Name: macrophage differentiation and polarization into an m2-like phenotype using a human monocyte-like thp-1 leukemia cell line
Uploaded: 2021-08-02T13:00:00
Description: Watch this Scientific Journal Video about Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line at JoVE.com

The Price Institute of Surgical Research, University of Louisville, is financially supported by the John W. Price and Barbara Thruston Atwood Price Trust. The funding sources had no role in the design and conduct of the study as well as in the collection, management, analysis, and interpretation of the data.

NOTE: An overview of the steps described in this protocol is shown in Figure 1. The human monocyte-like leukemia cell line THP-1 was purchased. Short tandem repeat analysis was performed to authenticate the THP-1 cell line. Perform all steps under sterile conditions. The THP-1 monocytic cell line grows in suspension and does not attach to cell culture surfaces. Adherence can be induced by differentiating monocytes into macrophage-like cells through, e.g., mechanical stress or specific treatm...

<ol>
	<li>Zhang, R., 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=Cancer-associated+fibroblasts+enhance+tumor-associated+macrophages+enrichment+and+suppress+NK+cells+function+in+colorectal+cancer.">Cancer-associated fibroblasts enhance tumor-associated macrophages enrichment and suppress NK cells function in colorectal cancer.</a> Cell Death and Disease. 10 (4), 273 (2019).</li><li>Wang, J., Li, D., Cang, H., Guo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosstalk+between+cancer+and+immune+cells:+Role+of+tumor-associated+macrophages+in+the+tumor+microenvironment.">Crosstalk between cancer and immune cells: Role of tumor-associated macrophages in the tumor microenvironment.</a> Cancer Medicine. 8 (10), 4709-4721 (2019).</li><li>Soncin, 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=The+tumor+microenvironment+creates+a+niche+for+the+self-renewal+of+tumor-promoting+macrophages+in+colon+adenoma.">The tumor microenvironment creates a niche for the self-renewal of tumor-promoting macrophages in colon adenoma.</a> Nature Communications. 9 (1), 582 (2018).</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> Nature Reviews Immunology. 8 (12), 958-969 (2008).</li><li>Mazzone, M., Menga, A., Castegna, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+and+TAM+functions-it+takes+two+to+tango.">Metabolism and TAM functions-it takes two to tango.</a> Federation of European Biochemical Societies Journal. 285 (4), 700-716 (2018).</li><li>Eum, H. 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=Tumor-promoting+macrophages+prevail+in+malignant+ascites+of+advanced+gastric+cancer.">Tumor-promoting macrophages prevail in malignant ascites of advanced gastric cancer.</a> Experimental and Molecular Medicine. 52 (12), 1976-1988 (2020).</li><li>Qian, B. Z., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+diversity+enhances+tumor+progression+and+metastasis.">Macrophage diversity enhances tumor progression and metastasis.</a> Cell. 141 (1), 39-51 (2010).</li><li>Scheurlen, K. M., Billeter, A. T., O'Brien, S. J., Galandiuk, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+dysfunction+and+early-onset+colorectal+cancer+-+how+macrophages+build+the+bridge.">Metabolic dysfunction and early-onset colorectal cancer - how macrophages build the bridge.</a> Cancer Medicine. 9 (18), 6679-6693 (2020).</li><li>Bosshart, H., Heinzelmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=THP-1+cells+as+a+model+for+human+monocytes.">THP-1 cells as a model for human monocytes.</a> Annals of Translational Medicine. 4 (21), 438 (2016).</li><li>. The American Type Culture Collection (ATCC) Available from: <a target="_blank" href="https://www.atcc.org/products/all/TIB-202.aspx#generalinformation">https://www.atcc.org/products/all/TIB-202.aspx#generalinformation</a> (2021)</li><li>Baxter, E. 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=Standardized+protocols+for+differentiation+of+THP-1+cells+to+macrophages+with+distinct+M(IFNgamma+LPS),+M(IL-4),+and+M(IL-10)+phenotypes.">Standardized protocols for differentiation of THP-1 cells to macrophages with distinct M(IFNgamma+LPS), M(IL-4), and M(IL-10) phenotypes.</a> Journal of Immunological Methods. 478, 112721 (2020).</li><li>Genin, M., Clement, F., Fattaccioli, A., Raes, M., Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M1+and+M2+macrophages+derived+from+THP-1+cells+differentially+modulate+the+response+of+cancer+cells+to+etoposide.">M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide.</a> BMC Cancer. 15, 577 (2015).</li><li>Starr, T., Bauler, T. J., Malik-Kale, P., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phorbol+12-myristate-13-acetate+differentiation+protocol+is+critical+to+the+interaction+of+THP-1+macrophages+with+Salmonella+Typhimurium.">The phorbol 12-myristate-13-acetate differentiation protocol is critical to the interaction of THP-1 macrophages with Salmonella Typhimurium.</a> PLoS One. 13 (3), 0193601 (2018).</li><li>Lund, M. E., To, J., O'Brien, B. A., Donnelly, 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+choice+of+phorbol+12-myristate+13-acetate+differentiation+protocol+influences+the+response+of+THP-1+macrophages+to+a+pro-inflammatory+stimulus.">The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus.</a> Journal of Immunological Methods. 430, 64-70 (2016).</li><li>Yin, Z., 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=IL-6/STAT3+pathway+intermediates+M1/M2+macrophage+polarization+during+the+development+of+hepatocellular+carcinoma.">IL-6/STAT3 pathway intermediates M1/M2 macrophage polarization during the development of hepatocellular carcinoma.</a> Journal of Cellular Biochemistry. 119 (11), 9419-9432 (2018).</li><li>O'Neill, L. A. J., Artyomov, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Itaconate:+the+poster+child+of+metabolic+reprogramming+in+macrophage+function.">Itaconate: the poster child of metabolic reprogramming in macrophage function.</a> Nature Reviews Immunology. 19 (5), 273-281 (2019).</li><li>Luig, M., 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=Inflammation-induced+IL-6+functions+as+a+natural+brake+on+macrophages+and+limits+gn.">Inflammation-induced IL-6 functions as a natural brake on macrophages and limits gn.</a> Journal of the American Society of Nephrology. 26 (7), 1597-1607 (2015).</li><li>Maruyama, K., Nemoto, E., Yamada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+regulation+of+macrophage+function+-+cyclic+tensile+force+inhibits+NLRP3+inflammasome-dependent+IL-1beta+secretion+in+murine+macrophages.">Mechanical regulation of macrophage function - cyclic tensile force inhibits NLRP3 inflammasome-dependent IL-1beta secretion in murine macrophages.</a> Inflammation and Regeneration. 39, 3 (2019).</li><li>Gatto, F., 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=PMA-Induced+THP-1+Macrophage+Differentiation+is+Not+Impaired+by+Citrate-Coated+Platinum+Nanoparticles.">PMA-Induced THP-1 Macrophage Differentiation is Not Impaired by Citrate-Coated Platinum Nanoparticles.</a> Nanomaterials (Basel). 7 (10), (2017).</li><li>Maess, M. B., Wittig, B., Cignarella, A., Lorkowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+PMA+enhances+the+responsiveness+of+transfected+THP-1+macrophages+to+polarizing+stimuli.">Reduced PMA enhances the responsiveness of transfected THP-1 macrophages to polarizing stimuli.</a> Journal of Immunological Methods. 402 (1-2), 76-81 (2014).</li><li>Daigneault, M., Preston, J. A., Marriott, H. M., Whyte, M. K., Dockrell, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+of+markers+of+macrophage+differentiation+in+PMA-stimulated+THP-1+cells+and+monocyte-derived+macrophages.">The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages.</a> PLoS One. 5 (1), 8668 (2010).</li><li>Raggi, F., 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=Regulation+of+human+macrophage+M1-M2+polarization+balance+by+hypoxia+and+the+triggering+receptor+expressed+on+myeloid+cells-1.">Regulation of human macrophage M1-M2 polarization balance by hypoxia and the triggering receptor expressed on myeloid cells-1.</a> Frontiers in Immunology. 8, 11097 (2017).</li><li>Neutelings, T., Lambert, C. A., Nusgens, B. V., Colige, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mild+cold+shock+(25+degrees+C)+followed+by+warming+up+at+37+degrees+C+on+the+cellular+stress+response.">Effects of mild cold shock (25 degrees C) followed by warming up at 37 degrees C on the cellular stress response.</a> PLoS One. 8 (7), 69687 (2013).</li><li>Kurashina, Y., 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=Enzyme-free+release+of+adhered+cells+from+standard+culture+dishes+using+intermittent+ultrasonic+traveling+waves.">Enzyme-free release of adhered cells from standard culture dishes using intermittent ultrasonic traveling waves.</a> Communications Biology. 2, 393 (2019).</li><li>Chen, S., So, E. C., Strome, S. E., 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=Impact+of+Detachment+Methods+on+M2+Macrophage+Phenotype+and+Function.">Impact of Detachment Methods on M2 Macrophage Phenotype and Function.</a> Journal of Immunological Methods. 426, 56-61 (2015).</li><li>Bailey, J. D., 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=Isolation+and+culture+of+murine+bone+marrow-derived+macrophages+for+nitric+oxide+and+redox+biology.">Isolation and culture of murine bone marrow-derived macrophages for nitric oxide and redox biology.</a> Nitric Oxide. 100-101, 17-29 (2020).</li></ol>

This protocol on differentiating and polarizing THP-1 monocyte-like cells within 14 days provides a method to obtain macrophages with a distinct M2-like phenotype due to long treatment incubation of cells with adequate resting periods between steps.
Certain steps are critical to this protocol. The doubling time of THP-1 monocytes is approximately 26 h. Cells can be split at a cell density of 9 x 105/mL and should be seeded at a density of 3 x 105/mL during every split. Th...

Tumor-associated macrophages (TAM) and their role in chronic inflammation, the onset of cancer, and tumor development are important targets in recent research1,2. Peripheral blood monocytes that are recruited to the tissue microenvironment of the developing tumor differentiate into macrophages and can be polarized into two main subtypes of macrophages3. The classically activated macrophage represents the primarily pro-inflammatory M1-like phenotype and the alternatively activated M2-like subtype shows predominantly anti-inflammatory characteristics4. Macrophages can switch dynamically between these two main phenotypes depending upon their cellular metabolism, with intermediate subtypes having both inflammatory and anti-inflammatory attributes5. TAM represents a heterogeneous population of both phenotypes. A tumor-promoting function and poor prognosis in different types of cancers is, however, particularly associated with M2-like macrophages6,7,8.
The functional profiles of macrophages and their interaction with other cells within the tumor microenvironment are complex and challenging to capture in a continuously changing environment during ongoing tumor development. Cell lines can provide a homogenous cell population with stable viability in culture, which can facilitate the process of demonstrating defined cellular and intercellular mechanisms. The monocyte-like THP-1 cell line is a legitimate model system for primary human monocytes9. This spontaneously immortalized cell line has been obtained from the peripheral blood of a one-year-old infant with acute monocytic leukemia9,10. The differentiation and polarization of THP-1 cells have been reported by several studies and have been performed in multiple different ways11,12,13,14. Activation and, therefore, the polarization of macrophages into an M1-like phenotype is followed by a compensatory anti-inflammatory rebound mechanism, promoting an M2-like phenotype through cytokines produced by inflammatory macrophages, such as interleukin 6 (IL-6) or itaconate15,16. This might serve as a break mechanism to attenuate an overshooting inflammatory response following cell activation17. The process of differentiating and polarizing monocytes and THP-1 monocyte-like cells into an anti-inflammatory M2-like phenotype is itself also accompanied by pro-inflammatory stimuli that must be overcome. An inflammatory cytokine response can be caused by mechanical stress18, such as changing media to refeed the cells, or adding chemical compounds to differentiate THP-1 cells, such as phorbol&#160;12-myristate 13-acetate (PMA), and induce production of tumor necrosis factor &#945; (TNF&#945;), interleukin 1&#946; (IL-1&#946;) or IL-619. This altered cytokine expression profile as a response to PMA can affect and prevent subsequent macrophage polarization20. Adequate resting periods, as reported before after PMA treatment, allow these inflammatory responses to decrease and facilitate cell polarization into a distinct M2-like phenotype21.
This protocol demonstrates a method to differentiate and polarize THP-1 monocyte-like cells into an M2-like phenotype of macrophages within 14 days.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4% trypan blue</td>
			<td>VWR, Radnor, USA</td>
			<td>152-5061</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>USA Scientific, Ocala, USA</td>
			<td>1615-5510</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipet</td>
			<td>VWR, Radnor, USA</td>
			<td>&#160;89130-898</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L TipOne pipet tips</td>
			<td>USA Scientific, Ocala, USA</td>
			<td>1111-2821</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL&#160; Centrifuge tube</td>
			<td>VWR, Radnor, USA</td>
			<td>89039-664</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;L TipOne pipet tips</td>
			<td>USA Scientific, Ocala, USA</td>
			<td>1120-1810</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L TipOne pipet tips</td>
			<td>USA Scientific, Ocala, USA</td>
			<td>1120-8810</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL serological pipet</td>
			<td>VWR, Radnor, USA</td>
			<td>&#160;89130-900</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL serological pipet</td>
			<td>VWR, Radnor, USA</td>
			<td>&#160;89130-896</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge tube</td>
			<td>VWR, Radnor, USA</td>
			<td>89039-662</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase solution 500 mL</td>
			<td>Sigma, St. Louis, USA</td>
			<td>A6964</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic Antimycotic Solution (100x), stabilized</td>
			<td>Sigma, St. Louis, USA</td>
			<td>A5955-100 mL</td>
			<td>with 10,000 units penicillin, 10 mg of streptomycin and 25 &#956;g of amphotericin B per mL, sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Binder CO2 Incubator</td>
			<td>VWR, Radnor, USA</td>
			<td>C170-ULE3</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoOne T-75cm flask with filter cap</td>
			<td>USA Scientific, Ocala, USA</td>
			<td>CC7682-4875</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (PBS)</td>
			<td>Sigma, St. Louis, USA</td>
			<td>D8537-500 mL</td>
			<td>PBS without calcium chloride and magnesium chloride should be used, since both can alter macrophage polarization</td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge 5804 R (refrigerated)</td>
			<td>Eppendorf, Enfield, USA</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol (70%)</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSCalibur flow cytometer</td>
			<td>BD Biosciences, San Diego, USA</td>
			<td>-</td>
			<td>The flow cytometer operates with CellQuest software (BD Biosciences)</td>
		</tr>
		<tr>
			<td>Falcon 24-well plate</td>
			<td>VWR, Radnor, USA</td>
			<td>353504</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ATCC, Manassas, USA</td>
			<td>30-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse Anti-Human CD14</td>
			<td>BD Biosciences, San Diego, USA</td>
			<td>555397</td>
			<td>Flow cytometry, myeloid cell marker (100 tests)</td>
		</tr>
		<tr>
			<td>FITC Mouse Anti-Human CD80</td>
			<td>BD Pharmingen, San Diego, USA</td>
			<td>557226</td>
			<td>Flow cytometry, M1 marker (100 tests)</td>
		</tr>
		<tr>
			<td>FITC Mouse IgG1 &#954; Isotype Control</td>
			<td>BD Pharmingen, San Diego, USA</td>
			<td>555748</td>
			<td>Flow cytometry, isotype control for CD80 (100 tests)</td>
		</tr>
		<tr>
			<td>FITC Mouse IgG2a, &#954; Isotype Control</td>
			<td>BD Biosciences, San Diego, USA</td>
			<td>553456</td>
			<td>Flow cytometry, isotype control for CD14 (100 tests)</td>
		</tr>
		<tr>
			<td>Human BD Fc Block</td>
			<td>BD Biosciences, San Diego, USA</td>
			<td>564220</td>
			<td>Flow cytometry, Fc block (0.25 mg)</td>
		</tr>
		<tr>
			<td>Human interleukin 13 (IL-13)</td>
			<td>R&#38;D, Minneapolis, USA</td>
			<td>IL-771-10 &#956;g</td>
			<td></td>
		</tr>
		<tr>
			<td>Human interleukin 4 (IL-4)</td>
			<td>R&#38;D, Minneapolis, USA</td>
			<td>SRP3093-20 &#956;g</td>
			<td></td>
		</tr>
		<tr>
			<td>Labconco Biosafety Cabinet (Delta Series 36212/36213)</td>
			<td>Labconco, Kansas City, USA</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine Solution, 200 mM</td>
			<td>ATCC, Manassas, USA</td>
			<td>30-2214</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharide (LPS) from E. coli 0111:B4</td>
			<td>Sigma, St. Louis, USA</td>
			<td>L2630-100 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Cell Scrapers</td>
			<td>Biotium, Fremont, USA</td>
			<td>22003</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer hemocytometer</td>
			<td>Fisher Scientific, Waltham, USA</td>
			<td>02-671-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse inverted microscope TS100</td>
			<td>Nikon, Melville, USA</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Invitrogen, Carlsbad, USA</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Light Microscope RH-2</td>
			<td>Microscope Central, Feasterville, USA</td>
			<td>40888</td>
			<td></td>
		</tr>
		<tr>
			<td>P10 variable pipet- Gilson</td>
			<td>VWR, Radnor, USA</td>
			<td>76180-014</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 variable pipet-Gilson</td>
			<td>VWR, Radnor, USA</td>
			<td>76177-990</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 variable pipet- Gilson</td>
			<td>VWR, Radnor, USA</td>
			<td>76177-988</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse Anti-Human CD11b</td>
			<td>BD Biosciences, San Diego, USA</td>
			<td>555388</td>
			<td>Flow cytometry, myeloid cell marker (100 tests)</td>
		</tr>
		<tr>
			<td>PE Mouse IgG1, &#954; Isotype Control</td>
			<td>BD Biosciences, San Diego, USA</td>
			<td>555749</td>
			<td>Flow cytometry, isotype control for CD11b (100 tests)</td>
		</tr>
		<tr>
			<td>PE-Cy 5 Mouse Anti-Human CD206</td>
			<td>BD Pharmingen, San Diego, USA</td>
			<td>551136</td>
			<td>Flow cytometry, M2 marker (100 tests)</td>
		</tr>
		<tr>
			<td>PE-Cy 5 Mouse IgG1 &#954; Isotype Control</td>
			<td>BD Pharmingen, San Diego, USA</td>
			<td>555750</td>
			<td>Flow cytometry, isotype control for CD206 (100 tests)</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma, St. Louis, USA</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>Powerpette Plus pipettor</td>
			<td>VWR, Radnor, USA</td>
			<td>75856-448</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Water bath (model 183)</td>
			<td>Precision Scientific, Chicago, USA</td>
			<td>66551</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium</td>
			<td>ATCC, Manassas, USA</td>
			<td>30-2001</td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1 cell line, American Type Culture Collection (ATCC)</td>
			<td>ATCC, Manassas, USA</td>
			<td>TIB-202</td>
			<td></td>
		</tr>
	</tbody>
</table>

macrophage differentiation and polarization into an m2-like phenotype using a human monocyte-like thp-1 leukemia cell line

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM to adapt to ongoing changes within the tumor microenvironment. Macrophages can have either primarily pro-inflammatory (M1-like) or anti-inflammatory (M2-like) attributes and can continually switch between these two main states. M2-like macrophages within the tumor environment are associated with cancer progression and poor prognosis in several types of cancer. Many different methods for inducing differentiation and polarization of THP-1 cells are used to investigate cellular and intercellular mechanisms and the effects of TAM within the microenvironment of tumors. Currently, there is no established model for M2-like macrophage polarization using the THP-1 cell line, and the results of expression and cytokine profiles of macrophages due to certain in vitro stimuli vary between studies. This protocol serves as detailed guidance to differentiate THP-1 monocyte-like cells into M0 macrophages and to further polarize cells into an M2-like phenotype within 14 days. We demonstrate the morphological changes of THP-1 monocyte-like cells, differentiated macrophages, and polarized M2-like macrophages using light microscopy. This model is the basis for cell line models investigating the anti-inflammatory effects of TAM and their interactions with other cell populations of the tumor microenvironment.

M2-like macrophages were characterized, and M2-polarization was validated using flow cytometry for Cluster of Differentiation markers (CD) CD14, CD11b, CD80 (M1-like marker), and CD206 (M2-like marker). Flow cytometry staining was performed according to the manufacturer&#39;s instructions. Macrophages were washed with PBS/5% FBS and incubated with Fc&#947;-receptor block to avoid unspecific binding. Cells were then stained with FITC-conjugated mouse anti-human CD14 and CD80 antibodies, with PE-conjugated mouse anti-human...

Watch this Scientific Journal Video about Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line at JoVE.com

Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line

M2-like tumor-associated macrophages (TAM) are associated with tumor progression and poor prognosis in cancer. This protocol serves as a detailed guide to reproducibly differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. This model is the basis to investigate the anti-inflammatory effects of TAM within the tumor microenvironment.

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM ...

Anti-inflammatory tumor associated macrophages are linked to progression and poor prognosis in cancer. This protocol is a guide to differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. Currently there is no established model for anti-inflammatory THP-1 polarization.This protocol uses adequate resting periods and a prolonged polarization process to create a distinct M2-like macrophage subtype. Tumor development, for example, in colon cancer and tissue remodeling, are controlled by anti-inflammatory macrophages. Among studies investigating their function, standardized creation of a distinct M2-like subtype ensures reproducible findings.Begin by setting a timer for 150 seconds. Remove the frozen vial containing the THP-1 cell line from the liquid nitrogen and thaw the vial immediately at 37 degrees Celsius in a clean water bath, starting the timer as soon as the vial is put into the water bath. Loosen the cap to release the pressure built up due to the thawing process, ensuring that the tube opening does not contact the water to avoid contamination.Thaw the cell suspension until an ice chip about four millimeters in size is left within the vial and proceed to the next step immediately. Transfer the liquid phase of the cell suspension to a 15 milliliter tube containing nine milliliters of warm growth media. Then transfer one milliliter of this warm medium cell suspension into the THP-1 vial and back to the 15 milliliter tube to melt the remaining ice chip, and flush the vial to ensure no cells are left behind.Mix the suspension gently by pipetting it up and down with a one milliliter pipette. Remove approximately 10 microliters of the sample to count the cells for viability using trypan blue. The cells with clear cytoplasm are viable, a blue cytoplasm indicates cell death.While counting, spin down the warm cell suspension at 200 times G for seven minutes at 37 degrees Celsius. Remove the supernatant completely and resuspend the cells in growth medium to achieve a cell density of 500, 000 cells per milliliter. Mix the suspension gently and transfer 22 milliliters into T75 cell culture flasks each.Store the flasks upright in an incubator at 37 degrees Celsius with a 5%carbon dioxide concentration and exchange the growth media every three to four days. Prepare the cell suspension to seed the cells at a density of 300, 000 per milliliter per well into 24-well cell culture plates. Mix the medium gently and a prepare aliquots of 26 milliliters in 50 milliliter tubes.Transfer one milliliter of the cell-containing medium into each well of a 24-well plate. Mix the media gently by pipetting it up and down between transfers. Keep the freshly prepared working solution of PMA on ice and add 100 nanograms of PMA per well.Incubate the plates in the incubator without any further treatment for 72 hours. After 72 hours, remove the growth medium and replace it with one milliliter of fresh growth medium, ensuring not to touch the bottom of the wells with pipette tips. Let the cells rest for another 96 hours in the incubator.Remove the growth medium and replace it with one milliliter of fresh growth medium. Add 20 nanograms of interleukin 4 and 20 nanograms of interleukin 13 per well. Then let the cells rest for 48 hours in the incubator.Repeat the whole process after 48 hours and let the cells rest for another 48 hours in the incubator. Remove the growth medium and replace it with one milliliter of fresh growth medium. Then let the cells rest for 48 hours in the incubator.Remove the warm cell medium from each well and replace it with a one milliliter mixture of ice cold PBS without calcium and magnesium and 5%FBS. Then immediately place the cell plate on ice for 45 minutes. After 45 minutes on ice, scrape off the cells using mini cell scrapers.Gently transfer the detached macrophages to cold PBS and 5%FBS in a 15 milliliter tube kept on ice. The macrophages derived from the polarization method show the expression of CD14, as well as CD11b markers. The monocyte and macrophage markers CD14 and CD11b were expressed in 70.9%and 74.7%of cells, respectively.Cells showed low surface levels of the M1-like marker CD80 in 0.2%of cells, and high surface levels of the M2-like marker CD206 in 62.6%of cells. As THP-1 cells in medium tend to sink to the bottom of a vial and are easy to activate through mechanical stress, gentle mixing and handling of the cells is important. M2-like macrophages are reproducibly created, and have been characterized using QR-TPCR, ELISA, and flow cytometry.This is the basis for investigating anti-inflammatory mechanisms in cancer development or tissue remodeling.

macrophage-differentiation-polarization-into-an-m2-like-phenotype

Research

JoVE Journal

Immunology and Infection

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

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 ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis and occlusive symptoms of headaches, stroke or vision loss. Macrophages and T-helper lymphocytes infiltrate the vascular wall and produce a pro-inflammatory response that lead to vessel damage and ischemia. To date, there is no GCA biomarker that can monitor disease activity and guide therapeutic response.
Folate receptor beta (FRB) is a glycosylphosphatidylinositol protein that is anchored on cell membranes and normally expressed in the myelomonocytic lineage and in the majority of myeloid leukemia cells as well as in tumor and rheumatoid synovial macrophages, where its expression correlates with disease severity. The ability of FRB to bind folate compounds, folic acid-conjugates and antifolate drugs has made it a druggable target in cancer and inflammatory disease research. This report describes the histopathologic and immunohistochemical methods used to assess expression and distribution of FRB in relation to GCAimmunopathology.
Formalin-fixed and paraffin-embedded temporal artery biopsies from GCA and normal controls were stained with Hematoxylin and Eosin to review tissue histology and identify pathognomonic features.Immunohistochemistry was used to detect FRB, CD68 and CD3 expression. A microscopic analysis was performed to quantify the number of positively stained cells on 10 selected high-power-field sections and their respective locations in the arterial wall.
Lymphohistiocytic (LH) inflammation accompanied by intimal hyperplasia and disrupted elastic lamina was seen in GCA with none found in controls. The LH infiltrate was composed of approximately 60% lymphocytes and 40% macrophages. FRB expression was restricted to macrophages, comprising 31% of the total CD68+ macrophage population and localized to the media and adventitia. No FRB was seen in controls.
This protocol demonstrated a distinct numerical and spatial pattern of the FRB macrophage relative to the vascular immune microenvironment in GCA.

The protocol illustrates the use of histopathologic examination and immunohistochemistry to profile the folate receptor beta macrophage and its relationship with the total immune cell infiltrate in temporal artery biopsies in giant cell arteritis.

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis ...