Hi all,
I'm working with primary, human monocyte-derived macrophages and have been polarizing them in vitro with LPS+IFNy or IL-4 (or medium alone). I've done this many times before in human and mouse, and I know from the literature that with LPS+IFNy treatment, CD206 should be low and CD86 and MHCII should be high while with IL-4 (50ng/mL) the opposite should be true.
Sometimes though I find that CD86 and MHCII expression is higher after IL-4 treatment compared to LPS+IFNy! I differentiate my macrophages in 50ng/mL M-CSF for 7 days in RPMI with 10% FBS. On day 7 I wash the cells, add my treatments in the same medium as above, then after 24 hours detach the cells with accutase EDTA and stain for flow cytometry after blocking with 8mg/mL human IgG.
Any thoughts would be much appreciated.
Hefin
Hello Oscar,
This isn't a direct answer, but it may help explain what you observe. A member of Joachim Schultze's group (LIMES, Bonn) presented a short talk at the Keystone symposium on dendritic cells and macrophages in Montreal two weeks ago. The experiment was to culture human blood monocytes in either GM-CSF or in GM-CSF plus IL-4, then to add IL-4 to both groups of cells and characterize their phenotypes. Overall, the second stimulation with IL-4 did not make the phenotypes more similar; instead, it exaggerated the differences initially created by prior IL-4 stimulation. I found this surprising and very interesting, because it seems the effect of IL-4 depended primarily on the activation state of the monocytes, not the canonical signaling pathways downstream of the IL-4 receptor. So, in your case, perhaps you're starting experiments with monocytes that sometimes have already received different activating signals before you begin, and so sometimes increase MHCII and B7.2 when you add IL-4.
Thanks for sharing you interesting observation, and good luck,
Luke
Macrophages polarized with IL4 are not very different than resting macrophages...have you checked the expression of CD86 and MHCII in your macrophages before been polarized? maybe you have initially expression of CD86 and MHCII...this could be donor dependent or maybe they are a bit activated already...
Oscar
Thanks for your answer Oscar. So I always include a control group of cells which have only received medium, nothing else. My IL-4-treated macrophages have higher CD86 and MHCII expression than these control cells (and also higher CD206 which I WOULD expect).
So from the day of blood isolation, they are in RPMI with serum and M-CSF for 7 days, then on day 7 I wash them and replace with RPMI with serum (if control) or RPMI with serum and my polarizing treatments (LPS+IFNy or IL-4). So there is no M-CSF while they are being polarized.
Hello Oscar,
This isn't a direct answer, but it may help explain what you observe. A member of Joachim Schultze's group (LIMES, Bonn) presented a short talk at the Keystone symposium on dendritic cells and macrophages in Montreal two weeks ago. The experiment was to culture human blood monocytes in either GM-CSF or in GM-CSF plus IL-4, then to add IL-4 to both groups of cells and characterize their phenotypes. Overall, the second stimulation with IL-4 did not make the phenotypes more similar; instead, it exaggerated the differences initially created by prior IL-4 stimulation. I found this surprising and very interesting, because it seems the effect of IL-4 depended primarily on the activation state of the monocytes, not the canonical signaling pathways downstream of the IL-4 receptor. So, in your case, perhaps you're starting experiments with monocytes that sometimes have already received different activating signals before you begin, and so sometimes increase MHCII and B7.2 when you add IL-4.
Thanks for sharing you interesting observation, and good luck,
Luke
There Hefin,
There are some publications showing that IL4 inducesa bit of expression of CD86 and MHCII...As these are not typical M2 markers, I reckon they have not been studied in detail during M2 polarization. Are their levels similar to M1 macrophages? Are you analyzing them by facs or mRNA? 24-48h after polarization?
I am still thinking that your macrophages are receiving any kind of "unknown polarizing signal"...Polarized macrophages are terminal differentiated cells and maybe few of them are starting to undergo apoptosis and releasing some inflammatory signals...
Do you also stain for CD11c? Could it be that you are driving their differentiation into monocyte derived dendritic cells? See link below.
http://www.bdbiosciences.com/documents/Tcell-Protocol-Cult-and-ID-of-Hu-Mono-Derived-Dendritic-Cell.pdf
Hi Hefin! I have a practical proposal. I work with endothelial cells and some experiments did not work because the 10% FBS (from a big shot company...) contained so much LPS that the cell were in a preactivated state. If your batch of FBS also contaminated with endotoxin, you probably have a complex LPS+IL-4 synergy. You can check the endotoxin content of the FBS, or use some serum-free media perhaps.
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