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Generation, Culture and Flow-Cytometric Characterization of Primary Mouse Macrophages

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Macrophages and Dendritic Cells

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 531))

Summary

Macrophages are not only host cells for many pathogens, but also fulfill several key functions in the innate and adaptive immune response, including the release of pro- and anti-inflammatory cytokines, the generation of organic and inorganic autacoids, the phagocytosis and killing of intracellular microorganisms or tumor cells, and the degradation and presentation of antigens. Several of these functions are shared by other immune cells, including dendritic cells, granulocytes, NK cells, and/or T lymphocytes. Thus, the analysis of macrophage functions in vitro using primary mouse cell populations requires standardized methods for the generation and culture of macrophages that guarantee high cell purity as well as the absence of stimulatory microbial contaminants. This chapter presents methodology to achieve these aims.

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References

  1. Bogdan C. Macrophages. Encyclopedia of Life Sciences 2006, Wiley, New York;DOI: 10.1038/npg.els.0004007.

    Google Scholar 

  2. Bogdan C. Reactive oxygen and reactive nitrogen metabolites as effector molecules against infectious pathogens. In: Kaufmann SHE, Medzhitov R, Gordon S, eds. The Innate Immune Response to Infection. Washington, DC: ASM; 2004, pp 357–96.

    Google Scholar 

  3. Gordon S. Macrophages and the immune response. In: Paul W, ed. Fundamental Immunology, 5th edn, Philadelphia: Lippincott Raven; 2003, pp 481–95.

    Google Scholar 

  4. Gordon S (2003). Alternative activation of macrophages. Nat Rev Immunol;3:23–35.

    Article  PubMed  CAS  Google Scholar 

  5. Paulnock DM (1992). Macrophage activation by T cells. Curr Opin Immunol;4:344–9.

    Article  PubMed  CAS  Google Scholar 

  6. Stout RD, Suttles J, Xu J, Grewal I, Flavell RA (1996). Impaired T cell-mediated macrophage activation in CD40 ligand-deficient mice. J Immunol;156:8–11.

    PubMed  CAS  Google Scholar 

  7. Andrade RM, Wessendarp M, Gubbels M-J, Striepen B, Subauste CS (2006). CD40 induces macrophage anti-Toxoplasma gondii activity by triggering autophagy-dependent fusion of pathogen-containing vacuoles and lysosomes. J Clin Invest;116:2366–77.

    Article  PubMed  CAS  Google Scholar 

  8. Stout RD. Macrophage activation by T cells: cognate and non-cognate signals (1993). Curr Opin Immunol;5:398–403.

    Article  PubMed  CAS  Google Scholar 

  9. Warschkau H, Kiderlen AF (1999). A monoclonal antibody directed against the murine macrophage surface molecule F4/80 modulates natural immune response to Listeria monocytogenes. J Immunol;163:3409–16.

    PubMed  CAS  Google Scholar 

  10. Baratin M, Roetynck S, Lepolard C, (2005). Natural killer cell and macrophage cooperation in MyD88-dependent innate responses to Plasmodium falciparum. Proc Natl Acad Sci U S A;102:14747–52.

    Article  PubMed  CAS  Google Scholar 

  11. Welte S, Kuttruff S, Waldhauer I, Steinle A (2006). Mutual activation of natural killer cells and monocytes mediated by NKp80-AICL interaction. Nat Immunol;12:1334–42.

    Article  Google Scholar 

  12. Tomura M, Yu W-G, Ahn H-J, (1999). A novel function of Va14+ CD4+ NKT cells: stimulation of IL-12 production by antigen-presenting cells in the innate immune system. J Immunol;163:93–101.

    PubMed  CAS  Google Scholar 

  13. Sköld M, Xiong X, Illarionov PA, Besra GS, Behar SM (2005). Interplay of cytokines and microbial signals in regulation of CD1d expression and NKT cell activation. J Immunol;175:3584–93.

    PubMed  Google Scholar 

  14. Hegde S, Chen X-H, Keaton JM, Reddington F, Besra GS, Gumperz JE (2007). NKT cells direct monocytes into a DC differentiation pathway. J Leukoc Biol;81:1224–35.

    Article  PubMed  CAS  Google Scholar 

  15. Mak NK, Leung KN, Ada GL (1982). The generation of cytotoxic macrophages in mice during infection with influenza A or Sendai virus. Scand J Immunol;15:553–61.

    Article  PubMed  CAS  Google Scholar 

  16. Siren J, Sareneva T, Pirhonen J, (2004). Cytokine and contact-dependent activation of natural killer cells by influenza A or Sendai virus-infected macrophages. J Gen Virol;85:2357–64.

    Article  PubMed  CAS  Google Scholar 

  17. Boorman GA, Luster MI, Dean JH, (1982). Peritoneal macrophage alterations caused by naturally occurring mouse hepatitis virus. Am J Pathol;106:110–7.

    PubMed  CAS  Google Scholar 

  18. Lamontagne L, Jolicoeur P (1994). Low-virulent mouse hepatitis viruses exhibiting various tropisms in macrophages, T and B cell subpopulations, and thymic stromal cells. Lab Anim Sci;44:17–24.

    PubMed  CAS  Google Scholar 

  19. Even C, Rowland RR, Plagemann PG (1995). Mouse hepatitis virus infection of mice causes long-term depletion of lactate dehydrogenase-elevating virus-permissive macrophages and T lymphocyte alterations. Virus Res;39:355–64.

    Article  PubMed  CAS  Google Scholar 

  20. Flano E, Husain SM, Sample JT, Woodland DL, Blackman MA (2000). Latent murine g-herpesvirus infection is established in activated B cells, dendritic cells and macrophages. J Immunol;165:1074–81.

    PubMed  CAS  Google Scholar 

  21. Munder PG, Modolell M, Wallach DFH (1971). Cell propagation on films of polymeric fluorocarbon as a means to regulate pericellular pH and pO2 in cultured monolayers. FEBS Lett;15:191–6.

    Article  PubMed  CAS  Google Scholar 

  22. Rutschman R, Lang R, Hesse M, Ihle JN, Wynn TA, Murray PJ (2001). Stat6-dependent substrate depletion regulates nitric oxide production. J Immunol;166:2173–7.

    PubMed  CAS  Google Scholar 

  23. El-Gayar S, Thüring-Nahler H, Pfeilschifter J, Röllinghoff M, Bogdan C (2003). Translational control of inducible nitric oxide synthase by IL-13 and arginine availability in inflammatory macrophages. J Immunol;171:4561–8.

    PubMed  CAS  Google Scholar 

  24. Fauve RM, Jusforgues H, Hevin B (1983). Maintenance of granuloma macrophages in serum-free medium. J Immunol Methods;64:345–51.

    Article  PubMed  CAS  Google Scholar 

  25. Chen T, Scott E, Morrison DC (1994). Differential effects of serum on lipopolysaccharide receptor-directed macrophage activation for nitric oxide production. Immunol Lett;40:179–87.

    Article  PubMed  CAS  Google Scholar 

  26. Nacy C, Pappas MG. Destruction of Leishmania. In: Adams DO, ed. Methods for Studying Mononuclear Phagocytes. Orlando, FL: Academic; 1981, pp 745–58.

    Google Scholar 

  27. Labadia M, Faanes RB, Rothlein R (1990). Role of adherence vs. spreading in the induction of membrane-associated interleukin-1 on mouse peritoneal macrophages. J Leukoc Biol;48:420–5.

    PubMed  CAS  Google Scholar 

  28. Hodge-Dufour J, Noble PW, Horton MR, (1997). Induction of IL-12 and chemokines by hyaluronan requires adhesion-dependent priming of resident but not elicited macrophages. J Immunol;159:2492–500.

    PubMed  CAS  Google Scholar 

  29. Schleicher U, Hesse A, Bogdan C (2005). Minute numbers of contaminant CD8+ T cells or CD11b+CD11c+ NK cells are the source of IFN-g in IL-12/IL-18-stimulated mouse macrophage populations. Blood;105:1319–28.

    Article  PubMed  CAS  Google Scholar 

  30. Ding AH, Nathan CF, Stuehr DJ (1988). Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J Immunol;141:2407–12.

    PubMed  CAS  Google Scholar 

  31. Bogdan C, Paik J, Vodovotz Y, Nathan C (1992). Contrasting mechanisms for suppression of macrophage cytokine release by transforming growth factor-b and interleukin-10. J Biol Chem;267:23301–8.

    PubMed  CAS  Google Scholar 

  32. Vodovotz Y, Bogdan C, Paik J, Xie Q-w, Nathan C (1993). Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor-b. J Exp Med;178:605–13.

    Article  PubMed  CAS  Google Scholar 

  33. Bogdan C, Thüring H, Dlaska M, Röllinghoff M, Weiss G (1997). Mechanism of suppression of macrophage nitric oxide release by IL-13. J Immunol;159:4506–13.

    PubMed  CAS  Google Scholar 

  34. Mattner J, Schindler H, Diefenbach A, Röllinghoff M, Gresser I, Bogdan C (2000). Regulation of type 2 NO synthase by type I interferons in macrophages infected with Leishmania major. Eur J Immunol;30:2257–67.

    Article  PubMed  CAS  Google Scholar 

  35. Schindler H, Lutz MB, Röllinghoff M, Bogdan C (2001). The production of IFN-γ by IL-12/IL-18-activated macrophages requires STAT4 signaling and is inhibited by IL-4. J Immunol;166:3075–82.

    PubMed  CAS  Google Scholar 

  36. Bogdan C, Vodovotz Y, Paik J, Xie Q-w, Nathan C (1993). Traces of bacterial lipopolysaccharide suppress IFN-γ-induced nitric oxide synthase gene expression in primary mouse macrophages. J Immunol;151:301–9.

    PubMed  CAS  Google Scholar 

  37. Hand WL, King-Thompson NL (1983). Effect of erythrocyte ingestion on macrophage antibacterial function. Infect Immun;40:917–23.

    Google Scholar 

  38. Hand WL (1984). Inhibition of cell-free oxidative bactericidal activity by erythrocytes and hemoglobin. Infect Immun;44:465–8.

    Google Scholar 

  39. Baron EJ, Proctor RA (1982). Elicitation of peritoneal polymorphonuclear neutrophils from mice. J Immunol Methods;49:305–13.

    Article  PubMed  CAS  Google Scholar 

  40. Fortier AH, Falk LA. Isolation of murine macrophages. In: Cooligan AMKea, JE, ed. Current Protocols in Immunology. Hoboken, NJ: Wiley; 1995:14.1.1–1.7.

    Google Scholar 

  41. Malorny U, Neumann C, Sorg C (1981). Influence of various detachment procedures on the functional state of cultured murine macrophages. Immunbiology;159:327–36.

    Article  CAS  Google Scholar 

  42. Tsunawaki S, Nathan CF (1984). Enzymatic basis of macrophage activation. Kinetic analysis of superoxide production in lysates of resident and activated mouse peritoneal macrophages and granulocytes. J Biol Chem;259:4305–12.

    PubMed  CAS  Google Scholar 

  43. Bogdan C, Donhauser N, Döring R, Röllinghoff M, Diefenbach A, Rittig MG (2000). Fibroblasts as host cells in latent leishmaniosis. J Exp Med;191:2121–9.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The preparation of this chapter as well as part of the experiments reported was supported by grants from the Deutsche Forschungsgemeinschaft (Bo996/3-1 and 3-2, SFB620 A9). We are grateful to Dr. Manuel Modolell and to Dr. Nicole Justies (Max Planck-Institute for Immunobiology, Freiburg, Germany) for introducing us into the “Teflon world” of macrophage culture.

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Authors and Affiliations

  1. Institute of Clinical Microbiology, Immunology and Hygiene, Institute of Medical Microbiology, Immunology and Hygiene,, University Clinic of Erlangen, WasserturmstraĂźe 3-5, D-91054, Erlangen, Germany

    Ulrike Schleicher & Christian Bogdan

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  1. Ulrike Schleicher
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  2. Christian Bogdan
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Correspondence to Christian Bogdan .

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    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

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    Schleicher, U., Bogdan, C. (2009). Generation, Culture and Flow-Cytometric Characterization of Primary Mouse Macrophages. In: Reiner, N. (eds) Macrophages and Dendritic Cells. Methods in Molecular Biology™, vol 531. Humana Press. https://doi.org/10.1007/978-1-59745-396-7_14

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    • DOI: https://doi.org/10.1007/978-1-59745-396-7_14

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    • Publisher Name: Humana Press

    • Print ISBN: 978-1-58829-972-7

    • Online ISBN: 978-1-59745-396-7

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