Hepatic Allograft Rejection Affected By The Function Of Kupffer Cells In Rats

Objective (1) To investigate the technique for performing orthotopic liver transplantation .(OLT) stably in Wistar→SD rats and observe the allogrft rejection occurred after OLT. (2) To study the effects of FasL expression , suppression on spleen lymphocyte proliferation and inducing lymphocyte apoptosis of Kupffer cells in different functional states, Which were induced by the pretreatment of gadolinium chloride (GdCl₃), lipopolysaccharide (LPS) and portal venous transfusion(PVT). (3) To investigate the hepatic allograft rejection affected by the functions of Kupffer cells in rats , explore its possible mechanisms and find out the exact roles of Kupffer cells in inducing the liver allograft tolerance .Methods (1) Orthotopic liver transplantation in Wistar→SD rats was performed with the two-cuff method that described by Kamada. OLT in Wistar→Wistar rats were used as control. The rats were killed at 3d, 5d and 7d after operation , liver tissues and blood samples were collected. Recipient survival, histopathological characteristics of hepatic allogfrats were investigated. Plasm levels of alanine aminotransferase (ALT), aspartase aminotransferase(AST), total bilirubin (TBIL), and albumin (Alb) were measured with an automatic biochemical analyser. (2) Pretreatment donor with gadolinium chloride (GdCl₃), lipopolysaccharide (LPS) and portal venous transfusion(PVT) respectively, OLT in Wistar → SD rats were performed. Kupffer cells in liver grafts were isolated 7days after OLT using the technique of Isopyknic gradient centrifugation with Percall. Isolated Kupffer cells were counted. Expression of Fas ligand protein in Kupffercells ,lymphocytes apoptosis in coculture with Kupffer cells were evaluated by flow cytometric assay (FCM) and the proliferation of lymphocytes was detected using MTT. (3) Another study was conducted to investigate the survival of recipients in which the function of Kupffer cells were altered. The liver function and histopathological characteristics of hepatic allogfrats were investigated. IL-10, TGF- |3 1, and TNF-a cytokine levels in plasma were also measured with enzyme linked immunosorbent assay (ELISA), respectively. Expression of FasL on Kupffer cells and apoptosis of lympocytes in liver grafts were detected using immunohistochemical staining and TUNEL.Data were expressed as means±SEM. Statistical analysis was performed using the Student t test. P value of O.05 was considered statistically significant.Results (1) Duration of operation is not significantly different between the two groups, 1 week survival were 87.5%. Liver transplantation from Wistar—SD result in acute rejection of hepatic graft and the survival of recipient rats were 9.63± 2. 13days. Banff RAI(rejection activity index) was 7. 1 ± 0. 85. (2) By pretreatment with GdCl3 ,LPS and PVT, the number and expression of FasL of Kupffer cells isolated from LPS or PVT pretreatments allografts 7d after OLT were much higher than the control (P<0.05 ) . The effects of suppression on spleen lymphocyte proliferation and inducing lymphocyte apoptosis by Kupffer cells were stronger also (P<0.05 ) .These effects could be abrogated by pretreatments with GdCb which can inhibit the function of Kupffer cells.(3) The survival of the GdCl3 group was 6.87+1. 25d, but prolonged in PVT group(25.75 + 5.44d) .Allograft rejection was enhanced by pretreatments with GdCb. IL-10, TGF- P 1, TNF-a cytokine levels in plasma and FasL expression of Kupffer cells were decreased together with the apoptosis of lymphocytes in the allografts(P<0. 05) .This effects were improved by pretreatments with PVT significantly.These results suggested that Kupffer cells-dependent T-cell deletion via the Fas/FasL pathway and immunoregualtion might play an important role in liver transplantation acceptance. Although pretreatments with LPS could bring some benefit as PVT, there was a decrease of survival. Probably it was related to the damage of high TNF-a level.Conclusions (1) Liver transplantation from donor Wistar rats to recipient SD rats can result in acute rejection of hepatic graft. It is a reliable combination for the research of hepatic allograft rejection and tolerance inducing. (2) Apoptosis of infiltrating lymphocytes, immunoregulation of cytokines IL-10/ TGF-P l could play an important role in mechanism of liver transplantation acceptance. Change the function of Kupffer cells by pretreatment could affect the Fas/FasL pathway and immunoregulation to induce liver transplantation tolerance. (3) Although enhanced Kupffer cells function could help to induce tolerance of hepatic allograft , there is still need to explore the effective, safe and applicable stimulation.