Cytotoxic Action Of Tumor Necrosis Factor Research Articles

The role of protein kinase C isozymes in TNF-alpha-induced cytotoxicity to a rat intestinal epithelial cell line.

Tumor necrosis factor (TNF)-alpha can induce cytotoxicity and apoptosis in a number of cell types and has been implicated in the regulation of many inflammatory processes. It has been suggested that protein kinase C (PKC) is one of the intracellular mediators of the actions of TNF-alpha. In the present study, the role of PKC isoforms in TNF-alpha-mediated cytotoxicity and apoptosis in intestinal cells was investigated using the rat epithelial cell line, IEC-18. Cells were incubated with TNF-alpha in the presence or absence of the transcription inhibitor actinomycin D (AMD). The extent of cell damage was enhanced when AMD was added to incubation medium, suggesting that new protein synthesis plays a role in the cytotoxic action of TNF. TNF-alpha also induced the translocation of PKC-alpha, -delta, and -epsilon from cytosol to the membrane fraction of the intestinal cells. Furthermore, the cytotoxic and apoptotic effects of TNF were reduced by pretreating the cells with the PKC-epsilon translocation inhibitor, PKC-epsilonV1-2. In contrast, although cells incubated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) also displayed an increase in cell injury, the extent of cytotoxicity and apoptosis was not enhanced by AMD. Furthermore, PMA-induced cell damage was reduced by rottlerin, a PKC-delta inhibitor. Caspase-3, an enzyme implicated in the mediation of apoptosis, was activated in cells in response to either TNF-alpha or PMA stimulation, and its effects on this activity were reduced by selective inhibition of PKC-epsilon and -delta, respectively. Furthermore, inhibition of caspase-3 activity reduced apoptosis. These data suggest that activation of selective PKC isoforms mediate the effects of TNF-alpha on intestinal epithelial cell injury.

The Oxidative Metabolism of Glutamine: A MODULATOR OF REACTIVE OXYGEN INTERMEDIATE-MEDIATED CYTOTOXICITY OF TUMOR NECROSIS FACTOR IN L929 FIBROSARCOMA CELLS

Treatment of the mouse fibrosarcoma cell line L929 with tumor necrosis factor (TNF) induces necrotic cell death. A crucial step in the cytotoxic action mechanism of TNF involves perturbation of mitochondrial functions leading to the formation of reactive oxygen intermediates (ROI). L929 cells have energy requirements adapted to a high proliferation rate. Glutamine (Gln) is utilized as a major energy source and drives mitochondrial ATP formation, while glucose is mainly converted to lactate through glycolysis. We investigated the role of the bioenergetic pathways involved in substrate utilization on the cytotoxic action of TNF and established a link between Gln oxidation and TNF-induced mitochondrial distress. Omission of Gln from the medium desensitizes the cells to TNF cytotoxicity, while the lack of glucose in the medium does not alter the TNF response. Sudden depletion of Gln from the culture medium results in a sharp decline in mitochondrial respiration in the cells, which might explain the decreased TNF responsiveness. However, when L929 cells are adapted to long term growth under conditions without Gln, these so-called L929/Gln- cells have restored respiration, but they still display a decreased sensitivity to TNF cytotoxicity. Thus the TNF responsiveness of L929 cells depends on bioenergetic reactions that are specifically involved in the oxidation of Gln. This is further confirmed by the desensitizing effect of specific inhibitors of these Gln-linked enzyme reactions on TNF cytotoxicity in the parental cells, but not in the L929/Gln- cells. Analysis of the induction of mitochondrial ROI formation by TNF in parental and L929/Gln- cells suggests that the effect of Gln on the sensitivity to TNF cytotoxicity involves a mechanism that renders the mitochondria more susceptible to TNF-induced mediators, resulting in enhanced ROI production and accelerated cytotoxicity.

Tnf-mediated cytotoxicity. importance of intracellular cgmp level for determining tnf-sensitivity

Previous work on the cytolytic action of activated macrophages indicated that tumor necrosis factor (TNF) showed synergistic cytolytic activity with NO, which has been shown to act as a cyclic GMP (cGMP) generator [Higuchi et al., J. Immun. 144, 1425–1431, (1990)]. In this study, we investigated the relationship between the accumulation of intracellular cGMP and the cytotoxic action of TNF. It was demonstrated that TNF-mediated cell lysis was closely related to the background level of intracellular cGMP, and that the accumulation of cGMP within TNF resistant cells induced TNF sensitivity. We reached these conclusions on the basis of the following results; (1) agents (sodium nitroprusside and isobutylmethylxantine) that cause the accumulation of cGMP intracellularly increased the TNF-sensitivity of TNF-resistant cells; (2) the addition of dibutyryl cGMP to TNF-resistant cells increased the TNF-sensitivity; and (3) treatment at 40°C or agents such as interferon gamma and actinomycin D, that synergistically kill tumor cells together with TNF, potentially increased the cGMP level. Therefore, intracellular cGMP may be one of the key molecules that lead to cell death caused by TNF.

Monoclonal anti-tumor necrosis factor (TNF) antibodies protect mouse and human cells from TNF cytotoxicity

Tumor necrosis factor (TNF) is a cytokine produced by macrophages which mediates septic shock. TNF is also associated with anti-tumor activity and is cytotoxic to many cell lines in vitro. While the clinical efficacy of anti-TNF antibodies has yet to be established, such antibodies may prevent TNF action. We have evaluated three murine monoclonal anti-TNF antibodies for their capacity to protect mouse and human cells from the cytotoxic effects of human TNF. Mouse WEHI 164 cells were tested because of their known high sensitivity to TNF. Several human cells lines were also examined: U937, HeLa, MRC5, and ME180. Two antibodies protected mouse and human cells from the cytotoxic action of TNF. The neutralizing activity was also present in the Fab fragment and insensitive to the presence of carbohydrate on the antibody. The third antibody had substantially less TNF-neutralizing activity. The relative ability of these monoclonal antibodies to protect both mouse and human cell lines from TNF-induced cell death may be an effective indicator of their in vivo efficacy.

Dexamethasone inhibits the cytotoxic activity of tumor necrosis factor

Effect of dexamethasone (DEX) on the cytotoxic activity of tumor necrosis factor (TNF) was examined using murine fibroblast cell line (L929 cells). DEX protected cells from the cytotoxic action of TNF. Protection of cytotoxic action was apparent when cells were pre-treated with DEX for 12h and no protection was observed in the presence of cycloheximide. These results suggested that de novo synthesis of new proteins was required for DEX-mediated protection. Moreover, prolonged simultaneous treatment with TNF and DEX resulted in the enhancement of cell growth, suggesting that TNF acted as a growth factor when cells were protected from the cytotoxic action of TNF. These results suggested that the signal transduction system for fibroblast growth enhancing and cytotoxic action of TNF were different from each other and that the interaction between TNF and glucocorticoids may play a modulating role in some inflammatory processes in vivo.

Demonstration of membrane receptors for human natural and recombinant 125I-labeled tumor necrosis factor on HeLa cell clones and their role in tumor cell sensitivity.

Samples of natural and recombinant tumor necrosis factor (TNF) were labelled with 125I by use of Iodobeads and were used to study TNF receptor expression on different HeLa clones. Both labeled preparations retained cytotoxic activity after 125I-labelling and were indistinguishable from their unlabeled counterparts on sodium dodecylsulfate/polyacrylamide gel electrophoresis. They bound specifically to TNF-sensitive cells but not to HeLa clones that were resistant to TNF. The binding of 125I-TNF was competed up to 90% by the addition of non-radiolabeled TNF. The binding reached a half-maximal level at 10 pM and saturation at 100 pM. These concentrations approximated those required for cell destruction. Scatchard analysis of the binding data yielded linear plots suggesting that TNF binds to homogeneous TNF receptor sites. The number of receptor sites ranged between 770 and 2200 sites on TNF-sensitive cells. The surface expression of these TNF receptors appeared to be necessary but not sufficient for the cells to become sensitive against the cytotoxic action of TNF. One of the HeLa clones was found to express the same number of TNF receptors as the most sensitive clone, but was nevertheless resistant to the toxic effect of TNF.