Has anyone specifically stimulated Raw264.7 orTHP-1 cells or primary Macs to get distinguished M2 phenotypes? LPS seems to activate M1 phenotypes. I have read IL4 and IL10 stimulates macrophages to secrete TGF, PGF, IGF and VEGF, I am unable to get a specific protocol, any suggestions or protocol would be greatly appreciated. Thanks, Brahma
IL4 or IL13 are commonly used. We have used 10ng/ml of each to induce M2 phenotypes in mouse microglia. Guess this should also work with macrophages.
I have used 20ng/ml of IL4 to stimulate M2 macrophages, they secrete CD163, CD206 and CD209
Try the search phrase "M1 M2 polarization in vitro" on pubmed and you will find a lot of specific literature examples with more useful info then I can type here. Note that many of these protocols are based on the use of monocytes isolated from peripheral blood, i.e. not yet differentiated macrophages. Cell lines or primary differentiated macrophages isolated from tissues are a different story, but I have seen that various tissue macs such as microglia and alveolar macrophages from lung can be polarized using similar techniques.
The typical reagents used in our labs for polarizing monocyte-derived macrophages are LPS and IFN-gamma for M1, IL-4 and IL-13 for M2. Concentrations can vary depending on cell source and experimental goals, check the literature for studies most similar to what you are trying to do - but ball park figures are 10-20 ng/ml.
Hi!
I have used IL-4 (5ng/mL) in bone marrow cells. My cells expressed Arginase-1 and CD206 (90-95%). I hope this helps!
There is more that one "type" of M2 macrophages. Some basic types are m2a, m2b and m2c and they have different triggers and differentially express various genes. TO make it more complicated, macrophages are highly plastic and can form intermediate phenotypes. This is what we probably are dealing with most of the time when macropahges are collected from the tissues at various circumstances:-tumor, infection, etc.
For the purpose of the in vitro studies M2a are the Th2-type M2 macrophages and are IL-4/IL-13 driven, most of the time when you see the M2 in articles it is meant to be M2a. Depending what type of disease or cellular interaction you trying to model, you may need another subset of M2 and the triggers will be different.
The most recent paper that makes these distinctinctions is here http://www.ncbi.nlm.nih.gov/pubmed/24631966
We usually do a dose-response with 1, 10 and 100 ng/ml of recombinant IL-4 for 24hrs. You can look for secretion of Relm-a or induction of Relm-a and/or YM1 mRNA. They are usually highly elevated.
They do not respond that well to IL-13 since the expression of IL-13Ra1 on their surface is pretty low. Nevertheless, if you stimulate with IL-13 (10 or 100 ng/ml)- you should be able to see induction of Relm-a.
Another readout, which is easy to assess is CCL17 (by ELISA)- its induced by IL-4 but not IL-13.
Good luck!
20ng/ml IL-4 in BMDM induces M2 phenotype after 24h. We noticed increased expression of: Arginase-1, Mrc1, Ym1, Ccl22, Il10
Good luck
Hi Brahma,
For primary macrophages, your best choice depends on which receptors are expressed. IL-4 and/or IL-13 are the canonical, STAT6-dependent stimuli for pushing macrophages into an "alternative" or "M2" activation state, but there's little point in using a cytokine that the cells in your experiment can't detect. Do you know the levels of IL-4Ralpha, IL-13Ralpha1, and the common gamma chain receptor in the starting macrophage population? If IL-13Ra1 is too low, then IL-13 won't be useful. If IL-4Ra is too low, then you could boost its expression by adding IL-10. You can also shift the cells' activation state by your choice of media (RPMI versus DMEM) or growth factors (G-CSF versus GM-CSF). Good luck!
M2s have 3 different subcategories: M2a, M2b, M2c. It really depends which phenotype
For M2a, use IL-4, and M2c, use IL-10.
We also use IL-4 (10 ng/ml) for M2a (CD206 as M2a marker) and IL-10 (10 ng/ml) for M2c (CD163 as M2c marker). For one M1 we use 1000 U/ml IFNg. Our Medium contains M-CSF (10 ng/ml). M-CSF is important to promote monocyte survival. BUT: M-CSF leads to expression of a substantial part of the M2 transcriptome, suggesting that under homeostatic conditions a default shift toward M2 occurs ... alternatively on can use GM-CSF BUT: GM-CSF can polarize monocytes towards the M1 macrophage ... so one has to decide ... at least it depends on the experimental requirements. One has to be careful if one has to choose the phenotype markers because it all depends on the medium and the stimulation conditions.
Please find an attached our previous paper. I do hope you will find answers to your question.
Members of our research group working on macrophages in asthma pathogenesis use IL-4 to generate M2 phenotype macrophages, particularly from mouse bone marrow derived macrophages, but also other cell types.
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