Also, at equivalent amounts, whole gram-negative bacteria may deliver more lipopolysaccharide to the macrophage as compared with free lipopolysaccharide and would also stimulate other pathways (Nau et al., 2003). Nonetheless, it is striking CDK assay how strongly every one of our treatments induced RCAN1-4, suggesting a common downstream pathway and mechanism of induction. This appears to involve calcium and ROS because both mediate gram-negative lipopolysaccharide effects (Figs 2 and 3), and we also observed calcium and ROS involvement in limited studies with our gram-positive agonists (data not shown). It is also important to note that commercial LTA and peptidoglycan

have been reported to contain TLR2 contaminants. These reports include evidence that lipoprotein-like compounds are responsible for the activity of the LTA fraction of Enterococcus hirae and S. aureus (Hashimoto et al., 2007); that bacterial compounds reported as TLR2 agonists are more likely contaminated with highly active natural lipoproteins and/or lipopeptides that are the true TLR2 agonists (Zähringer et al., 2008); and that proteoglycan

effects are actually due to the presence of LTAs (Travassos et al., 2004). Thus, we do not know for sure how much contribution either LTA or peptidoglycan provides in RCAN1-4 induction in our studies. Nonetheless, these contaminants, if present at significant levels in our peptidoglycan and LTA, are Selleck Ivacaftor still acting as TLR2 ligands, further

supporting that RCAN1-4 is induced by TLR2 stimulation. The in vivo studies revealed a strong effect of knocking out RCAN1, namely, cytokine induction in the lung. All of these same cytokines except IL-6 were also increased in day 7 spleen (data not shown). Interestingly, the cytokines analyzed (MCP-1, TNF-α, IL-6, and IFN-γ) can be upregulated by the calcineurin–NFAT pathway (Kiani et al., 2001; Satonaka et al., 2004; Keller et al., 2006), although these elevations appear to be cell and condition dependent as other systems show different responses (Ryeom et al., 2003; Keller et al., 2006). Nonetheless, our observation that MCP-1, TNF-α, IL-6, and IFN-γ are upregulated in the KOs are consistent with those reports that demonstrate their induction by this pathway Unoprostone because in the KO, the loss of RCAN1 and its inhibitory action would lead to elevated calcineurin activity and stimulation of target cytokine expression. Before our studies, there has only been limited characterization of RCAN1 regulation of cytokine expression. Specifically, Ryeom et al. (2003) observed decreased IFN-γ production in RCAN1 KO T-lymphocytes, although this was associated with a dying (FAS overexpressing) phenotype. Interestingly, they also observed that this effect was specific to Th1 T-helper cells, and that these cells had lower activation thresholds for IL-2, IFN-γ, and IL2 receptor as compared with WT cells.