Lots of murine M1 and M2 macrophage markers have been described in literature. However, the same markers are not translated in human. The markers described for human M2 macrophages include CD163, CD200R, CD206, and CD80 for M1. In my culture system (IL-4/IL-13 for M2, IFNg/LPS for M1) all these markers overlap. Using cell surface markers it is hard to define the borders of macrophage subsets. Can anyone suggest any subset specific marker, or any other approaches (such as, q-PCR of any genes, cytokines) by which these 2 macrophages subsets can be discriminated?
I have used CD80 for M1 and CD163 for M2. CD80 expression is almost exclusively on M1. CD163 may be expressed less brightly on M1 but is quite bright on M2. If you use MFI it is quite clear. It also depends on how you make your M1 and M2 cells. This staining pattern is very dependable when using GMCSF to make M1 cells and MCSF for M2. If you use MCSF and then LPS+IFN for M1 you are really making an entirely difirent M1 cell. This is an issue in the literature that does not seem to be acknowledged. If you would like to discuss with me feel free to contact me.
Look at the microarray analysis from Martinez FO et al., PMID: 17082649
Among which I like MRC1, DC-SIGN and CD163 for M2, and CXCL1, TNF, IL6, Serpine1. Of these, MRC1 and CD163 are the best one that works well for human samples. Of note, MRC1 is better for M2a, CD163 for M2c, and CCL1 for M2b (mouse).
It's very hard to find definitive markers for M1 and M2. As soon as somebody shouts 'Eureka' somebody else says 'I found it in x, y and z too'. All I can suggest is that you try a suite of markers, as you would need to if you were characterising a stem cell, for example. Those cells or patients with predominantly M1 characteristics........well, you understand.
Keep in mind just how much overlap there is between M1 and M2 (See Huang et al., PLoS One. 2012;7(8):e42507). Of course one reason for that is that M2 ('alternatively activated Mac') is just a convenient classification for anything that isn't classically activated! It's a rather diverse grouping - not surprising, given the variety of environments and signals that macrophages can be exposed and required to respond to.
Hi Abdullah, I do not know if you got your answer yet. If you are using flow cytometry, then you can use intracellular staining to differentiate between M1 and M2 macrophages. The markers of a classically activated macrophage (i.e. M1, with a ligand like LPS) would be expression of the inducible nitric oxide synthase gene (iNOS). The alternately activated macrophage (M2, stimulated with IL-4, e.g.) will have Arginase 1 expression shooting up. Another way is to look at MAP kinases and transcription factors. For example, p38 MAP kinase is a sure bet of classical activation, whereas the transcription factor STAT6 is a signature M2 marker.
These could be useful:
Alternative Activation of Macrophages: An Immunologic Functional Perspective
Annual Review of Immunology
Vol. 27: 451-483 (Volume publication date April 2009)
First published online as a Review in Advance on December 23, 2008
DOI: 10.1146/annurev.immunol.021908.132532
Fernando O. Martinez,1 Laura Helming,2 and Siamon Gordon1
Nature Reviews Immunology 5, 953-964 (December 2005) | doi:10.1038/nri1733
Monocyte and macrophage heterogeneity
Siamon Gordon & Philip R. Taylor
Good Luck!!!!
In response to Ratnadeep, his suggestions concerning the iNOS and ARG1 markers for M1 and M2 macrophages, respectively, are only suited for murine macrophages. Unfortunately, unless you are working directly on tissue samples (e.g. immunohistochemistry), the iNOS/ARG1 paradigm cannot be applied to any culture sytems to derived human macrophages; there is something missing in the in vitro system that does not elicit NO production in these cells.
I can also recommend this article: Ambarus CA, Krausz S, van Eijk M, Hamann J, Radstake TR, Reedquist KA, Tak PP, Baeten DL. Systematic validation of specific phenotypic markers for in vitro polarized human macrophages. J Immunol Methods. 2012; 375(1-2): 196-206.
We have also characterized how various in vitro conditions can induce the type of macrophages present in human first trimester decidua, comparing expression of various genes and proteins after culture with LPS+IFN-g, IL-4/IL-13, IL-10, M-CSF and GM-CSF: Judit Svensson, Maria Jenmalm, Andreas Matussek, Robert Geffers, Göran Berg and Jan Ernerudh: Macrophages at the fetal-maternal interface express markers of alternative activation and are induced by M-CSF and IL-10. Journal of Immunology, 2011; 187(7): 3671-3682.
http://www.jimmunol.org/content/187/7/3671.long
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