Dysfunction Of Macrophage-natural Killer Cells Crosstalk In The Development Of Biliary Atresia In Newborn Mice

Part 1 The changes of the cytokines and ligangds produced by liver macrophages that can modulate the function of liver NK cellsObjective:To investigate the change of the cytokines and ligands produced by kupffer cells following the stimulation of RRV.Methods:The supernatant from adult or neonatal Kupffer cells was collected and the concentration of cytokines such as IL-12,IL-23,IL-10 and TNF-α were tested,and the positive numbers of kupffer cells expressing Rae-1 and CD40 were tested by FACS after the RRV stimulation for 12h,24h,36h,48h and 60h.Results:The concentration of IL-12、IL-23、TNF-α and the positive cells expressing CD40 in neonatal liver macrophages was lower than the counterpart in adult kupffer cells(p<0.05).The IL-10 and Rae-1 produced by neonatal kupffer cells was higher than that in adult kupffer cells(p<0.05).The peak concentration of IL-12,IL-23,IL-10 and TNF-α were 24h,24h,60h,12h in neonatal kupffer cells stimulated by RRV,respectively.The peak concentration of IL-12,IL-23,IL-10 and TNF-a were 36h,36h,36h and 48h in adult kupffer cells,respectively.The point that the neonatal kupffer cells expressed Rae-1 and CD40 with the highest positive number was stimulated by RRV for 24h,while the highest expression in adult kupffer cells were 48h and 36h,respectively.Conclusion:The peak concentration of IL-12,IL-23 and TNF-α produced by neonatal kupffer cells were lower than that in adult kupffer cells under the stimulation of RRV,the expression of Rae-1 and CD40 shared the similar phenomenon.The peak concentration of IL-10 produced by neonatal liver macrophages was higher than that in adult kupffer cells following the stimulation of RRV.Part 2 To investigate the key cytokines which liver macrophages can modulate the function of liver NK cells in neonatal miceObjective:To investigate the key cytokines produced by kupffer cells that can modulates the function of NK cells in liver after the stimulation of RRV.Methods:The neonatal or the adult Kupffer cells were stimulated by RRV,and the IL-10 and 11-12 was blocked when they at the point with the highest concentration,then the liver NK cells were added and co-cultured with the Kupffer cells for 24h.CD69,TNF-α and IFN-γ expressed by NK were tested by FACS after stimulated by RRV.Results:When IL-12 had been blocked,the CD69 expression decreased both in adult and newborn liver NK cells.The concentration of TNF-α decreased than that in neonatal kupffer cells.There was no significant difference when comparing the IFN-y expressed in NK when the control group vs.the blocked group in adult kupffer cells.When IL-10 had been blocked,the expression of CD69 and the IFN-γ increased highly in neonatal mice,and the TNF-α increased greatly in adult kupffer cells.Conclusion:Both IL-10 and IL-12 played a more effective role in modulating the liver NK cells in neonatal mice when compared with the adult mice.IL-12 is not the key point in determining the IFN-γ production in NK cells.IL-10 produced by Kupffer cells cannot provide efficient modulation on liver NK cells may be an important factor in biliary atresia pathogenesis.Part 3 To investigate the key ligands which liver macrophages can modulate the function of liver NK cells in neonatal miceObjective:To investigate the key ligands expressed on kupffer cells that can modulate the function of NK cells in liver after the stimulation of RRV.Methods:The neonatal or the adult Kupffer cells were stimulated by RRV,and the Rae-1 and CD40 was blocked when they at the point with the highest expression,then the liver NK cells were added and co-cultured with the Kupffer cells for 24h,CD69,TNF-α and IFN-y expressed by NK cells were tested by FACS.Results:When CD40 had been blocked,the CD154 and TNF-a expression decreased both in adult and newborn liver NK cells.There was no significant difference when comparing the IFN-y expressed in NK when the control group vs.the blocked group in neonatal or adult kupffer cells.When Rae-1 had been blocked,the CD69 and TNF-a expression presented no significant differences both in adult and newborn liver NK cells,and the NKG2D expression presented significant difference both in adult and newborn liver NK cells.Conclusion:The contact between the Kupffer cells and NK cells contributes to the expression of IFN-γ and TNF-α,Rae-1/NKG2D expressed highly in neonatal mice my be an may be another important factor in biliary atresia pathogenesis.