OBJECTIVETo explore the effects of Vitamin C (Vit C) on the apoptosis and proliferation inhibition of human peripheral blood mononuclear cells (HPBMCs) induced by deoxynivalenol (DON) in vitro.

METHODSThe effects of Vit C pretreatment at different dosages (25 micromol/L and 100 micromol/L) on apoptosis, apoptosis related genes expression and proliferation inhibition of HPBMCs induced by DON were evaluated with cell culture, flow cytometric DNA analysis and Western blotting.

RESULTSFlow cytometry (FCM) analysis showed that the apoptosis rate of HPBMCs in 2000 microg/L DON group was (28.82 +/- 1.67)%, which was significantly higher than that in control group (14.07 +/- 0.70, P < 0.05). Compared with DON group, the apoptosis rate of HPBMCs in 25 micromol/L Vit C pretreatment group was significantly decreased (28.82 +/- 1.67)% vs (22.39 +/- 1.05)%, P < 0.05, while that in 100 micromol/L Vit C pretreatment group was obviously increased (36.07 +/- 2.92)%, P < 0.05. Western blotting analysis showed that the expression of Bax and Caspase-3 up-regulated by DON was markedly decreased, while the expression of Bcl-2 down-regulated by DON was increased by 25 micromol/L Vit C pretreatment (P < 0.05). 100 micromol/L Vit C pretreatment could further increase the expression of Bax and Caspase-3 of HPBMCs induced by DON, while no significant effects on the Bcl-2 expression induced by DON were seen. FCM analysis showed that the proliferation index of HPBMCs in Vit C pretreatment groups at different dosages was all dramatically increased as compared with that in DON groups (P < 0.05).

CONCLUSION25 micromol/L Vit C pretreatment could at certain extent inhibit the apoptosis and reverse the abnormal expression of apoptosis related genes of HPBMCs induced by DON in vitro, while 100 micromol/L Vit C pretreatment could further increase the apoptosis rate of HPBMCs induced by DON. Vit C pretreatment could reverse the proliferation inhibition of HPBMCs induced by DON in vitro.

Apoptosis ; drug effects ; Ascorbic Acid ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Humans ; Monocytes ; cytology ; drug effects ; Trichothecenes ; pharmacology

OBJECTIVETo examine the effect of ginsenoside Rb1 (GRb1) on the immune maturation of monocyte-derived dendritic cells (DCs) induced by oxidized low-density lipoprotein (OX-LDL).

METHODSHuman monocytes purified by CD14+ immuno-magnetic beads were differentiated and induced into immature DCs, which were randomly divided into 6 groups, Group A treated with PBS, Group B treated with OX-LDL, Group C and D treated respectively with GRb1 and ciglitazone, Group E and F were pretreated with the two testing drugs respectively followed by OX-LDL. The immuno-phenotypic expression (CD40, CD1a, and HLA-DR) and endocytosis function of DCs were examined using flow cytometry, the concentration of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) in the culture supernatants were measured with ELISA.

RESULTSCompared with Group B, Group E showed significantly lowered immuno-phenotypic expression of DCs in terms of CD40 (67.4 +/- 1.62 vs. 145.69 +/- 14.86), CD1a (79.64 +/- 3.04 vs. 159.89 +/- 6.09), and HLA-DR (46.43 +/- 2.85 vs. 99.33 +/- 17.11), as well as higher endocytosis level (88.13% +/- 1.06% vs. 25.90% +/- 5.77%, all P < 0.01). Meantime, the serum levels of IL-12 (88.65 +/- 5.59 ng/L vs. 716.69 +/- 36.35 ng/L) and TNF-alpha (133.27 +/- 11.98 ng/L vs. 968.10 +/- 36.42 ng/L) obviously decreased (P < 0.01). The surface molecular expression of DCs and the secretion of inflammatory factors in Group F also obviously decreased, showing insignificant difference from Group E (P > 0.05).

CONCLUSIONGRb1 could obviously inhibit the OX-LDL-induced maturation of DCs, showing similar effects to ciglitazone.

Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; immunology ; Flow Cytometry ; Ginsenosides ; pharmacology ; Humans ; Lipoproteins, LDL ; Monocytes ; cytology ; drug effects ; immunology

Dendritic cells (DC) are highly efficient antigen-presenting cells that initiate the primary immune response. Several laboratories have developed culture systems for human DC from peripheral blood monocytes. Most of these studies have used fetal calf serum (FCS) containing culture conditions that are inappropriate for human application. GM-CSF and IL-4 were used to make immature DC. The monocyte-conditioned medium (MCM) was used to induce the final maturation of DC. Using the previously described methods, the quality of MCM has unpredictable variations. Therefore using a defined cocktail of growth factors for the generation of mature DC would be advantageous for experimental as well as clinical purposes. In this study, it is suggested that combinations of both GM-CSF/IL-4 or GM-CSF/IL-13 could be used as the first-step culture to produce immature DC, and that cytokine cocktail (GM-CSF, IL-4, IL-1beta, TNF-alpha, IL-6, PGE2) was as efficient as MCM for the second step-culture to produce fully maturated DC. Here, we have generated an easily reproducible culture system for DC that allows for the generation of large amounts of immature and mature DC, and we also now have established the method in a FCS-free system that is suitable for clinical use.
Cell Division/drug effects ; Culture Media/pharmacology ; Cytokines/pharmacology ; Cytological Techniques* ; Dendritic Cells/cytology* ; Human ; Monocytes/cytology*

OBJECTIVETo study the effects of Lipopolysaccharide (LPS) on the maturation and secretion of human peripheral dendritic cells (DCs).

METHODSDCs from healthy human peripheral monocytes (PBMCs) were induced in vitro with rhGM-CSF, rhIL-4, Flt3-L and TNFalpha. The subjects were divided into 3 groups: the long-term group stimulated with LPS 1 microg/ml at day 1, 4, 7, 9 post culture; the short-term group stimulated with LPS 1 microg/ml at day 7 and 8 post culture, and the DCs without LPS stimulation was control group. After 10 days of culture, the morphologic features of DCs were observed by light and electron microscopes, the phenotypic patterns were characterized by flow cytometry, the proliferation of T cell were evaluated with mixed leukocytes reaction (MLR) and the levels of IL-12 and IFNgamma produced by DCs were analyzed with ELISA.

RESULTSCompared with the short-term group, the expressions of HLA-DR (65.81%+/-10.96%), CD86 (48.81%+/-18.13%), CD80 (13.56%+/-5.48%), CD83 (11.52%+/-5.09%), the secretions of IFNgamma(15.60+/-5.83 pg/ml) and IL-12 (51.77+/-11.02 pg/ml) by the DCs in long-term group were decreased obviously (P is less than 0.05) and the proliferation of homogenic lymphocyte cells (1.548+/-0.365) stimulated by DCs was also impaired (P < 0.05).

CONCLUSIONLong-term LPS stimulation can suppress the maturation and secretion of DCs, which might be the reason of poor immunity in the patients with intestinal endotoxemia.

Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Humans ; Interleukin-12 ; biosynthesis ; Lipopolysaccharides ; pharmacology ; Monocytes ; cytology ; metabolism

OBJECTIVETo observe the effect of Huanglian Jiedu Decoction (HLJDD) in in vivo regulating differentiation of monocytes in an apolipoprotein E knockout (ApoE(-/-)) mouse model, and to observe the effect of HLJDD-containing serum in in vitro regulating differentiation of macrophages and foam cells.

METHODSFifteen apoE(-/-) mice were randomly divided into the common diet group, the hyperlipidemia group, and the hyperlipidemia +HLJDD treatment group, 5 in each group. Mice in the common diet group were fed with a chow diet. Mice in the hyperlipidemia group were fed with high cholesterol wild diet (WD). Those in the hyperlipidemia +HLJDD treatment group were fed with high cholesterol WD supplemented with HLJDD. All mice were fed for 4 weeks. Five C57BL/6 wild types were recruited as the wild common diet control group. HLJDD was administered to mice in the hyperlipidemia + HLJDD treatment group by gastrogavage at the daily dose of 5 g/kg. Equal volume of purified water was given by gastrogavage to mice in the rest 3 groups. Four weeks later, subtypes of monocytes in the peripheral blood were detected by FACS. HLJDD administered to another 30 SD rats by gastrogavage at the daily dose of 5 g/kg, once for every 12 h for 5 times in total, thereby preparing 5% HLJDD containing serum to intervene the differentiation of in vitro primary bone marrow-derived macrophage (BMDM) and foam cells. The M2 subtype surface receptor CD206 of macrophages and foam cells were detected by FACS. The expression of Nos2 and Arg1 genes were assayed by Real-time PCR.

RESULTSThe ratio of inflammatory subset of monocytes (Ly6C(high)) increased in the peripheral blood after ApoE(-/-) mice were fed with high fat diet for 4 weeks. HLJDD significantly decreased the ratio of inflammatory subset of monocytes (P < 0.05). Compared with the vehicle serum, 5% HLJDD containing serum significantly increased differentiation of CD206 + M2 BMDM (P = 0.034). Results of real-time quantitative PCR showed that the expression level of Arg1 mRNA could be up-regulated by HLJDD containing serum (P < 0.05), and that of Nos2 mRNA down-regulated (P = 0.017). ox-LDL induced the differentiation of M2 subtype foam cells from BMDM, and HLJDD containing serum could further elevate the ratio of CD206 + M2 foam cells and increase the Arg1 mRNA expression level (both P < 0.01). HLJDD containing serum could inhibit the inversion of M2 subtype of foam cells to M1 subtype induced by Th1 factors, significantly elevate the Arg1 mRNA expression level, and decrease the Nos2 mRNA expression level (all P < 0.01).

CONCLUSIONSHLJDD could lower hyperlipidemia induced inflammatory monocyte subtype ratios in the peripheral blood of ApoE(-/-) mice. HLJDD containing serum promoted in vitro differentiation of M2 macrophages and foam cells. HLJDD attenuated and inhibited the occurrence and development of atherosclerosis induced by hyperlipidemia possibly through regulating the functional differentiation of monocytes, macrophages, and foam cells.

Animals ; Apolipoproteins E ; genetics ; Cell Differentiation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Foam Cells ; cytology ; drug effects ; Macrophages ; cytology ; drug effects ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Monocytes ; cytology ; drug effects

OBJECTIVEDendritic cells an hyperinsulinemia are both implicated in the pathogenesis of atherosclerosis. The aim of this study is to explore the effect of high concentration of insulin on the maturation of monocyte-derived dendritic cells (MoDCs) and related signal transduction pathways.

METHODSHuman monocytes were purified (over 98%) using Anti-CD14 micro-beads and cultured for 5 days with DC Cellgro medium containing rhGM-CSF (100 microg/L) and rhIL-4 (20 microg/L). Immature DC were then incubated with insulin of various concentrations (0, 1, 10, 100 nmol/L) for 24 hours in the presence or absence of LY294002 (PI3K inhibitor) or PD98059 (MAPK inhibitor). Immunophenotypic expression of CD86 and CD83 were detected using flow cytometry. Endocytosis function of the MoDCs was evaluated using FITC-Dextran and MoDCs secretion IL-12, IFN-gamma and TNF-alpha were measured by ELISA.

RESULTSInsulin induced significantly higher CD83 and CD86 expressions on MoDCs in a dose-dependent manner. The endocytosis function of MoDCs were significantly inhibited and cytokine secretions of IL-12, IFN-gamma and TNF-alpha significantly increased by 10 nmol/L and 100 nmol/L insulin. These effects could be blocked by the LY294002 and PD98059.

CONCLUSIONHyperinsulinemia contributed to atherosclerosis via stimulating immune maturation of MoDCs via both PI3K and MAPK pathways.

Cell Differentiation ; drug effects ; immunology ; Cells, Cultured ; Cytokines ; metabolism ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Humans ; Insulin ; administration & dosage ; pharmacology ; Monocytes ; cytology ; Phagocytosis ; drug effects ; Signal Transduction

BACKGROUNDThe adhesion of monocytes to the endothelium following accumulation of low-density lipoprotein (LDL) in subendothelial spaces is an important step in the development of intimal hyperplasia in arterially implanted vein grafts and atherosclerosis in both animals and humans. However, it is not well known how serum factors affect the adhesion of monocytes.

METHODSWe have studied the effect of fetal calf serum (FCS), which we considered a source of LDL, on the adhesion of monocytes to endothelial cells (ECs) by using human monocytic THP-1 cells and both a monolayer of cultured bovine aortic endothelial cells (EC monoculture) and a co-culture with bovine aortic smooth muscle cells (EC-SMC co-culture).

RESULTSIt was found that the addition of FCS to the medium greatly affected the adhesion of THP-1 cells, and the higher the concentration of FCS in the medium, the greater the adhesion of THP-1 cells to endothelial cells. Adhesion of THP-1 cells to an EC-SMC co-culture was approximately twofold greater than that to an EC monoculture, and after adhering to endothelial cells, many THP-1 cells transmigrated into the layer of smooth muscle cells.

CONCLUSIONThe results suggest that the elevation of the LDL (cholesterol) level in blood provides a favorable condition for the development of intimal hyperplasia and atherosclerosis by promoting the adhesion of monocytes to the endothelium and their subsequent migration into subendothelial spaces.

Animals ; Cell Adhesion ; drug effects ; Cells, Cultured ; Coculture Techniques ; methods ; Culture Media, Conditioned ; Endothelial Cells ; cytology ; Humans ; Monocytes ; cytology ; Myocytes, Smooth Muscle ; cytology ; Serum

The study was aimed to investigate the mechanism of mannan-binding lectin (MBL) on bacterial lipopolysaccharide (LPS)-induced human peripheral blood monocyte-derived dendritic cell (DC) maturation. The monocytes were prepared from the peripheral blood of healthy adult volunteers. The immature dendritic cells (imDC) were induced by 5-day-culture in medium supplemented with rhGM-CSF and rhIL-4. FACS was used to investigate the interaction of MBL with imDC and the impact of MBL on LPS binding to imDC. ELISA and Western blot was used to analyze the interaction of MBL with soluble TLR4 ectodomain protein (sTLR4); Western blot was used to detect LPS-induced NF-κB translocation in imDC. The results showed that MBL could directly bind to imDC in the presence of calcium. sTLR4 protein or LPS could competitively inhibit the binding of MBL to imDC. ELISA and Western blot showed that MBL could evidently bind to sTLR4 protein in a concentration-dependent manner. FACS showed that MBL could competitively inhibit the binding of LPS to imDC by binding to imDC directly. Western blot showed that MBL decreased LPS-induced NF-κB translocation in imDC. It is concluded that MBL may competitively inhibit the binding of LPS to imDC by binding to TLR4 expressed on imDC, resulted in inhibition of LPS-induced DC maturation, suggesting that MBL can regulate DC maturation through ligand-binding. This study provides the good foundation to clarify the mechanism of MBL inhibiting the LPS-induced DC maturation.
Cell Differentiation ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Humans ; Ligands ; Lipopolysaccharides ; adverse effects ; Mannose-Binding Lectin ; pharmacology ; Monocytes ; cytology ; metabolism ; Toll-Like Receptor 4 ; metabolism

OBJECTIVETo observe the expression and localization of endogenous C-reactive protein (CRP) in cells from different tissues under different conditions.

METHODSMacrophages differentiated from THP-1 monocytes with phorbol ester (PMA) induction and human LO2 hepatocytes were stimulated with lipopolysaccharide (LPS). The culture supernatant of the LPS-stimulated THP-1 cells was collected and added into LO2 cell culture, and after incubation, the cells were lysed to extract the proteins for SDS-PAGE and Western blotting. The stimulated cells were also examined immunocytochemically for CRP expression.

RESULTSWestern blotting detected CRP in both of the unstimulated cell lysates, but in neither of the two cell supernatants. After LPS stimulation, CRP expression was significantly increased in the cell lysate of THP-1 cells with also a small amount present in the supernatant, but CRP expression and release in the LO2 cells showed no significant variation. Treatment of the LO2 cells with the culture supernatant of LPS-stimulated THP-1 cells resulted in positivity of CRP in the cell lysate and the culture supernatant. Immunocytochemistry identified CRP expression throughout the THP-1 cell body (most obvious in the nuclei), which increased after LPS stimulation. In LO2 hepatocytes, CRP expression was found only outside the nuclei and increased after stimulation with the culture supernatant of LPS-treated THP-1 cells, especially obvious around the membrane.

CONCLUSIONCRP can not be up-regulated directly by LPS treatment in LO2 cells, but can be induced by certain cytokines (IL-6) secreted from LPS-stimulated THP-1 cells. The localization of CRP represents the characteristics of secreted protein in LO2 cells, but in THP-1 cells, CRP is found mainly in the cell nuclei.

Blotting, Western ; C-Reactive Protein ; biosynthesis ; Cell Differentiation ; drug effects ; Cell Line ; Culture Media, Conditioned ; pharmacology ; Hepatocytes ; cytology ; drug effects ; metabolism ; Humans ; Immunohistochemistry ; Lipopolysaccharides ; pharmacology ; Macrophages ; cytology ; Monocytes ; cytology ; drug effects ; metabolism

OBJECTIVETo observe the effects of Tongguan Capsule on the mobilization and differentiation of bone marrow mononuclear cells (BMMNCs) to the injured carotid arteries.

METHODSThe rat model of injured carotid arteries was established. Mononuclear cells were separated from the bone marrow of donor rats, which were labeled by PKH 26 and then injected through the tail vein to the recipient rats with injured carotid arteries. The rats were randomly divided into two groups. Tongguan Capsule suspension was administered by gastrogavage to rats in the experiment group, while equal volume of normal saline was given to rats in the control group. Four weeks later the injured carotid arteries were harvested and frozen sections were made to observe the differences of intima area/media area ratio (Ai/Am). The difference of BMMNCs migrating to the injured carotid arteries between the two groups was observed under fluorescence microscope. The difference of the vascular endothelial growth factor (VEGF) and stromal derived factor-1 (SDF-1) levels in serum were detected by ELISA.

RESULTSThe PKH 26 labeled cells appeared in the experiment group, while only little scattered fluorescent light points was presented in the control group. The intima area and the ratio of Ai/Am of the experiment group decreased more obviously than that of the control group (P<0.01). Two and 4 weeks later the VEGF and SDF-1 levels in serum of the experiment group were more obviously enhanced than those of the control group (P<0.01).

CONCLUSIONTongguan Capsule could promote the BMMNCs transplanting towards the intima of injured carotid arteries, and take part in the repair of the intima of injured carotid arteries.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; Chemokine CXCL12 ; blood ; Drugs, Chinese Herbal ; pharmacology ; Endothelium, Vascular ; Monocytes ; cytology ; drug effects ; Rats ; Rats, Wistar ; Tunica Intima ; cytology ; drug effects ; pathology ; Vascular Endothelial Growth Factor A ; blood