There can be several explanantions...

What type of cells are in your culture? Is mixed or just macrophages?

Complete experimental conditions (cell culture and treatment) could help in identification of a problem(s).

Dear Prof. Shen-An Hwang and Pof. Divaker Choubey ,

Here are the details:

I am using nHDF cell line (Human Dermal Fibroblast). After seeding cells (1*10^4) into 96 well plate, I grew them O/N before treatment. I did pre-treatment with only inhibitors while post-treatment after 1 hr with inhibitors plus recombinant TNF-alpha. After 24 hours of post-treatment, I collected medium from wells and centrifuged at 250xg for 5 min to get rid of cell debris. Then I used supernatent for cytokine detection by following the standard ELISA protocol given by company. I am using hIL-8 ELISA kit from Invitrogen and hIL-6 ELISA kit from Biolegend company. The final concentrations of inhibitors are 1uM, 10uM, 20uM while that of recombinant TNF-alpha is 1ng/ml.

I"m not familar with fibroblast cell lines, so this may or may not help.

The most likely answer is that this cell line just naturally produces a LOT of IL-6. This is seen quite often in cell lines, and I have seen it from macrophage/monocyte cell lines. No matter what you do, there is one cytokine that will not change. Granted, if htis is true, your complete negative controls (no TNFa and no inhibitor) will also have high IL-6.

If the above isn't true, then maybe your are looking at the inhibitor binding to something else that is stimulating IL-6. And 1uM is at saturation already.

There maybe other explanations... but these may be the most likely. Hopefully someone who has worked with this cell line can give you better answers.

Dear Prof. Shen-An Hwang,

Thank you for your reply. Actually, this cell line does not produce high IL-6 without stimulation (confirmed by negative control). By second case, if compound has non-specific binding resulting in IL-6 secretion then will not be there IL-6 for 20uM? But I couldn't see IL-6 for 20uM.

The only reason I'm thinking that it's binding to something else is because of the toxic effects. It's just strange for a specific receptor blocker that shouldn't induce a signal. (I'm assuming that you have done westerns of the TNFa signaling cascade to prove the this actually blocks signaling?) Since your 10uM is already toxic, maybe at 20uM the cells just can't produce any before the toxic effects takes over? Do you know by what mechanism the cells die? Have you looked at any of the cell death markers (either intracellular or the surface markers) with just the compound?

On the other hand, there are a lot examples of compounds that are stimulatory at lower concentrations and inhibitory at higher concentrations.

I'm also assuming that you have done titrations of the compound, and this is why you ended with the 1, 10 and 20uM?

Anyway... sorry that I'm haven't been too much help. It's always difficult to do this over the web since I don't have the complete picture of everything that you have tried or what has been done with the compound.

Dear prof. Shen-An Hwang,

I also assume that 20uM is too toxic for the cells to synthesize the cytokine. This compound is confirmed for TNF-alpha inhibition but death mechanism is not disclosed and this is also beyond my main goal. Thank you for your worthy views!

Sure, there can be several explanantions, as mentioned by others. A simple expanation is the quality of the used stimulus. Did you ever checked activity of the stimuli?