Name: effect of anti-c-fms antibody on osteoclast formation and proliferation of osteoclast precursor in vitro
Uploaded: 2019-03-18T13:30:00
Description: Watch this Scientific Journal Video about Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro at JoVE.com

This work was supported in part by a JSPS KAKENHI grant from the Japan Society for the Promotion of Science (No. 16K11776 to H. K., No. 17K17306 to K. S., No. 16K20637 to K. K., No. 16K20636 to M. S., No. 18K09862 to I. M.).&#160;

All animal procedures and animal care were performed according to Tohoku University rules and regulations.
1. Murine Hindlimb Dissection and Handling
<ol>
	<li>Before dissection, fill a plate with approximately 50 mL of &#945;-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 &#945;-MEM containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin G,...

<ol>
	<li>Crotti, T. N., Dharmapatni, A. A., Alias, E., Haynes, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoimmunology:+Major+and+Costimulatory+Pathway+Expression+Associated+with+Chronic+Inflammatory+Induced+Bone+Loss.">Osteoimmunology: Major and Costimulatory Pathway Expression Associated with Chronic Inflammatory Induced Bone Loss.</a> Journal of Immunology Research. 2015, 281287 (2015).</li><li>Teitelbaum, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+resorption+by+osteoclasts.">Bone resorption by osteoclasts.</a> Science. 289 (5484), 1504-1508 (2000).</li><li>Azuma, Y., Kaji, K., Katogi, R., Takeshita, S., Kudo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor-alpha+induces+differentiation+of+and+bone+resorption+by+osteoclasts.">Tumor necrosis factor-alpha induces differentiation of and bone resorption by osteoclasts.</a> Journal of Biological Chemistry. 275 (7), 4858-4864 (2000).</li><li>Kobayashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor+alpha+stimulates+osteoclast+differentiation+by+a+mechanism+independent+of+the+ODF/RANKL-RANK+interaction.">Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction.</a> Journal of Experimental Medicine. 191 (2), 275-286 (2000).</li><li>Kim, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+differentiation+independent+of+the+TRANCE-RANK-TRAF6+axis.">Osteoclast differentiation independent of the TRANCE-RANK-TRAF6 axis.</a> Journal of Experimental Medicine. 202 (5), 589-595 (2005).</li><li>Redlich, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclasts+are+essential+for+TNF-alpha-mediated+joint+destruction.">Osteoclasts are essential for TNF-alpha-mediated joint destruction.</a> Journal of Clinical Investestigation. 110 (10), 1419-1427 (2002).</li><li>Abu-Amer, Y., Ross, F. P., Edwards, J., Teitelbaum, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-stimulated+osteoclastogenesis+is+mediated+by+tumor+necrosis+factor+via+its+P55+receptor.">Lipopolysaccharide-stimulated osteoclastogenesis is mediated by tumor necrosis factor via its P55 receptor.</a> Journal of Clinical Investestigation. 100 (6), 1557-1565 (1997).</li><li>Zha, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-alpha+contributes+to+postmenopausal+osteoporosis+by+synergistically+promoting+RANKL-induced+osteoclast+formation.">TNF-alpha contributes to postmenopausal osteoporosis by synergistically promoting RANKL-induced osteoclast formation.</a> Biomedicine &amp; Pharmacotherapy. 102, 369-374 (2018).</li><li>Kitaura, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-CSF+mediates+TNF-induced+inflammatory+osteolysis.">M-CSF mediates TNF-induced inflammatory osteolysis.</a> Journal of Clinical Investigation. 115 (12), 3418-3427 (2005).</li><li>Kimura, K., Kitaura, H., Fujii, T., Hakami, Z. W., Takano-Yamamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-c-Fms+antibody+inhibits+lipopolysaccharide-induced+osteoclastogenesis+in+vivo.">Anti-c-Fms antibody inhibits lipopolysaccharide-induced osteoclastogenesis in vivo.</a> FEMS Immunology and Medical Microbiology. 64 (2), 219-227 (2012).</li><li>Kimura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+anti-c-Fms+antibody+inhibits+osteoclastogenesis+in+a+mouse+periodontitis+model.">An anti-c-Fms antibody inhibits osteoclastogenesis in a mouse periodontitis model.</a> Oral Diseases. 20 (3), 319-324 (2014).</li><li>Kitaura, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+M-CSF+receptor+c-Fms+antibody+inhibits+mechanical+stress-induced+root+resorption+during+orthodontic+tooth+movement+in+mice.">An M-CSF receptor c-Fms antibody inhibits mechanical stress-induced root resorption during orthodontic tooth movement in mice.</a> Angle Orthodontist. 79 (5), 835-841 (2009).</li><li>Kitaura, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+anti-c-Fms+antibody+inhibits+orthodontic+tooth+movement.">An anti-c-Fms antibody inhibits orthodontic tooth movement.</a> Journal of Dental Research. 87 (4), 396-400 (2008).</li><li>Bettina, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-CSF+Mediates+Host+Defense+during+Bacterial+Pneumonia+by+Promoting+the+Survival+of+Lung+and+Liver+Mononuclear+Phagocytes.">M-CSF Mediates Host Defense during Bacterial Pneumonia by Promoting the Survival of Lung and Liver Mononuclear Phagocytes.</a> Journal of Immunology. 196 (12), 5047-5055 (2016).</li><li>Dougall, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RANK+is+essential+for+osteoclast+and+lymph+node+development.">RANK is essential for osteoclast and lymph node development.</a> Genes &amp; Develpoment. 13 (18), 2412-2424 (1999).</li><li>Wiktor-Jedrzejczak, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+absence+of+colony-stimulating+factor+1+in+the+macrophage-deficient+osteopetrotic+(op/op)+mouse.">Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse.</a> Proceedings of the National Academy of Sciences of the United States of America. 87 (12), 4828-4832 (1990).</li><li>Yang, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+of+macrophage+colony-stimulating+factor+in+ankylosing+spondylitis+and+rheumatoid+arthritis.">Increased expression of macrophage colony-stimulating factor in ankylosing spondylitis and rheumatoid arthritis.</a> Annals of the Rheumatic Diseases. 65 (12), 1671-1672 (2006).</li><li>Takei, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+macrophage-colony+stimulating+factor+levels+in+synovial+fluid+of+loose+artificial+hip+joints.">High macrophage-colony stimulating factor levels in synovial fluid of loose artificial hip joints.</a> The Journal of Rheumatology. 27 (4), 894-899 (2000).</li></ol>

In this study, we investigated the effect of anti-c-fms antibody on RANKL-induced osteoclast formation, TNF-&#945;-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-&#945; induced osteoclast formation and M-CSF-induced proliferation of osteoclast precursors is different.
RANKL mediates osteoclastogenesis in the presenc...

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. Another factor which has been shown to induce osteoclast formation from bone marrow macrophages in vitro is tumor necrosis factor alpha (TNF-&#945;)3,4,5. TNF-&#945; mediated osteoclast formation has been shown to be critical for osteolysis in destructive bone diseases such as rheumatoid arthritis6, periodontal disease7, and postmenopausal osteoporosis8.
C-fms is the membrane receptor of M-CSF and mediates its action. The role of c-fms is well documented in the literature as it was demonstrated that the administration of an antibody against c-fms (anti-c-fms antibody) completely arrested osteoclastogenesis in an arthritis model as well as in TNF-&#945; induced bone erosions while osteoclastogenesis distant from the inflamed joint was still robust9. Administration of anti-c-fms antibody inhibited osteoclast formation and bone resorption induced by lipopolysaccharide (LPS) in mouse calvariae10 as well as in LPS-induced&#160; periodontitis model11. Moreover, anti-c-fms antibody inhibited mechanical stress-induced root resorption during orthodontic tooth movement12, as well as orthodontic tooth movement and bone resorption associated with orthodontic tooth movement13.
M-CSF has been reported to have other functions which are essential to the host immune response. The absence of M-CSF in op/op mice with bacterial pneumonia lead to the increase of bacterial burden and bacterial dissemination to the liver with hepatic necrosis14. Therefore, M-CSF is important for immunological response in the protection from infection.
This study demonstrates the effect of anti-c-fms antibody on M-CSF and RANKL or TNF-&#945; induced osteoclast formation in vitro and M-CSF induced proliferation of osteoclast precursors. This protocol demonstrates the isolation of murine bone marrow cells from long bones, and the steps to generate bone marrow macrophages (BMM) which are regarded as osteoclast precursors and to induce the differentiation of BMM into multinuclear osteoclasts by two methods; either RANKL or TNF-&#945;. The protocol also compares the arrest of osteoclastogenesis in both methods using anti-c-fms antibody.
The process by which bone marrow is extracted and subsequently used to generate osteoclast precursors is reliable in producing large quantities of pure osteoclast cultures which can be used in multiple downstream applications such as drug testing. The use of either RANKL or TNF-&#945; to induce osteoclastogenesis in this protocol distinguishes osteoclastogenesis as a physiologic process (RANKL) from osteoclastogenesis as a pathologic process (TNF-&#945;), which in turn provides two alternative procedures to guide the decision of which one is superior in terms of the reagents to be used in downstream applications. This protocol also provides a method by which we can determine a suitable concentration of anti-c-fms antibody that can be safely used for later studies involved in bone resorption and osteolytic diseases.

<table>
	<tbody>
		<tr>
			<td>Anti-c-Fms antibody</td>
			<td></td>
			<td></td>
			<td>AFS98, a rat monoclonal, antimurine, c-Fms antibody (IgG2a)</td>
		</tr>
		<tr>
			<td>TNF-&#945;</td>
			<td></td>
			<td></td>
			<td>Recombinant murine TNF-&#945; prepared in our laboratory. TNF-&#945; 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-&#945; was expressed in Escherichia coli BL21 cells cells (Stratagene, La Jolla, CA). The cells were lysed under nondenaturing conditions and GST-TNF-&#945; was purified over a glutathione-Sepharose column. GST was cleaved off by PreScission Protease (Amersham Biosciences) by manufacturer&#8217;s directions and was removed by a glutathione-Sepharose column.</td>
		</tr>
		<tr>
			<td>RANKL</td>
			<td>PEPROTECH</td>
			<td>315-11</td>
			<td>Recombinant Murine sRANK Ligand, Source: E.coli</td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td></td>
			<td></td>
			<td>Recombinant human M-CSF. 1/10 vol of CMG14&#8211;12 cell line culture supernatant at 5X106&#160;cells in a 10-cm suspension culture dish.</td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Wako</td>
			<td></td>
			<td>with L-Glutamine and phenol red</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>s1820-500</td>
			<td>Fetal Bovine Serum French Origin</td>
		</tr>
		<tr>
			<td>Culture dish</td>
			<td>Corning</td>
			<td></td>
			<td>100 mm x 20 mm style dish</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Thermofischer Scientific</td>
			<td></td>
			<td>Nun clon Delta surface</td>
		</tr>
		<tr>
			<td>Cell counting kit-8</td>
			<td>Dojindo, Kumamoto, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Sunrise REMOTE; Tekan Japan, Kawasaki, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>Corning</td>
			<td></td>
			<td>40 &#956;m Nylon</td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>Corning</td>
			<td></td>
			<td>50 mL CentriStar cap</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Wako</td>
			<td></td>
			<td>Polyoxyethylene (10) Octyphenyl ether</td>
		</tr>
	</tbody>
</table>

effect of anti-c-fms antibody on osteoclast formation and proliferation of osteoclast precursor in vitro

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-&#945;) 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-&#945; 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.

The purpose of this protocol is to evaluate the effect of anti-c-fms antibody on osteoclast formation in the presence of RANKL or TNF-&#945;, and to determine the effect of M-CSF on osteoclast precursors proliferation. In this protocol, we have provided a reliable process by which large quantities of pure osteoclast cultures are generated. We have also provided a way to test anti-c-fms antibody suitable concentration for inhibiting osteoclast formation under different culture conditions.<...

Watch this Scientific Journal Video about Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro at JoVE.com

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

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-&#945;) and their arrest by anti-c-fms antibody, M-CSF receptor.

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 ...

This experiment highlights the therapeutic value of Anti-c-fms antibody in diseases of inflammatory bone disruption, such as rheumatoid arthritis and periodontitis. The process by which bone marrow is extracted and used to generate osteoclast precursors, yields large quantities of pure osteoclasts, which can be used in multiple downstream applications. This protocol provide two alternative methods of osteoclastogenesis, either using RANKL ligand or TNF alpha.In addition to that, this method provides insight into the effect of Anti-c-fms antibody on osteoclastogenesis. Visual demonstration of this procedure familiarizes the researcher with the process of dissection and obtaining bone marrow cells in large amounts. To begin fill a plate that will serve as a collection container with approximately 50 milliliters of alpha MEM, depending on the size of the container, and place it on ice.Use pins to pierce through the palms and feet of a euthanized mouse to fix it in supine position, and spray thoroughly with 70%ethanol. Use sterile surgical scissors and tweezers to make a skin incision at the level of the hip and extend it down to the feet, exposing the muscles. Locate and cut the tendon attaching the quadriceps muscle.Peel away the muscle and cut it at the level of the hip. Then locate the tendon attaching the hamstring muscle to the bone. Peel away the muscles exposing the bone, and free the hamstring from its attachment, making sure not to cut into the bone.Position the scissors between the head of the femur and the joint space. Cut into it freeing the femur, but keeping the bone intact, which will enable detachment of the bones without risk of exposing the bone marrow. Cutaway the muscles attached to the tibia in a similar fashion.Then cut the tibia free from its attachment at the ankle joint, while keeping the bone intact to avoid contamination. Use the scissor blades and lab wipes to scrape any remaining soft tissue from the femur and tibia and place them in the prepared plate with alpha MEM placed on ice. The dissection should be carried out carefully to minimize contamination.That is why thorough soft tissue removal and bone localization are crucial. Fill a 30 gauge needle syringe with alpha MEM. Disconnect the tibia and femur away from the knee joint, and cut the ends of the long bone from both sides using scissors.Use tweezers to hold the bone from the shaft. Insert the needle into the marrow space to the bone and slowly flush the content of the marrow into a six centimeter culture dish and repeat for all bones. The bone will turn white, indicating the extraction of all bone marrow content.Use a 40 micrometer nylon cell strainer to strain the bone marrow extract into a 50 milliliter conical centrifuge tube. Centrifuge the tube at 300 times G for five minutes. Discard the supernatant.To wash, add five milliliters of alpha MEM to the pellet. Pipette the cells to detach them, centrifuge, and repeat the wash for a total of two times. After counting these isolated cells, as described in the manuscript, seed 10 million cells per 10 milliliters, in a 10 centimeter culture dish, and add M-CSF to the cells.Incubate the culture at 37 degrees Celsius, 5%carbon dioxide for three days. Remove the medium after three days, and wash the cells twice vigorously with 10 milliliters of PBS to remove nonadherent cells. Add five milliliters of room temperature 0.02%trypsin-EDTA and PBS, and incubate at 37 degrees Celsius, 5%carbon dioxide for five minutes.Thoroughly pipette the cells to detach them. Observe the culture under a microscope to make sure the cells have been detached and appear rounded and floating in media. When the cells have been detached, inactivate the reaction by adding five milliliters of the alpha MEM.Collect the cells into a 50 milliliter conical tube and centrifuge at 300 times G for five minutes. After discarding the supernatant, wash with five milliliters of alpha MEM, and centrifuge again at 300 times G for five minutes. Re-suspend the pellet in 10 milliliters of alpha MEM.Seed 1 million cells per 10 milliliters in a 10 centimeter culture dish, and add M-CSF. Incubate the culture at 37 degrees Celsius, 5%carbon dioxide for three days. Harvest the attached cells, which represent BMMs as osteoclast precursors after three days, as done previously during generation of BMMs.Seed the BMMs at 50, 000 cells per 200 micro liters of alpha MEM in a 96 well plate per well. To each well, add desired stimuli. And then add M-CSF for a final concentration of 100 nanograms per milliliter.Add Anti-c-fms antibody to each well. Incubate the plate at 37 degrees Celsius, 5%carbon dioxide for four days, while changing media every other day for four days. And then proceed with staining and assays, as described in the manuscript.The osteoclast formation in M-CSF and RANKL treated culture, with Anti-c-fms antibody, was evaluated. With 101, 000 nanograms per milliliter Anti-c-fms antibody, a significant decrease in the number of osteoclasts was observed compared to control. Whereas, with one in 10 nanograms per milliliter Anti-c-fms antibody, there was no significant decrease.Then, the osteoclast formation in M-CSF and TNF alpha treated culture, with Anti-c-fms antibody, was evaluated. With 10, 100, 1, 000 nanograms per milliliter Anti-c-fms antibody, a significant decrease in the number of osteoclasts was observed compared to control, with no significant decrease at one nanogram per milliliter. And the M-CSF culture with 1, 10, 100 nanograms per milliliter Anti-c-fms antibody, there was no significant decrease in the number of osteoclast precursors compared to control.However, at 1, 000 nanograms per milliliter, there was a significant decrease in the number of osteoclast precursors, indicating that a concentration as high as 1, 000 nanograms per milliliter is needed to significantly inhibit proliferation of osteoclast precursors. Make sure to free the bonds while keeping the bone intact, which will minimize the chance of contamination.

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