Isolation and Purification of Human Intestinal Macrophages

Lesley E. Smythies1, Larry M. Wahl2, Philip D. Smith1

1 University of Alabama at Birmingham, Birmingham, Alabama, 2 National Institute of Dental and Craniofacial Research/NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.6B
DOI:  10.1002/0471142735.im0706bs70
Online Posting Date:  January, 2006
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Abstract

The gastrointestinal mucosa contained within the lamina propria is the largest reservoir of macrophages in the human body. The isolation and study of this population of cells is important for understanding host defense and the pathogenesis of inflammation in the gastrointestinal mucosa. This unit describes methods that can be used to isolate and purify intestinal macrophages. Sources of intestinal tissue that can be used for this isolation include human subjects undergoing gastrojejunostomy for obesity, organā€transplantation donors, or the noninflamed margin of resected segments of small intestine from subjects undergoing resection for surgically indicated reasons.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of Intestinal Mononuclear Cells by Enzyme Digestion
  • Basic Protocol 2: Purification of Intestinal Macrophages by Counterflow Centrifugal Elutriation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Intestinal Mononuclear Cells by Enzyme Digestion

  Materials
  • Resected segments of human intestinal tissue (about 30 g wet weight) containing no detectable Peyer's patches as judged by examination with a dissecting microscope (histologic section shown in Fig. , panel A): normal jejunum or ileum (e.g., from patients undergoing gastrojejunostomy or from transplantation donors) or noninflamed margin of resected segments from patients undergoing resection for surgically indicated reasons
  • Storage medium (see recipe)
  • Rinse buffer 1 (see recipe)
  • Rinse buffer 2 (see recipe)
  • Isolation medium (see recipe)
  • Elutriation buffer (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 120‐ml specimen cup
  • Dissecting instruments including forceps and scissors
  • Cell strainers (40 µm, nylon)
  • Refrigerated centrifuge (e.g., Beckman centrifuge with GH‐3.7 rotor)
  • Additional reagents and equipment for Ficoll‐Hypaque gradient separations (unit 7.4)

Basic Protocol 2: Purification of Intestinal Macrophages by Counterflow Centrifugal Elutriation

  Materials
  • Elutriation buffer (see recipe)
  • Lamina propria mononuclear cells (see protocol 1)
  • Phosphate‐buffered saline (PBS; appendix 2A), low pyrogen and without Ca and Mg
  • DMEM (e.g., Invitrogen) supplemented with 2 mM L‐glutamine and 50 µg/ml gentamycin
  • 50% bleach
  • Coulter Counter with Channelyzer
  • 50‐ml conical polypropylene centrifuge tubes
  • Refrigerated centrifuge with JE‐6B rotor (Beckman)
  • Additional reagents and equipment for counterflow centrifugal elutriation (unit 7.6) and flow cytometry (Chapter 5)
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Figures

  •   FigureFigure 7.6.1 Isolation of jejunal lamina propria mononuclear cells. (A) whole intestinal tissue section; (B) section of dissected mucosa with epithelium present; (C) section of dissected mucosa (lamina propria) with epithelium removed; (D) lamina propria stroma; (E) resident mononuclear cells (macrophages and lymphocyte).
  •   FigureFigure 7.6.2 Purification of macrophages from jejunal lamina propria mononuclear cells.
  •   FigureFigure 7.6.3 Intestinal macrophage morphology and flow cytometric analysis for purity. Inserts show positive control staining for CD3, CD20, and CD69 using blood mononuclear cells, and CD83 using monocyte‐derived dendritic cells.

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Literature Cited

Literature Cited
   Lee, S.H., Starkey, P.M., and Gordon, S. 1985. Quantitative analysis of total macrophage content in adult mouse tissues. Immunochemical studies with monoclonal antibody F4/80. J. Exp. Med. 161:475‐489.
   Smith, P.D., Janoff, E.N., Mosteller‐Barnum, M., Merger, M., Orenstein, J.M., Kearney, J.F., and Graham, M.F. 1997. Isolation and purification of CD14‐negative mucosal macrophages from normal human small intestine. J. Immunol. Methods 202:1‐11.
   Smith, P.D., Smythies, L.E., Mosteller‐Barnum, M., Sibley, D.A., Russell, M.W., Merger, M., Sellers, M.T., Orentstein, J.M., Shimada, T., Graham, M.F., and Kubagawa, H. 2001a. Intestinal macrophages lacd CD14 and CD89 and consequently are down‐regulated for LPS‐ and IgA‐mediated activities. J. Immunol. 167:2651‐2656.
   Smith, P.D., Smythies, L.E., and Wahl, S.M. 2001b. Macrophage effector function. In Clinical Immunology: Principles and Practice, 2nd. ed. (R.R. Rich, T.A. Fleisher, W.T. Shearer, B.I. Kotzin, and H.W. Schroeder Jr., eds.) pp. 19.1‐19.9. Mosby, New York.
   Smythies, L.E., Sellers, M., Clements, R.H., Mosteller‐Barnum, M., Meng, G., Benjamin, W.H., Orenstein, J.M., and Smith, P.D. 2005. Human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal acitivity. J. Clin. Invest. 115:66‐75.
   Wahl, L.M., Katona, I.M., Wilder, R.L., Winter, C.C., Haraoui, B., Scher, I., and Wahl, S.M. 1984. Isolation of human mononuclear cell subsets by counterflow centrifugal elutriation (CCE). I. Characterization of B‐lymphocyte‐, T‐lymphocyte‐, and monocyte‐enriched fractions. Cell. Immunol. 85:373‐383.
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