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Cell-culture studies are our main source of knowledge of the various forms of programmed cell death. Yet genetic perturbations of death-protein function in animal models are almost the only source of our knowledge of the physiological roles of these programs. Shortcomings in the state of knowledge acquired by these two experimental approaches are exemplified in this Perspective by reference to research on the contribution of apoptosis to lymphocyte development, a subject on which there is already much knowledge, and on the role of necroptosis in inflammation, about which information is just beginning to emerge. To address these shortcomings, there is need to find ways to verify the notions obtained through the current experimental approaches by directly monitoring death programs within specific cells in vivo. Cell-culture studies are our main source of knowledge of the various forms of programmed cell death. Yet genetic perturbations of death-protein function in animal models are almost the only source of our knowledge of the physiological roles of these programs. Shortcomings in the state of knowledge acquired by these two approaches are exemplified in this Perspective by reference to research on the contribution of apoptosis to lymphocyte development, a subject on which there is already much knowledge, and on the role of necroptosis in inflammation, about which information is just beginning to emerge. To address these shortcomings, there is need to find ways to verify the notions obtained through the current experimental approaches by directly monitoring death programs within specific cells in vivo.

Until recently, programmed cell death was conceived of as a single set of molecular pathways. We now know of several distinct sets of death-inducing mechanisms that lead to differing cell-death processes. In one of them--apoptosis--the dying cell affects others minimally. In contrast, programmed necrotic cell death causes release of immunostimulatory intracellular components after cell-membrane rupture. Defining the in vivo relevance of necrotic death is hampered because the molecules initiating it [such as receptor-interacting protein kinase-1 (RIPK1), RIPK3, or caspase-1] also serve other functions. Proteins that participate in late events in two forms of programmed necrosis [mixed lineage kinase domain-like protein (MLKL) in necroptosis and gasdermin-D in pyroptosis] were recently discovered, bringing us closer to identifying molecules that strictly serve in death mediation, thereby providing probes for better assessing its role in inflammation.

A signaling pathway that induces programmed necrotic cell death (necroptosis) was reported to be activated in cells by several cytokines and various pathogen components. The major proteins participating in that pathway are the protein kinases RIPK1 and RIPK3 and the pseudokinase mixed lineage kinase domain-like protein (MLKL). Recent studies have suggested that MLKL, once activated, mediates necroptosis by binding to cellular membranes, thereby triggering ion fluxes. However, our knowledge of both the sequence of molecular events leading to MLKL activation and the subcellular sites of these events is fragmentary. Here we report that the association of MLKL with the cell membrane in necroptotic death is preceded by the translocation of phosphorylated MLKL, along with RIPK1 and RIPK3, to the nucleus.

Emerging evidence indicates that necrotic cell death can be regulated by a specific set of signaling molecules. Studies showing that the same signaling molecules also trigger inflammation, and that when cells die necrotically some of the molecules they release facilitate inflammation, raised the possibility that the death induced by these signaling molecules ("necroptosis") serves to trigger inflammation. Here we briefly discuss the work done on the anti-inflammatory function of caspase-8 and its relation to the inhibitory effect of this enzyme on the induction of necroptosis. The studies imply that caspase-8 and the other proximal signaling proteins known to participate in the induction and regulation of necroptosis are too pleiotropic to serve as reliable molecular probes for determining the relative contribution of this death mode to in vivo processes. (C) 2014 Published by Elsevier Ltd.

Emerging evidence indicates that the molecular mechanisms of cell death have regulatory roles in inflammation and that the molecular changes that are associated with different forms of cell death affect the course of inflammation in different ways. In this Timeline article, we discuss how our understanding of the mechanisms and functional roles of tissue injury and cell death in inflammation has evolved on the basis of almost two centuries of study. We describe how such ideas have led to our current models of cell death and inflammation, and we highlight the remaining gaps in our knowledge of the subject.

Keywords: DEATH DOMAIN; PROTEIN; RECEPTOR; MICE; APOPTOSIS; INTERACTS; FAS

Expression of enzymatically inactive caspase-8, or deletion of caspase-8 from basal epidermal keratinocytes, triggers chronic skin inflammation in mice. Unlike similar inflammation resulting from arrest of nuclear factor. B activation in the epidermal cells, the effect induced by caspase-8 deficiency did not depend on TNF, IL-1, dermal macrophage function, or expression of the toll-like receptor adapter proteins MyD88 or TRIF. Both interferon regulatory factor (IRF) 3 and TANK-binding kinase were constitutively phosphorylated in the caspase-8-deficient epidermis, and knockdown of IRF3 in the epidermis-derived cells from these mice abolished the expression of up-regulated genes. Temporal and spatial analyses of the alterations in gene expression that result from caspase-8 deficiency reveal that the changes are initiated before birth, around the time that cornification develops, and occur mainly in the suprabasal layer. Finally, we found that caspase-8-deficient keratinocytes display an enhanced response to gene activation by transfected DNA. Our findings suggest that an enhanced response to endogenous activators of IRF3 in the epidermis, presumably generated in association with keratinocyte differentiation, contributes to the skin inflammatory process triggered by caspase-8 deficiency.

Early in the exploration of the chemical nature of life, it was widely believed that the molecules of living organisms, by their very nature, differ from those of inorganic material molecules and possess a vital force ('elan vital'). Similarly, early scientific thinking on the subject of cell death and its induction by cytotoxic cells of the immune system was pervaded by a sense that the molecules mediating these functions possess intrinsic deadly activity and are dedicated exclusively to death-related tasks. This impression was also reflected in the initial notions of the mode of action of intracellular proteins that signal for death. It is now gradually becoming clear, however, that proteins participating in death induction also have functions unrelated to death. Nevertheless, as exemplified by studies of the function of caspase-8 (an enzyme that signals both for activation of the extrinsic cell-death pathway and for non-death-related effects), analysis of the mechanistic basis for such heterogeneity might allow identification of distinct structural determinants in the proteins participating in death induction that do bear death specificity.

Caspase-8, the proximal enzyme in the death-induction pathway of the TNF/nerve growth factor receptor family, is activated upon juxtaposition of its molecules within the receptor complexes and is then self-processed. Caspase-8 also contributes to the regulation of cell survival and growth, but little is known about the similarities or the differences between the mechanisms of these nonapoptotic functions and of the enzyme's apoptotic activity. In this study, we report that in bacterial artificial chromosome-transgenic mice, in which the aspartate residue upstream of the initial self-processing site in caspase-8 (D387) was replaced by alanine, induction of cell death by Fas is compromised. However, in contrast to caspase-8-deficient mice, which die in utero at mid-gestation, the mice mutated at D387 were born alive and seemed to develop normally. Moreover, mice with the D387A mutation showed normal in vitro growth responses of T lymphocytes to stimulation of their Ag receptor as well as of B lymphocytes to stimulation by LPS, normal differentiation of bone marrow macrophage precursors in response to M-CSF, and normal generation of myeloid colonies by the bone marrow hematopoietic progenitors, all of which are compromised in cells deficient in caspase-8. These finding indicated that self-processing of activated caspase-8 is differentially involved in the different functions of this enzyme: it is needed for the induction of cell death through the extrinsic cell death pathway but not for nonapoptotic functions of caspase-8.

Keywords: Biochemistry & Molecular Biology; Cell Biology; Immunology

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NF kappa B-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.

Recruitment of the NF-kappa B-activating IKK signaling complex to the TNF receptor is shown to be driven by induced binding of NEMO, a regulatory component of this complex, to K63-linked polyubiquitin chains attached to RIP1, a receptor-associated adaptor protein.

Knockout of caspase-8, a cysteine protease that participates in the signaling for cell death by receptors of the TNF/nerve growth factor family, is lethal to mice in utero. To explore tissue-specific roles of this enzyme, we established its conditional knockout using the Cre/loxP recombination system. Consistent with its role in cell death induction, deletion of caspase-8 in hepatocytes protected them from Fas-induced caspase activation and death. However, application of the conditional knockout approach to investigate the cause of death of caspase-8 knockout embryos revealed that this enzyme also serves cellular functions that are nonapoptotic. Its deletion in endothelial cells resulted in degeneration of the yolk sac vasculature and embryonal death due to circulatory failure. Caspase-8 deletion in bone-marrow cells resulted in arrest of hemopoietic progenitor functioning, and in cells of the myelomonocytic lineage, its deletion led to arrest of differentiation into macrophages and to cell death. Thus, besides participating in cell death induction by receptors of the TNF/nerve growth factor family, caspase-8, apparently independently of these receptors, also mediates nonapoptotic and perhaps even antiapoptotic activities.

NF-kappaB transcription factors have key roles in inflammation, immune response, oncogenesis and protection against apoptosis (1,2). In most cells, these factors are kept inactive in the cytoplasm through association with IkappaB inhibitors. After stimulation by various reagents, IkappaB is phosphorylated by the IkappaB kinase (IKK) complex(3) and degraded by the proteasome, allowing NF-kappaB to translocate to the nucleus and activate its target genes. Here we report that CYLD, a tumour suppressor that is mutated in familial cylindromatosis(4), interacts with NEMO, the regulatory subunit of IKK5,6. CYLD also interacts directly with tumour-necrosis factor receptor (TNFR)-associated factor 2 (TRAF2), an adaptor molecule involved in signalling by members of the family of TNF/ nerve growth factor receptors. CYLD has deubiquitinating activity that is directed towards non-K48-linked polyubiquitin chains, and negatively modulates TRAF-mediated activation of IKK, strengthening the notion that ubiquitination is involved in IKK activation by TRAFs and suggesting that CYLD functions in this process. Truncations of CYLD found in cylindromatosis result in reduced enzymatic activity, indicating a link between impaired deubiquitination of CYLD substrates and human pathophysiology.

David Wallach of the Weizmann Institute, Zhaodan Cao of Tularik Inc., Michael Karin of the University of California, San Diego, and Ebrahim Zandi of the University of Southern California, Norris Cancer Center, discuss how protein kinases regulate NF-kappa B activity.

FIP-3 (14.7K interacting protein) was discovered during a search for cell proteins that could interact with an adenovirus protein (Ad E3-14.7K) that had been shown to prevent tumor necrosis factor (TNF)-alpha-induced cytolysis, FIP-3, which contains leucine zippers and a zinc finger domain, inhibits both basal and induced transcriptional activity of NF-kappa B and causes a late-appearing apoptosis with unique morphologic manifestations. Ad E3-14.7K can partially reverse apoptotic death induced by FIP-3, FIP-3 also was shown to bind to other cell proteins, RIP and NIK, which previously had been described as essential components of TNF-alpha-induced NF-kappa B activation. In addition, FIP-3 inhibited activation of NF-kappa B induced by TNF-alpha, the TNFR-1 receptor, RIP, NIK, and IKK beta, as well as basal levels of endogenous NF-kappa B in 293 cells. Because the activation of NF-kappa B has been shown to inhibit apoptosis, FIP-3 appears both to activate a cell-death pathway and to inhibit an NF-kappa B-dependent survival mechanism.

The Epstein-Barr virus oncoprotein latent infection membrane protein 1 (LMP1) is a constitutively aggregated pseudo-tumor necrosis factor receptor (TNFR) that activates transcription factor NF-kappa B through two sites in its C-terminal cytoplasmic domain. One site is similar to activated TNFRII in associating,vith TNFR-associated factors TRAF1 and TRAF2, and the second site is similar to TNFRI in associating with the TNFRI death domain interacting protein TRADD. TNFRI has been recently shown to activate NF-kappa B through association with TRADD, RIP, and TRAF2; activation of the NF-kappa B-inducing kinase (NIK); activation of the I kappa B alpha kinases (IKK alpha and IKK beta); and phosphorylation of I kappa B alpha. I kappa B alpha phosphorylation on Ser-32 and Ser-36 is followed by its degradation and NF-kappa B activation. In this report, we show that NF-kappa B activation by LMP1 or by each of its effector sites is mediated by a pathway that includes NIK IKK alpha, and IKK beta. Dominant negative mutants of NIK, IKK alpha, or IKK beta substantially inhibited NF-kappa B activation by LMP1 or by each of its effector sites.

CD27 is a member of the tumor necrosis factor (TNF) receptor superfamily and is expressed on T, B, and NK cells. The signal via CD27 plays pivotal roles in T-T and T-B cell interactions. Here we demonstrate that overexpression of CD27 activates NF-kappa B and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). Deletion analysis of the cytoplasmic domain of CD27 revealed that the C-terminal PIQEDYR motif was indispensable for both NF-kappa B and SAPK/JNK activation and was also required for the interaction with TNF receptor-associated factor (TRAF) 2 and TRAF5, both of which have been implicated in NF-kappa B activation by members of the TNF-R superfamily. Co-transfection of a dominant negative TRAF2 or TRAF5 blocked NF-kappa B and SAPK/JNK activation induced by CD27. Recently, a TRAF2-interacting kinase has been identified, termed NF-kappa B-inducing kinase (NIK). A kinase-inactive mutant NIK blocked CD27-, TRAF2-, and TRAF5-mediated NF-kappa B and SAPK/JNK activation. These results indicate that TRAF2 and TRAF5 are involved in NF-kappa B and SAPK/JNK JNK activation by CD27, and MK is a common downstream kinase of TRAF2 and TRAF5 for NF-kappa B and SAPK/JNK activation.

We previously demonstrated that p38 MAPK is a crucial mediator in the NF-kappa B-dependent gene activation induced by TNF. Here, we have studied the role of several TNF receptor-associated proteins and caspases in p38 MAPK activation by TNF. The latter appears to be dependent on TRAF2, but independent of FADD or caspases. Remarkably, p38 MARK activation by TNF proceeds independently of the TRAF2-associated NF-kappa B-inducing kinase NIK, which is known to bind and activate two recently identified I kappa B kinases. These results demonstrate that two kinase pathways involved in NF-kappa B regulation, viz, NIK and p38 MAPK-mediated, diverge at the level of TRAF2. (C) 1998 Federation of European Biochemical Societies.

Objective: To investigate whether serum and amniotic fluid (AF) levels of soluble tumor necrosis factor receptors and interleukin-6, markers of immune activation and endothelial dysfunction, are altered in patients with severe preeclampsia. Methods: Plasma was collected before induction of labor, at delivery, and postpartum from 19 patients with severe preeclampsia. Amniotic fluid was also obtained in early labor from these patients. Similar samples were obtained from an antepartum control group matched for gestational age and a term control group without preeclampsia. All plasma and AF samples were assayed or p55 and p75 soluble tumor necrosis factor receptors and for interleukin-6 by specific enzyme-linked immunoassays. Levels in preeclamptic patients and the control groups were compared. Results: Levels of both receptors were significantly elevated in AF and all maternal plasma samples except those collected 24 hours postpartum for patients with preeclampsia relative to levels in controls. Interleukin-6 was detected more frequently and in higher concentrations in the plasma collected before labor for preeclamptic patients compared with controls, but no difference was noted in interleukin-6 detection rates or plasma concentrations at delivery. Conversely, AF concentrations of interleukin-6 were significantly reduced in patients with preeclampsia. Conclusion: The increased levels of soluble tumor necrosis factor receptors found in patients with severe preeclampsia may represent a protective response to increased turner necrosis factor activity and be a marker for immune activation. Increased interleukin-6 concentrations in maternal plasma before labor suggest the involvement of this cytokine as well in the altered immune response and its contribution to endothelial cell dysfunction.

Fas/APO-1 and p55 tumor necrosis factor (TNF) receptor (p55-R) activate cellular mechanisms that result in cell death. Upon activation of these receptors, Fas/APO-1 binds a protein called MORT1 (or FADD) and p55-R binds a protein called TRADD. MORT1 and TRADD can also bind to each other. We have cloned a novel protein, MACH, that binds to MORT1. This protein exists in multiple isoforms, some of which contain a region that has proteolytic activity and shows marked sequence homology to proteases of the ICE/CED-3 family. Cellular expression of the proteolytic MACH isoforms results in cell death. Expression of MACH isoforms that contain an incomplete ICE/CEDE region provides effective protection against the cytotoxicity induced by Fas/APO-1 or p55-R triggering. These findings suggest that MACH is the most upstream enzymatic component in the Fas/APO-1- and p55-R-induced cell death signaling cascades.

Purpose: To study the role of tumor necrosis factor (TNF) in the male reproductive system by examining the occurrence, source, and possible functional significance of soluble TNF receptors in seminal fluids of normal and infertile men. Materials and Methods: Concentrations of soluble TNF receptors (p55-sTNF-R and p75-sTNF-R) were measured by ELISA in human sera, seminal fluids, prostatic fluid and fluid obtained from an epididymal spermatocele. Results: The level of p55-sTNF-R in seminal fluids of normospermic men was approximate to 20-fold higher than in normal serum (13.9 +/- 6.9 ng./ml. versus 0.7 +/- 0.2 ng./ml.). In contrast, p75-sTNF-R, which occurs in serum at amounts higher than p55-sTNF-R, was almost indiscernible in the seminal fluids (

OBJECTIVES: We assessed maternal plasma and second-trimester amniotic fluid for levels of the p55 and p75 soluble tumor necrosis factor receptors. STUDY DESIGN: Blood was drawn from 61 healthy pregnant women (group A) before second-trimester genetic amniocentesis, and an aliquot of amniotic fluid was also obtained for this study. An additional blood sample was obtained from 13 of these patients at 36 to 40 weeks' gestation. Twenty-three healthy, nonpregnant women of reproductive age donated blood as a control group (group B). All plasma and amniotic fluid specimens were collectively assayed for the p55 and p75 soluble tumor necrosis factor receptors by specific enzyme-linked immunoassays. Additionally, tumor necrosis factor-alpha concentrations were measured in second-trimester plasma and amniotic fluid of 22 patients in group A and in all 23 of the nonpregnant women. RESULTS: The p55 and p75 soluble tumor necrosis factor receptors were detectable in all plasma samples from both groups of patients. The concentrations of both soluble receptors were significantly higher in second-trimester plasma compared with nonpregnant measurements (p

High levels of circulating soluble tumor necrosis factor receptors (sTNF-R) are associated with HIV-1 infection and disease. To understand better this association, we have investigated p55 and p75 TNF-R expression on peripheral blood mononuclear cell (PBMC) subsets and in the promonocytic cell line U937, with or without HIV infection, Using flow cytometry and monoclonal antibodies both to sTNF-R and to PBMC subsets, TNF-R were found to be expressed mostly by monocytes and in decreasing amounts and intensity in the following order: CD14(+) cells > CD8(+) cells > CD4(+) cells, Expression of TNF-R was higher on cells obtained from HIV-infected than from noninfected subjects, and expression of p75 sTNF-R was much higher than that of p55 sTNF-R. Studying the U937 cells revealed that over 80% of the cells expressed both sTNF-R, but with greater fluorescence intensity in the HIV-1 chronically infected cells (U-937-IIIB), Treatment of the cells with PMA caused an accelerated release into the medium of both sTNF-R, with a sharp decline in their cell surface expression, Basal levels of mRNA transcripts for p75 TNF-R were higher in the U-937-IIIB cells than in the uninfected cells, but p55 TNF-R mRNA was expressed only in the HIV-l-infected cells, These findings show that HIV-1 infection is accompanied by predominant elevation of p75 TNF-R surface expression on monocytes and CD8(+) lymphocytes, and results in both increased message and expression of these receptors in monocytes, It is very likely that increased shedding of these receptors into the serum accounts for the increased serum levels of both sTNF-R found in HIV-infected people.

The aim of this study was (a) to measure soluble tumor necrosis factor receptors (sTNF-Rs) and soluble interleukin-6 receptor (sIL-6-R) in coelomic and amniotic fluids, cord and maternal sera in pregnancy and labor, (b) to examine whether the changes in concentrations of biologically active TNF and IL-6 are related to changes in their soluble receptors, and (c) to determine if levels of soluble receptors in pre-eclamptic disorders differ from normal pregnancies at delivery. Materials collected from 206 women during pregnancy and at delivery were analyzed for soluble receptors by enzyme-linked immunosorbent assay (ELISA). All receptors were present in higher concentrations in coelomic than in th corresponding amniotic fluid. Concentrations increased in amniotic fluid from first to second trimester. The level of sIL-6-R then remained unchanged to term, but there was a decrease in the sTNF-Rs which might account for the simultaneous appearance of bioactive TNF. Labor did not affect the concentration of any receptor in amniotic fluid. In maternal serum, sTNF-Rs increased with gestational age and labor in parallel with IL-6. The origin and physiological importance of these soluble receptors are still unknown. In pre-eclamptic disorders p55 sTNF-R was elevated in maternal serum before initiation of labor compared to normal pregnancy.

Objective: To examine the effect of glycosylated recombinant human tumor necrosis factor binding protein-1 (r-hTNF binding protein-1), the extracellular domain of the tumor necrosis factor receptor p55 produced in mammalian cells, in a rabbit model of circulatory shock due to Escherichia coli. Design: Prospective, randomized, controlled trial. Setting: University hospital research laboratory. Subjects: Eighteen female, New Zealand white rabbits. Interventions: Anesthetized rabbits, infused with E. coli (10(9) organisms/kg), were pretreated with either r-hTNF binding protein-1 or saline. Mean arterial pressure, central venous pressure, cardiac output, and heart rate were recorded every 20 mins for 1 hr before, and for 4 hrs after, the infusion off. coli. Blood samples were obtained at 1-hr intervals for platelet count and white blood cell count, r-hTNF binding protein-1, and tumor necrosis factor (TNF) measurements. Measurements and Main Results: Administration of r-hTNF binding protein-1 resulted in improvement of mean arterial pressure, cardiac output, and systemic vascular resistance, as compared with the vehicle-treated group (p

Tumor necrosis factor (TNF) functions both as a soluble molecule and as a cell surface 26 kDa transmembrane protein, from which the soluble form is proteolytically derived. The 26 kDa TNF molecules isolated from P-31 labeled HeLa cells that had been transfected with the cDNA of a partially cleavable TNF mutant were found labeled. Phosphorylated 26 kDa TNF molecules could also be isolated from human LPS stimulated monocytic Mono Mac 6. Phosphoaminoacid analysis revealed that the labeled phosphate is bound to serine residues. No label was found incorporated in soluble 17 kDa TNF, indicating that the phosphorylated residue(s) of membrane-associated TNF occur in the cytoplasmic portion of the molecule. Phosphorylation of the intracellular domain of the 26 kDa TNF molecules may play a role in the regulation of expression or proteolytic processing of TNF, modulate TNF bioactivity, or take part in intracellular signal ing by cell-surface TNF. (C) 1995 Wiley-Liss, Inc.

Signaling by the p55 tumor necrosis factor (TNF) receptor and by the structurally related receptor Fas/APO1 is initiated by receptor clustering. Data presented here and in other recent studies (Wallach, D., Boldin, M., Varfolomeev, E. E., Bigda, Y., Camonis, H. J. and Mett, I. (1994) Cytokine 6, 556; Song, H. Y., Dunbar, J. D., and Bonner, D. B. (1994) J. Biol. Chem. 269, 22492-22495) indicate that part of that region within the intracellular domains of the two receptors that is involved in signaling for cell, death, as well as for some other effects (the ''death domain'', specifically self-associates. We demonstrate also the expected functional consequence of this association; a mere increase in p55 TNF receptor expression, or the expression just of its intracellular domain, is shown to trigger signaling for cytotoxicity as well as for interleukin 8 gene induction, while expression of the intracellular domain of Fas/APO1 potentiates the cytotoxicity of co-expressed p55 TNF receptor. These findings indicate that the p55 TNF and Fas/APO1 receptors play active roles in their own clustering and suggest the existence of cellular mechanisms that restrict the self-association of these receptors, thus preventing constitutive signaling.

Induced shedding of the p55 tumor necrosis factor receptor (p55-R) was previously shown 60 be independent of the amino acid sequence properties of the intracellular domain of this receptor. We now find it also independent of the sequence properties of the transmembrane domain and of the cysteine-rich region that constitutes most of the extracellular domain of the receptor. The shedding is shown to depend solely on the sequence properties of a small region within the spacer that Links the cysteine-rich region in the extracellular domain to the transmembrane domain. Detailed tests of effects of mutations in the spacer on the shedding indicate that the process is independent of the amino acid side-chain identity in this region except for a limited dependence on the identity of 1 residue (Val-173), located downstream to the putative major cleavage site of the receptor. It is strongly affected, however, by some mutations that seem to change the conformation of the spacer region. These findings suggest that a short amino acid sequence in the p55-R is essential and sufficient for its shedding and that the shedding is mediated either by a protease with limited sequence specificity or by several different proteases that recognize different amino acid sequences, yet it strictly depends on some conformational features of the cleavage region in the receptor.

Activated glial cells observed in the substantia nigra in Parkinson's disease may participate in the mechanism of nerve cell death by producing toxic substances such as cytokines. Among these compounds, tumor necrosis factor-alpha. (TNF) is of interest because it can provoke cell death. We detected TNF-immunoreactive glial cells in the substantia nigra of parkinsonian patients but not in those of control subjects. Immunoreactivity for TNF receptors was found in cell bodies and processes of most dopaminergic neurons of control and parkinsonian subjects, suggesting that nigral dopaminergic neurons might be sensitive to TNF produced in Parkinson's disease. These results suggest that TNF may participate in the degenerative processes occurring in Parkinson's disease, at least after a primary insult inducing a reactive gliosis.

Certain inflammatory cytokines and growth factors have been previously shown to interact with glycosaminoglycan moieties of the extracellular matrix (ECM). We have examined the association of the pleiotropic cytokine TNF-alpha with glycoprotein constituents of ECM. TNF-alpha interacted with fibronectin (FN) and laminin, and to a lesser degree with collagen. The major binding site for TNF-alpha on FN was localized to its 30-kDa N-terminal fragment (FN-N') with a K(i) in the sub-nM range. The binding of I-125-labeled TNF-alpha to immobilized FN or FN-N' persisted for at least 24 h, and was specifically inhibited by antibodies to FN, mAb directed against the FN-N' domain, unlabeled TNF-alpha, and by the truncated forms of TNF-alpha receptors. Once bound to immobilized FN or FN-N', the cytokine could not be released by the soluble TNF-alpha-receptors, although it could be released by anti-TNF-alpha Ab. TNF-alpha was also found to interact with soluble FN, although with a lower affinity. Similar to the soluble cytokine, the FN-bound TNF-alpha appears to be functional; it augmented the beta1-integrin-mediated adhesiveness of activated CD4+ human T cells to the glycoprotein. Hence, binding of TNF-alpha to immobilized FN, which modifies its functional accessibility to soluble TNF-alpha receptors, does not abolish but rather may locally restrict its activity. This study suggests that a major ECM glycoprotein can present, in a restricted manner, a functional adhesion-modulating cytokine to immune cells, and that ECM glycoproteins may regulate their intrinsic cell-adhesive properties by associating with cytokines.

Objective. To determine the value of measurement of serum soluble tumor necrosis factor receptor (sTNFR), compared with established parameters such as anti-double-stranded DNA, in monitoring systemic lupus erythematosus (SLE) disease activity, and to determine whether serum sTNFR are bioactive and can effectively inhibit TNF bioactivity. Methods. Fifty-three consecutive ambulatory or hospitalized SLE patients and 140 consecutive healthy subjects were enrolled in a prospective cohort study. Serum levels of sTNFR were measured by a unique 2-sided capture enzyme-linked immunosorbent assay using mouse monoclonal antibodies and rabbit antisera against the sTNFR. Results, The mean +/- SD concentrations of both the p55 (type I) and p75 (type II) soluble receptors were significantly higher in a group of 46 SLE patients than in controls: 1.89 +/- 0.89 ng/ml versus 0.77 +/- 0.19 ng/ml and 7.25 +/- 3.89 ng/ml versus 3.02 +/- 0.57 ng/ml, respectively (P

Two distinct types of soluble tumor necrosis factor a receptors (sTNFRs), which are felt to represent proteolytic cleavage products of the extracellular domains of membrane-bound TNFRs of molecular mass, 55 and 75 kDa, are found in the serum and urine of humans. We have measured the serum concentrations of these receptors in eight patients with metastatic renal cell carcinoma during treatment with interleukin-2 (IL-2)-based immunotherapy. The mean pretreatment concentration of sTNFR-55 kDa (p

Objective. Recently, 2 classes of cytokine inhibitors have been defined at the molecular level. The largest group comprises the extracellular domains of cell surface cytokine receptors, and includes both tumor necrosis factor receptors (TNF-R). The present study was conducted to investigate the role of TNF inhibitors in arthritis. Methods. We measured p55 and p75 soluble TNF-R (sTNF-R) in serum and synovial fluid (SF) samples from patients with rheumatic diseases and compared their levels with levels of soluble interleukin-2 receptors (sIL-2R). Sensitive enzyme-linked immunosorbent assays (ELISA), specific for p55 and p75 sTNF-R and for sIL-2R, were used. Results. Serum levels of p75 sTNF-R were 3-4-fold higher than levels of p55 sTNF-R, and both were significantly elevated in patients with osteoarthritis (OA) and rheumatoid arthritis (RA) compared with healthy controls. RA SF levels of sTNF-R were 4-5-fold higher than levels in serum, suggesting local production in the joint, and were significantly higher than levels in the SF of patients with seronegative arthropathy or OA. Furthermore, levels of p55 and p75 sTNF-R, but not sIL-2R or TNFalpha measured by ELISA, were increased in the SF of patients with clinically active RA. The soluble TNF-R in RA and OA SF were functional since they inhibited TNF activity in a cytotoxicity assay in proportion to the levels of inhibitor present. Evaluation of serially obtained serum samples suggested that sTNF-R may be a useful parameter for monitoring RA disease activity. Conclusion. Biologically active soluble TNF-R are up-regulated in patients with rheumatic disease and are produced locally in the joints. Measurement of serum levels of TNF-R may be useful for monitoring of disease, and determination of SF levels could be of diagnostic value.

Iodinated natural human urinary tumor necrosis factor binding protein I (I-125-uTBP) was iv injected into BALB/c mice, and its pharmacokinetics and tissue distribution were assessed during a short-term (0-1 hr) and for a long-term (0-24 hr) period. The blood I-125-uTBP concentration displayed a biphasic pattern that was adequately described by a biexponential function with estimated half-lives of 0.1 and 3.8 hr. The apparent volume of distribution (V(c)) of the central compartment was 3 ml, which approximated the mouse blood volume. The clearance (CL) derived either from a model-dependent or a model-independent method of analysis was 2.5 and 2.9 ml/hr, respectively. One hr after the iv administration of I-125-uTBP, the radioactivity accumulated in the major organs and tissues. The highest concentrations in terms of pg per organ were seen in the skin and in the liver. When expressed as pg I-125-uTBP per mg organ, the distribution was the highest in the gallbladder, bladder, kidneys, and lungs. At 24 hr, the distribution of I-125-uTBP represented about 10% of the amount measured at 1 hr. The rank order of accumulation of the radiolabeled uTBP in the major organs, expressed as pg per organ at 24 hr was skin > liver > kidneys > lungs > gut > spleen > gallbladder.

Expression of the two known receptors for TNF was studied in the promyelocytic leukemia cell line HL-60 before and after differentiation of the cells along the granulocyte lineage (induced by incubation with retinoic acid), or along the macrophage lineage (induced by incubation with the phorbol diester, PMA). The extent of inhibition of TNF binding by receptor-specific antisera, as well as the size of the complexes formed after cross-linking TNF to its receptors on intact cells, indicated that both receptor species were expressed on the surface of the undifferentiated HL60 cells. Differentiation into granulocyte-like cells resulted in some increase in TNF binding. The increase was apparently due to enhanced expression of the 75-kDa TNF-R, whereas the amounts of the 55-kDa TNF-R did not change significantly. In contrast, in HL-60 cells induced to differentiate into macrophage-like cells, expression of the 55-kDa TNF-R species was completely abolished. The pattern of TNF-R expression in the differentiated HL-60 cells was similar to that observed in leukocytes isolated from peripheral blood: on granulocytes, there were about equal amounts of both receptor species, whereas on monocytes the 75-kDa receptor was predominant. The loss of 55-kDa receptors during differentiation of HL-60 cells into macrophage-like cells was accompanied by a pronounced decrease in the level of the mRNA for that receptor, suggesting that at least part of the change in TNF-R expression is due to mechanisms that control the amounts of receptor mRNA. Although little is yet known regarding the functional differences between the two receptor species, marked changes in the pattern of their expression, as observed during HL-60 cell differentiation, are likely to alter the kind of response of the cells to TNF and may therefore play an important role in the coordination of TNF effects in the organism.

To localize the protease(s) involved in shedding of tumor necrosis factor receptors (TNF-R) from activated neutrophils (PMN) (Porteu, F., and C. Nathan (1990) J. Exp. Med. 172, 599-607), we tested subcellular fractions from PMN for their ability to cause loss of TNF-R from intact cells. Exposure of PMN to sonicated azurophil granules at 37-degrees-C resulted in inhibition of I-125-TNF binding; 50% inhibition ensued when PMN were treated for approximately 1 min with azurophil granules equivalent to 2-3 PMN per indicator cell. The TNF-R-degrading activity in azurophil granules was identified as elastase by its sensitivity to diisopropyl fluorophosphate (DFP), alpha-1-antitrypsin and N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone (MSAAPV-CK), and by the ability of purified elastase to reproduce the effect of azurophil granules. Elastase preferentially acted on the 75-kDa TNF-R, reducing by 85-96% the binding of I-125-TNF to mononuclear cells expressing predominantly this receptor, while having no effect on endothelial cells expressing almost exclusively the 55-kDa TNF-R. Elastase-treated PMN released a 32-kDa soluble fragment of p75 TNF-R that bound TNF and reacted with anti-TNF-R monoclonal antibodies. In contrast, fMet-Leu-Phe-activated PMN shed a 42-kDa fragment from p75 TNF-R, along with similar amounts of a 28-kDa fragment from p55 TNF-R. Shedding of both TNF-Rs by intact activated PMN was more extensive than shedding caused by elastase and was completely resistant to DFP and MSAAPV-CK. Thus, the TNF-R-releasing activity of azurophil granules is distinct from that operative in intact stimulated PMN and could provide an additional mechanism for the control of cellular responses to TNF at sites of inflammation.

Modulation of cellular responsiveness to tumor necrosis factor (TNF was studied in the human SV-80 cells. A marked cytocidal effect is exhibited by these cells at about 4 to 8 h after application of TNF together with protein synthesis inhibitors. Sensitivity of the cells to TNF toxicity was shown to be markedly decreased following their pretreatment with TNF itself or with interleukin (IL) 1 in the absence of protein synthesis inhibitors. The SV-80 cells respond to TNF also with enhanced phosphorylation of the small heat-shock protein, HSP27. This TNF effect is much more rapid than the cytocidal effect; it is observed within minutes of TNF application. The response to this effect, just like the response to the cytocidal effect, is markedly decreased following preexposure of the cells to either TNF or IL 1. Responsiveness to both effects of TNF is regained at the same time, about 15 to 20 h following removal of TNF or IL 1. The decrease in responsiveness after pretreatment with TNF or IL 1 does not reflect an inability of the pretreated cells to bind TNF. Although there is an initial decrease in TNF binding after such pretreatment, it is fully reserved already about 5 h following removal of the cytokines. The rate of uptake of TNF by the pretreated cells is also normal. In view of the rapidity of the effect of TNF on the phosphorylation of HSP27, it seems likely that the observed hyporesponsiveness reflects impairment of an early step in a signaling pathway, perhaps common to both the stimulation of phosphorylation and the induction of cell death by TNF. By restricting the duration of the effects of TNF this desensitization mechanism may safeguard against harmful consequences of these effects.

Keywords: TNF-ALPHA

Two proteins which specifically bind tumor necrosis factor (TNF) have recently been isolated from human urine in our laboratory. The two proteins cross‐react immunologically with two species of cell surface TNF receptors (TNF‐R). Antibodies against one of the two TNF binding proteins (TBPI) were found to have effects characteristic of TNF, including stimulating phosphorylation of specific cellular proteins. Oligonucleotide probes designed on the basis of the NH2‐terminal amino acid sequence of TBPI were used to clone the cDNA for the structurally related cell surface type 1 TNF‐R. It is notable that although this receptor can signal the phosphorylation of cellular proteins, it appears from its amino acid sequence to be devoid of intrinsic protein kinase activity. The extracellular domain of the receptor is composed of four internal cysteine‐rich repeats, homologous to structures repeated four times in the extracellular domains of the nerve growth factor receptor and the B lymphocytes surface antigen CDw40. The amino acid composition and size of the extracellular domain of the type I TNF‐R closely resemble those of TBPI. The COOH‐terminal amino acid sequence of the four cysteine rich repeats within the extracellular domain of the type I TNF‐R matches the COOH‐terminal sequence of TBPI. Amino acid sequences in the extracellular domain also fully match other sequences found in TBPI. On the other hand, amino acid sequences in the soluble form of the type II TNF‐R (TBPII), while indicating a marked homology of structure, did not suggest any identity between this protein and the extracellular domain of the type I TNF‐R. CHO cells transfected with type I TNF‐R cDNA produced both cell surface and soluble forms of the receptor. The receptor produced by CHO cells was recognized by several monoclonal antibodies against TBPI, reacting with several distinct epitopes in this molecule. These data suggest that the soluble forms of the TNF‐Rs are structurally identical to the extracellular cytokine binding domains of these receptors and are consistent with the notion that the soluble forms are, at least partly, derived from the same transcripts that encode the cell surface receptors.

Keywords: Chemistry, Analytical

Two proteins which specifically bind tumor necrosis factor (TNF) were isolated from human urine by ligand (TNF)-affinity purification, followed by reversed phase high performance liquid chromatography. The molecular weights of the two proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were similar (about 30,000). Both proteins provided protection against the cytocidal effect of TNF in vitro and both bound TNF-alpha more effectively than TNF-beta. Antibodies raised against each of the proteins had an inhibitory effect on the binding of TNF to cells, suggesting that both proteins are structurally related to the TNF receptors. However, the two proteins differed in NH2-terminal amino acid sequences: Asp-Ser-Val-Cys-Pro- in one and Val-Ala-Phe-Thr-Pro- in the other. The NH2-terminal sequence of the former protein was invariable, while that of the latter was truncated to varying degrees. The two proteins were also immunologically distinct. The relative efficacy of anti-sera against the two proteins in inhibiting the binding of TNF to cells varied markedly from one line of cells to another. Evidence has been presented recently for the existence of two distinct molecular species of cell surface receptors for TNF and for differential expression of those two receptors by cells of different lines. The findings presented in this study are consistent with the notion that the urinary TNF-binding proteins constitute soluble forms of the two molecular species of the cell surface TNF receptors.

Unfractionated preparations of the proteins of human urine provided protection against the in vitro cytocidal effect of tumor necrosis factor (TNF). In certain cells, the proteins decreased expression of the receptors for TNF in a temperature-dependent way. In all cells examined, the proteins were found to interfere also with the binding of both TNF and interleukin-1 when applied directly into the binding assays. That effect could be observed in the cold, suggesting that it was independent of cellular metabolism. A protein which protects cells against the cytotoxicity of TNF was purified from human urine by chromatography on CM-Sepharose followed by high performance liquid chromatography on Mono Q and Mono S columns and reversed phase high performance liquid chromatography. This protein is a very minor constituent of normal urine, with an apparent molecular weight of about 27,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Homogeneity of the purified protein was confirmed by microsequence analysis which revealed a single N-terminal sequence: Asp-Ser-Val-Cys-Pro-. The protein protected cells from TNF toxicity at concentrations of a few nanograms per ml and interfered with the binding of both TNF-alpha and TNF-beta to cells, when applied simultaneously with the cytokines. However, unlike crude preparations of the urinary proteins, the purified protein did not induce in cells a decrease in ability to bind TNF nor did it interfere with the binding of interleukin-1 to its receptor. Direct, specific binding to the protein of TNF-alpha and, to a lesser extent, also TNF-beta, but not of interleukin-1 nor interferon-gamma could be demonstrated. It is suggested that this protein blocks the function of TNF by competing for TNF with the TNF receptor and not by interacting with the target cell.

Bloksmu: I have some comments on the induction of haemorrhagic necrosis in Meth A tumours transplanted subcutaneously into the abdominal skin of syngeneic BALB/c mice. Endotoxin-induced tumour necrosis has been described in the literature as haemorrhage with necrosis, or haemorrhagic necrosis (Shear & Perrault 1943, Parr et a1 1973, Freudenberg et al 1984). However, morphological studies on the induction of necrosis in nine-day-old Meth A tumours (diameter about 7mm) by various agents have prompted us to propose that Meth A can show two distinct types of necrosis, as judged by pathogenesis and ultimate appearance. We have designated these as haemorrhagic necrosis (HN) and coagulation necrosis (CN), which is defined by cell decay with preservation of cell contours, nuclear condensation and nuclear disintegration. Both kinds of necrosis can be considered as different forms of coagulation necrosis. Evidence for the distinction has been compiled in Table 1. [first paragraph]