Role of damaged mitochondria in Vibrio vulnificus induced dendritic cell apoptosis

AIM： To investigate the role of damaged mitochondria in dendritic cell (DC) apoptosis induced by Vibrio vulnificus (Vv) and its possible mechanism. METHODS：DC2.4 cells were co-cultured with Vv 1.1758 strain. Fluorescent probes DCFH-DA and Fluo-8-AM were used to detect reactive oxygen species (ROS) and intracellular Ca2+ concentration in the invaded cells, respectively. The cellular apoptotic rates and mitochondrial membrane potential (Δψm) were measured by flow cytometry. The expression of nuclear factor-kappa B p65 (NF-κB p65) and tumor necrosis factor-alpha (TNF-α) was detected by Western blotting. RESULTS： Vv 1.1758 induced DC2.4 cell apoptosis. Vv 1.1758 bacteria invaded into the DC2.4 cells by binding with cellular membrane though the end of the body. In the invaded DC2.4 cells, the visible mitochondrial damage, elevated ROS and intracellular Ca2+ levels, and declined Δψm were presented. After 1 h of co-culture, NF-κB p65 began to rise and reached the peak at 5 h, and then slightly decreased at 6 h. The TNF-α level increased after 2 h of co-culture and reached the peak at 6 h. CONCLUSION： The damaged mitochondria play an important role in DC apoptosis induced by Vv, and its possible mechanism may associate with the elevation of ROS and intracellular Ca2+ level, and the declined Δψm. Meanwhile, NF-κB p65 and TNF-α are potential critical signaling molecules in the process of apoptosis.     

Effects of progesterone on the maturation and immunologic function of dendritic cells from human peripheral blood

AIM: To study the effects of progesterone (P₄) on the maturation and immunologic function of dendritic cells (DCs) from human peripheral blood. METHODS: Cultured DCs were treated with P₄ at doses of 10-7 mol/L and 10-6 mol/L. The morphologic changes were observed under the scanning electronic microscope. The immunophenotypes of DCs in control and treated groups were analyzed by flow cytometry. IL-10 and IL-12 production in culture supernatant was examined by ELISA assay. The capability of the stimulatory activity of the DCs on allogeneic T cells in mixed reaction was tested by incorporation of ［3H］-TdR. RESULTS: Compared with control group, cultured DCs in the presence of P₄ displayed less dendritic pseudopod, expressed low levels of MHC-II, CD40, CD80 and CD86, and exhibited weakly activity in stimulating the proliferation of allogeneic T cells. Increase in IL-10 production and decrease in IL-12 production were observed. CONCLUSION: P₄ exerts negative effect on the maturation and immunologic function in dendritic cells from human peripheral blood.     

Function of dendritic cells in cord blood

AIM: To explore the function of dendritic cells in cord blood.METHODS: Dendritic cell precursor subsets pDC/pDC(CD11c+CD123- /CD11c-CD123+) in cord blood and adult peripheral blood were analyzed gated by CD34-Lin- HLA-DR+ cells and the levels of IL-12p40,IL-10,IFN-γ,IL-4 in the serums were tested by ELISA.RESULTS: The level of IL-12p40 in cord blood serum was higher than that in peripheral blood.pDC1/pDC2,IL-10,IFN-γ,IL-4 in cord blood were similar to that in peripheral blood.CONCLUSION: Function development of dendritic cells in cord blood may be consummate.     

TGF β1 inhibits the maturation of dendritic cells and down-regulates TLR4 expression

AIM: To investigate the effects of transforming growth factor β1 (TGF-β1) on murine-derived dendritic cells (DC). METHODS: Murine bone marrow cells were cultured with GM-CSF and TGF-β1 to develop TGF β-DC. Then they were stimulated by lipopolysaccharide (LPS). Their phenotypes were assessed by flow cytometry (FCM). The allogeneic stimulating capacity of DC was measured by mixed lymphocyte reaction (MLR) using BrdU ELISA method. IL-12 p70 protein was detected by ELISA and the expressions of Toll like receptor 4 (TLR4) on DCs were measured by semi-quantitative RT-PCR and FCM. RESULTS: Compared to immature DC (imDC) cultured with GM-CSF alone, the expressions of CD80, CD86, I-Ab and CD40 in TGF β-DC were lower. The TGF β-DC was resistant to maturation by LPS. Maturation resistance was evident from a failure to up-regulate CMs, to stimulate larger T cell proliferation and to increase secretion of IL-12 p70. Down-regulation of TLR4 expression on TGF β-DC was also found. CONCLUSION: TGF-β1 inhibits the expression of co-stimulatory molecules on DC. It is resistant to maturation stimulus (LPS) and might be linked with TLR4 down-regulation.     

Effect of propolis water extract on apoptosis of human umbilical vein endothelial cells in vitro

AIM: To explore the effect of water extract propolis (WEP) from Taishan on apoptosis of human umbilical vascular endothelial cells (HUVECs) induced in vitro by tumor necrosis factor-α (TNF-α). METHODS: HUVECs were collect by digestion-perfusion and cultivated. TNF-α at concentration of 50 μg/L was administrated to induce the apoptosis of HUVECs. After injury, HUVECs were treated with WEP at concentrations of 50, 100, and 200 mg/L, respectively, for 24 h. Apoptosis was evaluated by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) and flow cytometry (FCM). RESULTS: Apoptosis index in injured group was significantly higher than that in control group, and decreased significantly after treating with WEP at concentrations of 50, 100, and 200 mg/L, respectively (P<0.01). CONCLUSION: WEP may be useful for protection of vascular endothelial cells by inhibiting apoptosis.     

Effect of adriamycin combined with sensitized dendritic cells on cervical tumor-bearing mice

AIM：To explore the immunotherapeutic effect of adriamycin (ADM) combined with frozen-thawed antigen-sensitized dendritic cells (DCs) on cervical tumor-bearing mice. METHODS：The U14 cervical cancer model of Kunming mice was established by subcutaneous implantion of U14 cells in axillary fossa. DCs vaccine was prepared by U14 cervical cancer cell frozen-thawed antigen-sensitized mouse bone marrow-derived DCs. Mature phenotype of sensitized DCs was identified by flow cytometry. Tumor-bearing mice were randomly divided into 4 groups and treated for 3 cycles with PBS (control), DCs vaccine, ADM and ADM combined with DCs vaccine, respectively. The tumor volume was evaluated. The tumor weight and the levels of interleukin-2 (IL-2), IL-12 and interferon γ (IFN-γ) in the serum were determined by ELISA on the 21st day. RESULTS：Cancer cell frozen-thawed antigen-sensitized DCs had higher expression levels of CD11C, CD80 and CD86. The volume and weight of the tumor in ADM combined with DCs vaccine group were less than those in ADM group, DCs vaccine group and control group. The tumor inhibitory rate in combination group was higher than that in the other 3 groups. Compared with the other 3 groups, the serum levels of IL-2, IL-12 and IFN-γ in combination group significantly increased. CONCLUSION：ADM combined with tumor antigen-sensitized DCs vaccine can strengthen the animal antitumor immune response and effectively inhibit the growth of tumor in cervical tumor-bearing mice.     

Role of dendritic cells in myocardial infarction and cardiac remodeling

Myocardial infarction （MI） is one of the leading causes of death worldwide. One of the primary reasons is that the rupture of atherosclerotic plaque leads to the formation of thrombosis, and then interrupts the coronary blood flow, thus finally causing the death of myocardial cells and cardiac dysfunction. A large number of researches have revealed that dendritic cells （DCs） play an essential role in immune inflammatory responses in the occurrence and development of atherosclerosis and MI. This article reviews the role of DCs in atherosclerosis, myocardial ischemia/reperfusion injury and cardiac remodeling after MI, and shows the potential values of DCs as an immunotherapeutic strategy for MI.     

Effect of heat shock protein on dendritic cell maturation

AIM: To study the effect of heat shock protein (HSP) on the maturation of dendritic cells (DCs) and observe the morphological changes of DCs dynamically. METHODS: The HSP (gp96)-peptide complex was purified from the tissues of hepatocellular carcinoma. Hepatoma cells were treated by heat shock for preparation of the HSP expressed on cell surface and then marked with DiI fluorescence. Immature DCs from peripheral blood mononuclear cells were cocultured with two types of HSPs. The morphological changes of DCs were observed dynamically and the effects of HSPs on the maturation of DCs analyzed by Flowcytometer. RESULTS: The morphological changes and the processes of antigen capture of DCs cocultured with DiI marked tumor cells were well showed. Four ways to capture antigens of DCs were observed, including direct contact, besieging, forming bubbles and extending pseudopodia with bubbles on the terminals. Results also indicated that both types of HSP could promote the maturation of DCs. CONCLUSION: DiI is a good fluorescent dye suitable for the morphological studies of DCs. Four ways for DCs to capture antigens were indicated in this paper. Different types of HSP, purifed from tumor tissues or expressed on tumor cells surface, promote DC maturation, and the purified HSP is more effective.     

Role of CCL17 and CCL22 expression in dendritic cells in maternal-fetal immune tolerance

AIM: To determine the expression of CCL17 and CCL22 in dendritic cells (DC) from human decidua and endometria. METHODS: The decidua were collected from normal pregnant women undergoing induced abortion and recurrent spontaneous abortion (RSA) women undergoing early abortion.The endometria were cllected from non-pregnant women undergoing abdominal hysterectomy.The mononuclear cells in the decidua and endometria were isolated. DC were induced by GM-CSF and IL-4, cultured in vitro and identified. The expression of CCL17 and CCL22 in DC at mRNA and protein levels was analyzed by real-time PCR and ELISA. RESULTS: The mRNA levels of CCL17 and CCL22 in decidual DC in normal pregnancy group were 3.04?0.40 and 1.83?0.24, respectively, significantly higher than those in endometrial DC in non-pregnancy group (0.85?0.24 and 0.31?0.08, respectively, P<0.01) and those in decidual DC in RSA group (1.65?0.14 and 0.96?0.09,respectively,P<0.01). Decidual DC continually and strongly secreted CCL17 and CCL22. The levels of CCL17 and CCL22 in normal pregnancy group were significantly higher than those in non-pregnancy group and RSA group at the same culture time point (P<0.01). CONCLUSION: The expression of CCL17 and CCL22 in decidual DC in pregnant woman increases. This may attract more CD4⁺CD25⁺ regulatory T cells to decidua and play an important role in the establishment of maternal-fetal immune tolerance.     

Recent advances in the correlations between dendritic cells and regulatory T cells

Dendritic cells (DCs), representing a heterogenous population of professional antigen-presenting cells, are the initiators and modulators of the immune responses. Studies indicate that regulatory T cells contribute to immune nullipotency and immune suppression via cell-cell contact or cytokine secretion. These two kinds of cells may be valuable tools for modulating immunity in the setting of auto-immunity, cancer, chronic viral infections and graft rejection, etc. Here we discuss the current knowledge on the functions of regulatory T cells and denditic cells-based immunoregulation and the applications.     

The expression of CD14 in rat Kupffer cells

AIM: To explore the expression of CD14 in rat Kupffer cells (KCs). METHODS: In rat KCs induced by LPS or the mediators from KCs induced by LPS, the changes of CD14 expression were measured by RT-PCR and immunohistochemistry.The expressions of TNFα mRNA、IL-6 mRNA or the concentrations of TNFα、IL-6 were estimated by in situ hybridization and radioimmunoassay, respectively. RESULTS: LPS increased the expression of CD14 in KCs in a dose-dependent fashion (LPS, 1 μg/L-10 mg/L) and in a time-dependent fashion(0.5 h-24 h, peaked at 3-6 hours). While the expression of CD14 in KCs stimulated by the active mediators from KCs which had been exposed to LPS 1 hour were obviously increased. CONCLUSIONS: There was a close relationship between LPS or the active mediators from KCs induced by LPS and the expressions of CD14. It is implied that the increase in CD14 expression may be induced by LPS and the cytokines produced by KCs, it also reveals that there is a auto-regulated loop in CD14 expression.     

A novel mechanism of RhoA activation induced by tumor necrosis factor-α in mouse cerebral microvascular endothelial cells

AIM: To investigate the effects of tumor necrosis factor α (TNF-α) on RhoA activity in mouse cerebral microvascular endothelial cells.METHODS: The bEnd.3 cells, a mouse brain microvascular endothelial cell line, were cultured. RhoA activity was analyzed by pull-down assay 10 min, 30 min and 60 min after TNF-α treatment. Expression of RhoA protein was determined by Western blotting 1 h, 3 h, 6 h, 12 h and 24 h after TNF-α treatment. Small interfering RNA (siRNA) targeting to p115RhoGEF or control nsRNA was transfected into bEnd.3 cells. The expression of p115RhoGEF was determined by Western blotting, and RhoA activity was detected by pull-down assay 30 min after TNF-α treatment.RESULTS: RhoA activity peaked at 30 min after TNF-α treatment(P<0.01) . TNF-α significantly increased the protein expression of RhoA at 12 h and 24 h (P<0.05). Knock-down of p115RhoGEF by siRNA in bEnd.3 cells attenuated TNF-α-induced RhoA activation (P<0.05).CONCLUSION: TNF-α up-regulates RhoA activity and expression. p115RhoGEF may play a role in TNF-α-induced activation of RhoA.     

Sinomenine inhibits TNF-α-induced VCAM-1 expression on human umbilical vein endothelial cells

AIM: To investigate whether sinomenine(SN) can decrease TNF-α-induced VCAM-1 expression in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were isolated from freshly collected umbilical cords.Positive control samples were stimulated with TNF-α, omitting SN. Negative control samples were treated in the same way, omitting TNF-α and SN. Experiment samples were co-cultured with TNF-α and SN at different concentration (0.25, 0.5, and 1.0 mol/L),or TNF-α and dexamethasone(Dex) at concentration of 1.0×10^-6mol/L.Cells were harvested after cultivation with the drugs for 12 hours. VCAM-1 mRNA expression was detected by real-time quantitative PCR, and VCAM-1 expression was detected by flow cytometry (FCM).RESULTS: VCAM-1 mRNA and VCAM-1 were induced by TNF-α. Compared with the positives, the relative VCAM-1 mRNA expression decreased to varying degrees in the experiment groups (P<0.05), and SN at concentration of 0.5 mol/L and 1.0 mol/L inhibited expression of VCAM-1 (P<0.05). SN at concentration of 1.0 mol/L decreased VCAM-1 expression by 28.8%(P<0.05), and SN at concentration of 0.5 mol/L reduced VCAM-1 expression by 21.68%(P<0.05). But SN at concentration of 0.25 mol/L and Dex at concentration of 1.0×10^-6mol/L didnt depress expression of VCAM-1. CONCLUSION: SN may inhibit TNF-α-induced VCAM-1 expression in HUVECs in vitro.     

Study on the immunological function of sodium butyrate-induced immature human monocyte-derived dendritic cells

AIM: To investigate the immunological function of sodium butyrate-induced immature dendritic cells in vitro.METHODS: The human monocyte-derived dendritic cells were induced in the presence of human granulocyte macrophage-colony stimulating factor(GM-CSF) and interleukin-4 (IL-4), combined with sodium butyrate. The immunological function of sodium butyrate-induced dendritic cells was detected by the FCM, endocytic activity, T cells stimulatory proliferation capacity, and interleukin-12 (IL-12) production.RESULTS: Sodium butyrate could down-regulate the major histocompatibility complex(MHC) class II and costimulatory molecules of dendritic cells, increase the endocytic activity, induce a stage of T-cell anergey, and inhibit the T helper cell type 1-skewing factor IL-12 production. CONCLUSION: Sodium butyrate inhibits the maturation of dendritic cells and induces production of immature dendritic cells, which may help to explore the machenism of its epigenitic modification.     

Role of p38 MAPK signaling pathway in inflammatory effects on cerulein-induced pancreatic cells

AIM: To investigate the effect of p38 MAPK signal pathway on cerulein-treated pancreatic acinar AR42J cells.METHODS: AR42J cells were divided into control group, cerulein group (treated with 10^-8 mol/L of cerulein), and SB203580 group (treated with 10 μmol/L of SB203580 and 10^-8mol/L of cerulein).The cells were harvested 3 h after treatment.Secretion rate of amylase was measured.The translocation of p-p38 MAPK to nuclei was imaged by immunofluorescence.The protein expression levels of p-p38 MAPK and TNF-α were detected by Western blotting.The activation of NF-κB was measured by electrophoretic mobility assay.RESULTS: Compared with control group, cerulein resulted in increases in the secretion rate of amylase and protein level of TNF-α (P<0.01), as well as the expression levels of p-p38 MAPK and NF-κB (P<0.01).Cerulein induced nuclear translocation of p-p38 MAPK.Compared with cerulein group, the secretion rate of amylase and protein level of TNF-α in SB203580 group decreased significantly (P<0.01).The expression of p-p38 MAPK and NF-κB also decreased greatly (P<0.05).Nuclear translocation of p-p38 MAPK was inhibited by SB203580.CONCLUSION: The p38 MAPK pathway involves in cerulein-induced pancreatic inflammatory response via regulating NF-κB.     

Effect of G-CSF on dendritic cell subsets in donors mobilized peripheral blood cells

AIM: To explore the effect of granulocyte colony stimulating factor (G-CSF) on donor’s dendritic cell （DC）subsets in peripheral blood cells (PBC). METHODS: The subsets of dendritic cells in PBC were analyzed by CD34^-Lin^- HLA-DR⁺ cells and the levels of IL-12p40, IL-10, IFN-γ, IL-4 in the serum were tested by ELISA before (25 cases) or after G-CSF mobiling. The relationship between the ratio of DC₁/DC₂ (CD11c⁺CD123^-/CD11c^-CD123⁺) and CD34⁺/MNC was explored. RESULTS: CD34⁺/MNC in PBC harvests was above 0.4% in 23 cases, and the ratio of DC₁/DC₂ was lower, the HLA-DR expression of DC₂ was enhanced after G-CSF mobiling than before, but the levels of IL-12p40, IL-10, IFN-γ, IL-4 in donors serum and CD83 expression of DC₂ were not changed by G-CSF. CONCLUSION: DC₂ increased accompanied by the increase in CD34⁺ cells in the PBC harvests. Although the expression of HLA-DR in DC₂ was up-regulated by G-CSF, these DC₂ did not regulate Th2 cells to excrete inhibitor cell factors and kept on the precursor characters.     

Immune function of dendritic cells in patients with hepatocellular carcinoma

AIM: To investigate the immune function of dendritic cells (DCs) in patients with hepatocellular carcinoma(HCC). METHODS: The DCs were cultured from human peripheral blood mononuclear cells (PBMC) by using GM-CSF and IL-4 for 7 days. Surface molecules CD86, HLA-DR of DCs were detected by flow cytometry. IL-12 production by DCs and IFN-γ production by T cells was measured with ELISA and ELISPOT, respectively. Allogenic mixed lymphocyte reaction was detected by MTT assay. RESULTS: CD86 expression in DCs in HCC patients were markedly lower than that in health control (91.7％ vs 83.5%, P<0.05). IL-12 production by DCs had no significant difference between the HCC patients and the health control ［(324.6±171.0)ng/L vs (436.5±142.7)ng/L, P>0.05］. However, after stimulated DCs with LPS, IL-12 production in HCC patients was significantly lower than that in health control ［(478.6±142.7)ng/L vs (630.0±151.9)ng/L, P<0.05］. IFN-γ production by T cells and T cell proliferation index (PI) in the HCC patients were all significantly lower than those in health control ［(IFN-γ: 133.4±51.2)103U/L vs (183.0±60.2)103U/L, P<0.05; PI: 2.3±0.7 vs 3.5±0.8, P<0.01］. CTL killing activity assay indicated that DC-induced CTL killing activity in HCC group was significantly lower than that in health group when the E/T ratio was 10∶1 and 20∶1, respectively. CONCLUSION: The immune function of DCs and T cells in the patients with HCC are significantly decreased. The CTL killing activity of HCC group is also markedly decreased.     

Effects of CpG ODN on dendritic cells and its mechanisms

Oligodeoxynucleotide containing unmethylated cytosine phosphate-guanosine motif(CpG ODN) may induce high expression of CD80, CD86, CD83, HLA I and HLAⅡ molecules on dendritic cells(DC) and stimulate DC to produce high level of IL-6, IL-12, TNF-α and IFN-α. CpG ODN is demonstrated in vivo to be a very potent adjuvant for Th1 cells, regulating Th0 cells to develop toward Th1 cells. Its role for DC is characteristics of CpG ODN sequence specificity and species specificity. CpG ODN is, at present, considered as a pathogen associated molecular pattern which binds its specific receptor，Toll-like receptor 9，then functions through TLR/IL-1R signaling pathway. It may represent a new therapeutic drug for broad applications in infectious disease, autoimmune disease, allergy and cancer therapy.