Detection of alternatively spliced variant messages of Fas gene and mutational screening of Fas and Fas ligand coding regions in peripheral blood mononuclear cells derived from silicosis patients

Silicosis is clinically characterized not only by respiratory disorders but by immunological abnormalities such as the appearance of autoantibodies and complications of autoimmune diseases. Dysregulation of apoptosis, particularly in the Fas/Fas ligand (FasL) pathway, has been considered to play a role in the pathogenesis of autoimmune diseases. It has been found that serum soluble Fas (sFas) levels are elevated in silicosis patients (SIL) and the sFas message is dominantly expressed in peripheral blood mononuclear cells (PBMC) derived from these individuals. In the present study, one tried to detect alternatively spliced variant messages including typical sFas message and found four that were highly and frequently expressed, and which possess a signal peptide domain, but not transmembrane and signal transducing domains, in PBMC derived from SIL. Functional mutations were not detected in Fas and FasL genes in silicosis PBMC. Still, alternative spliced variants of the Fas gene including typical sFas message appear to play an important role in the immunological dysregulation in SIL.     

Serum levels of soluble Fas ligand in patients with silicosis

Certain patients with silicosis have been reported to exhibit immunological abnormalities such as the appearance of antinuclear antibodies and the occurrence of autoimmune diseases. Fas ligand (FasL) is a type II membrane protein which induces apoptosis by binding to its membrane receptor, Fas. FasL is converted to a soluble form by a metalloproteinase-like enzyme. We have already found serum soluble Fas (sFas) levels in silicosis patients as well as in patients with systemic lupus erythematosus (SLE) to be significantly higher than those in healthy volunteers. To examine further the role of the Fas/FasL system in silica-induced immunological abnormalities, we investigated serum soluble FasL (sFasL) levels in silicosis patients with no clinical symptoms of autoimmune diseases, using ELISA for sFasL. Although the serum sFasL levels in patients with SLE were significantly higher than those in healthy volunteers and showed a slight positive correlation with serum sFas levels, those in silicosis patients exhibited no significant difference from those in healthy volunteers, and there was no correlation with serum sFas levels. However, sFasL levels were elevated in silicosis patients with slight dyspnoea or normal PCO2 among various clinical parameters of silicosis. It may be speculated that the immunological disturbances presented by the abnormalities of apoptosis-related molecules in silicosis patients do not occur with a similar degree of respiratory involvement. Further studies are required to clarify which kinds of factors are involved in silicosis patients who exhibit immunological abnormalities.     

Elevated serum decoy receptor 3 with enhanced T cell activation in systemic lupus erythematosus

Decoy receptor 3 (DcR3/TR6) is a decoy receptor for the Fas ligand (FasL) and can inhibit FasL-induced apoptosis. It has been reported recently that DcR3 can induce T cell activation via co-stimulation of T cells, suggesting that DcR3 may be involved in the pathophysiology of autoimmune diseases. This study aims to analyse the serum DcR3 in patients with systemic lupus erythematosus (SLE) and to investigate the role of DcR3 in the pathogenesis of SLE. Significantly elevated serum DcR3 was observed in SLE patients, and the mean serum DcR3 level was significantly higher for those with active disease [SLE disease activity index (SLEDAI) >/= 10] compared with that in patients with inactive disease (SLEDAI < 10). In addition to reducing activation-induced cell death in activated T cells via neutralization of the FasL, soluble DcR3-Fc enhanced T cell proliferation and increased interleukin-2 and interferon-gamma production via co-stimulation of T cells. Moreover, enhanced T cell reactivity to DcR3-induced co-stimulation was demonstrated in lymphocytes from patients with SLE, suggesting the elevated serum DcR3 may associate with enhanced T cell activation in vivo. These findings are the first to demonstrate that serum DcR3 concentrations are increased in SLE patients, and this may imply a possible role of DcR3 in the pathogenesis of SLE via enhanced T cell hyperreactivity and reduced apoptosis in activated T cells.     

Elevated soluble Fas/APO-1 (CD95) levels in silicosis patients without clinical symptoms of autoimmune diseases or malignant tumours

Soluble Fas (sFas) is produced as translation products of alternative mRNA splicing, and antagonizes the membranous Fas molecule in Fas/Fas ligand interactions. We investigated the serum sFas levels in 64 Japanese silicosis patients with no clinical symptoms of autoimmune diseases or malignant tumours, using ELISA for sFas. The serum sFas levels in the silicosis patients were significantly higher than those in healthy volunteers. Elevated serum sFas levels were also detected in patients with systemic lupus erythematosus but, unexpectedly, no difference was observed in sFas levels between progressive systemic sclerosis patients and healthy volunteers. On the other hand, there was no significant difference in the expression of Fas on peripheral blood lymphocytes between the patients with silicosis and age-matched healthy volunteers. These observations provided the first evidence that serum sFas levels are elevated in silicosis patients without clinical symptoms of autoimmune diseases or malignant tumours. It remains to be clarified whether patients with elevated sFas levels have a tendency to develop autoimmune diseases later, or whether some other distinct factor(s) is necessary to initiate the progression of autoimmune diseases.     

Evaluation of cases with silicosis using the parameters related to Fas-mediated apoptosis.

To establish a new clinical index for immunological abnormalities occurring in silicosis, several clinical parameters related to Fas-mediated apoptosis; i.e., membrane Fas expression on peripheral blood lymphocytes (mFas), serum soluble Fas levels (sFas), serum soluble Fas ligand levels (sFasL), and soluble/membrane Fas mRNA expression ratios (s/mFas ExR) in peripheral blood mononuclear cells (PBMC) were investigated. Fifty-eight silicosis patients with no clinical symptoms of autoimmune diseases were the subjects of this study. Factor analysis was performed using 12 clinical parameters including four parameters related to Fas-mediated apoptosis. Two common factors were identified. Factor 1 which consisted of the following parameters; duration of exposure, symptomatic dyspnea, PO2, PCO2, and A-aDO2, should be designated as the respiratory factor for cases with silicosis. The parameters of factor 2 were serum IgG, sFas with high factor loading, titer of ANA, sFasL, and s/mFas ExR. These parameters of factor 2 are indicative of the immunological disorders occurring in silicosis cases. Some cases exhibited abnormalities in parameters of factor 2 but not factor 1. The factor analysis clearly demonstrated that the parameters related to Fas-mediated apoptosis should be the most beneficial for predicting the pre-clinical status of complicated autoimmune diseases in silicosis.     

Correlation between expression of DcR3 on tumor cells and sensitivity to FasL

To investigate the correlation between sensitivity to Fas ligand （FasL） and expression level of decoy receptor 3 （DcR3） on tumor cell surface, Fas/DcR3 mRNA expression was detected by RT-PCR. Anti-DcR3 mAb was used to detect expression level of DcR3 on surface of tumor cells by flow cytometry. Caspase-8, caspase-9, caspase-3, Bcl-2 expressions were analyzed by Western blot, respectively. Sensitivity to apoptosis induced by FasL was determined by Annexin V apoptosis kit. The expressions of DcR3 on the surface of tumor cells from high to low were approximately 35.3% in BGC823 cells, and 21.6% in MCF-7 cells, respectively. The apoptotic rates induced by FasL from low to high were 15.6% in BGC823 cells, and 58.2% in MCF-7 cells, respectively. There was a significant correlation between the expression levels of DcR3 with FasL-inducing apoptosis. Cellular ＆ Molecular Immunology.     

Serum levels of soluble Fas/APO-1 (CD95) and its molecular structure in patients with systemic lupus erythematosus (SLE) and other autoimmune diseases

There are two major forms of the Fas molecule, membranous Fas and soluble Fas (sFas). To clarify the clinical significance of sFas in autoimmune diseases, we designed a sandwich ELISA to determine serum concentrations of sFas and its molecular structure, and we then analysed the correlation between levels of sFas and laboratory findings in patients with SLE and other autoimmune diseases. The levels of serum sFas were significantly higher in SLE patients than in subjects with other autoimmune diseases and in healthy donors, and the frequency of a positive serum sFas was much greater in SLE patients with high SLE disease activity index scores than in those with low scores. In addition, sFas-positive SLE patients showed a significant difference in various laboratory parameters from sFas-negative SLE patients. Serial measurements of serum sFas levels in SLE patients with active disease revealed that the elevated level of sFas dramatically decreased with improvement in clinical and laboratory findings, following corticosteroid therapy. We propose that the serum level of sFas can serve as an appropriate marker for evaluating SLE disease activity. Serum sFas is heterogeneous with respect to molecular structure, thus several mechanisms are involved in the generation of sFas.     

DcR3 对肝纤维化动物模型的影响

目的:研究DcR3 基因对肝纤维化大鼠的预防性治疗的作用。方法:SPF 级健康雄性Wistar 大鼠30 只,体重范围180 ~220 g,随机分为3 组,每组10 只,分别为正常对照组、DcR3 基因预防性治疗组(1% DMN+DcR3 组)、造模组(1%DMN 组)。采用1%DMN 诱导大鼠肝纤维化模型,腹腔注射DcR3 质粒进行预防性干预。分别采用HE 染色和Masson 染色观察肝组织病理情况;qRT-PCR 和Western blot 法检测DcR3、Fas、FasL、-SMA 和TGF-1 的mRNA 和蛋白表达水平。结果:与模型组相比,DcR3 预防治疗组大鼠肝组织炎性细胞浸润及胶原类物质沉积有所改善;DcR3 基因可显著降低肝纤维化大鼠Fas、FasL、 SMA 和TGF-1 mRNA 和蛋白表达水平,DcR3 基因预防性治疗组与对照组和造模组有显著性差异(P<0.05);同时DcR3 基因预防性治疗组DcR3 mRNA 和蛋白表达水平显著升高(P<0.05)。结论:DcR3 基因可有效防治大鼠肝纤维化,其作用机制可能是DcR3 通过降低肝脏炎症反应,减少胶原类物质沉积,抑制 SMA 和TGF-1 表达,从而抑制HSC 活化;下调Fas和FasL 表达,抑制Fas/ FasL 途径诱导的肝细胞凋亡。     

Idiopathic pulmonary fibrosis fibroblasts become resistant to Fas ligand‐dependent apoptosis via the alteration of decoy receptor 3

Idiopathic pulmonary fibrosis (IPF) is an irreversible lethal lung disease with an unknown etiology. IPF patients' lung fibroblasts express inappropriately high Akt activity, protecting them in response to an apoptosis‐inducing type I collagen matrix. FasL, a ligand for Fas, is known to be increased in the lung tissues of patients with IPF, implicated with the progression of IPF. Expression of Decoy Receptor3 (DcR3), which binds to FasL, thereby subsequently suppressing the FasL–Fas‐dependent apoptotic pathway, is frequently altered in various human disease. However, the role of DcR3 in IPF fibroblasts in regulating their viability has not been examined. We found that enhanced DcR3 expression exists in the majority of IPF fibroblasts on collagen matrices, resulting in the protection of IPF fibroblasts from FasL‐induced apoptosis. Abnormally high Akt activity suppresses GSK‐3β function, thereby accumulating the nuclear factor of activated T‐cells cytoplasmic 1 (NFATc1) in the nucleus, increasing DcR3 expression in IPF fibroblasts. This alteration protects IPF cells from FasL‐induced apoptosis on collagen. However, the inhibition of Akt or NFATc1 decreases DcR3 mRNA and protein levels, which sensitizes IPF fibroblasts to FasL‐mediated apoptosis. Furthermore, enhanced DcR3 and NFATc1 expression is mainly present in myofibroblasts in the fibroblastic foci of lung tissues derived from IPF patients. Our results showed that when IPF cells interact with collagen matrix, aberrantly activated Akt increases DcR3 expression via GSK‐3β–NFATc1 and protects IPF cells from the FasL‐dependent apoptotic pathway. These findings suggest that the inhibition of DcR3 function may be an effective approach for sensitizing IPF fibroblasts in response to FasL, limiting the progression of lung fibrosis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Detection, epitope-mapping and function of anti-Fas autoantibody in patients with silicosis

Dysregulation of apoptosis through the Fas-Fas ligand pathway is associated with the onset of autoimmune disease. Since autoantibodies directed against unknown antigens are present in the sera of these patients, sera samples were examined for the presence of autoantibodies directed against the Fas molecule. Using Western blotting and a ProteinChip analysis, autoantibodies against Fas were detected in patients with silicosis, systemic lupus erythematosus (SLE) and systemic sclerosis (SSc), and weakly detected in healthy individuals. Using epitope mapping employing 12-amino-acid polypeptides with the SPOTs system, a minimum of four epitopes and a maximum of 10 epitopes were found. Several amino acid residues involved in binding FasL, such as C66, R87, L90, E93 and H126, were presented within the epitopes. Serum containing a large amount of anti-Fas autoantibody from silicosis patients inhibited the growth of a Fas-expressing human cell line, but did not inhibit the growth of a low Fas-expresser nor a Fas-expresser in which the Fas gene had been silenced by small interference RNA. All epitopes in the intracellular region of Fas were located in the death domain. The possible roles of anti-Fas autoantibody detected in healthy volunteers and patients with silicosis or autoimmune diseases are discussed here.     

Graves病和桥本甲状腺炎患者外周血T细胞表面及血清中Fas/FasL的表达特点

目的探讨Graves病(GD)和桥本甲状腺炎(HT)患者外周血中Fas+、FasL+细胞占总T淋巴细胞的比例(Fas%、FasL%)以及血清中sFas、sFasL、TGAb、TPOAb等指标的变化及意义。方法选择GD患者36例、HT患者32例、对照组20例。用流式细胞术检测外周血T细胞表面Fa(sCD95)、FasL(CD178)的表达特点,用双抗体夹心法(ELISA)测定血清中可溶性Fas及FasL的含量,用化学发光法测定相关抗体TGAb、TPOAb的含量。结果 GD及HT患者外周血Fas%均高于对照组(P<0.05),且以HT组更为显著,而各组均未检测到FasL的表达;GD及HT患者血清中sFas含量均高于对照组,尤以GD组显著;各组间均可检测到sFasL,但差异无统计学意义(P>0.05)。结论 Fas及其配体介导的凋亡在GD和HT的自身免疫反应过程中起着重要的作用。流式细胞术的应用,可为探讨AITD的发病机制提供新的方法。     

DcR3 as a diagnostic parameter and risk factor for systemic lupus erythematosus

In this study, we investigated the diagnostic value of serum death decoy receptor 3 (DcR3) for systemic lupus erythematosus (SLE). The possible pathogenic role of DcR3 in SLE was also assessed. Serum DcR3 levels of 90 SLE patients, 11 patients with rheumatic conditions and 123 healthy controls were determined by ELISA. In all, 43% of the SLE patients, 9% of patients with rheumatic conditions and 2.4% of the normal healthy individuals presented elevated serum DcR3 levels. A higher percentage of DcR3-positive SLE patients, compared with DcR3-negative SLE patients, showed abnormally high serum IgE levels, a surrogate marker of T(h)2-type immune responses. To determine the cause and effect relationship of DcR3 expression and a T(h)2-prone status, we studied young DcR3 transgenic (Tg) mice, whose transgene was driven by an actin promoter. These mice had IL-4 overproduction and augmented serum IgE levels, signs of dominant T(h)2 immune responses. To determine possible SLE pathogenic roles of DcR3, the T-cell-depleted bone marrow of DcR3 Tg mice was transplanted into lethally irradiated syngeneic C57BL/6 female mice. The recipients developed an SLE-like syndrome. They presented anti-dsDNA and anti-nuclear antibodies, along with renal and liver pathology compatible with that of SLE. In total, 90% of Tg bone marrow-transplanted mice, compared with 20% of wild-type bone marrow-transplanted mice, perished within 12 months after the transplantation. Our results showed that serum DcR3 could serve as an additional parameter for SLE diagnosis and that DcR3 secreted from cells of hematopoietic origin was SLE pathogenic in mice.     

Fas、Caspase-3和抗核小体抗体在参与SLE患者淋巴细胞凋亡紊乱中的作用

目的探讨SLE患者免疫功能紊乱与淋巴细胞凋亡信号传导途径异常之间的关系。方法应用流式细胞术测定SLE患者淋巴细胞表面Fas、FasL及细胞质中活化caspase-3的表达率,并测定凋亡细胞百分率（AnnexinV^＋PI^-）和坏死细胞百分率（AnnexinV^＋PI^＋）。应用ELISA方法测定血清中抗核小体抗体浓度。结果与健康对照组相比,稳定期和活动期SLE患者组淋巴细胞中凋亡细胞和坏死细胞百分率均显著增加（P〈0．05）,淋巴细胞表面Fas、FasL及细胞质中活化caspase-3的表达率也显著增加（P〈0．05）。与稳定期SLE患者组相比,活动期SLE患者组淋巴细胞中坏死细胞百分率显著增加（P〈0．05）,凋亡细胞百分率略有增加但无统计学意义（P〉0．05）。活动期患者组淋巴细胞表面Fas、FasL以及细胞质中活化caspase-3的表达率略有增加但无统计学意义（P〉0．05）。活动期SLE患者组抗核小体抗体浓度显著高于健康对照组和稳定期患者组（P〈0．05）。SLE患者凋亡细胞百分率和活化caspase-3的表达率与补体C3浓度水平呈负相关关系（P〈0．05）。结论Fas信号传导通路在SLE患者淋巴细胞凋亡紊乱中发挥了重要作用。caspase-3的活化是早期提示淋巴细胞凋亡的重要信号。SLE患者淋巴细胞凋亡活化程度与疾病活动程度和免疫效应功能紊乱密切相关,而淋巴细胞凋亡异常程度与抗核小体抗体水平的高低密切相关。淋巴细胞凋亡加速在SLE患者免疫病理损伤加重和免疫细胞调控紊乱中扮演了重要角色。     

肾综合征出血热患者外周血淋巴细胞Fas/FasL表达及与血清sFas/sFasL的相关性研究

目的　探讨Fas FasL系统介导的细胞凋亡在肾综合征出血热 (HFRS)致病机理中的作用。方法　采用SABC免疫组化法检测HFRS病人外周血淋巴细胞Fas FasL的表达 ;采用ELISA法测定HFRS病人血清sFas sFasL的浓度。结果　HFRS病人发热期、低血压休克期、少尿期及多尿期外周血淋巴细胞Fas的表达明显增强 ,与正常对照组比较差异有极显著性 (P <0 .0 0 1) ;发热期、低血压休克期及少尿期淋巴细胞FasL的表达明显增强 ,与正常对照组比较差异有极显著性 (P <0 .0 0 1) ;HFRS病人各期血清sFas的浓度增高 ,与正常对照组比较差异有显著性 (P <0 .0 1) ,其中少尿期sFas的浓度最高 ,与其它各期比较差异有显著性 (P <0 .0 1) ;发热期与低血压休克期血清sFasL的浓度增高 ,与其它各期及正常对照组比较差异有显著性 (P <0 .0 1) ;淋巴细胞Fas及FasL的表达经直线相关分析(r=0 .885 5 ,P <0 .0 0 1) ,呈高度正相关 ;淋巴细胞FasL的表达与血清sFasL浓度经直线相关分析(r=0 .480 3,P <0 .0 1) ,呈高度正相关。结论　HFRS的致病过程中存在Fas FasL介导的细胞凋亡 ,sFas浓度的高低与机体损害的程度有关 ;淋巴细胞FasL的高表达及血清sFasL的浓度增高 ,由此而引起的细胞凋亡是机体清除病毒的主要机制之一     

Increased serum IL-10 in lupus patients promotes apoptosis of T cell subsets via the caspase 8 pathway initiated by Fas signaling

We sought to investigate the expression of Fas and FasL on T cell surface and caspase 8 involvement in T cell apoptosis promoted by serum IL-10 in systemic lupus erythematosus(SLE) patients.Cells and sera were obtained from 35 SLE patients.Apoptosis of T cells in patients with SLE was increased and associated with the SLE disease activity index(SLEDAI).Elevated expression of Fas and FasL on T cell surface contributed to increased apoptosis of T cells.Increased IL-10 in the sera of SLE patients was capable of inducing Fas and FasL expression on CD4~+T cell surface,promoting apoptosis of this cell subset.Decreased IL-10 serum levels and low expression of Fas were found in 5 patients of the first follow-up group after 2-month treatment.In another group with one-year treatment,the SLEDAI declined to inactive scores.Serum IL-10 was decreased significantly,and expression of Fas and FasL on T cells was also reduced.Declined apoptosis was predominant only in CD4~+T cell subset.When sera with high level of IL-10 were used to culture PBMCs from healthy controls,activated caspase 8 was elevated in CD3~+T,CD4~+T and CD8~+T cells.The study showed that serum IL-10 induced apoptosis of T cell subsets via the caspase8 pathway initiated by Fas signaling.Increased apoptosis of T cells contributes to autoantigen burden,which is pathogenic in the development of SLE.     

Combination of molecular mimicry and aberrant autoantigen expression is important for development of anti-Fas ligand autoantibodies in patients with systemic lupus erythematosus

We have reported previously that circulating anti-Fas ligand (FasL) autoantibodies able to inhibit Fas/FasL-mediated apoptosis were present in patients with systemic lupus erythematosus (SLE). In the present study, we describe the epitopes recognized by these anti-FasL autoantibodies. Rabbit antihuman antibody, raised against a FasL fragment consisting of amino acids (aa) 103-179 (fragment 2.0), inhibited Fas/FasL-mediated apoptosis, whereas an antibody against a FasL aa 103-146 fragment (fragment 1.0) did not. This suggested that an epitope around aa 146-179 was important for Fas/FasL interaction. Epitope mapping of anti-FasL autoantibodies using deletion mutants indicated that the epitope was located around aa 163-179. Three-dimensional molecular modelling of the Fas/FasL complex revealed that the aa 162-169 region was located on the outermost side of FasL, which suggested that the anti-FasL autoantibody would easily have access to the epitope. FasL point mutants involving aa positions 162-169 resulted in complete loss of apoptosis-inducing capability, which suggested that the aa 162-169 region was important for Fas/FasL interaction. A synthetic FasL peptide consisting of aa 161-170 blocked the binding of anti-FasL autoantibodies to FasL fragment 2.0 (aa 103-179). The FasL aa 161-170 sequence was found to be highly homologous with aa sequences from several infectious agents. Synthetic peptides derived from some of these microorganisms cross-reacted with the epitope recognized by the autoantibodies, suggesting that several foreign infectious agent-derived proteins may share an epitope with human FasL. As lymphocytes from SLE patients aberrartly expressed FasL, it is possible that infection by one of several infectious agents may trigger cross-reactive antibody responses, after which aberrantly expressed endogenous FasL might induce the shift from a cross-reactive response to an authentic autoimmune response. Therefore, a combination of molecular mimicry and aberrant autoantigen expression may be important for the development of anti-FasL autoantibodies in SLE patients.