Name: assessment of antibody-based drugs effects on murine bone marrow and peritoneal macrophage activation
Uploaded: 2017-12-26T15:00:00
Description: Watch this Scientific Journal Video about Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation at JoVE.com

L.K. is the recipient of the University of British Columbia 4-year fellowship (4YF) graduate studentship award. L.M.S. is the recipient of a Canadian Association of Gastroenterology/ Crohn's and Colitis Canada/ CIHR New Investigator Salary Award and is a Biomedical Scholar of the Michael Smith Foundation for Health Research. This work was supported by a Project Grant from Canadian Blood Services, in collaboration with the Canadian Institutes of Health Research (grant # CIHR2016-LS).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of British Columbia.
1. Bone Marrow Macrophage Derivation and Activation with Antibodies
<ol>
	<li>Perform euthanasia using CO2 asphyxiation.
	<ol>
		<li>Place mouse in induction chamber. Euthanize mouse with 5% isoflurane anesthesia, for 60 - 90 s, until immobile and breathing is deep and slow.</li>
		<li>Turn off isoflurane anesthesia and administer 6 - 8 L/...

<ol>
	<li>Murray, P. J., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+and+pathogenic+functions+of+macrophage+subsets.">Protective and pathogenic functions of macrophage subsets.</a> Nat Rev Immunol. 11 (11), 723-737 (2011).</li><li>Martinez, F. O., Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+M1+and+M2+paradigm+of+macrophage+activation:+time+for+reassessment.">The M1 and M2 paradigm of macrophage activation: time for reassessment.</a> F1000Prime Rep. 6, 13 (2014).</li><li>Mosser, D. M., Edwards, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+full+spectrum+of+macrophage+activation.">Exploring the full spectrum of macrophage activation.</a> Nat Rev Immunol. 8 (12), 958-969 (2008).</li><li>Edwards, J. P., Zhang, X., Frauwirth, K. A., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+functional+characterization+of+three+activated+macrophage+populations.">Biochemical and functional characterization of three activated macrophage populations.</a> J Leukoc Biol. 80 (6), 1298-1307 (2006).</li><li>Mosser, D. M., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+murine+macrophages.">Activation of murine macrophages.</a> Curr Protoc Immunol. , (2008).</li><li>Gallo, P., Gon&#231;alves, R., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+IgG+density+and+macrophage+Fc+(gamma)+receptor+cross-linking+on+phagocytosis+and+IL-10+production.">The influence of IgG density and macrophage Fc (gamma) receptor cross-linking on phagocytosis and IL-10 production.</a> Immunol Lett. 133 (2), 70-77 (2010).</li><li>Vos, A. 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=Anti-tumor+necrosis+factor-&#945;+antibodies+induce+regulatory+macrophages+in+an+Fc+region-dependent+manner.">Anti-tumor necrosis factor-&#945; antibodies induce regulatory macrophages in an Fc region-dependent manner.</a> Gastroenterology. 140 (1), 221-230 (2011).</li><li>Kozicky, L. 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=Intravenous+immunoglobulin+skews+macrophages+to+an+anti-inflammatory,+IL-10-producing+activation+state.">Intravenous immunoglobulin skews macrophages to an anti-inflammatory, IL-10-producing activation state.</a> J Leukoc Biol. 98 (6), 983-994 (2015).</li><li>Sutterwala, F. S., Noel, G. J., Salgame, P., Mosser, D. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERK+activation+following+macrophage+FcgammaR+ligation+leads+to+chromatin+modifications+at+the+IL-10+locus.">ERK activation following macrophage FcgammaR ligation leads to chromatin modifications at the IL-10 locus.</a> J Immunol. 175 (1), 469-477 (2005).</li><li>Murray, P. J., 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=Macrophage+activation+and+polarization:+nomenclature+and+experimental+guidelines.">Macrophage activation and polarization: nomenclature and experimental guidelines.</a> Immunity. 41 (1), 14-20 (2014).</li><li>Anderson, C. F., Gerber, J. S., Mosser, D. 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Macrophage activation states play an important role in both tissue homeostasis and disease22. Macrophages can have distinct as well as overlapping activation states, depending on cues in their microenvironment3. They have distinct roles in all stages of the inflammatory response: defense against pathogens, wound healing and tissue restitution, and also have a distinct anti-inflammatory activation state that is important for turning off the inflammatory response<sup class="x...

Macrophages are innate immune cells, which play multiple roles in the immune response to infection or injury. Macrophages are responsible for initiating an immune response to infection or tissue damage, stopping the inflammatory response, and promoting the healing response1. Examples of the three best studied macrophage activation states are: 1) macrophages treated with interferon gamma (IFN&#947;) and bacterial lipopolysaccharide (LPS), designated M(IFN&#947; + LPS), which contribute to the inflammatory response; 2) macrophages stimulated with interleukin 4 (IL-4), M(IL-4), which are associated with the healing response; 3) macrophages stimulated with immune complexes (IC) and LPS, M(IC + LPS), which have the ability to turn off the inflammatory response2,3. M(IC + LPS) are distinct from M(IL-4) wound healing macrophages, and do not express the enzyme arginase (Arg-1) or FIZZ14. The best marker for these anti-inflammatory macrophages is their cytokine production5. Macrophages have multiple roles in maintaining health, but also contribute to inflammatory diseases and cancer3. Because of this, macrophages are a key therapeutic target for the treatment of a wide variety of diseases. It is important to investigate the effects of antibodies on their activation state to develop macrophage-based treatments for disease.
The focus of this paper is on the use of murine bone marrow derived macrophages (BMDMs) and peritoneal macrophages to test the effect of antibody drugs on inflammatory responses in vitro and in vivo. Recently, there have been multiple studies showing the effects of antibodies on macrophage activation6,7,8. Macrophages co-activated with immune complexes, which are antibodies complexed with an antigen, and LPS, a normally inflammatory stimulus, produce very high levels of the anti-inflammatory cytokine, IL-10, and very low levels of the pro-inflammatory cytokine, interleukin 12 (IL-12)9. In addition, infliximab, a monoclonal antibody against TNF&#945;, has been found to work, in part, by inducing anti-inflammatory macrophages through its fragment crystallizable (Fc) region7. We have reported that IVIg + LPS induce anti-inflammatory macrophage activation that is similar to M(IC + LPS), wherein co-stimulated macrophages produce large amounts of IL-10 and low amounts of the pro-inflammatory cytokine subunit Interleukin 12 or 23 p40 (IL-12/23p40), interleukin- 6 (IL-6), and TNF8. IVIg is a drug comprised of polyclonal antibodies, primarily IgG, which has been pooled from the blood of more than 1,000 donors10. It is used to treat a wide variety of immunological diseases, such as idiopathic thrombocytopenic purpura and chronic demyelinating polyneuropathy, but its mechanism of action is not completely understood11. The effects of antibody based drugs on macrophage activation can be assessed using methods described herein.
The effects of specific biologics on macrophage activation can be tested in BMDMs and peritoneal macrophages. Using these macrophage sources permits assessment of primary cells. Preliminary testing of antibodies on cultured primary cells requires less time and monetary investment than other time consuming and expensive disease models. By injecting a drug into a healthy mouse in vivo, and isolating the cells and analyzing them ex vivo, one can determine if studies are warranted to assess whether treatment with biologics affects macrophage activation in disease models.
With few studies testing the effect of biologic therapies on macrophage activation in vitro and in vivo directly, our techniques provide an advantage over alternative techniques. Current techniques involve testing biologic drug effects on mixed cell populations in vitro, such as the effect of infliximab in a mixed lymphocyte reaction (MLR) or IVIg on human macrophages in peripheral blood mononuclear cells, where the effect cannot be attributed to a specific cell type7,12. Using BMDMs and peritoneal macrophages is advantageous over using cell lines, such as RAW264.7 cells, which do not produce the pro-inflammatory cytokine, IL-12, in response to LPS8,13. Testing the effect of an antibody-based drug on macrophage responses ex vivo has advantages because cytokine responses can be attributed directly to macrophages, rather than inferring macrophage responses by measuring serum cytokine levels14. BMDMs and peritoneal macrophages can be derived and isolated from genetically modified mice to determine the specific role of a protein in anti-inflammatory macrophage activation. For example, we have used Il10 deficient(-/-) BMDMs to demonstrate that IVIg-induced reduction of pro-inflammatory cytokine production is partially dependent on IL-108. A drug&#39;s mechanism of action can be investigated using western blotting, where the role of specific proteins and signaling events can be determined. Quantitative polymerase chain reaction (qPCR) can be performed on BMDMs or peritoneal macrophages to show patterns of gene expression that result from antibody activation. Disease models in mice can provide information on the potential efficacy of antibody-based biological therapies in models for diseases like inflammatory bowel disease, rheumatoid arthritis, and cancer15,16,17. However, the techniques described here will provide information on the mechanism of action of these biologics by determining whether they induce anti-inflammatory macrophage activity.

<table border="0" cellpadding="0" cellspacing="0" style="width:1312px;" width="1311">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Iscove&#8217;s modified Dulbecco&#8217;s medium (IMDM)</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">12440053</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">12483-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Recombinant murine macrophage colony stimulating factor (MCSF)</td>
			<td style="width:209px;">Stemcell technologies</td>
			<td>78059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Penicillin-streptomycin</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">15140148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Monothioglycerol (MTG)</td>
			<td style="width:209px;">Sigma</td>
			<td>88640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">1X red blood cell lysis buffer</td>
			<td style="width:209px;">eBioscience</td>
			<td style="width:408px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Cell dissociation buffer</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">13150016</td>
			<td>Enzyme-Free, Hanks&#39;s-based, EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Lipopolysaccharide (LPS)</td>
			<td style="width:209px;">Sigma aldrich</td>
			<td style="width:408px;">L 4516</td>
			<td>From E. coli 0127:B8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">IVIg (Gammunex)</td>
			<td style="width:209px;">Grifols</td>
			<td>Received from BC Children&#39;s Hospital, Transfusion Medicine</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">IVIg (Gammagard liquid)</td>
			<td style="width:209px;">Baxter Healthcare Corporation</td>
			<td>Received from BC Children&#39;s Hospital, Transfusion Medicine</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">IVIg (Octagam)</td>
			<td style="width:209px;">Octapharma</td>
			<td>Received from BC Children&#39;s Hospital, Transfusion Medicine</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Phosphate buffered saline (PBS) (sterile), pH 7.4</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">mouse IL-10 ELISA</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">555252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">mouse IL-12/23p40 ELISA</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">555165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">anti-Erk1/2 antibody</td>
			<td style="width:209px;">Cell signalling technology</td>
			<td style="width:408px;">9101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">anti-pp38 antibody</td>
			<td style="width:209px;">Cell signalling technology</td>
			<td style="width:408px;">9211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">anti-GAPDH antibody</td>
			<td style="width:209px;">Fitzgerald industries</td>
			<td style="width:408px;">10R-G109A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">26 g needle</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">305110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">1 mL syringe</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">10 mL syringe</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">15 mL conical tube</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">352096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">50 mL conical tube</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">microcentrifuge tube (1.7 mL)</td>
			<td style="width:209px;">Diamed</td>
			<td style="width:408px;">SPE155-N</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:453px;">75 cm2 tissue culture treated flask</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">353136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Cell scraper</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">353085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Forcepts</td>
			<td style="width:209px;">VWR</td>
			<td>82027-386</td>
			<td>fine tip, dissecting&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:453px;">Scissors</td>
			<td style="width:209px;">VWR</td>
			<td>82027-582</td>
			<td>Delicate, 4 1/2&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Brightfield microscope</td>
			<td style="width:209px;">Motic</td>
			<td style="width:408px;">AE31</td>
			<td>Inverted phase contrast</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Scale&#160;</td>
			<td style="width:209px;">Mettler&#160;</td>
			<td style="width:408px;">PE 3000</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of antibody-based drugs effects on murine bone marrow and peritoneal macrophage activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Murine bone marrow derived macrophages can be cultured from hematopoietic cell precursors in bone marrow aspirates. Bone marrow aspirates pooled from femurs and tibias of one C57BL/6 mouse typically yield 107 bone marrow derived macrophages, making them a convenient source of macrophages for experiments. BMDMs can be used to test antibody based drug responses when challenged with an inflammatory stimulus in vitro. Figure 1 shows that IVIg ...

Watch this Scientific Journal Video about Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation at JoVE.com

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation (Video) | JoVE

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Research

JoVE Journal

Immunology and Infection

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Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

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Bone Marrow-derived Macrophage Production

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Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

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Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

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