An Experimental Study On The Splenic Lymphocyte Cultured With Kupffer Cells Transduced With An Recombinant Adenoviral Vector Expressing Interleukin 12 In Vitro

Objectives:(1) To isolate and purify Kupffer Cell (KC) from Sprague-Dawley (SD) rat liver and receive highly purified and active KC.(2) To detect the expression of IL-12 and MHC class I antigen (H-2Kb)and MHC class II antigen (I-Ab) by KC transduced with murine IL-12 recombinant adenoviral vector in vitro . (3) To determine,with ELISA kits,the expression of IFN-γand IL-4 secreted by splenic T-lymphocyte cultured with KC modified with IL-12 gene and to determine, with the flow cytometry (FCM), the variation of CD4 and CD8 subsets of such T-cell.Methods: (1)A combined"collagenase IV & pronase E"perfusion, in situ and ex vivo , and then the discontinuous density gradient centrifugation using Nycodenz was performed to obtain the Nonparenchymal cells (NPC) from SD rat liver . Further, the method of selective adherence was used to purify and recover the KC from NPC. The activity and quantity of cells was judged by Trypan blue exclusion test. To identify the KC, we observed the phenomenon of auto-fluoresence and KC,s phagocytosis action and performed immunocytochemistry with the mouse anti-rat monoclonal antibody ED-2.(2) KC were seeded in 24-well plates at a density of 1×106/ml in DMEM and were divided into 3 groups,KC group & KC+Adv-EGFP group as controls and KC+Adv-IL-12 group, in triplicate. The supernatants were harvested in 24h and 48h respectively after ifection, then supernatant samples were testified with IL-12 ELISA kit to detect quantitatively the level of IL-12.In 48 hours, the cells of three wells of each group were harvested and used to detect the relative level of the mRNA of MHC class I antigen (H-2Kb)and MHC class II antigen (I-Ab) expressed by KC with RT-PCR test.(3) Splenic lymphocytes (SLC)of rat prepared as usual methods and cultured with stimulus of ConA,at the concentration of 5μg/ml, at a density of 2×10~6/ml for 72 hours in advance were co-cultured with KC which had been treated with the"recombined adenoviral"before and the ratio of SLC vs KC was 10:1.They were divided into 4 groups as, cultured with Adv-IL-12- KC(A-group)or with Adv-EGFP- KC (B-group)or with KC(C-group)and cultured alone (D-group) respectively. In 72 hours, the tissue supernatants were sampled and were used to detect quantitatively the level of IFN-γand IL-4 with ELISA kits. At the same time the SLC of each well were harvested and were used to determine the variation of CD4 and CD8 subsets of such cells with the FCM.Results:(1) The yield of KC per rat were 3.5-5.0×10~7 ,in which over 95% were positive for ED-2 and over 93% viable by Trypan blue exclusion test.(2) At 24h or 48h, uninfected KC and adv-EGFP-infected KC produced very low levels of IL-12 respectively,whereas a high level of secretion of IL-12 ,10-fold of which of controls,was detected in the adv-IL -12-infected KC.RT-PCR analysis showed that adv-IL -12-infected KC had an 1.5-fold (mRNA of H-2Kb)or 2-fold(mRNA of I-Ab)increase compared with uninfected KC concentration of IFN-γwere 2026.29±103.37(A-group),988.55±94.39 and adv-EGFP infected KC, whereas there was no difference between two of latter.(3) ELISA test demonstrated that the (B-group),1005.36±120.18(C-group),505.12±55.28 (D-group)pg/ml,whereas the concentration of IL-4 were 206 94±22.43,320.57±34.73,349.84±49.61,828.26±62.83 pg/ml in same turn.Such data demonstrated that the yield of cytokine of Th1 type increased markly and which of Th2 type decreased evidently in A-group. FCM analysis showed that the proportion of CD4 T-cell and which of T-cell(sum of CD4 T and CD8 T ) and the the ratio of CD4 T / CD8 T increased obviously,but the proportion of CD8 T-cell changed insignificant in A-group compared with B,C and D group.Conclusions: (1) The activity as well as purity of KC ,which be attained from SD rat by the methods of combined"collagenase IV & pronase E"perfusion and discontinuous density gradient centrifugation using Nycodenz and selective adherence were satisfactory and these KC can be used in further study. (2)The adenoviral vector can transfer effectually the IL-12 gene to KC in vitro and the modified KC highly expressed IL-12 protein and genetic transcription of MHC classⅠantigen & MHC classⅡantigen were up-regulated, which denoted an enhancement of the ability to present antigens.(3)The Splenic lymphocyte co-cultured with modified KC resulted in an evident increasing of the proportion of CD4 T-cell and the proportion of T-cell(sum of CD4T and CD8T ) and the ratio of CD4T / CD8T.Therefore ,the proportion of CD8T altered insignificant.The modified KC can also induced the T helper cells differentiate to Th1.