br Materials and methods br

Materials and methods
Results
Discussion In the present study, we found that CysLTs (LTD4 and LTC4) induced expression and release of the pro-inflammatory cytokine IL-8 by activating the CysLT2 receptor. Our results further indicate that the effect is mainly mediated through the ERK1/2-Egr-1 pathway. IL-8 is one of the representative pro-inflammatory mediators (Thompson et al., 2008, Nakamura et al., 1991), and can be used as an indicator of pro-inflammatory responses mediated by CysLT2 receptors (Brochu-Bourque et al., 2011, Thompson et al., 2008, Uzonyi et al., 2006). IL-8 transcription and release can be induced by other pro-inflammatory mediators, such as TNF-α and IL-1β (Hirota et al., 1992, Koch et al., 1991, Rathanaswami et al., 1993). The present study confirmed that CysLT2 receptors mediate IL-8 production induced by CysLTs, lipid-derived pro-inflammatory mediators, and investigated the signal pathway underlying this response. To compare the role of CysLT1 receptor, CysLT2 receptor and GPR17 in IL-8 induction in response to LTD4 and LTC4, we constructed hCysLT1-HEK293, hCysLT2-HEK293 and rGPR17-HEK293 cell line, respectively. By immunocytochemical staining we found that CysLT1 and CysLT2 receptors were mainly expressed in the nucleus, and GPR17 was in the cytoplasm. Our results are consistent with previous reports that show that CysLT1 receptor is expressed in the nucleus of colorectal adenocarcinoma NMS-1286937 receptor (Nielsen et al., 2005) and vascular smooth muscle cells (Eaton et al., 2012), CysLT2 receptor in the nucleus of LAD2 cells, a human MC line (Jiang et al., 2007). Then we determined IL-8 induction in response to LTD4 and LTC4 in the transfected HEK293 cells and found IL-8 promoter activity is induced only in hCysLT2-HEK293 cells, not in hCysLT1- and rGPR17-HEK293 cells (Fig. 1C). LTC4 induced IL-8 production as well as Egr-1 expression in hCysLT2-HEK293 cells but not in pcDNA-HEK293 cells (Fig. 5). The effects of the selective CysLT2 receptor antagonist HAMI3379 (Fig. 6) further supported the CysLT2 receptor mediates LTC4 induced IL-8 production. Also, larger responses induced by LTC4 for activating the CysLT2 receptor in induction of IL-8 promoter activity compared with LTD4 at one single concentration (Fig. 1, Fig. 3), which is consistent with findings in the same cell type (Thompson et al., 2008). On the other hand, CysLT1 receptor might not be involved in IL-8 production because LTC4 had no effect in hCysLT1-HEK293 cells (Fig. 1C), and the CysLT1 receptor antagonist montelukast did not block the effect of LTC4 in hCysLT2-HEK293 cells (Fig. 7). However, the non-selective antagonist Bay u9773 did not inhibit LTC4-induced IL-8 production and other responses excepting reduction in Egr-1 protein expression. Because Bay u9773 is a partial agonist possessing agonist and antagonist activities on CysLT2 receptors (Ito et al., 2008, Wikstrom Jonsson et al., 1998), its effect is complex and may vary on different responses. Because GPR17 is expressed in the cytoplasm in the rGPR17-HEK293 cell line, we cannot exclude the possibility that GPR17 plays a role in IL-8 promoter activity response to LTD4 and LTC4. However, it is clear that LTC4-induced IL-8 production is mediated by CysLT2 receptors in the transfected HEK293 cells and can be used as a model for investigating its signaling.