The defensive function of immune response in vivo is very important. Antigen presenting cells (APC) is the hinge in starting specific immune response. Dendritic cells (DC) are the most potent APC, which participate in double-regulating the immune response. Laser scanning confocal microscope (LSCM) becomes a powerful tool in the fields of basic medicine and clinical medicine with its high resolving power, high sensitivity, optical section, three-dimensional reconstruction and dynamic analysis. And it makes the study on DC more thoroughly and deeper. In this paper, we review the recent progress in the applications of LSCM to studies on the morphology, immunofluorescence localization and the functional analysis of dendritic cells.
Dendritic Cells ; physiology ; ultrastructure ; Fluorescent Antibody Technique ; Humans ; Microscopy, Confocal

Cutaneous dendritic cells (DCs), Langerhans cells (LCs) and dermal dendritic cells (DDCs), are present in an immature state. The maturation of DCs is crucial for initiating an immune response. Since HLA-DM has an important role for antigen presentation, an increase in HLA-DM expression according to the maturation of blood monocyte-derived dendritic cells (MoDCs), which have similar characteristics with DDCs, is expected. Therefore, the aim of this study was to determine whether or not HLA-DM expression in MoDCs is related to maturation at each culture day (from day 0 to day 13) by flow cytometry. This was compared with the functional changes related to the maturation of MoDCs. MoDCs were generated by culturing human peripheral blood monocytes in the presence of GM-CSF and IL-4 for 7 days, which were followed by subsequent treatment with a cytokine cocktail (GM-CSF, IL-4, IL-1beta, TNF-alpha, IL-6 and PGE2) for the maturation of MoDCs. The intracellular HLA-DM was expressed in the immature MoDC. A sudden 3 to 8 fold increase in the intracellular HLA-DM expression was observed after treatment with a cytokine cocktail. HLA-DM was weakly expressed on the surface of the immature MoDC, but it seemed to be decreased with maturation. This study indicated that the intracellular HLA-DM expression increased, but not on the MoDC surface during maturation. This was despite the fact that HLA-DM expression was noted not only on the surface but also in the intracellular in the MoDC.
Dendritic Cells/*immunology/physiology ; Endocytosis ; Flow Cytometry ; HLA-D Antigens/*analysis ; Human ; Monocytes/*physiology

Dendritic cells(DC) are specialized antigen-presenting cells that prime naive T cells to induce initial immune responses. The immature DC capture and process antigens in the periphery, then emigrate to lymphoid organs. There they complete their maturation by upregulating HLA-I, II molecules, costimulatory molecules (eg. CD80, CD86) and adhesive molecules (eg. CD50, CD54, CD58). More studies showed that in vitro only interferons type I (IFN-alpha, beta) accelerate DC maturation in a dose-dependent manner. The DC induced by IFN type I highly express HLA-A, B, C, HLA-DR, costimulatory molecules and adhesive molecules, and they express enhancing effect of T-cells stimulatory activity in vitro. Progress of research in this field was summarized in this paper.
Cells, Cultured ; Dendritic Cells ; drug effects ; immunology ; physiology ; Humans ; Immunophenotyping ; Interferon Type I ; pharmacology

To investigate the effect of mycobacterium phlei F.U.36 suspended liquor (Utilin"s", U) on the culture and proliferation of dendritic cells (DCs) derived from human umbilical cord blood in vitro, the mononuclear cells (MNCs) were isolated from human umbilical cord blood and cultured with RPMI 1640 in the control group. Test groups consisted of Utilin"s" group (only Utilin"s"), GTI group (GM-CSF, TNF-alpha, IL-4) and GTIU group (GM-CSF, TNF-alpha, IL-4 and Utilin"s"). MNCs in all test groups were cultured with RPMI-1640. The growth of DCs was observed by the light microscopy, the phenotypes of DCs were determined by flow cytometry on the 10th day of culture, and some harvest cells were stained with Wright-Giemsa, then observed and photographed under the oil immersion objective. The results showed that the test groups all displayed some number of typical DCs; both CD1a positive cell rate and HLA-DR positive cell rate of the Utilin"s" group were higher than those of the control; HLA-DR positive cell rate of GTIU group increased most significantly and much higher than that of the GTI group. It is concluded that mycobacterium phlei F.U.36 not only promotes the proliferation of DCs derived from human umbilical cord blood in vitro, but also co-operates with rhGM-CSF, rhTNF-alpha and rhIL-4 in promoting the maturity of DCs.
Cell Proliferation ; Cells, Cultured ; Dendritic Cells ; cytology ; Fetal Blood ; cytology ; Humans ; Mycobacterium phlei ; physiology

Dendritic cells (DCs), the most potent antigen-presenting cells, recognize pathogen by Toll-like receptors (TLRs) and serve as the bridge between the innate and the adaptive immune responses. TLRs-mediated signal transduction plays a crucial role in the functional maturation of DCs. This review summarizes the research progress on the expression and function of TLRs in DCs.
Dendritic Cells ; immunology ; metabolism ; Humans ; Immunity, Innate ; Signal Transduction ; Toll-Like Receptors ; metabolism ; physiology

The pathways leading to the development of different dendritic cell (DC) subsets have long been unclear. In recent years, a number of precursors on the route to DC development, both under steady state and inflammatory conditions, have been described, and the nature of these pathways is becoming clearer. In addition, the development of various knockout mouse models and an in vitro system modelling DC development have revealed the role of numerous cytokines and transcription factors that influence DC development. Here, we review recent findings on the factors important in DC development in the context of the developmental pathways that have been described.
Animals ; Cytokines ; metabolism ; Dendritic Cells ; metabolism ; Humans ; Signal Transduction ; physiology ; Transcription Factors ; metabolism

BACKGROUNDTo study the effects of dendritic cells (DC) transfected with recombinant adenovirus encoding Epstein-Barr virus(EBV)latent membrane protein 2A(LMP2A)gene, and to provide evidence for further investigation on the therapeutic vaccine against EBV-associated malignancies.

METHODSDCs were transfected with EBV-LMP2A recombinant adenovirus (Ad5-LMP2A), which was generated by homologous recombination. The expression of LMP2A protein on mature DC transfected with Ad5-LMP2A at different multiplicity of infection(MOI)was analyzed by fluorescence activated cell sorting(FACS), and the dead cells were counted by trypan blue staining. The alteration of surface markers on mature DC including CD1a, CD83, CD40, CD80, HLA?DR was detected by means of FACS before and after transfection. Meanwhile, the functions including stimulating allogenetic T cells reaction and expressing IL12 P40 mRNA on transfected DC were measured by methods of 3H-thymidine uptake and fluorescent semi?quantitative polymerase chain reaction (PCR), respectively.

RESULTSAbout 80% mature DC expressed LMP2A protein and >92% cells were viable after transfection at the MOI of 200 No. significant changes in the surface markers and the cytomorphology of mature DCs were detected during the transfection. Transfected DC still have strong potential to stimulate the proliferation of allogenetic T cells and to express IL-12 P40 mRNA.

CONCLUSIONEBV-LMP2A gene,which was carried by adenovirus vectors, could be transferred into DC with high efficiency. The function of mature DC was not affected significantly by the transfection of Ad5-LMP2A.

Adenoviridae ; genetics ; Cells, Cultured ; Dendritic Cells ; physiology ; Gene Expression ; Genetic Vectors ; Humans ; Recombination, Genetic ; Transfection ; Viral Matrix Proteins ; genetics

OBJECTIVETo investigate the effects of Mycobacterium tuberculosis on apoptosis of mouse dendritic cells (DC 2. 4) and the activation of caspase-3, caspase-8.

METHODSMycobacterium tuberculosis H37Rv strain was co-cultured with DC 2. 4 cells. The morphological changes of DC 2. 4 cells were observed with fluorescence microscope after DAPI staining and transmission electron microscope. The apoptosis of DC 2. 4 cells were examined by DNA agarose gel electrophoresis. The activities of caspase-3 and caspase-8 were detected by colorimetric assay.

RESULTSBacterial invasion was observed while DC 2. 4 cells and H37Rv were co-cultured for 2 h; and the rates of invasion were (16.1 ± 4.3)%, (35.8 ± 5.1)%, (50.2 ± 5.7)%, (58.3 ± 6.2)% and(65.9 ± 6.9)% at 4, 6, 8,10, 12 h, respectively. The phenomenon of nuclear condensation and marginalization were shown by DAPI staining and transmission electron microscope in DC 2. 4 cells at 6 h of co-cultivation with H37Rv. The characteristic bands of apoptosis by DNA electrophoresis were detected. The activities of caspase-3 and caspase-8 were increased in a time-dependent manner. The rates of DC 2. 4 cell apoptosis were (6.4 ± 2.5)%, (11.8 ± 5.3)% and (31.1 ± 8.7)% at 6 h,12 h and 24 h after co-cultivation with H37Rv, respectively. The maximal activities of intracellular caspase-3 and caspase-8 at 10 h and 6 h were (2.01 ± 0.09) U/μg and (2.40 ± 0.07)U/μg, respectively, which was significantly different compared with the control groups(P<0.05). The activation of caspase-8 was earlier than that of caspase-3.

CONCLUSIONMycobacterium tuberculosis can induce the apoptosis of DC 2. 4 cells, which is associated with the activation of intracellular caspase-3 and caspase-8.

Animals ; Apoptosis ; physiology ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Cells, Cultured ; Dendritic Cells ; metabolism ; pathology ; Mice ; Mycobacterium tuberculosis

OBJECTIVETo establish a method for in vitro amplification of immature dendritic cells from murine bone marrow, and to identify it with morphological, immunological phenotype determination, and functional examination.

METHODSDendritic cells from murine bone marrow were cultured with different dosage of rmGM-CSF. The suspending cells were examined with scanning electronic microscope, and the non-sensitized T lymphocyte proliferation was observed by mixed lymphocyte reaction.

RESULTSDendritic cells (DC) cultured in lower dosage of rmGM-CSF (Gm(low)DC) exhibited typical characteristics of DCs with high expression of CD11c and low expression of CD40 and I-A/I-E, and non-expression of B7-1 on the surface of the cells. The capacity of Gm(low)DC to stimulate the proliferation of non-sensitized T lymphocyte in vitro was weaker than that of Gm(high)DC.

CONCLUSIONGm(low)DC exhibited typical characteristics of DC, immature in cell phenotype and cell functions, suggesting that our methods of immature DCs culturing was feasible. The dosage of rm GM-CSF has direct relationship with the maturation degree of DC. Generally speaking, mature DC was mainly induced by high dosage of rmGM-CSF, while immature DC by low dosage.

Animals ; Bone Marrow Cells ; physiology ; Burns ; surgery ; Cell Division ; Dendritic Cells ; physiology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes ; immunology

The ability to generate dendritic cells (DCs) in sizeable numbers has enormous implications for the development of clinically-effective antigen presentation procedures for cancer immunotherapy. We evaluated the generation of immunostimulatory DCs from peripheral blood CD34+ cells collected from healthy donors. CD34+ cells purified from leukapheresis product were seeded at 1 x 10(4) cells/mL in complete medium supplemented with GM-CSF, TNF alpha, IL-4, c-kit ligand, and flt3 ligand (FL). By day 14 of culture in the presence of GM-CSF + TNF alpha, the total cell number increased by 23.4 +/- 5.4-fold compared to the starting number of CD34+ cells. When the c-kit and FL were added to GM-CSF and TNF alpha, the cell number increased by 109.8 +/- 11.2-fold without affecting the immunophenotype of recovered cells. Flow cytometric analysis indicated that cells with the markers of mature dendritic cells, i.e., CD1a +CD14 -HLA-DR+, and CD80+CD86+HLA-DR+, constituted 49.0% +/- 7.5%, and 38.9% +/- 6.5%, respectively. This pattern of expression of surface antigen was unchanged whether the c-kit ligand and/or FL was added. The irradiated CD1a+HLA-DR+ cells recovered from in vitro cultures elicit a vigorous proliferation of allogeneic peripheral blood T-cells, irrespective of cytokine combinations. These findings provide advantageous tools for the large-scale generation of DCs that are potentially usable for clinical protocols of immunotherapy or vaccination in patients undergoing cancer treatment.
Antigens, CD34/analysis* ; Dendritic Cells/physiology* ; Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology ; HLA-DR Antigens/analysis ; Hematopoietic Stem Cells/physiology* ; Human ; Interleukin-4/pharmacology ; Tumor Necrosis Factor/pharmacology