If you take at the look at the original gene array data published by Martinez et al. (J Immunol. 2006 Nov 15;177(10):7303-11.), you will find significantly increased CD14 mRNA in human monocyte-derived macrophages polarized toward an M2 phenotype. This would be consistent wit your finding in raw cells. Please keep in mind that there are substantial differences between human and murine myeloid cells that may also contribute to different findings using different cells types.

I have not used conditions designed to stimulate M1 vs M2, but we reported on CD14 clustering of CD14 (presumably in association with TLR4) on RAW 264.7 cells treated with LPS (see attached manuscript). However, one of the interesting features was that this only occurred in a percentage of the cells, not all of them. Thus, I wonder if CD14 is differentially expressed/regulated on existing subsets that have not yet been characterized even before using treatments to polarize to M1 and M2. Also, if I remember correctly, in flow cytometry, the profile of CD14 expression is not like a distinct, discrete population that all express at a similar level. It's like a normal distribution, so calling these cells positive or negative for CD14 can be a bit arbitrary. This may play into the changes that you see.

I work primarily with human macrophages and have not tried to differentiate them specifically into M1 and M2, although I think some people would regard them as M2-like because we mature them in a low concentration of M-CSF. When I have done flow cytometry for CD14 on these cells, I see a wide spread of CD14 expression, so as the above comment stated, it is hard to distinctly say that the cells are positive or negative. I would also comment that there is a lot of donor variation in the expression of CD14 on human macrophages, but as RAW264.7 are a cell line I suspect you would have better consistency.

How long are you treating the cells with LPS before examining them by flow? Because I would wonder if the increased CD14 you are seeing on M2 is actually a decrease in the CD14 available to be detected on the surface of the M1 cells? This is unlikely if you are treating for long periods, but if you are treating for shorter periods of time, or with a high enough concentration of LPS, it is possible that the LPS-CD14 interaction is resulting in internalization of the receptor.

Hi Peter. I treat the cells with respective cytokines (LPS + INFg for M1 and IL4 for M2) for 24 hours. Coming to the concentrations, I use 100ng/ml of LPS + 20ng/ml of IFNg or 20ng/ml of IL4.

Generally receptors which are recycled get back to the surface before 24 hours and when I use LPS I use 1 ng/mL, but I know 100 ng/mL, while high, is a commonly used concentration. From that information, it does not seem like your treatments would decrease CD14 surface expression, although you might look into CD14 receptor recycling patterns.

LPS induces NO production in Raw cells. NO can modify protein to affect CD14 recycling.

www.google.com/patents/US20130184202