I am trying to differentiate THP-1s to M1 and M2 macrophaes. After 48 hours of treatment with 25 nM or 50 nM PMA, I added 10 ng/ml IFN-g and 20 ng/ml IL-4 and IL-13 respectively but I could not see any significant TNF-a or IL-10 secretion respectively. I added those cytokines along with 100 ng/ml LPS, and now there are quite few amount of TNF-a and IL-10 but with no significant difference comparing to LPS alone. Can anyone help me with that?
Yes LPS will mask the effect of IL-4 and IL-13 cytokines. LPS will act synergistically and enhance IFNg effect.
Have you tried this experiment with primary human and/or mouse macrophages instead of PMA-differentiated THP1 cells? Becuase the THP-1 cells differentiated with PMA may not be a pysiologically relevant model system to study the M1 and M2 cells and PMA stimulation may also mask the effect of the cytokines you are using to differentiate the cells and this may depend on the time lapse before you add those cytokines (the time between PMA stimulation and cytokine stimulation). Adding LPS enhances the effect of cytokines either by priming the cells or simply providing another stimulation signal through TLR4. I would like to use primary macrophages to study M1 versus M2. If you trying to study the mechanisms for M1 and M2 differentiation, then cell lines may be a choice because of same background. Only a suggestion!
yes LPS is very powerful and can mask the effects of IL-4 or IFNgamma.
But it's also about the concentration. You could try really low conc. of LPS and test the effects of IFNg and IL-4.
See : Shuyi Zhang et al. Plos Negl. Trop. Dis. 2010, 4, e648 to convince yourself that the concept of M1 / M2 is far from being that simple dichotomy. In this paper the authors nicely show that once exposed to IFNg, bone marrow derived MØ and peritoneal MØ won't display similar gene expression (the same when exposed to IL-4). Accordingly, PMA differntiated THP-1 cells may not be the good tools to reach the aim you have. In most cases (again depending on the type of studied MØ), IFNg boosts LPS-induced TNF while IL-4 inhibits it. Not all MØ are able to produce IL-10. For example alveolar MØ don't (see Chignard et al.). So the results are highly dependent on the nature of the studied cell population and the compartment it derived from.
I have found that if you reduce the dose of LPS you can get IL-4 treated M2 macrophages to respond to LPS. You can't saturate them with LPS or they will polarize into a more M1 phenotype.
Make sure your IFNg is working properly by looking for rapid STAT1 phosphorylation. Also as stated by others, IFNg should strongly synergize with LPS unless you are using too high a dose of LPS
I had the same problem , PMA-induced activation, hope you serve this: http://www.ncbi.nlm.nih.gov/pubmed/24269601
and, the model:
http://www.ncbi.nlm.nih.gov/pubmed/23135314
@Gabriela thanks for your response, but I have already tried lower doses of PMA, and no difference. I think that it is more because of LPS. The paper about LPS induced THP-1 also have looked at the gene expression not cytokine secretion which I aim to gain. Have you seen IL-10 secretion from M2 polarized THP-1s?
Your LPS dose may be too high to see effects induced by IFNgamma. You will probably need to try 10-100 fold lower doses.
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