Abstract
The study aimed to investigate the relationship between tumor necrosis factor receptor-associated factor 6 (TRAF6) and a differentially mature dendritic cell (mDC) in collagen-induced arthritis (CIA) mice and to determine whether or not TRAF6 regulates the activation of an immature dendritic cell (iDC) and inhibits iDC maturation to induce immune tolerance. The mouse bone marrow stem cells were induced with recombinant granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant interleukin-4 (rmIL-4) to differentiate immature dendritic cells (DCs), which were divided into four groups with different maturation states: rmGM-CSF, rmIL-4; TNF-α; LPS; and FK506 group. The levels of the cell surfaces of CD80, CD86, and MHI-II were analyzed by flow cytometry to prove DCs at different levels of maturity. The expression of IL-12 in DCs at different maturation states was detected by enzyme-linked immunosorbent assay (ELISA). The expression of TRAF6 mRNA and protein in each group of DCs was detected by a reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. The results revealed that the differentiation of bone marrow cells into iDCs was significantly induced by cytokines (rmGM-CSF, IL-4). CD80, CD86, MHC-II were expressed in the four groups, and the difference between them was statistically significant (P<0.05). A higher degree of DC differentiation led to a gradual increase of IL-12 secretion in the four groups. The difference was statistically significant (P<0.05) for this secretion (group D, 10,620.73±276.73 pg/ml). The expression levels of TRAF6 mRNA were significantly higher in group D than those in the other three groups (P<0.01). Although there was no significant difference in the expression levels of TRAF6 mRNA between groups B and C, the expression levels of TRAF6 mRNA between groups B and C were higher than those of the control group. The TRAF6 protein expression was higher in group D than that in the other three groups (P<0.01), and the difference was statistically significant. There was a statistically significant difference in the TRAF6 protein expression between group A and groups B and C, but the expression in group C was higher than that in group B (P<0.01). In conclusion, the expression of co-stimulatory molecules gradually increased in the DCs of different maturation states, and the expression of IL-12, TRAF6 mRNA, and TRAF6 protein positively correlated with the degree of DC maturation. TRAF6 is important in iDC polarity and maturation.