I want to stimulate a murine endothelial cell line (sEnd.1) and test my drugs of interest to quantify anti-inflammatory potential. It is accepted for endothelial cells to be treated with TNF-α and/or IL-1β, but I have also found protocols which suggest addition of IFN-γ, as well. Can anybody feedback on these stimulation protocols in more detail?
First question is what type of drugs you will test, and whether you'd show the mechanism, i.e. the pathways they interact with? If these are novel drugs and you do not know what to expect I would suggest to use several proinflammatory agents, TNFalfa, IL-1b, and bacterial LPS. Each one stimulate another pathway within cell what give you broader view on mechanism of tested drugs. I also suggest to use macrophages and/or neutrophils instead of endothelial cells since these are not typical cells for tests of antiinflammatory effect of drugs, and first it is better to conduct 2-3 experiments by using macrophages line and then switch and repeat your experiments by using primary (human) cells.
I considered using HuVECs but I wanted a cell type that I can easily manipulate (e.g. transfection, gene knock down). I was between TNF-a alone ot TNF-a/IL1b stimuli as early pro-inflammatory mediators produced during inflammation but LPS sounds a good alternative (also less biased).
Theo - try reading Ramirez SH J Neuroscience (2012 32: 4004-16 - see you next year DRG
First question is what type of drugs you will test, and whether you'd show the mechanism, i.e. the pathways they interact with? If these are novel drugs and you do not know what to expect I would suggest to use several proinflammatory agents, TNFalfa, IL-1b, and bacterial LPS. Each one stimulate another pathway within cell what give you broader view on mechanism of tested drugs. I also suggest to use macrophages and/or neutrophils instead of endothelial cells since these are not typical cells for tests of antiinflammatory effect of drugs, and first it is better to conduct 2-3 experiments by using macrophages line and then switch and repeat your experiments by using primary (human) cells.
Hey Theo, I agree with Thomas that TNFa and IL1b are gold standards. But we use other things such as ADP, Thrombin e.t.c to activate endothelial cells.
Thomas, thanks for interesting discussion. In case of immune cell model the signal obtained after stimulation will enable demonstration of anti-inflammatory effect of tested drugs. I am afraid that the signal from stimulated endothelial cells may be insufficient for Theo's study purpose. And sure, it would be appreciable to check crosslinks to other functions linked to inflammation, but I suggest to split the experiments and study it in the next step and for this purpose co-culture system consisting of endothelium and immune cells would be valuable. First, however, I would focus on the experiments that will clarify whether the drug exerts or not anti-inflammatory effect.
Hi,
raw cell line can be used as model which will give you idea about your drug. It will be one of the best suitable model to compare your result.
All the best
Hi, I agree @Katarzyna with using murine macrophage cells.
@Thomas, LPS induced Inflammation is best among all methods to induce inflammatory condition in cell lines.
@Theo, I hope you got clear clarification in between the discussion..
Best of Luck Theo
Hi all,
thanks so much for your valuable feedback. I feel I need to clarify a few things to promote the discussion. Indeed the test drug I want to study has been shown to reduce E-sel and IL8 in HuVECs stimulated with TNF-a, IL-1b and IFN-g. I want to reveal the mechanism that links these two interesting findings, so I was wondering whether stimulation of endothelial cells with TNF-a and/or IL-1b would activate similar effects on selectins and chemokine levels upregulation with the cocktail of TNF-a/IL-1b/IFN-g. I would be happy if I could use an unbiased stimulus of an early stage of inflammation and thats why I was thinking that TNF-a might be the most appropriate agent for that as for instance IL-1b is induced upon TNF-a production in immune cells.
In parallel, I m carrying out experiments with BMDMs where use of this test drug doesnt seem to have an antiinflammatory effect upon stimulation of this macrophage population with TNF-a/IFN-g (however, there are a few more expepriments I need to perform to fully assess the drug's effects). Co-culture experiments under flow is one of the many future plans I have to study interactions of leukocyte-EC in presence/absence of my drug.
Theo
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