4b, there are greater quantities of intranuclear NF-B of DCs in W/BF1 mice than in B6 mice under LPS stimulation

4b, there are greater quantities of intranuclear NF-B of DCs in W/BF1 mice than in B6 mice under LPS stimulation. producer of TNF-, while both Mo/M? and dendritic cells (DCs) produce TNF- in W/BF1 mice. Because the number of DCs is higher in W/BF1 mice, the main producers of TNF- in W/BF1 mice are thought to be DCs. Moreover, administration of anti-TNF- antibodies rescued the W/BF1 mice from death induced by LPS, suggesting that TNF- is crucial for the effect of LPS. Although there is no significant difference in the expression of Toll-like receptor-4 (TLR-4) on DCs between B6 and W/BF1 mice, nuclear factor kappa b activity of DCs from W/BF1 mice is augmented under stimulation of LPS in comparison with that of normal mice. These results suggest that the signal transduction from TLR-4 is augmented in W/BF1 mice in comparison with normal mice, resulting in the hyperproduction of TNF- and reduced survival rate. The results also suggest that 2′-Deoxycytidine hydrochloride not only the quantity of endotoxin, but also the host conditions, the facility to translate signal from TLR, and so on, could reflect the degree of bacterial infections and prognosis. gene, which resides on the Y chromosome and accelerates autoimmune diseases [7]. W/BF1 mice show not only lupus nephritis and anti-DNA antibodies but also cardiac infarction, leucocytosis, hypertension, anti-cardiolipin antibodies and anti-platelet antibodies [6,8,9]. We have reported previously that the number of DCs increases in not only peripheral blood but also various organs of W/BF1 mice, and that the increased number of DCs could be 2′-Deoxycytidine hydrochloride related to the production of autoantibodies, at least anti-platelet antibodies [10]. In this paper, we show that W/BF1 mice are more sensitive to LPS than normal mice because of hyperproduction of TNF-, and that DCs are the main producers of TNF- 2′-Deoxycytidine hydrochloride in W/BF1 mice. Materials and methods Mice Female NZW mice and male BXSB mice were purchased from SLC (Shizuoka, Japan), and were inbred to obtain male W/BF1 mice. Otherwise, male W/BF1 mice were purchased from SLC. Male W W/BF1 mice more than 12 weeks old, which showed more than (++) proteinuria (Albustix?, Bayer Medical Ltd, Tokyo, Japan), were used for this experiment. Age-matched male C57/BL6 mice were purchased from SLC. Administration of LPS and anti-TNF- antibody to mice The LPS, purchased from Sigma Chemical Co. (St Louis, MO, USA), was injected into the peritoneal cavity of the mice. Anti-TNF- antibody (03 mg; Pierce Biotechnology Inc., Rockford, IL, USA) was administered 10 min before the LPS. Histological analyses W/BF1 mice were killed 12 h after LPS injection (15 mg/kg) and their organs were then fixed with buffered formalin. Haematoxylin and eosin staining and terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) assay of the organs were carried out. For the TUNEL assay, we used the apoptosis detection kit (Takara, Otsu, Japan). Rabbit polyclonal to FBXO42 Reverse transcriptionCpolymerase chain reaction to examine mRNA expression 2′-Deoxycytidine hydrochloride of TNF- in spleen cells cDNA from freshly isolated and cultured spleen cells was prepared as described previously [11]. After adjusting the quantity of cDNA, polymerase chain reaction was carried out using the cDNA and glyceraldehyde-3-phosphate dehydrogenase-specific primers (Toyobo, Tokyo, Japan) or mouse TNF–specific primers (Maxim Biotech, Inc., San Francisco, CA, USA). Preparation of DCs and Mo/M? from spleen For the preparation of DCs and Mo/M?, a single-cell suspension was prepared from spleen cells of W/BF1 mice and B6 mice, as described previously [10]. The cells were incubated with anti-CD11c coated MACS? magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). After 15 min 2′-Deoxycytidine hydrochloride incubation at 8C, the cells were passed through a magnetic affinity cell sorter midi column (Miltenyi Biotec) to obtain a CD11c+ enriched.