This study was conducted to get high quality and sufficient numbers of mature dendritic cells from healthy donor peripheral blood. The experiment began on culturing of plastic-adherent monocytes isolated from healthy donor peripheral blood with granulocyte-monocyte clony-stimulating factor (GM-CSF 150 ng/ml) and interleukin 4 (IL-4 800 U/ml) without fresh medium feeding and cytokines for 7 days. After 7 days, CD14+ monocytes not only differentiated into high purity DC but also expressed HLA-I and HLA-II molecules, costimulating molecules, adherent molecules and its progenitor marker CD14 molecule highly. These cells displayed all phenotypic and morphologic characteristics of mature dendritic cells and were most potent stimulatory cells in allogeneic mixed leukocyte reactions. The endocytosis ability of these DCs peaked at the third day in culture and decreased remarkably afterwards. These results provide evidence for the first time that CD14+ monocytes differentiated in vitro from peripheral blood monocytes exhibit dendritic cells characteristics and still express its progenitor marker CD14 molecules highly. The results of this experiments may facilitate further studies of CD14+ DC and its clinical applications.
Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Dendritic Cells ; cytology ; Granulocyte-Macrophage Colony-Stimulating Factor ; chemistry ; Humans ; Interleukin-4 ; chemistry ; Lipopolysaccharide Receptors ; metabolism ; Monocytes ; cytology

Mature dendritic cells are potent antigen-presenting cells that initiate primary immune responses, while immature dendritic cells have quite different properties from mature dendritic cells and are tolerance inducer actually. Here we describe the method of using monocyte condition medium to generate dendritic cells of different maturation phases from nonproliferating progenitors in human peripheral blood. The procedure involves two steps. The first step(or priming phase) is to work on a 6-7-day culture of plastic-adherent blood monocyte in medium supplement with GM-CSF and IL-4. The second step (or differentiation phase) requires the exposure to monocyte conditioned medium. Only the dendritic cells generated by the first step are actually immature, with strong immature dendritic cell features such as active endocytosis, the same expression of monocyte marker CD14, and much of the MHC class II still lies within intracellular compartments (MIIC). The second stage dendritic cells have all the features of mature dendritic cell, including a stellate shape, nonadherence to plastic, the expression of dendritic cells restricted marker CD83, and very strong T cell stimulatory function. All of these dendritic cell properties are retained for at least 3 days when the cytokines are removed, suggesting that these populations are stable and terminally differentiated. Since progression from immature to mature dendritic cell is entirely dependent on exogenously added growth factor such as monocyte condition medium, the peripheral blood monocyte may help to harness synchronized population of mature and immature dendritic cells for studies or therapies.
Cell Culture Techniques ; Cell Differentiation ; Culture Media, Conditioned ; chemistry ; Dendritic Cells ; cytology ; Granulocyte-Macrophage Colony-Stimulating Factor ; chemistry ; Humans ; Interleukin-4 ; chemistry ; Monocytes ; cytology

Adult ; Dendritic Cells ; pathology ; Diagnosis, Differential ; Humans ; Immunohistochemistry ; Keratins ; analysis ; Lymph Nodes ; pathology ; Male ; Prognosis ; Retroperitoneal Neoplasms ; chemistry ; pathology ; ultrastructure

OBJECTIVETo investigate immunomodulatory effect of Astragalus polysaccharides (APS) on IL-12-secreting dendritic cell (DC) subset CD11c(high)CD45RB(low) DC.

METHODSSpleen CD11c(high)CD45RB(low) DC and CD4(+)T lymphocytes in BALB/c mice were purified by magnetic beads sorting, and were treated with 0 (as control), 50, 100, 200 µg/mL APS. Immunofluorescence staining and flow cytometry were used to determine expressions of CD11c(high)CD45RB(low) DC surface molecules, including CD40, CD80, CD86, I-A/E, and Toll-like receptor (TLR) 4. IL-12 level in CD11c(high)CD45RB(low) DC culture supernatant was determined by ELISA. The CD4(+) T lymphocytes were divided into: normal control group, non-stimulation group (CD4(+)T lymphocytes cocultured with APS-unstimulated CD11c(high)CD45RB(low) DC), high-dose APS stimulation group (CD4(+)T lymphocytes cocultured with 200 µg/mL APS-stimulated CD11c(high)CD45RB(low) DC), high-dose APS stimulation+antibody 1 group (CD4(+)T lymphocytes cocultured with 200 µg/mL APS-stimulated CD11c(high)CD45RB(low) DC and IL-12 antibody), high-dose APS stimulation+ antibody 2 group (CD4(+)T lymphocytes cocultured with 200 µg/mL APS-stimulated CD11c(high)CD45RB(low) DC and IL-12 antibody isotype). Proliferation ability of CD4(+) T lymphocytes was determined with MTT method. IL-4 level as well as IFN-γ level in CD4(+)T lymphocyte culture supernatant was determined by flow cytometry. Data were processed with one-way analysis of variance.

RESULTSCompared with those in control, the expressions of CD11c(high)CD45RB(low) DC surface molecules (except for CD86) on CD11c(high)CD45RB(low) DC surface, as well as IL-12-secreting level with dose-dependence were increased in cells stimulated with 50, 100, 200 µg/mL APS. Proliferation ability of CD4(+)T lymphocytes in high-dose APS stimulation group was higher as compared with that in non-stimulation group (F = 13.438, P < 0.05). IFN-γ level in high-dose APS stimulation group \[(2784 ± 137) pg/mL\] was higher than that in non-stimulation group \[(1952 ± 101) pg/mL, F = 12.177, P < 0.05\]. IL-4 level in high-dose APS stimulation group was (172 ± 20) pg/mL, which was lower than that in non-stimulation group \[(193 ± 19) pg/mL, F = 11.963, P < 0.05\]. Proliferation ability of CD4(+) T lymphocytes, IFN-γ level, and IL-4 level in high-dose APS stimulation + antibody 1 group were all ameliorated when compared with those in non-stimulation group; while levels of the 3 indexes in high-dose APS stimulation + antibody 2 group were similar to those in high-dose APS stimulation group.

CONCLUSIONSAPS can activate IL-12-producing CD11c(high)CD45RB(low) DC, and further induce the activation of immune function of T lymphocyte with shifting of Th2 to Th1 in vitro. APS can enhance the immune response via promoting the phenotypic and functional maturation of CD11c(high)CD45RB(low) DC.

Animals ; Astragalus Plant ; chemistry ; Cell Differentiation ; Cells, Cultured ; Dendritic Cells ; drug effects ; immunology ; secretion ; Interleukin-12 ; metabolism ; Mice ; Mice, Inbred BALB C ; Polysaccharides ; pharmacology ; Th1 Cells ; immunology

Plasmacytoid dendritic cells (pDCs), also known as type I interferon (IFN)-producing cells, are specialized immune cells characterized by their extraordinary capabilities of mounting rapid and massive type I IFN response to nucleic acids derived from virus, bacteria or dead cells. PDCs selectively express endosomal Toll-like receptor (TLR) 7 and TLR9, which sense viral RNA and DNA respectively. Following type I IFN and cytokine responses, pDCs differentiate into antigen presenting cells and acquire the ability to regulate T cell-mediated adaptive immunity. The functions of pDCs have been implicated not only in antiviral innate immunity but also in immune tolerance, inflammation and tumor microenvironments. In this review, we will focus on TLR7/9 signaling and their regulation by pDC-specific receptors.
Animals ; Dendritic Cells ; cytology ; metabolism ; Humans ; Protein Transport ; Proteolysis ; Signal Transduction ; Toll-Like Receptor 7 ; chemistry ; metabolism ; Toll-Like Receptor 9 ; chemistry ; metabolism

This study was to establish the method of purifying heat shock protein GP96 from K562 cells and explore the differentiation and function of human DC influenced by heat shock prolein (HSP). Using ammonium sulfate precipitation, conA-sepharose affinity chromatography and DEAE-sephacel anion exchange chromatography GP96 from K562 cells lysate was isolated and purified. The identification of the purified protein was controlled by Western blot with anti-human GP96 IgG. Human dendritic cell derived from peripheral blood mononuclear cell were cultured with purified GP96. The phenotype changes of DC were analyzed by flow cytometry and MLR was detected by MTT. The results showed that 60-80 microg GP96 was purified successfully from 1 x 10(10) K562 cells. DC stimulated with HSP-GP96 had higher expression rates of CD83, CD86, HLA-DR and lower expression rates of CD1a and had stronger ability to induce T cells proliferation. It is concluded that heat shock protein GP96 can be isolated and purified from K562 cells and could induce maturation of dendritic cell. HSP-DC vaccine show stronger ability to induce T cell proliferation than DC.
Antigens, Neoplasm ; isolation & purification ; pharmacology ; Cancer Vaccines ; immunology ; Cell Differentiation ; drug effects ; Dendritic Cells ; cytology ; drug effects ; immunology ; Humans ; Immunophenotyping ; K562 Cells ; chemistry

Dendritic Cells ; pathology ; Histiocytes ; pathology ; Histiocytosis, Langerhans-Cell ; pathology ; Humans ; Keratins ; analysis ; Langerhans Cells ; pathology ; Lymphoma, Large B-Cell, Diffuse ; pathology ; Sarcoma ; chemistry ; classification ; pathology

Due to their high immunostimulatory ability as well as the critical role they play in the maintenance of self-tolerance, dendritic cells have been implicated in the pathogenesis of autoimmune diseases. The non-obese diabetic (NOD) mouse is an animal model of autoimmune type 1 diabetes, in which pancreatic beta cells are selectively destroyed mainly by T cell-mediated immune responses. To elucidate initiation mechanisms of beta cell-specific autoimmunity, we attempted to generate bone marrow-derived dendritic cells from NOD mice. However, our results showed low proliferative response of NOD bone marrow cells and some defects in the differentiation into the myeloid dendritic cells. NOD dendritic cells showed lower expressions of MHC class II, B7-1, B7-2 and CD40, compared with C57BL/6 dendritic cells. In mixed lymphocyte reactions, stimulatory activities of NOD dendritic cells were also weak. Treatment with LPS, INF-gamma and anti-CD40 stimulated NOD dendritic cells to produce IL-12p70. The amount of IL-12, however, appeared to be lower than that of C57BL/6. Results of the present study indicated that there may be some defects in the development of NOD dendritic cells in the bone marrow, which might have an impact on the breakdown of self tolerance.
Animal ; Autoimmune Diseases/pathology ; Autoimmune Diseases/immunology ; Bone Marrow Cells/pathology* ; Bone Marrow Cells/immunology* ; Bone Marrow Cells/chemistry ; Cell Differentiation/immunology ; Cell Differentiation/drug effects ; Dendritic Cells/pathology* ; Dendritic Cells/immunology* ; Dendritic Cells/chemistry ; Diabetes Mellitus, Insulin-Dependent/pathology* ; Diabetes Mellitus, Insulin-Dependent/immunology ; Enzyme-Linked Immunosorbent Assay ; Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology ; Interleukin-12/analysis ; Interleukin-4/pharmacology ; Lipopolysaccharides/pharmacology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Inbred NOD ; Obesity

OBJECTIVETo investigate the anti-atherosclerosis mechanism of Astragalus mongholicus polysaccharides (APS) by examining its effect on gene expression profiles of the dendritic cells (DCs) from healthy donors.

METHODSPeripheral blood DCs from healthy donors were incubated with 200 mg/L APS overnight, and changes in the gene expression profiles were investigated using microarray technique and RT-PCR.

RESULTSCompared with the control cells, APS-treated DCs showed significantly up-regulated expressions of CD36 (0.97 ± 0.23 vs 5.45 ± 1.14) and IL-27 (1.08 ± 0.22 vs 2.97 ± 0.61) and down-regulated expression of expression of IFI16 (0.98 ± 0.18 vs 0.46 ± 0.11).

CONCLUSIONSAPS can promote the maturation and differentiation of DCs by up-regulating CD36 and IL-27 and down-regulating IFI16, and thus positively affects the occurrence and progression of the atherosclerosis.

Astragalus Plant ; chemistry ; CD36 Antigens ; metabolism ; Cell Differentiation ; Dendritic Cells ; drug effects ; Humans ; Interleukins ; metabolism ; Nuclear Proteins ; metabolism ; Phosphoproteins ; metabolism ; Polysaccharides ; pharmacology ; Transcriptome

OBJECTIVETo investigate the feasibility of reduction in tumor antigen peptide dose by dendritic cell (DC)-presenting so as to elucidate the characteristics of modifying DC by heat shock protein (Hsp70) and antigen peptide.

METHODSAntigen peptide bound to Hsp70 was used to modify DC in vitro. The metabolism of the modified DC and the cytokine secreted thereby was determined. Then the activation of lymphocytes by the modified DC and Hsp70-H22 peptide was tested. The cytotoxicity of the activated lymphocytes to H22 tumor cells and the inhibition of tumor in mice by DC injection and Hsp70-H22 peptide was tested.

RESULTS0.15 micro g of H22 peptide bound to Hsp70 could mature 2 x 10(5) DC. 4 x 10(3) matured DC could activate 2 x 10(6) lymphocytes. The same amount of lymphocyte could be activated to produce similar cytotoxicity to tumor cells by either DC modified by 0.003 micro g of peptides bound with Hsp70 or by direct stimulation with 0.15 micro g of peptides bound to Hsp70. The dose of peptide could be reduced to 1/50 if the modified DC injection was used instead of direct Hsp70-peptide injection. Peptide from the normal hepatocytes, if bound to Hsp70, could not mature DC, nor could it activate lymphocytes through DC.

CONCLUSIONThe dose of Hsp70-H22 peptides can be reduced significantly by DC-presenting to activate lymphocytes. Peptides from normal cells, being unable to activate the lymphocytes by either Hsp70-presenting or DC-presenting, have little to offer in the induction of autoimmunity.

Animals ; Antigens, Neoplasm ; immunology ; Dendritic Cells ; immunology ; Disease Models, Animal ; HSP70 Heat-Shock Proteins ; chemistry ; immunology ; therapeutic use ; Immunity ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Neoplasms, Experimental ; prevention & control ; Peptides ; chemistry ; immunology ; therapeutic use ; Tumor Cells, Cultured