Femur bone marrow cells from these KO mice and WT littermates were isolated and differentiated into macrophages using L-cell-conditioned media (note: these type of cells were also used for the Fig. 1d study). These cells were then used to assess respiratory burst, an important functional activity of macrophages. Comparison of KO and WT bone marrow-derived macrophages revealed a modest increase in respiratory burst activity in the KO cells (Fig. 9). In these studies, we provide some of the first evidence that RCAN1 is involved in macrophage response, and that it regulates cytokine production in vivo. Combined with previous studies Inhibitor Library clinical trial by Ryeom et al. (2003) demonstrating its importance in T-cell activation and apoptosis,

and in our laboratory demonstrating its involvement in T-lymphocyte response to anti-CD3 plus anti-CD28 antibodies (Narayan et al., 2005), it is now clear that RCAN1 plays an important role in immune function. It is surprising that there have been so few studies to date on RCAN1 Acalabrutinib and the immune system in light of the known importance of calcineurin in T-lymphocyte activation, cytokine production, and apoptosis (Schreiber & Crabtree, 1992; Shibasaki & McKeon, 1995; Zhang et al., 1996; Rusnak & Mertz, 2000;

Hogan et al., 2003; Ryeom et al., 2003; Narayan et al., 2005). Nonetheless, the above studies establish RCAN1′s role in immunity and further suggest its importance in other immune cell types including B-lymphocytes, dendritic cells, natural killer cells, and regulatory T-cells. Numerous inducers of RCAN1-4 expression have now been described including, most notably, calcium-elevating agents (Crawford et al., 1997; Kingsbury & Cunningham, 2000; Lin et al., 2003) and cell receptor agonists as summarized (Van Riper et al., 2008). Interestingly, Exoribonuclease the cell receptor agonists that have been reported are quite varied including anti-CD3 and anti-CD28 to stimulate

T-cell activation; vascular endothelial growth factor (VEGF) to stimulate endothelial cell VEGF receptors; and angiotensin to stimulate angiotensin on rat smooth muscle cells (Mitchell et al., 2007). Our studies reveal a new class of cell receptor able to stimulate RCAN1-4 induction: toll-like receptors (TLRs). These receptors are well-known mediators of both gram-negative and gram-positive bacterial components (Aderem & Ulevitch, 2000; Takeda & Akira, 2004). TLR4 receptors are known to respond to gram-negative bacteria such as E. coli and their lipopolysaccharide endotoxin, whereas TLR2 respond to gram-positive bacteria such as S. aureus and their bioactive cell wall components LTA and peptidoglycan. These components prime the host and allow for the immune defense to build (Aderem & Ulevitch, 2000; Hume et al., 2001; Takeuchi & Akira, 2001; Takeda & Akira, 2004). It is difficult to compare each given treatment because the concentrations between these TLR agonists as purified components vs.