Novel muteins of human tumor necrosis factor α

For chemical synthesis of a gene coding for human necrosis factor α (TNF-α), DNA sequence predicted by the amino acid sequence of human TNF molecule was prepared. Codons were chosen according to the codon usage in Escherichia coli (E. coli). The 490 hp gene was assembled by enzymic ligation of 42 oligonucleotides and was cloned into vector (]KK223-3) for high expression of active TNF-α in E. coli. With use of site-directed mutagenesis on this DNA, five different muteins of TNF-α were synthesized. TNF-M1 and TNF-M4 have deletions of His-73 and Gln-102, respectively. These deletions didn't cause loss of the cytotoxic activity against L929 cells. TNF-M5, which has a substitution of Asp-10 to Arg, had the similar cytotoxic activity to that of TNF-α. The cytotoxic spectra against several tumor cells were not changed by this substitution. TNF-M3 has an amino acid substitution of Glu-116 to His which occupies this position in human TNF-β. This substitution didn't change the cytotoxicity. In addition, evidence was presented that the change of the carboxyl terminal residue doesn't always influence the cytotoxic activity of TNF-α. Many different muteins were also isolated by random mutagenesis with hydroxylamine-HCl. One of the muteins, which carries a mutation of His-15 to Tyr, lost the cytotoxic activity almost completely.     

Novel muteins of human tumor necrosis factor alpha

For chemical synthesis of a gene coding for human tumor necrosis factor alpha (TNF-alpha), DNA sequence predicted by the amino acid sequence of human TNF molecule was prepared. Codons were chosen according to the codon usage in Escherichia coli (E. coli). The 490 bp gene was assembled by enzymic ligation of 42 oligonucleotides and was cloned into a vector (pKK223-3) for high expression of active TNF-alpha in E. coli. With use of site-directed mutagenesis on this DNA, five different muteins of TNF-alpha were synthesized. TNF-M1 and TNF-M4 have deletions of His-73 and Gln-102, respectively. These deletions didn't cause loss of the cytotoxic activity against L929 cells. TNF-M5, which has a substitution of Asp-10 to Arg, had the similar cytotoxic activity to that of TNF-alpha. The cytotoxic spectra against several tumor cells were not changed by this substitution. TNF-M3 has an amino acid substitution of Glu-116 to His which occupies this position in human TNF-beta. This substitution didn't change the cytotoxicity. In addition, evidence was presented that the change of the carboxyl terminal residue doesn't always influence the cytotoxic activity of TNF-alpha. Many different muteins were also isolated by random mutagenesis with hydroxylamine-HCl. One of the muteins, which carries a mutation of His-15 to Tyr, lost the cytotoxic activity almost completely.     

Activation of human polymorphonuclear neutrophil functions by interferon-gamma and tumor necrosis factors

Recombinant human interferon-gamma (rHuIFN-gamma) and natural human tumor necrosis factor beta (nHuTNF-beta) (previously called lymphotoxin), purified to homogeneity, were used to assess their effects on certain functions of human polymorphonuclear neutrophils (PMN) in vitro. The treatment of PMN with 100 U of either rHuIFN-gamma or nHuTNF-beta for 20 min significantly increased their ability to phagocytize 1.5-microns latex beads as detected by flow cytometry. Preparations of recombinant human TNF-beta (rHuTNF-beta) showed activities similar to those of its natural counterpart in activating phagocytosis. In addition, a significant enhancement in PMN-mediated antibody-dependent cellular cytotoxicity was observed after treatment for 2 hr with IFN gamma and both TNF-alpha and TNF-beta. The enhancement by treatment with a combination of rHuIFN-gamma and nHuTNF-beta exceeded the enhancement caused by either agent alone. We also show that although lipopolysaccharide (LPS) is a potent stimulator of PMN function, polymyxin B can block LPS-induced but not lymphokine-induced activation. These data demonstrate new activities for both TNF-alpha and TNF-beta in augmenting the phagocytic and cytotoxic activities of PMN.     

The emitting species formed by the oxidation of hydrazides with hypohalites or N-halosuccinimides.

The chemiluminescence accompanying the oxidation of salicylic hydrazide (2-hydroxybenzoic acid hydrazide) with hypochlorite, hypobromite, N-chlorosuccinimide, N-bromosuccinimide or hydrogen peroxide with cobalt(II) matched the photoluminescence emission of salicylic acid. In a related reaction, the oxidation of a mixture of isoniazid and ammonia, a synergistic effect was observed. The chemiluminescence spectrum for this reaction matches that accompanying the oxidation of the hydrazide, rather than the oxidation of ammonia. These results were used to assess mechanisms proposed by previous authors.     

Inhibition by IL-1 of endothelial cell activation induced by tumor necrosis factor or lymphotoxin

Previous studies in this and other laboratories have demonstrated that IL-1, lymphotoxin (LT), and TNF rapidly stimulate a number of proinflammatory properties in cultured endothelial cells (EC) including cell-surface procoagulant activity and increased adhesivity for lymphocytes, monocytes, and polymorphonuclear leukocytes. In addition, we have demonstrated that LT and TNF, but not IL-1, stimulate increases in EC RNA synthesis, protein synthesis, and cellular volumes, changes which may correspond to the hypertrophy of EC seen at sites of inflammation in vivo. It is reported here that both human rIL-1 alpha and rIL-1 beta totally inhibit the increases in EC RNA synthesis, protein synthesis, and cell volumes induced by either TNF or LT. As little as 0.1 ng/ml of either IL-1 was sufficient to totally block the activation of EC induced by 100-fold higher concentrations (10 ng/ml) of either LT or TNF. The relevance of these findings to the regulation of inflammatory responses is discussed.     

Ornithine decarboxylase prevents tumor necrosis factor alpha-induced apoptosis by decreasing intracellular reactive oxygen species

Ornithine decarboxylase (ODC) plays an essential role in various biological functions, including cell proliferation, differentiation and cell death. However, how it prevents the cell apoptotic mechanism is still unclear. Previous studies have demonstrated that decreasing the activity of ODC by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, causes the accumulation of intracellular reactive oxygen species (ROS) and cell arrest, thus inducing cell death. These findings might indicate how ODC exerts anti-oxidative and anti-apoptotic effects. In our study, tumor necrosis factor alpha (TNF-) induced apoptosis in HL-60 and Jurkat T cells. The kinetic studies revealed that the TNF- -induced apoptotic process included intracellular ROS generation (as early as 1 h after treatment), the activation of caspase 8 (3 h), the cleavage of Bid (3 h) and the disruption of mitochondrial membrane potential ( _m) (6 h). Furthermore, ROS scavengers, such as glutathione (GSH) and catalase, maintained _m and prevented apoptosis upon treatment. Putrescine and overexpression of ODC had similar effects as ROS scavengers in decreasing intracellular ROS and preventing the disruption of _m and apoptosis. Inhibition of ODC by DFMO in HL-60 cells only could increase ROS generation, but did not disrupt _m or induce apoptosis. However, DFMO enhanced the accumulation of ROS, disruption of _m and apoptosis when cells were treated with TNF- . ODC overexpression avoided the decline of Bcl-2, prevented cytochrome c release from mitochondria and inhibited the activation of caspase 8, 9 and 3. Overexpression of Bcl-2 maintained _m and prevented apoptosis, but could not reduce ROS until four hours after TNF- treatment. According to these data, we suggest that TNF- induces apoptosis mainly by a ROS-dependent, mitochondria-mediated pathway. Furthermore, ODC prevents TNF- -induced apoptosis by decreasing intracellular ROS to avoid Bcl-2 decline, maintain _m, prevent cytochrome c release and deactivate the caspase cascade pathway.     

The effect of tumour necrosis factor-alpha (TNF-alpha) muteins on human neutrophils in vitro

Tumour necrosis factor-alpha (TNF-alpha) has been implicated as an important inflammatory mediator. In vitro, TNF-alpha is reported to activate human polymorphonuclear neutrophils (PMN), inducing responses such as phagocytic activity, degranulation and oxidative metabolism. Biological responses to TNF-alpha are initiated by its binding to specific cell surface receptors, and various studies have shown that the major TNF receptor species on PMN is the 75 kDa receptor. To verify the suggestion that the receptor binding domain includes the region close to the N-terminus of the TNF-alpha molecule, four TNF-alpha derivatives termed muteins were constructed, using a synthetic cDNA fragment substituting the N-terminal 3-7 selected hydrophilic or hydrophobic amino acids in the original TNF-alpha genomic DNA. Binding of muteins to PMN was assessed using monoclonal antibodies recognizing either the 55 kDa (p55) or the 75 kDa (p75) TNF receptor subtypes. Blocking by muteins of anti-p75 antibody binding to PMN was as expected from their N-terminal amino acid composition and hydrophilic properties. Hydrophilic muteins competed well with anti-TNF receptor antibodies for binding to the p75 receptor. In contrast, hydrophobic muteins were unable to block anti-p75 binding. Similarly, degranulation, chemiluminescence or enhancement of the PMN response to specific stimuli by the muteins correlated with the hydrophilic properties of the muteins. The significance of these observations in relation to the molecular structure of TNF-alpha is discussed.     

Stimulation of neutrophils by tumor necrosis factor

Human recombinant tumor necrosis factor (TNF) was shown to be a weak direct stimulus of the neutrophil respiratory burst and degranulation. The stimulation, as measured by iodination, H2O2 production, and lysozyme release, was considerably increased by the presence of unopsonized zymosan in the reaction mixture, an effect which was associated with the increased ingestion of the zymosan. TNF does not act as an opsonin but, rather, reacts with the neutrophil to increase its phagocytic activity. TNF-dependent phagocytosis, as measured indirectly by iodination, is inhibited by monoclonal antibodies (Mab) 60.1 and 60.3, which recognize different epitopes on the C3bi receptor/adherence-promoting surface glycoprotein of neutrophils. Other neutrophil stimulants, namely N-formyl-methionyl-leucyl-phenylalanine, the Ca2+ ionophore A23187, and phorbol myristic acetate, also increase iodination in the presence of zymosan; as with TNF, the effect of these stimulants is inhibited by Mab 60.1 and 60.3, whereas, in contrast to that of TNF, their stimulation of iodination is unaffected by an Mab directed against TNF. TNF may be a natural stimulant of neutrophils which promotes adherence to endothelial cells and to particles, leading to increased phagocytosis, respiratory burst activity, and degranulation.     

Modulation of life and death by the tumor necrosis factor receptor-associated factors (TRAFs)

The TNF receptor-associated factor (TRAF) family is a group of adapter proteins that link a wide variety of cell surface receptors. Including the TNF and IL-1 receptor superfamily to diverse signaling cascades, which lead to the activation of NF-kappaB and mitogen-activated protein kinases. In addition, TRAFs interact with a variety of proteins that regulate receptor-induced cell death or survival. Thus, TRAF-mediated signals may directly induce cell survival or interfere with the death receptor-induced apoptosis.     

Signal transduction by tumor necrosis factor receptors

Tumor necrosis factor (TNF) is a key mediator in the inflammatory response which is implicated in the onset of a number of diseases. Research on TNF led to the characterization of the largest family of cytokines known until now, the TNF superfamily, which exert their biological effects through the interaction with transmembrane receptors of the TNFR superfamily. TNF itself exerts its biological effects interacting with two different receptors: TNFR1 and TNFR2. TNFR1 presents a death domain on its intracellular region. In contrast to TNFR1, TNFR2 does not have a death domain. Activation of TNFR1 implies the consecutive formation of two different TNF receptor signalling complexes. Complex I controls the expression of antiapoptotic proteins that prevent the triggering of cell death processes, whereas Complex II triggers cell death processes. TNFR2 only signals for antiapoptotic reactions. However, recent evidence indicates that TNFR2 also signals to induce TRAF2 degradation. TRAF2 is a key mediator in signal transduction of both TNFR1 and TNFR2. Thus, this novel signalling pathway has two important implications: on one hand, it represents an auto regulatory loop for TNFR2; on the other hand, when this signal is triggered TNFR1 activity is modified so that antiapoptotic pathways are inhibited and apoptotic reactions are enhanced.     

Dominance of resistance to the cytocidal effect of tumor necrosis factor in heterokaryons formed by fusion of resistant and sensitive cells

The mechanisms underlying differences in vulnerability to the cytocidal effect of TNF, among various cell lines and strains, were explored by examining the response to TNF of heterokaryons formed by fusing TNF-resistant and -sensitive cells. Several combination pairs of human and murine cells, differing significantly in response to TNF toxicity, yet expressing a similar level of TNF receptors, were examined. In all combinations tested, the heterokaryons exhibited resistance to TNF toxicity, comparable, in extent, to that of the more resistant of the two parental cell lines. This dominance of resistance suggests that differences in vulnerability to TNF toxicity reflect activities which are expressed by resistant cells and are deficient in vulnerable ones, activities which perhaps protect the cell against the cytocidal effect of TNF.     

Production of tumor necrosis factor-alpha by naive or memory T lymphocytes activated via CD28.

While it is well established that activated T cells can produce tumor necrosis factor alpha (TNF-alpha), it is less clear whether this function is confined to a given subset, e.g., memory cells. To approach this question, we investigated the production of TNF-alpha by human peripheral blood T lymphocytes activated with anti-CD28 mAb since this activation pathway is known to potentiate cytokine production. Under the culture conditions used, the amount of TNF-alpha produced was markedly enhanced compared to that obtained after activation with immobilized anti-CD3 mAb. The enhancement of TNF-alpha production was already apparent after incubation of T cells for 6 hr. Up to 5 ng/ml of TNF-alpha was measured on Day 2 in supernatants of cultures of 10(4) T lymphocytes. To determine the source of the cells producing high amounts of TNF-alpha, T lymphocytes were separated into two subpopulations, namely naive cells (expressing the CD45RA isoform) and memory cells (expressing the CD45RO isoform). While both subpopulations proliferated equally well after stimulation with anti-CD28 mAb, up to 90% of the TNF-alpha produced under these conditions originated from memory T cells. These results thus document that T cell activation via CD28 results in a marked increase in TNF-alpha production without affecting the functional disparity that exists between naive and memory T cells.     

Recombinant tumor necrosis factors mediate platelet cytotoxicity to Schistosoma mansoni larvae

The involvement of platelets in the immunity directed toward the parasite Schistosoma mansoni has been demonstrated in vitro and in vivo. The killing properties of platelets were shown previously to involve specific IgE after their binding to a membrane receptor. More recently, we have demonstrated that lymphokines released by S. mansoni Ag- or lectin-stimulated CD4+/CD8-T cells were able to induce the platelet cytotoxicity. The isoelectric focusing of the T lymphocyte supernatants showed that two factors were able to stimulate the platelet killing functions. One of them was clearly identified as being IFN-gamma. The present work demonstrates that the second lymphokine was TNF. Indeed the rTNF-beta and, to a lesser extent, rTNF-alpha, induce normal platelets into killer cells for the young larvae of schistosome. Moreover, an additive effect of the TNF-alpha and IFN-gamma has been observed.     

Regulation of tumor necrosis factor cytotoxicity by calcineurin

Cyclosporin (CsA) inhibits mitochondrial death signaling and opposes tumor necrosis factor (TNF)-induced apoptosis in vitro. However, CsA is also a potent inhibitor of calcineurin, a phosphatase that may participate in cell death. Therefore, we tested the hypothesis that calcineurin regulates TNF cytotoxicity in rat hepatoma cells (FTO2B). TNF-treated FTO2B cells appeared apoptotic by DNA fragmentation, nuclear condensation, annexin V binding, and caspase activation. We studied two calcineurin inhibitors, CsA and FK506, and found that each potently inhibited TNF cytotoxicity. Western blot demonstrated calcineurin in FTO2B homogenates. In a model of mitochondrial permeability transition (MPT), we found that CsA prevented MPT and cytochrome c release, while FK506 inhibited neither. In summary, we present evidence that calcineurin participates in an apoptotic death pathway activated by TNF. CsA may oppose programmed cell death by inhibiting calcineurin activity and/or inhibiting mitochondrial signaling.