When you stimulate human macrophages with bacterial lipopolysaccharides, early response genes, like TNF, are transcribted quite fast in between one hour. What's the reason for the delayed transcription of interleukin 6? Maybe the autocrin stimulation of macrophages by secreted TNF increases the interleukin 6 transcription? Or is it due to the delayed Myd88 independent TLR4 signaling pathway?
Do you have other genes that can serve as positive control for LPS stimulation? Maybe there is a problem with some of your conditions.
What kind of macrophages are you working with? MDM? monocytes? THP-1? There are differential transcription profiles depending on the cell type. I have observed monocytes to be the cells with lower transcriptional IL1b levels in response to LPS. You probably need to assess whether the autocrine loop of IL1b is the major inductor of IL1b gene upregulation in your cells. Have in mind that IL1b exists in the form of pro-IL1 and it is ready to release upon cleavage by caspase-1, that would mean no urgency for synthesis de novo.
This has been published in detail (last century: don't limit your PubMed search to the last five years ;-) by Sanceau et al. JBC 1995 and JI 1991, kinetics of IL-6 transcription in monocytic cells and cell lines depends on TNF...
Hope this helps,
Johan
IL-6 is not an early LPS response gene. You can get more information about LPS-induced gene expression from the following publications:
1) IFN-γ abrogates endotoxin tolerance by facilitating Toll-like receptor-induced chromatin remodeling. Chen J and Ivashkiv L Proc Natl Acad Sci U S A. 2010 Nov 9;107(45):19438-43 doi: 10.1073/pnas.1007816107 (For free access follow this link http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2984206/);
2) Ramirez-Carrozzi VR, et al. A unifying model for the selective regulation of inducible transcription by CpG islands and nucleosome remodeling. Cell. 2009;138:114–128. (For free access follow this link http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2712736/)
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