Fas ligand expression in the germinal centre.

Whereas the importance of the Fas/FasL system in the regulation of T-cell homeostasis is well established, it is not yet clear if FasL is involved in B-cell regulation, especially in the clonal selection of B lymphocytes in the germinal centre (GC). This study therefore investigated the expression of FasL protein in tonsils and lymph nodes with lymphofollicular hyperplasia by western blotting and immunohistochemistry. In all the samples examined, western blot analysis showed FasL proteins of 33 and 52 kD, which presumably correspond to membrane-bound and soluble forms of the FasL protein. Immunohistochemically, FasL was found in a limited number of cells confined to a cluster in the light zone of the GC. The signal showed a delicate meshwork-like pattern of branching processes enmeshing the centrocytes and the few centroblasts of the light zone. In serial sections, the immunostaining pattern for FasL was found largely to coincide with the CD23 staining of follicular dendritic cells (FDCs), which are typically located in the light zone. In contrast, the FasL signal did not correspond to the distribution of the CD4-positive GC T-cells. In conclusion, expression of FasL in lymphofollicular hyperplasia seems to be largely confined to the light zone of the GCs, where selection of FDC-associated centrocytes is known to occur. These observations thus suggest that FasL is involved in selection processes of the B-cell system.     

Alteration of Fas and Fas ligand expression during human visceral leishmaniasis

Several studies in murine systems have suggested a role of apoptosis in the pathogenesis of leishmaniasis. However, the role of apoptosis in visceral leishmaniasis in man has not been explored. In this study, we show that patients with visceral leishmaniasis demonstrate significant dysregulation of Fas and Fas ligand. Levels of soluble Fas (sFas) and soluble Fas ligand (sFasL) were elevated in plasma of patients with active visceral leishmaniasis (VL) and individuals co-infected with VL-HIV-1 compared to healthy controls. The levels of sFas and sFasL were normalized 6 months after successful treatment. In VL patients, the expression of membrane bound Fas, and to a lower extent FasL, were up-regulated on Leishmania donovani-infected spleen cells, the site of parasite multiplication. Expression of Fas and FasL on peripheral blood mononuclear cells was within normal range, probably reflecting that the blood is not a normal site of L. donovani infection. Furthermore, this is suggested by the finding that in vitro infection of macrophages with L. donovani up-regulated Fas expression on the surface of infected cells and enhanced the levels of sFasL in supernatants from infected cultures. How this dysregulation may affect the pathogenesis of human visceral leishmaniasis is discussed.     

人Fas配体蛋白在大肠杆菌中的融合表达与应用

目的：在大肠杆菌中融合表达人Fas配体蛋白。方法：应用RT-PCR技术，从激活的人外周血淋巴细胞中提取总RNA，扩增Fas配体cDNA，克隆入PCR2.1载体，测序验证后，克隆入带有组氨酸盒的表达载体pQE-31，在大肠杆菌中表达，经亲和层析柱纯化后，用SDS-PAGE和Western blot鉴定表达产物。结果：表达的融合蛋白为人Fas配体，其相对分子质量（Mr)为40000。；经透析复性后，具有诱导Jurkat细胞凋亡的作用，用该蛋白分子免疫BALB/c小鼠制备抗血清，以间接ELISA检测了部分自身免疫病与肿瘤患者血清中可溶性的F 苛的含量。结果与进口试剂盒的灵敏性相似。结论：获得FasL单克隆抗体，深入研究FasL的应用提供了材料。     

Fas ligand, Fas antigen and Bcl-2 expression in human endometrium during the menstrual cycle.

In this study, we investigate Fas ligand expression in the human endometrium during the menstrual cycle in relation to Fas antigen and Bcl-2 expression, using immunoelectron microscopy and Western blotting. Endometrial samples were obtained from 54 pre-menopausal non-pregnant women who underwent laparotomies for benign diseases. The Fas ligand, as well as the Fas antigen, were expressed on the surface of endometrial glandular cells throughout the menstrual cycle, whereas Bcl-2 showed a cyclic expression pattern, peaking during the late proliferative phase. A noteworthy finding was that both the Fas ligand and the Fas antigen were localized on Golgi apparatuses and vesicles, in addition to the cell membranes, during the late proliferative phase. These results indicate that the Fas ligand and Fas antigen which are localized on Golgi apparatus and vesicles during the late proliferative phase are incorporated into the cell membranes during the secretory phase, and are co-expressed on the cell membranes of endometrial glands throughout the menstrual cycle. The factors regulating Fas-mediated apoptosis in the human endometrium, including the level of expression of the Fas ligand and Bcl-2 are discussed.     

FasL N-端编码序列对其表达的影响

目的探讨Fas配体（Fas ligand,FasL）胞内区氨基酸组成对膜型FasL蛋白表达的影响。方法PCR技术扩增FasL胞内区产生不同缺失的cDNA,构建重组表达质粒;使用脂质体转染法将表达质粒导入靶细胞;RT-PCR分析FasL mRNA水平;荧光抗体染色、流式细胞仪和Western免疫印迹技术检测细胞中FasL蛋白的表达。结果FasL N-端缺失第2～16氨基酸残基后,FasL蛋白在细胞中的表达水平显著增加,但mRNA水平不变。结论FasL N-端第2～16氨基酸残基对FasL蛋白表达水平具有自我调节作用。     

Immunohistochemical analysis of Fas ligand expression in normal human tissues

Cross-linking of Fas and Fas ligand (FasL) induces apoptosis in Fas-bearing cells and regulates apoptosis. Fas is widely expressed in normal human tissues, but FasL expression has been considered to be restricted to lymphoid tissues. Recent studies have demonstrated that FasL is also expressed in some nonlymphoid tissues. To screen the in situ expression of FasL in normal human tissues, immunohistochemistry was performed using paraffin-embedded human tissues. FasL immunostaining was easily detected in testis, neurons, trophoblasts, tonsil, lymph node, Paneth cells, hepatocytes, renal tubular epithelium and bronchial epithelium, consistent with previous reports. Surprisingly, FasL was also expressed in many other cell types, including thymic medulla, skeletal muscle, cardiac muscle, pituitary gland, parathyroid gland, prostate glands, oocytes, epithelium of fallopian tube, endometrial glands, and gastric parietal cells. These findings demonstrate that FasL is widely expressed in human tissues and suggest that wide but cell-type specific expression of FasL may not only be implicated in the regulation of immune homeostasis but also in the regulation of cell death and life in many cell types in vivo.     

Frequent expression of soluble Fas and Fas ligand in Chinese stomach cancer and its preneoplastic lesions

To investigate the frequency and pattern of Fas and FasL expression in the gastric mucosa at different stages of gastrocarcinogenesis, the combined examinations of pathology, immunocytochemistry and Western blot hybridisation were performed on the cancer specimens as well as their preneoplastic and non-cancerous counterparts. The frequencies of Fas and FasL expression were found to be 6.3% (1/16) and 62.5% (10/16) in non-cancerous mucosa, 60% (6/10) and 80% (8/10) in atrophic gastritis, 75% (9/12) and 83% (10/12) in intestinal metaplasia, 100% in both dysplasia Grades II (20/20) and Grade III (15/15) and 4 types of gastric carcinomas (74/74). Two forms of FasL protein in 37 kDa and 26 kDa were detected in all FasL+ cases. Soluble Fas (30 kDa) but not the membrane-type (43 kDa) is predominantly expressed in the Fas+ cases. Our data thus suggest a close correlation of soluble Fas with stomach tumour progression P<0.01. The sFas protein, together with the tumor-derived soluble and membrane FasL, may confer on the transforming and transformed gastric epithelial cells an immune advantage enabling escape from endogenous and exogenous suicide signal(s).     

Glomerular expression of Fas ligand and Bax mRNA in lupus nephritis

The current studies were carried out to determine the expression of Fas ligand and Bax in kidneys from lupus nephritis as possible indicators of apoptosis. Twenty-four kidney biopsies from patients with lupus nephritis and 30 normal controls were studied for FasL and Bax gene expression by fluorescent in situ hybridization. Seventy percent of the lupus biopsies displayed FasL or Bax mRNAs. These genes were mainly expressed in biopsies with higher activity indices. In contrast, neither of these mediators was detected in normal glomeruli. These data suggest that FasL and Bax are up-regulated in lupus nephritis and may play a pathogenic role through apoptotic cascades.     

Fas ligand expression and its correlation with apoptosis and proliferation in Lobund-Wistar prostate carcinomas.

OBJECTIVE: The Fas (CD95) interaction with its receptor Fas ligand (FasL) is one of the main mechanisms of cell apoptosis. High expression of FasL has been consistently observed in a variety of human cancers. In this study, we evaluated FasL and its relationship with apoptosis and proliferation in Lobund-Wistar (L-W) cancers. The L-W rat strain develops spontaneous and induced adenocarcinomas in the anterior prostate and seminal vesicles. Although FasL expression has been observed in a subset of human prostate carcinomas, this multistage model allowed in vivo evaluation of subclones of malignant cells with a single genetic susceptibility. METHODS: Apoptosis was evaluated in spontaneous, induced and transplanted tumors as well as metastasis using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique and transmission electron microscopy. Proliferating cell nuclear antigen (PCNA) and FasL expression were detected using immunohistochemistry and analyzed according to the number of positive cells and intensity of staining using a semiquantitive method. RESULTS: Apoptotic indexes were significantly higher in spontaneous tumors compared to induced (p < 0.008), transplanted tumors (p < 0.0112) and metastases (p < 0.009). TUNEL-positive cells were frequently observed in the leukocytic infiltrate of the stroma in transplanted carcinomas and metastases. These findings were confirmed by electron microscopy. FasL expression was not uniformly localized in L-W carcinomas and its highest expression was observed in transplanted tumors and metastasis (p < 0.005). Moreover, PCNA indices were directly correlated with cancers showing high FasL total scores (Hscores). CONCLUSIONS: In this model, high FasL expression was associated with cells displaying low apoptotic indexes and high PCNA index. Therefore, analysis of FasL may have clinical relevance in detecting the malignant potential of prostate cancers.     

Fas and Fas ligand gene mutations in Hashimoto's thyroiditis

To clarify whether Fas and Fas ligand (FasL) mutations are involved in the pathogenesis of Hashimoto's thyroiditis (HT), we examined the open reading frame of Fas and FasL in 21 cases. Mutations of Fas and FasL genes were detected in 8 (38.1%) and 1 (4.8%) of 21 cases, respectively. All but one of the Fas mutations were frameshift mutations, which affect the cytoplasmic region (death domain) known to be involved in apoptotic signal transduction and thus could be loss-of-function mutations. FasL mutation in one case was a 46-bp deletion from nucleotide 349 to 394, which corresponded to exon 2. Lack of exon 2 results in a frameshift, which generates a stop codon at residue 128. This mutant encodes the protein that contains only a part of the intracellular domain, thus the abnormal protein might not be expressed on the cell surface. The cells with Fas mutations were confined to the mantle zone and the germinal center, as determined by microdissection methods. These findings suggest that the cells with Fas mutations might accumulate in those areas and might be involved in the pathogenesis of Hashimoto's thyroiditis.     

Significance of Fas and Fas ligand in tuberculous lymphadenitis

The Fas/Fas-ligand (FasL) system plays an important role in regulation of apoptosis and the immune response, and is exploited by mycobacteria to evade the immune response. This study was performed to investigate the distribution and levels of FasL and Fas in lymph node granulomas and sera of tuberculous lymphadenitis patients by immunohistochemistry and enzyme-linked immunosorbent assay. The validity of soluble Fas (sFas) or soluble FasL (sFasL) as a diagnostic tool was also examined. Levels of sFasL in serum were elevated among patients. The numbers of FasL stained cells in lymph node granulomas were higher than Fas. Children had significantly higher levels of sFasL as compared to adults. The human immunodeficiency virus (HIV)-tuberculosis (TB)-coinfected patients displayed no differences in the levels of sFasL or sFas compared with HIV-negative patients. The healthy controls from a high endemic tuberculosis country (having latent TB) had significantly higher levels of sFasL than from a country with no TB transmission. The sensitivity and specificity of the FasL and Fas test were low when compared with the culture results as the gold standard. However, by using histology as the gold standard, the sensitivity and specificity of the FasL test were increased to 66.7% and 100%, respectively, but for the Fas test remained low. In conclusion, sFasL and sFas cannot be used as diagnostic tests for tuberculous lymphadenitis. However, its utility in detecting latent TB and childhood tuberculous lymphadenitis remains to be evaluated. FasL seems to play a role in immune modulation and pathogenesis of TB. Modulators of Fas/FasL-mediated apoptosis may therefore be clinically useful.     

Fas and Fas ligand: lpr and gld mutations

Fas ligand (FasL) is a death factor that binds to its receptor, Fas, and induces apoptosis. Two mutations that accelerate autoimmune disease, lpr and gld, are known to correspond to mutations within genes encoding Fas and FasL, respectively. Here, Shigekazu Nagata and Takashi Suda summarize current knowledge of Fas and FasL, and discuss the physiological role of the Fas system in T-cell development, cytotoxicity and cytotoxic T lymphocyte (CTL)-mediated autoimmune disease.     

Expression of Fas and Fas ligand in cutaneous B-cell lymphomas

Primary cutaneous B-cell lymphomas (CBCLs) represent a rare, but distinct group of B-cell neoplasms with a different clinical behaviour to B-cell lymphomas secondarily involving the skin. Fas-Fas ligand (Fasl) expression was investigated in a group of primary and secondary CBCLs to gain an insight into the putative role of these apoptotic molecules in the clinical behaviour of these lymphomas. Frozen and paraffin sections from 32 patients with a CBCL were investigated for Fas and Fasl expression, using immunohistochemistry. This group included 24 primary CBCLs [14 primary cutaneous follicle centre cell lymphomas (PCFCCLs), six primary cutaneous large B-cell lymphomas (PCLBCLs) on the leg, and four primary cutaneous immunocytomas] and eight secondary CBCLs. The results were correlated with follow-up data, bcl-2, and ICAM-1 expression. High Fas expression and absent or low Fasl expression were detected in the vast majority of PCFCCLs and immunocytomas. The group of PCLBCLs on the leg, which have an intermediate prognosis, showed variable results with relatively higher Fasl expression. The highest Fasl expression was found in the more aggressive secondary CBCLs whereas in this group, Fas was undetectable in five of eight cases. Statistical analysis showed that Fas and ICAM-1 expression was strongly related to a favourable prognosis, whereas expression of Fasl and bcl-2 was related to a very poor prognosis. Although only type of CBCL and age, but not Fas, Fasl, bcl-2, and ICAM-1 expression, proved independent prognostic parameters using multivariate analysis, the results of this study suggest that differences in the expression of Fas and Fasl, as well as bcl-2 and ICAM-1, contribute to the differences in clinical behaviour between these different types of CBCL.