Simultaneous inhibition of JNK and Gi activation through Ptx treatment abol ished the PMT effect and restored the T cell buy inhibitor activating ability of LPS treated cells after PMTwt stimu lation. These data indicate that PMT inhibits LPS mediated IL 12p40 expression via Gi mediated sig nalling and the activation of JNK. PMT stimulated JNK activation involves PI3 kinase Our next experiments addressed the question Inhibitors,Modulators,Libraries how PMT activates JNK. Recent publications suggest PI3 kinase as a mediator between Gi proteins and Ras MAP kinase activation. Interestingly, PMT activates PI3 kinase through dissociation of GB subunits from the Gi subunit. Here, we demonstrate by western blot analysis that PMTwt induces the phosphorylation of the PI3 kinase target Akt in primary monocytes.

To check whether the activation of JNK depends on PI3 kinase activation we blocked PI3 kinase using wortman nin and observed a reduction of JNK phosphorylation in PMTwt and most notably in LPS PMTwt stimulated cells. Furthermore, the inhibition of Gi activation by treatment with Ptx diminished the sti mulated Akt activation downstream of PI3 kinase and impaired Inhibitors,Modulators,Libraries the activation of JNK. This suggests that PI3 kinase mediates PMTwt sti mulated JNK activation and that the B subunits of Gi and other PMT activated G proteins induce the activa tion of the kinase. ELISA experiments confirmed that the inhibition of JNK activation through wortmannin could reduce the PMT mediated IL 12p40 suppression. Finally, the inhibition of both, JNK and Gi mediated signalling Inhibitors,Modulators,Libraries through Ptx, totally restored the LPS induced production of the cytokine.

Performed MLRs confirmed the impact of the restored IL 12 production on the T cell proliferation. Taken together we propose the following model LPS stimulates TLR4 mediated activation of the NF?B pathway and modulates the production of TNF. IL 6 and IL 12p40. PMT inhibits the production of IL 12p40 through Gi mediated inhibition of adenylate cyclase and cAMP accumulation as well as by PI3kinase Inhibitors,Modulators,Libraries Akt and JNK activation mediated by the dissociated GB sub unit. Discussion Antigen presenting cells are crucial for the first line host defence and act as mediators between innate and adap tive immunity. One major pathway of these cells involves TLR mediated signalling events, which can detect and react to extracellular as well as intracellular microbial components.

To be effective, immune responses need the crosstalk between TLR mediated and other sig nalling cascades that play a role in immune responses or other cellular pathways, respectively. These interactions can Inhibitors,Modulators,Libraries have synergistic effects but can also antagonise responses in order to prevent excessive inflammation. thoroughly To modify and presumably escape the hosts immune re sponse pathogens have developed various strategies to interfere with signalling cascades of the host.