Role Of GITRL On Kupffer Cells In Inflammation And Acute Rejection Of Rat Liver Transplantation

Objective(1) To investigate the best isolated method of Kupffer cells (KCs), KCs were isolated by perfusion of collagenase in vivo, discontinuous density gradient centrifugation, and selective adherence. The yield and purity of KCs were analyzed in different group. (2)The cultured KCs were stimulated by lipopolysaccharide (LPS) in order to observe the expression of glucocorticoid-induced tumor necrosis factor-related protein ligand (GITRL). To study the role of GITRL and dexamethasone (Dex) on apoptosis, indoleamine 2, 3-dioxygenase (IDO) expression and cytokine secretion, GITRL gene silencing technique was used in LPS stimulated KCs. (3)To investigate the protective mechanism of Dex in LPS induced hepatic injury, mouse toxemia model was established. The hepatic injury, hepatic expression of GITRL and change of cytokines were observed, as well as, the role of Dex on the change was observed. (4) To investigate molecular mechanism of tacrolimus (FK506) acute rejection during rat liver transplantation, the rat model of acute rejection was established by liver transplantation with Lewis rat as donor and Brown Noway (BN) rat as recipient. The GITRL expression on liver and KCs was observed, as well as, the role of FK506 on the expression was observed.Methods(1) Mouse KCs were isolated by collagenase digestion and the method were randomly divided into 4 groups according to the specific steps: no use of collagenase in situ perfusion + gradient centrifugation of three layer group (A group), no use of collagenase in situ perfusion + gradient centrifugation of double layer group (B group), collagenase perfusion in situ + gradient centrifugation of three layer group (C group) and collagenase perfusion in situ + gradient centrifugation of double layer group (D group). With F4/80 (BM8) or CD163 (ED2) and swallowing ink immunostaining experiments to determine cell purity and function, using trypan blue dye test to determine cell viability, the survival time of KCs was observed and morphological changes were observed, as well as, the effect of different methods was observed. (2) Isolated KCs from mouse were randomly divided into 5 groups: Control group, cultured in medium only; LPS group, adding LPS (10μg / ml); LPS + Control siRNA group, treatment as the LPS group after transfected with Control siRNA; LPS + GITRL siRNA group, treatment as the LPS group after transfected with GTRL siRNA; LPS + Dex group, treatment as LPS group after dexamethasone (100μmol / L) pre treatment. The transfection efficiency KCs was identified by fluorescence microscopy after siRNA (FITC labeled) transfection for 24 h. 24 h after LPS stimulation, the GITRL protein was detected by immunocytochemistry, immunofluorescence and flow cytometry (FCM) and the expression of IDO protein was detected by protein stain method (Western blot), as well as the apoptosis of KCs was detected by FCM with ELISA detection of tumor necrosis factor (TNF)-α, interleukin (IL)-6, interferon (IFN)-γand IL-10 expression.(3) Mouse sepsis model was established by intraperitoneal injection of LPS and experimental animals were divided into 4 groups: Control group, normal saline; Dex group, intraperitoneal injection of Dex (3mg/kg); LPS group, intraperitoneal injection of LPS (10mg / kg); LPS + Dex group, intraperitoneal injection of Dex (3mg/kg) + LPS (3mg/kg). At 24 h after intraperitoneal injection, liver and blood samples were obtained to observe the structural changes in liver pathology, automatic biochemical analysis of serum alanine aminotransferase (ALT), aspartate aminotransferase bilirubin (AST), ELISA serum TNF-α, IL-6, IFN-γand IL-10 content. The expression of GITRL of the liver was detected by immunohistochemistry. Mortality of sepsis mice was observed. (4) With a modified "two-cuff method" of orthotopic liver transplantation in the rat animal model, experimental animals were divided into 3 groups: tolerance group, BN rats as donors and Lewis rats as recipients; rejection group, Lewis rats as donors and BN recipients; FK group, Lewis rats as donors and BN recipients, 1-7 d after receptor, FK506 (1 mg / kg) intramuscular injection. In addition to the survival rate of rats, at 7 d after surgery, liver and blood were observed on liver pathology, automatic determination of biochemical changes in serum ALT and AST, ELISA and cell culture supernatants of serum IFN-γand IL -10. The expression of GITRL in KCs and the liver was detected by immunostaining.Results(1) Fluorescence in the 328nm excitation, no luminescence was observed in KCs. KCs isolated freshly were just quasi-circular and l h after inoculation, cells were harvested at this time of high purity, but the cell yield is relatively low. 4 h after inoculation KCs were harvested with relatively high yield. 2 d and 3 d after cultivation, cells were adhesion and fully extended, showing that star or irregular in shape. The cells were cultured for 4 w still alive. KCs were seen to swallow a lot of ink particles by in vivo and in vitro experiments. Trypan blue staining showed the vitality of cells in each group were about 90%. Immunohistochemistry and immunofluorescence showed that cells isolated were KCs. Collagenase perfusion and gradient can increase the yield of KCs and gradient centrifugation of double layer can increase the purity of isolated KCs. (2) Twenty four h after transfection with GITRL siRNA, the transfection efficiency was 85% under fluorescent microscope identification. LPS stimulated cells significantly increased the expression of GITRL, dexamethasone pretreatment or GITRL gene silencing can effectively inhibit the expression of GITRL. Compared with the control group, IDO protein expression and cell apoptosis in LPS group and LPS + Control siRNA group were significantly higher, but the dexamethasone pretreatment or GITRL gene silencing can reduce LPS-induced IDO protein expression and apoptosis. The expression of IFN-γ, TNF-α, IL-6 and IL-10 was increased in cell culture medium by LPS stimulation, whereas GITRL gene silencing or dexamethasone pretreatment inhibited LPS-induced increase of TNF-αand IL-6. (3) Intraperitoneal injection of dexamethasone significantly reduced the mortality rate in mice, with improving liver function and reduced LPS-induced liver injury. Immunohistochemical staining showed that the expression of GITRL was wide in the liver after intraperitoneal injection of LPS for 24 h, whereas dexamethasone reduced the expression of GITRL in the liver. After injection of LPS for 24 h, serum TNF-α, IFN-γ, IL-6, and IL-10 levels were significantly higher, however dexamethasone injection reduced LPS-induced increase of TNF-α, IL-6 and IFN-γ. (4) In Rejection group, liver injury, GITRL expression in KCs and liver grafts, and serum TNF-αwere increased, and serum IL-10 expression in reverse. In FK group, the expression of GITRL in liver grafts and KCs, TNF-α, IFN-γ, and liver damage were reduced with prolonged the survival rate of recipients.Conclusion(1) In vivo perfusion of enzymes is important to improve the yield of KCs and separation of double layer can improve the isolated effect. (2) GITRL mediates LPS-induced IDO expression, KCs apoptosis and the secretion of proinflammatory cytokines, but dexamethasone can inhibit the IDO expression, KCs apoptosis, and proinflammatory cytokine secretion by down regulating GITRL. (3) GITRL plays an important role in LPS-induced liver injury and the protective mechanism of dexamethasone may be reduced the expression of GITRL. (4) In rejection group, the increased expression of GITRL correlated to immune rejection in liver transplantation, FK506 maintaining graft survival by inhibiting GITRL on KCs.