TNF combined with IFN-alpha accelerates NF-kappaB-mediated apoptosis through enhancement of Fas expression in colon cancer cells

Immunostaining and EMSA revealed that NF-kappaB was activated strongly by TNF/IFN-alpha compared to TNF alone in a human colon adenocarcinoma cell line, RPMI4788. Although inhibition of activated NF-kappaB, by using an NF-kappaB decoy, reduced cell viability after treatment with TNF only, NF-kappaB decoy resulted in recovery of cell viability after TNF/IFN-alpha treatment. Caspase-3 activity was increased in cells induced by TNF/IFN-alpha, while suppression of caspase-3 activity was observed in cells transfected with NF-kappaB decoy and then treated by TNF/IFN-alpha. On the other hand, Fas expression was strongly enhanced by TNF/IFN-alpha, and inhibition of TNF/IFN-alpha-induced NF-kappaB activation, by using NF-kappaB decoy, decreased Fas expression. Cell viability and caspase-3 activity decreased in cells treated with TNF/IFN-alpha and anti-FasL antibody. Taken together, our findings suggest that activated NF-kappaB induced by the crosstalk between TNF and IFN-alpha is a novel pro-apoptotic signal acting via enhancement of Fas expression.     

Restoration by dietary glutamine of reduced tumor necrosis factor production in a low-protein-diet-fed rat model

Tumor necrosis factor-alpha (TNF) production by peritoneal macrophages and its dietary modification were investigated by using rats fed on a low-protein diet. The rats were given a 20% casein (control) diet or a 3% casein diet for 21 days, and TNF production was measured in activated macrophages of these animals. TNF production was significantly lower in macrophages from rats fed on the low-protein diet than that in macrophages from rats fed on the control diet. Oral administration of a cabbage extract, a known modulator of TNF production, to the low-protein-diet-fed rats significantly enhanced TNF production by macrophages. Glutamine supplementation to the low-protein diet significantly enhanced TNF production as well as TNF mRNA expression. These results indicate that the 3%-casein-diet-fed rat would be useful as a model for reduced TNF production in protein malnutrition. These results also suggest that glutamine administration restored the reduced TNF production associated with protein malnutrition.     

Limiting dilution analysis of TNF producing cells in C3H/HeJ mice

A limiting dilution assay (LDA) that measures the frequency of TNF producing cells is described. LDA determination is based on the inhibition of growth of a highly TNF sensitive subline from the WEHI-164 fibrosarcoma by using a micro assay sensitive to single picogram amounts of recombinant murine TNF. Using such LDA, it was determined that the reported deficiency in LPS-induced TNF production in C3H/HeJ mice is a function of reduced frequency of TNF producing cells rather than a complete lack of responsiveness. In bulk culture, LPS-triggered TNF was produced by Thy-1.2 negative spleen cells with activity recovered in both G10 Sephadex adherent and nonadherent subpopulations. LPS stimulation of spleen cells from C3H/HeJ mice resulted in TNF mRNA expression as shown in both Northern blots and in situ hybridization. The frequency of TNF mRNA bearing cells in control of C3H/HeSnJ mice by in situ hybridization correlated with that found for TNF producing cells in LDA. In C3H/HeJ spleen, significantly higher numbers of TNF mRNA positive cells were found than were shown to produce TNF in LDA.     

Tumor necrosis factor is partly responsible for the differentiation of mouse myeloid leukemia cells

When mouse myeloid leukemia M1 cells were incubated with 0.1% TNS(serum containing tumor necrosis factor, TNF), they were induced to differentiate into macrophage-like cells within 14 h. The stability of the differentiation activity to heat treatment was very similar to that of the cytotoxicity of TNF for L929 cells. The differentiation activity could not be separated from the TNF activity by sequential chromatography procedures using DEAE-Sephadex and Sephacryl S-200 or by isoelectric focusing. These results suggest that the differentiation activity of TNS is due to TNF. Indeed, recombinant human TNF induced differentiation of M1 cells.     

Autocrine TNF is critical for the survival of human dendritic cells by regulating BAK, BCL-2, and FLIPL

The life span of dendritic cells (DCs) is determined by the balance of pro- and antiapoptotic proteins. In this study, we report that serum-free cultured human monocyte-derived DCs after TLR stimulation with polyinosinic acid-polycytidylic acid or LPS underwent apoptosis, which was correlated with low TNF production. Apoptosis was prevented by the addition of exogenous TNF or by concomitant stimulation with R-848, which strongly amplified endogenous TNF production. Neutralization of TNF confirmed that DC survival was mediated by autocrine TNF induced either by stimulation with R-848 or by ligation of CD40. DCs stimulated by polyinosinic acid-polycytidylic acid or IFN-β, another known inducer of DC apoptosis, were characterized by high levels and activation of the proapoptotic protein BAK. The ratio of antiapoptotic BCL-2 to BAK correlated best with the survival of activated DCs. Addition of TNF increased this ratio but had little effect on BAX and XIAP. Knockdown experiments using small interfering RNAs confirmed that the survival of activated and also of immature DCs was regulated by BAK and showed that TNF was protective only in the presence of FLIP(L). Together, our data demonstrate that the survival of DCs during differentiation and activation depends on autocrine TNF and that the inhibition of BAK plays an important role in this process.     

Single-chain TNF, a TNF derivative with enhanced stability and antitumoral activity

The inflammatory and proapoptotic cytokine TNF possesses a compelling potential as an antitumoral therapeutic agent. Possible target cells include the malignant cells themselves, the tumor vasculature, or the immune system. As the clinical use of TNF is limited by systemic toxicity, targeting strategies using TNF-based fusion proteins are currently used. A major obstacle, however, is that homotrimeric TNF ligands are prone to activity loss due to dissociation into their monomers. In this study, we report the construction of single-chain TNF molecule, a TNF mutant consisting of three TNF monomers fused by short peptide linkers. In comparison to wild-type TNF, single-chain TNF was found to possess increased stability in vitro and in vivo, displayed reduced systemic toxicity yet slightly enhanced antitumoral activity in mouse models. Creation of single-chain variants is a new approach for improvement of functional activity of therapeutics based on TNF family ligands.     

A flow cytometric and immunofluorescence microscopic study of tumor necrosis factor production and localization in human monocytes

The production and localization of tumor necrosis factor (TNF) in human monocytes were investigated by using monoclonal and polyclonal antibodies against recombinant human TNF together with flow cytometry and immunofluorescence microscopy. Lipopolysaccharide (LPS) induced a rapid and transient accumulation of TNF in perinuclear vesicles which was detected 20 min after the addition of LPS. The fluorescence intensity of the vesicles peaked at 40 min of LPS exposure, concomitantly with the release of TNF into the medium. Thus, our results indicate that the secretion of TNF is typical for secretory proteins as it involves passage through the secretory apparatus. Additional studies demonstrated that plasma membrane-associated TNF could not be detected in live monocytes not exposed to LPS. However, after 90 min with LPS, a small population of monocytes expressed membrane-associated TNF, and by 24 hr approximately 50% of the monocytes displayed TNF on the plasma membrane. Furthermore, our results indicate that plasma membrane-associated TNF does not represent released TNF bound back to its own receptor. Thus, our findings support the view that TNF exists as a surface trans-membrane protein in LPS-stimulated monocytes.     

Complex regulation of tumor necrosis factor mRNA turnover in lipopolysaccharide-activated macrophages

The turnover of tumor necrosis factor (TNF) mRNA in permanently transfected macrophages of the RAW 264.7 cell line was studied directly (by Northern blot analysis using a probe specific for TNF) and indirectly (through studies of the turnover of various reporter mRNAs, either containing or lacking the TNF 3' untranslated region (UTR)). The TNF mRNA was found to be very unstable in RAW 264.7 cells. Instability appeared to result from two distinguishable nucleolytic processes. The major degradative process involved was not specific for the TNF 3' UTR of reporter mRNAs, and was inhibited by actinomycin D pretreatment. It appeared to be expressed constitutively, in that cell activation by lipopolysaccharide (LPS) did not modify message stability. When cells were treated with actinomycin D, a minor nucleolytic activity was 'uncovered'. This minor activity was noted to increase with time following LPS activation. It also exhibited specificity, in that reporter mRNAs bearing the 3' UTR of TNF were more susceptible to degradation in the presence of actinomycin D than were constructs lacking the 3' UTR of TNF. Thus, TNF mRNA turnover appears complex, and depends upon at least two separable degradative pathways. The TNF 3' UTR apparently contributes only modestly to the instability of this mRNA under normal conditions.     

Histidine-15: an important role in the cytotoxic activity of human tumor necrosis factor

The amino acids that are required for the cytotoxic activity of recombinant human tumor necrosis factor-alpha (TNF) were investigated by chemical modification and oligonucleotide-directed site-specific mutagenesis. TNF contains three histidine residues, located at positions 15, 73 and 78. The histidine-specific reagent diethylpyrocarbonate (DEP) was used to chemically modify TNF. The chemical inactivation of the in vitro cytotoxic activity of this lymphokine (using murine L929 target cells) was found to be time- and dose-dependent. Inactivated TNF failed to compete with fully bioactive [125I]TNF for human MCF-7 target cell receptors. Mutant polypeptides of TNF were genetically engineered by oligonucoleotide-directed site-specific mutagenesis. The cytotoxicity of a double histidine mutant, in which histidine-73 and histidine-78 were replaced with glutamine, was not altered and was chemically inactivated by DEP. Substituting glutamine for histidine-15 resulted in 10-15% of the wild-type bioactivity. Replacing histidine-15 with either asparagine, lysine or glycine resulted in a biologically inactive molecule. The data show that the histidine residue at position 15 is an amino acid that is required for the cytotoxic activity of TNF.     

Analysis of the structure-function relationship of tumour necrosis factor. Human/mouse chimeric TNF proteins: general properties and epitope analysis.

To analyse the structure-function relationship of tumour necrosis factor (TNF), a set of in-frame chimeric genes was constructed by coupling appropriate segments of the human and mouse TNF coding regions. Under control of the bacteriophage lambda inducible PL promoter high level expression of these chimeric genes was obtained in Escherichia coli. Although both human and mouse TNF were produced in E. coli as soluble proteins, a reduction of solubility was observed in some of the chimeric proteins. The specific activity was variable, but in some constructs comparable to human TNF, indicating that the structural conformation of these chimeric proteins resembled the human TNF structure. Neutralization analysis using two monoclonal antibodies directed against human TNF, indicated that the regions involved in the binding of these antibodies are distributed over multiple segments of the polypeptide. Further analysis by site-directed mutagenesis of one subregion allowed the identification of the Arg131 residue as involved in the binding of both neutralizing monoclonal antibodies; an Arg131----Gln replacement abolished antibody binding but did not affect the specific activity of TNF.     

Tumor necrosis factor-induced downregulation of its receptors in HeLa cells

Tumor necrosis factor (TNF) induced loss of TNF receptors in HeLa cells was studied using acid elution technique, which could distinguish surface occupancy and real loss of receptors. Exposure of HeLa cells to TNF resulted in a rapid reduction in the number of TNF receptors without affecting the apparent binding affinity. The binding of transferrin after treatment with unlabeled TNF was unaffected. The TNF-mediated decrease in receptor number on the cells was reversible. Following removal of TNF from growth medium, binding activity was restored within 3 h. Cycloheximide prevented the restoration of TNF receptors, suggesting that de novo synthesis of receptors was required to restore the binding activity.     

Necrotizing activity of tumor necrosis factor: histopathological investigation using Meth A sarcoma and granulation tissue

The action of tumor necrosis factor (TNF) was investigated histopathologically in mice using methylcholanthrene A (Meth A)-induced sarcomas and granulation tissue induced by autotransplantation of fragments of liver and spleen. Highly purified murine TNF caused hemorrhagic necrosis of both the tumors and the granulation tissue. Proliferation of tumor capillaries, demonstrated microangiographically, occurred 2 h after TNF administration and hyperemia of tumor vessels was obvious after 3-6 h. Hyperemia and capillary leakage were also observed in the granulation tissue 6 h after TNF injection and hemorrhage was noted in the epidermis after 12 h. These results strongly suggest that the in vivo necrotizing action of TNF is mainly related to capillary injury.     

Effects of TNF on human hepatocellular carcinoma cell lines and their modification by hyperthermia

Firstly, using HCC cell lines, the effects of r-h TNF were investigated. The authors had already confirmed that these cell lines were derived from human HCC. Each cell line showed a different growth curve on addition of TNF to the culture medium. JHH-4 exhibited enhancement of growth under the optimum concentration of TNF. On the other hand, growth of JHH-5 and JHH-7 was inhibited by TNF. JHH-7 were more sensitive to TNF than JHH-5, however, the direct effect of TNF on JHH-7 was not potent, as 10(4) u/ml TNF could not prevent proliferation of JHH-7. Morphological examinations were also performed. Phase-contrast microscopy showed that the JHH-4 cells were enlarged and tended to pile up after the addition of TNF to the culture medium. JHH-7 cells became detached from the culture dish due to cell death. Electron microscopy showed irregular proliferation of the rough endoplasmic reticulum of JHH-4 cells and increased number of lysosomes in JHH-7 cells. Furthermore, hyperthermia exhibited an interesting reciprocal action. Proliferation of JHH-4 was inhibited by low concentrations of TNF together with 41.4 degrees C hyperthermia in contrast to the effects of TNF alone. JHH-7 became more sensitive to TNF under hyperthermia at 41.4 degrees C. On the other hand, normal human fibroblast 'HAIN-55' were not affected by TNF at 37.0 degrees C, 41.4 degrees C or 42.5 degrees C. In this paper, the authors tried to study the effects of TNF and hyperthermia on human HCC cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of tumor necrosis factor secretion by purified influenza virus neuraminidase

We showed that purified neuraminidase (NA) of influenza virus, but not hemagglutinin (HA), possessed the potential to increase in vitro and in vivo the interleukin 1 (IL 1) activity of mouse peritoneal macrophages. In this study, we report the effect of NA and HA on the secretion of tumor necrosis factor (TNF) activity by murine peritoneal macrophages. TNF being a cytokine sharing many related and overlapping biological functions with IL 1. The two glycoproteins of the strain A/USSR/90/77 (H1N1) were purified electrophoretically and were tested in vitro at doses ranging from 0.5 to 5.0 micrograms using the adherent peritoneal macrophages of C3H/HeN mice elicited with thioglycolate. The TNF activity of culture supernatants, collected 24 hr after stimulation with viral protein, was evaluated by the standard cytolytic assay using L929 and WEHI.164 cells. No increase of the TNF activity was observed at 0.5 micrograms of NA (4.8 Units (U)/ml in the L929 assay and 20.4 U/ml in the WEHI assay) but further increase of NA to 1.0 microgram had a significant effect on the TNF activity (39.7 and 88.8 U/ml, respectively). Higher concentrations of NA (2.0 and 5.0 micrograms) did not improve the TNF activity. The addition of a rabbit anti-TNF-alpha serum to the assay system reduced the lysis of L929 cells by 85%, suggesting that the observed activity was due to TNF. In parallel, the enhancement of IL 1 activity due to NA was reverified using D10.G4.1 cells instead of the C3H/HeJ thymocytes assay used previously. NA augmented the IL 1 activity up to 1.0 micrograms (25.8 U/ml). The addition of monoclonal anti-IL 1 antibodies (100 neutralizing units) to the supernatants reduced the incorporation of [3H]-thymidine by 90 to 95%, suggesting that the observed activity was due to IL 1. Comparative results of NA and HA showed that only NA stimulated the TNF and IL 1 activities of murine macrophages.     

Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes

The mechanism of human peripheral blood monocyte-mediated cytotoxicity for tumor cells was investigated, using the A673 human rhabdomyosarcoma and HT-29 human colon adenocarcinoma lines as target cells. A673 cells were shown to be susceptible to the cytotoxic action of purified recombinant human tumor necrosis factor (TNF). A673 cells were also highly sensitive to the cytotoxic action of peripheral blood monocytes. Clones of A673 cells sensitive and resistant to TNF were isolated and characterized for their sensitivity to monocyte killing. A good correlation was found between the sensitivity of these clones to the cytotoxicity of TNF and their susceptibility to killing by monocytes. A TNF-specific neutralizing monoclonal antibody (MAb) reduced monocyte killing of parental A673 cells and of a TNF-sensitive clone of A673 cells. Inhibition of monocyte killing by this MAb was particularly pronounced at a low effector to target cell ratio. HT-29 cells were relatively resistant to the cytotoxic action of recombinant TNF and to monocyte killing. Treatment of HT-29 cells with recombinant human IFN-gamma increased their susceptibility to both TNF cytotoxicity and monocyte killing. In addition, MAb to TNF inhibited monocyte killing in HT-29 cells sensitized by incubation with IFN-gamma. Our data show that TNF is an important mediator of the cytotoxicity of human monocytes for tumor cells and that IFN-gamma can increase monocyte cytotoxicity by sensitizing target cells to the lytic action of TNF.