Essential role for hematopoietic Fas ligand (FasL) in the suppression of melanoma lung metastasis revealed in bone marrow chimeric mice

The outcome of cancer metastasis depends on multiple interactions between the malignant cell and the host environment. Such interactions can influence primary cancer growth and metastasis by altering the balance between tumor cell proliferation and death. We have previously reported that the pro-apoptotic protein FasL could potently suppress spontaneous lung metastasis of the Fas-sensitive melanoma, K1735-P. In this report, we have constructed bone marrow chimeric mice using wt and FasL-deficient animals to delineate the source of FasL (hematopoietic or nonhematopoietic) required to control spontaneous metastatic spread from a subcutaneous tumor. Using FasL-deficient animals (gld) reconstituted with wt FasL bone marrow (wt --> gld), and wt animals reconstituted with FasL-derived bone marrow (gld --> wt), we show, for the first time, an essential role for hematopoietic-derived FasL in the suppression of K1735-P metastasis. When FasL was expressed only in the nonhematopoietic compartment (gld --> wt), K1735-P spread was ineffectively controlled with a metastatic phenotype similar to that observed in animals completely lacking FasL (gld --> gld or gld controls). These studies provide evidence for the indispensable role for FasL+ bone marrow-derived cells in the control of melanoma and offer a strategy to target FasL expression in the prevention or therapy of metastatic disease.     

Defective function of Fas in T cells from paediatric patients with autoimmune thyroid diseases

Triggering of the Fas receptor induces T cell apoptosis and is involved in shutting-off the immune response. Inherited defects impairing Fas function cause the autoimmune lymphoproliferative syndrome, and may play a role in other autoimmune diseases. The aim of this work was to analyse the Fas function in paediatric patients with thyroid autoimmunities. We found that T cells from 24/28 patients with Graves' disease (GD) and 12/35 patients with Hashimoto's thyroiditis (HT) displayed defective Fas function. In HT, the defect was more frequent in patients requiring replacement therapy (11/20) than in those not requiring (1/15); moreover, in untreated HT the highest defect was displayed by patients with the highest levels of autoantibodies. Fas was always expressed at normal levels and no Fas mutations were detected. Analysis of the healthy parents of seven Fas-resistant patients showed that several of them were Fas-resistant, which suggests a genetic component. Fusion of Fas-resistant T cells with the Fas-sensitive HUT78 T cell line generated Fas-resistant hybrid cells, which suggests the presence of molecules exerting a dominant negative effect on Fas function. Analysis of Fas-induced activation of caspase-8 and -9 showed decreased activity of both caspases in HT, whereas activity of caspase-9 was increased and that of caspase-8 was decreased in GD. These data suggest that heterogeneous inherited defects impairing Fas function favour the development of thyroid autoimmunities.     

Agonistic antibodies to Fas induce a breach in the endothelial lining of the liver and a breakdown in B cell tolerance

Liver disease can be associated with a breakdown in self-tolerance and the production of autoantibodies such as rheumatoid factors (RF), which bind to IgG. Here we investigated whether primary, non-infectious liver damage was sufficient to induce autoantibody production. We established a model of targeted liver damage induced by weekly sublethal injections of pro-apoptotic anti-Fas (CD95) antibodies. Liver damage, monitored by measurements of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, was minimal 1 week after anti-Fas injection. However, the sublethal Fas stimulation was sufficient to trigger significant haemorrhage in the liver, as assessed by Evans Blue dye leakage into the organ 5 h after anti-Fas antibody injection. We observed an induction of RF in response to the weekly injections of sublethal anti-Fas antibodies but not of isotype control antibodies, indicating a breakdown of self-tolerance induced by Fas engagement. RF induction was unlikely to be due to direct activation of B cells, as splenocytes stimulated with anti-Fas antibodies in vitro did not produce RF. These studies show that sublethal damage to the liver by Fas engagement leads to liver haemorrhage and is sufficient to trigger the breakdown of self-tolerance.     

Fas/APO-1/CD95 in health and autoimmune disease: thymic and peripheral aspects

Fas is a relative of the tumor necrosis factor receptor super–family. The receptors of this family play an important role in decisions of survival and cell death by apoptosis. It has become clear that Fas has multiple roles in the regulation of the immune response. In the peripheral immune system, Fas/Fas ligand interactions control cell–mediated cytotoxicity, activation–induced cell death and could confer immune privilege. In the thymus, it may regulate apoptosis during negative selection in a thymocyte subset. Autoimmune diseases are often associated with deregulated Fas expression through different mechanisms: genetic defects, Fas–mediated cytoxicity or up–regulation of expression as a result of lymphocyte activation. Thus, in both the peripheral and central compartments, the Fas system controls the balance between lymphocyte life and death and failure to keep this balance may contribute to autoimmune disease.     

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.     

Regulation of cell surface APO-1/Fas (CD95) ligand expression by metalloproteases

APO-1/Fas (CD95) ligand (APO-1L) induces apoptosis in sensitive target cells. Activation-induced T cell death and Ca²⁺-independent cytotoxicity in perforin knockout mice are mediated by APO-1L. To define whether APO-1L is expressed on the surface of activated T cells and to investigate the mechanisms leading to the release of a soluble form, we developed rabbit anti-APO-1L antibodies (Ab). The purified rabbit Ab detected the mature forms of the human and mouse APO-1L of approximately 42 and 40kDa. In addition, the Ab recognized the non-glycosylated form of APO-1L of approximately 32-33 kDa. In activated human T cells, the soluble form of APO-1L was detectable with a moleculas mass of 26 kDa. Immunofluorescence of three human T lymphoblastoid cell lines showed that activation of these cells by phorbol 12-myristate 13-acetate/ionomycin induced a significant increase in cell surface APO-1L only in the presence of metalloprotease inhibitors. Zn²⁺, but not Ca²⁺, prevented the increase in surface APO-1L observed in the presence of 1,10-phenanthroline. Blocking of other classes of proteases (serine- and acid-proteases, chymotrypsin) had no effect. Increased expression of surface APO-1L by metalloprotease inhibitors was not dependent on T cell activation, as the metalloprotease inhibitors also modulated the low level of constitutive APO-1L expression. These results suggest that cell surface expression of human APO-1L is regulated by Zn²⁺-dependent metalloproteases. Cleavage of surface APO-1L may act as a regulatory mechanism to prevent accumulation of the membrane-bound form and may cause systemic effects of the APO-1L.     

Agonist antibody and Fas ligand mediate different sensitivity to death in the signaling pathways of Fas and cytoplasmic mutants

We have produced three forms of human Fas: full-length Fas, Fas with a C-terminal deletion, and a chimera between extracellular Fas and the intracellular domain of the tumor necrosis factor receptor I p55 subunit. We transfected cell lines with these constructs to compare the relative capacity of antibody agonists and the physiological Fas ligand (FasL) to stimulate death. With two agonistic antibodies, the chimera is 100- to 1000-fold more sensitive to induction of death than the full-length Fas. The C-terminal deletion mutant also shows greatly enhanced death in comparison to the wild-type receptor. In contrast, when FasL is used to trigger the Fas pathway, wild-type Fas and the deletion mutant are similarly sensitive, whereas the chimera is 100-fold less susceptible to ligand-mediated killing than Fas. This demonstrates that antibody agonists and natural ligand can stimulate different signaling pathways and emphasizes the limitations of defining physiologically important signaling pathways solely by antibody agonists.     

In vivo blockade of the Fas-Fas ligand pathway inhibits cyclophosphamide-induced diabetes in NOD mice.

There is compelling evidence to show that insulin dependent diabetes ensues from selective apoptosis of pancreatic beta-cells mediated by autoreactive T-lymphocytes. The respective implication in this phenomenon of the various apoptotic pathways driven by Fas, perforin, or tumor necrosis factor is still ill- defined. Here we took advantage of the cyclophosphamide-induced model of accelerated diabetes in NOD mice to explore the physiopathological role of the Fas-Fas Ligand pathway. A single injection of cyclophosphamide (200 mg/kg) to 7-8 week-old prediabetic NOD mice triggered diabetes within 10-15 days in 85-100% of the animals. Cyclophosphamide also induced a significant decrease in spleen T cells, that was most evident by days 6-10 after treatment, and selectively affected the CD3(+)CD62L(+)compartment that includes immunoregulatory T cells. To block the in vivo Fas-Fas ligand (Fas L) interaction we administered a biologically active recombinant fusion protein coupling mouse Fas to the Fc portion of human IgG1 (FAS-Fc). Mice treated with FAS-Fc (10 doses iv of 15 microg) starting on the day of cyclophosphamide injection up to day 22, were fully protected from disease. Unexpectedly this protective effect was not due to blockade of Fas-FasL-mediated beta-cell apoptosis but rather to the inhibition of the cyclophosphamide effect on T cells. Indeed FAS-Fc treatment prevented the drug-induced T cell depletion in general and that of immunoregulatory T cells in particular. Additionally, FAS-Fc administration limited to the phase of beta-cell destruction did not afford any protection.     

Immunolocalization of Fas and Fas ligand in the ovaries of women with polycystic ovary syndrome: relationship to apoptosis

Both Fas (APO-1, CD95), an apoptosis-inducing receptor, and its ligand, Fas ligand (FasL, CD95L), have been localized to the ovary. Granulosa cell apoptosis occurs in antral follicular atresia. In polycystic ovary syndrome (PCOS), antral follicles accumulate with some atretic features. The ovarian expression of Fas and FasL was examined in PCOS by immunohistochemistry and correlated with immunodetection of apoptotic cells. Fas immunostaining was present in pre-antral follicle oocytes, some primary and secondary pre-antral follicle granulosa cells, and both granulosa and theca of antral follicles. Thecal staining persisted with advancing atresia, while granulosa staining declined. In antral follicles, abundant Fas-positive cells co-localized with scattered nuclei immunopositive for apoptosis. Ovarian vascular myocytes were strongly Fas-immunopositive. FasL immunostaining was present in pre-antral follicles in oocytes and variably in granulosa. In antral follicles, granulosa and thecal FasL staining increased with advancing atresia. Normal control ovaries showed follicular Fas and FasL staining patterns similar to those in PCOS, but vascular staining was less prominent. In one healthy follicle, Fas immunostaining was seen in the oocyte and weakly in mural granulosa and theca interna. The results suggest that in PCOS, an alteration in Fas-mediated apoptosis, does not cause abnormal folliculogenesis, but may promote ovarian vascular remodelling.     

Propofol hemisuccinate suppression of experimental autoimmune encephalomyelitis

Propofol hemisuccinate is a prodrug water soluble form of the lipophilic, phenolic compound propofol (2,6-di-isopropylphenol), that is the active ingredient in the widely used anesthetic agent Diprovan. Propofol binds to GABA_A receptors but also has a phenolic structure that confers antioxidant properties to the molecule. The effects of propofol hemisuccinate in rat experimental autoimmune encephalomyelitis (EAE) were studied using different doses and time regimes. Propofol hemisuccinate, 100 mg/kg given three times a day from day 7 or day 12 until day 16 after disease initiation, significantly reduced maximal EAE score. Histology studies supported the clinical findings demonstrating reduction in the inflammatory response in the lumbar spinal cord in animals treated with propofol hemisuccinate. Decreased levels of nitrotyrosine and unchanged levels of induced nitric oxide synthase suggest propofol hemisuccinate crossed the blood brain barrier and exerted its effects by lowering reactive oxygen species levels. The results suggest that propofol hemisuccinate may provide an alternative mode of treatment for acute exacerbations of multiple sclerosis.     

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.     

Transplantation of olfactory ensheathing cells promotes partial recovery in rats with experimental autoimmune encephalomyelitis

Objective: This study was to investigate the efficacy of olfactory ensheathing cell (OEC) transplantation on experimental autoimmune encephalomyelitis (EAE). Methods: EAE models were established by guinea pig spinal cord homogenate (GPSCH) immunization in Lewis rats. OECs were purified and cultured from the olfactory nerve layer of SD rats, and then transplanted to the EAE models through the vena caudalis (Group A) or into the lateral cerebral ventricle (Group B). Neurological function scores and body weights were daily recorded following transplantation, and histological analysis was performed to assess the pathological changes in EAE rats. Results: Cultured cells mainly exhibited bipolar or tripolar morphology, and the majority of these cells were positive for NGFR p75 staining. Neurological function scoring and the body weight measurement showed that, OEC transplantation could significantly improve the performance of EAE rats, and similar results were observed for the transplantation through the vena caudalis and into the lateral cerebral ventricle. Moreover, the transplanted OECs accumulated to the lesions in the brains of EAE rats, in spite of the different transplantation approaches. However, no significant differences in histopathology (HE and LFB staining) were observed between the OEC-transplanted groups and the control group. Conclusion: OEC transplantation could exert beneficial effects in the treatment of EAE, no matter which the cells were transplanted through the vena caudalis or into the lateral cerebral ventricle. Our findings might provide evidence for the clinical treatment of multiple sclerosis with cell transplantation.     

Irgm1基因敲除对实验性自身免疫性脑脊髓炎小鼠CD4~+ T细胞亚群的影响

目的:探讨免疫相关GTP酶1(Irgm1)基因敲除对实验性自身免疫性脑脊髓炎(Experimental autoimmune encephalomyelitis,EAE)小鼠CD4+T细胞的影响。方法:C57BL/6纯系小鼠与Irgm1基因敲除杂合小鼠(Irgm1+/-)回交十代繁殖C57BL/6背景下Irgm1+/-小鼠,用C57BL/6 Irgm1+/-小鼠杂交获取Irgm1-/-、Irgm1+/-、Irgm1+/+三种基因型小鼠,PCR扩增DNA检测基因型。用髓鞘少突胶质糖蛋白(Myelin oligodendrocyte glycoprotein,MOG33-55)多肽与弗氏完全佐剂等量混合制成乳剂,免疫C57BL/6野生型(Wt.)和Irgm1基因敲除(Irgm1-/-)小鼠,建立EAE模型,并进行临床评分。MTT法测定MOG33-55免疫后7 d的EAE小鼠淋巴结细胞中MOG33-55特异性T细胞增殖分化水平。取MOG33-55免疫后14 d EAE小鼠脊髓切片,HE染色检测炎细胞浸润情况。取MOG33-55免疫后16 d的EAE-小鼠淋巴结、脊髓和脑组织,提取单个核细胞,用MOG33-55(20μg/ml)体外刺激培养7 d,收集细胞,用流式细胞仪分析EAE小鼠淋巴结、中枢神经系统浸润细胞中Th1、Th17的改变。结果:成功诱导了EAE小鼠模型;HE染色结果显示Wt.小鼠脊髓周围出现明显炎细胞浸润,而Irgm1-/-小鼠则无明显变化;MTT实验表明Irgm1-/-小鼠与Wt.小鼠相比,淋巴结中T细胞对MOG33-55特异性增殖能力降低;流式细胞分析表明相对于Wt.小鼠,Irgm1-/-小鼠EAE模型在淋巴结及中枢神经系统浸润细胞中Th1细胞亚群比例明显增高而Th17细胞亚群比例下降。结论:Irgm1基因敲除可部分保护EAE小鼠的脊髓功能及临床症状。在EAE发病早期,Irgm1可能起到了关键性的作用,因此Irgm1有可能成为EAE治疗的重要分子靶点。     

Inhibition of CD95 (Fas/Apo1)-mediated apoptosis by vaccinia virus WR

Stimulation of the CD95 (Apo-1/Fas) molecule either by the CD95 ligand or by monoclonal antibodies induces programmed cell death by apoptosis in a variety of cell lines and primary cells. In this study we observed that infection of B lymphoblast and T lymphoblast cell lines with vaccinia virus strain WR and recombinant vaccinia WR constructs, but not strain Copenhagen, rendered cells refractory to CD95-mediated apoptosis. In particular, vaccinia virus infection suppressed anti-CD95 antibody-induced membrane disintegration, apoptotic nuclear morphology of cells, and DNA fragmentation. Inhibition of apoptosis was not mediated by CD95 down-regulation or reduced binding of anti-CD95 antibody to infected cells, and occurred at a time point when cellular metabolism was not yet affected by the lytic vaccinia virus infection. Vaccinia virus (WR)-infected cells were resistant to CD95 ligand–CD95-mediated lysis by CD4⁺ and CD8⁺, T lymphocytes. Because cytolysis mediated by CD95 is one of two major mechanisms used by cytotoxic T lymphocytes to kill target cells, inhibition of CD95-mediated apoptosis may constitute a novel immune escape mechanism for this virus. Additionally, this mechanism may contribute to the higher pathogenicity of vaccinia virus strain WR compared with strain Copenhagen.     

Differential regulation of TRAIL and CD95 ligand in transformed cells of the T and B lymphocyte lineage

TRAIL (APO-2 ligand) and CD95L (CD95/APO-1/Fas ligand) share the highest homology among the TNF family members and the ability to induce apoptosis. These similarities raise the issue of a potential functional redundancy between the two ligands. We have previously shown that CD95L-resistant cells may be sensitive to TRAIL, even though apoptosis induced by both ligands is blocked by caspase inhibitors. Here we investigated TRAIL protein expression in cells of T and B origin and compared its regulation of expression with that of CD95L. A rabbit antibody (Ab) to a peptide sequence in the extracellular region of TRAIL identified recombinant TRAIL (rTRAIL) produced by Sf9 cells as a protein of approximately 32 – 33 kDa and soluble rTRAIL as a 19 – 20-kDa protein. In human and mouse cells, the Ab identified a 33 – 34-kDa and an additional 19 – 20-kDa protein only in human cells. Both transformed cells of the T and B lymphocyte lineage were found to react with the anti-TRAIL Ab by immunoblot analysis and surface staining. The majority of the cells analyzed co-expressed TRAIL and CD95L. Two cell lines showed a mirror-pattern, one being TRAIL^high CD95L^low and the other TRAIL^low CD95L^high, thus suggesting the existence of a cell type-specific regulation of expression of the two ligands. Differently from CD95L, surface TRAIL was not up-regulated by any of the metalloprotease inhibitors tested, independently of the cell type analyzed. Conversely, reactivity with the anti-TRAIL but not with the anti-CD95L Ab was enhanced by cysteine protease inhibitors. An in vitro cleavage assay showed that generation of soluble rTRAIL was dependent on the functional activity of cysteine proteases, as it was blocked by leupeptin and E64 but not by the metalloprotease inhibitor 1,10-phenanthroline. Thus, even though TRAIL and CD95L share structural and functional properties, they have unique properties as they differ in their regulatory pathways, i. e. cell-type-dependent expression and sensitivity to protease inhibitors.