Dexamethasone impairs the differentiation and maturation of murine dendritic cells by Toll-like receptor 4-nuclear factor-kappaB pathway.

BACKGROUND: Recent studies have demonstrated that dexamethasone (DEX) interferes with immune responses by targeting key functions of dendritic cells (DCs) at the earliest stage. However, the cellular and molecular mechanisms are still incompletely understood. This study aimed to explore the possible mechanisms by investigating the roles of DEX on differentiation, maturation & function of murine DCs and the effects of DEX on DCs via Toll-like receptor 4 (TLR4)-nuclear factor (NF)-kappaB mediated signal pathway. METHODS: Immature DCs (imDCs) were cultured from murine bone marrow (BM) cells. We added DEX into culture medium at different time. The expression of CD11c, CD86 and I-A(b) (mouse MHC class II molecule) was determined by flow cytometry. We determined the expression of NF-kappaB and its inhibitory protein I-kappaBalpha by electrophoretic mobility shift assay (EMSA) and Western blotting, respectively. The productions of interleukin (IL)-12p70 and IL-10 in cell culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: DEX impaired differentiation of DCs from murine bone marrow progenitors, and inhibited lipopolysaccharide (LPS) induced maturation of DCs. DEX significantly inhibited NF-kappaB expression of normal DCs, the higher the DEX concentration or the longer the DEX treatment time, the more obvious the effect. However, DEX had little effect on LPS-induced NF-kappaB activation, and partially impaired LPS-induced I-kappaBalpha degradation. DEX significantly decreased LPS induced IL-12p70 production by DCs. Interestingly, our results showed a synergistic effect between DEX and LPS on the production of IL-10 by DCs. CONCLUSIONS: DEX inhibits the differentiation and maturation of murine DCs involved in TLR4-I-kappaB-NF-kappaB pathway, and also indirectly impairs Th1 development and interferes with the Th1-Th2 balance through IL-12 and/or IL-10 secretion by DCs.

[The analysis in the expression of Toll-like receptor 4 mRNA and the effect of dexamethasone on mice dendritic cells by real-time PCR].

AIM: To establish a SYBR Green I quantitative real-time PCR method for detecting the expression of the TLR4 mRNA of mice and to monitor the dynamics for TLR4 mRNA level of mice bone marrow-derived dendritic cells (DC) in the process of the maturation and the effect of dexamethasone (DEX) on TLR4 expression. METHODS: (1) DC from mice bone marrow were induced by cytokines and separated by magnetic beads. (2) The combined plasmid pUCm-T/TLR4 and pUCm-T/beta-actin for the standard materials in the real-time PCR was reconstructed. (3) The dynamics for the express TLR4 mRNA of DC cultivated in vitro for 4, 6, 8, 10, 12 days was detected respectively. (4) The TLR4 mRNA expression between DC treated with or without DEX was detected and compared. RESULTS: (1) The DC with the purity over 90% were fully separated in success. (2) A SYBR Green I quantitative real-time PCR method for analyzing the TLR4 mRNA expression level of the mice was successful established. (3) TLR4 mRNA level was stable early during the culture of DC and then rise obviously at last. CONCLUSION: There exists a close relationship between the level of TLR4 mRNA and the period of maturation of DC and the expression of TLR4 could be increased by DEX.

[Effect of rapamycin and dexamethasone on differentiation and maturation of murine bone marrow-derived dendritic cells].

AIM: To observe the effect of rapamycin (Rap) and dexamethasone (Dex) on differentiation and development of murine bone marrow-derived dendritic cells (DC) in vitro. METHODS: DC cells generated from C57BL/6 murine bone marrow cells were induced by GM-CSF and IL-4. During the course, Rap or Dex was added to the culture and the cells were then stimulated by LPS. (1)Morphology development of DCs was observed by inverted microscope and scanning electron microscope.(2)The cells were analyzed by flow cytometry (FCM) to determine the proportion of CD11c(+) cells and the change of CD86 and MHC class II molecule.(3)The influence of DCs treated by Rap or Dex on the allogeneic T cell proliferation was studied by one-way MLR. RESULTS: (1)The morphology of DCs maintained in a durable state of immaturity after Rap or Dex pretreatment.(2)The expression of CD11c and MHC-II slightly decreased but CD86 dramatically reduced on Rap-treated DC cells. There was close relationship between the expression of CD11c on Dex-treated cells and the dosage of Dex. The surface expression of CD86 and MHC-II dramatically reduced on Dex-treated DCs. Moreover, DCs treated by Rap or Dex were both able to resist the maturation triggered by LPS.(3)Bone marrow-derived DCs cultured with Dex or Rap had a lower stimulatory effect on allogeneic T cells compared with that of mature DCs. CONCLUSION: Rap and Dex could keep DCs in durable immaturity. Compared with Dex, which inhibited the expression of co-stimulatory molecules, Rap hardly influenced the differentiation of DCs and the expression of MHC class II molecules.

Effects of oral administration of type II collagen on adjuvant arthritis in rats and its mechanisms.

OBJECTIVE: To investigate the effects of oral administration of type II collagen (CII) on adjuvant arthritis (AA) in rats and its mechanisms, and to compare the effects of CII with those of the Chinese traditional medicine Tripterygium Polyglycoside administered similarly. METHODS: Arthritis was induced in rats by immunization using Freund's complete adjuvant (FCA). After feeding rats either soluble CII or Tripterygium Polyglycoside, changes in degree of articular swelling and articular histological findings were observed in AA rats. Some correlative immunological indexes were measured, including delayed type hypersensitivity (DTH) reaction, anti-collagen and anti-Mycobacterium tuberculosis (MT) antibody in serum, and levels of IFN-gamma and TNF-alpha in articular steep in rats. RESULTS: Oral administration of CII was able to alleviate both distinctly articular and general symptoms in AA rats, suppress synovium hyperplasia and inflammatory cells infiltration in arthrosis capsule. The effects brought about by CII were stronger than those by Tripterygium Polyglycoside. Oral administration of CII inhibited antigen-specific immune response, such as DTH and antibody reaction to CII. In addition, the expression of IFN-gamma and TNF-alpha in joints were locally downregulated. CONCLUSIONS: The therapeutic effect of oral administration of CII is obvious on adjuvant arthritis in rats. Its remedial mechanisms are likely related to the downregulation of both IFN-gamma and TNF-alpha, and the suppression of cell immunity.