| BackgroundCoronary artery disease(CAD), which was also one of the most serious cardiovascular disease affected daily life quality, has become the third killer as a threat to human health worldwide. Recent studies have shown that inflammation plays a key role in CAD and other manifestations of atherosclerosis, while there were a large number of inflammatory cells exsiting within atherosclerotic plaque, such as monocyte, macrophage, dendritic cell, and T lymphocyte. The inflammatory response, which can turn stable plaque into vulnerable plaque, was extremely active in the plaque, that was the cause of acute coronary thrombosis. Therefore, inhibition of inflammatory cells activity and blockade of inflammatory response has become the hotspot and difficulty of immune therapy for CAD. As we all know, T lymphocyte activation is the central link in the inflammatory reaction. The PD-1/PD-L1pathway discovered recently plays an important role in regulation of T lymphocyte activity.Programmed cell death ligand1, also called PD-L1, B7-H1or D274, was type I transmemberane protein that comprised290amino acids. In1999, Dong’s research has described that PD-L1, which does not bind CD28, cytotoxic T-lymphocyte A4or ICOS (inducible co-stimulator), was the third member of the B7family. But, it binds programmed cell death receptor-1, also called CD279. Because it was initially found expressing highly on tumor cells, people speculated that PD-L1was involved with tumor infiltration. But besides tumor cells, PD-L1was found abundantly in various cells and tissues, such as T lymphocyte, B lymphocyte, macrophage, Kupffer cell, astrocyte, dendritic cell, vascular endothelial cell, mast cell, placental syncytiotrophoblast cells. A large number of clinical and experimental data suggested that PD-Ll/PD-1pathway was closely related to many diseases, for example transplant immune response, autoimmune disease, microbial infection (viral infection). In the recent year, a few theses have found that PD-1/PD-L1pathway contributes to atherosclerosis. Gotsman figured out that PD-L1could express in many of plaque when he studied coronary arteries plaques with fluorescence immunoassay technology. Lee found that the expression of PD-1and PD-L1was significantly down-regulated on T cells and myeloid dendritic cells in CAD patients than in healthy individuals, respectively. The stimulatory ability of dendritic cells on naive T cell proliferation in CAD was stronger than in healthy individuals.Studies carried out during the last few years have led to major progress in understanding the functions of PD-L1in vivo.But, no general pathway is known which controls PD-L1expression. Depending on stimulus and cell type, the expression of PD-L1was found to correlate with various signaling molecules: JAK/STAT, PI3K/Akt, MEK/Erk, NPM/ALK, IRF-1and STAT-3. P38mitogen-activated protein kinase, as well all know, which media cell differentiation, cell proliferation and apoptosis via phosphorylation of transcriptional factors, is activated by a wide range of cellular stresses as well as in response to inflammatory cytokines. But, the relationship between PD-L1expression and p38MAPK pathway remaied, however, unknew. So, here we aim to investigate wether p38MAPK pathway media the expression of PD-L1on dendritic cells. In this study, dendritic cells cultured in vitro as research object, was stimulated by LPS to imitate microbe invasion in vivo, and then expression of PD-L1on dendritic cells was measured. P38protein inhibitor SB203580was used to block the p38MAPK pathway to investigate the relationship between expression of PD-L1and p38MAPK pathway. We aim to clarify the molecular mechanism of the expression of PD-L1LPS-induced on dendritic cells, and perfect the mechanism of T cells negative regulation by PD-1/PD-L1pathway.Part one:lipopolysaccharide induce dendritic cells to express PD-L1Objective:Observe the influence of CD80, CD86and PD-L1phenotype on dendritic cells stimulated by LPS, to determine whether LPS can promote dendritic cells mutation and up-regulate the PD-L1expression.Object and Methods1、Object:Dendritic cells were cultured in vitro form peripheral blood mononuclear cells (PBMC).2、Methods2、1Preparation of PBMC and cultureBuffer coats of healthy donors were obtained according to institutional guidelines. PBMC were prepared by density gradient centrifugation using lymphocyte separation medium.PBMC were seeded (5×106cells) in six-well culture plate in3ml of RPMI1640medium supplemented with10%FCS,100U/ml penicillin and100μg/ml streptomycin, and incubated for2h at37℃in humidified5%CO2atmosphere. Nonadherent cells were removed by extensive washing, and the remaining adherent cells were recovered by scraping.2、2Culture of DCThe remaining adherent cells were subsequently cultured in six-well plates (5×106/well) in serum-free dendritic cell medium supplemented with20ng/ml GM-CSF,20ng/ml IL-4,100U/ml penicillin and100μg/ml streptomycin. Cells were refeed with1.5ml of fresh medium containing20ng/ml GM-CSF,20ng/ml IL-4on day3,5, and7. To get mature DC, the nonadherent cells (immature DC) were harvested on day7and stimulated with1.0μg/ml LPS.2、3Grouping(1)LPS group:DC were stimulated with LPS(1.0μg/ml) for24h.(2)NORMAL group:DC were cultured with DMSO(0.1%V/V) for24h.2、4Flow cytometric analysisCultured DC were washed, resuspended at5×105cells in50μl of cold PBS containing0.1%sodium azide,10mg/ml BSA, and200μg/ml mouse1gG, and incubated for15min on ice. Subsequent staining with labeled mAb was performed for30min on ice. Cells were then washed and resuspended in100μl of cold PBS containing0.1%sodium azide, and10mg/ml BSA. Stained cells were analyzed for four-color immunofluorescence with a FACSCalibur cell analyzer. A minimum of104cells were analyzed for each sample. Results were processed using CellQuest software.3、Statistical analysisStudent’s t test was performed wherever applicable. Mean±S.D. is shown unless otherwise stated. P value<0.05was considered significant.Results 1、Observation of morphology of DCSeveral phenomenons were observed under an inverted microscope. There were distinct dendritic structures and abundant cytoplasm in DC after cultured for7days.(1)LPS group:After culturing with LPS for6h, DC lost their characteristic morphology and readhered to the bottom of the culture wells. DC became fusiform like Macrophage. After culturing for24h, DC gradually resuspended and dendritic structures reappeared.(2)NORMAL group:DC remained round, suspend with pseudopodia on8day.2、Influence of phenotypeThe results showed that compared with normal group, there were higher expression of CD80, CD86and PD-L1on DCs in LPS group. Difference between treated group and control group were statistically significant (1492.46±82.65vs536.52±64.10, P<0.01;1136.73±81.62vs518.47±48.91, P<.0103665.89±261.66vs1093.38±115.54, P<0.01, sespectively).Conclusions1、The adherent cells of PBMC could be induced to be DCs after culturing with GM-CSF and IL-4for7days.2、LPS could prompt immature DC into mature DC, and up-regulate the expression of CD80and CD86.3、LPS could increase the expression of PD-L1on DC.Part two:p38MAPK pathway media the expression of PD-L1on dendritic cell ObjectiveTo investigate the relationship between PD-L1expression and p38MAPK pathwayObject and methods1、Object:Dendritic cells were cultured in vitro form peripheral blood mononuclear cells (PBMC).2、Methods2、1Preparation of PBMC and culture:the same as part one.2、2Culture of DC:the same as part one.2、3Grouping(1) LPS group:Treatment of immature DC with DMSO(0.1%V/V) was performed for1h, and then cultured with LPS(1μg/ml) for24h.(2) SB group:Treatment of immature DC with p38MAPK inhibitor SB203580(25μM) was performed for1h, and then cultured with LPS(1μg/ml) for24h.(3) NORMAL group:Treatment of immature DC with nothing was cultured for24h. LPS and SB203580were dissolved in DMSO.2、4Flow cytometric analysis:the same as part one.2、5Western blot analysisImmature DCs were exposed to various agonists for indicated periods of time. Thereafter, cells were wash twice with cold PBS and incubated with100μl lysis buffer. The homogenates were centrifuged at12000g/min for30min at4℃. Cell lysates were electrophoresed on12%SDS-PAGE gels, and transferred to nitrocellulose membranes for Western blot analysis. Briefly, nitrocellulose membranes were incubated in a blocking buffer for1h at room temperature, and then incubated overnight with Abs against PD-L1protein. The membranes were washed and incubated for1h with HRP-labeled goat anti-rabbit Abs. Immunoreactive bands were visualized by ECL detection reagent and quantified by scanning densitometry. 3、Statistical analysisThe software package SPSS13.0was applied for statistical analysis. Mean±S.D. is shown unless otherwise stated. Experimental data were analyzed by One-way ANOVA test and SNK-q test. P value<0.05was considered significant.Results1、Observation of morphology of DCMorphology of DC was observed under an inverted microscope.(1)LPS group: After culturing with LPS for6h, DC lost their characteristic morphology and readhered to the bottom of the culture wells. DC became fusiform and took on Macrophage properties. After culturing for24h, DC gradually resuspended and dendritic structures reappeared.(2)SB group:Dendritic cell kept naive with a few and short dendrites after SB203580stimulation.(3)NORMAL group:DC remained round, suspend with pseudopodia on8day.2、Influence of phenotype(1) There were no significant differences in the expression of CD11c among groups (LPS group:628.19±34.99, SB group:617.44±41.00, NORMAL group589.68±47.84, F=1.825, P=0.186)(2) There were significant differences in the expression of CD86among groups. SB203580treatment could significantly down-regulated the expression of CD86in dendritic cells as compared with LPS stimulated group (729.49±48.89vs873.01±71.24, P<0.05); As compared with NORMAL group, there was no significant difference (vs736.96±42.11, P>0.05).(3) SB203580treatment could significantly down-regulated the expression of PD-L1in dendritic cells as compared with other two groups, compared with LPS treatment group,P<0.01(3.03±0.08vs3.51±0.08),compared with NORMAL group, P<0.05(vs3.18±0.07).3、Western blot analysisThere were significant differences in the expression of PD-L1among groups. SB203580treatment could significantly down-regulated the expression of PD-L1in dendritic cells as compared with other two groups, compared with LPS treatment group,P<0.05(0.55±0.08vs1.24±0.11), compared with NORMAL group, P<0.05(vs0.95±0.14).Conclusions(1) p38MAPK pathway medias DC mutation induced by LPS.(2)p38MAPK pathway plays a crucial role in LPS-induced PD-L1expression on the monocye-derived dedritic cells. |
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