Name: assessment of antibody-based drugs effects on murine bone marrow and peritoneal macrophage activation
Uploaded: 2017-12-26T15:00:00.000+00:00
Duration: 10 min 35 s
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/min of CO2 until mouse has stopped breathing. Leave mouse with CO2 on for at least another 5 min. Verify that there is no longer a heartbeat or respiration. Perform a cervical dislocation to ensure death. 
		NOTE: 8 - 12 weeks old mice provide the highest yield of macrophages.</li>
	</ol>
	</li>
	<li>Spray mouse legs with 70% ethanol, and pin them with arms and legs stretched out in the supine position over a foam board. With scissors and forceps to hold the skin, make a shallow cut (0.2 cm) to remove the skin and fur from the surface of each of the hind legs. 
	NOTE: Perform this procedure and all tissue culture manipulations in a sterile hood.</li>
	<li>Trim muscle to make tibias and femurs visible. Remove the tibias and femurs, by cutting the bone and muscles just below the hip joint and above the ankles. Trim as much muscle off as possible.</li>
	<li>Spray bones with 70% ethanol and wait 1 min for it to evaporate, then place them in 6 well plate of bone flush medium (Iscove&#39;s modified Dulbecco&#39;s medium (IMDM), 10% fetal bovine serum (FBS)).</li>
	<li>Trim the ends of the bones by cutting 0.1 cm of bone off the ankle and top of the femur so that the bone cavity is exposed. Ensure that the marrow is visible and a 26 gauge needle can be placed in the bone cavity.</li>
	<li>Cut the bones and muscles to separate the tibias and femurs from the knee joints. Insert a 26 gauge needle attached to a 10 mL syringe into the cavity. Flush the bone marrow into a 50 mL conical tube with 5 mL of bone flush medium. Flush two tibias and two femurs, from one mouse, into each 50 mL tube.</li>
	<li>Pipette up and down several times or vortex the marrow at a slow speed to gently disperse clumps. Strings of marrow should be broken up into small (less than 0.5 mm) flecks of marrow. Fill the conical tube to 50 mL with bone flush medium. Pipette contents of the conical tube into a 75 cm2 tissue culture treated flask, and incubate at 37 &#176;C, 5% CO2 for 1 h. 
	NOTE: Mature macrophages and mesenchymal cells will become adherent to the flask and be discarded, while the desired hematopoietic progenitors remain in suspension.</li>
	<li>Pipette the medium with hematopoietic progenitors into a 50 mL conical tube. Centrifuge the tube at 300 x g at room temperature for 5 min. Discard the supernatant and resuspend the pellet in 5 mL of macrophage colony stimulating factor (MCSF) culture medium (IMDM, 10% FBS, 5 ng/mL MCSF, 100 U/mL penicillin/streptomycin, and 150 &#956;M monothioglycerol (MTG)).</li>
	<li>Count cells using a hemocytometer18. Add medium to resuspend cells to a concentration of 0.5 x 106 cells/mL, 1.5 x 107 cells/flask in 30 mL of medium, and pipette up and down gently. Pipette 30 mL of the suspension into a new 75 cm2 tissue culture treated flask.</li>
	<li>On day 4 and day 7 after initial culture in step 1.9, verify that the cells are adherent and slightly branched using an inverted phase contrast bright field microscope. Discard the medium containing the non-adherent cells. Wash the cells with IMDM one time and add 30 mL of MCSF culture medium.</li>
	<li>When the cells are mature by day 10 (&#62;95% positive for F4/80 and Mac-1), verify again that the cells are adherent and slightly.</li>
	<li>To plate cells for stimulations, remove the culture medium from the flask and add 8 mL of enzyme free, EDTA-based cell dissociation buffer (Table of Materials). Incubate the cells for 5 min at 37 &#176;C, 5% CO2.</li>
	<li>Scrape cells gently, with a small amount of pressure, using a sterile cell scraper. Check in the microscope that cells have detached from the flask surface. Pipette dissociated cells into a 50 mL conical tube. Rinse the flask 3 times with 5 mL of IMDM and pool the rinse solution with the cells that were harvested. Centrifuge cells at 300 x g at room temperature for 5 min.</li>
	<li>Resuspend the cell pellet in 3 mL of MCSF culture medium per 50 mL conical tube. Count viable cells using a hemocytometer18. Resuspend cells at a concentration of 1 x 106 cells/mL and plate 100 &#956;L of cell suspension (1 x 105 cells/well) per well of a 96-well tissue culture treated, flat bottom plate. 
	NOTE: Bones from one mouse will generate enough cells for 100 wells. If desired, 1 mL of cells can be plated in a 6-well plate (tissue culture treated, flat bottom). A larger number of cells (1 x 106 cells) may be useful for western blot analyses.</li>
	<li>Once adherent, stimulate duplicate or triplicate wells each with 10 ng/mL of LPS in IMDM, 30 mg/mL of IVIg, or IVIg + LPS. Doses of LPS or IVIg can be titrated to optimize responses. Leave duplicate or triplicate wells as unstimulated controls. Incubate them for 24 h (37 &#176;C, 5% CO2). 
	NOTE: Re-plated cells should adhere to tissue culture wells within 1 h.</li>
	<li>Collect cell supernatants from each well into individual 1.7 mL microcentrifuge tubes and remove any particulate matter by spinning at 10,000 x g for 5 min.</li>
	<li>Remove the cell supernatant and avoid disturbing the pellet. Place the clarified cell supernatant in a sterile microcentrifuge tube. 
	NOTE: Cell supernatants can be assayed for cytokines, IL-10, and cytokine subunit, IL-12/23p40, by enzyme-linked immunosorbent assay (ELISA) immediately, or stored at -80 &#176;C long term. Follow ELISA protocol from commercially available kit (Table of Materials). Other pro-inflammatory cytokines can be assayed, such as IL-6 and TNF, as they are also reduced by co-treatment with IVIg + LPS stimulation compared to stimulation with LPS alone. After stimulation, adherent cells can be prepared for techniques such as western blotting or quantitative polymerase chain reaction (Q-PCR) 19,20.</li>
</ol>
2. Challenging Mice In Vivo with IVIg and Harvesting Peritoneal Macrophages
<ol>
	<li>Weigh each mouse. Calculate the volume of IVIg (100 mg/mL stock concentration) needed to provide a final dose of 2.5 g/kg body weight for each mouse. 
	NOTE: For example, for a 20 g mouse, 500 &#181;L of IVIg is required.</li>
	<li>Draw the required amount of IVIg into a sterile 1 mL syringe fitted with a sterile 26 gauge needle. For control mice that will not receive IVIg, administer an equivalent dose volume of sterile phosphate buffered saline (PBS), pH 7.4 instead of IVIg.</li>
	<li>Scruff the mouse with one hand by grabbing its skin at the back of the neck with your thumb and index finger and holding its tail and hind legs with your remaining fingers. Tilt its body towards the ground.</li>
	<li>Place the needle at a 30 - 40&#176; angle relative to the mouse&#39;s abdomen, in the lower right quadrant, bevel up, and insert 1.5 cm of the needle. Inject the IVIg or PBS intraperitoneally. Wait 1 h.</li>
	<li>Euthanize mice with 5% isoflurane anesthesia followed by 6 - 8 L/min CO2 asphyxiation (see steps 1.1.1 - 1.1.2), or according to institution&#39;s ethical guidelines.</li>
	<li>Pin the mouse to a foam board, with limbs spread out. Spray the parts with 70% ethanol. 
	NOTE: Perform the procedure in a sterile hood.</li>
	<li>Make a shallow cut in the skin (0.2 cm) with scissors along the midline of the mouse, to avoid cutting the peritoneal cavity. Pull the abdominal skin off the mouse&#39;s belly using forceps, so that the lining of the intact peritoneal wall is visible.</li>
	<li>Fill a 5 mL syringe with 5 mL of sterile PBS (pH 7.4). Insert a 25 or 27 gauge needle at the top of the cavity from either the left or right side toward the center of the mouse with the bevel of the needle up. 
	NOTE: Place the needle carefully in the mouse so that you do not inject PBS into the organs, but rather, into the peritoneal space.</li>
	<li>Push the fluid into the peritoneal cavity. Pull the needle out of the cavity and massage the peritoneum for 10 s to dislodge any cells. Insert needle at the top of the cavity and avoid organs. Collect and place the lavage fluid recovered into a 15 mL conical tube on ice. 
	NOTE: For every 5 mL of fluid injected, approximately 3.75 mL will be recovered.</li>
	<li>Repeat injection and recovery (steps 2.8 - 2.9) 3 more times (total injection volume of 20 mL),to recover 15 mL of lavage fluid in total. Collect lavage from each mouse into a separate 15 mL conical tube. 
	NOTE: After the final injection, it may be easier to extract the fluid from the far left and right sides of the mouse, where fluid has accumulated. The organs will cause less interference with the needle at this position.</li>
	<li>Spin cells down at 300 x g for 5 min at 4 &#176;C. Resuspend the cells in 500 &#956;L of plating medium (IMDM, 10% FBS, and 100 U/mL penicillin/streptomycin) per mouse flushed. Count viable macrophages using a hemocytometer18. 
	NOTE: The macrophages will be slightly larger than the other peritoneal cells. Typical yield is 5 x 105 - 1 x 106 macrophages/mouse using a wild type C57BL/6 mouse. 
	Optional: If a large number of red blood cells are present in the cell pellet, perform a lysis step to remove them, using the 1x red blood cell lysis buffer according to manufacturer&#39;s instructions.</li>
	<li>Resuspend cells in plating medium at 1 x 106 macrophages/mL and plate 100 &#956;L per well in a 96-well flat bottom tissue culture treated plate. 
	NOTE: It may be necessary to pool cells from more than one mouse for an experiment if using triplicate wells or titrating stimulants. For example, if one mouse had 5 x 105 macrophages, 500 &#956;L of plating medium would be required. There would be enough cells for 5 wells, or 1 experiment in duplicate, with one LPS dose.</li>
	<li>Incubate cells for 1 h at 37 &#176;C, 5% CO2 to allow macrophages to become adherent. The adherent cells are the peritoneal macrophages. Remove cell supernatants and non-adherent cells. Rinse the wells twice with 200 &#956;L IMDM (pre-warmed to 37 &#176;C), wait 10 s and slowly tilt the plate. Replace the plating medium. Incubate the cells at 37 &#176;C, 5% CO2 for 30 min prior to stimulation.</li>
	<li>Stimulate the macrophages with 10 ng/mL of LPS in IMDM, or leave unstimulated, as a control. Incubate for 24 h, collect and clarify cell supernatants (see steps 1.16 - 1.17) for IL-10 cytokine analysis by ELISA. Follow ELISA protocol from commercially available kit (Table of Materials). 
	NOTE: After stimulation, adherent cells can be prepared for techniques such as western blot or PCR.</li>
</ol>
3. Challenging Mice in Vivo with IVIg + LPS and Harvesting Peritoneal Macrophages
<ol>
	<li>Weigh each mouse. Calculate the volume of IVIg (100 mg/mL stock concentration) needed to provide a final dose of 2.5 g/kg body weight. Calculate the amount of LPS (100 &#956;g/mL stock concentration in IMDM) needed to provide a final dose of 0.2 &#956;g/g body weight. 
	NOTE: For example, for a 20 g mouse, 500 &#181;L of IVIg stock solution and 40 &#956;L of a 100 &#956;g/mL solution of LPS is required.</li>
	<li>Draw the required amount of IVIg and LPS into a sterile 1 mL syringe fitted with a sterile 26 gauge needle. For control mice that will not receive IVIg, replace an equivalent volume of IVIg with sterile PBS pH 7.4.</li>
	<li>Scruff the mouse with one hand by grabbing its skin at the back of the neck with your thumb and index finger and holding its tail and hind legs with your remaining fingers. Tilt its body towards the ground.</li>
	<li>Place the needle at a 30 - 40&#176; angle relative to the mouse&#39;s abdomen, in the lower right quadrant, bevel up, and insert 1.5 cm. Inject the IVIg + LPS, or PBS + LPS, intraperitoneally as in step 2.4.</li>
	<li>After 1h, harvest peritoneal macrophages by performing steps 2.5 - 2.10.</li>
	<li>Spin cells down at 300 x g for 5 min at 4 &#176;C. Remove 1 mL of recovered lavage fluid and use for IL-10 and IL-12/23p40 analyses by ELISA according to manufacturer&#39;s instructions or freeze at -80 &#176;C for future analyses. 
	NOTE: To ensure accurate comparison between treatments for lavage fluid cytokine analysis, perform 5 mL flushs of the peritoneal cavity 4 times for a final volume of 15 mL. Not all fluid will be recovered from each flush.</li>
	<li>As in step 2.11, resuspend and count viable macrophages. 
	NOTE: The macrophages will be slightly larger than the other peritoneal cells. Typical yield is 5 x 105&#160;-&#160;1 x 106 macrophages/mouse using a wild type C57BL/6 mouse. 
	Optional: If a large number of red blood cells are present in the cell pellet, perform a lysis step according to manufacturer&#39;s instructions to remove them, as in step 2.11.</li>
	<li>As in step 2.12, re-suspend cells in plating medium. 
	NOTE: It may be necessary to pool cells from more than one mouse for an experiment. For example, if one mouse had 5 x 105 macrophages, 500 &#956;L of plating medium would be required.</li>
	<li>As in 2.13, incubate cells for 1 h at 37 &#176;C 5% CO2 to allow macrophages to become adherent.</li>
	<li>Collect and clarify conditioned medium, as per steps 1.14 - 1.15, from all peritoneal cells. 
	NOTE: Samples may be used immediately or frozen and stored at -80 &#176;C for cytokine analysis by ELISA.</li>
	<li>Perform step 2.11, but incubate cells for 24 h unstimulated. Collect and clarify cell supernatants, as per steps 1.16 - 1.17, for cytokine analysis by ELISA. 
	NOTE: Cells can be prepared for techniques such as western blot or PCR, as in steps 1.16 - 1.17 and 2.14.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+uses+of+intravenous+immunoglobulin.">Clinical uses of intravenous immunoglobulin.</a> Clin Exp Immunol. 142 (1), 1-11 (2005).</li><li>Murakami, 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+preparation+prevents+the+production+of+pro-inflammatory+cytokines+by+modulating+NF&#954;B+and+MAPKs+pathways+in+the+human+monocytic+THP-1+cells+stimulated+with+procalcitonin.">Intravenous immunoglobulin preparation prevents the production of pro-inflammatory cytokines by modulating NF&#954;B and MAPKs pathways in the human monocytic THP-1 cells stimulated with procalcitonin.</a> Inflamm Res. 63 (9), 711-718 (2014).</li></ol>

The authors have no conflicts of interest to disclose.

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><tbody><tr><td>Iscove&amp;rsquo;s modified Dulbecco&amp;rsquo;s medium (IMDM)</td><td>Life technologies</td><td>12440053</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life technologies</td><td>12483-020</td><td></td></tr><tr><td>Recombinant murine macrophage colony stimulating factor (MCSF)</td><td>Stemcell technologies</td><td>78059</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Life technologies</td><td>15140148</td><td></td></tr><tr><td>Monothioglycerol (MTG)</td><td>Sigma</td><td>88640</td><td></td></tr><tr><td>1X red blood cell lysis buffer</td><td>eBioscience</td><td></td><td></td></tr><tr><td>Cell dissociation buffer</td><td>Life technologies</td><td>13150016</td><td>Enzyme-Free, Hanks&#039;s-based, EDTA</td></tr><tr><td>Lipopolysaccharide (LPS)</td><td>Sigma aldrich</td><td>L 4516</td><td>From E. coli 0127:B8</td></tr><tr><td>IVIg (Gammunex)</td><td>Grifols</td><td>Received from BC Children&#039;s Hospital, Transfusion Medicine</td><td></td></tr><tr><td>IVIg (Gammagard liquid)</td><td>Baxter Healthcare Corporation</td><td>Received from BC Children&#039;s Hospital, Transfusion Medicine</td><td></td></tr><tr><td>IVIg (Octagam)</td><td>Octapharma</td><td>Received from BC Children&#039;s Hospital, Transfusion Medicine</td><td></td></tr><tr><td>Phosphate buffered saline (PBS) (sterile), pH 7.4</td><td>Life technologies</td><td>10010023</td><td></td></tr><tr><td>mouse IL-10 ELISA</td><td>BD biosciences</td><td>555252</td><td></td></tr><tr><td>mouse IL-12/23p40 ELISA</td><td>BD biosciences</td><td>555165</td><td></td></tr><tr><td>anti-Erk1/2 antibody</td><td>Cell signalling technology</td><td>9101</td><td></td></tr><tr><td>anti-pp38 antibody</td><td>Cell signalling technology</td><td>9211</td><td></td></tr><tr><td>anti-GAPDH antibody</td><td>Fitzgerald industries</td><td>10R-G109A</td><td></td></tr><tr><td>26 g needle</td><td>BD biosciences</td><td>305110</td><td></td></tr><tr><td>1 mL syringe</td><td>BD biosciences</td><td>309659</td><td></td></tr><tr><td>10 mL syringe</td><td>BD biosciences</td><td>309604</td><td></td></tr><tr><td>15 mL conical tube</td><td>BD biosciences</td><td>352096</td><td></td></tr><tr><td>50 mL conical tube</td><td>BD biosciences</td><td>352070</td><td></td></tr><tr><td>microcentrifuge tube (1.7 mL)</td><td>Diamed</td><td>SPE155-N</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2 &lt;/sup&gt;tissue culture treated flask</td><td>BD biosciences</td><td>353136</td><td></td></tr><tr><td>Cell scraper</td><td>BD biosciences</td><td>353085</td><td></td></tr><tr><td>Forcepts</td><td>VWR</td><td>82027-386</td><td>fine tip, dissecting&amp;nbsp;</td></tr><tr><td>Scissors</td><td>VWR</td><td>82027-582</td><td>Delicate, 4 &lt;sup&gt;1&lt;/sup&gt;/&lt;sub&gt;2&lt;/sub&gt;&quot;</td></tr><tr><td>Brightfield microscope</td><td>Motic</td><td>AE31</td><td>Inverted phase contrast</td></tr><tr><td>Scale&amp;nbsp;</td><td>Mettler&amp;nbsp;</td><td>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 ...

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

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.

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

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Immunology and Infection

8.2K Views.  University of British Columbia. 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.

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

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

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.

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 ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, and foreign pathogens. The phagocytic capacity of monocytes versus macrophages is a concept that is not well understood. Here, we aim to examine a difference in the phagocytosis of monocytes versus macrophages, specifically M1/M2 macrophages, against various opsonized red cells using a modified and updated version of the established monocyte monolayer assay (MMA). Peripheral blood mononuclear cells (PBMCs) were isolated from donor buffy coats. Using purified monocytes, inflammatory M1 and anti-inflammatory M2 macrophages were produced by in vitro culture and polarization. M1/M2 cells were harvested and used in an MMA-like assay, which we refer to as the M-MA, to decipher clinically significant phagocytosis of various red cell antibodies. A phagocytic index (PI) &#62; 5 was deemed clinically significant phagocytosis with the use of monocytes. A phagocytic index (PI) &#62; 12 was deemed clinically significant phagocytosis with the use of M1/M2 macrophages. M2 macrophages demonstrate an increased ability to phagocytose opsonized RBCs compared to monocytes and M1s. The same weak antibody (anti-S) yields significant phagocytosis with only M2 macrophages (PI=43) but not M1s (PI=2) or monocytes (PI=0), and this was demonstrated repeatedly using various antibodies. The use of M2 macrophages instead of monocytes may allow for more accurate results as these cells are more phagocytic, offering further clinical relevance to the assay. Further studies with different antibodies to red blood cells, including validation of the monocyte-macrophage assay (M-MA) with antibodies having known clinical significance, may show the M-MA&#160;more useful to help predict clinically significant red cell alloantibodies and transfusion reactions. This method will advance the field of transfusion medicine and immunology.

We have developed enhancements and updated methods for the existing monocyte-monolayer assay (MMA), in which macrophages are used to help better predict the clinical relevance of red cell alloantibodies in transfusion medicine and immunology. This assay is named the monocyte-macrophage assay (M-MA).

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, ...

Activation of Mouse Bone Marrow-Derived Macrophages in Response to Antibodies

Source: Kozicky, L. et al., <a href="https://app.jove.com/v/56689">Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the activation of bone marrow-derived macrophages in response to bacterial lipopolysaccharide and intravenous immunoglobulin.

Source: Kozicky, L. et al., Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation. J. Vis. Exp. (2017)
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Host Defense Mechanism

In Vivo Activation of Peritoneal Macrophages in Response to Antibodies in a Murine Model

Source: Kozicky, L., et al., <a href="https://app.jove.com/v/56689">Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the activation of a mouse&#39;s peritoneal macrophages using intravenous immunoglobulin and bacterial lipopolysaccharide. The co-stimulation activates the peritoneal macrophages into anti-inflammatory macrophages possessing immune regulatory activity.

Source: Kozicky, L., et al., Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation.&#160;J. Vis. Exp. ...