Summary
Diversity in macrophage responsiveness to inflammatory stimuli has resulted in the description of a new paradigm wherein macrophages are referred to as polarized into one of two distinct phenotypes, classically activated (M1) macrophages and alternatively activated (M2) macrophages. Classically activated, M1 or “killer” macrophages are thought to play a critical role in destroying foreign organisms and tumor cells, while alternatively activated M2 or “healer” macrophages are thought to be important in debris scavenging, wound healing, and angiogenesis. M2 macrophages may also play key roles in chronic infections, tumorigenesis, and tumor metastasis. It is therefore important to establish models of M1 and M2 polarized macrophages to study their characteristics and amenability to manipulation. M1 macrophages are typically derived from myeloid progenitors with murine macrophage-colony-stimulating factor (M-CSF, also known as CSF-1), while M2 macrophages are thought to be derived from mature M1 macrophages by treatment with interleukin-4 (IL-4) or IL-13. M2 macrophages can also be isolated from SH2-containing inositol 5′-phosphatase (SHIP)−/− mice by harvesting macrophages from peritoneal lavage fluids or they can be derived from SHIP−/− bone marrow aspirate cells with addition of 5% human serum. Upon stimulation with lipopolysaccharide (LPS), M1 macrophages produce high levels of proinflammatory cytokines, low levels of anti-inflammatory cytokines, and high levels of inducible nitric oxide synthase (iNOS), which leads to nitric oxide (NO) production. M2 macrophages, on the other hand, express high levels of M2 markers Ym1 and arginase I (ArgI) and, upon stimulation with LPS, produce relatively lower levels of proinflammatory cytokines and NO and higher levels of anti-inflammatory cytokines. In this chapter, we describe methods used in our laboratory to generate and characterize alternatively activated (M2) macrophages.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Gordon, S. (2003) Alternative activation of macrophages. Nat Rev Immunol 3, 23–35.
Mantovani, A., Schioppa, T., Biswas, S. K., Marchesi, F., Allavena, P., and Sica, A. (2003) Tumor-associated macrophages and dendritic cells as prototypic type II polarized myeloid populations. Tumori 89, 459–468.
Rauh, M. J., Ho, V., Pereira, C., Sham, A., Sly, L. M., Lam, V., Huxham, L., Minchinton, A. I., Mui, A., and Krystal, G. (2005) SHIP represses the generation of alternatively activated macrophages. Immunity 23, 361–374.
Fremond, C. M., Yeremeev, V., Nicolle, D. M., Jacobs, M., Quesniaux, V. F., and Ryffel, B. (2004) Fatal Mycobacterium tuberculosis infection despite adaptive immune response in the absence of MyD88. J Clin Invest 114, 1790–1799.
Mantovani, A., Sica, A., Sozzani, S., Allavena, P., Vecchi, A., and Locati, M. (2004) The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 25, 677–686.
Mantovani, A., Sica, A., and Locati, M. (2005) Macrophage polarization comes of age. Immunity 23, 344–346.
Mosser, D. M. (2003) The many faces of macrophage activation. J Leukocyte Biol 73, 209–212.
Stein, M., Keshav, S., Harris, N., and Gordon, S. (1992) Interleukin-4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation. J Exp Med 176, 287–292.
El-Gayar, S., Thuring-Nahler, H., Pfeilschifter, J., Rollinghoff, M., and Bogdan, C. (2003) Translational control of inducible nitric oxide synthase by IL-13 and arginine availability in inflammatory macrophages. J Immunol 171, 4561–4568.
Welch, J. S., Escoubert-Lozach, L., Sykes, D. B., Liddiard, K., Greaves, D. R., and Glass, C. K. (2002) TH2 cytokines and allergic challenge induce Yml expression in macrophages by a STAT6-dependent mechanism. J Biol Chem 277, 42821–42829.
Nair, M. G., Cochrane, D. W., and Allen, J. E. (2003) Macrophages in chronic type 2 inflammation have a novel phenotype characterized by the abundant expression of Yml and Fizzl that can be partly replicated in vitro. Immunol Lett 85, 173–180.
Raes, G., Noel, W., Beschin, A., Brys, L., de Baetselier, P., and Hassanzadeh, G. H. (2002) FIZZ1 and Ym as tools to discriminate between differentially activated macrophages. Dev Immunol 9, 151–159.
Raes, G., De Baetselier, P., Noel, W., Beschin, A., Brombacher, F., and Hassanzadeh, G. H. (2002) Differential expression of FIZZ1 and Yml in alternatively activated versus classically activated macrophages. J Leukoc Biol 71, 597–602.
Biswas, S. K., Gangi, L., Paul, S., Schioppa, T., Saccani, A., Sironi, M., Bottazzi, B., Doni, A., Vincenzo, B., Pasqualini, F., Vago, L., Nebuloni, M., Mantovani, A., and Sica, A. (2006) A distinct and unique transcriptional program expressed by tumor-associated macrophages (defective NF-kappaB and enhanced IRF-3/STAT1 activation). Blood 107, 2112–2222.
Ghassabeh, G. H., De Baetselier, P., Brys, L., Noel, W., Van Ginderachter, J. A., Meerschaut, S., Beschin, A., Brombacher, F., and Raes, G. (2006) Identification of a common gene signature for type II cytokine-associated myeloid cells elicited in vivo in different pathologic conditions. Blood 108, 575–583.
Rodriquez, P. C., Quiceno, D. G., Zabaleta, J., Oritz, B., Zea, A. H., Piazuelo, M. B., Delgado, A., Correa, P., Brayer, J., Sotomayor, E. M., Antonia, S., Ochoa, J. B., and Ochoa, A. C. (2004) Arginase I production in the tumor microenvironment by mature myeloid cells inhibits T-cell receptor expression and antigen-specific T-cell responses. Cancer Res 64, 5839–5849.
Dal-Pizzol, F. (2004) Alternative activated macrophage: a new key for systemic inflammatory response syndrome and sepsis treatment? Crit Care Med 32, 1971–1972.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the bacteriophage T4. Nature 227, 680–685.
Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Biotechnology 24, 145–149.
Morrison, A. C., and Correll, P. H. (2002) Activation of the stem cell-derived tyrosine kinase/RON receptor tyrosine kinase by macrophage-stimulating protein results in the induction of arginase activity in murine peritoneal macrophages. J Immunol 168, 853–860.
Kleinbongard, P., Rassaf, T., Dejam, A., Kerber, S., and Kelm, M. (2002) Griess method for nitrite measurement of aqueous and protein-containing samples. Methods Enzymol 359, 158–168.
Acknowledgments
The authors would like to thank Vivian Lam for technical assistance, and Michael J. Rauh and Dr. Gerald Krystal for their pioneering work establishing the macrophage phenotype from the SHIP knockout mouse as a genetic model of M2 polarized macrophages.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Ho, V.W., Sly, L.M. (2009). Derivation and Characterization of Murine Alternatively Activated (M2) 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_12
Download citation
DOI: https://doi.org/10.1007/978-1-59745-396-7_12
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-972-7
Online ISBN: 978-1-59745-396-7
eBook Packages: Springer Protocols