OBJECTIVETo investigate the effect of down-expression of inhibitor of differentiation-1 (Id-1) on the differentiation of dendritic cell sarcoma (DCS) cells in vitro.

METHODSDown-regulation of the expression of Id-1 in DCS cells was performed by RNAi, and confirmed by protein and mRNA quantitative analyses. Cellular differentiation and biological behavior including malignant phenotypes of the cells were evaluated. All experiments included negative (no treatment group and no-target siRNA) and positive (induction-differentiation drug sodium butyrate) controls.

RESULTSWhen the expression of Id-1 was down regulated, the DCS cells showed more mature morphology including cell enlargement, longer cellular extensions, more branches, and decreased nuclear/plasma ratio. Differentiation marker expression (Id-2 and CD86) was also increased. RNAi treated cells at 24 and 48 hours, showed increase percentage of cells at G0/G1 phase and less cells at S phase (P < 0.01). Importantly, the abilities of cell proliferation, colony formation and invasiveness were significantly decreased (P < 0.01), as evidenced by MTT, colony formation and transwell assays respectively.

CONCLUSIONRNAi inhibition of Id-1 protein can induce differentiation of malignant solid tumor cells along with reversion of their malignant phenotype.

Animals ; Cell Differentiation ; drug effects ; physiology ; Cell Proliferation ; drug effects ; Dendritic Cells ; cytology ; drug effects ; Down-Regulation ; Inhibitor of Differentiation Proteins ; pharmacology ; Mice ; Tumor Cells, Cultured

OBJECTIVETo explore the influence of oxidized high-density lipoprotein (oxHDL) on the maturation and migration of bone marrow-derived dendritic cells (BMDCs) from C57BL/6J mice.

METHODSThe C57BL/6J mice bone marrow cell suspension was prepared and purified. Recombinant granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant interleukin-4 (rmIL-4) were used to promote monocytes to differentiate and suppress lymphocytes. Then 50 microg/mL oxHDL was added to stimulate BMDCs, using 50 microg/mL high-density lipoprotein (HDL) as homologous protein control, PBS as negative control, and 1 microg/mL lipopolysaccharide (LPS) as positive control. The CD86 and MHCII expression rates were detected with fluorescence-activated cell sorting (FACS). Liquid scintillation counting (LSC) was used in mixed lymphocyte reactions (MLRs) to reflect the ability of BMDCs in stimulating the proliferation of homologous T cells. Levels of cytokines IL-12 and IL-10 were detected by ELISA. The cell migration was evaluated with the transwell system.

RESULTSCompared with PBS group, the expressions of CD86 and MHCII, counts per minute of MLRs, secretion of IL-12 and IL-10, and number of migrated cells in oxHDL group and LPS group significantly increased (all P<0.05), while the increment was less in oxHDL group than LPS group. The number of migrated cells in oxHDL group was about twice of that in HDL group.

CONCLUSIONOxHDL may promote the maturation and migration of BMDCs in vitro.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; physiology ; Cell Differentiation ; drug effects ; Cell Movement ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; physiology ; Humans ; Lipoproteins, HDL ; metabolism ; pharmacology ; Lipoproteins, LDL ; metabolism ; pharmacology ; Mice ; Mice, Inbred C57BL

BACKGROUNDDendritic cells are professional antigen-presenting cells found in an immature state in epithelia and interstitial space, where they capture antigens such as pathogens or damaged tissue. Matrix metallopeptidase 13 (MMP-13), a member of the collagenase subfamily, is involved in many different cellular processes and is expressed in murine bone marrow-derived dendritic cells (DCs). The function of MMP-13 in DCs is not well understood. Here, we investigated the effect of MMP-13 on DC maturation, apoptosis, and phagocytosis.

METHODSBone marrow-derived dendritic cells were obtained from C57BL/6 mice. One short-interfering RNA specific for MMP-13 was used to transfect DCs. MMP-13-silenced DCs and control DCs were prepared, and apoptosis was measured using real-time polymerase chain reaction and Western blotting. MMP-13-silenced DCs and control DCs were analyzed for surface expression of CD80 and CD86 and phagocytosis capability using flow cytometry.

RESULTSCompared to the control DCs, MMP-13-silenced DCs increased expression of anti-apoptosis-related genes, BAG1 (control group vs. MMP-13-silenced group: 4.08 ± 0.60 vs. 6.11 ± 0.87, P = 0.008), BCL-2 (control group vs. MMP-13-silenced group: 7.54 ± 0.76 vs. 9.54 ± 1.29, P = 0.036), and TP73 (control group vs. MMP-13-silenced group: 4.33 ± 0.29 vs. 5.60 ± 0.32, P = 0.001) and decreased apoptosis-related genes, CASP1 (control group vs. MMP-13-silenced group: 3.79 ± 0.67 vs. 2.54 ± 0.39, P = 0.019), LTBR (control group vs. MMP-13-silenced group: 9.23 ± 1.25 vs. 6.24 ± 1.15, P = 0.012), and CASP4 (control group vs. MMP-13-silenced group: 2.07 ± 0.56 vs. 0.35 ± 0.35, P = 0.002). Protein levels confirmed the same expression pattern. MMP-13-silenced groups decreased expression of CD86 on DCs; however, there was no statistical difference in CD80 surface expression. Furthermore, MMP-13-silenced groups exhibited weaker phagocytosis capability.

CONCLUSIONThese results indicate that MMP-13 inhibition dampens DC maturation, apoptosis, and phagocytosis.

Animals ; Apoptosis ; drug effects ; physiology ; Bone Marrow Cells ; cytology ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Female ; Lipopolysaccharides ; pharmacology ; Matrix Metalloproteinase 13 ; metabolism ; physiology ; Mice ; Mice, Inbred C57BL ; RNA, Small Interfering

Atherosclerosis ; etiology ; prevention & control ; Cytokines ; secretion ; Dendritic Cells ; drug effects ; immunology ; Endocytosis ; Fenofibrate ; pharmacology ; Humans ; Immunophenotyping ; Lipoproteins, LDL ; toxicity ; Monocytes ; cytology ; PPAR alpha ; agonists ; physiology

OBJECTIVEThis study is aimed at developing a simple and easy way to generate dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) in vitro.

METHODSPBMCs were isolated directly from white blood cell rather than whole blood and purified by patching methods (collecting the attached cell and removing the suspension cell). DCs were then generated by culturing PBMCs for six days with 30 ng/ml recombinant human granulocyte-macrophage stimulating factor (rhGM-CSF) and 20 ng/ml recombinant human interleukin-4 (rhIL-4) in vitro. On the sixth day, TNF-alpha (TNFalpha) 30 ng/ml was added into some DC cultures, which were then incubated for two additional days. The morphology was monitored by light microscopy and transmission electronic microscopy, and the phenotypes were determined by flow cytometry. Autologous mixed leukocyte reactions (MLR) were used to characterize DC function after TNFalpha or lipopolysaccharide (LPS) stimulations for 24 h.

RESULTSAfter six days of culture, the monocytes developed significant dendritic morphology and a portion of cells expressed CD1a, CD80 and CD86, features of DCs. TNFalpha treatment induced DCs maturation and up-regulation of CD80, CD86 and CD83. Autologous MLR demonstrated that these DCs possess potent T-cell stimulatory capacity.

CONCLUSIONThis study developed a simple and easy way to generate DCs from PBMCs exposed to rhGM-CSF and rhIL-4. The DCs produced by this method acquired morphologic and antigenic characteristics of DCs.

Cell Culture Techniques ; methods ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; physiology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Interleukin-4 ; pharmacology ; Leukocytes, Mononuclear ; cytology ; drug effects ; physiology ; Phenotype ; Recombinant Proteins ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology

To investigate whether the dendritic cells (DC) could grow up in cultural system with umbilical cord serum (UCS), the UCS was used in the culture instead of fetal calf serum. The phenotype of dendritic cells was detected by flow cytometry and the antigen presenting ability of DC in allo-MLR was measured by MTT assay. The results showed that DC grown in UCS (UCS-DC) had higher expression rate of CD86, CD83 and HLA-DR than that in grown in FCS (FCS-DC). (P < 0.05), and their expression of CD1a was lower than that of FCS-DC. The ability to induce T cell proliferation had no difference between UCS-DC and FCS-DC. It is suggested that dendritic cells with more mature phenotype had been produced in the medium containing UCS than those in the medium containing FCS, and UCS-DC possessed potent ability to stimulate proliferation of allogeneic T cells.
Animals ; Antigens, CD ; immunology ; B7-2 Antigen ; Cattle ; Cell Culture Techniques ; methods ; Cell Division ; drug effects ; Culture Media ; pharmacology ; Dendritic Cells ; cytology ; drug effects ; immunology ; Fetal Blood ; physiology ; HLA-DR Antigens ; immunology ; Humans ; Immunoglobulins ; immunology ; Membrane Glycoproteins ; immunology

AIMTo investigate the role of 1 alpha, 25-dihydroxyvitamin D3 (calcitriol) and its analogues tacalcitol and 24, 25(OH)2D3 on the phagocytosis of human monocyte-derived dendritic cells (MoDC).

METHODSMoDC were generated in vitro by differentiating monocytes in the presence of GM-CSF and IL-4 for 5 days. Expression of mannose receptor (MR) and Fc gamma receptors (Fc gamma Rs) by MoDC was analysed by flow cytometry. Zymosan ingestion was measured to assess the phagocytosis of MoDC.

RESULTSMoDC expressed high level of MR and Fc gamma Rs and showed the capacity of zymosan ingestion. Calcitriol and tacalcitol but no 24, 25(OH)2D3 not only upregulated the expression of MR and Fc gamma Rs on MoDC but also correspondingly enhanced their phagocytosis by increasing zymoasan ingestion. Furthermore, the upregulatory role occurred in the early stage of MoDC differentiation and was irreversible. The upregulatory role of calcitriol was dose dependent.

CONCLUSIONCalcitriol and its analogue tacalcitol may play an important role in dendritic cell binding and capturing foreign antigens at the initiation of immune response.

Calcitriol ; pharmacology ; Calcium Channel Agonists ; pharmacology ; Dendritic Cells ; drug effects ; metabolism ; physiology ; Dihydroxycholecalciferols ; pharmacology ; Humans ; Lectins, C-Type ; metabolism ; Mannose-Binding Lectins ; metabolism ; Monocytes ; cytology ; Phagocytosis ; drug effects ; Receptors, Cell Surface ; metabolism ; Receptors, IgG ; metabolism

Taxol has been used effectively in cancer therapies. Our previous study demonstrated that taxol induced altered maturation and improved viability of dendritic cells (DCs). However, the effects of taxol on DC viability have not been fully elucidated. In the present study, flow cytometric analyses revealed that taxol treatment significantly increased the number of viable DCs and the expression levels of a representative anti-apoptotic protein Bcl-xL. Furthermore, mobilization of the p65 subunit of nuclear factor-kappaB (NF-kappaB) from the cytosol to the nucleus in DCs was observed by confocal microscopy. An inhibition assay using N-p-tosyl-L-phenylalanine chloromethyl ketone confirmed that NF-kappaB was intimately involved in the effects of taxol on DC viability. In addition, we investigated the mechanisms of taxol enhancement of DC viability. Since taxol is a popular anticancer agent used in clinic, this study may provide a rationale for the use of taxol in DC immunotherapy to treat cancer patients. Taken together, these results confirm that taxol increases DC viability, and this information may provide new insights for new clinical applications of both taxol and DCs.
Animals ; Antineoplastic Agents, Phytogenic/pharmacology ; Blotting, Western ; Cell Survival/drug effects/physiology ; Dendritic Cells/cytology/*drug effects/physiology ; Female ; Flow Cytometry ; Interleukin-12/physiology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Microscopy, Confocal ; Paclitaxel/*pharmacology ; Tosylphenylalanyl Chloromethyl Ketone/pharmacology ; Transcription Factor RelA/antagonists & inhibitors/*physiology ; Tumor Necrosis Factor-alpha/physiology ; bcl-X Protein/*physiology

OBJECTIVETo investigate the effects of transforming growth factor beta1 (TGF-beta1) on dendritic cells (DC).

METHODSMurine bone marrow cells were cultured with different cytokine combinations to develop immature DC (imDC, GM-CSF only) and TGFbeta-DC (GM-CSF + TGF-beta1), and their responses to lipopolysaccharide (LPS) stimulation were observed. The cell ultrastructure was observed by transmission electron microscopy and their phenotypes were assessed by flow cytometry (FCM). The allogeneic stimulating capacity of DC was assayed by mixed lymphocyte reaction (MLR) with BrdU incorporation. IL-12p70 protein was detected by ELISA and the expressions of Toll-like receptor 4 (TLR4) on DCs were analyzed with semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSCompared to imDC, the TGFbeta-DC had no significant alterations in ultrastructure after LPS stimulation. The expressions of CD80, CD86 were lower on TGFbeta-DC than on imDC [(4.14 +/- 0.95)% vs (13.90 +/- 7.22)%; (8.60 +/- 0.75)% vs (20.63 +/- 5.03)%, P < 0.05, both]. The TGFbeta-DC kept their immature morphology after LPS stimulation, but the expressions of I-Ab and CD80 were slightly increased. After 96 h MLR, TGFbeta-DC had weaker stimulating capacity than imDC did, especially when DC/T cells ratios were 1:4 and 1:1 (P < 0.05, both). TGFbeta-DC showed impaired IL-12p70 production and down-regulation of TLR4 expression.

CONCLUSIONSTGF-beta1 can inhibit the expression of co-stimulatory molecules on DC. The TGFbeta-DC is resistant to maturation stimulus (LPS) and might be linked with TLR4 down-regulation.

Animals ; Bone Marrow Cells ; cytology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dendritic Cells ; drug effects ; physiology ; Down-Regulation ; drug effects ; Lipopolysaccharides ; pharmacology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Receptors, Cell Surface ; immunology ; metabolism ; Toll-Like Receptor 4 ; Transforming Growth Factor beta ; pharmacology ; Transforming Growth Factor beta1

Anti-cancer drug bleomycin (BLM) can cause acute lung injury (ALI) which often results in pulmonary fibrosis due to a failure of resolving acute inflammatory response. The aim of this study is to investigate whether toll-like receptor (TLR) 2 mediates BLM-induced ALI, inflammation and fibrosis. BLM-induced dendritic cells (DCs) maturation was analyzed by flow cytometry and cytokine secretion was detected by the ELISA method. The expression and activity of p38 and ERK MAPK were determined with Western blotting. The roles of TLR2 in ALI, inflammation and fibrosis were investigated in C57BL/6 mice administered intratracheally with BLM. The results demonstrated that BLM-administered mice had higher expression of TLR2 (P<0.001) and its signaling molecules. Blocking TLR2 significantly inhibited the maturation of DCs and reversed BLM-stimulated secretion of cytokines in DCs, such as IL-6 (P<0.001), IL-17 (P<0.05) and IL-23 (P<0.05). TLR2 inhibition attenuated BLM-induced increase of inflammatory cells in bronchoalveolar lavage fluid (BALF), and reversed the immunosuppressive microenvironment by enhancing TH1 response (P<0.05) and inhibiting TH2 (P<0.001), Treg (P<0.01) and TH17 (P<0.01) responses. Importantly, blocking TLR2 in vivo significantly protected BLM-administered mice from pulmonary injury, inflammation and fibrosis and subsequently increased BLM-induced animal survival (from 50% to 92%). Therefore, TLR2 is a novel potential target for ALI and pulmonary fibrosis.
Acute Lung Injury ; chemically induced ; metabolism ; pathology ; Animals ; Bleomycin ; toxicity ; Bronchoalveolar Lavage Fluid ; Cells, Cultured ; Cytokines ; secretion ; Dendritic Cells ; cytology ; metabolism ; Inflammation ; chemically induced ; metabolism ; pathology ; Interleukin-17 ; secretion ; Interleukin-23 ; secretion ; Interleukin-6 ; secretion ; Lung ; metabolism ; MAP Kinase Signaling System ; Male ; Mice ; Mice, Inbred C57BL ; Pulmonary Fibrosis ; chemically induced ; metabolism ; pathology ; T-Lymphocytes, Regulatory ; drug effects ; Th1 Cells ; drug effects ; Th2 Cells ; drug effects ; Toll-Like Receptor 2 ; metabolism ; physiology