Matrix metalloproteinases and soluble Fas/FasL system as novel regulators of apoptosis in children and young adults on chronic dialysis

The system of membrane receptor Fas and its ligand FasL compose one of the main pathways triggering apoptosis. However, the role of their soluble forms has not been clarified yet. Although sFasL can be converted from the membrane-bound form by matrix metalloproteinases (MMPs), there are no data on relations between sFas/sFasL, MMPs and their tissue inhibitors (TIMPs) in patients on chronic dialysis—neither children nor adults. The aim of our study was to evaluate serum concentrations of sFas, sFasL, and their potential regulators (MMP-2, MMP-7, MMP-9, TIMP-1, TIMP-2), in children and young adults chronically dialyzed. Twenty-two children on automated peritoneal dialysis (APD), 19 patients on hemodialysis (HD) and 30 controls were examined. Serum concentrations of sFas, sFasL, MMPs and TIMPs were assessed by ELISA. Median values of sFas, sFasL, sFas/sFasL ratio, MMP-2, MMP-7, MMP-9, TIMP-1 and TIMP-2 were significantly elevated in all dialyzed patients vs. controls, the highest values being observed in subjects on HD. A single HD session caused the decrease in values of all parameters to the levels below those seen in children on APD. Regression analysis revealed that MMP-7 and TIMP-1 were the best predictors of sFas and sFasL concentrations. Children and young adults on chronic dialysis are prone to sFas/sFasL system dysfunction, more pronounced in patients on hemodialysis. The correlations between sFas/sFasL and examined enzymes suggest that MMPs and TIMPs take part in the regulation of cell death in the pediatric population on chronic dialysis, triggering both anti- (sFas) and pro-apoptotic (sFasL) mechanisms.     

Soluble Fas/Apo-1 (CD95) levels during T cell activation in the presence of acute myelogenous leukemia accessory cells; contributions from local release and variations in systemic levels

Fas (CD95/Apo-1) exists both in membrane-bound and in biologically active soluble (s) forms. Ligation of membrane-expressed Fas can induce apoptosis, and Fas-mediated signaling seems to be involved in T-cell-induced apoptosis of human acute myelogenous leukemia (AML) blasts. The local release of sFas by AML blasts may then function as a protective mechanism by competing with membrane-bound Fas for binding sites on the common Fas ligand (FasL). sFas was released by AML blasts during in vitro culture, and this release was modulated by several cytokines that can be secreted by activated T cells. Increased levels of sFas could be detected during in vitro activation of T cells in the presence of native AML accessory cells, and this was observed both for (i) mitogenic activation of CD4⁺ and CD8⁺ T cell clones derived from acute leukemia patients with therapy-induced leukopenia and (ii) allostimulated activation of T cells derived from normal donors. However, local in vivo levels of sFas will also be influenced by variations in systemic levels. High serum levels of sFas were detected in acute leukemia patients during chemotherapy-induced cytopenia, but these levels decreased during complicating bacterial infections. In contrast, serum levels of sFasL were normal in leukopenic patients. The present results support the hypothesis that local release of sFas can function as a protective mechanism against AML-reactive T cells, but the effects of this local release are, in addition, modulated by variations in systemic levels of sFas (but not sFasL). Received: 9 March 2000 / Accepted: 25 May 2000     

Temporal evolution of soluble Fas and Fas ligand in patients with orthotopic liver transplantation

Aim: The aim of this study was to analyze the expression levels of plasma soluble Fas (sFas) and soluble Fas ligand (sFasL) in patients with orthotopic liver transplantation (OLT) procedures routinely performed without venovenous bypass. Methods: The sFas and sFasL were analyzed in the blood of 20 consecutive patients who underwent transplantation. Blood samples were drawn from the radial artery at serial time points before, during, and after surgery. Plasma levels of sFas and sFasL were detected by Enzyme Linked-Immuno-Sorbent Assay. Plasma aspartate transaminase (AST) and alanine transaminase (ALT) were assayed by routine clinical chemistry testing. Results: Marked elevation of plasma AST and ALT were detected at the reperfusion and postoperation time points (P < 0.001), with a peak on the first postoperative day. The mean plasma concentration of sFas and sFasL remained unchanged from preoperative to anhepatic phase (T1 to T3) (P  0.268). The sFas and sFasL concentrations were significantly higher at 15 and 60 min after reperfusion compared to the preoperative value (P  0.048). Postoperatively, sFas and sFasL concentration were decreased to preoperative levels on the first postoperative day (P  0.127). Conclusion: The sFas and sFasL seem to be involved in reperfusion injury during OLT. The understanding of Fas may provide new insights into the mechanisms of ischemia/reperfusion injury during OLT.     

Serum soluble Fas ligand (sFasL) in patients with primary squamous cell carcinoma of the esophagus

Esophageal carcinomas have been shown to express Fas ligand (FasL) and down-regulate Fas to escape from host immune surveillance. Circulating soluble FasL (sFasL) has been suggested to provide protection from Fas-mediated apoptosis. The aim of this study was to assess serum sFasL levels in esophageal cancer. The pretreatment levels of sFasL in the serum of 100 patients with esophageal squamous cell cancer and 41 healthy volunteers were determined by ELISA. Probability of survival was calculated according to the method of Kaplan-Meier. The prognostic influence of high and low level of sFasL was analyzed with the log-rank test. The mean serum level of sFasL in patients with esophageal cancer was significantly higher than that in healthy donors (1.567+/-1.786 vs 0.261+/-0.435, p<0.0001). The levels of serum sFasL were significantly higher in advanced stages (II vs IV p<0.034; III vs IV p<0.041; except II vs III p=0.281), patients with lymph node (N0 vs N1 p<0.0389) or distant (M0 vs. M1 p<0.0388) metastases and significantly lower in patients with well differentiated tumors (G1 vs G2 p<0.0272). The serum levels of soluble FasL were not related to gender, age, tumor size, T-stage, tobacco smoking and history of chronic alcohol intake. The survival difference between pretreatment high and low level of sFasL in surgery and chemio- and/or radiotherapy group was not statistically significant (p=0.525; p=0.840). Our results indicate that elevated serum sFasL levels might be associated with a disease progression in patients with esophageal squamous cell carcinoma.     

The metalloproteinase matrilysin proteolytically generates active soluble Fas ligand and potentiates epithelial cell apoptosis

BACKGROUND: The Fas ligand/Fas receptor (FasL/Fas) system is an important mediator of apoptosis in the immune system where the juxtaposition of cells expressing the cell-surface ligand induces the apoptotic pathway in Fas-expressing lymphocytes. The FasL/Fas system has also been shown to be involved in apoptosis in epithelial tissues, including the involuting rodent prostate. FasL can be shed through the action of an hitherto unidentified metalloproteinase to yield soluble FasL (sFasL), although the biological activity of sFasL has been disputed. RESULTS: Here we report that the matrix metalloproteinase matrilysin can process recombinant and cell-associated FasL to sFasL, and that matrilysin-generated sFasL was effective at inducing apoptosis in a target epithelial cell population. In the involuting mouse prostate, FasL and matrilysin colocalized to the cell surface in a restricted population of epithelial cells. Mice deficient in matrilysin demonstrated a 67% reduction in the apoptotic index in the involuting prostate compared with wild-type animals, implicating matrilysin in this FasL-mediated process. CONCLUSIONS: The results show that a functional form of sFasL was generated by the action of the metalloproteinase matrilysin, and suggest that matrilysin cleavage of FasL is an important mediator of epithelial cell apoptosis.     

Serum Levels of Soluble Fas and Fas Ligand in Iranian Women with Pre-eclampsia

Background:The precise responsible mechanism of pre-eclampsia remains controversial however, recent data suggest a main role of the abnormal activation of the adaptive immune system and Apoptosis. In this study, we have measured serum levels of Fas/Fasl as two important members of extrinsic apoptotic pathway in patient with pre-eclampsia.Methods:207 participants including 99 pre-eclampsia patients and 108 age and sex-matched normal pregnant women were involved in the case-control study. Plasma sample from each participant was collected and stored at –20 °C until batch processing.Serum levels of Fas and Fas ligand were measured by ELISA for each participant including 99 pre-eclampsia patients and 108 normal pregnant women. Following a test of statistical normality, nonparametric data were analyzed by Mann-Whitney.Results:sFas levels in case group was significantly higher than controls; 584 (397-892) pg/ml in cases opposed to 341 (213-602) pg/ml in controls (p value< 0.01). sFasL in pre-eclampsia women was a little lower than controls; 255 (173-318) pg/ml and in case group compared to 265.5 (184-381.5) pg/ml in controls.Conclusion:We have found the increased levels of sFas in patients with pre-eclampsia in compare with the healthy pregnant women. It seems that abnormality in sFAS is related with pre-eclampsia.Key Words: Pre-eclampsia, Pregnancy, sFAS, sFASL, Apoptosis     

Soluble Fas ligand inhibits angiogenesis in rheumatoid arthritis

The characteristics of rheumatoid arthritis (RA) pathology include the infiltration of inflammatory leukocytes, the proliferation of synovial cells, and the presence of extensive angiogenesis, referred to as rheumatoid pannus. Fas ligand is critical to the homeostatic regulation of the immune response, but its role in the angiogenic process of RA remains to be defined. In this study, we investigated whether soluble Fas ligand (sFasL) induces synoviocyte apoptosis and regulates angiogenesis of endothelial cells in RA. The levels of sFasL were elevated in the synovial fluids of RA patients when compared to those of osteoarthritis (OA) patients, and they correlated inversely with vascular endothelial growth factor₁₆₅ (VEGF₁₆₅) concentrations. sFasL, ranging from 10 to 100 ng/ml, induced the apoptosis of RA fibroblast-like synoviocytes (FLS) in vitro, and thereby decreased VEGF₁₆₅ production. In addition, sFasL inhibited VEGF₁₆₅-induced migration and chemotaxis of endothelial cells to basal levels in a manner independent of the Fas-mediated cell death. sFasL dose-dependently suppressed the VEGF₁₆₅-stimulated increase in pAkt expression in endothelial cells, which might be associated with its anti-migratory effect on endothelial cells. Moreover, sFasL strongly inhibited neovascularization in the Matrigel plug in vivo. Our data suggest that sFasL shows anti-angiogenic activity within RA joints not only by inducing apoptosis of VEGF₁₆₅-producing cells but also by blocking VEGF₁₆₅-induced migration of endothelial cells, independent of Fas-mediated apoptosis.     

Hsp27 as a marker of cell damage in children on chronic dialysis

Intracellular heat shock protein (Hsp) 27 is a potent anti-apoptotic factor that, among other activities, prevents the binding of membrane receptor Fas to its ligand FasL. However, the potential role of extracellular Hsp27 and possibilities to control it have not been clarified. Moreover, there are no data on relations between Hsp27, sFas/sFasL system, matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in patients with chronic kidney disease (CKD)—neither children nor adults. The aim of this study was to evaluate serum concentrations of Hsp27 and their potential regulators (sFas, sFasL, MMP-7, TIMP-1) in children with CKD and on chronic dialysis. Twenty-six CKD children stage 5 still on conservative treatment, 19 patients on hemodialysis (HD), 22 children on automated peritoneal dialysis (APD), and 30 controls were examined. Serum concentrations of Hsp27, sFas, sFasL, MMP-7, and TIMP-1 were assessed by ELISA. Median values of Hsp27 were significantly elevated in all dialyzed patients vs. those in pre-dialysis period and vs. controls, the highest values being observed in subjects on HD. Regression analysis revealed that MMP-7, TIMP-1, sFas, and sFasL were the best predictors of Hsp27 concentrations in dialyzed patients. Children with CKD are prone to Hsp27 dysfunction, aggravated by the dialysis commencement, and more pronounced in patients on hemodialysis. Correlations between Hsp27 and examined parameters suggest the potential role for Hsp27 as a marker of cell damage in the pediatric population on chronic dialysis.     

Neutrophils induce apoptosis of lung epithelial cells via release of soluble Fas ligand

Neutrophils release soluble Fas ligand (sFasL), which can induce apoptosis in certain Fas-bearing cell types (Liles WC, Kiener PA, Ledbetter JA, Aruffo A, and Klebanoff SJ. J Exp Med 184: 429-440, 1996). We hypothesized that neutrophils could induce alveolar epithelial apoptosis via release of sFasL. A549 pulmonary adenocarcinoma cells expressed surface Fas and underwent cell death (10 +/- 7% viability) and DNA fragmentation (354 +/- 98% of control cells) when incubated with agonistic CD95/Fas monoclonal antibody (P < 0.05). Coincubation with human neutrophils induced significant A549 cell death at 48 (51 +/- 9% viability; P < 0.05) and 72 h (25 +/- 10%; P < 0.05) and increased DNA fragmentation (178 +/- 42% of control cells; P < 0.05), with morphological characteristics of apoptosis. The addition of antioxidants did not inhibit apoptosis. sFasL concentrations were maximally increased in coculture medium at 24 h (4.9 +/- 0.7 ng/ml; P < 0.05). Neutrophil-induced A549 cell apoptosis was blocked by inhibitory anti-Fas (42 +/- 6% of control cells; P < 0.05) and anti-FasL monoclonal antibodies (29 +/- 3%; P < 0.05). Human neutrophils and Fas similarly affected murine primary alveolar epithelial cell bilayers, and caspase activation occurred in response to Fas exposure. We conclude that neutrophils undergoing spontaneous apoptosis induce A549 cell death and DNA fragmentation, independent of the oxidative burst, that is mediated by sFasL.     

Inhibition of metalloproteinase cleavage enhances the cytotoxicity of Fas ligand

The Fas ligand (FasL)/Fas receptor (CD95) pathway is an important mediator of apoptosis in the immune system and can also mediate cancer cell death. Soluble FasL (sFasL), shed from the membrane-bound form of the molecule by a putative metalloproteinase (MP), may function to locally regulate the activity of membrane-bound FasL. Using a replication-defective recombinant adenovirus-expressing FasL (RAdFasL), we identified a variable ability of different carcinoma cells to respond to FasL-induced cytotoxicity and to shed sFasL. Blockade of FasL cleavage with an MP inhibitor significantly enhanced RAdFasL-induced apoptosis suggesting that sFasL may antagonize the effect of membrane-bound FasL. In support of this concept, a recombinant adenovirus expressing a noncleavable form of FasL (RAdD4) was found to be a potent inducer of apoptosis even at very low virus doses. Our results highlight the therapeutic potential of noncleavable FasL as an antitumor agent and emphasize the important role of MP via the production of sFasL in regulating the response of the Fas pathway. Moreover, these findings have general implications for the therapeutic exploitation of TNF family ligands and for the possible impact of MP-based therapies on the normal physiology of Fas/TNF pathways.     

The abnormal apoptosis of T cell subsets and possible involvement of IL-10 in systemic lupus erythematosus

To study the apoptosis of lymphocyte subpopulations in systemic lupus erythematosus (SLE) patients and the possible role of IL-10 in this apoptosis involved in the pathogenesis of SLE, three color fluorescence and flow cytometry were used to investigate the early apoptosis of lymphocyte subsets from freshly separated or cultured peripheral blood mononuclear cells (PBMCs). ELISA was employed to detect the levels of IL-10 in serum and the levels of sFas and sFasL in cultured PBMC supernatants, and the results of sFas and sFasL were confirmed by real-time PCR of Fas and FasL mRNA. The results showed that in cells from SLE patients, the apoptosis of CD3+, CD4+, and CD8+ T cells was distinctly increased, and the percentage of CD4+ cells and the CD4/CD8 ratio was significantly decreased, as compared with normal controls. The apoptosis of T lymphocytes cultured with SLE serum was markedly higher than that of cells cultured with control's serum. Blockade of interleukin-10 (IL-10) activation by an anti-IL-10 antibody reduced the SLE serum induced apoptosis of CD4+ and CD8+ T cells. The levels of sFas and sFasL in the culture supernatant and Fas and FasL mRNA expressions in cultured cells were significantly higher in the SLE serum-cultured groups, but decreased evidently in the presence of the anti-IL-10 antibody. Above findings suggested that SLE cells showed abnormally high apoptosis of T lymphocytes, especially of the CD4+ subpopulation, resulting in a decreased CD4/CD8 ratio. The high percentage of apoptotic T cells in SLE patients may be related to the high levels of IL-10 in SLE serum, as IL-10 may induce the abnormally activated T cells to trigger apoptosis via the Fas-FasL pathway.     

Osteoprotegerin Induces Apoptosis of Osteoclasts and Osteoclast Precursor Cells via the Fas/Fas Ligand Pathway

Osteoprotegerin (OPG) is known to inhibit differentiation and activation of osteoclasts (OCs) by functioning as a decoy receptor blocking interactions between RANK and RANKL. However, the exact role of OPG in the survival/apoptosis of OCs remains unclear. OPG caused increased rates of apoptosis of both OCs and osteoclast precursor cells (OPCs). The expression of Fas and activated caspase-8 was increased by both 20 ng/mL and 40 ng/mL of OPG, but was markedly decreased at 80 ng/mL. Interestingly, we noted that while levels of Fas ligand (FasL) increased with increasing doses of OPG, the soluble form of FasL in the supernatant decreased. The results of a co-immunoprecipitation assay suggested that the decrease of sFasL might be caused by the binding of OPG. This would block the inhibition of the apoptosis of OCs and OPCs. Furthermore, changes in expression levels of Bax/Bcl-2, cleaved-caspase-9, cleaved-caspased-3 and the translocation of cytochrome c, illustrated that OPG induced apoptosis of OCs and OPCs via the classic Fas/FasL apoptosis pathway, and was mediated by mitochondria. Altogether, our results demonstrate that OPG induces OCs and OPCs apoptosis partly by the Fas/FasL signaling pathway.     

Severe malarial anemia associated with increased soluble Fas ligand (sFasL) concentrations in Gabonese children

To investigate if severe malarial anemia is associated with specific cytokine overproduction, we evaluated serum levels of soluble Fas ligand (sFasL), tumor necrosis factor (TNF-alpha) and interleukin-10 (IL-10) from three groups of young children with Plasmodium falciparum infection (asymptomatic cases, uncomplicated malaria cases and severe malarial anemia cases), in a hyperendemic area of Gabon. In uncomplicated cases, only TNF levels were significantly (p < 0.001) increased in comparison to asymptomatic cases with P. falciparum infection. High levels of sFasL, TNF-alpha and IL-10 were associated with low hemoglobin concentrations, sFasL levels were significantly higher in children with severe malarial anemia (p < 0.001) as compared to both other groups. The parasite density was positively correlated with IL-10, TNF-alpha and sFasL levels. TNF-alpha and sFasL, but not IL-10 or parasitemia, were independent predictors of hemoglobin concentrations. These results suggest that, in malaria, a specific dysregulation of the cytokine balance may lead to complications such as severe anemia.     

一种小鼠可溶性Fas cDNA的克隆与表达

为获得调节鼠Fas Fas配体系统诱导凋亡的作用 ,根据GenBank中小鼠Fas基因碱基序列 ,设计扩增可溶性Fas(solubleFas ,sFas)引物 ,用RT PCR方法从幼鼠胸腺组织中克隆出与人可溶性Fas类似的新的鼠sFas (FasC)cDNA序列 .该序列缺乏Fas基因的跨膜区片段 ,但不改变其阅读框 .采用定向克隆的方法将之插入pUC19中间载体 ,DNA序列测定证实该片段序列与预期序列完全一致 .利用亚克隆的方法将鼠FasC的cDNA片段克隆到真核表达载体pCA13上 ,构建出重组载体pCA13 FasC ,通过脂质体LF2 0 0 0转染至 2 93细胞 .RT PCR和Western印迹证实 ,鼠FasC在 2 93细胞获得高效表达 .凋亡诱导实验表明 ,鼠FasC的表达可阻断Fas诱导凋亡的作用 ,证实了所转染鼠FasC的生物活性 .     

Soluble Fas ligand induces epithelial cell apoptosis in humans with acute lung injury (ARDS).

The goals of this study were to determine whether the Fas-dependent apoptosis pathway is active in the lungs of patients with the acute respiratory distress syndrome (ARDS), and whether this pathway can contribute to lung epithelial injury. We found that soluble Fas ligand (sFasL) is present in bronchoalveolar lavage (BAL) fluid of patients before and after the onset of ARDS. The BAL concentration of sFasL at the onset of ARDS was significantly higher in patients who died. BAL from patients with ARDS induced apoptosis of distal lung epithelial cells, which express Fas, and this effect was inhibited by blocking the Fas/FasL system using three different strategies: anti-FasL mAb, anti-Fas mAb, and a Fas-Ig fusion protein. In contrast, BAL from patients at risk for ARDS had no effect on distal lung epithelial cell apoptosis. These data indicate that sFasL is released in the airspaces of patients with acute lung injury and suggest that activation of the Fas/FasL system contributes to the severe epithelial damage that occurs in ARDS. These data provide the first evidence that FasL can be released as a biologically active, death-inducing mediator capable of inducing apoptosis of cells of the distal pulmonary epithelium during acute lung injury.     

Transforming growth factor beta1 and soluble Fas serum levels in hepatocellular carcinoma

In this study we assessed the usefulness of serum Transforming Growth Factor-beta1 (TGF-beta1) and soluble Fas (sFas) in distinguishing liver cirrhosis (LC) with and without hepatocellular carcinoma (HCC) as compared with alpha-fetoprotein (AFP). Serum TGF-beta1 and sFas levels were measured by ELISA in 51 LC patients, 54 patients with HCC and 30 healthy donors. Considering as a cut-off limit (mean+1SD of controls) 74 pg/ml and 637 pg/ml for TGF-beta1 and sFas, respectively, we computed serum concentrations of TGF-beta1 and sFas as a score (mean+/-SD). The positive frequency of serum TGF-beta1 levels in HCC patients (54%) was greater than in LC patients (26%) and healthy donors (3%). TGF-beta1 levels were higher in HCC (1.6+/-0.5) than in LC (1.1+/-0.2) (P<0.0001) and healthy donors (0.6+/-0.2). Using a cut-off limit of 82 pg/ml (mean+2SD), the positive frequency of TGF-beta1 was 20% in HCC patients. None of the controls and LC patients had TGF-beta1 levels higher than 82 pg/ml. The positive frequency of serum sFas levels was 100% in HCC patients, 98% in LC patients and 3% in healthy controls. Serum sFas levels were higher in HCC (2.5+/-0.7) than in LC (1.9+/-0.5) (P<0. 001) and healthy donors (0.6+/-0.3). No significant change of positive frequency was obtained by setting sFas cut-off at higher levels. sFas values did not correlate with TGF-beta1 levels. No relationship was found between TGF-beta1 amounts and AFP levels. However, in the 23% of HCC patients, with normal AFP values TGF-beta1 levels were higher than the cut off. These findings suggest the potential usefulness for TGF-beta1 assay in AFP-negative HCC.     

A membrane-bound Fas decoy receptor expressed by human thymocytes

Human thymocytes at several stages of maturation express Fas, yet resist apoptosis induction through its ligation. A proximal step in apoptotic signaling through Fas is implicated in this resistance, as these cells undergo normal levels of apoptosis induction after exposure to tumor necrosis factor-alpha. We studied the Fas receptors expressed in human thymocytes to search for mechanisms of receptor-mediated inhibition of Fas signaling in these cells. We describe here a unique, membrane-bound form of Fas receptor that contained a complete extracellular domain of Fas but that lacked a death domain due to alternative splicing of exon 7. This Fas decoy receptor (FDR) was shown to have nearly wild-type ability to bind native human Fas ligand and was expressed predominantly at the plasma membrane. Unlike soluble forms of Fas receptor, FDR dominantly inhibited apoptosis induction by Fas ligand in transfected human embryonic kidney cells. Titration of FDR in Fas-expressing cells suggests that FDR may operate through the formation of mixed receptor complexes. FDR also dominantly inhibited Fas-induced apoptosis in Jurkat T cells. In mixing experiments with wild-type Fas, FDR was capable of inhibiting death signaling at molar ratios less than 0.5, and this relative level of FDR:wild type message was observed in at least some thymocytes tested. The data suggest that Fas signal pathways in primary human cells may be regulated by expression of a membrane-bound decoy receptor, analogous to the regulation of tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis by decoy receptors.     

Age related changes of soluble Fas, Fas ligand and Bcl-2 in autoimmune thyroid diseases

INTRODUCTION: Apoptosis plays a pivotal role in the regulation of immune mechanisms in the pathogenesis of autoimmune thyroid diseases (AITD). The prevalence of AITD increases with age. Purpose to compare soluble Fas, FasL and Bcl-2 in Graves disease (GD) and Hashimoto thyroiditis (HT) in relation to the age of the studied patients. MATERIAL AND METHODS: 3 groups of subjects: 1/25 patients with GD in euthyreosis on methimazol 2/27 patients with ChH in euthyreosis on levothyroxine. 3/12 healthy volunteers age and sex-matched to group 1-2. The serum levels of Fas, FasL and Bcl-2 were determined by the ELISA kit. RESULTS: We found positive correlations between sFas and age in GD patients (r = 0.35; p < 0.05). In GD patients we found a negative correlation between sFasL and age in all studied patients with AITD (r = -0.34; p < 0.01). We also found a negative correlation between sBcl-2 and age in HT patients (r = -0.42; p < 0.05). CONCLUSIONS: Fas/FasL and Bcl-2 signaling pathways seem to be age-related and may explain, at least in part, milder course of Graves disease in elderly patients and increased prevalence of Hashimoto disease in this group of subjects.     

Non-apoptotic signaling pathways activated by soluble Fas ligand in serum-starved human fibroblasts. Mitogen-activated protein kinases and NF-kappaB-dependent gene expression

Many Fas-expressing cells do not undergo cell death upon Fas stimulation. In the normal human diploid cell line GM6112, the addition of soluble Fas ligand (sFasL) leads to morphological signs of cell death in less than 1% of cells. Treatment of serum-starved GM6112 fibroblasts with sFasL resulted in a rapid and transient phosphorylation of ERK1/2 without a significant increase in JNK and p38 activities. Unless co-treated with the protein synthesis inhibitor anisomycin, sFasL did not show gene-inducing activity in cells maintained in complete medium. However, when cells were serum-starved for 4 days, treatment with sFasL alone induced interleukin-6 gene expression and, less strongly, interleukin-8 gene expression. Sensitization of the gene-inducing activity by serum starvation correlated with NF-kappaB activation by sFasL. Furthermore, we found that the expression of FADD and caspase-8 was significantly reduced in serum-starved cells, whereas the level of cFLIP remained unchanged. Transfection of GM6112 cells with the antisense caspase-8 expression construct sensitized cells toward sFasL-induced NF-kappaB-dependent reporter activation. Our results support the notion that a change in the ratio of cFLIP and caspase-8 may be responsible for turning on the Fas-activated NF-kappaB pathway, which otherwise is supplanted by the death-inducing pathway.