OBJECTIVETo study the effects of Lipopolysaccharide (LPS) on the maturation and secretion of human peripheral dendritic cells (DCs).

METHODSDCs from healthy human peripheral monocytes (PBMCs) were induced in vitro with rhGM-CSF, rhIL-4, Flt3-L and TNFalpha. The subjects were divided into 3 groups: the long-term group stimulated with LPS 1 microg/ml at day 1, 4, 7, 9 post culture; the short-term group stimulated with LPS 1 microg/ml at day 7 and 8 post culture, and the DCs without LPS stimulation was control group. After 10 days of culture, the morphologic features of DCs were observed by light and electron microscopes, the phenotypic patterns were characterized by flow cytometry, the proliferation of T cell were evaluated with mixed leukocytes reaction (MLR) and the levels of IL-12 and IFNgamma produced by DCs were analyzed with ELISA.

RESULTSCompared with the short-term group, the expressions of HLA-DR (65.81%+/-10.96%), CD86 (48.81%+/-18.13%), CD80 (13.56%+/-5.48%), CD83 (11.52%+/-5.09%), the secretions of IFNgamma(15.60+/-5.83 pg/ml) and IL-12 (51.77+/-11.02 pg/ml) by the DCs in long-term group were decreased obviously (P is less than 0.05) and the proliferation of homogenic lymphocyte cells (1.548+/-0.365) stimulated by DCs was also impaired (P < 0.05).

CONCLUSIONLong-term LPS stimulation can suppress the maturation and secretion of DCs, which might be the reason of poor immunity in the patients with intestinal endotoxemia.

Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Humans ; Interleukin-12 ; biosynthesis ; Lipopolysaccharides ; pharmacology ; Monocytes ; cytology ; metabolism

MicroRNAs (miRNAs) are small, single-stranded and noncoding RNA molecules of about 22 nucleotides (nt) in length that regulate mRNA by binding to 3' untranslated regions (3'UTR) of target mRNA, inducing digestion, degradation and/or translational repression of the latter. Posttranscriptional regulation of gene expression by miRNA is critical for a wide range of physiologic and pathologic processes, including cell proliferation, differentiation, apoptosis, development and oncogenesis. Dendritic cells (DC) are professional antigen presenting cells that have a pivotal role in controlling immune responses. The latest studies indicated that miRNA are indispensable in regulation of development, differentiation and functions of DC. This review discusses the latest studies of miRNA controlling DC biological properties in order to deep understand the regulatory mechanism of DC, therefore, provide a new thinking for the therapeutic strategies of DC-associated immunological disorders.
Animals ; Cell Differentiation ; Cell Proliferation ; Dendritic Cells ; cytology ; metabolism ; Humans ; MicroRNAs ; metabolism

This study was aimed to investigate the effect of dendritic cells (DC) on the proliferation capability, immunophenotype changes, level of secreted cytokines and activity against leukemia of cytokine-induced killer (CIK) cells in vitro. DCs and CIK cells were induced from peripheral blood mononuclear cells of healthy volunteers. They were co-cultured meanwhile CIK cells were cultured alone as controls. Increased number of cells were counted by trypan-blue staining; the killing activity was detected by MTT assay; immunophenotype changes were analyzed by flow cytometry; the IL-12 and INF-gamma levels of the cultured supernatants were detected by ELISA kits. The results showed that the proliferation capability of DC-CIK cells was significantly higher than that of CIK cells (p < 0.05). Under the same condition, the ratio of double positive cells such as CD3(+) CD8(+), CD3(+) CD56(+) in CIK cells was significantly enhanced by co-cultured with DC cells (p < 0.05). The levels of IL-12 and INF-gamma in cultured supernatants of DC-CIK cells increased noticeably on day 3 as compared with CIK cells cultured alone (p < 0.01, p < 0.05). Within the effector-target ratio range between 5:1 to 40:1, the activity of DC-CIK cells against leukemia cells were much higher than that of CIK cells (p < 0.05), and this effect showed a positive correlation with the effector-target ratio. It is concluded that the proliferation capability of DC-CIK cells, the level of their secreted cytokines and their activity against leukemia cells are significantly higher than those of CIK cells. This research may suggest an approach for clinical immunotherapy against leukemia with DC-CIK cells.
Cell Line, Tumor ; Cell Proliferation ; Coculture Techniques ; Cytokine-Induced Killer Cells ; cytology ; immunology ; metabolism ; Dendritic Cells ; cytology ; immunology ; metabolism ; Humans ; Interferon-gamma ; metabolism ; Interleukin-12 ; metabolism

This study was aimed to investigate the effect of dendritic cell-derived exosome (DCex) on in vitro osteoblast differentiation of human bone marrow mesenchymal stem cells (hBM-MSC). DCex were harvested from the DC culture supernatants by ultracentrifugation. The morphology of DCex was observed by using transmission electron microscopy and the surface marker expression was detected by flow cytometry. MSCs at passage 3 were used in this study. DCex incorporation into MSCs was observed under a confocal microscope. MSCs were either exposed to DCex (10 µg/ml) or the standard osteogenic induction condition. The cells cultured in complete medium were served as the control. The expression levels of Runt related transcription factor 2 (Runx2) were detected by real-time and standard PCR. The cellular alkaline phosphatase (ALP) activity was also detected. The results showed that the DCex were spherical or oval membrane vesicles with diameters of about 40-100 nm under transmission electron microscope. The DCex expressed surface molecules specific for DCs, including CD83, CD86, CD80, and HLA-DR. After cultured for 7 days, the MSCs treated with DCex highly expressed Runx2 as compared with the control group (P < 0.05). After cultured for 14 days, ALP activity of the DCex-treated MSCs was markedly higher than the control group (P < 0.01), though it was lower than that of MSCs treated with standard inductive agents. It is concluded that DCex can induce MSCs to differentiate into osteoblasts. The detailed investigations are needed to clarify the underlying mechanisms.
Alkaline Phosphatase ; metabolism ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Dendritic Cells ; cytology ; metabolism ; Exosomes ; metabolism ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Osteogenesis ; RNA, Messenger

The study was aimed to investigate the mechanism of mannan-binding lectin (MBL) on bacterial lipopolysaccharide (LPS)-induced human peripheral blood monocyte-derived dendritic cell (DC) maturation. The monocytes were prepared from the peripheral blood of healthy adult volunteers. The immature dendritic cells (imDC) were induced by 5-day-culture in medium supplemented with rhGM-CSF and rhIL-4. FACS was used to investigate the interaction of MBL with imDC and the impact of MBL on LPS binding to imDC. ELISA and Western blot was used to analyze the interaction of MBL with soluble TLR4 ectodomain protein (sTLR4); Western blot was used to detect LPS-induced NF-κB translocation in imDC. The results showed that MBL could directly bind to imDC in the presence of calcium. sTLR4 protein or LPS could competitively inhibit the binding of MBL to imDC. ELISA and Western blot showed that MBL could evidently bind to sTLR4 protein in a concentration-dependent manner. FACS showed that MBL could competitively inhibit the binding of LPS to imDC by binding to imDC directly. Western blot showed that MBL decreased LPS-induced NF-κB translocation in imDC. It is concluded that MBL may competitively inhibit the binding of LPS to imDC by binding to TLR4 expressed on imDC, resulted in inhibition of LPS-induced DC maturation, suggesting that MBL can regulate DC maturation through ligand-binding. This study provides the good foundation to clarify the mechanism of MBL inhibiting the LPS-induced DC maturation.
Cell Differentiation ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Humans ; Ligands ; Lipopolysaccharides ; adverse effects ; Mannose-Binding Lectin ; pharmacology ; Monocytes ; cytology ; metabolism ; Toll-Like Receptor 4 ; metabolism

OBJECTIVETo observe the changes in the phenotype characteristics and immune function after transfection of cord blood derived immature dendritic cells( imDC) with Adeasy-EGFP adenovirus vector, and to explore the function of IL-10 in inhibition of imDC maturation.

METHODSImmature dendritic cells were generated from human cord blood(CB) monocyte cultured with rhGM-CSF and rhIL-4. The recombinant adenovirus vector AdEASY-EGFP was transduced into immature dendritic cells on the third day with or without adding IL-10. The expression of cell maturation marker CD83, CD86 and HLA-DR were determined with flow cytometry. Allogeneic mixed leukocyte reaction( MLR) was used to examine the imDC's ability to promote T cell proliferation.

RESULTSThe expression of surface maturation markers of imDC after transfection with adenovirus were significantly up-regulated ( CD86:46+/-10; CD83: 38 +/- 7; HLA-DR: 82 + 12) , and its ability to promote T cell proliferation was also obviously increased( SI > 2. 0). However, the expression of surface maturation markers of imDC after IL-10 treatment had lower mature phenotypes expression after transduction (CD86:8 +/- 5; CD83: 9 +/- 3; HLA-DR:63 +/- 12), and T cell stimulating ability was decreased comparing with adenovirus transfection groups.

CONCLUSIONAdenovirus can be transduced into imDC with high efficiency, but transfection itself can promote imDC's maturation. IL-10 treatment can inhibit the tendency to maturation stimulated by adenovirus transduction efficiently.

Adenoviridae ; genetics ; Cell Differentiation ; Cells, Cultured ; Dendritic Cells ; cytology ; Genetic Vectors ; Humans ; Interleukin-10 ; pharmacology ; T-Lymphocytes ; metabolism ; Transfection

OBJECTIVETo detect the secretions of type I interferon and the expressions of phospho-IRF3 in murine liver dendritic cells intervened by HBV.

METHODSThe murine liver dendritic cells were isolated via anti-CD11c microbeads and were incubated in the presence of GM-CSF and IL-4 to induce the DC generation and proliferation in 24-well cell culture plates. HBV virions were isolated via ultracentrifugation and were detected by quantitative Realtime-PCR. The DCs were divided into two groups: one group was cultured with HBV virions for 24 hours, the other group was cultured without HBV as control group. The cells were harvested at Oh, 1h, 2h, 6h and 24h after being stimulated with poly I:C and the expressions of p-IRF3 and the concentration of IFN beta in supernatants were detected with western blot and ELISA respectively.

RESULTSThe IFN beta concentrations at 0 h, 6 h and 24 h in the supernatants of the HBV group and the control group were (12.38 +/- 3.71) pg/ml, (88.67 +/- 9.01) pg/ml and (69.89 +/- 5.80) pg/ml vs (10.83 +/- 4.11) pg/ml, (137.68 +/- 12.28) pg/ml and (72.25 +/- 8.61) pg/ml, respectively. No statistical differences found at 0 h (t = 0.8398, P > 0.05) and 24 h (t = 0.6820, P > 0.05) between the two groups except that at 6 h (t = 9.653, P < 0.01). The expressions of phospho-IRF3 in HBV group were lower than that in control group.

CONCLUSIONSThe type I interferon secretion and the phospho-IRF3 expression were decreased in murine liver dendritic cells when intervened by HBV.

Animals ; Cells, Cultured ; Coculture Techniques ; Dendritic Cells ; cytology ; metabolism ; Hepatitis B virus ; Hepatocytes ; cytology ; Interferon Regulatory Factor-3 ; metabolism ; Interferon Type I ; secretion ; Male ; Mice ; Mice, Inbred C57BL

OBJECTIVEThe impact of dendritic cells (DCs) and regulatory T cells (Tr) on the pathogenesis of asthma have been investigated over the past decades. As the professional antigen presenting cells, DCs not only prime immune response directing Th0 cells toward different T subtypes but also induce immune tolerance. As an immunoregulator, Bacillus Calmette-Guerin (BCG) has potential to be applied in allergic diseases such as asthma for prevention. Previous study showed that neonatal BCG vaccination could induce Th1/Tr1 development in mice in vivo. To further identify the mechanism of neonatal BCG vaccination on T cell subsets differentiation, the present study was designed to investigate the impact of BCG vaccination on splenic DCs development in neonatal mice.

METHODSNeonatal specific pathogen free (SPF) BALB/c mice (2-3 days) were divided into intraperitoneal BCG-treated group, subcutaneous BCG-treated group and control group; simultaneously adult SPF BALB/c mice (6-8 weeks) were divided into intraperitoneal BCG-treated and control group. The BCG-treated mice were inoculated with 1 x 10(5) CFU BCG, the mice in control group were not inoculated with any vaccine. Four weeks post BCG vaccination, spleen cells were isolated. With flow cytometry, subtype and maturity of splenic DCs were analysed. Moreover, cells were further separated into mononuclear cell by Ficoll solution. The mononuclear cells were stimulated by 1 microg/ml lipopolysaccharide (LPS) for 18 or 10(5) CFU /ml BCG for 48 hours at 37 degrees C in a humidified atmosphere containing 5% CO2 and cytokines concentration was detected by ELISA.

RESULTS(1) CD11c(+) CD8alpha(+) and CD11c(+) CD8alpha(-) DCs were found in spleen cells of the BALB/c mice. In comparison with the control group, the percent of CD11c(+) CD8alpha(-) DCs in intraperitoneal BCG group significantly declined (P < 0.01) and that of CD11c(+) CD8alpha(+) DCs significantly increased (P < 0.01), there were no significant difference in DC subtypes between intraperitoneal and subcutaneous BCG-vaccinated mice. In contrast, the percent of CD11c(+) CD8alpha(-)DCs markedly increased (P < 0.01) and that of CD11c(+) CD8alpha(+)DCs noticeably reduced (P < 0.01) in adult BCG-vaccinated mice. The percent of CD11c(+)CD8alpha(-)DC was significantly higher and that of CD11c(+) CD8alpha(+)DC was significantly lower in adult-vaccinated BALB/c mice than that of neonatal-vaccinated ones. (2) The expression of costimulatory molecules CD40 on CD11c(+) CD8alpha(-) DCs and CD86 on CD11c(+) CD8alpha(+) DCs in neonatal BCG-treated BALB/c mice was higher than the controls. There were no significant difference in expression of costimulatory molecules on DC between neonatal BCG-vaccinated mice. Compared with the control group, expression of CD40 and MHC-II molecules on CD11c(+) CD8alpha(-) and CD11c(+) CD8alpha(+)DC was significantly higher and that of CD86 was significantly lower in adult BCG-vaccinated mice. The expression of costimulatory molecules on DC had no significant difference between neonatal and adult BCG vaccinated BALB/c mice. (3) As compared with the control mice, concentration of IL-12p70 induced by LPS and IL-10 induced by BCG in vitro from spleen cells culture supernatant was noticeably elevated (P < 0.05) in neonatal BCG-treated BALB/c mice, but that of IL-6 did not change by LPS or BCG stimulation.

CONCLUSION(1) By up-regulating splenic CD8alpha(+)DCs and inducing IL-12p70 and IL-10 production in BALB/c mice, neonatal BCG vaccination promoted Th1/Tr1 development. (2) The effect of BCG vaccination on DC was different between neonates and adult BALB/c mice.

Animals ; Animals, Newborn ; BCG Vaccine ; immunology ; Cell Differentiation ; Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; metabolism ; Immunophenotyping ; Mice ; Mice, Inbred BALB C ; Spleen ; cytology ; immunology ; metabolism

OBJECTIVETo explore a simple, rapid and efficient way to generate dendritic cells from leukemic cells.

METHODSK562 cells were cultured with calcium ionosphere A23187 alone, A23187 plus GM-CSF, or a DC differentiation cocktail consisting of GM-CSF, IL-4 and TNF-alpha, respectively. The expression of surface markers of induced DCs was analyzed by flow cytometry. The K562-DCs stimulating the proliferation of allo-genetic naive T cells and inducing cytotoxicity of T cells were determined by MTT assay.

RESULTSMicroscopic examination revealed that under all the three culture conditions, K562 cells became displaying DC morphology. At 72 hours in the two culture systems containing A23187, there were higher proportions of cells with dendritic morphology [(69.5 +/- 17.2)% and (73.1 +/- 13.9)%, respectively] than that in the cocktail system [(28.5 +/- 12.3)%] (P < 0.05). And the same did when cultured for 7 days [(69.5 +/- 17.2)%, (73.1 +/- 13.9)% respectively vs (51.2 +/- 10.7)%, P < 0.05]. In the 7-day cultures, the percentage of CD1a expressing cells was lower [(8.2 +/- 2.3)% and (10.3 +/- 5.1)% vs (17.2 +/- 1.6)%, respectively] while the CD83 expressing cells was higher [(85.6 +/- 8.8)% and (82.4 +/- 9.1)% vs (77.4 +/- 12.9)%, respectively] compared with that in the cocktail system (P < 0.05). No significant difference was found in the allogeneic T cell proliferation response and induced T cell cytotoxicity between A23187 containing and cocktail groups (P > 0.05).

CONCLUSIONSA23187 treatment is a simple, rapid and efficient in vitro strategy for inducing dendritic cell from leukemic cells.

Calcimycin ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Dendritic Cells ; cytology ; immunology ; metabolism ; Humans ; K562 Cells ; cytology ; drug effects

Dendritic cells (DCs) are key modulators that shape the immune system. In mucosal tissues, DCs act as surveillance systems to sense infection and also function as professional antigen-presenting cells that stimulate the differentiation of naive T and B cells. On the basis of their molecular expression, DCs can be divided into several subsets with unique functions. In this review, we focus on intestinal DC subsets and their function in bridging the innate signaling and adaptive immune systems to maintain the homeostasis of the intestinal immune environment. We also review the current strategies for manipulating mucosal DCs for the development of efficient mucosal vaccines to protect against infectious diseases.
Animals ; Dendritic Cells/*immunology/metabolism ; Humans ; Immunity, Mucosal ; Intestinal Mucosa/cytology/*immunology ; T-Lymphocytes, Helper-Inducer/immunology