Characterization of Human Blood Monocytes and Intestinal Macrophages

Evida A. Dennis1, Tanya O. Robinson2, Lesley E. Smythies1, Phillip D. Smith3

1 Department of Medicine (Gastroenterology), VA Medical Center, Birmingham, Alabama, 2 Department of Pediatrics (Rheumatology), VA Medical Center, Birmingham, Alabama, 3 VA Medical Center, Birmingham, Alabama
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.3
DOI:  10.1002/cpim.30
Online Posting Date:  August, 2017
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Abstract

Monocytes and macrophages play fundamental roles in defense against microbes, clearance of senescent and dead cells, and immunoregulation. Although blood monocytes are the source of intestinal macrophages in the developed mucosal immune system, blood monocytes and intestinal macrophages from healthy human subjects display distinct phenotypic and functional differences. Blood monocytes can be induced to polarize into M1 and M2 macrophages, whereas intestinal macrophages appear to be terminally differentiated and are unable to undergo such inducible polarization. Nevertheless, in response to local conditions, monocytes differentiated into intestinal macrophages display phenotypic and functional characteristics that enhance their capacity to provide non‐inflammatory host defense and participate in local immunoregulation. Using the protocols described here, this unit presents the key phenotypic and functional differences between human blood monocytes and intestinal macrophages, as well as between mouse and human intestinal macrophages. © 2017 by John Wiley & Sons, Inc.

Keywords: monocytes; intestinal macrophages; phenotype; function; M1 and M2 polarization

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Strategy for Phenotyping Human Blood Monocytes and Intestinal Macrophages
  • Basic Protocol 2: Strategy for Phenotyping Human Blood Monocytes and Intestinal Macrophages Exposed to Polarization Stimuli
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Strategy for Phenotyping Human Blood Monocytes and Intestinal Macrophages

  Materials
  • PBMCs fractionated by density gradient sedimentation and the resultant CD14+ blood monocytes purified from PBMCs by CD14+ magnetic beads (unit 14.36; Steinhäuser et al., )
  • Intestinal MNLs isolated from resected segments of normal jejunum or ileum by enzyme digestion and gradient sedimentation (unit 7.1; Fuss, Kanof, Smith, & Zola, )
  • Elutriated intestinal macrophages purified from the intestinal MNLs by counterflow centrifugal elutriation (unit 7.6; Smythies, Wahl, & Smith, )
  • RPMI Complete culture medium: RPMI 1640 medium supplemented with 1% penicillin/streptomycin, 1% L‐glutamine and 10% heat‐inactivated human AB serum (all from Mediatech)
  • BD Pharmingen Stain Buffer (BD Biosciences)
  • Live/dead fixable dead cell stain Kit (e.g., Live/Dead Stain from Invitrogen)
  • Phosphate‐buffered saline (PBS; Corning Life Sciences)
  • 70% ethanol
  • Cell surface marker antibodies: the following antibodies were used to characterize blood MNLs, blood monocytes, intestinal MNLs, and intestinal macrophages for the studies shown in Figures , , , and HLA‐DR‐PerCP, CD11b‐APC, CD11c‐V450, CD13‐PE, CD14‐V450 (eBioScience), CD14‐APC, CD14‐FITC, CD16‐FITC, CD32‐FITC, CD33‐APC, CD36‐FITC, CD45‐APC‐Cy7, CD64‐FITC, CD80‐Brilliant Violet 605, CD86‐Brilliant Violet 605, CD116‐FITC, CD163‐PerCP‐Cy5.5, CD209‐APC and CX3CR1‐FITC, as well as appropriate isotype antibody controls (all BD Biosciences, unless otherwise indicated)
  • Isotype control antibodies
  • Cytofix (BD Biosciences)
  • 12 × 75–mm polystyrene tubes for FACS analysis (e.g., Corning Falcon)
  • Refrigerated centrifuge
  • LSRII flow cytometer (BD Biosciences)
  • FlowJo 7.6.5 software (TreeStar)

Basic Protocol 2: Strategy for Phenotyping Human Blood Monocytes and Intestinal Macrophages Exposed to Polarization Stimuli

  Materials
  • PBMCs fractionated by density gradient sedimentation and the resultant CD14+ blood monocytes purified from PBMCs by CD14+ magnetic beads (unit 14.36; Steinhäuser et al., )
  • Intestinal MNLs isolated from resected segments of normal jejunum or ileum by enzyme digestion and gradient sedimentation (unit 7.1; Fuss, Kanof, Smith, & Zola, )
  • Elutriated intestinal macrophages purified from the intestinal MNLs by counterflow centrifugal elutriation (unit 7.6)
  • RPMI Complete culture medium: RPMI 1640 medium supplemented with 1% penicillin/streptomycin, 1% L‐glutamine and 10% heat‐inactivated human AB serum (all from Mediatech)
  • Differentiating cytokines: human GM‐CSF and M‐CSF (R&D Systems)
  • Polarizing cytokines: IFN‐γ and IL‐4 (R&D Systems)
  • Lipopolysaccharide (LPS; Sigma Aldrich)
  • 24‐ and 48‐well culture plates (Corning Life Sciences)
  • Tissue culture hood (Biosafety cabinet, level 2)
  • Coulter counter and channelyzer
  • Additional reagents and equipment for harvesting, staining, and evaluating monocytes and intestinal macrophages ( protocol 1, steps 1 to 8) and ELISA (Hornbeck, )
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Figures

Videos

Literature Cited

Literature Cited
  Bain, C. C., Bravo‐Blas, A., Scott, C. L., Gomez Perdiguero, E., Geissmann, F., Henri, S., … Mowat, A. M. (2014). Constant replenishment from circulating monocytes maintains the macrophage pool in the intestine of adult mice. Nature Immunology, 15, 929–937. doi: 10.1038/ni.2967.
  Bain, C. C., Scott, C. L., Uronen‐Hansson, H., Gudjonsson, S., Jansson, O., Grip, O., … Mowat, A. M. (2013). Resident and pro‐inflammatory macrophages in the colon represent alternative context‐dependent fates of the same Ly6Chi monocyte precursors. Mucosal Immunology, 6, 498–510. doi: 10.1038/mi.2012.89.
  Benoit, M., Desnues, B., & Mege, J. L. (2008). Macrophage polarization in bacterial infections. Journal of Immunology, 181, 3733–3739. doi: 10.4049/jimmunol.181.6.3733.
  Bogunovic, M., Ginhoux, F., Helft, J., Shang, L., Hashimoto, D., Greter, M., … Merad, M. (2009). Origin of the lamina propria dendritic cell network. Immunity, 31, 513–525. doi: 10.1016/j.immuni.2009.08.010.
  Cassol, E., Cassetta, L., Alfano, M., & Poli, G. (2010). Macrophage polarization and HIV‐1 infection. Journal of Leukocyte Biology, 87, 599–608. doi: 10.1189/jlb.1009673.
  Chu, H., & Mazmanian, S. K. (2013). Innate immune recognition of the microbiota promotes host‐microbial symbiosis. Nature Immunology, 14, 668–675. doi: 10.1038/ni.2635.
  Denning, T. L., Wang, Y. C., Patel, S. R., Williams, I. R., & Pulendran, B. (2007). Lamina propria macrophages and dendritic cells differentially induce regulatory and interleukin 17‐producing T cell responses. Nature Immunology, 8, 1086–1094. doi: 10.1038/ni1511.
  Fuss, I. J., Kanof, M. E., Smith, P. D., & Zola, H. (2009). Isolation of whole mononuclear cells from peripheral blood and cord blood. Current Protocols in Immunology, 85, 7.1.1–7.1.8. doi: 10.1002/0471142735.im0701s85.
  Gordon, S. (2003). Alternative activation of macrophages. Nature Reviews Immunology, 3, 23–35. doi: 10.1038/nri978.
  Herbein, G., Gras, G., Khan, K. A., & Abbas, W. (2010). Macrophage signaling in HIV‐1 infection. Retrovirology, 7, 34. doi: 10.1186/1742‐4690‐7‐34.
  Hornbeck, P. (2015). Enzyme‐linked immunosorbent assays. Current Protocols in Immunology, 110, 2.1.1–2.1.23. doi: 10.1002/0471142735.im0201s110.
  Kelsall, B. L., Leon, F., Smythies, L. E., & Smith, P. D. (2005). Antigen handling and presentation by mucosal dendritic cells and macrophages. In J. Mestecky, J. Bienenstock, M. E. Lamm, L. Mayer, J. R. McGhee, & W. Strober (Eds.). Mucosal Immunology (3rd Ed.). San Diego: Elsevier.
  Lee, Y. K., & Mazmanian, S. K. (2010). Has the microbiota played a critical role in the evolution of the adaptive immune system? Science, 330, 1768–1773. doi: 10.1126/science.1195568.
  Lee, S. H., Starkey, P. M., & Gordon, S. (1985). Quantitative analysis of total macrophage content in adult mouse tissues. Immunochemical studies with monoclonal antibody F4/80. The Journal of Experimental Medicine, 161, 475–489. doi: 10.1084/jem.161.3.475.
  Medina‐Contreras, O., Geem, D., Laur, O., Williams, I. R., Lira, S. A., Nusrat, A., … Denning, T. L. (2011). CX3CR1 regulates intestinal macrophage homeostasis, bacterial translocation, and colitogenic Th17 responses in mice. Journal of Clinical Investigation, 121, 4787–4795. 10.1172/JCI59150.
  Mosser, D. M., & Edwards, J. P. (2008). Exploring the full spectrum of macrophage activation. Nature Reviews Immunology, 8, 958–969. doi: 10.1038/nri2448.
  Rivollier, A., He, J., Kole, A., Valatas, V., & Kelsall, B. L. (2012). Inflammation switches the differentiation program of Ly6Chi monocytes from antiinflammatory macrophages to inflammatory dendritic cells in the colon. The Journal of Experimental Medicine, 209, 139–155. doi: 10.1084/jem.20101387.
  Robinson, T. O., Zhang, M., Ochsenbauer, C., Smythies, L. E., & Cron, R. Q. (2017). CD4 regulatory T cells augment HIV‐1 expression of polarized M1 and M2 monocyte derived macrophages. Virology, 504, 79–87. doi: http://doi.org/10.1016/j.virol.2017.01.018.
  Schenk, M., Bouchon, A., Birrer, S., Colonna, M., & Mueller, C. (2005). Macrophages expressing triggering receptor expressed on myeloid cells‐1 are underrepresented in the human intestine. Journal of Immunological Methods, 174, 517–524. doi: 10.4049/jimmunol.174.1.517.
  Schulz, O., Jaensson, E., Persson, E. K., Liu, X., Worbs, T., Agace, W. W., & Pabst, O. (2009). Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymph and serve classical dendritic cell functions. The Journal of Experimental Medicine, 206, 3101–3114. doi: 10.1084/jem.20091925.
  Schulz, C., Gomez Perdiguero, E., Chorro, L., Szabo‐Rogers, H., Cagnard, N., Kierdorf, K., … Geissmann, F. (2012). A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science, 336, 86–90. doi: 10.1126/science.1219179.
  Shen, R., Richter, H. E., Clements, R. H., Novak, L., Huff, K., Bimczok, D., … Smith, P. D. (2009). Macrophages in vaginal but not in intestinal mucosa are monocyte‐like and permissive to HIV‐1. Journal of Virology, 83, 3258–3267. doi: 10.1128/JVI.01796‐08.
  Sica, A., & Mantovani, A. (2012). Macrophage plasticity and polarization: In vivo veritas. The Journal of Clinical Investigation, 122, 787–795. doi: 10.1172/JCI59643.
  Smith, P. D., Smythies, L. E., Mosteller‐Barnum, M., Sibley, D. A., Russell, M. W., Merger, M., … Kubagawa, H. (2001). Intestinal macrophages lack CD14 and CD89 and consequently are down‐regulated for LPS‐ and IgA‐mediated activities. Journal of Immunology, 167, 2651–2656. doi: 10.4049/jimmunol.167.5.2651.
  Smith, P. D., Smythies, L. E., Shen, R., Greenwell‐Wild, T., Gliozzi, M., & Wahl, S. M. (2011). Intestinal macrophages and response to microbial encroachment. Mucosal Immunology, 4, 31–42. doi: 10.1038/mi.2010.66.
  Smythies, L. E., Denning, T. L., & Smith, P. D. (2015). Mucosal macrophages in defense and regulation. In J. Mestecky, W. Strober, M. W. Russell, B. L. Kelsall, H. Cheroutre, & B. N. Lambrecht (Eds.). Mucosal immunology (Vol. 1, 4th Edn.). Boston: Elsevier.
  Smythies, L. E., Maheshwari, A., Clements, R. H., Eckhoff, D., Novak, L., Mosteller‐Barnum, M., … Smith, P. D. (2006a). Mucosal IL‐8 and TGF‐b recruit blood monocytes: Evidence for cross‐talk between the lamina propria stroma and myeloid cells. Journal of Leukocyte Biology, 80, 492–499. doi: 10.1189/jlb.1005566.
  Smythies, L. E., Sellers, M., Clements, R. H., Mosteller‐Barnum, M., Meng, G., Benjamin, W. H., … Smith, P. D. (2005). Human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal activity. The Journal of Clinical Investigation, 115, 66–75. doi: 10.1172/JCI200519229.
  Smythies, L. E., Shen, R., Bimczok, D., Novak, L., Clements, R. H., Eckhoff, D. E., … Smith, P. D. (2010). Inflammation anergy in human intestinal macrophages is due to Smad‐induced IkBa expression and NF‐kB inactivation. The Journal of Biological Chemistry, 285, 19593–19604. doi: 10.1074/jbc.M109.069955 [pii] M109.069955.
  Smythies, L. E., Wahl, L. M., & Smith, P. D. (2006b). Isolation and purification of human intestinal macrophages. Current Protocols in Immunology, 70, 7.6B.1–7.6B.9. doi: 10.1002/0471142735.im0706bs70.
  Springer, M. S., & Murphy, W. J. (2007). Mammalian evolution and biomedicine: New views from phylogeny. Biological Reviews of the Cambridge Philosophical Society, 82, 375–392. doi: 10.1111/j.1469‐185X.2007.00016.x.
  Steinhäuser, C., Dallenga, T., Tchikov, V., Schaible, U. E., Schütze, S., & Reiling, N. (2014). Immunomagnetic isolation of pathogen‐containing phagosomes and apoptotic blebs from primary phagocytes. Current Protocols in Immunology, 105, 14.36:14.36.1–14.36.26. doi: 10.1002/0471142735.im1436s105.
  Tiemessen, M. M., Jagger, A. L., Evans, H. G., van Herwijnen, M. J., John, S., & Taams, L. S. (2007). CD4+CD25+Foxp3+ regulatory T cells induce alternative activation of human monocytes/macrophages. Proceedings of the National Academy of Sciences of the United States of America, 104, 19446–19451. doi: 10.1073/pnas.0706832104.
  Varol, C., Mildner, A., & Jung, S. (2015). Macrophages: Development and tissue specialization. Annual Review of Immunology, 33, 643–675. doi: 10.1146/annurev‐immunol‐032414‐112220.
  Zigmond, E., Bernshtein, B., Friedlander, G., Walker, C. R., Yona, S., Kim, K. W., … Jung, S. (2014). Macrophage‐restricted interleukin‐10 receptor deficiency, but not IL‐10 deficiency, causes severe spontaneous colitis. Immunity, 40, 720–733. doi: 10.1016/j.immuni.2014.03.012.
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