The Study Of Time Sequence Of Dendritic Cells’ Activation In Lung And Immigration In Celiac Lymph Nodes After Disseminated Murine Cytomegalovirus Infection

Objectives:1. To set up the mice disseminated Murine Cytomegalovirus infection model;2. To observe the time sequence of the dendritic cells’ activation and their surfacemarkers’ expression levels in lung as well as celiac lymph nodes dendritic cells’immigration after disseminated Murine Cytomegalovirus infection.Methods:1. Set up the mice disseminated Murine Cytomegalovirus infection model: FemaleBALB/c mice aged4.5-week-old was inoculated intraperitoneally with salivary glandhomogenates containing5×103PFU of MCMV Smith strain to establish the model ofdisseminated MCMV infectio（nMCMV infected group）. Built Normal and LPS groupsat the same time;2. At the time points of1d,3d and7d after infection, harvested the lung and lymph nodesof all experimental groups. After making single cell suspensions of both tissues, usemonoclonal antibodies directly conjugated with phycoerythrin (PE), allophycocyanin(APC) and fluorescein isothiocyanate (FITC) to label dendritic cells’surface markersCD11c, MHCⅡ, CD86respectively. Then all cell analyses were performed using aFACSCalibur (BD Biosciences) flow cytometer to get the datum of dendritic cells’percentage and surface markers’expression levels.Results:1. Flow cytometry of lung1) Dendritic cells’percentage: In MCMV group, the percentage has a slight rise in1day after MCMV infection, reached the peak at3dpi (3day past infection) and thendescend to7dpi which still higher than the Normal group. Meanwhile in the group ofLPS, the percentage of CD11chiMHC-IIhicells retained stable;2) Dendritic cells’expression levels of CD11c: In MCMV group, both1dpi and3dpishowed no distinct difference but ascend apparently at7dpi. In the group of LPS, theexpression levels was first lower than normal in1dpi and then rose to normal;3) Dendritic cells’expression levels of CD86: In MCMV group, the expression levels hadno difference with normal at the time of1dpi, however it reached the peak at3dpi thendropt back to normal at7dpi. In the group of LPS, it expressed the highest value at1dpibut with no statistical difference, and then fell to normal;4) Dendritic cells’expression levels of MHCⅡ: In MCMV group, there was no changesin1dpi, and then downgraded the expression levels at3dpi, after that rised to normal at7dpi. In the group of LPS, at the time of1dpi and3dpi showed a slight drop and thenrose to Normal;5) The CD11cintpopulatin in lung: At7dpi, we found a large population of cells withintermedial expression of CD11c which appears in groups of MCMV while there wereno significant emerges in Normal and LPS. When3dpi the CD11cintpopulation had justa little rose and we cannot distinguish them however the distinction has became largerat7dpi. Besides the remaining group have no such display. After analysing of CD11cintpopulation, we found out that the cells expressed none/low CD86and MHCⅡlevelfrom none to feeble (CD11cintMHCII–/loCD86–/lo).2. Flow cytometry of lymph nodes: In MCMV group, no significant difference of CD11c+population was found both in1dpi and3dpi compared with Normal group, but itincresed highter than Normal in7dpi. There were no significant changes in the wholeprocess in LPS group.Conclusions:1. Compared with LPS group, the time of lung dendritic cells’activation and immigration in MCMV group has been delayed. In lung of MCMV group, when the dendritic cells’percentage reached the peak the expression of CD86has a slightly increased whileMHCII dropt. At the same time the dendritic cells’activation has been inhibited whichmighet related to the infection of dendritic cells by MCMV;2. The groups infected with MCMV Smith strain have showed a large population of cellswith intermedial expression of CD11c which increased obviously at7dpi. These cellsexpressed low CD86and MHCⅡlevel from none to feeble (CD11cintMHCII–/loCD86–/lo) which might be plasmacytoid DC (pDC) and/or IFN-producing killer dendritic cells(IKDC). Both of them might related to the antiviral immunity of host;3. DCs in lymph nodes showed great increase in7dpi, which indicated that peripheraltissues’dendritic cells had immigrated to lymph nodes.