lysed under nondenaturing conditions and GST-TNF-α was purified over a glutathione-Sepharose column. GST was cleaved off by PreScission Protease (Amersham Biosciences) by manufacturer’s directions and was removed by a glutathione-Sepharose column.
RANKL	PEPROTECH	315-11	Recombinant Murine sRANK Ligand, Source: E.coli
M-CSF			Recombinant human M-CSF. 1/10 vol of CMG14–12 cell line culture supernatant at 5X106 cells in a 10-cm suspension culture dish.
α-MEM	Wako		with L-Glutamine and phenol red
Fetal Bovine Serum	Biowest	s1820-500	Fetal Bovine Serum French Origin
Culture dish	Corning		100 mm x 20 mm style dish
96-well plate	Thermofischer Scientific		Nun clon Delta surface
Cell counting kit-8	Dojindo, Kumamoto, Japan
Microplate reader	Sunrise REMOTE; Tekan Japan, Kawasaki, Japan
Cell strainer	Corning		40 μm Nylon
Centrifuge tube	Corning		50 mL CentriStar cap
Triton X-100	Wako		Polyoxyethylene (10) Octyphenyl ether