Measuring Opsonic Phagocytosis via Fcγ Receptors and Complement Receptors on Macrophages

David M. Mosser1, Xia Zhang1

1 University of Maryland and The Maryland Pathogen Research Institute, College Park, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.27
DOI:  10.1002/0471142735.im1427s95
Online Posting Date:  November, 2011
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Abstract

Phagocytosis is a cellular process that plays crucial roles in the removal of dead or dying cells, tissue remodeling, and host defense against invading pathogens. Most eukaryotic cells are decorated with glycoproteins containing terminal sialic acids, whose negative charges tend to repel cells, making so‐called “nonspecific” phagocytosis a relatively inefficient process. Professional phagocytes are so designated because they express two major classes of receptors on their surfaces that are primarily involved in phagocytosis. Paradoxically, these receptors do not recognize microbes directly, but rather endogenous proteins that become tethered to microbes and target them for destruction. These are the Fcγ receptors that bind to the Fc portion of IgG and the complement receptors (CRs), which bind primarily to cleavage products of the third component of complement, C3. This unit describes assays that are used to measure these two types of macrophage phagocytosis. Curr. Protoc. Immunol. 95:14.27.1‐14.27.11. © 2011 by John Wiley & Sons, Inc.

Keywords: phagocytosis; opson; complement receptors; macrophages

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of FcγR‐ or Complement Receptor–Mediated Phagocytosis
  • Alternate Protocol 1: Measurement of Receptor‐Mediated Binding Activity Following the Addition of Phagocytosis Inhibitors
  • Support Protocol 1: Preparation of IgG‐SRBC and E‐IgMC
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Measurement of FcγR‐ or Complement Receptor–Mediated Phagocytosis

  Materials
  • Mice of interest (also see unit 14.1)
  • Culture medium for macrophages (unit 14.1)
  • Macrophage activating agent of choice (unit 14.2)
  • Opsonized SRBC suspension ( protocol 3)
  • ACK lysis solution (see recipe)
  • 0.1% (w/v) sodium dodecyl sulfate (SDS)
  • 2,7‐diaminofluorene (DAF) working solution (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe), ice cold
  • 2.5% (v/v) glutaraldehyde in PBS (see recipe for PBS), freshly prepared, ice cold
  • Giemsa stain (Sigma; GS‐500)
  • 96‐well flat‐bottom tissue culture plates (e.g., Falcon or Costar)
  • 12‐channel pipettor (to deliver 50‐ and 100‐µl aliquots) and disposable reservoirs (PGC Scientific)
  • Inverted microscope
  • 96‐well microtiter plates (e.g., Falcon or Costar)
  • Spectrophotometer with microtiter plate reader
  • Additional reagents and equipment for preparation of macrophages from bone marrow or peritoneal exudate (unit 14.1), activation of macrophages (unit 14.2), and preparation of opsonized SRBC ( protocol 3)

Alternate Protocol 1: Measurement of Receptor‐Mediated Binding Activity Following the Addition of Phagocytosis Inhibitors

  • 30 µM cytochalasin D (Calbiochem, cat no. 250255)
  • 10 µM latrunculin A (BIOMOL‐Enzo Life Sciences, http://www.biomol.com, cat. no. T119)
  • 24‐well flat‐bottom tissue culture plates (e.g., Costar or Falcon)

Support Protocol 1: Preparation of IgG‐SRBC and E‐IgMC

  Materials
  • Sheep red blood cells, washed and preserved (MP Biomedicals, cat no. 55876)
  • Phosphate‐buffered saline (PBS; see recipe)
  • GVB+ buffer (see recipe)
  • Anti‐SRBC IgG (one of the following):
    • Rabbit Polyclonal IgG against SRBC MP Biomedicals, cat no. 55806, http://www.mpbio.com)
    • Mouse monoclonal IgG2a against SRBC (ATCC number TIB‐111; hybridoma clone S.S‐1)
    • Mouse monoclonal IgG2b against SRBC (ATCC number TIB‐109; hybridoma clone N‐S.8.1)
  • Anti‐SRBC IgM:
    • Rabbit IgM antibody to SRBC is obtained from Cedarlane Laboratories (cat no. CL9000‐M)
    • Alternatively a hybridoma secreting IgM monoclonal antibody to SRBC is obtained from the American Type Culture Collection (ATCC number TIB‐112, hybridoma clone S.S‐3
  • Human complement component C5‐deficient serum: Quidel Corporation, cat. no. A501; http://www.quidel.com, or mouse C5‐deficient serum can be obtained from C5‐deficient mice such as AKR/J mouse (Jackson Laboratories, cat. no. 00648)
  • 50‐ml conical plastic centrifuge tubes (e.g., Falcon)
  • Refrigerated centrifuge (Eppendorf Centrifuge 5810R or equivalent)
  • Mini‐rotator (end‐over‐end; Glas‐Col)
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
   Aderem, A. and Underhill, D.M. 1999. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17:593‐623.
   de Oliveira, C.A. and Mantovani, B. 1988. Latrunculin A is a potent inhibitor of phagocytosis by macrophages. Life Sci. 43:1825‐1830.
   Greenberg, S. and Grinstein, S. 2002. Phagocytosis and innate immunity. Curr. Opin. Immunol. 14:136‐145.
   He, J.Q., Wiesmann, C., and van Lookeren Campagne, M. 2008. A role of macrophage complement receptor CRIg in immune clearance and inflammation. Mol Immunol. 45:4041‐4047.
   Helmy, K.Y., Katschke, K.J. Jr., Gorgani, N.N., Kljavin, N.M., Elliott, J.M., Diehl, L., Scales, S.J., Ghilardi, N., and van Lookeren Campagne, M. 2006. CRIg: A macrophage complement receptor required for phagocytosis of circulating pathogens. Cell 124:915‐927.
   Montaño, R.F. and Morrison, S.L. 1999. A colorimetric‐enzymatic microassay for the quantitation of antibody‐dependent complement activation. J. Immunol. Methods 222:73‐82.
   Mosser, D.M. and Edwards, J.P. 2008. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8:958‐969.
   Nimmerjahn, F. and Ravetch, J.V. 2006. Fcgamma receptors: Old friends and new family members. Immunity 24:19‐28.
   Oliveira, C.A., Kashman, Y., and Mantovani, B. 1996. Effects of latrunculin A on immunological phagocytosis and macrophage spreading‐associated changes in the F‐actin/G‐actin content of the cells. Chem. Biol. Interact. 100:141‐153.
   Pearse, R.N., Feinman, R., and Ravetch, J. 1992. Characterization of the promoter of the human gene encoding the high‐affinity IgG receptor: Transcriptional induction by ‐interferon is mediated through common DNA response elements. Proc. Natl. Acad. Sci. U.S.A. 88:11305‐11309.
   Politis, A.D. and Vogel, S.N. 1990. Pharmacologic evidence for the requirement of protein kinase C in IFN‐induced macrophage Fc receptor and Ia antigen expression. J. Immunol. 145:3788‐3795.
   Swanson, J.A. and Hoppe, A.D. 2004. The coordination of signaling during Fc receptor‐mediated phagocytosis. J. Leukocyte Biol. 76:1093‐1103.
   van Lookeren Campagne, M., Wiesmann, C., and Brown, E.J. 2007. macrophage complement receptors and pathogen clearance. Cell. Microbiol. 9:2095‐2102.
   Vogel, S.N., Finbloom, D.S., English, K.E., Rosenstreich, D.L., and Langreth, S.G. 1983. Interferon‐induced enhancement of macrophage Fc receptor expression: Interferon treatment of C3H/HeJ macrophages results in increased numbers and density of Fc receptors. J. Immunol. 130:1210‐1214.
   Worthington, R.E., Bossie‐Codreanu, J., and Van Zant, G. 1987. Quantitation of erythroid differentiation in vitro using a sensitive colorimetric assay for hemoglobin. Exp. Hematol. 15:85‐92.
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