Effect of lung cancer cells transfected with interleukin 18 gene on the immunological activity of dendritic cells

AIM: To study the effect of interleukin 18 gene transfected lung cancer cells on the phenotype and immunological activity of dendritic cells (DC). METHODS: A secretive IL-18 expression vector containing IL-12 P40 signal sequence was constructed and transfected into NCI-H460 lung cancer cells. DC induced from human peripheral blood were divided into 4 groups (NT, PV, GT and PD). DC were stimulated by non-transfected NCI-H460 cells, pure vector transfected NCI-H460 cells and IL-18 transfected NCI-H460 cells respectively for group NT, PV, GT, and non-stimulated DC for group PD. CD54, CD80, CD83 and CD86 on DC in the 4 groups were detected with flow cytometry. T cell proliferation stimulated by DC in the 4 groups was assayed with MTT method. IL-12 release in cultured DC supernatant was measured by ELISA. RESULTS: Sequencing result of the secretive IL-18 was correct. The transfected cells expressed IL-18 fusion gene and 18 kD IL-18 protein. DC in GT group expressed more surface molecules than those in other 3 groups. T cell proliferation and IL-12 secretion in GT group were higher than those in other 3 groups. CONCLUSION: IL-18 gene transfected NCI-H460 cell increases surface molecule expressions on DC. It also enhances immunological activity and IL-12 secretion in DC.     

Inhibitive effect of cytomegalovirus infection on immunological functions of dendritic cells

AIM: To investigate the effect of cytomegalovirus (CMV) infection on the immunological functions of dendritic cells (DC) derived from monocytes. METHODS: RT-PCR assay was used to detect the mRNA expression of CMV immediate early antigen (IEA) and glyceraldehyde phosphate dehydrogenase (GAPDH) genes in immature and mature dendritic cell (cmv-imDC, cmv-mDC) infected by 50-folds median tissue culture infective dose (TCID50) of CMV. The expression of early antigen (EA) in cmv-imDC and cmv-mDC was analyzed by indirect immunofluorescence assay. CMV late antigen pp65 was determined by flow cytometry. The allogeneic stimulating capacity of cmv-DC was assayed by mixed lymphocyte reaction (MLR) with BrdU incorporation. RESULTS: The expression of IE mRNA in cmv-mDC was lower than that in cmv-imDCs at 12 h after infection (0.102±0.020 and 0.862±0.124, respectively, P<0.05). EA, primarily localized in nucleus, was found in cmv-imDC (62.32±14.20)% and cmv-mDC (10.78±3.04)% at 24 h (P<0.01). pp65 positive cells in cmv-imDC and cmv-mDC at 72 h were 4.86% and 0.82%, respectively. Compared with untreated mDC, cmv-imDC showed depressed antigen presentation even after stimulated with maturation signal factor LPS (both P<0.05), while cmv-mDC had weaker stimulating capacity only when DC/T cell ratio was 1∶1 (P<0.05). CONCLUSION: CMV efficiently infectes and replicates in imDC. CMV suppresses the antigen presenting capacity of cmv-DC.     

Effects of progesterone on the maturation and immunologic function of dendritic cells from human peripheral blood

AIM: To study the effects of progesterone (P₄) on the maturation and immunologic function of dendritic cells (DCs) from human peripheral blood. METHODS: Cultured DCs were treated with P₄ at doses of 10-7 mol/L and 10-6 mol/L. The morphologic changes were observed under the scanning electronic microscope. The immunophenotypes of DCs in control and treated groups were analyzed by flow cytometry. IL-10 and IL-12 production in culture supernatant was examined by ELISA assay. The capability of the stimulatory activity of the DCs on allogeneic T cells in mixed reaction was tested by incorporation of ［3H］-TdR. RESULTS: Compared with control group, cultured DCs in the presence of P₄ displayed less dendritic pseudopod, expressed low levels of MHC-II, CD40, CD80 and CD86, and exhibited weakly activity in stimulating the proliferation of allogeneic T cells. Increase in IL-10 production and decrease in IL-12 production were observed. CONCLUSION: P₄ exerts negative effect on the maturation and immunologic function in dendritic cells from human peripheral blood.     

Effect of uric acid on the maturation and the biological function of BMDCs

AIM: To investigate the effect of uric acid on the maturation and the biological function of murine bone-marrow derived dendritic cells (BMDCs) in vitro.METHODS: BMDCs were cultured with GM-CSF, IL-4, LPS and uric acid. Cell phenotypes were analyzed by flow cytometry. MTT was used to detect the effect of uric acid on the function of BMDCs in stimulating the proliferation of T cells. IL-12 released by BMDCs was also detected. RESULTS: BMDCs were cultured and identified. Uric acid at concentrations of 200 mg/L and 400 mg/L increased the expression of the molecules (CD11c, CD83, CD86, IA/IE) on BMDCs surface and the IL-12 level in the culture supernatants (P<0.05), promoted the proliferation of T cells at the T: DC rate 5∶1, 10∶1, 20∶1 (P<0.05). However, uric acid at concentration of 70 mg/L had no effect on above molecule expression (P>0.05), no effect on T cell proliferation with BMDCs (P>0.05) was observed. CONCLUSION: Uric acid promotes the differentiation, maturation, the expression of co-stimulatory molecules, the IL-12 production in BMDCs and enhances the ability of BMDCs to stimulate the proliferation of T cell in a specical dose range.     

Immune function of dendritic cells in patients with hepatocellular carcinoma

AIM: To investigate the immune function of dendritic cells (DCs) in patients with hepatocellular carcinoma(HCC). METHODS: The DCs were cultured from human peripheral blood mononuclear cells (PBMC) by using GM-CSF and IL-4 for 7 days. Surface molecules CD86, HLA-DR of DCs were detected by flow cytometry. IL-12 production by DCs and IFN-γ production by T cells was measured with ELISA and ELISPOT, respectively. Allogenic mixed lymphocyte reaction was detected by MTT assay. RESULTS: CD86 expression in DCs in HCC patients were markedly lower than that in health control (91.7％ vs 83.5%, P<0.05). IL-12 production by DCs had no significant difference between the HCC patients and the health control ［(324.6±171.0)ng/L vs (436.5±142.7)ng/L, P>0.05］. However, after stimulated DCs with LPS, IL-12 production in HCC patients was significantly lower than that in health control ［(478.6±142.7)ng/L vs (630.0±151.9)ng/L, P<0.05］. IFN-γ production by T cells and T cell proliferation index (PI) in the HCC patients were all significantly lower than those in health control ［(IFN-γ: 133.4±51.2)103U/L vs (183.0±60.2)103U/L, P<0.05; PI: 2.3±0.7 vs 3.5±0.8, P<0.01］. CTL killing activity assay indicated that DC-induced CTL killing activity in HCC group was significantly lower than that in health group when the E/T ratio was 10∶1 and 20∶1, respectively. CONCLUSION: The immune function of DCs and T cells in the patients with HCC are significantly decreased. The CTL killing activity of HCC group is also markedly decreased.     

Effect of ginsenoside Rg1 and Rh1 onthe anti-tumor activity of dendritic cell

AIM: To study the effect of ginsenoside Rg1 and Rh1 on the anti-tumor activity of dendritic cells (DC). METHODS: Effect of Rg1 and Rh1 on the production of IL-12 p40 protein was detected by ELISA, and the IL-12 p40 mRNA level of DC was monitored by RT-PCR. Anti-tumor activity of DC-LPAK was determined by neutral red staining assay. RESULTS: The results of ELISA showed that Rg1 and Rh1 significantly enhanced the production of IL-12 p40 of DC. Rg1 at 1 mg/L and Rh1 at 100 mg/L upregulated the IL-12 p40 mRNA level. Rg1 and Rh1 enhanced the anti-tumor ability of DC, induced lymphokine and PHA activated killer (DC- LPAK) on human papillate tumor cell line. Each dose of Rg1 can obviously accelerate the cytotoxity to L929 at the E∶T ratio of 5∶1(P<0.05,0.01), while only Rh1 10 mg/L enhanced the cytotoxity ability of DC-LPAK (P<0.05). CONCLUSION: Rg1 and Rh1 enhanced the production of IL-12 p40. This effect may be mediated by the increase in the mRNA level. As a result, Rg1 and Rh1 promote the ability of DC to stimulate the cytotoxitic acticity of DC-LPAK.     

The molecular mechanism of tolegenic dendritic cells induced by IL-10 in mouse

AIM:To investigate the relation of tolerogenic dendritic cells (DC) induced by interleukin-10 (IL-10) and the paired immunoglobin-like receptor (PIR) A and B (PIR-A and PIR-B) in mouse. METHODS:The mouse dendritic cell line, DC2.4 cells were cultured with the IL-10 to develope the IL-10-DC and were stimulated by lipopolysaccharide (LPS) for 48 h to induce the mature dendritic cells (LPS-DC). Special small inference RNA (siRNA) molecule of PIR-B was chemically synthesized and was transfected into IL-10-DC by Lipofectamine 2000 (Si-DC). The expression of PIR A and PIR B on DC2.4 cells were measured by semi-quantitative RT-PCR and flow cytometry (FCM). The allogeneic stimulating capacity of DC was measured by mixed lymphocyte reaction (MLR) using ［3H］-thymidine incorporation test. The concentration of IFN-γ in supernatants of MLR from distinct groups was analyzed by ELISA. RESULTS:IL-10 up-regulated the PIR-B and down-regulated the PIR-A by semi-quantitative RT-PCR. On the contrary, LPS down-regulated the PIR-B expression and up-regulated the PIR-A expression. The expression of PIR, which is the common extra-membrane of PIR-A and PIR-B, was increased in both the IL-10-DC and the LPS-DC groups by FCM detection, but the higher expression was found in IL-10-DC group than that in LPS group. The IL-10 induced the higher PIR-B expression, inhibited allogenetic T cell proliferation and down-regulated the IFN-γ secretion. Special siRNA molecules of PIR-B in IL-10 group promoted the T cell proliferation and enhanced the IFN-γ secretion in MLR. CONCLUSION:IL-10 up-regulates the PIR-B expression and makes DC tolerance. Up-regulated PIR-B expression may be the molecular mechanism of tolerogenic dendritic cells induced by IL-10 in mouse.     

Role of TRAF6 in maturation of human PBMC-derived dendritic cells

AIM: To investigate the immunological effect of tumor necrosis factor receptor-associated factor 6 (TRAF6) on the maturation of dendritic cells in vitro.METHODS: The human peripheral blood mononuclear cell (PBMC)-derived dendritic cells (DCs), induced by recombined human granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, were stimulated to mature with TNF-α, LPS or cocktail of cytokines (TNF-α, IL-6, IL-1β and PGE₂).The cell surface markers, T-cell stimulatory proliferation capacity and IL-12 p40 production of the DCs were determined by the methods of flow cytometry, ELISA and mixed lymphocyte reation (MLR), respectively.The mRNA expression of TRAF6 was detected by real-time PCR.RESULTS: The expression of TRAF6 was observed in all groups, which in cocktail group was the highest in the DCs with optimum state of maturation.Furthermore, TRAF6 enhanced the production of IL-12 and the ability of T-cell stimulation of mature DCs.CONCLUSION: TRAF6 might play an important role in inducing the maturation of human PBMC-derived DCs.     

Recent advances in the correlations between dendritic cells and regulatory T cells

Dendritic cells (DCs), representing a heterogenous population of professional antigen-presenting cells, are the initiators and modulators of the immune responses. Studies indicate that regulatory T cells contribute to immune nullipotency and immune suppression via cell-cell contact or cytokine secretion. These two kinds of cells may be valuable tools for modulating immunity in the setting of auto-immunity, cancer, chronic viral infections and graft rejection, etc. Here we discuss the current knowledge on the functions of regulatory T cells and denditic cells-based immunoregulation and the applications.     

Effects of CpG ODN on dendritic cells and its mechanisms

Oligodeoxynucleotide containing unmethylated cytosine phosphate-guanosine motif(CpG ODN) may induce high expression of CD80, CD86, CD83, HLA I and HLAⅡ molecules on dendritic cells(DC) and stimulate DC to produce high level of IL-6, IL-12, TNF-α and IFN-α. CpG ODN is demonstrated in vivo to be a very potent adjuvant for Th1 cells, regulating Th0 cells to develop toward Th1 cells. Its role for DC is characteristics of CpG ODN sequence specificity and species specificity. CpG ODN is, at present, considered as a pathogen associated molecular pattern which binds its specific receptor，Toll-like receptor 9，then functions through TLR/IL-1R signaling pathway. It may represent a new therapeutic drug for broad applications in infectious disease, autoimmune disease, allergy and cancer therapy.     

Effect of injecting immature dendritic cell intratumorally after microwave ablation under different temperature on the activity of cytotoxic T lymphocytes

AIM: To investigate whether the injection of immature dendritic cell (iDC) after ablation induce and upregulate tumor specific cytotoxic T lymphocytes (CTL).METHODS: The model of hepatoma was established and the tumors were ablated with microwave under (45±2) ℃, (50±2) ℃, (55±2) ℃, (60±2) ℃ and void ablation, after which the bone marrow derived iDC was injected intratumorally. The activity of CTL was detected, and the levels of IFN-γ and IL-4 secreted by activated spleic lymphocytes were measured.RESULTS: Compared with iDC injection intratumorally after the ablation of (45±2) ℃, (55±2) ℃, (60±2) ℃ and void ablation, the cytotoxic effect of CTL towards Hepa l-6 was heightened by injecting iDC intratumorally 72 hours after ablation of (50±2) ℃ (P<0.05), so was the secretion of IFN-γ (P<0.05). The level of IL-4 decreased after ablation of (50±2) ℃ subsequently with injecting iDC intratumorally (P<0.05). The cytotoxic effect of CTL towards Hepa l-6 was higher than that towards B16 lymphadenoma cell after iDC injection intratumorally and ablation of (50±2) ℃ and (55±2) ℃ (P<0.05).CONCLUSION: Thermal ablation of (50-55) ℃ and subsequent injection of iDC intratumorally may induce tumor specific CTL by the way of promoting the antigenicity of ablated tumor tissue and augmenting the presentation of TAA to effector cells.     

Effects of CpG ODN on dendritic cells in anti-hepatocarcinoma action

AIM: To study the effect of oligonucleotides containing unmethylated CpG motif (CpG ODN) on mouse bone marrow-derived dendritic cells (BMDCs) in anti-HcaF cytotoxicity. METHODS: BMDCs were stimulated by CpG ODN in combination with tumor antigen (TAg). The expression of CD80 on BMDC surface was analyzed by FCAS. Level of IL-12 (p70) in supernatants of BMDC culture was detected by ELISA. The proliferation of T cells was examined by MTT assay. Cytotoxicity of CTL induced by CpG ODN combining with TAg was detected by MTT assay. RESULTS: CpG ODN combining or not combining with TAg up-regulated the expression of CD80 on BMDC surface and stimulated BMDCs to produce a high level of IL-12. CpG ODN-activated BMDC promoted the proliferation of T cells. CTL induced by CpG ODN in combination with TAg appeared strong specific cytotoxicity on Hca-F cells. CONCLUSION: CpG ODN may effectively induce the functional maturation of mouse BMDC in vitro. CpG ODN in combination with TAg can enhance the anti-HcaF cytotoxicity of CTL.     

Effects of interleukin -13 on interleukin-12 production in mesangial cells induced by LPS

AIM: To investigate the effect of interleukin-13 (IL-13) on interleukin-12 (IL-12) production in mesangial cells.METHODS: The protein synthesis of IL-12 in mesangial cells was measured by ELISA.The expression of IL-12 mRNA in mesangial cells was evaluated by RT-PCR.RESULTS: The production of IL-12 in mesangial cells stimulated by lipopolysaccharide(LPS) was significantly increased (P<0.01).IL-13 (1-100 μg/L) inhibited the protein and mRNA expression of IL-12 in a dose-dependent manner (P<0.05 or P<0.01).CONCLUSION: IL-13 inhibits IL-12 expression induced by LPS in mesangial cells.IL-13 may regulate immune responses by balancing Th1/Th2 in glomerulonephritis.     

Influence of stimulation by LPS and CD40 ligandization in vitro on the TLR4-MD2 expression and IL-12 production in DCs modified by sCD40 gene

AIM: To study the influence of stimulation by LPS and CD40 ligandization in vitro on the TLR4-MD2 expression and IL-12 production in dendritic cells (DCs) modified by sCD40 gene and provide the experimental clues of inducing dornor-specific immune tolerance.METHODS: Plasmid pEGFP-N1/sCD40 and pEGFP-N1 was transfected into DC2.4 cell line with lipofectamine.After 6 h of treatment with LPS and anti-CD40mAb,the expression of TLR4-MD2 on DCs was determined with flow cytometry (FCM) and RT-PCR.IL-12p70 protein was detected by ELISA.RESULTS: LPS treatment of DCs down-regulated surface expression of TLR4-MD2,LPS treatment along with anti-CD40mAb significantly up-regulated TLR4-MD2 surface expression.CD40 ligandization did not affect TLR4-MD2 mRNA expression in DCs but partly increased its low level induced by LPS and markly enhanced IL-12p70 secretion after LPS stimulation.DCs modified by sCD40 gene inhibited the above effect.CONCLUSION: After treatment with LPS and anti-CD40mAb,DCs modified by sCD40 gene down-regulate surface expression of TLR4-MD2 and IL-12p70 secretion decreases significantly,which might be linked with the interruption of TLR4-MD2 transportation from cytoplasm.     

Effect of monoclonal antibody to CD47 molecule on dendritic cell differentiation and function

AIM: To explore the influence of CD47 molecules on the maturation and function of cultured dendritic cells (DCs). METHODS: Monocyte cell-derived DCs were propagated in granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS) plus interleukin (IL)-4, in the presence or absence of anti CD47 monoclonal antibodies (anti-CD47 mAbs). Flow cytometry was used to detect the cell surface phenotype. The concentration of IL-12P70 in supernatant was measured by ELISA technique. The antigen-presenting functions of DCs were determined in one-way mixed leukocyte reaction by Brdu-ELISA. Electrophoretic mobility shift assays (EMSA) was used to examine NF-κB activity. RESULTS: The anti-CD47 mAbs markedly suppressed the expression of CD80,CD86,CD83,CD1a,HLA-DR on the surface of DCs (P＜0.05). The data of mixed leukocyte reaction and IL-12P70 production were consistent with the flow cytometry results (P＜0.01). Nuclear extracts from the anti-CD47 mAb-treated DCs revealed a decrease in NF-κB binding activity. CONCLUSION: The anti-CD47 mAb exerts a negative effect on the maturation and function of in vitro cultured DCs via inhibiting of NF-κB binding activity.     

Function of dendritic cells in cord blood

AIM: To explore the function of dendritic cells in cord blood.METHODS: Dendritic cell precursor subsets pDC/pDC(CD11c+CD123- /CD11c-CD123+) in cord blood and adult peripheral blood were analyzed gated by CD34-Lin- HLA-DR+ cells and the levels of IL-12p40,IL-10,IFN-γ,IL-4 in the serums were tested by ELISA.RESULTS: The level of IL-12p40 in cord blood serum was higher than that in peripheral blood.pDC1/pDC2,IL-10,IFN-γ,IL-4 in cord blood were similar to that in peripheral blood.CONCLUSION: Function development of dendritic cells in cord blood may be consummate.     

Renal tumor cell lysate antigen induces the activation of dendritic cells

AIM:To investigate the effects of renal tumor cell lysates and other cytokines on the activation of immature dendritic cells (iDCs), and to develop DCs vaccines for stimulation of renal tumour-specific immunity.METHODS:DCs induced from peripheral blood mononuclear cells of healthy volunteers were cultured and propagated in vitro using rhGM-CSF and rhIL-4, and then were cocultured with renal tumor cell lysates and different cytokines. A group: only with renal tumor cell lysates; B group: with renal tumor cell lysates and TNF-α; C group: with renal tumor cell lysates and IL-1β; D group: renal tumor cell lysates and TNF-α+IL-1β. RESULTS:iDCs were induced to mature in all four groups, and high level expressions of CD86, CD80 and HLA-DR were observed. Compared to other groups, DCs in D group expressed CD83 and CD54 at higher level (P<0.05), secreted higher quantity of IL-12 (P<0.01). Moreover, mDCs in D group induced multiplication of lymphopoiesis more effectively (P<0.05).CONCLUSION:Renal tumor cell lysates and TNF-α+IL-1β induce the mature phenotype and IL-12 production in DCs and synergistically promote the stimulatory effects of lymphopoiesis.     

Effect of adriamycin combined with sensitized dendritic cells on cervical tumor-bearing mice

AIM：To explore the immunotherapeutic effect of adriamycin (ADM) combined with frozen-thawed antigen-sensitized dendritic cells (DCs) on cervical tumor-bearing mice. METHODS：The U14 cervical cancer model of Kunming mice was established by subcutaneous implantion of U14 cells in axillary fossa. DCs vaccine was prepared by U14 cervical cancer cell frozen-thawed antigen-sensitized mouse bone marrow-derived DCs. Mature phenotype of sensitized DCs was identified by flow cytometry. Tumor-bearing mice were randomly divided into 4 groups and treated for 3 cycles with PBS (control), DCs vaccine, ADM and ADM combined with DCs vaccine, respectively. The tumor volume was evaluated. The tumor weight and the levels of interleukin-2 (IL-2), IL-12 and interferon γ (IFN-γ) in the serum were determined by ELISA on the 21st day. RESULTS：Cancer cell frozen-thawed antigen-sensitized DCs had higher expression levels of CD11C, CD80 and CD86. The volume and weight of the tumor in ADM combined with DCs vaccine group were less than those in ADM group, DCs vaccine group and control group. The tumor inhibitory rate in combination group was higher than that in the other 3 groups. Compared with the other 3 groups, the serum levels of IL-2, IL-12 and IFN-γ in combination group significantly increased. CONCLUSION：ADM combined with tumor antigen-sensitized DCs vaccine can strengthen the animal antitumor immune response and effectively inhibit the growth of tumor in cervical tumor-bearing mice.