Measurement of Macrophage‐Mediated Killing of Intracellular Bacteria, Including Francisella and Mycobacteria

Karen L. Elkins1, Siobhán C. Cowley1, J. Wayne Conlan2

1 Laboratory of Mycobacterial Diseases and Cellular Immunology, Division of Bacterial, Parasitic, and Allergenic Products, CBER/U.S. FDA, Rockville, Maryland, 2 National Research Council of Canada, Institute for Biological Sciences, Ottawa, Ontario, Canada
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.25
DOI:  10.1002/0471142735.im1425s93
Online Posting Date:  April, 2011
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Macrophages activated by T cell cytokines are a critical defense mechanism against intracellular bacterial pathogens. This unit presents two general methods for assessing the capacity of mouse macrophages, activated with either soluble cytokines or whole immune T lymphocytes, to control or reduce numbers of intracellular bacteria residing within them. “Measurement of killing” is inferred from a reduction in the number of colony‐forming units (cfu) of bacteria at the end of a culture period, compared to the input numbers of cfu at initiation of culture, to the peak numbers of cfu measured during culture, or to a control group in which killing is expected to be poor. Curr. Protoc. Immunol. 93:14.25.1‐14.25.13. © 2011 by John Wiley & Sons, Inc.

Keywords: Francisella; Mycobacteria; intracellular bacteria; macrophage; cytokine; intracellular killing; T lymphocytes

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Table of Contents

  • Introduction
  • Basic Protocol 1: IFN‐γ/TNF‐α‐Mediated Activation of Mouse Macrophages for Intracellular Killing of F. tularensis LVS
  • Basic Protocol 2: T Cell–Mediated Control of Intramacrophage Growth of F. tularensis LVS
  • Alternate Protocol 1: T Cell–Mediated Control of Intramacrophage Growth of Mycobacterium tuberculosis
  • Support Protocol 1: Preparation of F. tularensis LVS Bacterial Stocks
  • Support Protocol 2: Generation of LVS‐Immune T Lymphocytes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: IFN‐γ/TNF‐α‐Mediated Activation of Mouse Macrophages for Intracellular Killing of F. tularensis LVS

  • Confluent monolayer of mouse BMM (unit 14.1), cultured without any antibiotics in the medium for at least three days before beginning the assay.
  • Complete tissue culture medium (for BMM and cocultures) with 10% FBS (see recipe), with and without 50 µg/ml gentamicin sulfate (e.g., Sigma)
  • Wash buffer: phosphate‐buffered saline (PBS; appendix 2A) with 2% fetal bovine serum (FBS; appendix 2A)
  • Frozen stock of F. tularensis LVS bacteria ( protocol 4)
  • Recombinant IFN‐γ (various sources, e.g., BD Pharmingen)
  • Recombinant TNF‐α (various sources, e.g., BD Pharmingen)
  • Mueller‐Hinton agar plates (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 24‐well tissue culture plates
  • 37°C, 5% CO 2 humidified incubator
NOTE: Use distilled water, preferably water for injection (WFI), where water is called for.

Basic Protocol 2: T Cell–Mediated Control of Intramacrophage Growth of F. tularensis LVS

  • Source of naive, control, and LVS‐immune lymphocytes, e.g., splenic lymphoctyes from LVS‐immune mice ( protocol 5; also see unit 19.14)
  • Additional reagents and equipment for measuring intracellular killing of F. tularensis LVS by mouse macrophages (see protocol 1)

Alternate Protocol 1: T Cell–Mediated Control of Intramacrophage Growth of Mycobacterium tuberculosis

  • Frozen stocks of M. tuberculosis. bacteria (unit 19.5)
  • Distilled water (preferably WFI, water for injection) with 0.01% (w/v) saponin
  • 7H11 agar plates (unit 19.5)
  • PBS with 0.05% (v/v) Tween 80
  • Source of naive, control, and M. tuberculosis—immune lymphocytes ( protocol 5), e.g., splenic lymphoctyes from M. bovis BCG–vaccinated mice (unit 19.5)
  • Phosphate‐buffered saline (PBS; appendix 2A)
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Literature Cited

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   Bosio, C.M. and Elkins, K.L. 2001. Susceptibility to secondary Francisella tularensis LVS infection in B cell deficient mice is associated with neutrophilia but not with defects in specific T cell mediated immunity. Infect. Immun. 69:194‐203.
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   Cowley, S.C. and Elkins, K.L. 2003a. CD4+ T cells mediate IFN‐gamma‐independent control of Mycobacterium tuberculosis infection both in vitro and in vivo. J. Immunol. 171:4689‐4699.
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   Elkins, K.L., Cooper, A., Colombini, S.M., Cowley, S.C., and Kieffer, T.L. 2002. In vivo clearance of an intracellular bacterium, Francisella tularensis LVS, is dependent on the p40 sub‐ of Interleukin‐12 (IL‐12) but not on IL‐12 p70. Infect. Immun. 70:1936‐1948.
   Elkins, K.L., Cowley, S.C., and Bosio, C.M. 2003. Innate and adaptive immune responses to an intracellular bacterium, Francisella tularensis live vaccine strain. Microbes Infect. 5:132‐142.
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Key References
   Bosio and Elkins, 2001. See above.
  This publication describes the original development of the coculture assay using LVS, and includes data regarding choices in many of the key parameters.
   Cowley and Elkins, 2003a. See above.
  This publication describes the original development of the coculture assay using M. tuberculosis.
   Elkins et al., 2002. See above.
  This publication compares the activities of T cells from IL‐12 p35 and p40 knockout mice in the coculture assay, illustrating minimal differences in the ability of each type to control intramacrophage LVS growth despite the dramatic difference in vivo in the outcome of intradermal LVS infection.
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