IFN-alpha in vivo enhances tumor necrosis factor receptor levels on hairy cells

Hairy cell leukemia (HCL) is a preplasma B cell neoplasia that is characterized by monocytopenia and responds to IFN-alpha therapy. We investigated the expression of receptors for TNF-alpha, a monocyte-derived cytokine, on the surface of hairy cells from seven HCL patients before and after treating them with IFN-alpha. Iodinated TNF-alpha binding experiments showed the presence of high affinity TNF-alpha receptors in six patients, but no specific binding was detected in the seventh. Scatchard's analysis revealed the presence of a single class of receptors, with 130 to 1200 sites/cell (mean 420) and Kd values of 0.37 to 0.89 nM. The TNF-alpha-R complex was identified by cross-linking as a single band of 94 kDa. Treatment of the patients with 3 x 10(6) U of IFN-alpha 2a markedly increased the number of TNF-alpha-R at 24 or 48 h, without changing the Kd. In the patient who lacked receptor expression on his tumor cells, high affinity TNF-alpha-R were detected at 48 h after IFN injection. These results suggest that in hairy cells, IFN-alpha can interact in vivo with TNF-alpha, through the modulation of TNF-R.     

Chromosomal location of the human tumor necrosis factor receptor genes.

TNFR1 and TNFR2, the genes encoding the two forms of the human tumor necrosis factor receptor, were localized to normal human chromosomes by in situ hybridization and Southern blot analysis of a series of human x mouse hybrid cell lines. TNFR1 maps to 12p13 and TNFR2 maps to 1p36.     

Allelic variation of the type 2 tumor necrosis factor receptor gene

The nucleotide sequence data reported in this paper have been submitted to the EMBL Data library and have been assigned the accession number X76401.     

Oxidation of thioredoxin reductase in HeLa cells stimulated with tumor necrosis factor-alpha

Stimulation of cells with tumor necrosis factor-alpha (TNF-alpha) results in the increase in generation of H(2)O(2) in mitochondria that leads to apoptosis. The effect of H(2)O(2) produced by TNF-alpha on the redox status of selenocysteine (SeCys) residue essential for mitochondrial thioredoxin reductase (TrxR2) was investigated in HeLa cells. TNF-alpha caused accumulation of oxidized TrxR2 with a thioselenide bond. The conditional induction of SeCys-deficient TrxR2 resulted in the increased production of H(2)O(2) and apoptosis. These results suggest that the SeCys residue of TrxR2 plays a critical role in cell survival by serving as an electron donor for Trx-II and subsequent peroxiredoxin-III, which is a primary line of defense against H(2)O(2) in mitochondria.     

Tumor necrosis factor receptor signaling. A dominant negative mutation suppresses the activation of the 55-kDa tumor necrosis factor receptor.

To investigate the signaling mechanism of the 55-kDa tumor necrosis factor (TNF) receptor a functional transfection based assay was developed. The human 55-kDa TNF receptor, stably expressed in mouse L929 cells, was demonstrated to be activated specifically by agonist antibodies and to initiate a signal for cellular cytotoxicity. A deletion mutant of the human TNF receptor lacking most of the cytoplasmic domain was found to be completely defective in generating the signal for cytotoxicity. Additionally, expression of the truncated receptor substantially suppressed signaling by endogenous mouse TNF receptors in response to TNF, but not in response to specific anti-murine TNF receptor antibodies. These results suggest that aggregation of 55-kDa TNF receptor intracellular domains, which are not associated in the absence of ligand, is an important component of the signal for cellular toxicity. This work also provides an example of a dominant negative mutation in a transmembrane receptor that lacks a tyrosine kinase domain, and suggests a more general utility of dominant negative mutations in the investigation of cytokine receptor function.     

Vascular endothelial growth factor KDR receptor signaling potentiates tumor necrosis factor-induced tissue factor expression in endothelial cells

Vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF-alpha) have been shown to synergistically increase tissue factor (TF) expression in endothelial cells; however, the role of the VEGF receptors (KDR, Flt-1, and neuropilin) in this process is unclear. Here we report that VEGF binding to the KDR receptor is necessary and sufficient for the potentiation of TNF-induced TF expression in human umbilical vein endothelial cells. TF expression was evaluated by Western blot analysis and fluorescence-activated cell sorting. In the absence of TNF-alpha, wild-type VEGF- or KDR receptor-selective variants induced an approximate 7-fold increase in total TF expression. Treatment with TNF alone produced an approximate 110-fold increase in total TF expression, whereas coincubation of TNF-alpha with wild-type VEGF- or KDR-selective variants resulted in an approximate 250-fold increase in TF expression. VEGF lacking the heparin binding domain was also able to potentiate TF expression, indicating that heparin-sulfate proteoglycan or neuropilin binding is not required for TF up-regulation. Neither placental growth factor nor an Flt-1-selective variant was capable of inducing TF expression in the presence or absence of TNF. Inhibition of protein-tyrosine kinase or protein kinase C activity significantly blocked the TNF/VEGF potentiation of TF up-regulation, whereas phorbol 12-myristate 13-acetate, a protein kinase C activator, increased TF expression. These data demonstrate that KDR receptor signaling governs both VEGF-induced TF expression and the potentiation of TNF-induced up-regulation of TF.     

Cell cycle specificity of tumor necrosis factor and its receptor

Phase specificity in the TNF cytotoxic effect and the number of TNF binding receptors was investigated using L-M cells incubated synchronously from the S phase. TNF cytotoxicity was observed to occur at various levels during the cell cycle, with peak effect in the G2-M phase. Analysis with 125I-labeled TNF to determine the number of receptors binding TNF in the various cell phases shewed a phase specificity with the maximum number occurring in the G2-M phase, similar to the peak in cytotoxicity. The results suggest the existence of a cell cycle specificity in the cytotoxicity of TNF which is apparently related to changes in the number of receptors capable of binding TNF.     

Binding of human tumor necrosis factor to high affinity receptors on HeLa and lymphoblastoid cells sensitive to growth inhibition

Purified human tumor necrosis factor (TNF) was iodinated to high specific activity with good retention of its biological activity, as determined by the cytotoxic titer on murine L929 cells. The binding of 125I-TNF to L929 and human HeLa S2 cells grown in monolayer was initially measured, but high levels of nonspecific binding were observed. Specific binding to high affinity receptors of HeLa S2 cells grown in suspension culture was demonstrated by competitive displacement experiments and analysis of the binding data with the LIGAND program. A KD of 2 X 10(-10) M and 6000 receptors/cell were calculated in this way. These observations provide the first direct evidence for a cellular receptor for TNF. The cell-bound 125I-TNF was internalized at 37 degrees C, presumably by receptor-mediated endocytosis, and subsequently degraded to acid-soluble products. Three lines of human lymphoblastoid cells were examined for sensitivity to the cytostatic effect of TNF and for the presence of high affinity receptors. Daudi and Raji cells were insensitive to TNF and showed very few specific binding sites when incubated with 125I-TNF. Jurkat cells were growth-inhibited by TNF and showed a significantly greater number of specific binding sites than the other lymphoblastoid cells. These findings suggest that the sensitivity of some cell lines to the biological effects of TNF may be correlated with the presence of a relatively high number of receptors for this factor.     

Signalling pathway of tumor necrosis factor in normal and tumor cells

Summary Several aspects of the activity and effects of tumor necrosis factor (TNF) were investigated to gain further insight into its cytotoxic mechanism. The relation between number of TNF receptors and TNF susceptibility of both tumor cells and normal cells was studied, utilizing a specific binding assay. Among the tumor cells, a fairly close correlation (r=0.855) was observed between receptor number and sensitivity to TNF. No cytotoxic effect by TNF was observed on any of the normal cells tested, even though TNF receptors were shown to be present, and cell proliferation was apparently stimulated by TNF in some cases. TNF internalization and intracellular distribution were studied by pulse-labelling and Percoll density gradient centrifugation. In L-M (murine tumorigenic fibroblasts, highly sensitive to TNF cytotoxicity) cells and HEL (human embryonic lung cells, non-sensitive to TNF cytotoxicity) cells, receptor-bound ¹²⁵I-labelled recombinant human TNF was rapidly internalized and delivered to lysosomes within 15–30 min, and this was followed by degradation and release into the culture medium. The presence of either a cytoskeletal disrupting agent or a lysosomotropic agent was observed to inhibit the cytotoxic effect of TNF, thus also indicating that TNF internalization, followed by delivery to lysosomes, is essential in the cytolytic mechanism of TNF.As observed by [³H]uridine incorporation, TNF did not affect RNA synthesis in L-R cells (TNF-resistant cell lines derived from L-M cells) and HEL cells, but markedly stimulated (by 3.5 times) RNA synthesis in L-M cells.     

Identification of a ligand for glucocorticoid-induced tumor necrosis factor receptor constitutively expressed in dendritic cells

Glucocorticoid-induced tumor necrosis receptor (GITR) has been implicated in regulation of T cell suppression by CD25(+)CD4(+) regulatory T cells (Tregs). We isolated a cDNA encoding GITR ligand (GITRL) from mouse endothelioma cells. When stably expressed in HEK293 cells, its specific interaction with GITR was confirmed by flow cytometry with the use of GITR-Fc. The interaction was greatly diminished by the addition of soluble GITRL. Consistent with this, soluble GITRL bound to the cell surface of the GITR-expressing HEK293 cells. Coexpression of GITR with GITRL or stimulation of the GITR-expressing cells with soluble GITRL led to activation of NF-kappaB, which was significantly reduced by anti-GITR. More importantly, GITRL was expressed by both immature and mature dendritic cells, suggesting that the interaction between GITR and GITRL may contribute to immune regulation of Tregs by dendritic cells. This isolated TNFRL represents a bona fide GITRL whose presence has been elusive until this time.     

Induction of interleukin 2 receptor (TAC) by tumor necrosis factor in YT cells

The effect of human recombinant tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) on interleukin 2 receptor (IL-2R) expression on YT cells was examined. IL-2R expression was assessed by flow cytometric analysis with a monoclonal antibody to IL-2R (anti-TAC). TNF-alpha, like IL-1 beta, induced increased levels of IL-2R expression on YT cells with similar kinetics of induction. Maximum induction occurred at 20 to 30 hr. On a molar basis. TNF was less active than IL-1 beta. RNA isolated from TNF-alpha- or IL-1 beta-treated YT cells contained increased levels of IL-2R-specific mRNA as indicated by slot blot analysis by using an IL-2R-specific mRNA probe. Kinetic and IL-1 beta mRNA expression studies indicated that the TNF effect was a direct one. Because IL-2R expression is known to be associated with lymphocyte activation, the present results suggest that TNF-alpha may play a role in the regulation of immune responses.     

Tumor necrosis factor receptors in HeLa cells and their regulation by interferon-gamma

Incubation of HeLa cell cultures with human interferon-gamma (IFN-gamma) increased the binding of radioiodinated human tumor necrosis factor (TNF) to specific cell surface receptors (TNF-R). IFN-gamma also produced a proportionate increase in receptor-mediated endocytosis of TNF. TNF-R expression was significantly increased after 6 h of exposure to IFN-gamma (100 units/ml), and it remained elevated in the continuous presence of IFN-gamma for at least 20 h. Incubation of cells with IFN-gamma in the presence of cycloheximide, followed by treatment with actinomycin D and reversal of the inhibition of protein synthesis, also resulted in increased TNF-R expression as compared to cultures subjected to the same treatments in the absence of IFN-gamma. These results suggest that IFN-gamma can directly stimulate accumulation of the mRNA for TNF-R and that TNF-R is among the cellular proteins whose synthesis is increased by IFN-gamma.     

Targeting of tumor necrosis factor to tumor cells: secretion by myeloma cells of a genetically engineered antibody-tumor necrosis factor hybrid molecule

The construction, synthesis and secretion of a genetically engineered antibody-cytokine fusion molecule is described. To target tumor necrosis factor (TNF) to tumor cells, recombinant antibody techniques were used to produce a Fab-like antibody-TNF conjugate. At the gene level, the heavy chain gene of an antitransferrin receptor antibody was linked to a synthetic TNF gene encoding human TNF. Transfection of the heavy chain-TNF gene into a myeloma derived cell line which was producing the light chain of the same antibody, allowed the isolation of a cell line secreting a fusion protein of the expected molecular weight and composition. The culture supernatant of the cell line contained TNF cytotoxic activity towards murine L929 cells and human MCF-7 cells. Cytotoxicity towards the human cancer cells was inhibited by an excess of the original antitransferrin receptor antibody, indicating that the antibody-TNF molecules are targeted to the transferrin receptor rich tumor cells. Since the antibody genes used are chimeric (i.e. composed of mouse variable and human constant regions) and since DNA encoding human TNF was used, the hybrid protein is an example of a humanized immunotoxin-like molecule. These results illustrate the possibilities of antibody engineering technology to create and produce improved agents for cancer therapy. Furthermore, they demonstrate for the first time the ability of myeloma cells to secrete an antibody-cytokine chimeric molecule.     

Decreased heat- and tumor necrosis factor-mediated hsp28 phosphorylation in thermotolerant HeLa cells

Heat shock or tumor necrosis factor rapidly stimulated the phosphorylation of the mammalian low molecular weight stress protein hsp28. We have found that both phenomena are greatly decreased in cells which are made tolerant to heat. This observation correlated with a better survival of thermotolerant cells exposed to either heat or TNF treatment. The results suggest that the phosphorylation of hsp28 may be linked to the resistance of the cells to the deleterious effects induced by either heat or a mediator of inflammation such as TNF.