I am planning to screen an array of potential anti-inflammatory and anti-oxidant agents in macrophage cell lines such as RAW 264.7 or J774. The cells will be stimulated with LPS to look for inflammatory cytokines and other mediators. However, I am apprehensive that there may be some compounds or agents which may not show any effect in LPS stimulated cells despite of the fact that they are actually antiinflammatory in nature and may have some other pathway of action (LPS independent).
I don't want to skip such compounds in the screening process, so I want to get some foolproof method which will eliminate any such possibilities..
I went across studies showing mycobacterium extract stimulation, but that too doesn't show better stimulation than LPS.
Please also comment about PMA stimulation and any other..
I am not sure if there exists any "fool-proof" approach to this, but have you considered cytomix (IL-1b + TNF-a + IFNg, at equal concentrations)? You could also use Poly I:C (synthetic analogue of viral RNA) which activates TLR3.
You could try any of the well described PAMPS. These like LPS induce 'innate activation' of macrophages. Poly IC is a good additional PAMP to try as it is Myd88 independent unlike LPS. Some people also use LPS in combination with IFN-gamma, often termed 'classical activation'. You might also wish to consider 'alternative activation' so you could use IL-4 or IL-4 in combination with LPS.
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Hey Debasis, You can use ligands for different TLRs, all of them can stimulate inflammatory response. LPS, LTA, flagelin, bacterial DNA or even entire bacterial lysates. Other then PAMS, you can also use DAMPS like HMGB1 or histones to stimulate RAW cells. These are milder but can induce some level of inflammatory response.
use msu crystals. we use here as an alternative to lps in thp1 cells with great results. same with atp salt or alum crystals, they all activate the nalp3-nlrp3 cascade, inflamasome formation, and immune cells tend to react strongly to it. expect loads of il-1beta and il-18. good luck.
You might be interested in this.
Rockel C and Hartung, T. Systematic review of membrane components of Gram-positive bacteria responsible as pyrogens for inducing human monocyte / macrophage cytokine release. Frontiers in Pharmacology, 2012, 3. DOI=10.3389/fphar.2012.00056. available at: URL=http://www.frontiersin.org/Journal/Abstract.aspx?s=926&name=predictive_toxicity&ART_ DOI=10.3389/fphar.2012.00056
Best,
Thomas
Please note that Raw 264.7 are deficient in ASC (universal inflammasome adaptor protein), so don't be surprised if you will not see any IL-1beta and IL-18 release.
Synthetic double strand RNA can mimic, to a certain extent, viral infections. Have used before and published the results. It induces nice IFN response in bronchial cells (primary culture and cell lines) via TLR3. Not sure about your cell lines. S
I have a long range of compounds to be screened so I would like to go for two to three parameters at most such as nitric oxide assay, since NO is readily released with the LPS stimulation, TNF-alpha (which is a prime cytokine and LPS stimulated). However, I am confused with the third one, for which I am going to use poly IC as suggested by Roberts. So, I am actually looking for the inflammatory mediator which is NOT produced by LPS stimulation but by poly IC alone. (Confusion is just because of many reports which suggest that both MyD 88 dependent and independent pathways result in the induction of same cytokines, though in varying concentrations).
Moreover, I would like to ask Gavrilin whether J774 is an appropriate cell line to use for IL1beta and IL18, whether alum stimulation will be best to check that, or poly IC will do...
Please suggest ....
Hey Debasis, I believe that indications above are substantial but so don't be surprised if depending of the stimulus the balance between pro-inflammatory or anti-inflammatory cytokines is different. I advise you to use LPS plus IFN-gamma or Poli:IC, but if the macrophages are derived from bone marrow cells you must induce M1 or M2 population before. Bone marrow-derived macrophages is better producer of NO when compared with citokines. This is my experience.
I never used J774 but it is published that these cells do release IL-1beta. Simply try. THP-1 cells are good for such screening purposes. Also, inflammasome activation is a two step process and you may need to use ATP as a complementary signal ( refer to our last article in JI, Ghonime et.al).
alum is good for NLRP3 inflammasome. I will be glad to discuss your experiments in details, if you wish.
Thank you Dr. Gavrilin.... Sure... I will be happy discussing my experiments in detail...
to mimic 'antibacterial' response you can use peptidoglycan or superantigen. for 'anti'viral' - poly I:C.
Not yet, I am still waiting for my compounds to reach.... however, I have thought of using LPS as the primary stimulant and poly IC and alum will be used to corroborate the results if I get similar results... Thank you with anticipation of your support in near future...
try sodium nitroprusside..
its a donor of NO, and hence induce NO mediated inflammatory responses.
Hi Saurabh, can you please send me any reference for sodium nitroprusside stimulation.
Hi Debasis,
Please find these links for SNP stimulation, i hope it will help
http://www.ncbi.nlm.nih.gov/pubmed/18507870
http://www.ncbi.nlm.nih.gov/pubmed/17067557
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