Role of P2z purinergic receptors in ATP-mediated killing of tumor necrosis factor (TNF)-sensitive and TNF-resistant L929 fibroblasts.

Two closely related cell lines were characterized in their responses to extracellular ATP (ATPo): the fibroblast cell line L929 and a TNF-resistant variant L929/R. Both lines showed ATPo-activated increases in intracellular Ca2+, inward current, and sustained depolarization of the plasma membrane, cell responses compatible with activation of purinergic receptors of the P2y, P2x, or P2z subtype; however, only the L929/R variant was susceptible to ATPo-dependent early permeabilization of the plasma membrane to hydrophilic solutes of M(r) below 900, a response uniquely caused by the activation of P2z receptors. Both cell types were susceptible to the cytotoxic effect of ATPo, but killing of the L929/R variant required much shorter incubations in the presence of this nucleotide. Morphologic examination of ATPo-challenged L929 and L929/R cells showed that cell death occurred by two alternative mechanisms: colloido-osmotic lysis or apoptosis. Occurrence of apoptosis was confirmed by agarose gel analysis of cellular DNA. Although ATPo caused a fast mobilization of intracellular Ca2+, neither colloido-osmotic lysis nor apoptosis were Ca2+ dependent. Our results show that the L929/R variant, but not the L929 parental fibroblast cell line, expresses functional purinergic receptors of the P2z subtype. The presence of P2z receptors confers to L929/R cells enhanced susceptibility to ATPo-mediated cytotoxicity.     

Cell-associated tumor necrosis factor (TNF) as a killing mechanism of activated cytotoxic macrophages

Different macrophage populations were investigated for their abilities to secrete tumor necrosis factor (TNF) and to lyse TNF-susceptible tumor cells. In this way we could demonstrate that TNF-secretion, although a feature of all activated macrophage populations, is no absolute requirement for the killing of the TNF-sensitive Wehi 164 target. Macrophage cytotoxicity against this cell but not against the TNF-resistant P815 mastocytoma, was completely inhibitable by a specific anti-TNF serum also in the absence of measurable secreted TNF. Moreover the TNF-dependent lysis of tumor cells could also be performed by activated macrophages that had been fixed with paraformaldehyde before the addition of the target cells. In the indirect radioimmunoassay, TNF could be demonstrated on the surface of fixed effector cells. Our results must be interpreted in terms of membrane-associated TNF as the lytic principle for TNF-susceptible tumor cells.     

A nonsecretable cell surface mutant of tumor necrosis factor (TNF) kills by cell-to-cell contact

In addition to the induction of tumor regression, tumor necrosis factor (TNF) has been implicated as the causative agent in a number of pathologies, including cachexia, septic shock, rheumatoid arthritis, autoimmunity, and induction of HIV expression. We propose that this complex physiology might be manifest by different forms of TNF: the 17 kd secretory component, the 26 kd transmembrane form, or both. To determine whether the 26 kd form of TNF was biologically active and whether its biology differed from that of the secretory component, we generated uncleavable and solely secretable mutants of TNF and studied their biological activities. We found that an uncleavable mutant of the 26 kd cell surface transmembrane form of TNF kills tumor cells and virus-infected cells by cell-to-cell contact, and that TNF need not be internalized by its target to kill. Thus, the 26 kd integral transmembrane form of TNF may function in vivo to kill tumor cells and other targets locally in contrast to the systemic bioactivity of the secretory component.     

Histamine antagonizes tumor necrosis factor (TNF) signaling by stimulating TNF receptor shedding from the cell surface and Golgi storage pool

Tumor necrosis factor (TNF) activates pro-inflammatory functions of vascular endothelial cells (EC) through binding to receptor type 1 (TNFR1) molecules expressed on the cell surface. The majority of TNFR1 molecules are localized to the Golgi apparatus. Soluble forms of TNFR1 (as well as of TNFR2) can be shed from the EC surface and inhibit TNF actions. The relationships among cell surface, Golgi-associated, and shed forms of TNFR1 are unclear. Here we report that histamine causes transient loss of surface TNFR1, TNFR1 shedding, and mobilization of TNFR1 molecules from the Golgi in cultured human EC. The Golgi pool of TNFR1 serves both to replenish cell surface receptors and as a source of shed receptor. Histamine-induced shedding is blocked by TNF-alpha protease inhibitor, an inhibitor of TNF-alpha-converting enzyme, and through the H1 receptor via a MEK-1/p42 and p44 mitogen-activated protein kinase pathway. Cultured EC with histamine-induced surface receptor loss become transiently refractory to TNF. Histamine injection into human skin engrafted on immunodeficient mice similarly caused shedding of TNFR1 and diminished TNF-mediated induction of endothelial adhesion molecules. These results both clarify relationships among TNFR1 populations and reveal a novel anti-inflammatory activity of histamine.     

Enhancement of cell-mediated cytotoxicity by recombinant tumor necrosis factor (TNF alpha)

The effects of recombinant tumor necrosis factor (rTNF alpha) on the immune responses were investigated. A single iv injection of rTNF alpha (6 x 10(3) U) caused regression of sarcoma-180 transplanted into BALB/c nu/+ mice, but failed to regress this tumor in nu/nu mice. A higher dose of rTNF alpha (2 x 10(4) U) was necessary to induce antitumor effect in nu/nu mice. A host-related factor seemed to be involved in mediating tumor regression. Therefore, the effects of rTNF alpha on various T-dependent immune responses, including delayed footpad reaction (DFR), cell mediated cytolysis (CMC), and plaque-forming cells (PFC) were examined in BALB/c mice, immunized ip with chicken erythrocytes (CRBC). A single injection of rTNF alpha, at the time of the antigen administration, induced the augmentation of CMC to CRBC in a dose-dependent manner. DFR and PFC were not affected in optimal immunization procedures. The TNF alpha injection, at or after the time of antigen administration, was more effective in inducing augmentation of CMC. The increase in CMC by TNF alpha was mediated by nonadherent, Thy 1.2, Lyt 2.2 positive cells and neutralization of TNF alpha by the anti-TNF alpha monoclonal antibody abolished the effect on CMC. These results indicated that the human recombinant TNF alpha induced changes in the T-cell-mediated responses.     

Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human

Ligands of the tumor necrosis factor superfamily (TNFSF) (4-1BBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA1, EDA2, FasL, GITRL, LIGHT, lymphotoxin alpha, lymphotoxin alphabeta, OX40L, RANKL, TL1A, TNF, TWEAK, and TRAIL) bind members of the TNF receptor superfamily (TNFRSF). A comprehensive survey of ligand-receptor interactions was performed using a flow cytometry-based assay. All ligands engaged between one and five receptors, whereas most receptors only bound one to three ligands. The receptors DR6, RELT, TROY, NGFR, and mouse TNFRH3 did not interact with any of the known TNFSF ligands, suggesting that they either bind other types of ligands, function in a ligand-independent manner, or bind ligands that remain to be identified. The study revealed that ligand-receptor pairs are either cross-reactive between human and mouse (e.g. Tweak/Fn14, RANK/RANKL), strictly species-specific (GITR/GITRL), or partially species-specific (e.g. OX40/OX40L, CD40/CD40L). Interestingly, the receptor binding patterns of lymphotoxin alpha and alphabeta are redundant in the human but not in the mouse system. Ligand oligomerization allowed detection of weak interactions, such as that of human TNF with mouse TNFR2. In addition, mouse APRIL exists as two different splice variants differing by a single amino acid. Although human APRIL does not interact with BAFF-R, the shorter variant of mouse APRIL exhibits weak but detectable binding to mouse BAFF-R.     

Poliovirus protein 3A inhibits tumor necrosis factor (TNF)-induced apoptosis by eliminating the TNF receptor from the cell surface

Viral infections often trigger host defensive reactions by activating intrinsic (intracellular) and extrinsic (receptor-mediated) apoptotic pathways. Poliovirus is known to encode an antiapoptotic function(s) suppressing the intrinsic pathway. Here, the effect of poliovirus nonstructural proteins on cell sensitivity to tumor necrosis factor (TNF)-induced (i.e., receptor-mediated) apoptosis was studied. This sensitivity is dramatically enhanced by the viral proteinase 2A, due, most likely, to inhibition of cellular translation. On the other hand, cells expressing poliovirus noncapsid proteins 3A and 2B exhibit strong TNF resistance. Expression of 3A neutralizes the proapoptotic activity of 2A and results in a specific suppression of TNF signaling, including the lack of activation of NF-kappaB, due to elimination of the TNF receptor from the cell surface. In agreement with this, poliovirus infection results in a dramatic decrease in TNF receptor abundance on the surfaces of infected cells as early as 4 h postinfection. Poliovirus proteins that confer resistance to TNF interfere with endoplasmic reticulum-Golgi protein trafficking, and their effect on TNF signaling can be imitated by brefeldin A, suggesting that the mechanism of poliovirus-mediated resistance to TNF is a result of aberrant TNF receptor trafficking.     

Effects of tumor necrosis factor (TNF) on lipolytic activities of rat heart

Summary Tumor Necrosis Factor (TNF) inhibits lipoprotein lipase activity in cultured myocytes and in the Langendorff rat heart after 3 h perfusion with TNF of glucocorticoid-pretreated rats. TNF acutely stimulates glyc(ogen)olysis and concomitantly endogenous lipolysis. The latter was significantly increased only when rats had been pretreated with glucocorticoid or fed a trierucate-rich diet. Under these conditions, contractile activity of the Langendorff hearts was acutely increased by TNF The mechanism of the actue increase of contractile function and the accompanying increased glycolytic and lipolytic activities, by TNF, may be explained by increased cytosolic Ca²⁺ and cAMP levels.     

Endogenous membrane tumor necrosis factor (TNF) is a potent amplifier of TNF receptor 1-mediated apoptosis

The heat shock protein 90 (Hsp-90) inhibitor, geldanamycin, and the proteasome inhibitor, MG-132, both inhibited tumor necrosis factor receptor 1 (TNF-R1)- but not TRAIL-induced apoptosis in Kym-1 cells, suggesting that TNF-R1-induced cell death is dependent on NF-kappaB activation in this model. Triggering of TNF-R1 by agonistic antibodies led to cell-type specific induction of endogenous TNF and apoptosis, the latter of which was abrogated by neutralizing TNF specific antibodies. TNF-R1-stimulated cells expressed TNF mainly in a cell-associated form, suggesting that the endogenously produced TNF act in its membrane-bound form. Geldanamycin failed to inhibit apoptosis induction by a combination of agonistic TNF-R1- and TNF-R2-specific antibodies, indicating that both TNF receptors co-operate in TNF-R1-triggered apoptosis in Kym-1 cells. Thus, TNF-R1 stimulation can elicit a strong and rapid apoptotic response via induction of membrane TNF and subsequent cooperation of TNF-R1 and TNF-R2. Moreover, we give evidence that this mechanism circumvents the need of the prolonged presence of exogenous soluble TNF for TNF-R1-mediated apoptosis induction.     

Effect of antisera against recombinant tumor necrosis factor and the monocyte-derived cytotoxin(s) on monocyte-mediated killing of various tumor cells

Antisera raised against recombinant tumor necrosis factor (TNF) and against the monocyte-derived cytotoxic/cytostatic protein factor (CF), which is related to recombinant TNF, have been compared with respect to their ability to inhibit monocyte-mediated killing of various types of cells which differ in their sensitivity to recombinant TNF. During 6 hr of coculturing monocytes and target cells, the recombinant TNF antiserum inhibited killing of the extremely TNF-sensitive WEHI 164 clone 13 cells and actinomycin D-treated WEHI 164 cells from which the clone 13 cells were derived (parental WEHI 164 cells (P-WEHI 164 cells]. The CF antiserum also inhibited monocyte-mediated killing of these cells during 6 hr of coculturing with monocytes, but on a per volume basis it was less potent than the recombinant TNF antiserum, consistent with the fact that the CF antiserum also was much less potent in inhibiting the cytotoxic activity of recombinant TNF. However, during 72 hr of coculturing with monocytes and target cells, the CF antiserum inhibited monocyte-mediated killing of P-WEHI 164 cells more efficiently than the recombinant TNF antiserum. Moreover, during 72 hr of coculturing with monocytes, only the CF antiserum was able to significantly inhibit monocyte-mediated killing of the relatively recombinant TNF-resistant K562 cells. This suggests that a factor immunologically different from recombinant TNF, perhaps a form of natural TNF differing somewhat immunologically from recombinant TNF, was involved in the killing of K562 cells, and possibly in the killing of P-WEHI 164 cells, during 72 hr of coculturing with monocytes. Although this factor was present extracellularly, it appears that it may act as a monocyte-associated factor in monocyte-mediated killing of K562 cells, since exposure to recombinant interferon-gamma (rIFN-gamma) in the absence of Escherichia coli endotoxin (lipopolysaccharide, LPS) activated the monocytes to mediate killing of K562 cells more efficiently than exposure to LPS alone, despite the fact that only little cytotoxic/cytostatic activity was released from the monocytes without the addition of LPS. The ability of rIFN-gamma and LPS to activate monocytes to produce and release CF has also been studied.     

Intracellular localization and transcriptional regulation of tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4).

To gain insight in the subcellular localization of tumor necrosis factor receptor-associated factor (TRAF4) we analyzed GFP chimeras of full-length TRAF4 and various deletion mutants derived thereof. While TRAF4-GFP (T4-GFP) was clearly localized in the cytoplasm, the N-terminal deletion mutant, T4(259-470), comprising the TRAF domain of the molecule, and a C-terminal deletion mutant consisting mainly of the RING and zinc finger domains of TRAF4 were both localized predominantly to the nucleus. Passive nuclear localization of T4(259-470) can be ruled out as the TRAF domain of TRAF4 was sufficient to form high molecular weight complexes. T4(259-470) recruited full-length TRAF4 into the nucleus whereas TRAF4 was unable to change the nuclear localization of T4(259-470). Thus, it seems that individual T4(259-470) mutant molecules are sufficient to direct the respective TRAF4-T4(259-470) heteromeric complexes into the nucleus. In cells forming cell-cell contacts, TRAF4 was recruited to the sites of contact via its C-TRAF domain. The expression of some TRAF proteins is regulated by the NF-kappaB pathway. Thus, we investigated whether this pathway is also involved in the regulation of the TRAF4 gene. Indeed, in primary T-cells and Jurkat cells stimulated with the NF-kappaB inducers TNF or phorbol 12-myristate 13-acetate (PMA), TRAF4-mRNA was rapidly up-regulated. In Jurkat T-cells deficient for I-kappaB kinase gamma (IKKgamma, also known as NEMO), an essential component of the NF-kappaB-inducing-IKK complex, induction of TRAF4 was completely inhibited. In cells deficient for RIP (receptor interactive protein), an essential signaling intermediate of TNF-dependent NF-kappaB activation, TNF-, but not PMA-induced up-regulation of TRAF4 was blocked. These data suggest that activation of the NF-kappaB pathway is involved in up-regulation of TRAF4 in T-cells.     

鲢（Hypophthalmichthys molitrix）肿瘤坏死因子基因的克隆与表达分析

肿瘤坏死因子(TNF)是一种炎症细胞因子,在非特异性免疫系统中发挥着重要作用.TNF由单核细胞或巨噬细胞产生,能直接造成肿瘤细胞的死亡,并参与机体炎症和免疫应答的调节.用RACE(rapid amplification of cDNAends)-PCR方法,从鲢总RNA反转录产物中获得了1 254 bpTNF cDNA全序列.该序列包含394 bp的5'端非编码区,398 bp的3'端非编码区和462 bp的开放阅读框.鲢TNF的开放阅读框编码239个氨基酸,其中包含构成一对二硫键的2个保守半胱氨酸.同时利用RT-PCR技术,对该基因在鲢鱼体内不同组织之间的表达差异进行了分析研究,结果表明鲢TNF mRNA主要在脑、鳃和头肾中表达,肠和脾中有少量表达,而在肝中几乎没有表达.     

Tumor necrosis factor (TNF)-alpha and TNF receptors in viral pathogenesis

Tumor necrosis factor-alpha (TNF-alpha) and TNF receptors (TNFR) are members of the growing TNF ligand and receptor families that are involved in immune regulation. The present report will focus on the role of the prototypic ligand TNF and its two receptors, TNFR1 and TNFR2, in viral pathogenesis. Although TNF was reported years ago to modulate viral infections, recent findings on the molecular pathways involved in TNFR signaling have allowed a better understanding of the molecular interactions between cellular and viral factors within the infected cell. The interactions of viral proteins with intracellular components downstream of the TNFR have highlighted at the molecular level how viruses can manipulate the cellular machinery to escape the immune response and to favor the spread of the infection. We will review here the role of TNF and TNFR in immune response and the role of TNF and TNFR signaling in viral pathogenesis.     

Recombinant 55-kDa tumor necrosis factor (TNF) receptor. Stoichiometry of binding to TNF alpha and TNF beta and inhibition of TNF activity.

The extracellular domain of the 55-kDa TNF receptor (rsTNFR beta) has been expressed as a secreted protein in baculovirus-infected insect cells and Chinese hamster ovary (CHO)/dhfr- cells. A chimeric fusion protein (rsTNFR beta-h gamma 3) constructed by inserting the extracellular part of the receptor in front of the hinge region of the human IgG C gamma 3 chain has been expressed in mouse myeloma cells. The recombinant receptor proteins were purified from transfected cell culture supernatants by TNF alpha- or protein G affinity chromatography and gel filtration. In a solid phase binding assay rsTNFR beta was found to bind TNF alpha with high affinity comparable with the membrane-bound full-length receptor. The affinity for TNF beta was slightly impaired. However, the bivalent rsTNFR beta-h gamma 3 fusion protein bound both ligands with a significantly higher affinity than monovalent rsTNFR beta reflecting most likely an increased avidity of the bivalent construct. A molecular mass of about 140 kDa for both rsTNFR beta.TNF alpha and rsTNFR beta.TNF beta complexes was determined in analytical ultracentrifugation studies strongly suggesting a stoichiometry of three rsTNFR beta molecules bound to one TNF alpha or TNF beta trimer. Sedimentation velocity and quasielastic light scattering measurements indicated an extended structure for rsTNFR beta and its TNF alpha and TNF beta complexes. Multiple receptor binding sites on TNF alpha trimers could also be demonstrated by a TNF alpha-induced agglutination of Latex beads coated with the rsTNFR beta-h gamma 3 fusion protein. Both rsTNFR beta and rsTNFR beta-h gamma 3 were found to inhibit binding of TNF alpha and TNF beta to native 55- and 75-kDa TNF receptors and to prevent TNF alpha and TNF beta bioactivity in a cellular cytotoxicity assay. Concentrations of rsTNFR beta-h gamma 3 equimolar to TNF alpha were sufficient to neutralize TNF activity almost completely, whereas a 10-100-fold excess of rsTNFR beta was needed for similar inhibitory effects. In view of their potent TNF antagonizing activity, recombinant soluble TNF receptor fragments might be useful as therapeutic agents in TNF-mediated disorders.     

Endotoxin-induced tumor necrosis factor (TNF): selective triggering of TNF and interleukin-1 production by distinct glucosamine-derived lipids

The isolated lipid A of Bordetella pertussis endotoxin (LipA) has been found to induce in vitro release of tumor necrosis factor (TNF) by murine macrophages, albeit much less efficiently than does the intact lipopolysaccharide. Synthetic analogs (monosaccharides M4 and M6) of both glucosamine units present in the LipA backbone induced production of TNF by peritoneal macrophages of Swiss mice. Macrophages from A/J mice gave higher responses than those from Swiss mice, while those of C3H/HeJ mice were unresponsive. Enhancement of TNF secretion was observed for all cells if they were pretreated with a calcium ionophore, and no otherwise inactive substance became active with cells thus treated. For synthetic monosaccharide derivatives, a phosphate group on O-4 was not required for, and a phosphate group on O-1 abolished, the TNF-inducing activity. Synthetic monosaccharides, chemically closely related to substructures recognized to be present in isolated lipid A preparations, could induce either TNF or interleukin-1 (IL-1) production, but not both simultaneously: the monosaccharides M4 and M6 were active TNF inducers, but did not initiate IL-1 production, while the monosaccharides M9 and lipid X efficiently elicited IL-1 production, but did not trigger TNF secretion. It should be noted, however, that the active synthetic compounds are considerably less efficient TNF inducers as is the intact B. pertussis endotoxin.