To investigate the effect of INF-γ on the expression of programmed death ligand 1 (PD-L1), epithelial-mesenchymal transition (EMT) and the potential mechanism in breast cancer cell line MDA-MB-231, the cells were treated with different concentrations of INF-γ. The expressions of proteins, including PD-L1, cell-migration-related proteins (E-cadherin, N-cadherin and vimentin), ERK, p-ERK, Jak2, and p-Jak2 were detected by Western blotting analysis and immunofluorescent staining assay. Cell migration was studied through cell wound healing assay and transwell assay. IFN-γ could up-regulate the expressions of PD-L1 in MDA-MB-231 cells. The cell migration rate was significantly increased after adding IFN-γ. The expression levels of vimentin and N-cadherin were increased whereas the expression of E-cadherin was decreased after adding IFN-γ. The expression levels of ERK, p-ERK, Jak2 and p-Jak2 were significantly increased and this phenomenon was inhibited when adding ERK inhibitor U0126 or Jak2 inhibitor AG490. These results demonstrate that IFN-γ could up-regulate the expression of PD-L1, promote cell migration and transmission, and facilitate epithelial-mesenchymal transformation of breast cancer cells and this process may be related with ERK and Jak2-STAT signaling pathways.
B7-H1 Antigen ; Breast Neoplasms ; Cell Line, Tumor ; Cell Movement ; Epithelial-Mesenchymal Transition ; Extracellular Signal-Regulated MAP Kinases ; Humans ; Interferon-gamma ; Janus Kinase 2 ; STAT Transcription Factors ; Signal Transduction

OBJECTIVETo investigate the trichostain A (TSA)-induced expression of costinmulatory molecules CD80 and CD86 in HL-60, K562 and mononuclear cells (MNC) of bone marrow in AML patients and its clinical significance.

METHODSThe TSA-induced expression of costimulatory molecules CD80, CD86 in HL-60, K562 and BMMNC, and the cell viability were detected by flow cytometry; the mRNA expression of CD80 and CD86 was detected by RT-PCR; after the TSA-induced HL-60 cells and K562 cells were irradiated with 75 Gy, the effect of these cells on proliferation of PBMNC from healthy volunteers was determined with CCK-8 method.

RESULTSThe HL-60 cells and BMMNC in AML patients expressed CD86, not expressed CD80, while the K562 cells not expressed CD86 and CD80. TSA could up-regulate the expression of CD86 in HL-60 cells and BMMNC of AML patients. The TSA-induced HL-60 cells expressing costimulatory molecule CD86 showed the proliferative effect on BMMNC from healthy volunteers.

CONCLUSIONThe TSA can induce the expression of costimulatory molecule CD86 in HL-60 cells and BMMNC in AML patients, and can improve the proliferation of PBMNC in healthy volunteers.

B7-1 Antigen ; B7-2 Antigen ; Cell Line, Tumor ; Cell Survival ; Flow Cytometry ; Humans ; Hydroxamic Acids ; Leukemia, Myeloid, Acute

OBJECTIVETo investigate the role of glycogen synthase kinase 3β (GSK-3β) in the maturation and function of murine bone marrow-derived dendritic cells (BMDCs).

METHODSMature DCs (mDCs) induced by LPS were examined for GSK-3β phosphorylation level with Western blotting before and after LPS exposure. To explore the role of GSK-3β in maturation and function of DCs, we added SB216763, a selective inhibitor of GSK-3β, in the cell culture of immature DCs (iDCs), and examined CD40 and CD86 expressions in the cells by flow cytometry and the expression of IL-6, IL-12 and IL-10 mRNA by real-time PCR; the changes of the immunogenicity of the cells was evaluated by mixed lymphocyte reaction. The expression of GSK-3β and RelB was examined by Western blotting in DC2.4 cells transfected with a lentiviral vector over-expressing murine GSK-3β gene.

RESULTSLPS exposure significantly lowered GSK-3β activity in iDCs as demonstrated by increased Ser9 phosphorylation and reduced Tyr216 phosphorylation. GSK-3β inhibition induced DC maturation by increasing the expression of surface costimulatory molecules CD40 and CD86, lowered the expressions of IL-6 and IL-12 while enhanced the expression of IL-10 in iDCs, and impaired mixed lymphocyte reaction of the cells. In DC2.4 cells, lentivirus-mediated over-expression of GSK-3β obviously down-regulated the expression of RelB.

CONCLUSIONSGSK-3β is a crucial enzyme involved in the differentiation and maintenance of an immature phenotype of DCs. GSK-3β is constitutively active in iDCs to inhibit their spontaneous maturation. DCs become phenotypically mature after inhibition of GSK-3β, which also executes a proinflammatory task in DC activation. The reduction of RelB protein levels as a result of GSK-3β overexpression supports GSK-3β as a new target for inducing tolerogenic DCs.

Animals ; B7-2 Antigen ; metabolism ; CD40 Antigens ; metabolism ; Cell Differentiation ; Cells, Cultured ; Culture Media ; chemistry ; Dendritic Cells ; enzymology ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Indoles ; chemistry ; Interleukin-10 ; metabolism ; Interleukin-12 ; metabolism ; Interleukin-6 ; metabolism ; Lentivirus ; Lymphocyte Culture Test, Mixed ; Maleimides ; chemistry ; Mice ; Myeloid Cells ; enzymology ; Phosphorylation ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Signal Transduction

B7-1 Antigen ; B7-2 Antigen ; Hematologic Neoplasms ; Humans

OBJECTIVETo investigate the mechanism of Mycobacterium tuberculosis invasion to mouse dendritic cells (DC).

METHODSMycobacterium tuberculosis strain H37Rv was co-cultured with mouse DC2.4 cells.The mRNA expression of Toll-like receptor 2/4(TLR2/4) in DC2.4 cells was detected by fluorescent quantitative real-time PCR and the protein expression of nuclear factor κB(NF-κB) was assessed by Western blotting.The extracellular concentration of tumor necrosis factor α(TNF-α) was measured by ELISA methods during Mycobacterium Tuberculosis invasion.Indirect immunofluorescent staining and flow cytometry assay were used to detect the expression of CD80 and CD86 on DC2.4 cells before and after invasion.

RESULTSThe invasion of Mycobacterium tuberculosis in DC2.4 cells was observed after 2 h of co-incubation.The rates of invasion were (37.9±5.6)%,(51.2±7.6)%,(57.2±8.9)% and(63.9±6.8)% at 6,8,10 and 12 h after co-incubation,respectively.The mRNA expression level of TLR2 /4 was significantly increased at 6 h but decreased at 10 h after co-incubation.The expressions of NF-κB p65 and TNF-α were higher in DC2.4 cells after being invaded by 6,8,and 10 h and then gradually decreased.CD80 and CD86 expression were increased on DC2.4 at 6 h after co-incubation.

CONCLUSIONInvasion of Mycobacterium tuberculosis strain H37Rv to DC might enhance its antigen-presenting function through activation of TLR2/4-NF-kB signaling pathway.

Animals ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; Cells, Cultured ; Dendritic Cells ; immunology ; metabolism ; Mice ; Mycobacterium tuberculosis ; NF-kappa B ; metabolism ; Signal Transduction ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptor 4 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

This study was aimed to elucidate the expression of costimulatory molecule CD80 and CD86 in HL-60 cells induced by proteasome inhibitor MG132 and its effect on allogeneic mixed lymphocyte reaction. Acute myelocytic leukemia cell line HL-60 and chronic myelocytic leukemia cell line K562 were cultured. The viability of the cells was measured by flow cytometry. Proteasome inhibitor MG132 at the concentrations of 2 or 3 µmol/L was used to stimulate the HL-60 cell cultured for 24 h and 48 h respectively, and the Annexin V/7-AAD staining and flow cytomotry were used to detect the apoptosis of the HL-60 cells. HL-60 and K562 cells were treated with 1 µmol/L MG132 for 24 h and 48 h respectively, then CD80 and CD86 antibodies were added, finally the expression of CD80 and CD86 was analysed by flow cytomery. The mRNA expression of CD86 in the HL-60 cells treated with 1 µmol/L MG132 was detected by RT-PCR. HL-60 and K562 cells were treated by 1 µmol/L MG132 and then underwent irradiation of 75 Gy (60)Co to kill the cells with their antigenicity preserved. Peripheral blood mononuclear cells (PBMNCs) of healthy volunteers, as reactive cells, were isolated and inoculated into the (60)Co irradiated HL-60 cells of different concentrations, as stimulating cells, CCK-8 was added and then the A value of absorbance was measured at the wave length of 450 nm in an enzyme labeling instrument. The results showed that the cell viability of the HL-60 cells treated with 1 µmol/L MG132 for 24 h an d 48 h was 92.95% and 85.87% respectively. The apoptotic rates of the HL-60 cells treated with MG132 increased in dose-and time-dependent manner. High-concentration of MG132 directly killed HL-60 cells. Before MG132 treatment K562 cells did not express CD86, but the CD86 expression of the HL-60 cells was up-regulated time-dependently after MG132 treatment (P < 0.01). The mRNA expression of CD86 in the HL-60 treated with MG132 was up-regulated time-dependently (P < 0.01). CCK-8 test showed that the proliferation level of PBMNC gradually increased along with the concentration of HL-60 cells treated with MG132 and reached its peak when the concentration of the HL-60 cells was 1×10(5) (P < 0.01). No remarkable proliferation of PBMNC was observed in the K562 groups no matter if the HL-60 cells had been treated with MG132. It is concluded that the high concentration of MG132 can directly kill HL-60 cells, low-concentration of MG132 can induce the expression of costimulatory molecule CD86 in HL-60 cells, also can improve the proliferation of PBMNC.
Apoptosis ; B7-2 Antigen ; immunology ; Cell Survival ; Flow Cytometry ; HL-60 Cells ; Humans ; K562 Cells ; Leukocytes, Mononuclear ; drug effects ; Leupeptins ; pharmacology ; Lymphocyte Culture Test, Mixed ; Proteasome Inhibitors ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Up-Regulation

OBJECTIVEAntigen-presenting cells such as monocytes and dendritic cells (DCs) stimulate T-cell proliferation and activation during adaptive immunity. This cellular interaction plays a role in the growth of atherosclerotic plaques. Tanshinone II A (TSN) had been shown to decrease the growth of atherosclerotic lesions. We therefore investigated the ability of TSN to inhibit human monocyte-derived DCs and their T-cellstimulatory capacity.

METHODSDCs derived from human monocytes cultured with recombinant human interleukin (IL)-4 and recombinant human granulocyte-macrophage colony-stimulating factor were co-cultured with TSN and lipopolysaccharide for 48 h. Phosphate-buffered saline was used as a negative control. Activation markers and the capacity of DCs for endocytosis were measured by flow cytometry, and proinflammatory cytokines were measured by enzyme-linked immunosorbent assays. DCs were co-cultured with lymphocytes to measure T-cell proliferation and IL-2 secretion by mixed lymphocyte reactions.

RESULTSTSN dose-dependently attenuated DC expression of costimulatory molecules (CD86), and decreased expression of major histocompatibility complex class II (human loukocyte antigen-DR) and adhesion molecules (CD54). Moreover, TSN reduced secretion of the proinflammatory cytokines IL-12 and IL-1 by human DCs, and restored the capacity for endocytosis. Finally, TSN-preincubated DCs showed a reduced capacity to stimulate T-cell proliferation and cytokine secretion.

CONCLUSIONSTSN inhibits DC maturation and decreases the expression of proinflammatory cytokines, while impairing their capacity to stimulate T-cell proliferation and cytokine secretion. These effects may contribute to the influence of TSN on the progression of atherosclerotic lesions.

Antigen-Presenting Cells ; drug effects ; Atherosclerosis ; immunology ; pathology ; B7-2 Antigen ; metabolism ; Cell Membrane ; drug effects ; metabolism ; Cytokines ; secretion ; Dendritic Cells ; drug effects ; immunology ; secretion ; Diterpenes, Abietane ; pharmacology ; Endocytosis ; drug effects ; Flow Cytometry ; Humans ; Immunity, Cellular ; drug effects ; Inflammation Mediators ; metabolism ; Lymphocyte Activation ; drug effects

OBJECTIVETo discuss the anti-tumor effect of Xihuang pill on tumor-bearing rats and its effect on the immune clearance function of tumor-bearing organisms.

METHODWalker256 tumor cells were adopted to establish the tumor-bearing rat model. The rats were randomly divided into five groups: the normal control group, the model control group, the lentinan group and Xihuang pill low dose, middle dose and high dose groups, with 10 rats in each group, and continuously treated and given drugs for 14 d after modeling. Blood and tumors were collected from abdominal aorta to calculate the tumor inhibition rate. The content of CD3+, CD4+, CD8+ T cells and adhesion molecule B7-1 (CD80) in peripheral blood were detected by flow cytometry (FCM). The expressions of IL-2 and IFN-gamma in were determined by ELISA.

RESULTThe tumor inhibition rate of the Xihuang pill high dose group was 33. 1 percent. Compared with the model group, the Xihuang pill large dose group showed significantly low IL-2, IFN-gamma, CD3+, CD4+, B7-1 in peripheral blood, with statistical significance in their differences (P < 0.05).

CONCLUSIONXihuang pill could show its anti-tumor effect by enhancing the immune clearance function and increasing IL-2, IFN-gamma, CD3+ T, CD4+ T, B7-1 in peripheral blood.

Animals ; Antineoplastic Agents ; administration & dosage ; B7-1 Antigen ; genetics ; immunology ; Breast Neoplasms ; drug therapy ; genetics ; immunology ; CD4-Positive T-Lymphocytes ; drug effects ; immunology ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Flow Cytometry ; Humans ; Immune System ; drug effects ; Immunologic Factors ; administration & dosage ; Interferon-gamma ; genetics ; immunology ; Interleukin-2 ; genetics ; immunology ; Rats ; Rats, Wistar ; Tumor Burden ; drug effects

Dendritic cells (DCs) are crucial for the induction and maintenance of tumor-specific immune responses. Studies have shown that tumor-associated DCs are immunosuppressed in some human tumors. However, phenotype and function of DCs in retinoblastoma (RB) remain unclear. RB cell supernatant (RBcs) was used to treat DCs in vitro to explore the effect of RB cells on DCs. DCs were generated from peripheral blood mononuclear cells of healthy donors. On day 5 of culture, DCs were treated with RBcs for 24 h, and then purified using magnetic beads. The maturation of DCs was induced by TNF-α or LPS. After treatment with RBcs, expression of co-stimulatory molecules CD80 and CD86 was elevated in DCs, accompanied by increased production of IL-12p70, TNF-α, IL-6, IL-1β, and IL-8 but decreased production of IL-10. RBcs neither inhibited DC maturation nor promoted DC apoptosis. Moreover, RBcs-exposed DCs stimulated allogenetic T cell proliferation and T cell-derived cytokine production. These results indicate that RBcs can improve DCs' antigen presenting function and capability to activate T cells, suggesting that RB cells may have an immunostimulatory effect on DCs, and DC-based immunotherapy may be adopted in the treatment of RB.
B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Culture Media, Conditioned ; pharmacology ; Cytokines ; metabolism ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Humans ; Lipopolysaccharides ; toxicity ; Retinal Neoplasms ; metabolism ; pathology ; Retinoblastoma ; metabolism ; pathology ; T-Lymphocytes ; cytology ; immunology ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo compare the effects of Bushen Jiedu Recipe (BJR) and Jianpi Jiedu Recipe (JJR) containing plasma on dendritic cells (DCs) of chronic hepatitis B virus (HBV) infection patients under different immune states.

METHODSRecruited were 36 chronic HBV infection outpatients from First Affiliated Hospital of Hunan University of Traditional Chinese Medicine from April 2010 to January 2011. They were assigned to the immune tolerance group (18 cases) and the immune clearance group (18 cases).Another 10 healthy subjects were recruited as the healthy control group. Their anticoagulated peripheral venous blood was respectively collected. The peripheral blood mononuclear cells (PBMCs) were isolated and further extracted for incubating DCs. The DCs were intervened by BJR and JJR containing plasma. The morphology of DCs was identified. The expressions of CD1alpha, CD80, CD86, and HLA-DR were detected. The level of interferon-alpha (IFN-alpha) in the supernatant was observed by ELISA.

RESULTSThe CD80 expression level was lower in the immune clear group than in the healthy control group before intervention (P < 0.05). The expression levels of CD80, CD86, and HLA-DR were lower in the immune tolerance group than in the healthy control group before intervention (P < 0.05).The IFN-alpha expression level was lower in the immune tolerance group and the immune clearance group than in the healthy control group before intervention (P < 0.05). The expression levels of CD80, HLA-DR, and IFN-alpha were lower in the immune tolerance group than in the immune clearance group before intervention (P < 0.05). Compared with the same group before intervention, the CD80 expression significantly increased in each treatment group (P < 0.05). After intervention the expression levels of CD80 and HLA-DR were higher in the immune tolerance group than in the immune clearance group in the same time phase, and the CD86 expression level was higher in the BJR group than in the immune clearance group in the same time phase, showing statistical difference (P < 0.05).

CONCLUSIONSThe middle dose BJR and the small dose JJR both could promote the recovery of DCs in chronic HBV infection patients. Besides, BJR showed more prominent effects on the function of DCs in chronic HBV infection patients in the immune tolerance stage.

Adult ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; Case-Control Studies ; Dendritic Cells ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; HLA-DR Antigens ; metabolism ; Hepatitis B, Chronic ; blood ; drug therapy ; immunology ; Humans ; Immune Tolerance ; drug effects ; Interferon-alpha ; metabolism ; Male ; Phytotherapy ; Plasma ; Young Adult