Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors.

Viruses have evolved many distinct strategies to avoid the host's apoptotic response. Here we describe a new family of viral inhibitors (v-FLIPs) which interfere with apoptosis signalled through death receptors and which are present in several gamma-herpesviruses (including Kaposi's-sarcoma-associated human herpesvirus-8), as well as in the tumorigenic human molluscipoxvirus. v-FLIPs contain two death-effector domains which interact with the adaptor protein FADD, and this inhibits the recruitment and activation of the protease FLICE by the CD95 death receptor. Cells expressing v-FLIPs are protected against apoptosis induced by CD95 or by the related death receptors TRAMP and TRAIL-R. The herpesvirus saimiri FLIP is detected late during the lytic viral replication cycle, at a time when host cells are partially protected from CD95-ligand-mediated apoptosis. Protection of virus-infected cells against death-receptor-induced apoptosis may lead to higher virus production and contribute to the persistence and oncogenicity of several FLIP-encoding viruses.

TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1-TRAF2 complex to sensitize tumor cells to TNFalpha.

Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor kappaB (NF-kappaB) signaling, and sensitize cells to tumor necrosis factor alpha (TNFalpha). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1-Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1-TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-kappaB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFalpha-induced death occurs. TWEAK-induced loss of the cIAP1-TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFalpha-induced death, whereas primary cells remain resistant. Conversely, cIAP1-TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFalpha sensitization. Lysosomal degradation of cIAP1-TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.

Persistent inhibition of FLIP(L) expression by lentiviral small hairpin RNA delivery restores death-receptor-induced apoptosis in neuroblastoma cells.

Neuroblastoma represents the most common and deadly solid tumour of childhood, which disparate biological and clinical behaviour can be explained by differential regulation of apoptosis. To understand mechanisms underlying death resistance in neuroblastoma cells, we developed small hairpin of RNA produced by lentiviral vectors as tools to selectively interfere with FLIP(L), a major negative regulator of death receptor-induced apoptosis. Such tools revealed highly efficient in interfering with FLIP(L) expression and function as they almost completely repressed endogenous and/or exogenously overexpressed FLIP(L) protein and fully reversed FLIP(L)-mediated TRAIL resistance. Moreover, interference with endogenous FLIP(L) and FLIP(S) significantly restored FasL sensitivity in SH-EP neuroblastoma cell line. These results reveal the ability of lentivirus-mediated shRNAs to specifically and persistently interfere with FLIP expression and support involvement of FLIP in the regulation of death receptor-mediated apoptosis in neuroblastoma cells. Combining such tools with other therapeutic modalities may improve treatment of resistant tumours such as neuroblastoma.

SOCS-1 protein prevents Janus Kinase/STAT-dependent inhibition of beta cell insulin gene transcription and secretion in response to interferon-gamma.

In the pathogenesis of type I diabetes mellitus, activated leukocytes infiltrate pancreatic islets and induce beta cell dysfunction and destruction. Interferon (IFN)-gamma, tumor necrosis factor-alpha and interleukin (IL)-1 beta play important, although not completely defined, roles in these mechanisms. Here, using the highly differentiated beta Tc-Tet insulin-secreting cell line, we showed that IFN-gamma dose- and time-dependently suppressed insulin synthesis and glucose-stimulated secretion. As described previously IFN-gamma, in combination with IL-1 beta, also induces inducible NO synthase expression and apoptosis (Dupraz, P., Cottet, S., Hamburger, F., Dolci, W., Felley-Bosco, E., and Thorens, B. (2000) J. Biol. Chem. 275, 37672--37678). To assess the role of the Janus kinase/signal transducer and activator of transcription (STAT) pathway in IFN-gamma intracellular signaling, we stably overexpressed SOCS-1 (suppressor of cytokine signaling-1) in the beta cell line. We demonstrated that SOCS-1 suppressed cytokine-induced STAT-1 phosphorylation and increased cellular accumulation. This was accompanied by a suppression of the effect of IFN-gamma on: (i) reduction in insulin promoter-luciferase reporter gene transcription, (ii) decrease in insulin mRNA and peptide content, and (iii) suppression of glucose-stimulated insulin secretion. Furthermore, SOCS-1 also suppressed the cellular effects that require the combined presence of IL-1 beta and IFN-gamma: induction of nitric oxide production and apoptosis. Together our data demonstrate that IFN-gamma is responsible for the cytokine-induced defect in insulin gene expression and secretion and that this effect can be completely blocked by constitutive inhibition of the Janus kinase/STAT pathway.

Effects of ciliary muscle plasmid electrotransfer of TNF-alpha soluble receptor variants in experimental uveitis.

Intraocular inflammation has been recognized as a major factor leading to blindness. Because tumor necrosis factor-alpha (TNF-alpha) enhances intraocular cytotoxic events, systemic anti-TNF therapies have been introduced in the treatment of severe intraocular inflammation, but frequent re-injections are needed and are associated with severe side effects. We have devised a local intraocular nonviral gene therapy to deliver effective and sustained anti-TNF therapy in inflamed eyes. In this study, we show that transfection of the ciliary muscle by plasmids encoding for three different variants of the p55 TNF-alpha soluble receptor, using electrotransfer, resulted in sustained intraocular secretion of the encoded proteins, without any detection in the serum. In the eye, even the shorter monomeric variant resulted in efficient neutralization of TNF-alpha in a rat experimental model of endotoxin-induced uveitis, as long as 3 months after transfection. A subsequent downregulation of interleukin (IL)-6 and iNOS and upregulation of IL-10 expression was observed together with a decreased rolling of inflammatory cells in anterior segment vessels and reduced infiltration within the ocular tissues. Our results indicate that using a nonviral gene therapy strategy, the local self-production of monomeric TNF-alpha soluble receptors induces a local immunomodulation enabling the control of intraocular inflammation.

Five new CYLD mutations in skin appendage tumors and evidence that aspartic acid 681 in CYLD is essential for deubiquitinase activity.

Brooke-Spiegler syndrome, familial cylindromatosis, and familial trichoepithelioma are autosomal-dominant genetic predispositions for benign tumors of skin appendages caused by mutations in the CYLD gene localized on chromosome 16q12-q13. The encoded protein functions as ubiquitin-specific protease (UBP), which negatively regulates NF-kappaB and c-Jun N-terminal kinase (JNK) signaling. We investigated five families affected with these skin neoplasms and identified four premature stop codons and the novel missense mutation D681G in a family in which 11 of 12 investigated tumors were trichoepitheliomas. CYLD protein harboring this missense mutation had a significant reduced ability to inhibit TNF receptor-associated factor (TRAF)2- and TRAF6-mediated NF-kappaB activation, tumor necrosis factor-alpha (TNFalpha)-induced JNK signaling, and to deubiquitinate TRAF2. CYLD-D681G was coimmunoprecipitated by TRAF2, but was unable to cleave K63-linked polyubiquitin chains. Aspartic acid 681 is highly conserved in CYLD homologues and other members of the UBP family, but does not belong to the Cys and His boxes providing the CYLD catalytic triad (Cys601, His871, and Asp889). As reported previously, the homologous residue D295 of HAUSP/USP-7 forms a hydrogen bond with the C-terminal end of ubiquitin and is important for the enzymatic activity. These results underline that D681 in CYLD is required for cleavage of K63-linked polyubiquitin chains.

Caspase-induced inactivation of the anti-apoptotic TRAF1 during Fas ligand-mediated apoptosis.

The activation of the transcription factor NF-kappaB often results in protection against apoptosis. In particular, pro-apoptotic tumor necrosis factor (TNF) signals are blocked by proteins that are induced by NF-kappaB such as TNFR-associated factor 1 (TRAF1). Here we show that TRAF1 is cleaved after Asp-163 when cells are induced to undergo apoptosis by Fas ligand (FasL). The C-terminal cleavage product blocks the induction of NF-kappaB by TNF and therefore functions as a dominant negative (DN) form of TRAF1. Our results suggest that the generation of DN-TRAF1 is part of a pro-apoptotic amplification system to assure rapid cell death.

Tumor necrosis-like weak inducer of apoptosis as a proinflammatory cytokine in human adipocyte cells: up-regulation in severe obesity is mediated by inflammation but not hypoxia.

CONTEXT Adipose tissue hypoxia and endoplasmic reticulum (ER) stress may link the presence of chronic inflammation and macrophage infiltration in severely obese subjects. We previously reported the up-regulation of TNF-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) axis in adipose tissue of severely obese type 2 diabetic subjects. OBJECTIVES The objective of the study was to examine TWEAK and Fn14 adipose tissue expression in obesity, severe obesity, and type 2 diabetes in relation to hypoxia and ER stress. DESIGN In the obesity study, 19 lean, 28 overweight, and 15 obese nondiabetic subjects were studied. In the severe obesity study, 23 severely obese and 35 control subjects were studied. In the type 2 diabetes study, 11 type 2 diabetic and 36 control subjects were studied. The expression levels of the following genes were analyzed in paired samples of sc and visceral adipose tissue: Fn14, TWEAK, VISFATIN, HYOU1, FIAF, HIF-1a, VEGF, GLUT-1, GRP78, and XBP-1. The effect of hypoxia, inflammation, and ER stress on the expression of TWEAK and Fn14 was examined in human adipocyte and macrophage cell lines. RESULTS Up-regulation of TWEAK/Fn14 and hypoxia and ER stress surrogate gene expression was observed in sc and visceral adipose tissue only in our severely obese cohort. Hypoxia modulates TWEAK or Fn14 expression in neither adipocytes nor macrophages. On the contrary, inflammation up-regulated TWEAK in macrophages and Fn14 expression in adipocytes. Moreover, TWEAK had a proinflammatory effect in adipocytes mediated by the nuclear factor-kappaB and ERK but not JNK signaling pathways. CONCLUSIONS Our data suggest that TWEAK acts as a pro-inflammatory cytokine in the adipose tissue and that inflammation, but not hypoxia, may be behind its up-regulation in severe obesity.

Enhanced heat shock protein 70 expression alters proteasomal degradation of IkappaB kinase in experimental acute respiratory distress syndrome.

OBJECTIVES: Acute respiratory distress syndrome is a common and highly lethal inflammatory lung syndrome. We previously have shown that an adenoviral vector expressing the heat shock protein (Hsp)70 (AdHSP) protects against experimental sepsis-induced acute respiratory distress syndrome in part by limiting neutrophil accumulation in the lung. Neutrophil accumulation and activation is modulated, in part, by the nuclear factor-kappaB (NF-kappaB) signal transduction pathway. NF-kappaB activation requires dissociation/degradation of a bound inhibitor, IkappaBalpha. IkappaBalpha degradation requires phosphorylation by IkappaB kinase, ubiquitination by the SCFbeta-TrCP (Skp1/Cullin1/Fbox beta-transducing repeat-containing protein) ubiquitin ligase, and degradation by the 26S proteasome. We tested the hypothesis that Hsp70 attenuates NF-kappaB activation at multiple points in the IkappaBalpha degradative pathway. DESIGN: Laboratory investigation. SETTING: University medical center research laboratory. SUBJECTS: Adolescent (200 g) Sprague-Dawley rats and murine lung epithelial-12 cells in culture. INTERVENTIONS: Lung injury was induced in rats via cecal ligation and double puncture. Thereafter, animals were treated with intratracheal injection of 1) phosphate buffer saline, 2) AdHSP, or 3) an adenovirus expressing green fluorescent protein. Murine lung epithelial-12 cells were stimulated with tumor necrosis factor-alpha and transfected. NF-kappaB was examined using molecular biological tools. MEASUREMENTS AND MAIN RESULTS: Intratracheal administration of AdHSP to rats with cecal ligation and double puncture limited nuclear translocation of NF-kappaB and attenuated phosphorylation of IkappaBalpha. AdHSP treatment reduced, but did not eliminate, phosphorylation of the beta-subunit of IkappaB kinase. In vitro kinase activity assays and gel filtration chromatography revealed that treatment of sepsis-induced lung injury with AdHSP induced fragmentation of the IkappaB kinase signalosome. This stabilized intermediary complexes containing IkappaB kinase components, IkappaBalpha, and NF-kappaB. Cellular studies indicate that although ubiquitination of IkappaBalpha was maintained, proteasomal degradation was impaired by an indirect mechanism. CONCLUSIONS: Treatment of sepsis-induced lung injury with AdHSP limits NF-kappaB activation. This results from stabilization of intermediary NF-kappaB/IkappaBalpha/IkappaB kinase complexes in a way that impairs proteasomal degradation of IkappaBalpha. This novel mechanism by which Hsp70 attenuates an intracellular process may be of therapeutic value.

Cytotoxic T cells deficient in both functional fas ligand and perforin show residual cytolytic activity yet lose their capacity to induce lethal acute graft-versus-host disease.

Graft-versus-host disease (GVHD) is the main complication after allogeneic bone marrow transplantation. Although the tissue damage and subsequent patient mortality are clearly dependent on T lymphocytes present in the grafted inoculum, the lethal effector molecules are unknown. Here, we show that acute lethal GVHD, induced by the transfer of splenocytes from C57BL/6 mice into sensitive BALB/c recipients, is dependent on both perforin and Fas ligand (FasL)-mediated lytic pathways. When spleen cells from mutant mice lacking both effector molecules were transferred to sublethally irradiated allogeneic recipients, mice survived. Delayed mortality was observed with grafted cells deficient in only one lytic mediator. In contrast, protection from lethal acute GVHD in resistant mice was exclusively perforin dependent. Perforin-FasL-deficient T cells failed to lyse most target cells in vitro. However, they still efficiently killed tumor necrosis factor alpha-sensitive fibroblasts, demonstrating that cytotoxic T cells possess a third lytic pathway.

Inflammatory responses in aggregating rat brain cell cultures subjected to different demyelinating conditions.

To study inflammatory reactions occurring in relation to demyelination, aggregating rat brain cell cultures were subjected to three different demyelinating insults, i.e., (i) lysophosphatidylcholine (LPC), (ii) interferon-gamma combined with lipopolysaccharide (IFN-gamma+LPS), and (iii) anti-MOG antibodies plus complement (alpha-MOG+C). Demyelination was assessed by measuring the expression of myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), and the activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP). The accompanying inflammatory reactions were examined by the quantification of microglia-specific staining, by immunostaining for glial fibrillary acidic protein (GFAP), and by measuring the mRNA expression of a panel of inflammation-related genes. It was found that all three demyelinating insults decreased the expression of MBP and MOG, and induced microglial reactivity. LPC and alpha-MOG+C, but not IFN-gamma+LPS, decreased CNP activity; they also caused the appearance of macrophagic microglia, and increased GFAP staining indicating astrogliosis. LPC affected also the integrity of neurons and astrocytes. LPC and IFN-gamma+LPS upregulated the expression of the inflammation-related genes IL-6, TNF-alpha, Ccl5, Cxcl1, and iNOS, although to different degrees. Other inflammatory markers were upregulated by only one of the three insults, e.g., Cxcl2 by LPC; IL-1beta and IL-15 by IFN-gamma+LPS; and IFN-gamma by alpha-MOG+C. These findings indicate that each of the three demyelinating insults caused distinct patterns of demyelination and inflammatory reactivity, and that of the demyelinating agents tested only LPC exhibited general toxicity.

Pancreatic stone protein as an early biomarker predicting mortality in a prospective cohort of patients with sepsis requiring ICU management.

ABSTRACT: INTRODUCTION: Biomarkers, such as C-reactive protein [CRP] and procalcitonin [PCT], are insufficiently sensitive or specific to stratify patients with sepsis. We investigate the prognostic value of pancreatic stone protein/regenerating protein (PSP/reg) concentration in patients with severe infections. METHODS: PSP/reg, CRP, PCT, tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL1-^6;), IL-6 and IL-8 were prospectively measured in cohort of patients ≥ 18 years of age with severe sepsis or septic shock within 24 hours of admission in a medico-surgical intensive care unit (ICU) of a community and referral university hospital, and the ability to predict in-hospital mortality was determined. RESULTS: We evaluated 107 patients, 33 with severe sepsis and 74 with septic shock, with in-hospital mortality rates of 6% (2/33) and 25% (17/74), respectively. Plasma concentrations of PSP/reg (343.5 vs. 73.5 ng/ml, P < 0.001), PCT (39.3 vs. 12.0 ng/ml, P < 0.001), IL-8 (682 vs. 184 ng/ml, P < 0.001) and IL-6 (1955 vs. 544 pg/ml, P < 0.01) were significantly higher in patients with septic shock than with severe sepsis. Of note, median PSP/reg was 13.0 ng/ml (IQR: 4.8) in 20 severely burned patients without infection. The area under the ROC curve for PSP/reg (0.65 [95% CI: 0.51 to 0.80]) was higher than for CRP (0.44 [0.29 to 0.60]), PCT 0.46 [0.29 to 0.61]), IL-8 (0.61 [0.43 to 0.77]) or IL-6 (0.59 [0.44 to 0.75]) in predicting in-hospital mortality. In patients with septic shock, PSP/reg was the only biomarker associated with in-hospital mortality (P = 0.049). Risk of mortality increased continuously for each ascending quartile of PSP/reg. CONCLUSIONS: Measurement of PSP/reg concentration within 24 hours of ICU admission may predict in-hospital mortality in patients with septic shock, identifying patients who may benefit most from tailored ICU management.

Fas ligand elicits a caspase-independent proinflammatory response in human keratinocytes: implications for dermatitis.

Fas ligand (FasL) causes apoptosis of epidermal keratinocytes and triggers the appearance of spongiosis in eczematous dermatitis. We demonstrate here that FasL also aggravates inflammation by triggering the expression of proinflammatory cytokines, chemokines, and adhesion molecules in keratinocytes. In HaCaT cells and in reconstructed human epidermis (RHE), FasL triggered a NF-kappaB-dependent mRNA accumulation of inflammatory cytokines (tumor necrosis factor-alpha, IL-6, and IL-1beta), chemokines (CCL2/MCP-1, CXCL1/GROalpha, CXCL3/GROgamma, and CXCL8/IL-8), and the adhesion molecule ICAM-1. Oligomerization of Fas was required both for apoptosis and for gene expression. Inhibition of caspase activity abolished FasL-dependent apoptosis; however, it failed to suppress the expression of FasL-induced genes. Additionally, in the presence of caspase inhibitors, but not in their absence, FasL triggered the accumulation of CCL5/RANTES (regulated on activation normal T cell expressed and secreted) mRNA. Our findings identify a novel proinflammatory role of FasL in keratinocytes that is independent of caspase activity and is separable from apoptosis. Thus, in addition to causing spongiosis, FasL may play a direct role in triggering and/or sustaining inflammation in eczemas.

Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule.

Cell death is achieved by two fundamentally different mechanisms: apoptosis and necrosis. Apoptosis is dependent on caspase activation, whereas the caspase-independent necrotic signaling pathway remains largely uncharacterized. We show here that Fas kills activated primary T cells efficiently in the absence of active caspases, which results in necrotic morphological changes and late mitochondrial damage but no cytochrome c release. This Fas ligand-induced caspase-independent death is absent in T cells that are deficient in either Fas-associated death domain (FADD) or receptor-interacting protein (RIP). RIP is also required for necrotic death induced by tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL). In contrast to its role in nuclear factor kappa B activation, RIP requires its own kinase activity for death signaling. Thus, Fas, TRAIL and TNF receptors can initiate cell death by two alternative pathways, one relying on caspase-8 and the other dependent on the kinase RIP.

Efficiency of recombinant human TNF in human cancer therapy.

Recombinant human tumour necrosis factor (TNF) has a selective effect on angiogenic vessels in tumours. Given that it induces vasoplegia, its clinical use has been limited to administration through isolated limb perfusion (ILP) for regionally advanced melanomas and soft tissue sarcomas of the limbs. When combined with the alkylating agent melphalan, a single ILP produces a very high objective response rate. In melanoma, the complete response (CR) rate is around 80% and the overall objective response rate greater than 90%. In soft tissue sarcomas that are inextirpable, ILP is a neoadjuvant treatment resulting in limb salvage in 80% of the cases. The CR rate averages 20% and the objective response rate is around 80%. The mode of action of TNF-based ILP involves two distinct and successive effects on the tumour-associated vasculature: first, an increase in endothelium permeability leading to improved chemotherapy penetration within the tumour tissue, and second, a selective killing of angiogenic endothelial cells resulting in tumour vessel destruction. The mechanism whereby these events occur involves rapid (of the order of minutes) perturbation of cell-cell adhesive junctions and inhibition of alphavbeta3 integrin signalling in tumour-associated vessels, followed by massive death of endothelial cells and tumour vascular collapse 24 hours later. New, promising approaches for the systemic use of TNF in cancer therapy include TNF targeting by means of single chain antibodies or endothelial cell ligands, or combined administration with drugs perturbing integrin-dependent signalling and sensitizing angiogenic endothelial cells to TNF-induced death.