Role of CCL17 and CCL22 expression in dendritic cells in maternal-fetal immune tolerance

AIM: To determine the expression of CCL17 and CCL22 in dendritic cells (DC) from human decidua and endometria. METHODS: The decidua were collected from normal pregnant women undergoing induced abortion and recurrent spontaneous abortion (RSA) women undergoing early abortion.The endometria were cllected from non-pregnant women undergoing abdominal hysterectomy.The mononuclear cells in the decidua and endometria were isolated. DC were induced by GM-CSF and IL-4, cultured in vitro and identified. The expression of CCL17 and CCL22 in DC at mRNA and protein levels was analyzed by real-time PCR and ELISA. RESULTS: The mRNA levels of CCL17 and CCL22 in decidual DC in normal pregnancy group were 3.04?0.40 and 1.83?0.24, respectively, significantly higher than those in endometrial DC in non-pregnancy group (0.85?0.24 and 0.31?0.08, respectively, P<0.01) and those in decidual DC in RSA group (1.65?0.14 and 0.96?0.09,respectively,P<0.01). Decidual DC continually and strongly secreted CCL17 and CCL22. The levels of CCL17 and CCL22 in normal pregnancy group were significantly higher than those in non-pregnancy group and RSA group at the same culture time point (P<0.01). CONCLUSION: The expression of CCL17 and CCL22 in decidual DC in pregnant woman increases. This may attract more CD4⁺CD25⁺ regulatory T cells to decidua and play an important role in the establishment of maternal-fetal immune tolerance.     

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.     

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.     

Anticancer effect of mouse marrow-derived dendritic cells transfected by pcDNA3-hCEA

AIM: To prepare the vaccine of DCs(pcDNA3-hCEA) and observ the immunity effect of the DCs(pcDNA3-hCEA) inoculating on CT26(hCEA⁺) loaded in BALB/c mice. METHODS: DCs were generated from bone marrow in the presence of rmGM-CSF and rmIL-4. A new recombinant plasmid, pcDNA3-hCEA, reformed with inserting a 2.4 kb human CEA cDNA into pcDNA3. DC vaccine was prepared by transfection with pcDNA3-hCEA using lipofectamine. CEA mRNA expressed in DCs(pcDNA3-hCEA) was confirmed by RT-PCR, CEA expression level was detected with RIA method, and CEA specific CTL was induced in vitro. After vaccination of DCs(pcDNA3-hCEA), the survival time of the BALB/c mice challenged with critical loading CT26(hCEA⁺) was observed. RESULTS: G₄₁₈ test showed that about14% DCs were transfected with pcDNA3-hCEA. And CEA mRNA and protein could be detected respectively by RT-PCR and RIA in the genetically modified DCs. Furthermore, the DCs coud be targeted by specific CTL, the survival time of the mice challenged with CT26(hCEA⁺) was prolonged1-4 weeks. CONCLUSION: These results demonstrate that specific antitumor immune responses could be induced efficiently by vaccination of DCs(pcDNA3-hCEA), which is transfected eukaryotic expression vector encoding tumor antigen gene.     

Study on the immunological function of sodium butyrate-induced immature human monocyte-derived dendritic cells

AIM: To investigate the immunological function of sodium butyrate-induced immature dendritic cells in vitro.METHODS: The human monocyte-derived dendritic cells were induced in the presence of human granulocyte macrophage-colony stimulating factor(GM-CSF) and interleukin-4 (IL-4), combined with sodium butyrate. The immunological function of sodium butyrate-induced dendritic cells was detected by the FCM, endocytic activity, T cells stimulatory proliferation capacity, and interleukin-12 (IL-12) production.RESULTS: Sodium butyrate could down-regulate the major histocompatibility complex(MHC) class II and costimulatory molecules of dendritic cells, increase the endocytic activity, induce a stage of T-cell anergey, and inhibit the T helper cell type 1-skewing factor IL-12 production. CONCLUSION: Sodium butyrate inhibits the maturation of dendritic cells and induces production of immature dendritic cells, which may help to explore the machenism of its epigenitic modification.     

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.     

TGF β1 inhibits the maturation of dendritic cells and down-regulates TLR4 expression

AIM: To investigate the effects of transforming growth factor β1 (TGF-β1) on murine-derived dendritic cells (DC). METHODS: Murine bone marrow cells were cultured with GM-CSF and TGF-β1 to develop TGF β-DC. Then they were stimulated by lipopolysaccharide (LPS). Their phenotypes were assessed by flow cytometry (FCM). The allogeneic stimulating capacity of DC was measured by mixed lymphocyte reaction (MLR) using BrdU ELISA method. IL-12 p70 protein was detected by ELISA and the expressions of Toll like receptor 4 (TLR4) on DCs were measured by semi-quantitative RT-PCR and FCM. RESULTS: Compared to immature DC (imDC) cultured with GM-CSF alone, the expressions of CD80, CD86, I-Ab and CD40 in TGF β-DC were lower. The TGF β-DC was resistant to maturation by LPS. Maturation resistance was evident from a failure to up-regulate CMs, to stimulate larger T cell proliferation and to increase secretion of IL-12 p70. Down-regulation of TLR4 expression on TGF β-DC was also found. CONCLUSION: TGF-β1 inhibits the expression of co-stimulatory molecules on DC. It is resistant to maturation stimulus (LPS) and might be linked with TLR4 down-regulation.     

Effect of IL-10 on IL-1β-induced cyclooxygenase-2 expression and prostaglandin E2 release in human mesangial cells

AIM: To investigate the effect of IL-10 on IL-1β-induced prostaglandin E₂(PGE₂) release and cyclooxygenase-2(COX-2) expression in human mesangial cells and to examine whether IL-10 has effect on the biological function of IL-1β.METHODS: The PGE₂ concentration in supernatants of HMC was measured by radioimmunoassay. The COX-2 mRNA and protein were measured by RT-PCR and Western blot, respectively. RESULTS: PGE₂ and COX-2 were significantly increased after treatment with IL-1β(P＜0.01 for both) in cultured human mesangial cells. IL-10 had no effects on basical production of COX-2 and PGE₂(P＞0.05, respectively), while it inhibited IL-1β-elicited PGE₂ production, as well as COX-2 mRNA and protein expression in a concentration-dependent fashion. CONCLUSIONS: These results indicated that IL-10 depressed the IL-1β-induced release of PGE₂ and expression of COX-2. These data suggested that IL-10 could exert anti-inflammatory actions at several levels, not only by inhibiting the production of pro-inflammatory cytokines but also by suppressing their biological function.     

Dendritic cells transfected with recombinant adenovirus carrying IL-24 gene induces CTLs to promote apoptosis of cervical cancer cell line CaSki

AIM：To investigate the therapeutic potential of interleukin-24 (IL-24) in cancer treatment, we observed the inductive effect of cytotoxic T-lymphocytes (CTLs) against human cervical cancer cell line CaSki using dendritic cells (DCs) transfected with recombinant adenovirus carrying IL-24 gene (Ad-IL-24-DCs). METHODS：Immature mouse DCs were isolated and cultured. The DCs were infected with recombinant adenovirus carrying IL-24 gene. The CaSki cell lysate-loaded autologous DCs vaccine was also prepared. The cell proliferation was analyzed by MTT assay. The cell apoptosis was determined by flow cytometry with PE-Annexin V staining. The cleaved caspase-3 in the Ad-IL-24-DCs was measured by Western blotting. Colony-forming assay was used to detect the colony number of Ad-IL-24-DCs. The tumorigenic capacity of CaSki cells in vivo in the presence of Ad-IL-24-DCs was observed by tumor-burdened model. RESULTS：IL-24 was highly expressed in the Ad-IL-24-DCs. In the presence of Ad-IL-24-DCs, the apoptosis of CaSki cells was significant increased. Additionally, the protein level of cleaved caspase-3 was increased in CaSki cells in the presence of Ad-IL-24-DCs, suggesting that the CaSki cells with Ad-IL-24-DCs induced apoptosis. CTLs induced by Ad-IL-24-DCs inhibited the tumorigenic capacity of CaSKi cells in vivo. CONCLUSION：Genetically modified DCs are successfully prepared by infection with Ad-IL-24 and show a significant effect on triggering specific CTLs against CaSki cells.     

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.     

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.     

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.     

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.     

Induction of tumor-specific cytotoxic T lymphocytes in vitro by dendritic cells pulsed with different glioma antigens

AIM: The goal of this study was to compare different methods for tumor antigen preparation, to observe the induction of tumor-specific cytotoxic T lymphocytes in rats by dendritic cells (DCs) pulsed with different tumor antigens. METHODS: The precursors of dendritic cells were isolated from bone marrow of rats, stimulated in vitro with recombinent rat granulocyte-macrophage colony-stimulating factor (rrGM-CSF) and interleukin-4 (rrIL-4). Then rat DCs were pulsed with C6 tumor cell antigens prepared with different methods: freeze-thaw, boiling or total protein extracted from ultrasonic crushed tumor cell. Subsequently primed DCs were cocultured with T lymphocytes isolated from spleen to induce CTL. Lymphocyte chemoattractant factor from DCs and cytokine IFN-γ release were determined by ELISA, the cytotoxicity of CTL was assayed by JAM test. RESULTS: DCs pulsed with boiled tumor cell in vitro induced an enhanced ability of T-cell proliferation and cytotoxic T lymphocyte activity.CONCLUSION: Our results demonstrated that DCs primed with boiled tumor cell may represent a method for inducing immune responses against the entire repertoire of tumor antigens of malignancies.     

Effects of interleukin-10 on the calcineurin activity in cultured cardiac fibroblasts induced by arginine vasopressin

AIM: The purpose of the present study was to investigate the effect of interleukin-10 (IL-10) on the proliferation and calcineurin (CaN) activity in cultured cardiac fibroblasts (CFs) induced by arginine vasopressin (AVP).METHODS: The CFs of left ventricle in neonatal Sprague-Dawley rats were isolated and cultured by trypsin digestion and selective plating technique. Then the proliferation rates of cells were determined by using the MTT ［3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide］ assay (A490 value). Cell cycle distribution was determined with flowcytometry technique. The CaN activity was measured by ultra-violet spectrophotography.RESULTS: (1) MTT colorimetry showed that 10-7 mol/L AVP significantly increased A490 value of CFs in comparison with control group (P<0.01). IL-10 attenuated the A490 value of AVP group in a concentration dependent manner. The A490 value of the 10-8, 10-7, 10-6, 10-5 g/L IL-10+10-7 mol/L AVP groups was 0.201±0.007, 0.187±0.006, 0.173±0.010 and 0.157±0.029 respectively, all data significantly lower than those in the presence of AVP alone (P<0.05 or P<0.01). (2) The percentage of the cells in S stage and proliferation index were markedly increased in 10-7mol/L AVP group compared with the control (P<0.01, respectively). In the 10-6 g/L IL-10+10-7 mol/L AVP group, the percentage cells in S stage and proliferation index were significantly lower than those in AVP group (P<0.01, respectively). IL-10 itself had no effect on fibroblast proliferation, but reduced AVP-induced fibroblast proliferation. (3) There was a significantly increase in CaN activity in AVP group compared with control (P<0.01). In the 10-8, 10-7, 10-6 and 10-5 g/L IL-10＋10-7 mol/L AVP groups, the CaN activity was 3.22±0.04, 3.06±0.06, 2.53±0.04 and 2.22±0.04, respectively. IL-10 dose-dependently down-regulated the CaN activity induced by AVP (P<0.01, respectively). However, the CaN activity was still higher in IL-10+AVP group than that in control group (P<0.05 or P<0.01).CONCLUSION: Our data indicate that IL-10 regulates the CaN activity of CFs in the cell proliferation induced by AVP, suggesting that IL-10 plays a role in the regression of cardiac remodeling.     

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.     

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.     

Signaling mechanism of dendritic cell maturation stimulated by uric acid

AIM: To investigate the effect of uric acid on the signal molecule expression involved in MAPKs and NF-κB pathways during the maturation of dendritic cells (DCs). METHODS: DCs were obtained from murine bone-marrow and cultured in vitro. After the immature DCs were stimulated with uric acid (200 mg/L) and NF-κB inhibitor PDTC, or MAPKs inhibitors SB203580, PD98059 or SP600125 for 15 min, 30 min or 45 min, the cytoplasmic and nuclear extracts of the cells were collected and were subject to immunoblot analysis with the antibodies specific for NF-κB p65 or phosphorylated forms of p38, ERK1/2 and JNK. The cell lysates from DCs treated with LPS or DMSO served as controls. After treated with uric acid and PDTC, SB203580, PD98059 or SP600125 for 48 h, DCs were collected. The cell surface markers were analyzed by flow cytometry. The production of IL-12 p70 in the culture supernatants was detected by ELISA. RESULTS: Within 15 min of uric acid conditioning in the immature DCs, increased expression of NF-κB p65 and the phosphorylation of p38, ERK1/2 and JNK in the nuclear or cytoplasmic extracts of DCs were observed. The expression of these proteins reached their peak at 30 min after stimulation. Pretreatment of DCs with PDTC, SB203580, SP600125 or PD98059 blocked the expression of NF-κB p65 and phosphorylation of p38, ERK1/2 and JNK in response to uric acid stimulation. Treatment of DCs with SB203580, SP600125 or PDTC reduced the uric acid-induced up-regulation of CD83, CD86 and IA/IE, and inhibited the effect of uric acid on the secretion of IL-12 p70 (P<0.05 or P<0.01). SB203580 and PDTC possessed a significant inhibitory effect on uric acid. Nevertheless, PD98059 increased the up-regulation of CD83, CD86, IA/IE and IL-12 p70 induced by uric acid (P<0.05). CONCLUSION: Uric acid controls the balance of signal molecule phosphorylation of p38 MAPK, ERK1/2 and JNK, and NF-κB pathways. A possible mechanism of the DCs maturation stimulated by uric acid may be the modulation of the threshold and duration of MAPKs and NF-κB signaling.     

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.