Effects Of Rapamycin And Paclitaxel On Development And Maturation And Immune Function Of Human Monocyte Derived Dendritic Cells

ObjectAtherosclerosis(AS)has been considered as a chronic inflammation/autoimmune disease,as inflammation and immune response might play a central role in the pathogenesis of AS.One class of immune cells-the dendritic cells(DCs)-have been found in normal arterial walls.Recent studies observed that the number of DCs increased significantly in atherosclerosis plaques,suggesting an important role of DCs in the inflammation of AS.In the present manuscript,we studied the effects of rapamycin and paclitaxel on the monocyte-derived DCs, including the mature of surface markers,its antigen processing function, secretion of cytokines,and apoptosis.We also discussed the role of rapamycin and paclitaxel in the differentiation,mature and immune function of monocyte-derived DCs.We further studied the effects of rapamycin and paclitaxel in the inflammation of atherosclerosis.Methods1.DC Culture:White blood cell suspension which was contributed by normal donors was contained from blood center of Zhejiang Province. Peripheral blood monouclear cells(PBMCs)were harvested from interface with lymphocyte separating medium.CD14~+ cells were separated by means of immunomagnetic technique(MACS)with＞98%purity.Then those cells were plated in RPMI 1640 containing 15%fetal bovine serum supplemented with granulocyte-macrophage colony-stimulating factor(rhGM -CSF,20ng/ml)and interleukin-4 (rhIL-4,2ng/ml).Cultures were fed every other day by half.On day 5, buoyant cells were harvested as immature DC(imDC),then LPS(100 ng/ml)was added to imDC and hatched 2 days as mature DC(mDC).2.DC were exposed to rapamycin(1.0 ug/ml,0.5 ug/ml,0.1 ug/ml), paclitaxel(100 nmol/L,10 nmol/L,1 nmol/L),LPS(100ng/ml)for 24h.The control group is added none of the above.3.Allogeneic T cell activation by DCs was tested by mixed lymphocyte reaction(MLR).ELISA was used to detect the expression of IL-10,IL-12,TNF-a.4.The Des immunophenotypic expression:after DCs were exposed for 24 h,the fluorescent labeled antibody of CD1a,CD83,CD86,HLA-DR were added to DCs and hatched for 30min,then washed with PBS twice, FITC labeled antibody was added to DCs,and hatched for 20min,washed with PBS twice again,and was measured by flow cytometry.5.Endocytic activity of DCs:after DCs were exposed for 24h, FITC-Dextran at the concentration of 1mg/ml were added to DCs and hatched for 30min at 37℃washed with PBS including 5%FBS twice,then PBS(200ul)was added to DC,it was measured by flow cytometry.6.DCs Apoptosis:after DCs were exposed for 24h,Binding Buffer(500ul),Annexin-FITC(5ul)and Propidium Iodide(5ul)were added to DCs,avoided the light for 10min,then it was measured by flow cytometry in an hour necessary.Results1.mDC expressed high level of CD86,CD83,HLA-DR,moderate levels of CDla,the level of CD86,CDla,HLA-DR in the treatment group is lower than those in the control group(P＜0.01),however,the level of CD83 in the treatment group is no lower than that in the control group. 2.The endocytic activity of mDCs is higher than that of irnDCs; rapamycin and paclitaxel modulate the endocytic activity of imDCs, however,the Rapamycin group showed no statistical difference compared with the paclitaxel group.3.Mature DCs with high concentration were more effective than control in stimulating T-cell proliferation(P＜0.01),Rapamycin with lower concentration and paclitaxel groups showed statistical difference compared with the control group(P＜0.01).4.The secretion of IL-10,IL-12 and TNF-a of mDC by ELISA was higher than those of the imDC(P＜0.01);the secretion of IL-10,IL-12 and TNF-a of imDC in treatment group was lower than those of the control groupimDC(P＜0.01).5.Rapamycin and paclitaxel both induced imDC apoptosis(P＜0.01), but rapamycin showed statistical difference compared with paclitaxe; mDC showed no statistical difference compared with the imDC.Conclusion1.Rapamycin and paclitaxel are able to inhibit maturing imDCs and lowering the endocytic activity of imDCs.2.mDCs were more effective than imDCs in stimulating T-cell proliferation,and rapamycin with lower concentration and paclitaxel are able to inhibit imDCs in stimulating T-cell proliferation. 3.The endocytic activity of imDCs is higher than that of mDCs,the endocytic activity of imDCs in treatment group is lower than those of the control group.4.Rapamycin and paclitaxel both induced imDC apoptosis,however,the activity of rapamycin is higher than that of paclitaxel...