I appriciate any answer.

Hi shokoufeh ,of course no. Cytokines are pleiotropic ... some Studies designed to determine the role of TNF in the animal models or cell culture system using pure recombinant molecules have revealed that TNF never operates by itself, but instead operates within a network of cytokines. Each cytokine has a matching cell -surface receptor. Upon binding, intracellular signaling and gene expression regulations are altered, leading to the production of other cytokines, surface receptors, or feedback inhibition.

But for a better and definitive answer, you can look at the path-way studio site.

I think it will be useful to you

The answer is most likely no. (Although you can differentiate the sigaling by looking at specific intracellular signaling proteins for each cytokine.)

Every cytokine does not have it's own signaling pathway. The pathways are usually grouped by receptor family.

And while the direct intracellular signaling events may be somewhat separated... depending on how close to the receptor you are looking at... There are a lot of major signaling molecules that cross receptor families. And a lot of stuff ends up activating NFkb.

Dear, most cytokines are pleiotropic in nature and work in synergistic manner to perform their functions.

Thank you so much Behnoush Nasr Zanjani, Shen-An Hwang, Emmanuel Ifeanyi Obeagu.

I have another question. I work with a co/culture model. I stimulated the cells with IL6, I checked the expression of TNF-a , INF-a, and IL-8, none of them has been upregulated . When I look at IL-6 signaling path is differed from TNF-a. I am thinking that's why didn't see the upregulation of TNF-a. I appreciate any answer from you.

Totally agree with fantastic answer of Zanjani.

Frida Gorreja hi dear , Pathway Studio İs an exhaustive resource of easily searchable data from biology articles describing interactions between molecules, cell processes, and diseases. It also helps biological researchers import and analyze their experimental data using statistical tools.

Firstly you can creatr an account then choose pathway mammalplus or plant . you can get valuable information

Pls fallow this link : https://www.pathwaystudio.com/

What are the cells in the co-culture?

Amended on 15 March 2019

I must sadly say that humans has no JAK-STAT signal pathway; i.e., both Janus kinase/Just another kinase and Signal transducer and activator of transcription/STAT are absent in my human protein database (please see files; HepG2 Fucoidin and JMBT Alopecia). This may due to Species differences between Humans and Mouse and/or utilization of un-reliable PCR method. Further, Nerve growth factor/NGF is also absent in my human protein database. This also may due to Species differences. Further, MS, NMR, ELISA (Immunologic methods), Western blot, and Flow cytometry are also not quantitative. HPLC-photometry is uniquely quantitative, when utilized under the high-recovery conditions. RP-HPLC should be used under the Gradient elution (please see file; Lysozyme by RP-HPLC). HPLC-SEC should be performed by non-ionic detergent (please see file; JCB Fucoidin Transport). Direct utilization of photmeter without purification is also not quantitative due to violence against Beer-Lambert law; i.e., ELISA, MTT, Nanodrop, and Flow cytometry are non-quantitative.

Furthermore, IL-6 and IL-8 are absent in humans. IL-7 is present in humans. This is due to false-inducing Immunological methods (RIA and/or ELISA), un-reliable PCR method, and/or un-reliable Western blotting method.

Therefore, human liver cancer/hepatocellular carcinoma/HCC is occurring by the co-infection of HIV-1 and HCV. HIV-1 and HCV is very species specific to humans.

I have now estimating that some Cytokine receptors and some Hormone receptors (Growth hormone receptor, Prolactin receptor, and IRS2/Insulin receptor substrate 2) seem to use some of such tyrosine kinase proteins and transcription factors/TFs as shown below; i.e.,

Tyrosine-protein kinase ABL1/Proto-oncogene c-Abl/Abelson murine leukemia viral oncogene homolog 1, Receptor tyrosine-protein kinase erbB-2/Proto-oncogene Neu/CD340/Neuregulin-receptor complex 185 kDa, Ring3 protein/Really interesting new gene 3 protein/Bromodomain-containing protein 2, Transcriptional activator GLI3/Gli3 protein, Tyrosine protein kinase receptor EPH/Ephrin type-A receptor 1, Tyrosine-protein kinase ITK/TSK/Interleukin-2-inducible T-cell kinase/T-cell-specific kinase/Tyrosine protein kinase LYK, Tyrosine protein kinase Lyn/Lck/Yes-related novel protein tyrosine kinase, Tyrosine-protein kinase BTK/Bruton tyrosine kinase, Breast cancer anti-estrogen resistance 2/Transcriptional-regulating factor 1, Zinc finger protein 366/Dendritic cell-specific transcript protein, Zinc finger protein 614, and/or Zinc finger protein 84/Zinc finger protein HPF2.

By the way, human blood has following Cytokine-related proteins as assessed by reliable PDMD method; i.e.,

Interleukin-9 receptor/CD129/Cluster of Differentiation 129 at 4.5, Epidermal growth factor receptor/EGFR/Proto-oncogene c-ErbB-1/Receptor tyrosine-protein kinase erbB-1 at 3.6, Antisense basic fibroblast growth factor B/Nucleoside diphosphate-linked moiety X motif 6/Nudix motif 6 at 6.4, Tumor necrosis factor receptor superfamily member 1A/p55/TNF-R1/Tumor necrosis factor receptor 1 at 1.6, Metallothionein-1F at 11.3, Growth differentiation factor 8/GDF8/Myostatin at 11.9, C-C motif chemokine 17/CCL17/CC chemokine TARC/Small-inducible cytokine A17/Thymus and activation-regulated chemokine at 6.2, Fibroblast growth factor 13/Fibroblast growth factor homologous factor 2 at 4.0, Intestine and liver tetraspan membrane protein/Transmembrane 4 L6 family member 4 at 2.3, Unnamed protein product at 29.5, Putative testis-specific prion protein/Protein M8 at 5.0, Steroidogenic acute regulator protein, mitochondrial at 3.1, GTPase IMAP family member 7/Immunity-associated nucleotide 7 protein at 11.3, Interleukin-31 receptor subunit alpha at 3.5, and Protein Wnt5a at 9.1 μg/mg of serum protein, respectively.

Further, LC and HCC liver tissues have following Interferon and interleukin; i.e., LC tissue (with leprosy) has Interferon alpha-14/Interferon lambda-2-H at 1.3, Interleukin-32/Natural killer cells protein 4 at 1.5, and Interleukin-4 /B-cell stimulatory factor 1 at 0.97 μg/mg of tissue protein, respectively. HCC tissue (with PBC) has Interferon beta/Fibroblast interferon at 1.4, and Interferon alpha/beta receptor 1/IFN-alpha/beta receptor 1 at 9.5 μg/mg of tissue protein, respectively. LC tissue (named as No.6) has Interferon-induced protein with tetratricopeptide repeats 1/Interferon-induced 56 KD protein/IFI-56K at 2.0, and Interleukin-1 alpha/IL-1 alpha at 2.1 μg/mg of tissue protein, respectively. HCC, tissue (No.6) has Interferon-alpha receptor/IFN-alpha-Rec at 0.41, and Interferon delta-1 at 0.89, and Interleukin-7 receptor alpha chain/IL-7RA at 0.29 μg/mg of tissue protein, respectively. On the other hand, control normal liver (with pseudo liver cancer) has no IF and IL related proteins.

Thus, the human liver cancer/HCC has been proven to be induced by co-infection of virus (HIV-1 and HCV) through the results that "LC and HCC tissues has higher content of defending proteins of IF and IL than normal liver tissue" (please see file again; HepG2 Fucoidan).

By the way, your estimation that "Each human cytokine has its own signaling pathway" is surely right. Signaling pathway hypothesis seems to be depended onto five Jewish biochemists Drs. William Aaron Nierenberg, Gerald Maurice Edelman, Alfred Goodman Gilman, Martin Rodbell, and Rita Levi-Montalcini, who do not believe the presence of Species differences. Then, this Signaling pathway hypothesis seems to be not applicable to humans.

We have previously presented about the presence of the Racial differences (please see file; Racial difference BIN Urine). The possible explanation of this Racial gene differeces may have been occurred by the Geographical infectious differences by virus and bacteria has been still remained. Because, we have estimated that IDDM seems to be induced by Mumps rubulavirus/Mumps virus/MuV (Rubulavirus), which possesses Neuraminidase. Then, co-infection of Influenza virus/Flu and Avian avulavirus 1/Newcastle disease virus/NDV (Avulavirus) and/or Measles morbillivirus/Measles virus/MeV (Morbillivirus) also seem to be the cause of IDDM. Then, serum biotinidase of IDDM patient shows changed glycochain structure (changed substrate specificity), which suggests the infection of some virus may have previously been occurred in IDDM patients (About the changed glycochains in serum biotinidase of IDDM rat has presented by us; Kou Hayakawa et al., Effect of a Biotin-Supplemented Diet on Biotinidase Activity in Streptozotocin-induced Diabetic Rats, Vitamins (Japan), 72 (1998) 49-54). Syreptozotocin is a Glucosamin containing antibiotic. Sialic acid/Neuraminic acid of glycoprotein enzyme biotinidase is important to recognize biotinyl substrate (our unpublished observation and please see files; JMBT alopecia and Wide range of Biotin).

Therefore, we firstly has discovered that early changes in glycoprotein biotinidase in kidney is occurring (not albumin (non-glycoprotein) metabolism) suggesting that glycochain metabolism influenced by virus is already changed in IDDM (please see file; Dr. Terentyeva Urine BIN).

I am grateful to Dr. Shokoufeh Karimi (Department of Chemical and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden) for leading me to this important result.

Thank you very much for all answers. Dear Shen-An Hwang, the cell line are Caco2 and Hep G2.

Thanks a lots for your valuble knowledge that yoj are sharing with me . Still did not get a direct answer to my question. Can added IL6 to cell culture medium .upregulate TNF-a in cell culture model.

So... if you were using a macrophage/monocyte cell lines, I would have said yes, adding IL-6 will stimulate measureable TNFa production. Although this would also depend on the time point.

With these cell lines... I'm not too sure. It might not.

Further, added on 14 March 2019

I have determined and compared the defending proteins between non-cancer and cancer tissues.

Cancer (n=3);

HCC tissue (with PBC) has Interferon beta/Fibroblast interferon at 1.4, Interferon alpha/beta receptor 1/IFN-alpha/beta receptor 1 at 9.5, Mast/stem cell growth factor receptor Kit at 3.0, Growth hormone receptor/GH receptor at 12.4, and Vimentin at 11.5 μg/mg of tissue protein, respectively. Total defence proteins becomes to be 37.4 μg/mg of tissue protein.

HCC tissue (named as No.6; has survived) has Interferon-alpha receptor/IFN-alpha-Rec at 0.41, Interferon delta-1 at 0.89, Interleukin-7 receptor alpha chain /IL-7RA at 0.29, Prolactin receptor type 2 at 6.4, Transforming growth factor beta receptor type 3/TGF-beta receptor type III/TGFR-3/Betaglycan at 5.0 μg/mg of tissue protein, respectively. Total defence proteins becomes to be 12.99 μg/mg of tissue protein.

Hepatoma HepG2 (cultured without edible Japanese Fucoidin) has Insulin receptor substrate 2/IRS-2 at 15.04, Transforming growth factor beta receptor type 3/TGF-beta receptor type III/TGFR-3/Betaglycan at 2.7, B-lymphocyte activator macrophage expressed/SLAM family member 8; at 0.19, Tumor suppressor candidate 2 at 0.34, SH2 (Src homology 2) domain-containing protein 4A at 0.9, Nucleotide-binding site protein 1/PYRIN domain and NACHT domain-containing protein 1/Leucine-rich repeats (LRRs), ribonuclease inhibitor (RI)-like subfamily at 2.7, Macrophage mannose receptor 2/C-type mannose receptor 2/CD206 at 6.9, Leukosialin/Leukocyte sialoglycoprotein/CD43 at 1.4, Uromodulin/THP/Tamm-Horsfall urinary glycoprotein at 2.0, and Uromodulin-like 1/Olfactorin at 0.32 μg/mg of cell protein, respectively. Total defence proteins becomes to be 32.49 μg/mg of cell protein. 　　

Normal (n=4);

Healed hepatocyte HepG2 (cultured with edible Japanese Fucoidin at 0.102 mg/mL for 3 days) has EGF-like module-containing, mucin-like, hormone receptor-like 2 at 0.85, Interleukin-21/IL-21 at 0.09, and Stabilin-1 /Fasciclin, EGF-like, laminin-type EGF-like and link domain-containing scavenger receptor 1 at 10.1 μg/mg of cell protein, respectively. Total defence proteins becomes to be 11.04 μg/mg of cell protein. 　　

LC tissue (named as No.6) has Interferon-induced protein with tetratricopeptide repeats 1/Interferon-induced 56 KD protein/IFI-56K at 2.0, Interleukin-1 alpha/IL-1 alpha/Hematopoietin-1 at 2.1, Insulin-degrading enzyme/Insulinase/IDE at 4.0, and Amyloid beta A4 protein/Alzheimer's disease amyloid A4 protein/PN-II at 0.25 μg/mg of tissue protein, respectively. Total defence proteins becomes to be 8.35 μg/mg of tissue protein.

LC tissue (with leprosy) has Interferon alpha-14/Interferon lambda-2-H/IFN at 1.3, Interleukin-4/B-cell stimulatory factor 1 at 0.97, Interleukin-32/Natural killer cells protein 4 at 1.5, and Interferon-induced, double-stranded RNA-activated protein kinase/P68 kinase at 3.6 μg/mg of tissue protein, respectively. Total defence proteins becomes to be 9.47 μg/mg of tissue protein.

Normal liver (with pseudo liver cancer) has Interferon-regulated resistance GTP-binding protein Mx1/Myxovirus resistance protein 1 at 4.3 μg/mg of tissue protein. Total defence protein becomes to be 4.3 μg/mg of tissue protein.

Therfore, Cancer (HCC) tissues have higher amount of Defence proteins than Normal and LC tissues (p < 0.05; one-sided test, Mann-Whitney's U test; n1 = 3, n2 = 4). Thus, cancer tissue has higher amount of virus and bacteria than normal tissue. Then, the consideration that the hepatocellular carcinoma (HCC) is occurring by the co-infection of HIV-1 and HCV seems to be logically true. This logical truth has leaded us to find that Japanese edible Fucoidin and/or Kinu-Mozuku is surely the drug to liver cancer (please see file; Rat DEN Np-Fucoi). Fucoidin seems to up-rgulate the immune system or defending system against invading microbes (virus and bacteria).

I am grateful again to Dr. Shokoufeh Karimi (Department of Chemical and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden) for leading me to this important finding.

Dear Karimi,

Did you add soluble IL-6 receptor (sIL-6R) with IL-6 in CACO2 culture medium? If you have not done yet, I would like to recommend. IL-6 needs IL-6R expressed on the surface of cells or sIL-6R. In addition, you should also check the expression of IL-6R of CACO2.

All the best,

Kunihiko

Thank you very much Professor Kou Hayakawa for providing valuable knowledge.

Thanks Dr. Shen-An Hwang, do you have any suggestion why IL6 can stimulate TNF-a in macrophage and monocyte cell lines but not other cell lines?

Thank you Dr.Kunihiko Umekita.

No I have not added. But this cell lines express IL6, they might already have possibility of expression of the IL6R.What do you suggest?

Dr.Shen-An Hwang can you put the link for articles showing the upregulation of TNF-a in monocyte and macrophage cell lines after IL6 stimulation please?

Dear Karimi,

I would like to recommend to investigate the expression of IL-6 receptor or gp130 on your cell lines when you could not get any responses induced by IL-6.

Best wishes,

Kunihiko

Article Interleukin-6 trans-signaling increases the expression of ca...

In this article the presence of IL6R has been already identified.

I know that IL-6 is a pleiotropic cytokine , I dont know if it is what I see in my experiment, that cant upregulatet TNF-a, IL-8 and INF-a?

Dear Kunihiko Umekita, I hope I give you an proper snwer to your comment.

I take that back... I guess I must've remembered wrong. IL-6 can enhance TNF-a in the presence of something else (mostly LPS). That would explain why I kept thinking that macrophages might be more able to be stimulated with IL-6.

The only link I could find where using only IL-6 can stimulate TNFa is in vivo.

Article A Novel Small-Molecule Inhibitor Targeting the IL-6 Receptor...

Here's one in vitro... but apparently IL-6 may be more antagonistic to TNF production by itself without a primary stimulant.

https://ars.els-cdn.com/content/image/1-s2.0-S0925443917300108-gr4.jpg

Article IL-6 promotes M2 macrophage polarization by modulating purin...

Thanks Dr. Shen-An Hwang .

NO. IL-6 can induce TNF-a expression and vice versa. Even more other proinflammatory cytokines (e.g. IL-1) van also induce TNF-a and/or IL-6 expression nad chemokines were abloe to induce proinflammatory cytokine expression, too...and so on!

Dear Andreas Eisenreich Thank you. Can you send a link to a paper suggesting the fact above :IL6 can induce TNF-a.

e.g. involving RNA interaction :

Zhuang YT, Xu DY, Wang GY, Sun JL, Huang Y, Wang SZ.

Eur Rev Med Pharmacol Sci. 2017 Jan;21(2):302-309.

Other examples 4 an increasing activity:

-> Caiello I, Minnone G, Holzinger D, Vogl T, Prencipe G, Manzo A, De Benedetti F, Strippoli R. PLoS One. 2014 Oct 1;9(10):e107886. doi: 10.1371/journal.pone.0107886. eCollection 2014.

-> Ogawa H, Mukai K, Kawano Y, Minegishi Y, Karasuyama H.

Biochem Biophys Res Commun. 2012 Mar 30;420(1):114-8. doi: 10.1016/j.bbrc.2012.02.124. Epub 2012 Mar 3.

And:

Modulation of TNF-alpha mRNA production in rat C6 glioma cells by TNF-alpha, IL-1beta, IL-6, and IFN-alpha: in vitro analysis of cytokine-cytokine interactions.

Gayle D, Ilyin SE, Miele ME, Plata-Salamán CR.

Brain Res Bull. 1998 Oct;47(3):231-5.

Thank you so much Anders,

Really nice to see the involvement of IL-6 in inducing TNF-a.

I would like to give you more information that we have used IL-6 to stimulate the epithelial and liver cells and then we checked the TNF-a , IL-8 and INF-a. we used 10 ng /ml of IL-6 as final dose. but none of these cytokines has been upregulated. what do you suggest?

As you can see the impact of IL-6 on TNF-alpha depends on the experimental setting. Maybe, in your setting there is no impact at the concentration of 10 ng/mL or lower. Another reason may be the quality of the stimulus. HAve you checke activity of IL-6 used in your experiments as well as specificity of your analytical tools ans setting (qPCR, assays etc.)?

Moreover, did you checked successful transition of EpC to liver cells as well as expression of corresponding receptors, needed for IL-6-sependent signaling, and downstream effects (e.g. transcitional modification of potentail targets)?

Thank you so much Andreas Eisenreich for sharing your valuable knowledge with me. I have seen in many signalling pathways for cytokines that they have thier own pathways, and very few share partially small common path e.g. IL-10 and IL6....stat3 .... I am puzzeled if with seperate paths for TNF´-a and IL6 how and where in signalling pathway they effect each other. I know in cytokines network they may do that. Any idea?

This strongly depends on your experimental setting (cell types used, receptors and signaling pathways available etc.). One potential signaling pathway linking TNF-alpha and IL-6 expression is NFkappaB activation (e.g. see Clin Immunol. 2012 May; 143(2): 188–199 ). However, for further insights in this issue you have to check literature and advace your experimental setting.

Best wishes

Andreas

Dear Andreas, thank you so much. One final question as in my experiment I see upregulation of IL10 in cells treated with our samples. How can I mechanistically study this upregulation ?

I gratefully thank your reply in advance, Andreas Eisenreich.

In some disease settings (includig inflammation) IL-6 and IL-10 expression are linked (in the same direction). I hope this helps you finding adequate information from existing literature (e.g. see: Cell Mol Immunol. 2019 Mar;16(3):275-287. doi: 10.1038/cmi.2018.5. Epub 2018 Mar 19. ).

Best wishes Shokoufeh

Thank you so much Andreas.

I am really thankful :)

You`re welcome Shokoufeh.

Hi Shokoufeh,

IL-6 can trigger a signaling cascade involving different kinases and transcription factors, some of which are regulators of TNF-Alpha expression as well. In particular, IL-6 binding on its receptor transduce a JAK-dependent signaling leading to STAT3 activation (this is one of the more studied pathway but really there can be more downstream effectors). STAT3 is involved in several cellular processes, including inflammation, oncogenesis and cell proliferation. Other cytokines are able to induce a JAK-STAT signaling, similarly to IL-6. In fact cytokines commonly present redundant properties.

Thank you so much dear Valentino Bezzerri.

I believe that the body is so complicated and we are not able to understand exact mechanism. There are a number of active pathways and it is depending on different factors such as species, tissue, model (in vitro or in vivo), time of exposure and dosage of treatment. Don’t forget that gene expression is like taking a picture from tissue in a particular time, and cell is so intelligent, therefore it can be changed based on conditions. On the other hand, most of genes especially transcription factors are multifunction and have a small effect on phenotype, so it is difficult to say how and where they affect each other. Additionally, we should consider other factors such as non-coding RNAs. May be we have some up-regulated genes but we are not able to detect or measure their mRNA level because of microRNAs effect.

Dear Mojtaba Najafi thank you so much, I totally agree with you.

Some effects caused by IL-6 are similar to those observed under the action of IL-1 and TNF. However, the main effect of IL-6 is associated with its participation as a cofactor in the differentiation of B-lymphocytes, their maturation and transformation into plasma cells secreting immunoglobulins. In addition, IL-6 promotes the expression of the IL-2 receptor on activated immunocytes, and also induces the production of IL-2 by T-cells.

Thank you so much O. Raksha-Slusareva