Isolation and Purification of Human Intestinal Macrophages
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, organ‐transplantation donors, or the noninflamed margin of resected segments of small intestine from subjects undergoing resection for surgically indicated reasons.
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
Figure 7.6.2 Purification of macrophages from jejunal lamina propria mononuclear cells.
Figure 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.
|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.|