Effect Of Heat Shock Protein 60-loaded Dendritic Cells On Atherosclerosis In Apolipoprotein E～(-/-) Mice

Part 1 Effect of heat shock protein 60 on murine dendritic cell maturation and immunologic function in vitroObjective To investigate the effect of heat shock protein 60 (HSP60), significant inflammatory factor in atherogenesis, on the maturation of murine dendritic cells (mDCs) in vitro. Methods Myeloid mDCs were obtained in vitro from C57BL/6J mouse marrow and incubated with OVA, two concertrations of mHSP60 or PBS 24 hours, observed the morphological changes; detected the variation of CD80 and CD86 phenotype with flow cytometric analysis (FCA); the stimulating T-cell capacity determined in allogenic mixed lymphocyte reaction (MLR); ELISA was used to analyze the level of cytokines, such as IL-4, IL-12 and IFN-γin the medium of MLR. Results Compared to control group, mDC showed more protrusions in OVA and mHSP60 incubated groups. The expression of CD86, CD80 on OVA-DCs and mHSP-DCs up-regulated markedly. The stimulating T-cell capacity of OVA-DCs and mHSP-DCs was higher. Higher levels of IFN-γ, IL-12 but no significant difference of IL-4 was displayed in supernatant of MLR and the ratio of IFN-γ/ IL-4 was higher. Above effects were evident on OVA-DCs and higher mHSP60 concentration. Conclusion mHSP60 promotes the maturation of mDC and the capacity of immune activity dose-dependently in vitro. Part 2 Target Effect of Heat Shock Protein 60-loaded Dendritic Cells on Progression of Atherosclerosis in Apolipoprotein E-/- MiceObjective To investigate whether murine dendritic cells (mDCs) loaded with heat shock protein 60(HSP60) could enhance atherosclerotic plaque in Apolipoprotein E-null mice. Methods Myeloid mDCs were extracted and incubated in vitro then incubated with PBS, OVA and HSP60 to maturation respectively and observed the morphological changes and detected the variation of CD80 and CD86 phenotype with flow cytometric analysis (FCA) in vitro to know the degree of mDCs maturation. Homogenous mice were fed high-cholesterol diet 16 weeks to form plaque. ApoE-/- mice were treated with mDCs marked by fluorescent CFSE subcutaneously for three times at a one–week interval. 48 hours after the last treated, aortas were harvested for Haematoxylin-Eosin stained and anti-CD4+T cell immunostained. Serum cytokines (IL-10, IFN-γ) were analyzed by ELISA assay. Results Compared to PBS-DC group, HSP60-DC and OVA-DC group expressed higher levels of CD80 and CD86. After treatment of mice with DCs, serum IFN-γlevel was increased in HSP60-DC and OVA-DC group (P<0.01), while IL-10 no significance (P>0.05), and IFN-γ/IL-10 ratio also rose (P<0.01). HSP60-DC made inflammatory cells infiltration in atherosclerotic plaques greatly, and plaques tended to vulnerability. Then OVA-DC had no significance to plaque (P>0.05). And HSP60-DC migrated to atherosclerotic plaques more greatly than OVA-DC and PBS-DC (P<0.01). Conclusion DC loaded HSP60 enhances inflammatory reaction of atherosclerotic plaque and induces cytokines release, leads to immune deviation.