Assays for Membrane Complement Receptors

Lynda D. O'Rear1, Gordon D. Ross1

1 University of Louisville, Louisville, Kentucky
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 13.4
DOI:  10.1002/0471142735.im1304s10
Online Posting Date:  May, 2001
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Abstract

Leukocytes express several types of receptors for activated products of the complement (C) system. This unit describes assays for a subset of these receptors: those specific for fixed fragments of C3 (CR1, CR2, CR3, and CR4). Although the assays have been designed for tests of human C receptors, they can often be carried out with rodent (e.g., mouse, rat, or guinea pig) cells by substitution of rodent‐specific reagents. Two basic protocols are presented for rosette assay of CR1, CR2, CR3, and CR4 on adherent and nonadherent cells. Isolated leukocytes may be tested for rosette formation in suspension or adherent to a surface. Nonadherent cells such as lymphocytes, NK cells, or cells from an undifferentiated leukocyte cell line must be tested in suspension. The unit also contains three support protocols for formation and quantitation of sheep erythrocyte‐C3 complexes for use in the two basic protocols.

     
 
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Table of Contents

  • Basic Protocol 1: Rosette Assay of Adherent Neutrophils or Monocyte/Macrophages
  • Basic Protocol 2: Rosette Assay of Neutrophils, Monocytes, and Lymphocytes in Suspension
  • Support Protocol 1: Preparation of Sheep Erythrocyte–C3 Complexes (EC3)
  • Support Protocol 2: Radioiodination of Antibodies Using Iodo‐Gen
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Rosette Assay of Adherent Neutrophils or Monocyte/Macrophages

  Materials
    For recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • Hanks balanced salt solution (HBSS; appendix 2A) with 1% (w/v) Cohn fraction V BSA (H‐BSA), 37°C
  • protocol 3Sheep erythrocyte–C3 complexes (EC3): EC3b and EC3bi (first protocol 3support protocol)
  • Soybean trypsin inhibitor (STI)
  • Four‐well slide chambers (LabTek, Miles Laboratories)
  • 22 × 50–mm coverslips
  • Pencil with new eraser
  • Clear nail polish or nail hardener
  • 10 × 75–mm plastic tubes (e.g., Falcon tubes)
  • Phase‐contrast microscope with 40× to 50× objective lens
  • Additional materials for isolation of human peripheral blood neutrophils (unit 7.23) or mononuclear cell fraction (unit 7.1)

Basic Protocol 2: Rosette Assay of Neutrophils, Monocytes, and Lymphocytes in Suspension

  Materials
    For recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • Leukocyte suspension: neutrophils (unit 7.23), monocytes (unit 7.21), or lymphocytes (unit 7.21)
  • Hanks balanced salt solution (HBSS; appendix 2A) with 1% (w/v) Cohn fraction BSA (H‐BSA), ice cold
  • Soybean trypsin inhibitor (STI)
  • protocol 3Sheep erythrocyte–C3 complexes: EC3b, EC3bi, EC3d, and EC3dg (first protocol 3support protocol)
  • 10 × 75–mm plastic tubes (e.g., Falcon tubes)
  • 37°C incubator with tube rotator at near‐horizontal angle
  • Glass microscope slides with frosted end for label
  • 22‐mm‐square coverslips
  • Phase‐contrast microscope with 40× to 50× objective lens
  • Clean soft pencil eraser
  • Clear nail polish or nail hardener

Support Protocol 1: Preparation of Sheep Erythrocyte–C3 Complexes (EC3)

  Materials
    For recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • Sheep blood in Alsevers solution (unit 13.1)
  • Phosphate‐buffered saline (PBS; appendix 2A), ice cold
  • recipeGVB+ − buffer (see recipe)
  • Trypsin
  • Soybean trypsin inhibitor (STI)
  • GVBNi+ buffer (see recipe), ice cold
  • Purified C3 (unit 13.3), >10 mg/ml, 95% pure, and hemolytically active
  • Factors B, D, H, and I (Quidel)
  • recipeBDVA buffer (see recipe), ice cold
  • recipeBDVEA buffer (see recipe), ice cold
  • Soluble recombinant CR1 (rCR1)
  • 125I‐labeled anti‐C3c, anti‐C3bi‐neo, and anti‐C3d (second protocol 4support protocol)
  • Mineral oil mixture: 4:1 dibutylphthalate/dinonyl phthalate
  • 50‐ml conical plastic centrifuge tubes (e.g., Falcon)
  • Sorvall RC‐3B centrifuge and H6000A rotor, or equivalent
  • Circulating 37°C water bath
  • 12 × 75–mm plastic tubes with corks
  • 400–µl microcentrifuge tubes (Bio‐Rad)

Support Protocol 2: Radioiodination of Antibodies Using Iodo‐Gen

  Materials
    For recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • Iodo‐gen (Pierce)
  • Methylene chloride
  • MAbs to C3c, C3d, and C3bi‐neoantigen (C3bi‐neo; all from Quidel)
  • 17.4 Ci/mg [125I]sodium iodide, carrier‐free (Du Pont NEN)
  • 35% (w/v) BSA solution (e.g., Sigma)
  • Sephadex G‐25 M disposable PD‐10 columns (Pharmacia Biotech)
  • 19‐G hypodermic and butterfly needles
  • 3/32 × 5/32–in. Tygon tubing
  • Tubing adapter
  • Hemostat
  • 12 × 75–mm plastic tubes
  • Disposable methyl acrylate spectrophotometer cuvettes for UV wavelengths
CAUTION: This procedure should be performed only at NRC‐licensed sites by personnel trained in the proper use of 125I. Standard precautions to prevent excessive exposure and radioactive contamination of personnel and equipment should be followed at all times.
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Figures

Videos

Literature Cited

Literature Cited
   Anderson, D.C., Schmalstieg, F.C., Shearer, W., Becker‐Freeman, K., Kohl, S., Smith, C.W., Tosi, M.F., and Springer, T. 1987. Leukocyte LFA‐1, OKM1, p150,95 deficiency syndrome: Functional and biosynthetic studies of three kindreds. Fed. Proc. 44:2671‐2677.
   Arnaout, M.A. 1990. Structure and function of the leukocyte adhesion molecules CD11/CD18. Blood 75:1037‐1050.
   Becherer, J.D., Alsenz, J., and Lambris, J.D. 1989. Molecular aspects of C3 interactions and structural/functional analysis of C3 from different species. Curr. Top. Microbiol. Immunol. 153:45‐72.
   Bianco, C., Patrick, R., and Nussenzweig, V. 1971. A population of lymphocytes bearing a membrane receptor for antigen‐antibody‐complement complexes. I. Separation and characterization. J. Exp. Med. 132:702‐720.
   Cooper, N.R., Moore, M.D., and Nemerow, G.R. 1988. Immunobiology of CR2, the B lymphocyte receptor for Epstein‐Barr virus and the C3d complement fragment. Annu. Rev. Immunol. 6:85‐113.
   Cooper, N.R., Bradt, B.M., Rhim, J.S., and Nemerow, G.R. 1990. CR2 complement receptor. J. Invest. Dermatol. 94(Suppl.):112S‐117S.
   Dukor, P., Bianco, C., and Nussenzweig, V. 1970. Tissue localization of lymphocytes bearing a membrane receptor for antigen‐antibody‐complement complexes. Proc. Natl. Acad. Sci. U.S.A. 67:991‐997.
   Fearon, D.T. and Ahearn, J.M. 1989. Complement receptor type 1 (C3b/C4b receptor; CD35) and complement receptor type 2 (C3d/Epstein‐Barr virus receptor; CD21). Curr. Top. Microbiol. Immunol. 153:83‐98.
   Fishelson, Z. 1991. Complement C3: A molecular mosaic of binding sites. Mol. Immunol. 28:545‐552.
   Henson, P.M. 1969. The adherence of leucocytes and platelets induced by fixed IgG antibody or complement. Immunology 16:107‐121.
   Krych, M., Atkinson, J.P., and Holers, V.M. 1992. Complement receptors. Curr. Opin. Immunol. 4:8‐13.
   Lachmann, P.J., Pangburn, M.K., and Oldroyd, R.G. 1982. Breakdown of C3 after complement activation. Identification of a new fragment, C3g, using monoclonal antibodies. J. Exp. Med. 156:205‐216.
   Lay, W.H. and Nussenzweig, V. 1968. Receptors for complement on leukocytes. J. Exp. Med. 128:991‐1007.
   Myones, B.L., Dalzell, J.G., Hogg, N., and Ross, G.D. 1988. Neutrophil and monocyte cell surface p150,95 has iC3b‐receptor (CR4) activity resembling CR3. J. Clin. Invest. 82:640‐651.
   Rosen, H. and Law, S.K.A. 1989. The leukocyte cell surface receptor(s) for the iC3b product of complement. Curr. Top. Microbiol. Immunol. 153:99‐122.
   Ross, G.D. 1989. Complement and complement receptors. Curr. Opin. Immunol. 2:50‐62.
   Ross, G.D. 1992a. Function of neutrophils in host defense and immunity. In Immune System Accessory Cells (L. Fornusek and V. Vetvicka, eds.) pp. 263‐286. CRC Press, Boca Raton, Fla.
   Ross, G.D. 1992b. Complement receptor type 1. Curr. Top. Microbiol. Immunol. 178:31‐44.
   Ross, G.D. 1992c. Complement receptors. In Encyclopedia of Immunology, Vol. 1 (I.M. Roitt and P.J. Delves, eds.) pp. 388‐391. Academic Press, London.
   Ross, G.D. and Polley, M.J. 1975. Specificity of human lymphocyte complement receptors. J. Exp. Med. 141:1163‐1180.
   Ross, G.D. and Vetvicka, V. 1993. CR3 (CD11b,CD18): A phagocyte and NK cell membrane receptor with multiple ligand specificities and functions. Clin. Exp. Immunol. 92:181‐184.
   Ross, G.D. and Winchester, R.J. 1980. Methods for enumerating lymphocyte populations. In Manual of Clinical Immunology (N.R. Rose, and H. Friedman, eds.) pp. 213‐228. American Society for Microbiology, Washington, D.C.
   Ross, G.D., Polley, M.J., Rabellino, E.M., and Grey, H.M. 1973. Two different complement receptors on human lymphocytes. One specific for C3b and one specific for C3b inactivator–cleaved C3b. J. Exp. Med. 138:798‐811.
   Ross, G.D., Newman, S.L., Lambris, J.D., Devery‐Pocius, J.E., Cain, J.A., and Lachmann, P.J. 1983. Generation of three different fragments of bound C3 with purified factor I or serum. II. Location of binding sites in the C3 fragments for factors B and H, complement receptors, and bovine conglutinin. J. Exp. Med. 158:334‐352.
   Ross, G.D., Yount, W.J., Walport, M.J., Winfield, J.B., Parker, C.J., Fuller, C.R., Taylor, R.P., Myones, B.L., and Lachmann, P.J. 1985. Disease‐associated loss of erythrocyte complement receptors (CR1, C3b‐receptors) in patients with systemic lupus erythematosus and other diseases involving autoantibodies and/or complement activation. J. Immunol. 135:2005‐2014.
   Ross, G.D., Walport, M.J., and Hogg, N. 1989. Receptors for IgG Fc and fixed C3. In Human Monocytes (G.L. Asherson and M. Zembala, eds.) pp. 123‐139. Academic Press, London.
   Ross, G.D., Reed, W., Dalzell, J.G., Becker, S.E., and Hogg, N. 1992. Macrophage cytoskeleton association with CR3 and CR4 regulates receptor mobility and phagocytosis of iC3b‐opsonized erythrocytes. J. Leukocyte Biol. 51:109‐117.
   Todd, R.F. III and Freyer, D.R. 1988. The CD11/CD18 leukocyte glycoprotein deficiency. Hematol. Oncol. Clin. N. Am. 2:13‐31.
   Tsoukas, C.D. and Lambris, J.D. 1993. Expression of EBV/C3d receptors on T cells: Biological significance. Immunol. Today 14:56‐59.
   Venkatesh, Y.P. and Levine, R.P. 1988. The esterase‐like activity of covalently bound human third complement protein. Mol. Immunol. 25:821‐828.
   Walport, M.J. and Ross, G.D. 1989. Deficiency of the LFA‐1 family of molecules. In Human Monocytes (G.L. Asherson, and M. Zembala, eds.) pp. 417‐427. Academic Press, London.
   Walport, M.J., Ross, G.D., Mackworth‐Young, C., Watson, J.V., Hogg, N., and Lachmann, P.J. 1985. Family studies of erythrocyte complement receptor type 1 levels: Reduced levels in patients with SLE are acquired, not inherited. Clin. Exp. Immunol. 59:547‐554.
   Weis, J.J., Tedder, T.F., and Fearon, D.T. 1984. Identification of a 145,000 Mr membrane protein as the C3d receptor (CR2) of human B lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 81:881‐885.
   Wong, W.W. 1990. Structural and functional correlation of the human complement receptor type 1. J. Invest. Dermatol. 94 Suppl:64S‐67S.
Key References
   Bianco, C., Patrick, R., and Nussenzweig, V. 1971. See above.
  Basic references to techniques for assaying CR1, CR2, CR3, and CR4 by rosette formation with C3 fragment–coated sheep erythrocytes.
   Fearon, D.T. 1980. Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte. J. Exp. Med. 152:20‐30.
   Iida, K., Nadler, L., and Nussenzweig, V. 1983. Identification of the membrane receptor for the complement fragment C3d by means of a monoclonal antibody. J. Exp. Med. 158:1021‐1033.
   Myones, B.L., Dalzell, J.G., Hogg, N., and Ross, G.D. 1988. See above.
   Ross, G.D. and Lambris, J.D. 1982. Identification of a C3bi‐specific membrane complement receptor that is expressed on lymphocytes, monocytes, neutrophils, and erythrocytes. J. Exp. Med. 155:96‐110.
   Ross, G.D., Polley, M.J., Rabellino, E.M., and Grey, H.M. 1973. See above.
   Ross, G.D., Newman, S.L., Lambris, J.D., Devery‐Pocius, J.E., Cain, J.A., and Lachmann, P.J. 1983. See above.
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