Measurement of Bacterial Ingestion and Killing by Macrophages

Douglas A. Drevets1, Beth P. Canono2, Priscilla A. Campbell3

1 Department of Medicine, University of Oklahoma Health Sciences Center and the Veterans Affairs Medical Center, Oklahoma City, Oklahoma, 2 Central Translational Research Center, National Jewish Health, Denver, Colorado, 3 National Jewish Health, Denver, Colorado
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.6
DOI:  10.1002/0471142735.im1406s109
Online Posting Date:  April, 2015
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Abstract

This unit presents assays that allow accurate measurement of phagocytosis and killing of bacteria by macrophages. The first basic protocol describes how to measure the ability of macrophages to ingest bacteria. Importantly, because macrophage phagocytosis entails separate binding and internalization steps, assays are described here that will also determine the extent to which bacteria bound to the macrophage are in fact internalized. Two effective methods to do this are described in alternate protocols. Both of these alternate protocols rely on enumeration of differentially labeled bacteria by fluorescence microscopy to distinguish intracellular from extracellular bacteria. The unit also presents two protocols to measure the ability of a macrophage to kill bacteria it has internalized. The second basic is a straightforward assay in which bacterial colonies are enumerated before and after a killing period. Bactericidal activity is evidenced by reduced CFU bacteria on agar plates. Because it is critical to remove residual extracellular organisms, the protocol presents two alternative steps to accomplish this: a washing procedure and a more stringent method in which cells are sedimented through sucrose. An alternate protocol describes a way to measure bacterial viability based on bacterial metabolism, in which the ability of bacterial dehydrogenases to mediate the reduction of a tetrazolium salt to purple formazan is monitored by measuring absorbance spectrophotometrically. © 2015 by John Wiley & Sons, Inc.

Keywords: macrophages; internalization; ingestion of bacteria; phagocytosis; bacterial killing

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Bacterial Phagocytosis by Macrophages
  • Alternate Protocol 1: Discrimination Between Extracellular and Intracellular Bacteria Using FITC Labeled Bacteria
  • Alternate Protocol 2: Discrimination Between Extracellular and Intracellular Bacteria Using Antibody Staining
  • Basic Protocol 2: Measurement of Macrophage Killing of Bacteria
  • Alternate Protocol 3: Colorimetric Determination of Macrophage Killing of Bacteria
  • Support Protocol 1: Preparation of Listeria monocytogenes
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of Bacterial Phagocytosis by Macrophages

  Materials
  • Balanced salt solution (BSS; see recipe)
  • Monocytes/macrophages: e.g., cultured macrophage cell line, murine peritoneal exudate macrophages (unit 14.1; Zhang et al., ), or human PBMC (unit 7.1; Wahl et al., )
  • Overnight bacterial culture (e.g., Listeria monocytogenes EGD), live or heat‐killed (see protocol 6Support Protocol)
  • Normal serum (see recipe), fresh or freshly thawed and kept on ice
  • 30% (w/v) sucrose in PBS (filter sterilize and store for months at 4°C unless contaminated)
  • PBS ( appendix 2A) containing 5% (v/v) FBS, ice‐cold
  • Hema 3 (Fischer Scientific)
  • Centrifuge
  • 10 × 75–mm polypropylene snap‐top tubes
  • Microscope slides and cover slips
  • Labquake Shaker (Barnstead Thermolyne)
  • CytoSpin 4 Cytocentrifuge (Thermo Scientific)

Alternate Protocol 1: Discrimination Between Extracellular and Intracellular Bacteria Using FITC Labeled Bacteria

  Additional Materials (also see protocol 1)
  • Heat‐killed bacteria (e.g., Listeria monocytogenes EGD; see protocol 6Support Protocol), 109 cells/ml
  • FITC/NaHCO 3: 0.1 mg/ml FITC (fluorescein isothiocyanate) isomer 1 (Sigma) in 0.1 M NaHCO 3, pH 9.0
  • HBSS with calcium and magnesium containing 5% FCS
  • Ethidium bromide (Sigma)
  • 70°C water bath
  • Fluorescence microscope equipped with standard fluorescein optics using a long‐pass filter and oil immersion
  • CAUTION: Ethidium bromide is a strong teratogen; it should be handled with gloves and disposed of properly.

Alternate Protocol 2: Discrimination Between Extracellular and Intracellular Bacteria Using Antibody Staining

  Additional Materials (also see protocol 1)
  • 2% neutral buffered formalin
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 5% heat‐inactivated mouse serum (Jackson ImmunoResearch Laboratories, cat. no. 015‐000‐120) and 5% heat‐inactivated donkey serum (Jackson ImmunoResearch Laboratories, cat. no. 017‐000‐121)
  • Rabbit anti‐Listeria monocytogenes O poly antiserum (BD Difco)
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 3% (w/v) bovine serum albumin (BSA; Sigma, cat. no. A2153)
  • PBS ( appendix 2A) containing 0.5% BSA (Sigma, cat. no. A2153) and 10 mM NaN 3
  • Cy2‐conjugated donkey anti‐rabbit IgG (Jackson Immunoresearch Laboratories)
  • PBS ( appendix 2A) containing 0.5% BSA (Sigma, cat. no. A2153)
  • Triton X‐100 (Sigma) diluted to 0.2% in PBS
  • 300 nM DAPI (Invitrogen)
  • Texas Red donkey anti–rabbit IgG (Jackson Immunoresearch Laboratories)
  • ProLong antifade mounting medium (Life Technologies)
  • Glass cover slips6‐well plates
  • Fluorescence microscope equipped with oil‐immersion lens, digital camera and imaging software, and standard band‐pass filters for DAPI, fluorescein/CY2, and Texas Red
  • Immersion oil

Basic Protocol 2: Measurement of Macrophage Killing of Bacteria

  Materials
  • Live log‐phase bacterial culture (e.g., L. monocytogenes, E. coli, or Staphylococcus sp.), cultured overnight after inoculation of liquid medium from frozen stock (see protocol 6Support Protocol)
  • Balanced salt solution (BSS; see recipe)
  • Monocytes/macrophages: e.g., cultured macrophage cell line, murine peritoneal exudate macrophages (unit 14.1; Zhang et al., ), or human PBMC (unit 7.1; Wahl et al., )
  • Normal serum (see recipe), fresh or freshly thawed and kept on ice
  • BSS/5% normal serum, ice‐cold
  • Tryptic Soy Agar culture plates (BD Difco)
  • Sterile water
  • 2.0‐ml conical polypropylene tubes with O‐ring screw cap (Sarstedt)
  • 12 × 75–mm snap‐cap polypropylene tubes, sterile
  • Labquake shaker (Barnstead Thermolyne)
  • 12 × 75–mm polypropylene or polystyrene tubes, sterile

Alternate Protocol 3: Colorimetric Determination of Macrophage Killing of Bacteria

  Additional Materials (also see protocol 4)
  • RPMI‐5: RPMI 1640 (Life Technologies) without phenol red, supplemented with 5% autologous normal serum (see recipe)
  • Amphyl (Thermo Scientific) diluted 1/20 in water
  • 5% (w/v) saponin (Sigma; filter sterilize and store 3 to 6 months at room temperature in ≤10‐ml aliquots to minimize contamination)
  • 2.95% (w/v) tryptose phosphate broth (Difco; autoclave and store in 5‐ml aliquots in glass screw‐cap tubes ≤1 year at 4°C)
  • 5 mg/ml MTT in PBS (Sigma; filter sterilize and store in light‐resistant container 3 to 6 months at 4°C)
  • 1 N HCl (optional)
  • Flat‐bottom 96‐well plates, sterile (Fisher or Falcon)
  • 37°C, 10% CO 2 incubator
  • Microplate centrifuge carriers (e.g.,M‐20 Microplate Swinging Bucket Rotor for Sorvall™ Legend™ X1 Centrifuge Series)
  • Microplate reader (e.g., BioTek EL312e)
  • CAUTION: MTT is a carcinogen and teratogen. Use appropriate precautions as suggested on the bottle.
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Figures

Videos

Literature Cited

Literature Cited
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  Czuprynski, C.J., Henson, P.M., and Campbell, P.A. 1984. Killing of Listeria monocytogenes by inflammatory neutrophils and mononuclear phagocytes from immune and nonimmune mice. J. Leukoc. Biol. 35:193‐208.
  Donovan, J. and Brown, P. 2006. Blood Collection. Curr. Protoc. Immunol. 73:1.7.1‐1.7.9.
  Drevets, D.A. and Campbell, P.A. 1991a. Macrophage phagocytosis: Use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria. J. Immunol. Methods 142:31‐38.
  Drevets, D.A. and Campbell, P.A. 1991b. Roles of complement and complement receptor type 3 in phagocytosis of Listeria monocytogenes by inflammatory mouse peritoneal macrophages. Infect. Immun. 59:2645‐2652.
  Drevets, D.A. and Elliott, A.M. 1995. Fluorescence labeling of bacteria for studies of intracellular pathogenesis. J. Immunol. Methods 187:69‐79.
  Drevets, D.A., Canono, B.P., and Campbell, P.A. 1992. Listericidal and nonlistericidal mouse macrophages differ in complement receptor type 3‐mediated phagocytosis of L. monocytogenes and in preventing escape of the bacteria into the cytoplasm. J. Leukoc. Biol. 52:70‐79.
  Drevets, D.A., Leenen, P.J., and Campbell, P.A. 1993. Complement receptor type 3 (CD11b/CD18) involvement is essential for killing of Listeria monocytogenes by mouse macrophages. J. Immunol. 151:5431‐5439.
  Drevets, D.A., Leenen, P.J., and Campbell, P.A. 1996. Complement receptor type 3 mediates phagocytosis and killing of Listeria monocytogenes by a TNF‐alpha‐ and IFN‐gamma‐stimulated macrophage precursor hybrid. Cell Immunol. 169:1‐6.
  Drevets, D.A., Jelinek, T.A., and Freitag, N.E. 2001. Listeria monocytogenes‐infected phagocytes can initiate central nervous system infection in mice. Infect. Immun. 69:1344‐1350.
  Drevets, D.A., Canono, B.P., Leenen, P.J., and Campbell, P.A. 1994. Gentamicin kills intracellular Listeria monocytogenes. Infect. Immun. 62:2222‐2228.
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  Leenen, P.J., Canono, B.P., Drevets, D.A., Voerman, J.S., and Campbell, P.A. 1994. TNF‐alpha and IFN‐gamma stimulate a macrophage precursor cell line to kill Listeria monocytogenes in a nitric oxide‐independent manner. J. Immunol. 153:5141‐5147.
  Mege, J.L., Mehraj, V., and Capo, C. 2011. Macrophage polarization and bacterial infections. Curr. Opin. Infect. Dis. 24:230‐234.
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  Zhang, X., Goncalves, R., and Mosser, D.M. 2008. The isolation and characterization of murine macrophages. Curr. Protoc. Immunol. 83:14.1.1‐14.1.14.
Key References
  Drevets and Campbell 1991b. See above.
  Describes method for determining critically whether bacteria are merely bound to the surface of bacteria, or have been internalized—a question clearly important for bactericidal activity as well, as bacteria bound to macrophage surfaces are not killed by the macrophage unless internalized.
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