In vitro activation of murine peritoneal macrophages by recombinant YopJ: production of nitric oxide, proinflammatory cytokines and chemokines.
Recently it was reported that 3 μg/ml of recombinant YopJ induced apoptosis in murine peritoneal macrophages in vitro. However, in this study, we report the activation of murine peritoneal macrophages in vitro on treatment with sub-apoptotic dose of recombinant YopJ protein (1 μg/ml). The activation involves enhanced production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), IL-12, and IL-6. Production of NO and IL-6 was found to peak at 24 h of rYopJ treatment, whereas IL-12 and IFN-γ production peaked at 18 h of rYopJ treatment. Increased mRNAs expression of nitric oxide, IL-12, IL-6 and IFN-γ molecules, was also observed in rYopJ-treated macrophages by RT-PCR. rYopJ induced the enhanced activity of protein tyrosine kinases which was inhibited by pharmacological inhibitor genestein, wortmanin and H-7 suggesting the role of tyrosine kinases, PI3K and PKC in the above process. rYopJ also induced increased enhanced production chemokines MIP-1α, MCP-1, and RANTES in macrophages. Significantly, increased expression of TLR-2, TLR-6, MyD 88 and IRAK-1 was also observed by immunoblotting in rYopJ-treated macrophages. rYopJ induced production of NO, TNF-α and IL-6 was significantly inhibited in macrophages pretreated with pharmacological inhibitor wortmanin, genestein and H-7 demonstrating the probable involvement of protein tyrosine kinases in the above process.
Sodhi
Ajit
A
School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi 221005, India. ajit.sodhi@lycos.com
Pandey
Ashok Kumar
AK
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000276:immunology*; D018925:Chemokines / Q000096:biosynthesis; D016207:Cytokines / Q000096:biosynthesis*; D019833:Genistein / Q000494:pharmacology; D015151:Immunoblotting; D007249:Inflammation / Q000276:immunology; D007371:Interferon-gamma / Q000096:biosynthesis; D018664:Interleukin-12 / Q000096:biosynthesis; D015850:Interleukin-6 / Q000096:biosynthesis; D008262:Macrophage Activation; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000276:immunology; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D000077191:Wortmannin
D000730:Androstadienes; D001426:Bacterial Proteins; D018925:Chemokines; D016207:Cytokines; D015850:Interleukin-6; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C109227:YopP protein, Yersinia; D018664:Interleukin-12; D009569:Nitric Oxide; D007371:Interferon-gamma; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D000077191:Wortmannin