The Role Of Lung Dendritic Cells In Immunologic Activation And Tolerance In Multiple Organs Dysfunction Syndrome

Objective:1. To explore the relationship between immunophenotype and immunologic function of lung dendritic cells (DCs) and excessive inflammation and immunosuppression in multiple organs dysfunction syndrome (MODS).2. To confirm the immunologic tolerance of lung DCs in the stage of MODS and to explore its effect on immunosuppression.3. To observe the effects of alleviating lung damage and improving lung function by applying N-acetyl-L-cysteine (NAC) and fms-like tyrosine kinase 3 ligand(Flt3L) to regulate DCs'function in excessive inflammation and immunosuppression in MODS respectively.Methods:The model of MODS was replicated by zymosan injecting into the peritoneal cavity of Balb/c mice. The experimental groups were mainly divided as below:(1) acute damage groups (n=30). (2)NAC treating groups (n=30). (3)MODS groups (n=30). (4)Flt3L treating groups (n=20). (5)Control groups (n=20). The subcutaneous administration of NAC in dose of 100mg/kg body weight was at 3h, 6h,12h,18h,24h and 36h after zymosan injection. The subcutaneous administration of Flt3L in dose of 10μg/per day and per mouse, consecutive injection in 7 days was at 48h after zymosan injection. Symptom and mortality of the mice were observed. Biochemical indicator of CO2, ALT, AST and creatinine were detected by automatic biochemical analysator. MPO in lung tissue was measured by biochemical analysis. Pathological changes of lung were observed by light microscope. Specific surface markers CD205 and CDllc of lung DCs were detected by immunohistochemistry and immunofluorescence. Lung low density mononuclear cells were separated by density gradient centrifugation and then DCs were isolated by MACS microbeads. Purity identification and DCs'phenotypes of I-Ad, CD86 and PD-L1 were analyzed by flow cytometry. Ultrastructure of DCs was observed by scanning and transmission electron microscope. The protein and activity of NF-κB in the DCs' nuclear were detected electrophoretic mobility shift assay. The expression of CCR5/CCR7mRNA in DCs was measured by by real-time quantitative PCR. TNF-a from lung tissue homogenate and serum and IL-12p70 and IL-10 ELISA in supernatant were determined by ELISA kits.Results:In acute damage groups(comparing with control groups), the proportion of CO2 in blood decreased. The level of TNF-a, MPO and water content in lung increased dramatically. The numbers of CD11c+DCs rise. The significantly elevated expression of I-Ad, CD86 and CCR5 in DCs were detected. The activity of NF-κB in the DCs' nuclear increased notably. In NAC treating groups (comparing with acute damage groups), the level of TNF-a, MPO and water content in lung decreased notably, and the proportion of CO2 in blood increased. The remarkable decreased expression of I-Ad, CD86, CCR5 and CCR7 in DCs were measured. The activity of NF-κB in the DCs'nuclear declined. The mortality of the mice declined 20%. In MODS groups, the level of ALT, AST, lipase, amylase and creatinine increased dramatically and glucose decreased in serum comparing with control groups. The proportion myeloid and plasmacytoid DCs and CD205+DCs declined, but lymphoid DCs increased with comparison to that of control groups. The remarkable decreased expression of I-Ad and CD86 in DCs were measured, and DCs expressed high-level tolerant molecule PD-L1 markedly. DCs appeared morphologic change of apoptosis and degeneration. DCs secreted more IL-10 and less IL12-p70 after LPS stimulation in vitro contrast to control and acute damage groups. In Flt3L treating groups (comparing with MODS groups), the proportion myeloid and plasmacytoid DCs and CD205+DCs increased, and lymphoid DCs decreased. The remarkable increased expression of I-Ad and CD86 in DCs were detected, and DCs expressed low-level tolerant molecule PD-L1. DCs secreted more IL12-p70 after LPS stimulation in vitro. The mortality of the mice declined 12%. Conclusions:1.Isolated by density gradient centrifugation and MACS microbeads, Lung DCs acquired high purity and activity and stable phenotypes.2. In the stage of acute damage, the activation and maturation of lung DCs induced excessive immune response and inflammation, which caused inflammatory damage in lung.3. NAC had the effect on lightening lung damage in the stage of acute damage by inhibiting the maturation and migration of DCs with less NF-κB shift to nuclear.4. In the stage of MODS, lung DCs presented inactivition and induce immunologic tolerance. DCs excreted overmuch IL-10 to have effect on negative immunologic regulation and participated in immunosuppression.5. After Flt3L administration in vivo, lung DCs presented amplification and induce positive immunologic regulation. Flt3L reversed DCs'immunologic tolerance, which lightening lung injury and protect organ function.