Measurement of Fcγ Receptor–Mediated Binding and Phagocytosis

Alexander D. Politis1, Stefanie N. Vogel2

1 National Institute of Health, Bethesda, Maryland, 2 University of Maryland, Baltimore, Baltimore, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.8
DOI:  10.1002/0471142735.im1408s68
Online Posting Date:  September, 2005
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Abstract

This unit describes functional assays that quantify macrophage FcγR surface expression. In each assay, IgG‐opsonized sheep red blood cells (SRBC) labeled with Na251CrO4 are used to detect interaction with macrophages. The Basic Protocol measures phagocytosis of target cells following treatment of macrophages with agents known to modulate FcγR expression. The Alternate Protocol describes a method that can be used to measure FcγR‐mediated binding of target cells to macrophages. A choice of opsonin in both methods allows for some discrimination between subclasses of FcγRs that are responsible for the observed results.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of FcγR‐Mediated Phagocytosis
  • Alternate Protocol 1: Measurement of FcγR‐Mediated Binding
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of FcγR‐Mediated Phagocytosis

  Materials
  • Primary macrophage cultures or macrophage cell lines (units 7.6& 14.1)
  • Stimulus (e.g., murine IFN‐α/β, RND Systems; see unit 14.2 for other activating agents)
  • Culture medium (e.g., complete RPMI‐1640; appendix 22)
  • Sheep red blood cells (SRBC) diluted 1:1 (v/v) in Alsever's solution ( appendix 22)
  • Saline solution: 0.9% (w/v) NaCl
  • 200 to 900 Ci/g Na 251CrO 4 in saline solution (30,000 Ci/mmol, ICN Biomedicals or DuPont NEN)
  • Polyclonal IgG against SRBC (e.g., Diamedix) or a monoclonal IgG2a FcγRI‐specific opsonin (hybridoma clone S.S‐1, ATCC)
  • ACK lysis solution (see recipe)
  • 0.5% (w/v) SDS solution
  • 8‐channel pipettor (to deliver 100‐µl aliquots) and disposable reservoirs (PGC Scientific)
  • 96‐well flat‐bottom tissue culture plates (e.g., Falcon or Costar)
  • Gamma (γ) counter and tubes
  • 15‐ and 50‐ml conical centrifuge tube
  • Centrifuge (clinical model)
  • 37°C water bath
  • Supernatant Collecting System (Skatron SCS; Molecular Devices)
  • 8‐well manifold aspirator (Drummond Scientific)
  • Inverted microscope
  • Additional reagents and equipment for counting cells with a hemacytometer ( appendix 3A)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: Measurement of FcγR‐Mediated Binding

  • 150 mM iodoacetic acid (IAA; prepare fresh and store wrapped in foil to protect from light)
  • 24‐well flat‐bottom tissue culture plates (e.g., Costar or Falcon)
  • 4‐well manifold aspirator (Drummond Scientific)
  • Microtiter plate shaker (optional)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Figures

Videos

Literature Cited

   Fischer, D.G. and Koren, H.S. 1981. Quantitative immune phagocytosis by macrophages. In Manual of Macrophage Methodology: Collection, Characterization, and Function (H.B. Herscowitz, H.T. Holden, J.A. Bellanti, and A. Ghaffar, eds.) pp. 259‐264. Marcel Dekker, New York.
   Kabat, E.A. 1976. The reagents of immunology. Detection of antigen and antibody interaction in solution and on cells. In Structural Concepts in Immunology and Immunochemistry (E.A. Kabat, ed.) pp. 48‐56. Holt, Rinehart, and Winston, New York.
   Leu, R.W., Rummage, J.A., and Horn, M.J. 1989. Characterization of murine macrophage Fc receptor‐dependent phagocytosis and antibody‐dependent cellular cytotoxicity during in vitro culture with interferons‐gamma, alpha/beta and/or fetal bovine serum. Immunobiology 178:340‐350.
   Nimmerjahn, F., Bruhns, P., Horiuchi, K., and Ravetch, J.V. Immunity. In press.
   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 γ‐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 Fcγ receptor and Ia antigen expression. J. Immunol. 145:3788‐3795.
   Politis, A.D., Sivo, J., and Vogel, S.N. 1993. Multiple pathways of interferon‐induced gene expression in murine macrophages. J. Leukocyte Biol. 53:583‐590.
   Ravetch, J.V. and Bolland, S. 2001. IgG Fc receptors. Annu. Rev. Immunol. 19:275‐290.
   Ravetch, J.V. and Kinet, J.‐P. 1991. Fc receptors. Annu. Rev. Immunol. 9:457‐492.
   Sears, D.W., Osman, N., Tate, B., McKenzie, I.F.C., and Hogarth, P.M. 1990. Molecular cloning and expression of the mouse high affinity Fc receptor for IgG. J. Immunol. 144:371‐378.
   Suzuki, T. 1991. Signal transduction mechanisms through Fcγ receptors on the mouse macrophage surface. FASEB J. 5:187‐193.
   Swanson, J.A. and Hoppe, A.D. 2004. The coordination of signaling during Fc receptor‐mediated phagocytosis. J. Leukocyte Biology 76:1093‐1103.
   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: β‐interferon treatment of C3H/HeJ macrophages results in increased numbers and density of Fc receptors. J. Immunol. 130:1210‐1214.
   Weinshank, R.L., Luster, A.D., and Ravetch, J. 1988. Function and regulation of a murine macrophage‐specific IgG Fc receptor, FcgR‐α. J. Exp. Med. 167:1909‐1925.
Key Reference
   Ravetch and Kinet, 1991. See above.
  Reviews murine and human Fc receptors.
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