Measuring the Killing of Intracellular Pathogens: Leishmania

S. Stenger1, G. van Zandbergen1

1 Institute for Medical Microbiology and Hygiene, University Hospital of Ulm, Ulm, Germany
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.23
DOI:  10.1002/0471142735.im1423s93
Online Posting Date:  April, 2011
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Abstract

Macrophages are professional phagocytes serving as a first line of defence against pathogenic organisms. Macrophages are equipped with efficient effector functions to kill invading microorganisms. The first important mechanism of macrophage host‐defence is phagocytosis of pathogens. Subsequently, internalized pathogens are targeted for destruction in maturating phagolysosomal compartments. This process is mediated by lysosomal proteases and an acidified compartment. To investigate macrophages' killing potential in this chapter, we describe an assay based on human primary cells infected with the obligatory intracellular parasite Leishmania. For this pathogen the macrophage has a dual role. The parasite can use macrophages for its intracellular multiplication, but at the same time host macrophages, upon stimulation, can kill the parasite. Curr. Protoc. Immunol. 93:14.23.1‐14.23.12. © 2011 by John Wiley & Sons, Inc.

Keywords: macrophages; infection; apoptosis; Leishmania; killing assay; phagocytes

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Measuring the Killing of Intracellular Fluorescent Leishmania by Flow Cytometry
  • Alternate Protocol 1: Measurement of Leishmania Killing by Macrophages Using Limiting Dilution
  • Support Protocol 1: Preparation of Virulent Leishmania Promastigote Cultures
  • Support Protocol 2: Isolation of Leishmania Amastigotes from Host Macrophages
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measuring the Killing of Intracellular Fluorescent Leishmania by Flow Cytometry

  Materials
  • Human anti‐inflammatory type II macrophages generated by 5‐day stimulation of adherent monocytes with 10 ng/ml MCSF (PeproTech, cat. no. 300‐25) (Xu et al., )
  • Stationary‐phase eGFP‐ or DsRED‐transfected Leishmania promastigotes (see Strategic Planning and protocol 3; Misslitz et al., ) taken from biphasic Novy‐Nicolle‐McNeal (NNN) blood agar/Leishmania medium (World Health Organization, ); alternatively use macrophage‐derived amastigotes (see protocol 4)
  • Wash medium: phosphate‐buffered saline (PBS; Invitrogen) containing 5% (v/v) complete medium (see recipe for complete medium)
  • Complete medium (see recipe)
  • Miltefosine (positive killing control; Calbiochem, cat. no. 475841)
  • Flow cytometry buffer: phosphate‐buffered saline (PBS; Invitrogen) containing 1% fetal bovine serum (FBS; Sigma)
  • 24‐well cell culture plates (BD Falcon, cat. no. 35.3047)
  • Centrifuge with plate adaptor
  • 12‐ml tubes
  • Cell scraper, 16 cm (Sarstedt, cat. no. 83.1832)
  • Flow cytometry tubes (BD Falcon, cat. no. 352052)
  • Flow cytometer
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise indicated.

Alternate Protocol 1: Measurement of Leishmania Killing by Macrophages Using Limiting Dilution

  • Stationary‐phase Leishmania promastigotes ( protocol 3) or axenic amastigotes ( protocol 4)
  • Novy‐Nicolle‐McNeal (NNN) blood agar medium (World Health Organization, ) (see recipe)
  • Leishmania medium (see recipe)
  • 96‐well cell culture plates
  • Humidified 27°C 5% CO 2 incubator
  • Additional reagents and equipment for counting cells ( appendix 3A)

Support Protocol 1: Preparation of Virulent Leishmania Promastigote Cultures

  Materials
  • Leishmania parasites: the authors' original strain is L. major (MHOM/IL/81/FEBNI)
  • Novy‐Nicolle‐McNeal (NNN) blood agar medium (World Health Organization, ) (see recipe)
  • Leishmania medium (see recipe)
  • Schneider's medium (see recipe; optional)
  • 96‐well cell culture plates
  • Humidified 27°C 5% CO 2 incubator

Support Protocol 2: Isolation of Leishmania Amastigotes from Host Macrophages

  Materials
  • Leishmania‐infected macrophages
  • RPMI 1640 medium, unsupplemented
  • RPMI 1640 medium supplemented with 0.008% sodium dodecyl sulfate (SDS)
  • Complete medium (see recipe)
  • Centrifuge
  • Additional reagents and equipment for Diff‐Quick staining of cytospin slides (unit 14.1)
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Figures

Videos

Literature Cited

   Aga, E., Katschinski, D. M., van Zandbergen, G., Laufs, H., Hansen, B., Muller, K., Solbach, W., and Laskay, T. 2002. Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major. J. Immunol. 169:898‐905.
   Alexander, J., Satoskar, A.R., and Russell, D.G. 1999. Leishmania species: Models of intracellular parasitism. J. Cell. Sci. 18:2993‐3002.
   Allenbach, C., Zufferey, C., Perez, C., Launois, P., Mueller, C., and Tacchini‐Cottier, F. 2006. Macrophages induce neutrophil apoptosis through membrane TNF, a process amplified by Leishmania major. J. Immunol. 176:6656‐6664.
   Bogdan, C. and Rollinghoff, M. 1998. The immune response to Leishmania: Mechanisms of parasite control and evasion. Int. J. Parasitol. 28:121‐134.
   Bogdan, C., Donhauser, N., Doring, R., Rollinghoff, M., Diefenbach, A., and Rittig, M.G. 2000. Fibroblasts as host cells in latent leishmaniosis. J. Exp. Med. 191:2121‐2130.
   Diefenbach, A., Schindler, H., Rollinghoff, M., Yokoyama, W.M., and Bogdan, C. 1999. Requirement for type 2 NO synthase for IL‐12 signaling in innate immunity. Science 284:951‐955.
   Fahrer, J., Rieger, J., and van Zandbergen, G., and Barth, H. 2010. The C2‐streptavidin delivery system promotes the uptake of biotinylated molecules in macrophages and T‐leukemia cells. Biol. Chem. 391:1315‐1325.
   Freitas Balanco, J.M., Moreira, M.E., Bonomo, A., Bozza, P.T., Amarante‐Mendes, G., Pirmez, C., and Barcinski, M.A. 2001. Apoptotic mimicry by an obligate intracellular parasite downregulates macrophage microbicidal activity. Curr. Biol. 11:1870‐1873.
   Hendricks, L. and Wright, N. 1979. Diagnosis of cutaneous leishmaniasis by in vitro cultivation of saline aspirates in Schneider's Drosophila Medium. Am. J. Trop. Med. Hyg. 28:962‐964.
   Lippuner, C., Paape, D., Paterou, A., Brand, J., Richardson, M., Smith, A.J., Hoffman, K., Brinkmann, V., Blackburn, C., and Aebisher, T. 2009. Real‐time imaging of Leishmania mexicana–infected early phagosomes: A study using primary macrophages generated from green fluorescent protein‐Rab5 transgenic mice. FASEB J. 23:483‐491.
   Misslitz, A., Mottram, J.C., Overath, P., and Aebischer, T. 2000. Targeted integration into a rRNA locus results in uniform and high level expression of transgenes in Leishmania amastigotes. Mol. Biochem. Parasitol. 107:251‐261.
   Pearson, R.D., Wheeler, D.A., Harrison, L.H., and Kay, H.D. 1983. The immunobiology of leishmaniasis. Rev. Infect. Dis. 5:907‐927.
   Peters, N.C., Egen, J.G., Secundino, N., Debrabant, A., Kimblin, N., Kamhawi, S., Lawyer, P., Fay, M.P., Germain, R.N., and Sacks, D. 2008. In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science 321:970‐974.
   Ritter, U. and Moll, H. 2000. Monocyte chemotactic protein‐1 stimulates the killing of Leishmania major by human monocytes, acts synergistically with IFN‐gamma and is antagonized by IL‐4. Eur. J. Immunol. 30:3111‐3120.
   Ritter, U., Mattner, J., Rocha, J.S., Bogdan, C., and Korner, H. 2004. The control of Leishmania (Leishmania) major by TNF in vivo is dependent on the parasite strain. Microbes Infect. 6:559‐565.
   Solbach, W. and Laskay, T. 2000. The host response to Leishmania infection. Adv. Immunol. 74:275‐317.
   van Zandbergen, G., Hermann, N., Laufs, H., Solbach, W., and Laskay, T. 2002. Leishmania promastigotes release a granulocyte chemotactic factor and induce interleukin‐8 release but inhibit gamma interferon‐inducible protein 10 production by neutrophil granulocytes. Infect. Immun. 70:4177‐4184.
   van Zandbergen, G., Klinger, M., Mueller, A., Dannenberg, S., Gebert, A., Solbach, W., and Laskay, T. 2004. Cutting edge: Neutrophil granulocyte serves as a vector for Leishmania entry into macrophages. J. Immunol. 173:6521‐6525.
   van Zandbergen, G., Bollinger, A., Wenzel, A., Kamhawi, S., Voll, R., Klinger, M., Müller, A., Hölscher, C., Herrmann, M., Sacks, D., Solbach, W., and Laskay, T. 2006. Leishmania disease development depends on the presence of apoptotic promastigotes in the virulent inoculum. Proc. Natl. Acad. Sci. U.S.A. 103:13837‐13842.
   van Zandbergen, G., Solbach, W., and Laskay, T. 2007. Apoptosis driven infection. Autoimmunity 40:349‐352.
   Verreck, F.A., de Boer, T., Langenberg, D.M., Hoeve, M.A., Kramer, M., Vaisberg, E., Kastelein, R., Kolk, A., de Waal‐Malefyt, R., and Ottenhoff, T.H. 2004. Human IL‐23‐producing type 1 macrophages promote but IL‐10‐producing type 2 macrophages subvert immunity to (myco)bacteria. Proc. Natl. Acad. Sci. U.S.A. 101:4560‐4565.
   Wanderley, J.L., Pinto da Silva, L.H., Deolindo, P., Soong, L., Borges, V.M., and Prates, D.B. 2009. Cooperation between apoptotic and viable metacyclics enhances the pathogenesis of leishmaniasis. PLoS One 4:e5733.
   World Health Organization. 1984. The leishmaniases: Report of a WHO Expert Committee. World Health Organ. Tech. Rep. Ser. 701:1‐140.
   Xu, W., Roos, A., Schlagwein, N., Woltman, A. M., Daha, M. R., and van Kooten, C. 2006. IL‐10‐producing macrophages preferentially clear early apoptotic cells. Blood 107:4930‐4937.
Key References
   Misslitz et al., 2000. See above.
  Elegantly describes a method to generate fluorescent protein–expressing Leishmania.
   van Zandbergen et al., 2004. See above.
  Describes how neutrophils can be misused as a Trojan horse for parasite transfer to macrophages.
   van Zandbergen et al., 2006. See above.
  Describes how Leishmania can silence host phagocytes by an altruistic behavior of the parasite population: the apoptotic death of a subpopulation silences neutrophils and enables the survival of infective promastigotes.
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