Kupffer细胞中GITRL促进肝脏炎症与移植排斥反应机制的实验研究
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摘要
目的
(1)本试验采用在体酶灌注、不连续密度梯度离心、选择性贴壁三步法分离Kupffer细胞(KCs),比较不同分离方法的KCs得率及纯度,以寻求分离小鼠肝KCs的最佳方法。(2)采用体外培养的小鼠KCs,观察在脂多糖(1ipopolysaccharide , LPS)激活的KCs上GITRL(glucocorticoid induced tumor necrosis factor related protein ligand)的表达情况,采用GITRL基因沉默技术探讨GITRL以及地塞米松(Dex)在KCs激活、凋亡和IDO表达中的作用。(3)建立小鼠毒血症动物模型,观察肝脏损伤、肝脏内GITRL的表达和细胞因子的变化,观察Dex在这些变化中的作用,探讨Dex对毒血症小鼠的保护机制。(4)建立Lewis到Brown Noway(BN)大鼠急性排斥反应肝移植模型,观察肝脏及KCs内GITRL的表达,观察他克莫司(tacrolimus, FK506)对GITRL的影响,探讨FK506抑制急性排斥反应的分子机制。
方法
(1)胶原酶消化法分离小鼠KCs,根据具体步骤方法不同随机分为4组:不采用胶原酶原位灌注+三层梯度离心组(A)、不采用胶原酶原位灌注+双层梯度离心组(B)、胶原酶原位灌注+三层梯度离心组( C)和胶原酶原位灌注+双层梯度离心组(D)。采用F4/80(BM8)或CD163(ED2)免疫染色及吞墨实验判断细胞纯度和功能,采用台盼蓝拒染实验判断细胞的活力,观察KCs的生存时间及形态变化,观察不同方法的分离效果。(2)分离小鼠KCs并随机分为5组:Control组,只加培养基; LPS组,加入LPS(10μg/ml); LPS+Control siRNA组,转染Control siRNA后同LPS组; LPS+GITRL siRNA组,转染GITRL siRNA后同LPS组; LPS+Dex组,地塞米松(100μmol/L)处理后同LPS组。siRNA(异硫氰酸荧光素标记)转染KCs后24 h通过荧光显微镜鉴定转染效果。LPS刺激24 h后,免疫细胞化学染色、免疫荧光及流式细胞术(FCM)检测GITRL蛋白表达,蛋白质斑迹法(Western blot)检测IDO蛋白表达,FCM检测KCs的凋亡变化,ELISA检测KCs培养上清液肿瘤坏死因子(TNF)-α、白介素(IL)-6、干扰素(IFN)–γ和IL-10表达情况。(3)采用LPS腹腔注射建立小鼠毒血症模型,实验动物分为4组:Control组,腹腔注射生理盐水; Dex组,腹腔注射Dex (3mg/kg);LPS组,腹腔注射LPS (10mg/kg); LPS+Dex组,腹腔注射Dex (3mg/kg)+ LPS(10mg/kg)。于术后腹腔注射后24 h获取肝脏及血液标本,观察肝脏组织病理结构变化,全自动生化分析法测定血清谷丙转氨酶(ALT)、天冬氨酸转氨酶胆红素(AST)、ELISA检测血清TNF-α、IL-6、IFN–γ和IL-10含量,免疫组化法检测肝脏GITRL的表达。观察毒血症小鼠的死亡率。(4)用改良的“两袖套法”建立大鼠原位肝移植动物模型,实验动物分为3组:耐受组, BN大鼠为供体Lewis大鼠为受体;排斥组, Lewis大鼠为供体BN大鼠为受体;FK组,Lewis大鼠为供体BN大鼠为受体,受体术后1-7天以FK506(1 mg/kg)肌肉注射。除观察大鼠存活率外,于术后7 d获取肝脏及血液标本,观察肝脏组织病理变化,全自动生化分析法测定血清ALT和AST变化,ELISA检测血清及细胞培养上清液IFN-γ和IL-10含量,免疫组化检测肝脏及KCs内GITRL的表达。
结果
(1)在328nm荧光激发下,未见KCs发出荧光。刚分离的KCs细胞近似圆形,接种l h后,此时收获细胞纯度较高,但细胞得率相对较低。KCs培养4 h后得率相对较高,培养2 d及3 d后,细胞贴壁,且充分伸展,可见星形或不规则形,细胞培养4 w仍能存活。体内及体外吞墨实验均见KCs吞噬大量碳素颗粒。台盼蓝染色显示各组细胞的活力均在90%左右。免疫组化染色及免疫荧光显示分离的细胞为KCs。胶原酶灌注和双层梯度离心可以增加KCs的得率,双层梯度离心法可以增加分离KCs的纯度。(2) GITRL siRNA能够高效转染KCs,转染后24 h荧光显微镜下鉴定转染效果达85%。LPS刺激的细胞上GITRL表达明显升高,地塞米松预处理或者GITRL基因沉默可以有效地抑制GITRL的表达。与对照组比较,LPS组及LPS+Control siRNA组IDO蛋白表达及细胞凋亡明显增高,而地塞米松预处理及GITRL基因沉默可以降低LPS诱导的IDO蛋白表达及细胞凋亡。LPS刺激后,细胞培养液的IFN-γ、TNF-α、IL-6和IL-10表达水平明显升高,GITRL基因沉默或者地塞米松预处理后,抑制了LPS诱导的TNF-α和IL-6升高。(3)地塞米松腹腔注射明显降低了小鼠的死亡率、改善肝脏的功能和减轻了LPS诱导的肝损伤。免疫组化染色检测GITRL的表达结果显示腹腔内注射LPS 24 h后肝脏广泛表达GITRL,地塞米松降低了GITRL在肝脏的表达。LPS注射后24 h,血清TNF-α、IFN-γ、IL-6和IL-10水平明显升高,地塞米松注射减低了LPS诱导的TNF-α、IL-6和IFN-γ升高。(4)排斥组肝脏损伤重,KCs和肝脏移植物内GITRL的表达升高,和血清TNF-α及表达一致,与血清IL-10表达相反。FK组KCs及肝脏移植物内GITRL的表达降低,和TNF-α及IFN-γ的降低一致,肝脏损伤减轻,大鼠的生存率延长。
结论
(1)在体酶灌注对提高KCs得率较为重要,双层分离可改善分离效果。(2)GITRL介导了LPS诱导的IDO表达、KCs的凋亡和促炎细胞因子的分泌,地塞米松可以通过下调GITRL抑制IDO表达、KCs的凋亡和促炎细胞因子的分泌。(3)GITRL在内毒素血症相关的肝损伤中具有重要作用,地塞米松在LPS诱导的肝损伤中的保护机制可能是通过下调GITRL的表达来实现的。(4)在免疫排斥组的移植肝脏中GITRL的表达升高和免疫排斥具有相关性, FK506通过抑制KCs上GITRL以维持移植肝脏的成活。
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.
(1)本试验采用在体酶灌注、不连续密度梯度离心、选择性贴壁三步法分离Kupffer细胞(KCs),比较不同分离方法的KCs得率及纯度,以寻求分离小鼠肝KCs的最佳方法。(2)采用体外培养的小鼠KCs,观察在脂多糖(1ipopolysaccharide , LPS)激活的KCs上GITRL(glucocorticoid induced tumor necrosis factor related protein ligand)的表达情况,采用GITRL基因沉默技术探讨GITRL以及地塞米松(Dex)在KCs激活、凋亡和IDO表达中的作用。(3)建立小鼠毒血症动物模型,观察肝脏损伤、肝脏内GITRL的表达和细胞因子的变化,观察Dex在这些变化中的作用,探讨Dex对毒血症小鼠的保护机制。(4)建立Lewis到Brown Noway(BN)大鼠急性排斥反应肝移植模型,观察肝脏及KCs内GITRL的表达,观察他克莫司(tacrolimus, FK506)对GITRL的影响,探讨FK506抑制急性排斥反应的分子机制。
方法
(1)胶原酶消化法分离小鼠KCs,根据具体步骤方法不同随机分为4组:不采用胶原酶原位灌注+三层梯度离心组(A)、不采用胶原酶原位灌注+双层梯度离心组(B)、胶原酶原位灌注+三层梯度离心组( C)和胶原酶原位灌注+双层梯度离心组(D)。采用F4/80(BM8)或CD163(ED2)免疫染色及吞墨实验判断细胞纯度和功能,采用台盼蓝拒染实验判断细胞的活力,观察KCs的生存时间及形态变化,观察不同方法的分离效果。(2)分离小鼠KCs并随机分为5组:Control组,只加培养基; LPS组,加入LPS(10μg/ml); LPS+Control siRNA组,转染Control siRNA后同LPS组; LPS+GITRL siRNA组,转染GITRL siRNA后同LPS组; LPS+Dex组,地塞米松(100μmol/L)处理后同LPS组。siRNA(异硫氰酸荧光素标记)转染KCs后24 h通过荧光显微镜鉴定转染效果。LPS刺激24 h后,免疫细胞化学染色、免疫荧光及流式细胞术(FCM)检测GITRL蛋白表达,蛋白质斑迹法(Western blot)检测IDO蛋白表达,FCM检测KCs的凋亡变化,ELISA检测KCs培养上清液肿瘤坏死因子(TNF)-α、白介素(IL)-6、干扰素(IFN)–γ和IL-10表达情况。(3)采用LPS腹腔注射建立小鼠毒血症模型,实验动物分为4组:Control组,腹腔注射生理盐水; Dex组,腹腔注射Dex (3mg/kg);LPS组,腹腔注射LPS (10mg/kg); LPS+Dex组,腹腔注射Dex (3mg/kg)+ LPS(10mg/kg)。于术后腹腔注射后24 h获取肝脏及血液标本,观察肝脏组织病理结构变化,全自动生化分析法测定血清谷丙转氨酶(ALT)、天冬氨酸转氨酶胆红素(AST)、ELISA检测血清TNF-α、IL-6、IFN–γ和IL-10含量,免疫组化法检测肝脏GITRL的表达。观察毒血症小鼠的死亡率。(4)用改良的“两袖套法”建立大鼠原位肝移植动物模型,实验动物分为3组:耐受组, BN大鼠为供体Lewis大鼠为受体;排斥组, Lewis大鼠为供体BN大鼠为受体;FK组,Lewis大鼠为供体BN大鼠为受体,受体术后1-7天以FK506(1 mg/kg)肌肉注射。除观察大鼠存活率外,于术后7 d获取肝脏及血液标本,观察肝脏组织病理变化,全自动生化分析法测定血清ALT和AST变化,ELISA检测血清及细胞培养上清液IFN-γ和IL-10含量,免疫组化检测肝脏及KCs内GITRL的表达。
结果
(1)在328nm荧光激发下,未见KCs发出荧光。刚分离的KCs细胞近似圆形,接种l h后,此时收获细胞纯度较高,但细胞得率相对较低。KCs培养4 h后得率相对较高,培养2 d及3 d后,细胞贴壁,且充分伸展,可见星形或不规则形,细胞培养4 w仍能存活。体内及体外吞墨实验均见KCs吞噬大量碳素颗粒。台盼蓝染色显示各组细胞的活力均在90%左右。免疫组化染色及免疫荧光显示分离的细胞为KCs。胶原酶灌注和双层梯度离心可以增加KCs的得率,双层梯度离心法可以增加分离KCs的纯度。(2) GITRL siRNA能够高效转染KCs,转染后24 h荧光显微镜下鉴定转染效果达85%。LPS刺激的细胞上GITRL表达明显升高,地塞米松预处理或者GITRL基因沉默可以有效地抑制GITRL的表达。与对照组比较,LPS组及LPS+Control siRNA组IDO蛋白表达及细胞凋亡明显增高,而地塞米松预处理及GITRL基因沉默可以降低LPS诱导的IDO蛋白表达及细胞凋亡。LPS刺激后,细胞培养液的IFN-γ、TNF-α、IL-6和IL-10表达水平明显升高,GITRL基因沉默或者地塞米松预处理后,抑制了LPS诱导的TNF-α和IL-6升高。(3)地塞米松腹腔注射明显降低了小鼠的死亡率、改善肝脏的功能和减轻了LPS诱导的肝损伤。免疫组化染色检测GITRL的表达结果显示腹腔内注射LPS 24 h后肝脏广泛表达GITRL,地塞米松降低了GITRL在肝脏的表达。LPS注射后24 h,血清TNF-α、IFN-γ、IL-6和IL-10水平明显升高,地塞米松注射减低了LPS诱导的TNF-α、IL-6和IFN-γ升高。(4)排斥组肝脏损伤重,KCs和肝脏移植物内GITRL的表达升高,和血清TNF-α及表达一致,与血清IL-10表达相反。FK组KCs及肝脏移植物内GITRL的表达降低,和TNF-α及IFN-γ的降低一致,肝脏损伤减轻,大鼠的生存率延长。
结论
(1)在体酶灌注对提高KCs得率较为重要,双层分离可改善分离效果。(2)GITRL介导了LPS诱导的IDO表达、KCs的凋亡和促炎细胞因子的分泌,地塞米松可以通过下调GITRL抑制IDO表达、KCs的凋亡和促炎细胞因子的分泌。(3)GITRL在内毒素血症相关的肝损伤中具有重要作用,地塞米松在LPS诱导的肝损伤中的保护机制可能是通过下调GITRL的表达来实现的。(4)在免疫排斥组的移植肝脏中GITRL的表达升高和免疫排斥具有相关性, FK506通过抑制KCs上GITRL以维持移植肝脏的成活。
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.
引文
[1] Thomson AW, Knolle PA. Antigen-presenting cell function in the tolerogenic liver environment [J]. Nat Rev Immunol. 2010, 10(11):753-766.
[2] Tiegs G, Lohse AW. Immune tolerance: what is unique about the liver [J]. J Autoimmun. 2010, 34(1): 1-6.
[3] Traeger T, Mikulcak M, Eipel C, et al. Kupffer cell depletion reduces hepatic inflammation and apoptosis but decreases survival in abdominal sepsis [J]. Eur J Gastroenterol Hepatol. 2010, 22(9):1039-1049.
[4] Wittebole X, Castanares-Zapatero D, Laterre PF. Toll-like receptor 4 modulation as a strategy to treat sepsis [J]. Mediators Inflamm. 2010; 2010: 568396.
[5] Stebbings R, Findlay L, Edwards C, et al. "Cytokine storm" in the phase I trial of monoclonal antibody TGN1412: better understanding the causes to improve preclinical testing of immunotherapeutics [J]. J Immunol. 2007, 179(5): 3325-3331.
[6] Rudd CE, Taylor A, Schneider H. CD28 and CTLA-4 coreceptor expression and signal transduction [J]. Immunol Rev. 2009, 229(1): 12-26.
[7] Koehn BH, Ford ML, Ferrer IR, et al. PD-1-dependent mechanisms maintain peripheral tolerance of donor-reactive CD8+ T cells to transplanted tissue. J Immunol [J]. 2008, 181(8): 5313-5322.
[8] rdona ID, Goleva E, Ou LS, et al. Staphylococcal enterotoxin B inhibits regulatory T cells by inducing glucocorticoid-induced TNF receptor-related protein ligand on monocytes [J]. J Allergy Clin Immunol. 2006, 117(3): 688-695.
[9] Hwang H, Lee S, Lee WH, et al. Stimulation of glucocorticoid-induced tumor necrosis factor receptor family-related protein ligand (GITRL) induces inflammatory activation of microglia in culture [J]. J Neurosci Res. 2010, 88(10): 2188-2196.
[10] Scumpia PO, Delano MJ, Kelly-Scumpia KM, et al. Treatment with GITR agonistic antibody corrects adaptive immune dysfunction in sepsis [J]. Blood. 2007, 110(10): 3673-81.
[11] Fujiki M, Esquivel CO, Martinez OM, et al. Induced tolerance to rat liver allografts involves the apoptosis of intragraft T cells and the generation of CD4 (+) CD25 (+) FoxP3 (+) T regulatory cells [J]. Liver Transpl. 2010, 16: 147-154.
[12] Grohmann U, Volpi C, Fallarino F, et al. Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy [J]. Nat Med. 2007, 13: 579-586.
[13] Fábrega E, Unzueta MG, Cobo M, et al. Value of soluble CD30 in liver transplantation [J]. Transplant Proc. 2007, 39: 2295-2296
[14] Miyagawa-Hayashino A, Tsuruyama T, Egawa H, et al. FasL expression in hepatic antigen-presenting cells and phagocytosis of apoptotic T cells by FasL+ Kupffer cells are indicators of rejection activity in human liver allografts [J]. Am J Pathol. 2007, 171: 1499-1508.
[15] Qin L, Guan HG, Zhou XJ, et al. Blockade of 4-1BB/4-1BB ligand interactions prevents acute rejection in rat liver transplantation [J]. Chin Med J (Engl). 2010, 123:212-215.
[16]夏仁品,卢实春,赖威,等.针对OX40小干扰RNA供体转染抗大鼠肝移植排斥反应[J].中华实验外科杂志. 2008, 25: 860- 862.
[17] Cardona ID, Goleva E, Ou LS, et al. Staphylococcal enterotoxin B inhibits regulatory T cells by inducing glucocorticoid-induced TNF receptor-related protein ligand on monocytes [J]. J Allergy Clin Immunol. 2006, 117: 688-695.
[18] Shimizu J, Yamazaki S, Takahashi T, et al. Stimulation of CD25 (+) CD4 (+) regulatory T cells through GITR breaks immunological self-tolerance [J]. Nat Immunol. 2002, 3: 135-142.
[19] Adams DH, Ju C, Ramaiah SK, et al. Mechanisms of immune-mediated liver injury [J]. Toxicol Sci. 2010, 115(2): 307-321.
[20] Baffy G. Kupffer cells in non-alcoholic fatty liver disease: the emerging view [J]. J Hepatol. 2009, 51(1):212-23.
[21] Ramadori G, Moriconi F, Malik I, et al. Physiology and pathophysiology of liver inflammation, damage and repair [J]. J Physiol Pharmacol. 2008, 59 Suppl 1:107-117.
[22] Valatas V, Xidakis C, Roumpaki H, et al. Isolation of rat Kupffer cells: a combined methodology for highly purified primary cultures [J]. Cell Biol Int. 2003, 27(1): 67-73.
[23] Folch E, Prats N, Hotter G, et al. P-selectin expression and Kupffer cell activation in rat acute pancreatitis [J]. Dig Dis Sci. 2000, 45(8): 1535-1544.
[24] Smedsr?d B, Pertoft H. Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence [J]. J Leukoc Biol. 1985, 38(2): 213-230.
[25] Tsujimoto T, Kawaratani H, Kitazawa T, et al. Decreased phagocytic activity of Kupffer cells in a rat nonalcoholic steatohepatitis model [J]. World J Gastroenterol. 2008, 14(39): 6036-6043.
[26] Olynyk JK, Clarke SL. Isolation and primary culture of rat Kupffer cells [J]. J Gastroenterol Hepatol. 1998, 13(8): 842-845.
[27] Knook DL,Blansjaar N,Sleyster EC. Isolation and characterization of kupffer and endothelial cells from the rat liver [J]. Exp Cell Res. 1997, 109(2): 317-329.
[28] Kindberg GM, Tolleshaug H, Roos N, et al. Hepatic clearance of Sonazoid perfluorobutane microbubbles by Kupffer cells does not reduce the ability of liver to phagocytose or degrade albumin microspheres [J]. Cell Tissue Res. 2003, 312(1): 49-54.
[29] Dan C,Wake K.Modes of endoeytosis of latex particles in sinusoidal endothelialand kupffer cells of normal and perfused rat liver [J]. Exp Cell Res. 1985, 158: 75-85.
[30] Klotz C, Frevert U. Plasmodium yoelii sporozoites modulate cytokine profile and induce apoptosis in murine Kupffer cells [J]. Int J Parasitol. 2008, 38(14): 1639-1650.
[31] Ten-Hagan TL, van-Vianen W, Bakker WA.Isolation and characterization of routine kupffer cells and splenic macrophages [J]. J Immunol Method. 1996, 19: 81-91.
[32] Van Furth R. Production and migration of monocytes and kinetics of macrophages [M] . In Van Furth, R. (ed.), Mononuclear Phagocytes . Kluwer Academic Publishers,Norwell,MA,1992,3-12
[33] Xu FL, You HB, Li XH, et al. Glycine attenuates endotoxin-induced liver injury by down regulating TLR4 signaling in Kupffer cells [J]. Am J Surg. 2008, 196: 139-48.
[34] Higuchi N, Kato M, Kotoh K, et al. Methylprednisolone injection via the portal vein suppresses inflammation in acute liver failure induced in rats by lipopolysaccharide and d-galactosamine [J]. Liver Int. 2007, 27: 1342-8.
[35] Tsao CM, Ho ST, Liaw WJ, et al. Combined effects of propofol and dexamethasone on rats with endotoxemia [J]. Crit Care Med. 2008, 36: 887-94.
[36] Shimizu J, Yamazaki S, Takahashi T, et al. Stimulation of CD25 (+) CD4 (+) regulatory T cells through GITR breaks immunological self-tolerance [J]. Nat Immunol. 2002, 3 (2): 135-142.
[37] Liu B, Li Z, Mahesh SP, et al. Glucocorticoid-induced tumor necrosis factor receptor negatively regulates activation of human primary natural killer (NK) cells by blocking proliferative signals and increasing NK cell apoptosis [J]. J Biol Chem. 2008, 283 (13): 8202-8210.
[38] Huttunen R, Syrj?nen J, Aittoniemi J, et al. High activity of indoleamine 2,3 dioxygenase enzyme predicts disease severity and case fatality in bacteremic patients[J]. Shock. 2010, 33(2): 149-154.
[39]魏思东,杨慷,龚建平. GITR/GITRL信号系统在单核巨噬细胞免疫调节中的作用[J].细胞与分子免疫学杂志. 2009, 25 (12): 1207-1209.
[40] Albertson TE. Controversies in the treatment of sepsis [J]. Semin Respir Crit Care Med. 2010, 31(1):66-78.
[41] Miyashita M. Controversy of corticosteroids in septic shock [J]. J Nippon Med Sch. 2010, 77(2): 67-70.
[42] Dellinger RP, Levy MM, Carlet JM, et al. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008 [J]. Crit Care Med. 2008, 36(1): 296-327.
[43] d'Emmanuele di Villa Bianca R, Lippolis L, Autore G, et al. Dexamethasone improves vascular hyporeactivity induced by LPS in vivo by modulating ATP-sensitive potassium channels activity [J]. Br J Pharmacol. 2003, 140(1): 91-96.
[44] JapiassúAM, Salluh JI, Bozza PT, et al. Revisiting steroid treatment for septic shock: molecular actions and clinical effects--a review [J]. Mem Inst Oswaldo Cruz. 2009, 104(4):531-548.
[45] Wang Y, Liu H, McKenzie G, et al. Kynurenine is an endothelium-derived relaxing factor produced during inflammation [J]. Nat Med, 2010, 16(3): 279-285.
[46] Tattevin P, Monnier D, Tribut O, et al. Enhanced indoleamine 2,3-dioxygenase activity in patients with severe sepsis and septic shock[J]. J Infect Dis. 2010, 201(6): 956-966.
[47] Jung ID, Lee MG, Chang JH, et al. Blockade of indoleamine 2, 3-dioxygenase protects mice against lipopolysaccharide induced endotoxin shock [J]. J Immunol. 2009, 182(5): 3146-3154.
[48] Grohmann U, Volpi C, Fallarino F, et al. Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy [J]. Nat Med. 2007, 13(5): 579-586.
[49] Cuesta N, Nhu QM, Zudaire E, et al. IFN regulatory factor-2 regulates macrophage apoptosis through a STAT1/3- and caspase-1-dependent mechanism [J]. J Immunol. 2007, 178 (6): 3602-3611.
[50] Ottonello L, Bertolotto M, Montecucco F, et al. Dexamethasone -induced apoptosis of human monocytes exposed to immune complexes. Intervention of CD95- and XIAP-dependent pathways [J]. Int J Immunopathol Pharmacol. 2005, 18 (3): 403-415.
[51] Fong CC, Zhang Y, Zhang Q, et al. Dexamethasone protects RAW264.7 macrophages from growth arrest and apoptosis induced by H2O2 through alteration of gene expression patterns and inhibition of nuclear factor-kappa B (NF-kappa B) activity [J]. Toxicology. 2007, 236 (1-2): 16-28.
[52] Bae EM. Reverse signaling initiated from GITRL induces NF-B activation through ERK in the inflammatory activation of macrophages [J]. Mol Immunol. 2008, 45(2): 523-533.
[53] Finkelstein RA, Li Y, Liu B, et al. Treatment with histone deacetylase inhibitor attenuates MAP kinase mediated liver injury in a lethal model of septic shock [J]. J Surg Res. 2010, 163: 146-154.
[54] Su GL. Lipopolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation [J]. Am J Physiol Gastrointest Liver Physiol. 2002, 283: G256-265.
[55] Higuchi N, Kato M, Kotoh K, et al. Methylprednisolone injection via the portal vein suppresses inflammation in acute liver failure induced in rats by lipopolysaccharide and d-galactosamine [J]. Liver Int. 2007, 27: 1342-1348.
[56] [4] Tsao CM, Ho ST, Liaw WJ, et al. Combined effects of propofol and dexamethasoneamethasone on rats with endotoxemia [J]. Crit Care Med. 2008, 36: 887-894.
[57] Wang X, Nelin LD, Kuhlman JR, et al. The role of MAP kinase phosphatase-1 in the protective mechanism of dexamethasone against endotoxemia [J]. Life Sci. 2008, 83: 671-680.
[58] Nocentini G, Cuzzocrea S, Bianchini R, et al. Modulation of acute and chronic inflammation of the lung by GITR and its ligand [J]. Ann N Y Acad Sci. 2007, 1107: 380-391.
[59] Kim WJ, Bae EM, Kang YJ, et al. Glucocorticoid-induced tumor necrosis factor receptor family related protein (GITR) mediates inflammatory activation of macrophages that can destabilize atherosclerotic plaques [J]. Immunology. 2006, 119(3): 421-429.
[60] Bae E, Kim WJ, Kang YM, et al. Glucocorticoid-induced tumor necrosis factor receptor-related protein-mediated macrophage stimulation may induce cellular adhesion and cytokine expression in rheumatoid arthritis [J]. Clin Exp Immunol. 2007, 148(3): 410-418.
[61] Cuzzocrea S, Nocentini G, Di Paola R, et al. Proinflammatory role of glucocorticoid-induced TNF receptor-related gene in acute lung inflammation [J]. J Immunol. 2006, 177(1): 631-641.
[62] Buras JA, Holzmann B, Sitkovsky M. Animal models of sepsis: setting the stage. Nat Rev Drug Discov. 2005, 4: 854-865.
[63] Xu FL, You HB, Li XH, et al. Glycine attenuates endotoxin-induced liver injury bydown regulating TLR4 signaling in Kupffer cells [J].Am J Surg. 2008, 196: 139-148.
[64] Chatterjee S, Premachandran S, Shukla J, et al. Synergistic therapeutic potential of dexamethasone and L-arginine in lipopolysaccharide-induced septic shock [J]. J Surg Res. 2007, 140(1): 99-108.
[65] Melgert BN, Weert B, Schellekens H, et al. The pharmacokinetic and biological activity profile of dexamethasone targeted to sinusoidal endothelial and Kupffer cells [J]. J Drug Target. 2003, 11(1):1-10.
[66] Melgert BN, Olinga P, Van Der Laan JM, et al. Targeting dexamethasone to Kupffer cells: effects on liver inflammation and fibrosis in rats [J]. Hepatology. 2001, 34(4 Pt 1):719-728.
[67] Muratore CS, Harty MW, Papa EF, et al. Dexamethasone alters the hepatic inflammatory cellular profile without changes in matrix degradation during liver repair following biliary decompression [J]. J Surg Res. 2009, 156(2):231-239.
[68] Horiguchi M, Kim J, Matsunaga N, Kaji Het al. Glucocorticoid-dependent expression of O(6)-methylguanine-DNA methyltransferase gene modulates dacarbazine-induced hepatotoxicity in mice [J]. J Pharmacol Exp Ther. 2010, 333(3):782-787.
[69] Müschen M, Warskulat U, Douillard P, et al. Regulation of CD95 (APO-1/Fas) receptor and ligand expression by lipopolysaccharide and dexamethasone in parenchymal and nonparenchymal rat liver cells [J]. Hepatology. 1998, 27(1): 200-208.
[70] Fischer L, Trune?ka P, Gridelli B, et al. Pharmacokinetics for once-daily versus twice-daily tacrolimus formulations in de novo liver transplantation: a randomized, open-label trial [J]. Liver Transpl. 2011, 17(2): 167-177.
[71]赖星,连峥嵘,李金政,等.组蛋白去乙酰化酶11在诱导大鼠肝移植免疫耐受中的作用[J].第三军医大学学报. 2011, 33(4): 342-344.
[72] Balibrea JM, García-Martín MC, Cuesta-Sancho S, et al. Tacrolimus modulates liver and pancreas nitric oxide synthetase and heme-oxygenase isoforms and cytokine production after endotoxemia [J]. Nitric Oxide. 2011, 24(2): 113-122.
[73] Shimizu J, Yamazaki S, Takahashi T, et al. Stimulation of CD25 (+) CD4 (+) regulatory T cells through GITR breaks immunological self-tolerance [J]. Nat Immunol, 2002, 3(2): 135-142.
[74] Chen Y, Yan T, Shi LJ, et al. Knockdown of interleukin-2 by shRNA-mediated RNA interference prolongs liver allograft survival [J]. J Surg Res. 2010, 159(1): 582-587.
[75] Hossain MA, Hamamoto I, Kobayashi S, et al. Immunosuppression in auxiliary partial liver transplantation with FK506 in rats. Transplant Proc. 1997, 29(8): 3617-3618.
[76] Sonawane SB, Kim JI, Lee MK, et al. GITR Blockade Facilitates Treg Mediated Allograft Survival [J]. Transplantation. 2009, 88(10): 1169-1177.
[77] Rintam?ki H, Salo HM, Vaarala O, et al. New means to monitor the effect of glucocorticoid therapy in children[J]. World J Gastroenterol. 2010, 16(9): 1104-1109.
[78] Moriuchi H, Kamohara Y, Eguchi S, et al.Diverse Effects of FK506 on the Apoptosis of Hepatocytes and Infiltrating Lymphocytes in an Allografted Rat Liver[J]. J Surg Res. 2009, 06:054.
[79] Zhan Y, Gerondakis S, Coghill E, et al. Glucocorticoid-induced TNF receptor expression by T cells is reciprocally regulated by NF-kappa B and NFAT [J]. JImmunol. 2008, 181(8):5405-5413.
[80] Chen Y, Liu Z, Liang S, et al. Role of Kupffer cells in the induction of tolerance of orthotopic liver transplantation in rats[J]. Liver Transpl. 2008, 14(6): 823-836.
[81] Rentsch M,Puellmann K,Sirek S,et a1.Benefit of Kupffer cell modulation with glycine versus Kupffer cell depletion after liver transplantation in the rat: effects on post ischemic reperfusion injury, apoptotic cell death graft regeneration and survival [J]. Transpl Int. 2005, 18(9): 1079-1089.
[82]陈丽红,刘景丰,邱明链,等.树突状细胞诱导大鼠肝移植免疫耐受的研究[J].中华实验外科杂志. 2008, 25(4): 463-467.
[1] Nocentini G, Giunchi L, Ronchetti S, et al. A new member of the tumor necrosis factor / nerve growth factor receptor family inhibits T cell receptor-induced apoptosis [J]. Proc Natl Acad Sci USA, 1997, 94(12): 6216-6221.
[2] Kwon B, Yu KY, Ni J, et al. Identification of a novel activation-inducible protein of the tumor necrosis factor receptor superfamily and its ligand [J]. J Biol Chem, 1999, 274(10): 6056-6061.
[3] Xu FL, You HB, Li XH, et al. Glycine attenuates endotoxin-induced liver injury by downregulating TLR4 signaling in Kupffer cells [J]. Am J Surg, 2008, 196(1):139-148.
[4] Nocentini G, Riccardi C. GITR: a multifaceted regulator of immunity belonging to the tumor necrosis factor receptor superfamily [J]. Eur J Immunol, 2005, 35(4): 1016-1022.
[5] Chattopadhyay K, Ramagopal UA, Brenowitz M, et al. Evolution of GITRL immune function: Murine GITRL exhibits unique structural and biochemical properties within the TNF superfamily [J]. Proc Natl Acad Sci USA, 2008, 105(2): 635-640.
[6] Zhou Z, Tone Y, Song X, et al. Structural basis for ligand-mediated mouse GITR activation [J]. Proc Natl Acad Sci USA, 2008, 105(2): 641-645.
[7] Kim WJ, Bae EM, Kang YJ, et al. Glucocorticoid-induced tumor necrosis factor receptor family related protein (GITR) mediates inflammatory activation of macrophages that can destabilize atherosclerotic plaques [J]. Immunology, 2006, 119(3): 421-429.
[8] Bae E, Kim WJ, Kang YM, et al. Glucocorticoid-induced tumor necrosis factor receptor-related protein-mediated macrophage stimulation may induce cellular adhesion and cytokine expression in rheumatoid arthritis [J]. Clin Exp Immunol, 2007, 148(3): 410-418.
[9] Cuzzocrea S, Nocentini G, Di Paola R, et al. Proinflammatory role of glucocorticoid - induced TNF receptor-related gene in acute lung inflammation[J]. J Immunol, 2006; 177(1): 631-641.
[10] Bae EM, Kim WJ, Suk K,et al. Reverse signaling initiated from GITRL induces NF-kappa B activation through ERK in the inflammatory activation of macrophages [J]. Mol Immunol, 2008, 45(2): 523-533.
[11] Shimizu J, Yamazaki S, Takahashi T, et al. Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance[J]. Nat Immunol, 2002, 3(2): 135-142.
[12] Esparza EM, Arch RH. Signaling triggered by glucocorticoid-induced tumor necrosis factor receptor family-related gene: regulation at the interface between regulatory T cells and immune effector cells [J]. Front Biosci, 2006, 11: 1448-1465.
[13]陈钰,钟江,徐健,等.超抗原SEB活化耐受性NKT细胞亚群的研究[J].细胞与分子免疫学杂志, 2008,24(10): 943-946.
[14] Shin HH, Kim SJ, Lee HS,et al. The soluble glucocorticoid-induced tumor necrosis factor receptor causes cell cycle arrest and apoptosis in murine macrophages [J]. Biochem Biophys Res Commun, 2004, 316(1):24-32.
[15] Kravchenko VV, Kaufmann GF, Mathison JC,et al. Modulation of gene expressionvia disruption of NF-kappa B signaling by a bacterial small molecule [J]. Science, 2008, 321(5886): 259-263.
[16] Kim EY, Battaile JT, Patel AC, et al. Persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease [J]. Nat Med, 2008, 14(6): 633-640.
[17] Kim S, Takahashi H, Lin WW, et al. Carcinoma-produced factors activate myeloid cells through TLR2 to stimulate metastasis [J]. Nature, 2009, 457(7225): 102-106.
[18] Weiser-Evans MC, Wang XQ, Amin J, et al. Depletion of Cytosolic Phospholipase A2 in Bone Marrow-Derived Macrophages Protects against Lung Cancer Progression and Metastasis [J]. Cancer Res. 2009, 69(5): 1733-1738.
[19] Cadili A, Kneteman N. The role of macrophages in xenograft rejection [J]. Transplant Proc, 2008, 40(10): 3289-3293.
[20] Yong Z, Chang L, Mei YX, et al. Role and mechanisms of CD4+CD25+ regulatory T cells in the induction and maintenance of transplantation tolerance[J]. Transpl Immunol, 2007, 17(2): 120-129.
[2] Tiegs G, Lohse AW. Immune tolerance: what is unique about the liver [J]. J Autoimmun. 2010, 34(1): 1-6.
[3] Traeger T, Mikulcak M, Eipel C, et al. Kupffer cell depletion reduces hepatic inflammation and apoptosis but decreases survival in abdominal sepsis [J]. Eur J Gastroenterol Hepatol. 2010, 22(9):1039-1049.
[4] Wittebole X, Castanares-Zapatero D, Laterre PF. Toll-like receptor 4 modulation as a strategy to treat sepsis [J]. Mediators Inflamm. 2010; 2010: 568396.
[5] Stebbings R, Findlay L, Edwards C, et al. "Cytokine storm" in the phase I trial of monoclonal antibody TGN1412: better understanding the causes to improve preclinical testing of immunotherapeutics [J]. J Immunol. 2007, 179(5): 3325-3331.
[6] Rudd CE, Taylor A, Schneider H. CD28 and CTLA-4 coreceptor expression and signal transduction [J]. Immunol Rev. 2009, 229(1): 12-26.
[7] Koehn BH, Ford ML, Ferrer IR, et al. PD-1-dependent mechanisms maintain peripheral tolerance of donor-reactive CD8+ T cells to transplanted tissue. J Immunol [J]. 2008, 181(8): 5313-5322.
[8] rdona ID, Goleva E, Ou LS, et al. Staphylococcal enterotoxin B inhibits regulatory T cells by inducing glucocorticoid-induced TNF receptor-related protein ligand on monocytes [J]. J Allergy Clin Immunol. 2006, 117(3): 688-695.
[9] Hwang H, Lee S, Lee WH, et al. Stimulation of glucocorticoid-induced tumor necrosis factor receptor family-related protein ligand (GITRL) induces inflammatory activation of microglia in culture [J]. J Neurosci Res. 2010, 88(10): 2188-2196.
[10] Scumpia PO, Delano MJ, Kelly-Scumpia KM, et al. Treatment with GITR agonistic antibody corrects adaptive immune dysfunction in sepsis [J]. Blood. 2007, 110(10): 3673-81.
[11] Fujiki M, Esquivel CO, Martinez OM, et al. Induced tolerance to rat liver allografts involves the apoptosis of intragraft T cells and the generation of CD4 (+) CD25 (+) FoxP3 (+) T regulatory cells [J]. Liver Transpl. 2010, 16: 147-154.
[12] Grohmann U, Volpi C, Fallarino F, et al. Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy [J]. Nat Med. 2007, 13: 579-586.
[13] Fábrega E, Unzueta MG, Cobo M, et al. Value of soluble CD30 in liver transplantation [J]. Transplant Proc. 2007, 39: 2295-2296
[14] Miyagawa-Hayashino A, Tsuruyama T, Egawa H, et al. FasL expression in hepatic antigen-presenting cells and phagocytosis of apoptotic T cells by FasL+ Kupffer cells are indicators of rejection activity in human liver allografts [J]. Am J Pathol. 2007, 171: 1499-1508.
[15] Qin L, Guan HG, Zhou XJ, et al. Blockade of 4-1BB/4-1BB ligand interactions prevents acute rejection in rat liver transplantation [J]. Chin Med J (Engl). 2010, 123:212-215.
[16]夏仁品,卢实春,赖威,等.针对OX40小干扰RNA供体转染抗大鼠肝移植排斥反应[J].中华实验外科杂志. 2008, 25: 860- 862.
[17] Cardona ID, Goleva E, Ou LS, et al. Staphylococcal enterotoxin B inhibits regulatory T cells by inducing glucocorticoid-induced TNF receptor-related protein ligand on monocytes [J]. J Allergy Clin Immunol. 2006, 117: 688-695.
[18] Shimizu J, Yamazaki S, Takahashi T, et al. Stimulation of CD25 (+) CD4 (+) regulatory T cells through GITR breaks immunological self-tolerance [J]. Nat Immunol. 2002, 3: 135-142.
[19] Adams DH, Ju C, Ramaiah SK, et al. Mechanisms of immune-mediated liver injury [J]. Toxicol Sci. 2010, 115(2): 307-321.
[20] Baffy G. Kupffer cells in non-alcoholic fatty liver disease: the emerging view [J]. J Hepatol. 2009, 51(1):212-23.
[21] Ramadori G, Moriconi F, Malik I, et al. Physiology and pathophysiology of liver inflammation, damage and repair [J]. J Physiol Pharmacol. 2008, 59 Suppl 1:107-117.
[22] Valatas V, Xidakis C, Roumpaki H, et al. Isolation of rat Kupffer cells: a combined methodology for highly purified primary cultures [J]. Cell Biol Int. 2003, 27(1): 67-73.
[23] Folch E, Prats N, Hotter G, et al. P-selectin expression and Kupffer cell activation in rat acute pancreatitis [J]. Dig Dis Sci. 2000, 45(8): 1535-1544.
[24] Smedsr?d B, Pertoft H. Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence [J]. J Leukoc Biol. 1985, 38(2): 213-230.
[25] Tsujimoto T, Kawaratani H, Kitazawa T, et al. Decreased phagocytic activity of Kupffer cells in a rat nonalcoholic steatohepatitis model [J]. World J Gastroenterol. 2008, 14(39): 6036-6043.
[26] Olynyk JK, Clarke SL. Isolation and primary culture of rat Kupffer cells [J]. J Gastroenterol Hepatol. 1998, 13(8): 842-845.
[27] Knook DL,Blansjaar N,Sleyster EC. Isolation and characterization of kupffer and endothelial cells from the rat liver [J]. Exp Cell Res. 1997, 109(2): 317-329.
[28] Kindberg GM, Tolleshaug H, Roos N, et al. Hepatic clearance of Sonazoid perfluorobutane microbubbles by Kupffer cells does not reduce the ability of liver to phagocytose or degrade albumin microspheres [J]. Cell Tissue Res. 2003, 312(1): 49-54.
[29] Dan C,Wake K.Modes of endoeytosis of latex particles in sinusoidal endothelialand kupffer cells of normal and perfused rat liver [J]. Exp Cell Res. 1985, 158: 75-85.
[30] Klotz C, Frevert U. Plasmodium yoelii sporozoites modulate cytokine profile and induce apoptosis in murine Kupffer cells [J]. Int J Parasitol. 2008, 38(14): 1639-1650.
[31] Ten-Hagan TL, van-Vianen W, Bakker WA.Isolation and characterization of routine kupffer cells and splenic macrophages [J]. J Immunol Method. 1996, 19: 81-91.
[32] Van Furth R. Production and migration of monocytes and kinetics of macrophages [M] . In Van Furth, R. (ed.), Mononuclear Phagocytes . Kluwer Academic Publishers,Norwell,MA,1992,3-12
[33] Xu FL, You HB, Li XH, et al. Glycine attenuates endotoxin-induced liver injury by down regulating TLR4 signaling in Kupffer cells [J]. Am J Surg. 2008, 196: 139-48.
[34] Higuchi N, Kato M, Kotoh K, et al. Methylprednisolone injection via the portal vein suppresses inflammation in acute liver failure induced in rats by lipopolysaccharide and d-galactosamine [J]. Liver Int. 2007, 27: 1342-8.
[35] Tsao CM, Ho ST, Liaw WJ, et al. Combined effects of propofol and dexamethasone on rats with endotoxemia [J]. Crit Care Med. 2008, 36: 887-94.
[36] Shimizu J, Yamazaki S, Takahashi T, et al. Stimulation of CD25 (+) CD4 (+) regulatory T cells through GITR breaks immunological self-tolerance [J]. Nat Immunol. 2002, 3 (2): 135-142.
[37] Liu B, Li Z, Mahesh SP, et al. Glucocorticoid-induced tumor necrosis factor receptor negatively regulates activation of human primary natural killer (NK) cells by blocking proliferative signals and increasing NK cell apoptosis [J]. J Biol Chem. 2008, 283 (13): 8202-8210.
[38] Huttunen R, Syrj?nen J, Aittoniemi J, et al. High activity of indoleamine 2,3 dioxygenase enzyme predicts disease severity and case fatality in bacteremic patients[J]. Shock. 2010, 33(2): 149-154.
[39]魏思东,杨慷,龚建平. GITR/GITRL信号系统在单核巨噬细胞免疫调节中的作用[J].细胞与分子免疫学杂志. 2009, 25 (12): 1207-1209.
[40] Albertson TE. Controversies in the treatment of sepsis [J]. Semin Respir Crit Care Med. 2010, 31(1):66-78.
[41] Miyashita M. Controversy of corticosteroids in septic shock [J]. J Nippon Med Sch. 2010, 77(2): 67-70.
[42] Dellinger RP, Levy MM, Carlet JM, et al. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008 [J]. Crit Care Med. 2008, 36(1): 296-327.
[43] d'Emmanuele di Villa Bianca R, Lippolis L, Autore G, et al. Dexamethasone improves vascular hyporeactivity induced by LPS in vivo by modulating ATP-sensitive potassium channels activity [J]. Br J Pharmacol. 2003, 140(1): 91-96.
[44] JapiassúAM, Salluh JI, Bozza PT, et al. Revisiting steroid treatment for septic shock: molecular actions and clinical effects--a review [J]. Mem Inst Oswaldo Cruz. 2009, 104(4):531-548.
[45] Wang Y, Liu H, McKenzie G, et al. Kynurenine is an endothelium-derived relaxing factor produced during inflammation [J]. Nat Med, 2010, 16(3): 279-285.
[46] Tattevin P, Monnier D, Tribut O, et al. Enhanced indoleamine 2,3-dioxygenase activity in patients with severe sepsis and septic shock[J]. J Infect Dis. 2010, 201(6): 956-966.
[47] Jung ID, Lee MG, Chang JH, et al. Blockade of indoleamine 2, 3-dioxygenase protects mice against lipopolysaccharide induced endotoxin shock [J]. J Immunol. 2009, 182(5): 3146-3154.
[48] Grohmann U, Volpi C, Fallarino F, et al. Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy [J]. Nat Med. 2007, 13(5): 579-586.
[49] Cuesta N, Nhu QM, Zudaire E, et al. IFN regulatory factor-2 regulates macrophage apoptosis through a STAT1/3- and caspase-1-dependent mechanism [J]. J Immunol. 2007, 178 (6): 3602-3611.
[50] Ottonello L, Bertolotto M, Montecucco F, et al. Dexamethasone -induced apoptosis of human monocytes exposed to immune complexes. Intervention of CD95- and XIAP-dependent pathways [J]. Int J Immunopathol Pharmacol. 2005, 18 (3): 403-415.
[51] Fong CC, Zhang Y, Zhang Q, et al. Dexamethasone protects RAW264.7 macrophages from growth arrest and apoptosis induced by H2O2 through alteration of gene expression patterns and inhibition of nuclear factor-kappa B (NF-kappa B) activity [J]. Toxicology. 2007, 236 (1-2): 16-28.
[52] Bae EM. Reverse signaling initiated from GITRL induces NF-B activation through ERK in the inflammatory activation of macrophages [J]. Mol Immunol. 2008, 45(2): 523-533.
[53] Finkelstein RA, Li Y, Liu B, et al. Treatment with histone deacetylase inhibitor attenuates MAP kinase mediated liver injury in a lethal model of septic shock [J]. J Surg Res. 2010, 163: 146-154.
[54] Su GL. Lipopolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation [J]. Am J Physiol Gastrointest Liver Physiol. 2002, 283: G256-265.
[55] Higuchi N, Kato M, Kotoh K, et al. Methylprednisolone injection via the portal vein suppresses inflammation in acute liver failure induced in rats by lipopolysaccharide and d-galactosamine [J]. Liver Int. 2007, 27: 1342-1348.
[56] [4] Tsao CM, Ho ST, Liaw WJ, et al. Combined effects of propofol and dexamethasoneamethasone on rats with endotoxemia [J]. Crit Care Med. 2008, 36: 887-894.
[57] Wang X, Nelin LD, Kuhlman JR, et al. The role of MAP kinase phosphatase-1 in the protective mechanism of dexamethasone against endotoxemia [J]. Life Sci. 2008, 83: 671-680.
[58] Nocentini G, Cuzzocrea S, Bianchini R, et al. Modulation of acute and chronic inflammation of the lung by GITR and its ligand [J]. Ann N Y Acad Sci. 2007, 1107: 380-391.
[59] Kim WJ, Bae EM, Kang YJ, et al. Glucocorticoid-induced tumor necrosis factor receptor family related protein (GITR) mediates inflammatory activation of macrophages that can destabilize atherosclerotic plaques [J]. Immunology. 2006, 119(3): 421-429.
[60] Bae E, Kim WJ, Kang YM, et al. Glucocorticoid-induced tumor necrosis factor receptor-related protein-mediated macrophage stimulation may induce cellular adhesion and cytokine expression in rheumatoid arthritis [J]. Clin Exp Immunol. 2007, 148(3): 410-418.
[61] Cuzzocrea S, Nocentini G, Di Paola R, et al. Proinflammatory role of glucocorticoid-induced TNF receptor-related gene in acute lung inflammation [J]. J Immunol. 2006, 177(1): 631-641.
[62] Buras JA, Holzmann B, Sitkovsky M. Animal models of sepsis: setting the stage. Nat Rev Drug Discov. 2005, 4: 854-865.
[63] Xu FL, You HB, Li XH, et al. Glycine attenuates endotoxin-induced liver injury bydown regulating TLR4 signaling in Kupffer cells [J].Am J Surg. 2008, 196: 139-148.
[64] Chatterjee S, Premachandran S, Shukla J, et al. Synergistic therapeutic potential of dexamethasone and L-arginine in lipopolysaccharide-induced septic shock [J]. J Surg Res. 2007, 140(1): 99-108.
[65] Melgert BN, Weert B, Schellekens H, et al. The pharmacokinetic and biological activity profile of dexamethasone targeted to sinusoidal endothelial and Kupffer cells [J]. J Drug Target. 2003, 11(1):1-10.
[66] Melgert BN, Olinga P, Van Der Laan JM, et al. Targeting dexamethasone to Kupffer cells: effects on liver inflammation and fibrosis in rats [J]. Hepatology. 2001, 34(4 Pt 1):719-728.
[67] Muratore CS, Harty MW, Papa EF, et al. Dexamethasone alters the hepatic inflammatory cellular profile without changes in matrix degradation during liver repair following biliary decompression [J]. J Surg Res. 2009, 156(2):231-239.
[68] Horiguchi M, Kim J, Matsunaga N, Kaji Het al. Glucocorticoid-dependent expression of O(6)-methylguanine-DNA methyltransferase gene modulates dacarbazine-induced hepatotoxicity in mice [J]. J Pharmacol Exp Ther. 2010, 333(3):782-787.
[69] Müschen M, Warskulat U, Douillard P, et al. Regulation of CD95 (APO-1/Fas) receptor and ligand expression by lipopolysaccharide and dexamethasone in parenchymal and nonparenchymal rat liver cells [J]. Hepatology. 1998, 27(1): 200-208.
[70] Fischer L, Trune?ka P, Gridelli B, et al. Pharmacokinetics for once-daily versus twice-daily tacrolimus formulations in de novo liver transplantation: a randomized, open-label trial [J]. Liver Transpl. 2011, 17(2): 167-177.
[71]赖星,连峥嵘,李金政,等.组蛋白去乙酰化酶11在诱导大鼠肝移植免疫耐受中的作用[J].第三军医大学学报. 2011, 33(4): 342-344.
[72] Balibrea JM, García-Martín MC, Cuesta-Sancho S, et al. Tacrolimus modulates liver and pancreas nitric oxide synthetase and heme-oxygenase isoforms and cytokine production after endotoxemia [J]. Nitric Oxide. 2011, 24(2): 113-122.
[73] Shimizu J, Yamazaki S, Takahashi T, et al. Stimulation of CD25 (+) CD4 (+) regulatory T cells through GITR breaks immunological self-tolerance [J]. Nat Immunol, 2002, 3(2): 135-142.
[74] Chen Y, Yan T, Shi LJ, et al. Knockdown of interleukin-2 by shRNA-mediated RNA interference prolongs liver allograft survival [J]. J Surg Res. 2010, 159(1): 582-587.
[75] Hossain MA, Hamamoto I, Kobayashi S, et al. Immunosuppression in auxiliary partial liver transplantation with FK506 in rats. Transplant Proc. 1997, 29(8): 3617-3618.
[76] Sonawane SB, Kim JI, Lee MK, et al. GITR Blockade Facilitates Treg Mediated Allograft Survival [J]. Transplantation. 2009, 88(10): 1169-1177.
[77] Rintam?ki H, Salo HM, Vaarala O, et al. New means to monitor the effect of glucocorticoid therapy in children[J]. World J Gastroenterol. 2010, 16(9): 1104-1109.
[78] Moriuchi H, Kamohara Y, Eguchi S, et al.Diverse Effects of FK506 on the Apoptosis of Hepatocytes and Infiltrating Lymphocytes in an Allografted Rat Liver[J]. J Surg Res. 2009, 06:054.
[79] Zhan Y, Gerondakis S, Coghill E, et al. Glucocorticoid-induced TNF receptor expression by T cells is reciprocally regulated by NF-kappa B and NFAT [J]. JImmunol. 2008, 181(8):5405-5413.
[80] Chen Y, Liu Z, Liang S, et al. Role of Kupffer cells in the induction of tolerance of orthotopic liver transplantation in rats[J]. Liver Transpl. 2008, 14(6): 823-836.
[81] Rentsch M,Puellmann K,Sirek S,et a1.Benefit of Kupffer cell modulation with glycine versus Kupffer cell depletion after liver transplantation in the rat: effects on post ischemic reperfusion injury, apoptotic cell death graft regeneration and survival [J]. Transpl Int. 2005, 18(9): 1079-1089.
[82]陈丽红,刘景丰,邱明链,等.树突状细胞诱导大鼠肝移植免疫耐受的研究[J].中华实验外科杂志. 2008, 25(4): 463-467.
[1] Nocentini G, Giunchi L, Ronchetti S, et al. A new member of the tumor necrosis factor / nerve growth factor receptor family inhibits T cell receptor-induced apoptosis [J]. Proc Natl Acad Sci USA, 1997, 94(12): 6216-6221.
[2] Kwon B, Yu KY, Ni J, et al. Identification of a novel activation-inducible protein of the tumor necrosis factor receptor superfamily and its ligand [J]. J Biol Chem, 1999, 274(10): 6056-6061.
[3] Xu FL, You HB, Li XH, et al. Glycine attenuates endotoxin-induced liver injury by downregulating TLR4 signaling in Kupffer cells [J]. Am J Surg, 2008, 196(1):139-148.
[4] Nocentini G, Riccardi C. GITR: a multifaceted regulator of immunity belonging to the tumor necrosis factor receptor superfamily [J]. Eur J Immunol, 2005, 35(4): 1016-1022.
[5] Chattopadhyay K, Ramagopal UA, Brenowitz M, et al. Evolution of GITRL immune function: Murine GITRL exhibits unique structural and biochemical properties within the TNF superfamily [J]. Proc Natl Acad Sci USA, 2008, 105(2): 635-640.
[6] Zhou Z, Tone Y, Song X, et al. Structural basis for ligand-mediated mouse GITR activation [J]. Proc Natl Acad Sci USA, 2008, 105(2): 641-645.
[7] Kim WJ, Bae EM, Kang YJ, et al. Glucocorticoid-induced tumor necrosis factor receptor family related protein (GITR) mediates inflammatory activation of macrophages that can destabilize atherosclerotic plaques [J]. Immunology, 2006, 119(3): 421-429.
[8] Bae E, Kim WJ, Kang YM, et al. Glucocorticoid-induced tumor necrosis factor receptor-related protein-mediated macrophage stimulation may induce cellular adhesion and cytokine expression in rheumatoid arthritis [J]. Clin Exp Immunol, 2007, 148(3): 410-418.
[9] Cuzzocrea S, Nocentini G, Di Paola R, et al. Proinflammatory role of glucocorticoid - induced TNF receptor-related gene in acute lung inflammation[J]. J Immunol, 2006; 177(1): 631-641.
[10] Bae EM, Kim WJ, Suk K,et al. Reverse signaling initiated from GITRL induces NF-kappa B activation through ERK in the inflammatory activation of macrophages [J]. Mol Immunol, 2008, 45(2): 523-533.
[11] Shimizu J, Yamazaki S, Takahashi T, et al. Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance[J]. Nat Immunol, 2002, 3(2): 135-142.
[12] Esparza EM, Arch RH. Signaling triggered by glucocorticoid-induced tumor necrosis factor receptor family-related gene: regulation at the interface between regulatory T cells and immune effector cells [J]. Front Biosci, 2006, 11: 1448-1465.
[13]陈钰,钟江,徐健,等.超抗原SEB活化耐受性NKT细胞亚群的研究[J].细胞与分子免疫学杂志, 2008,24(10): 943-946.
[14] Shin HH, Kim SJ, Lee HS,et al. The soluble glucocorticoid-induced tumor necrosis factor receptor causes cell cycle arrest and apoptosis in murine macrophages [J]. Biochem Biophys Res Commun, 2004, 316(1):24-32.
[15] Kravchenko VV, Kaufmann GF, Mathison JC,et al. Modulation of gene expressionvia disruption of NF-kappa B signaling by a bacterial small molecule [J]. Science, 2008, 321(5886): 259-263.
[16] Kim EY, Battaile JT, Patel AC, et al. Persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease [J]. Nat Med, 2008, 14(6): 633-640.
[17] Kim S, Takahashi H, Lin WW, et al. Carcinoma-produced factors activate myeloid cells through TLR2 to stimulate metastasis [J]. Nature, 2009, 457(7225): 102-106.
[18] Weiser-Evans MC, Wang XQ, Amin J, et al. Depletion of Cytosolic Phospholipase A2 in Bone Marrow-Derived Macrophages Protects against Lung Cancer Progression and Metastasis [J]. Cancer Res. 2009, 69(5): 1733-1738.
[19] Cadili A, Kneteman N. The role of macrophages in xenograft rejection [J]. Transplant Proc, 2008, 40(10): 3289-3293.
[20] Yong Z, Chang L, Mei YX, et al. Role and mechanisms of CD4+CD25+ regulatory T cells in the induction and maintenance of transplantation tolerance[J]. Transpl Immunol, 2007, 17(2): 120-129.