Glucocorticoid induced tumor necrosis factor receptor (GITR) is a new member of the tumor necrosis factor^nerve growth factor receptor superfamily of which the function has not been well studied. The extracellular domain of GITR was produced in Escherichia coli and purified as a single band of predicted M r of 18.0 kDa. GITR and GITR ligand were expressed constitutively on the surface of Raw 264.7 macrophage cell line and murine peritoneal macrophages. An extracellular domain of GITR can activate murine macrophages to express inducible nitric oxide synthase and to generate nitric oxide in a dose-and time-dependent manner. ß

The aqueous extract of Mori Fructus (MF) exerts a change of phenotype and a cytoprotective effect in macrophages. The present study was carried out to investigate the immunomodulating activity of MF on the expression of nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), co-stimulatory molecules and also interferon-gamma (IFN-gamma) in macrophages and splenocytes. Toll-like receptor 4 (TLR4) is a promising molecular target for immune-modulating drugs. It was hypothesized that one possible upstream signaling pathway leading to immunoregulation of MF may be mediated by TLRs. Multiple signaling molecules (NF-kappaB, ERK1/2, p38 and JNK) of the TLR4 signaling pathway were also detected. It was found that MF increased NO production and TNF-alpha secretion in RAW 264.7 and peritoneal macrophages, co-stimulatory molecules expression in peritoneal macrophages and IFN-gamma expression in splenocytes. Further studies indicated that MF could significantly induce the phosphorylation of signal molecules of MAPKs and the degradation of IkappaBalpha which finally led to the activation and nuclear translocation of nuclear factor-kappaB (NF-kappaB) for the target gene expression. All those notions disclosed that the aqueous extract MF is a new TLR4 activator, which induces a Th1 immune response as a consequence of induction of cytokines secretion, especially TNF-alpha and IFN-gamma.