The Chemotaxis And The Pro-mature Effect Of OxLDL And OxHDL On Bone Marrow Derived Dendritic Cells From C57BL/6J Mice

ObjectiveDendritic Cells (DCs) are regarded as a kind of antigen presenting cells to play a central role in controlling immunity by the unique ability to active the native T cells in vivo. Atherosclerosis(As) is believed as a chronic immuno- inflammatory disease related with dyslipoidemia. Recent researches display that DCs perhaps influences the beginning and development of atherosclerotic plaque. It has been proved that many antigens involved in As can induce the maturation of DCs, which is essential for DCs to bring into full play. We separated and cultured DCs from C57B/6J mouse bone marrow, and studied the chemotaxis and the pro-mature effect of oxLDL and oxHDL on DCs. The goal was to study the effects of oxLDL and oxHDL as autoantigens on migration, maturation and activation of DCs, which could result in local immune response and the initiation of As. MethodsKill mouse by cervical dislocation. Dissect both femurs and tibias and Remove most of the muscle tissues surrounding and the epiphysises. Wash the cavities of bones with RPMI-1640 medium to gain the cell suspension.We applied separating medium for animals and the difference of cell adhesion to remove off most macrophages and lymphocytes, adding rmGM-CSF and rmIL-4 to promote monocytes to differentiate and to suppress lymphocytes.Draw off a half of all the culture medium on other day, supplementing new medium and needed cytokines. At the seventh day, blow and harvest the immature bone marrow derived dendritic cells (BMDCs). Identification was performed from morphology, molecule marker expression and function:(1)observe the morphological changes of BMDCs during the course of growing and differentiation by microscope; scan mature and immature BMDCs with scanning electron microscope(SEM) and count the percentage of dendritelike cells;(2)detect expressions of more than 3 surface markers using immunofluorescence and fluorescence-activated cell sorting(FACS);(3)mixed lymphocyte reaction(MLR) reflected the ability of BMDCs to present antigens. 50μg/ml oxLDL and oxHDL were added to stimulate BMDCs, using 50μg/ml LDL and HDL as homologous protein controls , PBS and 1μg/ml LPS as negative and positive controls. In vitro the chemoattractant of oxLDL and oxHDL to BMDCs were observed with Transwell system. The CD86 and MHCⅡexpression percentages of 6 groups were detected with FACS. Liquid scintillation counting(LSC) was used to reflect the ability of BMDCs to stimulate homologous T cells proliferation. After stimulation, these BMDCs were injected into C57B/6J mouse abdominal cavities, then the percentages of BMDCs in the spleens were observed from FACS, which could illustrate the mature condition of BMDCs in vivo.ResultMononuclear cells from C57B/6J mouse bone marrow was stimulated with rmGM-CSF and rmIL-4, with the typical growing pattern of DCs. At the third day, the cells became round or roundlike, semi-adherent clustering just like clusters of grapes. And little mixed cells with long apophyses can been seen. At the seventh day, a great deal of cells got roundlike or irregular. After maturation with the stimulation of LPS, apophyses became obviously increased by SEM scanning; the expression percentages of 33D1 and CD11c exceeded 80% by immunofluorescence staining and those of CD86 and MHCⅡwere 99.0% and 32.5%; MLR showed that mature BMDCs could better stimulate the proliferation of homologous T cells. So the cells obtained accords with the needs of our experiments.Our study was carried out with 6 groups: PBS group, LDL group, oxLDL group, HDL group, oxHDL group and LPS group. Transwell system motify imitates course of corpuscular trans-endotheliocyte. Compared with PBS group, the number of migrated cells was larger in other groups except LDL group, which suggests that oxidized lipoproteins could better chemoattract BMDCs to move than homologous lipoproteins. Compared with the homologous lipoproteins, oxLDL and oxHDL could better promote the expressions of CD86 (84.7%vs68.7%; 90.8%vs73.8%) and MHCⅡ(26.0%vs15.2%;25.9%vs11.4%), better stimulate the proliferation of homologous T cells(cpm: 992±150.00 vs 645±21.17; 996.3±212.81 vs 652±50.59) and secretion of cytokine(sIL-12 and IL-10). These data suggested that BMDCs induced by oxLDL and oxHDL got more close to mature BMDCs stimulated by LPS, the expressions of molecule markers and MLR in LDL and HDL groups were both below those of LPS group, BMDCs stimulated by LDL and HDL could only remain a state of semi-maturation. In vivo, the number of cells migrated to the spleens were larger also in the oxidized lipoproteins than that in the homologous lipoproteins 24 hours'later(percentage:5%vs 2%; 8%vs2%).Conclusion(1) Compared with the homologous lipoproteins, chemoattractant of oxLDL and oxHDL could better promote BMDCs down to the the lower chamber of Transwell , which showed that oxidized lipoproteins might directly chemoattract DCs into the intima. (2)The expressions of CD86 and MHCⅡ, MLR and the secretions of cytokines(IL-12 and IL-10) had been all improved in the oxLDL group and the oxHDL group than the LDL group and the HDL group, which suggested that oxidized lipoproteins might induce BMDCs to mature.(3) After treated with these materials, the BMDCs ware injected back into C57B/6J mice, and found the number of the cells migrated into the spleen obviously increased in oxLDL and oxHDL groups than the homologous lipoproteins goups. These results proved that oxLDL and oxHDL could induce C57B/6J mouse BMDCs to mature.