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.