Oxidative Metabolism of Murine Macrophages

Shawn J. Green1, Jacinta Aniagolu1, Jennifer J. Raney1

1 EntreMed, Inc., Rockville, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.5
DOI:  10.1002/0471142735.im1405s12
Online Posting Date:  May, 2001
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Abstract

This unit describes two simple and straightforward microassays that can be used to measure the levels of NO2 and O2, respectively that are generated by a small number of immunologically‐stimulated macrophages. Detection of these products may be used to identify cytokine(s), microbe(s), or microbial products(s) that regulate oxidative metabolism and effector activity. Although a number of other reliable and sensitive methods are available for assaying these two oxidative metabolites, the microassays described here require little time, technical expertise, or materials. It is not clear at present whether human monocytes/macrophages can also produce NO2. These protocols are therefore restricted to mouse macrophages.

     
 
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Table of Contents

  • Basic Protocol 1: Measuring Nitrite in Tissue Culture Supernatants
  • Basic Protocol 2: Measuring Superoxide Anion in Tissue Culture Supernatants
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measuring Nitrite in Tissue Culture Supernatants

  Materials
  • Murine resident peritoneal cells (unit 14.1)
  • DMEM‐5: Dulbeccos modified Eagle medium (GIBCO/BRL) containing 5% heat‐inactivated FBS (HyClone)
  • 1 mM NG‐monomethyl‐L‐arginine acetate (L‐NMA; Calbiochem or Research Biochemicals) in sterile PBS (store at 4°C; stable)
  • Murine recombinant interferon gamma (IFN‐γ; Genzyme)
  • Lipopolysaccharides (LPS) from E. coli serotype 0111:B4 (Sigma), tumor necrosis factor alpha (TNF‐α; Genzyme), or other triggering agent
  • 2 mM NaNO 2 (Sigma) in DMEM‐5 (store at 4°C; stable)
  • recipeGreiss reagent solutions (see recipe)
  • 12 × 75–mm snap‐cap polypropylene tissue culture tubes (Falcon)
  • 96‐well flat‐bottom microtiter plates (Costar)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Measuring Superoxide Anion in Tissue Culture Supernatants

  Materials
  • Murine resident peritoneal cells (unit 14.1)
  • DMEM‐5: Dulbeccos modified Eagle medium (GIBCO/BRL) containing 5% heat‐inactivated FBS (HyClone)
  • Murine recombinant interferon gamma (IFN‐γ; Genzyme)
  • Hanks balanced salt solution (HBSS), phenol red–free with Ca2+ and Mg2+ ( appendix 2A)
  • recipeFerricytochrome c solution (Fe3+ cytochrome c; see recipe)
  • recipeSuperoxide dismutase (SOD) solution (see recipe)
  • recipe10 µg/ml phorbol 12‐myristate 13‐acetate (PMA; see recipe)
  • 1 mg/ml sodium dithionite (Sigma) in phenol red–free HBSS with Ca2+ and Mg2+ (make solution fresh)
  • 12 × 75–mm snap‐cap polypropylene tissue culture tubes (Falcon)
  • 96‐well flat‐bottom microcentrifuge plates (Costar)
  • Beckman centrifuge and swinging‐bucket rotor (e.g., JS‐4.2)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Figures

Videos

Literature Cited

Literature Cited
   Archer, S. 1993. Measurement of nitric oxide in biological models. FASEB J. 7:349‐360.
   Condino‐Neto, A., Muscara, M.N., Grumach, A.S., Carneiro‐Sampaio, M.M., and De Nucci, G. 1993. Neutrophils and mononuclear cells from patients with chronic granulomatous disease release nitric oxide. Br. J. Clin. Pharmacol. 35:485‐490.
   Ding, A.H. and Nathan, C.F. 1987. Trace levels of bacterial lipopolysaccharide prevent interferon‐gamma or tumor necrosis factor‐alpha from enhancing mouse peritoneal macrophage respiratory burst capacity. J. Immunol. 139:1971‐1977.
   Ding, A.H., Nathan, C.F., and Stuehr, D.J. 1988. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. J. Immunol. 141:2407‐2412.
   Granger, D.L., Hibbs, J.B., Prefect, J.R., and Durack, D.T. 1990. Metabolic fate of L‐arginine in relation to microbistatic capacity of murine macrophages. J. Clin. Invest. 85:264‐273.
   Green, S.J. and Nacy, C.A. 1993. Antimicrobial and immunopathologic effects of cytokine‐induced nitric oxide synthesis. Curr. Opin. Infect. Dis. 6:384‐396.
   Green, S.J., Crawford, R.M., Meltzer, M.S., and Nacy, C.A. 1990. Leishmania major amastigotes initiate the L‐arginine dependent killing mechanism in IFN‐γ‐stimulated macrophage induction of TNF‐α. J. Immunol. 145:4290‐4297.
   Green, S.J., Nacy, C.A., Schreiber, R.D., Granger, D.L., Crawford, R.M., Meltzer, M.S., and Fortier, A. 1993. Neutralization of IFNγ and TNFα blocks in vivo synthesis of nitrogen oxides from L‐arginine and protection against Francisella tularensis in BCG‐treated mice. Infect. Immun. 61:689‐698.
   Hibbs, J.B. Jr., Taintor, R.R., and Vavrin, Z. 1987. Macrophage cytotoxicity: Role for L‐arginine deiminase and imino nitrogen oxidation to nitrite. Science 235:473‐476.
   Hibbs, J.B., Vavrin, Z., and Taintor, R.R. 1988. L‐arginine is required for expression of the activated macrophage effector mechanisms causing selective metabolic inhibition in target cells. J. Immunol. 138:550‐565.
   Johnson, R.B. 1981. Secretion of superoxide anion. In Methods for Studying Mononuclear Phagocytes (D.O. Adams, P.J. Edelson, and H. Koren, eds.) pp. 489‐497. Academic Press, New York.
   Klebanoff, S.J. 1992. Oxygen metabolites from phagocytes. In Inflammation: Basic Principles and Clinical Correlates (J.I. Gallin, I.M. Goldstein, and R. Synderman, eds.) pp. 541‐588. Raven Press, New York.
   Martin, J.H.J. and Edwards, S.W. 1993. Changes in mechanisms of monocyte/macrophage‐mediated cytotoxicity during culture. J. Immunol. 150:3478‐3486.
   Mauel, J., Ransijn, A., and Buchmuller‐Rouiller, Y. 1991. Killing of Leishmania parasites in activated murine macrophages is based on an L‐arginine‐dependent process that produces nitrogen derivatives. J. Leukocyte Biol. 49:73‐82.
   Nacy, C.A. and Meltzer, M.S. 1991. T‐cell‐mediated activation of macrophages. Curr. Opin. Immunol. 3:330‐335.
   Nathan, C.F. 1981. Release of hydrogen peroxide. In Methods for Studying Mononuclear Phagocytes (D.O. Adams, P.J. Edelson, and H. Koren, eds.) pp. 498‐509. Academic Press, New York.
   Nathan, C.F and Gabay, J. 1992. Antimicrobial mechanisms of macrophages. In Mononuclear Phagocytes (R. van Furth, ed.) pp. 259‐265. Kluwer Academic Publishers, Netherlands.
   Pick, E. and Mizel, D. 1981. Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader. J. Immunol. Methods 46:211‐226.
   Rotrosen, D. 1992. The respiratory burst oxidase. In Inflammation: Basic Principles and Clinical Correlates (J.I. Gallin, I.M. Goldstein, and R. Synderman, eds.) pp. 589‐601. Raven Press, New York.
   Schneemann, M., Schoedon, G., Hofer, S., Blau, N., Guerrero, L., and Schaffner, A. 1993. Nitric oxide synthase is not a constituent of the antimicrobial armature of human mononuclear phagocytes. J. Infect. Dis. 167:1358‐1363.
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