, 2001; Peng et al., 2001; Crabtree & Olson, 2002; Ryeom et al., 2003; Zhu et al., 2003). Calcineurin is especially important in T-lymphocytes. Its stimulation of IL-2 transcription here is a key mediator of T-cell activation and the subsequent autocrine loop

proliferation find more that is so critical to adaptive immune response. This pathway is so important that clinically, it is a major target of immunosuppressants such as cyclosporin A (CsA) and FK506 for transplant and autoimmune patients (Liu et al., 1991, 1992; Schreiber & Crabtree, 1992). Ryeom et al. (2003) investigated the role of RCAN1 in T-cells by assessing the induction of calcineurin-dependent proinflammatory genes in RCAN1-deficient mouse T-lymphocytes. They observed decreased interferon-γ (IFN-γ) production, lower proliferation, and an overstimulation of FasL leading to apoptosis in RCAN1-deficient T-lymphocytes. Also, we observed that the stimulation of Jurkat and primary T-lymphocyte signaling leads to isoform 4 induction in a calcium, calcineurin, and reactive oxygen species (ROS)-dependent manner that is accompanied by IL-2 induction (Narayan et al., 2005). Despite these T-cell studies, however, there has been a surprisingly

lack of reports on the involvement of RCAN1 in immune function. The aim of the presented studies is to further investigate the role of RCAN1 in immune response, extending the above prior studies in T-lymphocytes. Because T-cells are involved in adaptive immunity, Doxorubicin mw we decided to initially ever investigate the role of RCAN1 in the other major defense system, innate immunity, and chose macrophages for these studies. Subsequently, we examined the role of RCAN1 in vivo by assessing the impact of deleting RCAN1 expression on the susceptibility of mice to bacterial (Fransicella tularensis) infection, especially the production of proinflammatory cytokines because calcineurin is an important regulator of these genes. Mouse macrophage RAW 264.7 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium plus 10% heat-inactivated fetal calf serum containing 50–100 U mL−1 penicillin and 50–100 μg mL−1 streptomycin, and maintained in a humidified

incubator atmosphere of 95% air and 5% carbon dioxide (CO2) at 37 °C. Mouse primary bone marrow macrophages (BMM) were flushed from 3-month-old WT and KO mice femur bone marrow using RPMI media. After centrifugation, red blood cells were lysed and the bone marrow cells were resuspended in bone marrow media for macrophage differentiation in L-cell-conditioned media for 7 days. After a change of media, the cells were then counted and plated in whole bone marrow media and maintained in a humidified incubator atmosphere of 95% air and 5% CO2 at 37 °C. Cells were grown to 60–80% confluency at the time of agonist addition. These agonist treatments included Escherichia coli lipopolysaccharide, Staphylococcus aureus lipoteichoic acid (LTA), and S. aureus peptidoglycan, all obtained from Sigma (St.