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.     

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.     

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.     

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.     

Expression and role of CD134 and NF-κB in renal tissue of lupus nephritis

AIM:To investigate the role of CD134 (OX40) and NF-κB in the pathogenesis of lupus nephritis (LN).METHODS:Renal in situ CD134 and NF-κB expression were examined in 40 biopsy specimens from LN patients by immunohistochemistry and microwave-based immunohistochemistry, respectively. The relationship between expression of CD134 and NF-κB was analyzed.RESULTS:The expression of glomerular and tubular CD134 and NF-κB in LN were higher than that in normal control, especially in class Ⅳ LN, where there was intense staining of endothelial cell, distal tubules, and interstitial mononuclear cell. The CD134 expression of glomerular and tubular was closely related to NF-κB expression, respectively (r=0.5542, P＜0.05;r=0.6279, P＜0.05). CONCLUSIONS:The abnormal expression of costimulatory molecule CD134 was well evidenced in LN. Strong expression of renal in situ NF-κB was likely mediated by CD134 signal pathway, which may play an important role in the pathogenesis of LN.     

Influence of complement bypass-activation on IL-8 expression in endothelial cells

AIM:To study the induction of IL-8 expression by bypass-activated complement in human umbilical vein endothelial cells (HUVECs) and regulatory effect of nuclear factor-kappa B on the expression of IL-8. METHODS:In vitro, zymosan-activated human serum(ZAHS) directly challenged the HUVECs monolayers. Following techniques were used in the experiment: ① RIA for measurement of IL-8,ISH for measurement of their mRNA.② EMSA for measurement of nuclear factor-kappa B(NF-κB). RESULTS:①After HUVECs monolayers were stimulated with ZAHS, the level of IL-8 increased significantly at 4 h. ②The NF-κB activity began upregulated within 30 min after ZAHS stimulation, maximal NF-κB activity was observed at 120 min. Pretreatment of endothelial monolayers with PDTC (20 μmol/L) significantly inhibited the secretion of IL-8 (P＜0.05). CONCLUSION:Bypass-activated complement directly challenged HUVECs to secret IL-8. Cytoplasma to nuclear translocation of NF-κB was necessary for this response.     

Effects of 17β-estradiol on the maturation and immunologic function of dendritic cells from human peripheral blood

AIM: To investigate the effects of 17β-estradiol (E2) on the maturation and immunologic function of dendritic cells from human peripheral blood. METHODS: Cultured DCs were treated with E2 at doses of 10-7 mol/L and 10-6 mol/L. The morphologic changes were observed under the scanning electronic microscope. The immunophenotype of DCs in control and treated groups was analyzed by flowcytometry. 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, DCs cultured in the presence of E2 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 and reduction of IL-12 production. CONCLUSION: E2 exerts a negative effect on the maturation and immunologic function of dendritic cells from human peripheral blood.     

Expression of nuclear factor-κB in asthmatic guinea pigs and the effect of erigeron breviscapus on it

AIM:To explore the expression of nuclear factor-κB(NF-κB)in asthmatic guinea pigs,and the effect of erigeron breviscapus,a protein kinase C(PKC)inhibitor,on the expression of nuclear factor-κB(NF-κB).METHODS:48 guinea pigs were randomly divided into 6 groups(n=8).Airway resistance and eosinophilic inflammation of airway wall were examined,the expression of NF-κB in the lung tissue was detected by immunohistochemical staining.RESULTS:The expression of NF-κB was mainly found in airway epithelium,all the asthmatic animals showed significantly higher optical densities than that of the normal control group(P＜0.01),and the rats subjected therapeutic treatment for two weeks showed significantly lower NF-κB expression than those of the asthmatic groups(P＜0.01).Positive correlation exist between the airway resistance and the percentage of cells expressing NF-κB in epithelium,and between the amount of eosinophil in airway wall and the percentage of cells expressing NF-κB in epithelium(P＜0101).CONCLUSION:The increased expression of NF-κB in airway epithelium of the asthmatic guinea pigs suggested that NF-κB may be involved in asthma.And result that the increased expression of NF-κB was inhibited significantly by the treatment of the erigeron breviscapus suggested that PKC may play a significant role in the pathogenesis of asthma through NF-κB.     

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.     

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.     

Role of Akt/NF-κB pathway in immune-complexes-induced MCP-1 and CSF-1 expression in murine glomerular mesangial cells

AIM: To explore the role of Akt/NF-κB pathway in immune-complexes-induced monocyte chemoattractant protein-1 (MCP-1) and colony stimulating factor-1 (CSF-1) expression in Mesangial Cells. METHODS: Primary murine glomerular mesangial cells were cultured in vitro and divided into control group, stimulation group and antisense, sense and mismatched oligodeoxynucleotide group. In control group, the cells were stimulated with monomeric IgG after treatment with 0.5% lipofectin for 8 h. In stimulation group, the cells, which had been treated with 0.5% lipofectin for 8 h, were stimulated with aggregated IgG. In antisense, sense and mismatched oligodeoxynucleotide group, being transduced antisense, sense and mismatched oligodeoxynucleotide respectively with 0.5% lipofectin 8 h, the cells were stimulated with AIgG. MCP-1 and CSF-1 in supernatant were deteced with ELISA. In addition, RT-PCR was used to determine MCP-1 and CSF-1 mRNA expression, and EMSA to investigated the activation of NF-κB. RESULTS: Mesangial cells cultured in vitro had a low level NF-κB activation and a low level constitutive expression of MCP-1 and CSF-1. Stimulated with AIgG, activation of NF-κB was markedly increased(0.35±0.06 vs 0.75±0.16, P＜0.01), expression of MCP-1 and CSF-1 mRNA (0.48±0.03 vs 0.72±0.02, P＜0.05; 0.44±0.01 vs 0.59±0.02, P＜0.05), MCP-1 and CSF-1 levels in supernatant(15.52±1.81 vs 43.05±3.18, P＜0.05; 389.06±13.75 vs 764.22±31.78, P＜0.05) were markedly increased. Akt1 antisense oligodeoxynucleotide markedly inhibited immune-complexes-induced NF-κB activation, MCP-1 and CSF-1 mRNA and protein expression. CONCLUSION: Akt/NF-κB pathway mediates immune-complexes-induced MCP-1 and CSF-1 expression in mesangial cells. It suggests that Akt/NF-κB pathway may be a new therapy target for macrophage recruitment and activation in immune complexes nephritis.     

Influence of bifidobacterium on NF-κB and I κBα of experimental large bowel carcinoma

AIM:To explore the antitumor mechanisms of bifidobacteria adolescence in vivo. METHODS:The activity of NF-κB and its inhibiting protein I κBα of large bowel carcinoma tissues was detected by using laser scanning confocal microscope and immunohistochemistry.RESULTS:The positive cell density of NF-κB of large bowel carcinoma transplantation tumors in bifidobacterium injection group was markedly lower than that in tumor control group(P＜0.01).The expression of I κBα was contary in the two group. The average fluorescent strength of I κBα of large bowel carcinoma in bifidobacterium injection group was significantly higher than that in tumor control group(P＜0.01).CONCLUSION:Bifidobacteria adolescence could inhibit the degrade of I κBα and the activition of NF-κB in large bowel carcinoma in vivo.     

Influence of histone deacetylase inhibitor romidepsin on phenotype and function of T lymphocytes in vitro

AIM: To explore the effects of romidepsin (FK228), a novel histone deacetylase inhibitor, on the effector and regulatory T cells in vitro.METHODS: As the reactive cells, lymphocytes, CD4⁺ T cells and CD8⁺ T cells were labelled with CFSE, and stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group), or PBS (placebo group).After 72 h, the proliferation of the cells was detected in different groups. The lymphocytes were stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group),or PBS (placebo group). After 72 h, the percentage of CD4⁺ Foxp3⁺ T cells and the levels of related cytokines were detected in different groups. RESULTS: The proliferation of CFSE-labelled lymphocytes, CD4⁺ T cells and CD8⁺ T cells triggered by anti-CD3 and anti-CD28 mAbs all were inhibited when cultured with romidepsin at concentrations of 1 μmol/L, 3 μmol/L and 5 μmol/L in a dose-dependent manner (P<0.05). Compared with placebo group, in the presence of anti-CD3 and anti-CD28 mAbs, 1 μmol/L romidepsin did not increase the percentage of CD4⁺ Foxp3⁺ T cells (P>0.05). When cultured with romidepsin at concentrations of 3 μmol/L and 5 μmol/L, the percentage of CD4⁺ Foxp3⁺ T cells was enhanced markedly (P<0.05). The levels of IL-10 and TNF-α in the supernatant were markedly increased in positive control group and 3 experimental groups (P<0.05), and the levels of cytokines in different experimental groups were gradually decreased with the elevation of FK228 concentration (P<0.05). The level of TGF-β was slightly increased in positive control group with no significant difference compared with placebo group (P>0.05). With the increase in the concentration of FK228 in different experimental groups, the TGF-β level was increased in a dose-dependent manner and there were significant differences in the 3 experimental groups. Meanwhile, significant differences existed between experimental groups and placebo group and between experimental groups and positive control group (P<0.05). CONCLUSION: Romidepsin inhibits the proliferation of CD4⁺ and CD8⁺ effector T cells and increases the percentage of CD4⁺ Foxp3⁺ regulatory T cells. It may be related to the increased level of TGF-β, but independent of IL-10.     

Effects of eicosapentaenoic acid on expression of nuclear factor κB and release of cytokines in human umbilical vein endothelial cells stimulated by lipopolysaccharide

AIM: To investigate the effects of eicosapentaenoic acid(EPA) on the expression of nuclear factor kappa B(NF-κB) and release of vascular endothelial growth factor(VEGF), IL-1α and IL-6 in cultured human umbilical vein endothelial cells(HUVECs) stimulated by lipopolysaccharide(LPS).METHODS: HUVECs were obtained from cell strain and cultured in vitro. HUVECs were divided into 4 groups: control group, LPS group, 0.030 g/L EPA treatment group and 0.050 g/L EPA treatment group. The cells were cultured with LPS alone in LPS group and incubated with EPA for 1 h in the EPA pretreatment groups at the concentrations of 0.030 g/L and 0.050 g/L before LPS stimulation. Twenty-four hours after stimulated by LPS, the protein expression of NF-κB p65 in HUVECs were assessed by Western blotting analysis at different time points. The production of VEGF, IL-1α and IL-6 in cultured HUVECs was evaluated by ELISA. The effects of EPA on the protein expression of NF-κB p65 and the production of VEGF, IL-1α and IL-6 in HUVECs challenged by LPS were also determined.RESULTS: Compared with control group, the protein expression of NF-κB p65 was significantly increased in HUVECs induced by LPS and was inhibited by EPA. Compared with control group, the protein expression of VEGF, IL-1α and IL-6 was dramatically increased in HUVECs induced by LPS and most of the increase was inhibited by EPA.CONCLUSION: LPS enhances the protein expression of NF-κB and the release of VEGF, IL-1α and IL-6. EPA inhibits the protein expression of NF-κB, and the production of VEGF and the inflammatory cytokines in cultured HUVECs stimulated by LPS, indicating that EPA may be useful for preventing and treating neovascular and inflammatory diseases.     

Effects of IL-32γ on proliferation and cell cycle of rat vascular smooth muscle cells

AIM: To observe the effects of interleukin-32γ (IL-32γ)on the proliferation and cell cycle of rat vascular smooth muscle cells (VSMCs). METHODS: The VSMCs were isolated from the thoracic aorta of SD rats by the method of tissue-piece inoculation. The cells were cultured and treated with different concentrations of IL-32γ. The proliferation of the cells was examined by MTT assay. The cell cycles were analyzed by flow cytometry. The protein levels of NF-κB p65 and cyclin D1 were detected by Western blotting. The expression of proliferating cell nuclear antigen (PCNA)was examined by immunocytochemical staining. RESULTS: Administration of IL-32γ at the concentrations of 10~50 μg/L for 24~48 h significantly promoted the proliferation of VSMCs in a dose- and time-dependent manner. After stimulation with IL-32γ at the concentration of 50 μg/L for 24 h, the cell cycle transition from G₁ phase to S/G₂ phase was accelerated and the expression levels of NF-κB p65, cyclin D1 and PCNA increased as compared with those in control group. CONCLUSION: IL-32γ promotes the proliferation of rat VSMCs and accelerates the cell cycle transition via upregulating the expression of NF-κB p65 and cyclin D1.     

Early apoptotic T lymphocytes induce DCs from bone marrow to tolerogenic DCs

AIM: To study the immunosuppressive effects of early apoptotic T lymphocytes.METHODS: Early apoptotic spleen T cells were induced by ultraviolet irradiation for 5 min.After irradiation,spleen T cells were incubated at 37 ℃ with 5% CO₂ for 2 h and thus early apoptotic T lymphocytes were obtained.Three to four freeze thaw cycles resulted in disruption of the spleen T cells into fragments.imdendribic cells(DCs) were prepared from red cells and T cells depleted bone marrow cells.The imDCs were divided into five groups: group A: necrotic spleen T cells were added to imDCs;group B: early apoptotic spleen T cells were added to imDCs;group C: supernatants from early apoptotic spleen T cells alone with necrotic spleen T cells were added to imDCs;group D: TGFβ₁ neutralizing antibody along with early apoptotic T lymphocytes were added to imDCs;group E: immature dendridic cells culture in RPMI-1640 for 5 days were used as negative control.Flow cytometry was employed to analyze the expression of MHCⅡ,CD40,CD80 and CD86 on DCs in each group.ELISA was employed to assay the IL-12 p70 produced by DCs in different groups.The amounts of TGFβ₁ released by early apoptotic T lymphocytes were also determined by ELISA.T cells proliferation assay was employed to study DCs T cells stimulatory capacity.RESULTS: The DCs expressed high level of MHCⅡ,CD40,CD80 and CD86 when exposed to necrotic cells while early apoptotic cells did not.The supernatants from early apoptotic spleen T cells suppressed the expression of MHCⅡ,CD40,CD80 and CD86 on DCs exposed to necrotic spleen T cells.When TGFβ₁ neutralizing antibody along with early apoptotic spleen T were added to imDCs,the expression of MHCⅡ,CD40,CD80 and CD86 was increased significantly.The necrotic spleen T cells increased IL-12 p70 production by DCs,while apoptotic spleen T cells at early stage did not (P<0.01,group B vs group A or B;P>0.05,group B vs group E).Only the DCs that exposed to necrotic spleen T cells gained significant T lymphocytes stimulatory capacity,while DCs exposed to apoptotic cells at early stage did not.The amounts of TGFβ₁ released by early apoptotic spleen T cells were much higher than those released by viable spleen T cells.CONCLUSION: Apoptotic spleen T cells at early stage have the capacity to induce the generation of tolerogenic DCs.     

The immunity effect of B7-H1 blockade on immature dendritic cells

AIM: To investigate the immune stimulation capacity of B7-H1 blockade on immature dendritic cells (DCs) in vitro. METHODS: The human monocyte-derived dendritic cells were induced in the presence of cytokine GM-CSF and IL-4. The expression of B7-H1 was detected by FCM. On blockade of B7-H1, the maturation and endocytic activity, T cells stimulatory proliferation capacity, IL-12 production, T cell differentiation effect of DCs were detected by FCM, MTT assay, ELISA and ELISPOT, respectively. RESULTS: The expression of B7-H1 was increased with the induction of DCs. On day 7, the positive expression was 54.12%, and the TNF-α induced mature DCs had the positive expression rate of 83.64%. The blockade of B7-H1 on immature DCs had sharply increased their T cells stimulatory proliferation capacity and IL-12 production, and efficiently induced the development of Th1/Tc1 cells, but had no effect on their maturation and endocytic activity. CONCLUSION: The blockade of B7-H1 on immature DCs increases its immune stimulation activity. It is valuable to investigate the antitumor immune responses of DCs vaccine with B7-H1 blockade.     

Differentiation of dendritic cells from embryonic stem cells

AIM：To investigate the method of directed differentiation dendritic cells (DC) from embryonic stem cells,and to amplify high purity dendritic cells in vitro for immunolgical therapy.METHODS：E14 embryonic stem cell line was generated ES-DC in complete medium supplemented with GM-CSF and IL-3.Flow cytometry was used to determine CD11c,CD80,CD86,MHC-II cell-surface phenotype in immatured ES-DC.Lipopolysaccharide (LPS) was added to induce the ES-DC maturation.The matured ES-DC was harvested 24 hours later to identifying with morphology and transmission electron microscopy.The phenotype of matured ES-DC was analyzed by flow cytometry and compared with the immatured ES-DC.The antigen presenting was evaluated by mixed lymphocyte responses (MLR).RESULTS：The ES-DC had obviously dendritic processes under scanning electron microscope.The immature DCs expressed low level of CD11c (4.33±0.23)%,CD80 (7.62±0.19) %,CD86 (4.77±1.22) % and MHC-II (9.68±0.15) %.The mature DCs expressed higher level of CD11c (47.36±2.68)%,CD80 (74.40±1.47) %,CD86 (29.77±2.00) % and MHC-II (87.56±2.75) %.MLR showed that ES-DCs effectively stimulated lymphocyte proliferation.CONCLUSIONS：These results provide evidence that dendritic cells can be generated from E14 embryonic cells with the stimulations of GM-CSF and IL-3.The differentiated cells expresse high level of CD11c,CD80,CD86,MHC-II and effectively stimulate lymphocytes to proliferate.ES cells may become new origin of DC for immunotherapy.     

Effect of glucocorticoid-treated dendritic cells on asthmatic Th2 response

AIM: To investigate the effect of dexamethasone-treated dendritic cells (DCs) on Th2 cytokine production from autologous T cells in asthmatic patients and explore the mechanisms by studying the effect of dexamethasone on differentiation, maturation and function of DCs from patients with asthma. METHODS: Human peripheral blood monocyte-derived DCs generated from asthmatic patients and healthy subjects were cultured in the absence or presence of dexamethasone. The phenotypic characterization of DCs was analyzed by flow cytometry. The mature DCs were harvested, washed, and then cocultured in vitro with autologous T cells purified by a nylon cotton column. The DC-T coculture supernatants were collected after 72 h incubation and analyzed for levels of IL-5 and IFN-γ by ELISA. RESULTS: The concentrations of IL-5 in the culture supernatants of DC-T coculture were significantly up-regulated in patients with asthma compared with that in healthy controls ［(145.13±89.76) ng/L vs (50.28±22.37) ng/L, P<0.01］. The level of IFN-γ in the DC-T coculture supernatants tended to be decreased in asthmatic patients than that in healthy controls, although this difference did not achieve statistical significance ［(197.58±76.32) ng/L vs (220.46±65.34) ng/L, P>0.05)］. There were significantly decreased levels of IL-5 by autologous T cells primed by dexamethasone-treated mature DCs from asthmatic patients ［(45.39±19.61) ng/L vs (145.13±89.76) ng/L, P<0.01］, alterations not observed from healthy controls (P>0.05). IFN-γ production was decreased by autologous T cells primed by dexamethasone-treated mature DCs from both asthmatic patients and healthy controls ［asthma group: (40.21±22.89) ng/L vs (197.58±76.32) ng/L, P<0.01; healthy controls: (56.78±20.37) ng/L vs (220.46±65.34) ng/L, P<0.01］. Dexamethasone-treated DCs exhibited decreased expression of CD83 (P<0.01) and increased expression of CD14 (P<0.01) in both asthmatic patients and healthy controls. CONCLUSION: DCs of asthmatic patients induce a Th2-skewed cytokine production from autologous T cells. Dexamethasone-treated DCs inhibit the Th2 reactions, and this effect is probably mediated through the pathway that dexamethasone inhibits DCs maturation and skews the macrophage/DC balance towards the macrophage side and thus directs the development more towards the macrophage lineage.     

Role of B cells in CD45RB antibody-induced transplantation immune tolerance

AIM: To investigate the role of B cells in CD45RB antibody-induced transplantation immune tolerance. METHODS: Single cell suspension was made from the spleen of BALB/c nude mice disposed by CD45RB antibody, then mixed cultured with T cells of BALB/c mice and spleen cells of C57BL/6 mice. The Th1, Th2, Treg and Tm cells were monitored by flow cytometry during the culture process. The skin graft model was set up with B6.μMT^-/- mice as receptors and BALB/c mice as donors. CD45RB antibody was intraperitoneally injected into the receptors after transplantation and then CD3⁺CD45RB^hi cells were detected by flow cytometry. In another mixed lymphocyte culture, CD45RB antibody was added, and then B cells were isolated and injected into B6.μMT^-/- mice through the tail vein. The heart transplantation model was established with B6.μMT^-/- mice as receptors and BALB/c mice as donors, and then the survival and the migration of B cells to the thymus were observed. RESULTS: When T lymphocytes were co-cultured with B lymphocytes treated with anti-CD45RB monoclonal antibody(mAb) in vivo, the percentages of Th2 and Treg cells were up-regulated and Th1 cells were down-regulated, but Tm cells were not altered as compared with the control. In vivo without B lymphocytes, anti-CD45RB mAb also down-regulated the expression of CD45RB in T lymphocytes. The reduction was faster and the percentage of CD3⁺CD45RB^hi T cells was not altered as compared with the control. The B lymphocytes treated with anti-CD45RB mAb in vitro prolonged the lifetime of receptor in heart transplantation model but failed to induce complete tolerance. After recieving B cells treated with anti-CD45RB mAb and allogeneic heart transplantation, B cells migrated to the thymus in B6.μMT^-/- mice. CONCLUSION: B lymphocytes play a definite role in the transplantation immune tolerance induced by anti-CD45RB mAb through their affection on T-cell subgroups and also in the central tolerance. However, the induction of immune tolerance can not only rely on B cells.