Here, we investigated how the activation of heterotrimeric G proteins through the bacterial toxin PMT modulates TLR4 mediated activation of human blood derived monocytes and how this potentially affects cells of the adaptive immune system such as T cells. Our data on the expression of MHC and costimulatory B7 family members show that PMT alone does not acti vate monocytes by itself often and only slightly enhances LPS triggered cell activation. However, PMT is able to modu late the TLR4 mediated cytokine production of these cells significantly leading to an almost complete block of IL 12p40 production. Bioactive IL 12 is a potent activa tor of natural killer cell and T cells and, together with the B7 CD28 interaction between antigen present ing cells and T cells, IL 12 has a strong synergistic effect in inducing T cell proliferation.
Our data show that due to the absence of IL 12, PMT suppresses Inhibitors,Modulators,Libraries T cell activation induced by LPS activated hBDMs. This effect strictly depends Inhibitors,Modulators,Libraries on IL 12, as activity could be restored by adding IL 12 containing supernatants of LPS stimulated hBDMs or recombinant IL 12 to the mixed lymphocyte reaction. Although IL 10 is a known sup pressor of IL 12 activity, a dominant role of IL 10 could be excluded, since PMT only slightly enhanced the LPS induced IL 10 production. Bagley et al.demonstrated that PMT also suppresses LPS stimulated IL 12 release of human macrophage derived dendritic cells and they speculate that this block ade is caused by Gq mediated Phospholipase C B activa tion and subsequent intracellular calcium mobilization, but they did not further investigate this.
For human monocytes Inhibitors,Modulators,Libraries we suggest that PMT prevents TLR4 mediated IL 12p40 Inhibitors,Modulators,Libraries production through Gi and B mediated signalling cascades, respectively. PMT activates Gi and thereby inhibits adenylate cyclase activity and cAMP accumulation, independently of Gq and G12 13. Our data point to Gi signalling as the Gi agonist mastoparan mimicked the suppression of LPS induced IL 12p40 production. Recently it became clear that data obtained from experiments using mastoparan have to be interpreted with care as mastoparan can bind the toxic lipid A portion of the LPS molecule. Nevertheless, as in our studies mastoparan only blocked LPS stimulated IL 12p40 production and did not block TLR4 mediated IL 6 and TNF expression, we assign the result primarily to Gi mediated signalling.
Additionally, the Gi inhibi tor Pertussis toxin reversed the PMT mediated blockage of IL Inhibitors,Modulators,Libraries 12 release and restored the T cell activating ability of LPS treated monocytes. In the past it has been shown that both Gi and Gs, although having opposite function and acting as negative regulators for each other, are able to downregulate IL 12 production. This suggests that alterations in intra cellular cAMP metabolism in general play a role in modulating the IL 12 production. selleckchem Carfilzomib The molecular mech anism, however, is only poorly understood.