Role of soluble Fas ligand in autoimmune diseases

AIM: To investigate the role of soluble Fas ligand in autoimmune diseases. METHODS: RT-PCR was performed to amplify sFasL cDNA from the total RNA extracted from activated human peripheral blood lymphocytes. DNA fragments were cloned into PCR vector. After sequenced, sFasL gene fragments were inserted into pQE-31 vector and expressed in E. Coli M15 respectively. Proteins were purified through affinity chromatography column with ligand of 6XHis tag and identified by SDS-PAGE and Western blot. Mice were immunized with sFasL protein and specific anti-serum was harvested 6 wk after immunization. Monoclonal anti-human FasL antibody was made from the immunized mice. Serum level of sFasL in different patients was detected using anti-FasL antibodies from the immunized mice. RESULTS: The protein expressed was 24 ku by SDS-PAGE electrophrosis. The protein was specially bound to anti-human FasL antibody by Western blot analysis. The sFasL protein could induce Jurket cell apoptosis in vitro. The concentration of serum sFasL in patients with autoimmune diseases was higher than that in normal individuals. sFasL could reduce arthritis in collagen induced arthritis (CIA) mice model by subcutaneous injection. CONCLUSION: sFasL may be involved in either induction of apoptosis or autoimmune diseases. Furthermore, sFasL may have potential application in treatment of autoimmune diseases.     

Fas/FasL系统与胃癌

Fas抗原是细胞表面重要的死亡受体之一,Fas与相应配体（FasL)相结合是诱导细胞凋亡的重要途径之一,而肿瘤的发生不仅有细胞增殖的异常,也存在细胞凋亡的异常,近年来为了阐明胃癌发生过程中细胞凋亡异常的分子机制,对Fas/FasL系统在胃癌及癌前病变中的表达和作用进行了深入研究,现将有关的研究进展综述如下。     

Role of serum soluble Fas/soluble Fas ligand and TNF-alpha on response to interferon-alpha therapy in chronic hepatitis C

AIMS/BACKGROUND: To determine the relationship between host factors and host response to interferon (IFN) therapy, serum soluble Fas (sFas), soluble Fas ligand (sFas ligand), and tumor necrosis factor-alpha (TNF-alpha) were analyzed in 41 patients with chronic hepatitis C (CH-C) treated with IFN-alpha. METHODS: Serum levels of sFas, sFas ligand, and TNF-alpha were measured at 0, 4, and 24 weeks of IFN therapy. RESULTS: Eighteen patients were complete responders (CR) and 23 patients were non-responders (NR). Serum levels of sFas and TNF-alpha in patients with CHC were significantly higher than those in healthy controls (p<0.01 and p<0.01, respectively). Serum sFas ligand levels were significantly lower in CH-C patients than in healthy controls (p<0.01). Before IFN therapy, serum levels of sFas in NR were significantly higher than those in CR (p<0.05). At 4 weeks of IFN therapy, serum levels of sFas of CR were significantly elevated compared with levels before IFN therapy (p<0.05). Serum levels of sFas correlated with the histological activity of the liver (p<0.05) and alanine aminotransferase (p<0.05). None of the three parameters, serum sFas, sFas ligand, or TNF-alpha levels, correlated with each other, with HCV-RNA genotype or with serum HCV-RNA load. Multiple logistic regression analysis showed that serum sFas levels before IFN therapy were a contributive factor to predict efficacy of IFN therapy. CONCLUSIONS: Serum sFas/sFas ligand and TNF-alpha play a possible role in pathogenesis of CH-C and also in IFN therapy. Serum sFas levels before IFN therapy may be one of the host-related factors used for evaluating the response of CH-C patients to IFN therapy.     

Apoptosis of nur77/N10-transgenic thymocytes involves the Fas/Fas ligand pathway.

The orphan nuclear receptor Nur77/N10 has recently been demonstrated to be involved in apoptosis of T cell hybridomas. We report here that chronic expression of Nur77/N10 in thymocytes of transgenic mice results in a dramatic reduction of CD4+CD8+ double-positive as well as CD4+CD8- and CD4-CD8+ single-positive cell populations due to an early onset of apoptosis. CD4-CD8- double-negative and CD25+ precursor cells, however, are unaffected. Moreover, nur77/N10-transgenic thymocytes show increased expression of Fas ligand (FasL), while the levels of the Fas receptor (Fas) are not increased. The mouse spontaneous mutant gld (generalized lymphoproliferative disease) carries a point mutation in the extracellular domain of the FasL gene that abolishes the ability of FasL to bind to Fas. Thymuses from nur77/N10-transgenic mice on a gld/gld background have increased cellularity and an almost normal profile of thymocyte subpopulations. Our results demonstrate that one pathway of apoptosis triggered by Nur77/N10 in double-positive thymocytes occurs through the upregulation of FasL expression resulting in increased signaling through Fas.     

Severe necroinflammatory reaction caused by natural killer cell-mediated Fas/Fas ligand interaction and dendritic cells in human hepatocyte chimeric mouse

The necroinflammatory reaction plays a central role in hepatitis B virus (HBV) elimination. Cluster of differentiation (CD)8-positive cytotoxic T lymphocytes (CTLs) are thought to be a main player in the elimination of infected cells, and a recent report suggests that natural killer (NK) cells also play an important role. Here, we demonstrate the elimination of HBV-infected hepatocytes by NK cells and dendritic cells (DCs) using urokinase-type plasminogen activator/severe combined immunodeficiency mice, in which the livers were highly repopulated with human hepatocytes. After establishing HBV infection, we injected human peripheral blood mononuclear cells (PBMCs) into the mice and analyzed liver pathology and infiltrating human immune cells with flow cytometry. Severe hepatocyte degeneration was observed only in HBV-infected mice transplanted with human PBMCs. We provide the first direct evidence that massive liver cell death can be caused by Fas/Fas ligand (FasL) interaction provided by NK cells activated by DCs. Treatment of mice with anti-Fas antibody completely prevented severe hepatocyte degeneration. Furthermore, severe hepatocyte death can be prevented by depletion of DCs, whereas depletion of CD8-positive CTLs did not disturb the development of massive liver cell apoptosis. CONCLUSION: Our findings provide the first direct evidence that DC-activated NK cells induce massive HBV-infected hepatocyte degeneration through the Fas/FasL system and may indicate new therapeutic implications for acute severe/fulminant hepatitis B.     

Fas and Fas ligand gene expression in autoimmune thyroiditis in BB/W rats

OBJECTIVE: Apoptosis via the Fas pathway is a potential mechanism for thyroid tissue destruction leading to clinical hypothyroidism in Hashimoto's thyroiditis (HT). Recent studies reported contradictory results regarding the regulation of Fas/Fas ligand (FasL) expression by cytokines in vitro. We therefore determined the Fas and FasL gene expression in the BioBreeding/Worcester (BB/W) rat thyroiditis model, which can be regarded as a model for HT. METHODS: In order to obtain BB/W rats with spontaneous, iodine-induced or without lymphocytic thyroiditis (LT), rats were divided into 3 groups: 55-day-old rats after 24 days of iodine administration, 75-day-old rats after 45 days of iodine administration, and 101-day-old rats respectively. The gene expression of Fas, FasL, and interleukin (IL)-1beta was determined by Genescan fragment analysis using reverse polymerase chain reaction. Serum thyroglobulin (TG) antibody concentrations were measured and the extent of lymphocytic infiltration of one thyroid lobe was histologically graded. RESULTS: Fas and FasL gene expression was significantly higher in rats with LT and correlated with the extent of lymphocytic infiltration and the TG antibody level. There was no evidence that the expression of IL-1beta or other cytokines is related to the expression of Fas or its ligand. CONCLUSIONS: The increased expression of Fas and FasL in LT of BB/W rats suggests the involvement of the Fas pathway in the pathogenesis of LT in BB/W rats. However, in contrast to results of recent in vitro studies, in the BB/W rat Fas/FasL expression is not regulated by IL-2, -4, -6, -10, -12, interferon gamma, and tumor necrosis factor alpha.     

Activation of the Fas/Fas ligand pathway in hypertensive renal disease in Dahl/Rapp rats

Background  

Hypertensive nephrosclerosis is the second most common cause of end-stage renal failure in the United States. The mechanism by which hypertension produces renal failure is incompletely understood. Recent evidence demonstrated that an unscheduled and inappropriate increase in apoptosis occurred in the Dahl/Rapp rat, an inbred strain of rat that uniformly develops hypertension and hypertensive nephrosclerosis; early correction of the hypertension prevents the renal injury. The present study examined the role of the Fas/FasL pathway in this process.     

Mechanism of counterattack of colorectal cancer cell by Fas/Fas ligand system

AIM: To determine the role of Fas/Fas ligand (FasL) in the immune escape of colon cancer cells. METHODS: Immunohistochemistry was used to observe the expression of Fas and FasL in the tissues of colon cancer patients. In situ hybridization was used to detect the localization of FasL mRNA expression in cancer tissues. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay and CD45 staining were performed to detect the apoptosis of tumor-infiltrating lymphocytes (TILs). Co-culture assays of colon cancer cells (SW480) and Jurkat cells (Fas-sensitive cells) were performed to observe the counterattack of colon cancer cells to lymphocytes. RESULTS: Of 53 cases of colon carcinomas, 23 cases (43.4%) expressed Fas which was significantly lower as compared to the normal colonic mucosa (73.3%, P<0.01), and 45 cases (84.9%) of colon carcinomas expressed FasL, whereas only two cases (3.75%) in normal mucosa expressed FasL. FasL expression in the colon cancer cells was found to be associated with increased cell death of TILs. The apoptotic rate of TIL in the FasL-positive staining regions of tumor cells was significantly higher than that in the FasL-negative staining region (54.84±2.79% vs 25.73±1.98%, P<0.01). The co-culture of SW480 cells and Jurkat cells confirmed the function of FasL on the SW480 cells. The apoptotic rates of Jurkat cells were found to be related with the amount of SW480 cells. CONCLUSION: Colon cancer cells can escape the immune surveillance and killing via decreasing Fas expression, and can counterattack the immune system via increasing FasL expression. Fas/FasL can serve as potential targets for effective antitumor therapy.     

Prevalence of HLA-DRB1 genotype and altered Fas/Fas ligand expression in adrenocortical carcinoma

A distinctive feature of malignant adrenocortical neoplasms is decreased major histocompatibility complex (MHC) class II molecule expression. However, it is unknown whether there exists a restriction to certain MHC genotypes and whether this involves alterations of the Fas/Fas ligand system and thereby affects tissue homeostasis. Therefore, MHC class II phenotype and genotype and expression patterns of the Fas/Fas ligand system were investigated in 24 adrenocortical tumors (n(Adenomas) = 14, n(Carcinomas) = 10) and an adrenal cancer cell line (NCI-H295). No MHC class II antigen expression was detected in carcinomas. The DRB1*01 genotype was found in 54.5% of patients with carcinoma (P = 0.046). No prevalence of any genotype could be detected in patients with adenomas, which exhibited varying levels of antigen expression. Fas receptor expression was 75.0% in adenomas compared with 20.0% in carcinomas (P = 0.0196), whereas ligand expression was 37.7% in adenomas and reached almost 100% in the carcinomas investigated in this study (P = 0.0033). In summary, the DRB1*01 genotype may be correlated to a higher risk for malignancy. Additional studies on MHC class II genotype and phenotype and the altered Fas/Fas ligand system in adrenal neoplasms may help to identify mechanisms of immune escape and suggest new diagnostic approaches.     

sFas与sFasL在自身免疫疾病中的意义

目的　研究sFas与sFasL在系统性红斑狼疮 (SLE)等自身免疫疾病中的意义及抗单链DNA(ssDNA)抗体与sFas和sFasL介导凋亡的相关性。方法　采用夹心ELISA方法检测 31例SLE病人 ,32例类风湿关节炎 (RA)病人 ,2 0例 1型糖尿病 (IDDM )病人及 5例多发性硬化病 (MS)病人血清中sFas与sFasL含量及抗ssDNA抗体水平。结果　在SLE、RA、IDDM及MS患者血清中的sFas含量 (pg/ml)分别为 2 881± 16 5 3 ,988± 6 96 ,135 2± 413 ,15 40± 5 6 6 ,明显高于正常对照 (P <0 0 0 2 ) ,SLE病人sFas含量高于RA ,MS ,IDDM病人。SLE、RA患者血清sFasL含量 (pg/ml)分别为5 35± 431、12 38± 1184,明显高于正常对照 (P <0 0 2 ) ,MS、IDDM患者血清sFasL含量 (pg/ml)分别为 2 5 1± 140 ,2 11± 73 ,低于正常对照 (P >0 0 5 )。在SLE、RA病人中 ,高浓度sFasL者伴有高浓度sFas。在SLE病人中 ,所有抗ssDNA抗体阳性者均伴有高浓度sFas,所有抗sFas阴性者 ,ssDNA抗体也为阴性。结论　在SLE等疾病中sFas水平明显高于正常人 ,可作为疾病进展与治疗效果的判断指标。抗ssDNA抗体与sFas具有关联性。sFas与sFasL在疾病中的相互作用及动态变化有待进一步研究     

Role of CD95/Fas and its ligand in the regulation of the growth of human CD34(++)CD38(-) fetal liver cells.

The functional significance of CD95/Fas expressed by candidate hematopoietic stem cells (HSCs) from human fetal liver was studied by testing the effect of agonistic anti-CD95 monoclonal antibody (mAb) CH-11 and soluble CD95 ligand (sCD95L) on the growth of CD34(++)CD38(-)lineage cells in vitro. Candidate fetal HSCs exhibited a dose-dependent proliferative response to CH-11 as well as to sCD95L when combined with kit ligand (KL) + interleukin 3 (IL-3) under serum-deprived culture conditions. CH-11 mAb increased, in a synergistic fashion, the number of myeloid colony-forming unit culture (CFU-C) generated by candidate HSCs in liquid cultures with the cytokine combinations KL + IL-3, KL + granulocytemacrophage colony-stimulating factor, and KL + IL-6. CH-11 mAb and sCD95L also enhanced erythropoiesis supported by KL + IL-3 + erythropoietin (Epo). Furthermore, sCD95L was able to increase the number of megakaryocytes, granulocytes, and CD34- cells generated in the presence of KL + IL-3 + Epo + thrombopoietin. An analysis performed using Western blotting revealed that the membrane-bound CD95L (mCD95L) was expressed by both immature (total CD34+/++) and mature (CD34-) hematopoietic lin(-) FL cells. Among the CD34(++)lin(-)cells, both the freshly isolated CD38+ and CD38 subsets as well as CD95+ and CD95- cells constitutively expressed mCD95L, demonstrating that the CD95/CD95L system represents a paracrine and potentially autocrine regulator of early hematopoiesis. To study the role of the endogenously produced CD95L, we determined the effects of a neutralizing anti-CD95L NOK-1 on the growth of candidate HSCs. By blocking the endogenous CD95L with NOK-1 mAb, we observed an increase in CFU-C generated by candidate HSCs. We conclude that the endogenous CD95L has an inhibitory effect on fetal candidate HSCs, which can be blocked by sCD95L and CH-11 mAb.