FasL Shedding Research Articles

Subcellular localization and activation of ADAM proteases in the context of FasL shedding in T lymphocytes

The “A Disintegrin And Metalloproteinases” (ADAMs) form a subgroup of the metzincin endopeptidases. Proteolytically active members of this protein family act as sheddases and govern key processes in development and inflammation by regulating cell surface expression and release of cytokines, growth factors, adhesion molecules and their receptors. In T lymphocytes, ADAM10 sheds the death factor Fas Ligand (FasL) and thereby regulates T cell activation, death and effector function. Although FasL shedding by ADAM10 was confirmed in several studies, its regulation is still poorly defined. We recently reported that ADAM10 is highly abundant on T cells whereas its close relative ADAM17 is expressed at low levels and transiently appears at the cell surface upon stimulation. Since FasL is also stored intracellularly and brought to the plasma membrane upon stimulation, we addressed where the death factor gets exposed to ADAM proteases. We report for the first time that both ADAM10 and ADAM17 are associated with FasL-containing secretory lysosomes. Moreover, we demonstrate that TCR/CD3/CD28-stimulation induces a partial positioning of both proteases and FasL to lipid rafts and only the activation-induced raft-positioning results in FasL processing. TCR/CD3/CD28-induced FasL proteolysis is markedly affected by reducing both ADAM10 and ADAM17 protein levels, indicating that in human T cells also ADAM17 is implicated in FasL processing. Since FasL shedding is affected by cholesterol depletion and by inhibition of Src kinases or palmitoylation, we conclude that it requires mobilization and co-positioning of ADAM proteases in lipid raft-like platforms associated with an activation of raft-associated Src-family kinases.

Differential Surface Expression of ADAM10 and ADAM17 on Human T Lymphocytes and Tumor Cells

A disintegrin and metalloproteases (ADAMs) have been implicated in many processes controlling organismic development and integrity. Important substrates of ADAM proteases include growth factors, cytokines and their receptors and adhesion proteins. The inducible but irreversible cleavage of their substrates alters cell-cell communication and signaling. The crucial role of ADAM proteases (e.g. ADAM10 and 17) for mammalian development became evident from respective knockout mice, that displayed pre- or perinatal lethality with severe defects in many organs and tissues. Although many substrates for these two ADAM proteases were identified over the last decade, the regulation of their surface appearance, their enzymatic activity and their substrate specificity are still not well understood. We therefore analyzed the constitutive and inducible surface expression of ADAM10 and ADAM17 on a variety of human T cell and tumor cell lines. We demonstrate that ADAM10 is constitutively present at comparably high levels on the majority of the tested cell types. Stimulation with phorbol ester and calcium ionophore does not significantly alter the amount of surface ADAM10, except for a slight down-regulation from T cell blasts. Using FasL shedding as a readout for ADAM10 activity, we show that PKC activation and calcium mobilization are both prerequisite for activation of ADAM10 resulting in a production of soluble FasL. In contrast to ADAM10, the close relative ADAM17 is detected at only low levels on unstimulated cells. ADAM17 surface expression on T cell blasts is rapidly induced by stimulation. Since this inducible mobilization of ADAM17 is sensitive to inhibitors of actin filament formation, we propose that ADAM17 but not ADAM10 is prestored in a subcellular compartment that is transported to the cell surface in an activation- and actin-dependent manner.

Fas/FasL-dependent and -independent activation of caspase-8 in doxorubicin-treated human breast cancer MCF-7 cells: ADAM10 down-regulation activates Fas/FasL signaling pathway

The contribution of Fas-mediated death pathway to doxorubicin-induced death of MCF-7 cells is not unambiguously elucidated. Thus, this study was conducted to explore doxorubicin-induced Fas/FasL signaling pathway activation in MCF-7 cells and doxorubicin-resistant MCF-7 (MCF-7/Dox) cells. Doxorubicin-induced caspase-8 activation was found to be mediated through Akt/ERK inactivation and FasL-independent Fas pathway in MCF-7 cells, while caspase-8 activation in MCF-7/Dox cells depended exclusively on FasL-stimulated Fas pathway. Suppression of caspase-8 activation restored the viability of doxorubicin-treated MCF-7 cells and MCF-7/Dox cells. Contrary to FasL surface expression exclusively detected in MCF-7/Dox cells, intracellular FasL expression was noted with MCF-7 cells. Promotion of FasL translocation to the cell surface by lysophosphatidic acid evoked a FasL-activated Fas death pathway in MCF-7 cells. Doxorubixin-evoked β-TrCP up-regulation promoted Sp1 degradation, which subsequently suppressed ADAM10 expression in MCF-7 and MCF-7/Dox cells. Doxorubicin-induced down-regulation of ADAM10 reduced FasL shedding, leading to Fas pathway activation in MCF-7/Dox cells. Knock-down of ADAM10 induced death in MCF-7/Dox cells, but marginally reduced the viability of MCF-7 cells. Taken together, our data indicate that Akt/ERK-mediated caspase-8 activation and Fas/FasL-mediated caspase-8 activation mostly elucidate doxorubicin-induced death in MCF-7 cells and MCF-7/Dox cells, respectively. These observations suggest a promising therapeutic modality for overcoming doxorubicin-resistant breast cancer by targeting ADAM10 sheddase activity.

FasL shedding is reduced by hypothermia in experimental stroke

Protection by mild hypothermia has previously been associated with better mitochondrial preservation and suppression of the intrinsic apoptotic pathway. It is also known that the brain may undergo apoptotic death via extrinsic, or receptor-mediated pathways, such as that triggered by Fas/FasL. Male Sprague-Dawley rats subjected to 2 h middle cerebral artery occlusion with 2 h intraischemic mild hypothermia (33 degrees C) were assayed for Fas, FasL and caspase-8 expression. Ischemia increased Fas, but decreased FasL by approximately 50-60% at 6 and 24 h post-insult. Mild hypothermia significantly reduced expression of Fas and processed caspase-8 both by approximately 50%, but prevented ischemia-induced FasL decreases. Fractionation revealed that soluble/shed FasL (sFasL) was decreased by hypothermia, while membrane-bound FasL (mFasL) increased. To more directly assess the significance of the Fas/FasL pathway in ischemic stroke, primary neuron cultures were exposed to oxygen glucose deprivation. Since FasL is cleaved by matrix metalloproteinases (MMPs), and mild hypothermia decreases MMP expression, treatment with a pan-MMP inhibitor also decreased sFasL. Thus, mild hypothermia is associated with reduced Fas expression and caspase-8 activation. Hypothermia prevented total FasL decreases, and most of it remained membrane-bound. These findings reveal new observations regarding the effect of mild hypothermia on the Fas/FasL and MMP systems.

Tissue inhibitor of metalloproteinases-3 facilitates Fas-mediated neuronal cell death following mild ischemia

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a natural inhibitor of metalloproteinases involved in matrix degradation and ectodomain shedding of many cell-surface proteins, including death receptors and/or their ligands. In the present study, we examined the role of TIMP-3 in Fas-mediated neuronal cell death following cerebral ischemia, using both gene deletion and pharmacological approaches. In culture, exposure of primary cortical neurons to 2 h of oxygen-glucose deprivation (OGD) resulted in delayed neuronal cell death that was dependent on activation of the death receptor, Fas. Cortical cultures derived from timp-3(-/-) mice displayed partial resistance against OGD-induced neuronal cell death and also displayed increased shedding of Fas ligand (FasL) into the culture media, compared to wild-type control cultures. Both the increased neuroprotection and increased FasL shedding in timp-3(-/-) cultures were reversed by addition of exogenous metalloproteinase inhibitors, recombinant TIMP-3 or GM6001. In vivo, timp-3(-/-) mice showed marked resistance to a brief (30 min) middle cerebral artery occlusion (MCAO), but were not protected against more severe lesions induced by 90 min of MCAO. These studies demonstrate that TIMP-3 facilitates Fas-mediated neuronal cell death following OGD and plays a pro-apoptotic role in mild cerebral ischemia.

ADAM10 regulates FasL cell surface expression and modulates FasL-induced cytotoxicity and activation-induced cell death

The apoptosis-inducing Fas ligand (FasL) is a type II transmembrane protein that is involved in the downregulation of immune reactions by activation-induced cell death (AICD) as well as in T cell-mediated cytotoxicity. Proteolytic cleavage leads to the generation of membrane-bound N-terminal fragments and a soluble FasL (sFasL) ectodomain. sFasL can be detected in the serum of patients with dysregulated inflammatory diseases and is discussed to affect Fas-FasL-mediated apoptosis. Using pharmacological approaches in 293T cells, in vitro cleavage assays as well as loss and gain of function studies in murine embryonic fibroblasts (MEFs), we demonstrate that the disintegrin and metalloprotease ADAM10 is critically involved in the shedding of FasL. In primary human T cells, FasL shedding is significantly reduced after inhibition of ADAM10. The resulting elevated FasL surface expression is associated with increased killing capacity and an increase of T cells undergoing AICD. Overall, our findings suggest that ADAM10 represents an important molecular modulator of FasL-mediated cell death.

Chronic Exposure to a Trichloroethylene Metabolite in Autoimmune-Prone MRL+/+ Mice Promotes Immune Modulation and Alopecia

The industrial solvent trichloroethylene (TCE) is a widespread environmental contaminant known to impact the immune system. In the present study, female MRL+/+ mice were treated for 40 weeks with trichloroacetaldehyde hydrate (TCAH), a metabolite of TCE, in the drinking water. The results were compared with the data from an earlier study in which MRL+/+ mice were exposed to TCAH for 4 weeks. Following a 40-week exposure, the mice developed skin inflammation and dose-dependent alopecia. In addition, TCAH appeared to modulate the CD4(+) T-cell subset by promoting the expression of an activated/effector (i.e., CD62L(lo)) phenotype with an increased capacity to secrete the proinflammatory cytokine interferon-gamma. However, unlike what was observed after only 4 weeks of exposure, TCAH did not significantly attenuate activation-induced cell death (AICD) or the expression of the death receptor FasL in CD4(+) T cells. Some metalloproteinases (MMPs) are thought to play a role in susceptibility to AICD by inducing FasL shedding. Thus, both the 4- and 40-week sera were tested for MMP-7 levels in an attempt to explain the disparate results of TCAH on AICD and FasL expression. Serum MMP-7 levels were significantly higher in mice exposed to TCAH for 4 weeks. In contrast, the serum MMP-7 levels were increased in all the mice by 40 weeks when compared with a nonautoimmune strain. Taken together, a chronic exposure to TCAH promotes alopecia and skin inflammation. The early effects of TCAH on MMP-7 levels may provide a mechanism by which TCAH promotes skin pathology.

Exposure to a Metabolite of the Environmental Toxicant, Trichloroethylene, Attenuates CD4+ T Cell Activation-Induced Cell Death by Metalloproteinase–Dependent FasL Shedding

Long-term exposure to the environmental contaminant trichloroethylene (TCE) in drinking water has been shown to promote autoimmune disease in association with the expansion of activated CD4+ T cells. The effects of TCE on CD4+ T cells were linked in the present study to the ability of TCE metabolite, trichloroacetaldehyde hydrate (TCAH), to inhibit activation-induced cell death (AICD) in CD4+ T cells. TCAH attenuated AICD in CD4+ T cells by decreasing FasL (CD178) expression but not by altering Fas (CD95) expression or by interfering with Fas-signaling events following direct engagement of the Fas receptor. The TCAH-induced decrease in FasL expression did not appear to be mediated at the transcriptional level but was instead due to increased shedding of FasL from the surface of the CD4+ T cells. The ability of TCAH to cleave FasL and thereby decrease AICD appeared to be mediated by metalloproteinases and correlated with a TCAH-induced increase in matrix metalloproteinase-7. Thus, this study presents the novel finding that the environmental contaminant TCE works via its metabolite TCAH to attenuate AICD by increasing metalloproteinase activity that cleaves FasL from CD4+ T cells. This represents a mechanism by which an environmental trigger inhibits AICD in CD4+ T cells and may thereby promote CD4+ T cell-mediated autoimmune disease.

Downregulation of caspases and Fas ligand expression, and increased lifespan of neutrophils after transmigration across intestinal epithelium.

During inflammatory bowel diseases, commitment of extravased polymorphonuclear leucocytes (PMN) to apoptosis is required for the resolution of inflammation. To investigate the effect of transepithelial migration on PMN apoptotic rates, PMN transepithelial migration was reproduced in vitro using T84 intestinal monolayers. Transepithelial migration was found to delay neutrophil apoptosis, and this survival effect correlated with a downregulation of the surface expression of Fas ligand (FasL) and with a decrease in both procaspases-3, and -8 mRNA and procaspases-3, -6, -7 and -8 protein levels. Moreover, neutrophil survival and FasL shedding mediated by transepithelial migration were abrogated by a broad-spectrum metalloproteinase inhibitor, BB-94. Although Erk1/2 and p38 MAPK were activated in transmigrated PMN, inhibition of these MAP kinases did not impair transmigration-induced PMN survival. Taken together, our results show that trans-epithelial migration induces the downregulation of proapoptotic proteins expression in transmigrated PMN, which results in their increased lifespan.

SFas and sFas ligand and pediatric sepsis-induced multiple organ failure syndrome.

The Fas-Fas ligand system is important for apoptosis of activated immune cells. Perturbation of this system occurs in diseases with dysregulated inflammation. Increased soluble Fas (sFas) occurs in systemic inflammatory response syndrome (SIRS) and can block apoptosis. Increased shedding of FasL (sFasL) occurs in viral infection and hepatitis. Although dysregulated inflammation is associated with sepsis-induced multiple organ failure (MOF) in children, a role for Fas has not been established. We hypothesize that 1) sFas will be increased in children with severe and persistent sepsis-induced MOF and will correlate with inflammatory markers suggesting a role for sFas in inflammatory dysregulation in severe sepsis, and 2) sFasL will be increased when viral sepsis or sepsis-induced liver failure-associated MOF is present in children. Plasma sFas, sFasL, IL-6, IL-10, nitrite + nitrates, and organ failure scores were measured on d 1 and d 3 in 92 children with severe sepsis and 12 critically ill control children. sFas levels were increased in severe sepsis, continued to increase in persistent MOF and nonsurvivors, and were correlated with serum inflammatory markers (IL-6, IL-10, nitrite + nitrate levels). In contrast, sFasL was not increased in severe sepsis and did not correlate with inflammation. sFasL was, however, increased in liver failure-associated MOF and in nonsurvivors, and was associated with viral infection. At autopsy, hepatocyte destruction and lymphocyte infiltration were associated with increased sFas and sFasL levels. sFas may interfere with activated immune cell death and contribute to dysregulation of inflammation, worsening outcome from severe sepsis. sFasL may contribute to hepatic injury and the development of liver failure-associated MOF.

SFas and sFas Ligand and Pediatric Sepsis-Induced Multiple Organ Failure Syndrome

The Fas-Fas ligand system is important for apoptosis of activated immune cells. Perturbation of this system occurs in diseases with dysregulated inflammation. Increased soluble Fas (sFas) occurs in systemic inflammatory response syndrome (SIRS) and can block apoptosis. Increased shedding of FasL (sFasL) occurs in viral infection and hepatitis. Although dysregulated inflammation is associated with sepsis-induced multiple organ failure (MOF) in children, a role for Fas has not been established. We hypothesize that 1) sFas will be increased in children with severe and persistent sepsis-induced MOF and will correlate with inflammatory markers suggesting a role for sFas in inflammatory dysregulation in severe sepsis, and 2) sFasL will be increased when viral sepsis or sepsis-induced liver failure–associated MOF is present in children. Plasma sFas, sFasL, IL-6, IL-10, nitrite + nitrates, and organ failure scores were measured on d 1 and d 3 in 92 children with severe sepsis and 12 critically ill control children. sFas levels were increased in severe sepsis, continued to increase in persistent MOF and nonsurvivors, and were correlated with serum inflammatory markers (IL-6, IL-10, nitrite + nitrate levels). In contrast, sFasL was not increased in severe sepsis and did not correlate with inflammation. sFasL was, however, increased in liver failure–associated MOF and in nonsurvivors, and was associated with viral infection. At autopsy, hepatocyte destruction and lymphocyte infiltration were associated with increased sFas and sFasL levels. sFas may interfere with activated immune cell death and contribute to dysregulation of inflammation, worsening outcome from severe sepsis. sFasL may contribute to hepatic injury and the development of liver failure–associated MOF.

Metalloproteinase Shedding of Fas Ligand Regulates β-Amyloid Neurotoxicity

Extracellular deposits of β-amyloid (Aβ) peptide closely match areas of neuronal loss in, and are a postmortem diagnostic indicator of, Alzheimer's disease. Neuronal cultures treated with fibrillar Aβ can be protected from neurotoxicity by caspase-8 inhibition or the expression of dominant-negative FADD, both of which are components of the Fas death receptor pathway [1, 2], and neurons with defective Fas and FasL are resistant to Aβ neurotoxicity [3]. The receptor binding region of FasL can be shed from cells by metalloproteinases, and this process greatly reduces its proapoptotic activity [4, 5]. Here, we show that factors affecting the shedding of membrane-bound FasL significantly impact Aβ neurotoxicity. A broad-spectrum metalloproteinase inhibitor, GM6001/Ilomastat, acted synergistically with Aβ to enhance neurotoxicity through a FasL-dependent mechanism. The disruption of ADAM-based metalloproteinase activity was likely responsible, as MMP-inhibiting TIMPs had no such effect. In contrast, enhanced FasL shedding, by recombinant MMP-7, completely protected neurons from Aβ neurotoxicity. These findings suggest that factors that affect metalloproteinase-mediated shedding of FasL may play a role in the etiology of Alzheimer's disease and may provide an avenue for therapeutic intervention.

Fas (CD95)-Fas ligand interactions are responsible for monocyte apoptosis occurring as a result of phagocytosis and killing of Staphylococcus aureus.

Human peripheral blood monocytes become apoptotic following phagocytosis of Staphylococcus aureus. In this study, we investigated the mechanisms involved in this phenomenon. Cells exposed to bacteria were examined for the surface expression of Fas and Fas ligand (FasL). The level of soluble form of FasL was also measured in the culture supernatants. As Fas-mediated apoptosis involves the activation of caspases, the activities of caspase-8 and caspase-3 were determined. Finally, the involvement of oxidative stress in apoptosis of infected monocytes was investigated. The data indicated that as a consequence of phagocytosis of S. aureus, FasL is released from the monocyte surface and induces apoptosis of phagocytic monocytes and to some extent the bystander cells. The importance of this mechanism was confirmed by demonstrating that blockage of CD95 prevents S. aureus-induced apoptosis of monocytes. Cell death occurring after phagocytosis of S. aureus involves the activation of caspase-3-like proteases, as the specific caspase-3 inhibitor suppressed apoptosis of infected cells. The generation of reactive oxygen intermediates by phagocytic monocytes by itself is not sufficient as a death signal but rather acts in up-regulating FasL shedding and possibly in modulating caspase activity.

Hypoxia Stimulates Release of the Soluble Form of Fas Ligand That Inhibits Endothelial Cell Apoptosis

Fas ligand (FasL), an apoptosis-inducing cytokine, is constitutively expressed on endothelial cells (EC). Here, we report that the soluble form of FasL (sFasL) is released from EC and inhibits hypoxia-induced EC apoptosis. For hypoxia experiments, human EC were exposed to low oxygen tension in airtight chambers flushed with preanalyzed gas mixtures (1% oxygen, 5% CO2, 94% N2) at 37° C. Exposure of cultured EC to hypoxia transiently increased FasL mRNA and protein levels. The maximum increase was observed at 3 and 6 hours after exposure to hypoxia, respectively. Although sFasL protein was not detected in the supernatant from EC without hypoxia, sFasL protein level in the supernatant was transiently increased from 6 hours and disappeared again at 24 hours after the exposure to hypoxia. Interestingly, the supernatant from hypoxia-exposed EC inhibited EC apoptosis induced by hypoxia, which was abolished by a neutralizing antibody against FasL. In addition, incubation with KB8301, an inhibitor of metalloproteinase, suppressed the release of sFasL from EC and enhanced hypoxia-induced apoptosis in EC. Furthermore, exogenously added recombinant sFasL inhibited hypoxia-induced apoptosis. These findings indicate that sFasL released from EC may inhibit hypoxia-induced EC apoptosis. Therefore, the shedding of FasL could be a new therapeutic target in regulating hypoxia-induced EC injury.

Role of IFN-gamma produced after intraperitoneal transplantation of AK-5 cells in the induction of Fas ligand expression by tumor cells leading to immune evasion.

AK-5 tumor cells expressed Fas-L on their surface after intraperitoneal transplantation in syngeneic animals. Fas-L expression by AK-5 cells is involved in the killing of the effector cells. Thus, the tumor has developed an escape mechanism from immune attack. In the present study, we showed that Fas-L expression on AK-5 cells is regulated by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), as injection of antibodies against IFN-gamma downregulated the expression of Fas-L by tumor cells as determined by immunostaining and Northern hybridizations. Fas-L present on the tumor cells is biologically functional, as it induced DNA fragmentation in Fas+ YAC-1 cells. We have also shown shedding of Fas-L in cell-free ascitic fluid from tumor-bearing animals. These observations suggest that such cytokines as IFN-gamma and TNF-alpha play an important role in regulating the expression of Fas-L by AK-5 cells.

Downregulation of Fas ligand by shedding.

Apoptosis-inducing Fas ligand (FasL) is a type II membrane protein, predominantly expressed in the activated T cells. FasL is cleaved by a putative metalloproteinase to produce a soluble form. Here, we blocked the shedding of human FasL by deleting its cleavage site. Although human Jurkat cells and mouse primary hepatocytes that express a low level of Fas were resistant to the soluble form of FasL, they were efficiently killed by membrane-bound FasL. Furthermore, soluble FasL inhibited cytotoxicity of the membrane-bound FasL. These results indicate that the membrane-bound form of FasL is the functional form and suggest that shedding of FasL is to prevent the killing of the healthy bystander cells by cytotoxic T cells.

Membrane Fas ligand kills human peripheral blood T lymphocytes, and soluble Fas ligand blocks the killing.

It has been believed that the Fas expressed on human peripheral blood T cells (PBT) is nonfunctional, because these cells are insensitive to agonistic anti-Fas/Apo-1 mAbs that efficiently kill in vitro-activated T cells and many Fas-expressing cell lines. Here, we demonstrate that membrane-bound Fas ligand (FasL) kills both fresh and in vitro-activated PBT, indicating that the Fas expressed on fresh PBT is functional. In contrast, soluble FasL kills only the latter. Naive T cells in umbilical cord blood do not express Fas, but can be induced to express Fas by IFN-gamma or by a combination of IL-2 and anti-CD28 mAb, after which they acquire sensitivity to membrane but not to soluble FasL. Soluble FasL inhibited the killing of fresh PBT by membrane FasL. These results indicate that the shedding of FasL from the membrane is a mechanism for downregulating at least part of its killing activity.