Cytotoxic Effects Of TNF-alpha Research Articles

INDUCTION OF APOPTOSIS IN B16 MELANOMA CELLS UNDER THE ACTION OF DRUG CONTAINING TUMOR NECROSIS FACTOR ALPHA AS A PART OF VIRUS-LIKE PARTICLES <em>IN VITRO</em>

Aim : to evaluate the antitumor activity of the drug containing TNF-alpha and high-polymer double-stranded RNA (dsRNA) in the composition of virus-like particles (VLP-TNF-alpha) on B16-F10 melanoma cells. Material and Methods . Analysis of the anti-proliferative effect of VLP-TNF-alpha as well as its components, TNFalpha and dsRNA, was carried out using the MTT -test. Apoptosis of melanoma cells was assessed by flow cytofluorimetry with FITC-annexin V. Results . It was shown that the cytotoxic effect of the drug containing the combination of TNF-alpha and dsRNA on melanoma cells significantly exceeded the total cytotoxic effect of TNF-alpha or dsRNA alone (LD50 for combination drug was 0.05 μg/ml, TNF-alpha – 9.5 μg/ml, dsRNA>20 μg/ml). Conclusion . The drug containing TNF-alpha and dsRNA molecules may be a promising drug for the treatment of malignant tumors, including melanoma.

Lipopolysaccharide-induced cross-tolerance against renal ischemia–reperfusion injury is mediated by hypoxia-inducible factor-2α-regulated nitric oxide production

Although the protective effect of lipopolysaccharide (LPS) pretreatment on renal ischemia/reperfusion injury is known, a link to hypoxia-inducible factors (HIFs) has not been established. Here we show that LPS treatment led to HIF-2α accumulation in mouse kidneys and endothelial cells, a result of nuclear factor-κB activation. Inactivation of HIF-2α, rather than HIF-1α, completely negated LPS-mediated protection against renal ischemia/reperfusion injury. LPS-stimulated renoprotection was related to inducible/endothelial nitric oxide synthase (iNOS/eNOS) expression, increased production of nitric oxide, and enhanced postischemic microcirculatory recovery. All these effects were lost in HIF-2α knockout mice. Preischemic administration of a nitric oxide donor, rather than erythropoietin, restored the lost preconditioning effect of LPS in HIF-2α knockout mice. In vitro and in vivo studies demonstrated that HIF-2α in endothelial cells, rather than myeloid cells or hepatocytes, was responsible for the LPS-mediated effects. Thus, our results demonstrated that LPS preconditioning protected against renal ischemia/reperfusion injury by HIF-2α activation in endothelial cells that subsequently improved renal microvascular perfusion and reduced ischemic tubular damage.

Anti‐tumour necrosis factor‐α activity in Ixodes ricinus saliva

Tumour necrosis factor-alpha (TNF-alpha) is one of the most prominent inflammatory mediators playing a central role in starting off the inflammatory reactions of the innate immune system. We identified a TNF-alpha-inhibitory activity in the saliva and salivary gland extract (SGE) from partially fed Ixodes ricinus ticks. Using mouse and human TNF-alpha specific ELISA, we showed that tick saliva or SGE markedly reduced the level of detectable cytokine. Both saliva and SGE inhibited the cytotoxic effect of TNF-alpha in a bioassay. Elimination of the TNF-alpha-inhibitory activity in SGE by trypsin digestion demonstrated that the anti-TNF-alpha factor is a protein. Fast protein liquid chromatography fractionation of SGE showed one peak of TNF-alpha-inhibitory activity corresponding to a protein with estimated molecular mass 23 kDa. The likely mechanism of the inhibitory effect is a direct binding of the cytokine. The TNF-alpha-inhibitory molecule seems to play an important role in the anti-inflammatory effect of tick saliva at the tick feeding site, providing a gateway to the host for tick-borne pathogens.

Identification of p54nrb and the 14-3-3 Protein HS1 as TNF-α-Inducible Genes Related to Cell Cycle Control and Apoptosis in Human Arterial Endothelial Cells

TNF-alpha plays a pivotal role in inflammation processes which are mainly regulated by endothelial cells. While TNF-alpha induces apoptosis of several cell types like tumor cells, endothelial cells are resistant to TNFa mediated cell death. The cytotoxic effects of TNF-alpha on most cells are only evident if RNA or protein synthesis is inhibited, suggesting that de novo RNA or protein synthesis protect cells from TNF-alpha cytotoxicity, presumably by NF-kappaB mediated induction of protective genes. However, the cytoprotective genes involved in NF-kappaB dependent endothelial cell survival have not been sufficiently identified. In the present study, the suppression subtractive hybridization (SSH) method was employed to identify rarely transcribed TNF-alpha inducible genes in human arterial endothelial cells related to cell survival and cell cycle. The TNF-alpha-induced expression of the RNA binding protein p54(nrb) and the 14-3-3 protein HS1 as shown here for the first time may contribute to the TNF-alpha mediated cell protection of endothelial cells. These genes have been shown to play pivotal roles in cell survival and cell cycle control in different experimental settings. The concerted expression of these genes together with other genes related to cell protection and cell cycle like DnaJ, p21(cip1) and the ubiquitin activating enzyme E1 demonstrates the identification of new genes in the context of TNF-alpha induced gene expression patterns mediating the prosurvival effect of TNF-alpha in endothelial cells.

Tumor Necrosis Factor α Reduces Butyrate Oxidation In Vitro in Human Colonic Mucosa: A Link from Inflammatory Process to Mucosal Damage?

Butyrate produced by colonic bacterial fermentation is the main fuel for colonocytes, glucose being an alternative fuel. During inflammatory bowel disease, butyrate oxidation by colonocytes is impaired, and increased production of proinflammatory cytokines is detected in the colonic mucosa. We hypothesized that proinflammatory cytokines might reduce butyrate oxidation, and we assessed the in vitro effects of 3 proinflammatory cytokines on butyrate and glucose oxidation in colonic mucosa. Colonic biopsies were obtained from normal mucosa in 42 patients who underwent a colonoscopy. Biopsies were incubated in RPMI 1640 with [1-14C]-butyrate or [U-14C]-glucose with or without 1 of the 3 following proinflammatory cytokines: tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, and IL-6. For each cytokine, 4 different concentrations were tested in 8 subjects. Concentrations overlapped those commonly found in inflamed mucosa and had no cytotoxicity as assessed in preliminary experiments using both the trypan blue exclusion test and lactate dehydrogenase release. Production of 14CO2 (picomoles per microgram dry weight per hour) was measured after a 2-hour incubation and expressed as a percentage of the control [14C]-substrate oxidation without cytokines. Whereas glucose oxidation was not affected, butyrate oxidation was reduced significantly (P < 0.05) by TNFalpha at concentrations of 100 (-26 +/- 6%), 1000 (-32 +/- 7%), and 5000 pg/mL (-34 +/- 5%). IL-1beta (0, 500, 5000, and 25,000 pg/mL) and IL-6 (0, 100, 1000, and 5000 pg/mL) did not affect either substrate oxidation. TNFalpha at concentrations found in inflamed mucosa reduces butyrate oxidation in vitro in mucosa from healthy controls. This result is not caused by a cytotoxic effect of TNFalpha and is not balanced by an increased oxidation of glucose. Reduced butyrate oxidation results in a decreased energy supply to colonocytes and may explain, in part, mucosal damage occurring during attacks of inflammatory bowel disease.

Effect of Isoproterenol on the Cardiac Troponin I Degradation and Release during Early TNFα-Induced Ventricular Dysfunction in Isolated Rabbit Heart

We studied the consequences of an early phase of TNFalpha-induced LV dysfunction and of its treatment by isoproterenol on an isolated rabbit heart preparation. Two dosages of TNFalpha (2 and 4 microg) were infused, followed by isoproterenol (ISO), infused by increasing concentrations from 10 to 10 M. Left ventricular developed pressure (DP) was recorded. Creatine kinase (CKtot) and cardiac Troponin I (cTnI) were measured in the effluent perfusate. An anatomic score was calculated by histologic examination of the hearts while a structural analysis of cTnI was done. TNFalpha induced a dose-dependent decrease in DP (-43 +/- 18% for 4 microg) without change in coronary vascular resistances, which was not followed by biochemical or structural abnormalities. TNFalpha reduced the maximum effect (Emax) of ISO on DP (mean DeltaDPmaxISO = -40% for 4 microg) without change in the concentration leading to half Emax (ED50ISO). ISO treatment of TNFalpha (4 microg)-induced LV dysfunction resulted in a selective release of cTnI, myocardial tissue contraction bands, and a significant proteolysis of cTnI. Within the limits of the model, the myocardial injury reported during severe sepsis would not be related to an early cytotoxic effect of TNFalpha but could be attributed to an enhancement of the effects of isoproterenol by TNFalpha.

Regulative capacity of glutamine.

The amino acid glutamine plays a central role in nitrogen transport within the body and is a fuel for rapidly dividing cells, such as in the gut and the immune system. Plasma glutamine levels decline during critical illness, and therefore these cells suffer from glutamine starvation under these conditions. The present review summarizes data on the specific effect of extracellular glutamine on metabolism, function, stress response, and apoptosis of glutamine-utilizing cells. Glutamine starvation leads to an energy depletion that is associated with a reduced responsiveness to exogenous stimuli. In addition, glutamine-starving cells show a reduced expression of the 70000 M(r) heat shock protein, which is an important factor for cell survival, and contain a reduced level of the antioxidant glutathione. Recent findings show that the extracellular glutamine level affects the susceptibility of cells to different apoptosis triggers: whereas glutamine-starving cells are more sensitive to Fas ligand-mediated apoptosis, they are desensitized against the cytotoxic effects of TNF-alpha. In addition, this review summarizes current knowledge on the molecular mechanisms of glutamine sensing. It discusses the role of AMP-activated protein kinase, the cellular redox state, osmosignalling, the regulation of translation, and amino acyl transfer RNA synthetases. These data show that glutamine-utilizing cells possess molecular mechanisms to detect the availability of glutamine and to respond specifically to changes in the extracellular glutamine concentration.

Inhibition of nuclear factor-kappaB activation un-masks the ability of TNF-alpha to induce human eosinophil apoptosis.

Apoptosis renders eosinophils functionally effete and marks them for "silent" removal from inflamed sites by macrophages. We show, for the first time, that eosinophils exposed to TNF-alpha rapidly lose their cytoplasmic levels of IkappaBalpha, the inhibitory subunit of NF-kappaB. Consequently, TNF-alpha triggers NF-kappaB mobilization from the cytoplasm to the nucleus, as determined by tracking the NF-kappaB subunit p65 by immunofluorescence and Western blot analysis. Inhibition of TNF-alpha-mediated IkappaBalpha degradation and NF-kappaB activation by gliotoxin or the proteasome inhibitor MG-132 un-masks the caspase-dependent pro-apoptotic properties of TNF-alpha. In addition, cycloheximide similarly renders TNF-alpha pro-apoptotic, suggesting that NF-kappaB activation controls the production of a protein(s) that protects eosinophils from the cytotoxic effects of TNF-alpha. Evidence is presented suggesting that TNF-alpha triggered apoptosis is more susceptible to NF-kappaB inhibition than constitutive apoptosis, leading to the possibility of the specific targeting of apoptosis in eosinophil sub-populations. Prior to morphological signs of apoptosis, TNF-alpha-induced IL-8 synthesis is abrogated by inhibition of NF-kappaB. We propose that NF-kappaB activation plays a critical role in controlling eosinophil responsiveness and apoptosis, and speculate that selective inhibitors of eosinophil NF-kappaB activation may ultimately provide alternative therapeutic agents for the treatment of eosinophilic diseases, including asthma and allergic rhinitis.

Activation of NF-kappaB determines the sensitivity of human colon cancer cells to TNFalpha-induced apoptosis.

Tumor necrosis factor alpha (TNFalpha) generates a potent cytotoxic effect, however many cancer cells are resistant to TNFalpha-mediated killing and the cause of the differential sensitivity remains to be elucidated. In this study, we demonstrated that TNFalpha induced cell death in four different human colon cancer cell lines. The degree of cytotoxic effect was different in each cell line, in that HCT-15 was relatively sensitive, while DLD-1, HT-29 and WiDr were relatively resistant. TNFalpha induced apoptotic changes such as morphological changes, DNA fragmentation and activation of caspase-3 in HCT-15, but to a lesser degree in the others. Transcriptional expression of TNFR1(p55), as well as that of FLICE, Fas, FADD, DR3, FAF, TRADD, and RIP was similar in these cell lines, indicating that the susceptibility to TNFalpha-induced apoptosis may not be determined by the constitutive expression level of these factors. Interestingly, the cytotoxic effect of TNFalpha was well correlated with the DNA binding activity of NF-kappaB in the colon cancer cell lines. Further, the overexpression of a non-phosphorylated mutant form of IkappaBalpha enhanced the cytotoxicity of TNFalpha in the resistant cell line, DLD-1, indicating that NF-kappaB activity may determine the sensitivity of colon cancer cells to TNFalpha-induced apoptosis. Thus, our results indicate that modulation of NF-kappaB activity may provide a useful tool to sensitize colon cancer cells to TNFalpha treatment.

Prostaglandin-independent anovulatory mechanism of indomethacin action: inhibition of tumor necrosis factor alpha-induced sheep ovarian cell apoptosis.

Indomethacin, a nonsteroidal anti-inflammatory agent, is a potent inhibitor of ovulation in vertebrates. The presumptive obligate anovulatory mode of indomethacin action is via suppression of ovarian prostaglandin production. We report that a very high systemic dose of indomethacin (800 mg i.m.) is required to block ovulation in gonadotropin-treated anestrous ewes. A lower dose of indomethacin (200 mg), which negated the preovulatory rise in follicular prostaglandin (PGF(2alpha)) biosynthesis, did not prevent ovulation. Endothelial secretion of tumor necrosis factor (TNF)-alpha within the apical follicular wall (prospective site of rupture) was not altered by indomethacin; notwithstanding, the apoptosis (DNA-fragmentation)-inducing effect of TNF-alpha (a determinant of ovulatory stigma formation) was attenuated by 800 (but not 200) mg indomethacin. A suprapharmacological concentration of indomethacin also was necessary to protect ovarian surface epithelial cells from a (prostaglandin-independent) cytotoxic effect of TNF-alpha in vitro. It is concluded that indomethacin inhibits ovulation by anti-apoptotic mechanisms that can be dissociated from the paradigm of prostanoid down-regulation.

NF-κB Activation Is a Critical Regulator of Human Granulocyte Apoptosis in Vitro

During beneficial inflammation, potentially tissue-damaging granulocytes undergo apoptosis before being cleared by phagocytes in a non-phlogistic manner. Here we show that the rate of constitutive apoptosis in human neutrophils and eosinophils is greatly accelerated in both a rapid and concentration-dependent manner by the fungal metabolite gliotoxin, but not by its inactive analog methylthiogliotoxin. This induction of apoptosis was abolished by the caspase inhibitor zVAD-fmk, correlated with the inhibition of nuclear factor-kappa B (NF-kappaB), and was mimicked by a cell permeable inhibitory peptide of NF-kappaB, SN-50; other NF-kappaB inhibitors, curcumin and pyrrolidine dithiocarbamate; and the proteasome inhibitor, MG-132. Gliotoxin also augmented dramatically the early (2-6 h) pro-apoptotic effects of tumor necrosis factor-alpha (TNF-alpha) in neutrophils and unmasked the ability of TNF-alpha to induce eosinophil apoptosis. In neutrophils, TNF-alpha caused a gliotoxin-inhibitable activation of an inducible form of NF-kappaB, a response that may underlie the ability of TNF-alpha to delay apoptosis at later times (12-24 h) and limit its early killing effect. Furthermore, cycloheximide displayed a similar capacity to enhance TNF-alpha induced neutrophil apoptosis even at time points when cycloheximide alone had no pro-apoptotic effect, suggesting that NF-kappaB may regulate the production of protein(s) which protect neutrophils from the cytotoxic effects of TNF-alpha. These data shed light on the biochemical and molecular mechanisms regulating human granulocyte apoptosis and, in particular, indicate that the transcription factor NF-kappaB plays a crucial role in regulating the physiological cell death pathway in granulocytes.

Effect of gene transfer of tumor necrosis factor receptors into human lung carcinoma cell line.

The human lung adenocarcinoma cell line A549 is known to be resistant to tumor necrosis factor alpha (TNF‐α)‐mediated tumor cell lysis in spite of the expression of 55 kDa TNF receptor (TNF‐R55) mRNA and its cell surface protein. In this study, we investigated the mechanism of TNF‐α resistance and the role of two types of TNF receptors (TNF‐R55 and TNF‐R75 (75 kDa TNF receptor)). TNF‐R55 or TNF‐R75 cDNA was transfected into A549 cells. In addition, a C‐terminal deletion mutant of TNF‐R75 which lacks the intracellular domain of TNF‐R75 was also transfected into A549 cells. We assessed the TNF‐α‐mediated tumor cell lysis of these transfected clones, and found that the cytotoxic effect increased in transfected clones highly expressing TNF‐R55, but not in low‐expression clones. As for TNF‐R75, the cytotoxic effect of TNF‐α was observed in TNF‐R75‐transfected clones even when expression was low. Furthermore, the cytotoxic effect was also observed in clones transfected with the deletion mutant of TNF‐R75, as well as the complete TNF‐R75. These results indicate that a certain level of expression of TNF‐R55 is necessary for obtaining TNF‐α‐mediated tumor cell lysis in the absence of TNF‐R75. On the other hand, the expression of TNF‐R75 strongly induces TNF‐α‐mediated cytotoxicity through TNF‐R55 in the absence of an intracellular signal via TNF‐R75.

Soluble fractions of tumor necrosis factor-alpha, interleukin-6 and of their receptors in toxic epidermal necrolysis: a comparison with second-degree burns.

Drug-induced toxic epidermal necrolysis (TEN) is a rare bullous disease characterized by severe epidermal necrosis and sloughing. Soluble TNF-alpha(sTNF-alpha), soluble IL-6 (sIL-6) and their reactive soluble receptors (sTNF-Rp55 or-R1, sTNF-Rp75 or-R2, sIL-6R) were quantified in blister fluid and serum of 6 TEN patients and 13 cases of second-degree burn. The amounts of sTNF-alpha, sTNF-R1 and sTNF-R2 were significantly higher in TEN blisters than in burns reflecting the probable involvement of the TNF-alpha system in the specific pathomechanism of TEN. The ratio sTNF-alpha/sTNF-R2 was significantly lower in TEN blisters than in burns. The concentrations of sTNF-R2 in TEN blisters and serums were significantly greater than those of sTNF-R1. This suggests a potential important role for sTNF-R2 in TEN by enhancing the cytotoxic effect of TNF-alpha. In addition, both sTNF-R1 and sTNF-R2 were significantly more abundant in TEN blisters than in serums, indicating that the TNF-alpha processing was mainly a local event in the TEN skin. No significant difference could be established for sIL-6 and sIL-6R between TEN and burns. Although a role for IL-6 cannot be ruled out, its production has no specific characteristics in TEN compared to burns.

Stress proteins in inflammation.

Inflammation provides those searching in the field with a number of "models" allowing them to study, in vivo, in humans and in animals, the regulation and the functions of HSP, which are being considered as a new and promising marker for the severity and the prognosis of inflammatory diseases. HSP are differentially regulated according to the type of inflammation, whether acute or chronic, whether self-limiting (inflammatory cell elimination by apoptosis) or self-perpetuating (inflammatory cell death by necrosis). We propose that mitochondria are a key organelle in determining the outcome of inflammation, because they are both the cellular "switchboard" for apoptosis and a selective target for the protective effects of HSP against the cytotoxic effects of TNF alpha and ROS. On the other hand, HSP exert multiple protective effects in inflammation, including self/non-self discrimination, enhancement of immune responses, immune protection, thermotolerance and protection against the cytotoxicity of inflammatory mediators. The latter protective effects against the deleterious effects of the mediators of inflammation, including ROS and cytokines, open new avenues for the development of original anti-inflammatory therapies, such as non-toxic inducers of a complete HS response. It may well be that the "beneficial effects of fever" already described by Hippocrates actually relate to increased HSP expression during fever, and to their protective effects....

Hyaluronidase induces murine L929 fibrosarcoma cells resistant to tumor necrosis factor and Fas cytotoxicity in the presence of actinomycin D.

Actinomycin D (ActD) enhances the potency of tumor necrosis factor-alpha (TNF-alpha) in killing cancer cells. However, it is determined in this study that murine L929 fibrosarcoma cells, when pretreated with bovine testicular hyaluronidase for 12-24 h, became resistant to the cytotoxic effect of TNF-alpha in the presence of DNA intercalators, such as ActD, doxorubicin, and daunorubicin. Monoclonal anti-Fas antibody-mediated apoptosis in the presence of ActD was also blocked in hyaluronidase-pretreated L929 cells. Hyaluronidase failed to up- or downregulate the expression of apoptosis regulatory proteins, including Bcl-2, Bcl-xL, ICH-1, and TIAR, suggesting that these proteins were not involved in the hyaluronidase-induced resistance to TNF/ActD. A semisynthetic polysulfated hyaluronic acid (HA) inhibited the increased TNF/ActD resistance, whereas unmodified HA, dextran sulfate, and naturally polysulfated glycosaminoglycans had no effect. Evidence is provided here that the induced resistance is related to serum fetuin and a novel intracellular 35-kDa TNF-binding protein (intra TBP). Under serum-free conditions, L929 became refractory to TNF/ActD cytotoxicity and hyaluronidase reversed the resistance. Exogenous fetuin increased L929 cell spreading and proliferation, and restored hyaluronidase-induction of TNF/ActD resistance in these serum-starved cells. Hyaluronidase failed to reduce the expression of TNF-receptors and their binding of TNF-alpha. However, binding and Western-blotting analyses revealed that hyaluronidase downregulated the intra-TBP. Overall, these observations suggest that serum fetuin and intraTBP are involved in the hyaluronidase induction of TNF/ ActD resistance.

OVERCOMING TNF-ALPHA AND CDDP RESISTANCE OF A HUMAN OVARIAN-CANCER CELL-LINE (C30) BY TREATMENT WITH BUTHIONINE SULFOXIMINE IN COMBINATION WITH TNF-ALPHA AND OR CDDP

Previous studies have demonstrated that glutathione (GSH) plays an important role in a wide range of cellular functions including protection, detoxification, transport and metabolism. GSH has been implicated in tumor cell resistance to drugs and/or cytotoxic factors. Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, depletes intracellular GSH and thus could reverse resistance. The present study investigated the effect of BSO used in combination with tumor necrosis factor-alpha (TNF-alpha) or cisdiamminedichloroplatinum (II) (CDDP) on cytotoxicity of a TNF-alpha and CDDP resistant human ovarian cancer cell line (C30). Cytotoxicity was monitored by the MTT assay. Treatment of C30 cells with BSO and CDDP or BSO and TNF-alpha resulted in overcoming resistance and a synergistic cytotoxic effect was obtained. Pretreatment of the tumor cells by either agent for 4 h and wash and followed by the addition of the second agent for 20 h resulted in the same cytotoxicity as observed in the presence of the two agents. Furthermore, combination treatment with BSO, CDDP and TNF-alpha further augmented the synergistic cytotoxic activity achieved by two agents against C30 cells. The protective effect of GSH was shown for TNF-alpha but not for CDDP as treatment of C30 cells with TNF-alpha in combination with GSH or N-acetyl-cysteine (NAC) reduced the cytotoxic effect of TNF-alpha. One mechanism of resistance to TNF-alpha in tumor cells is through the induction of TNF-alpha mRNA and/or protein. The C30 cells did not constitutively express TNF-alpha mRNA, however, treatment of C30 cells with TNF-alpha upregulated the expression of TNF-alpha mRNA. When BSO was used in combination with TNF-alpha, the level of TNF-alpha mRNA induced by TNF-alpha was markedly reduced. Further, incubation of C30 cells with TNF-alpha in conjunction with GSH or NAC also downregulated the expression of TNF-alpha mRNA induced by TNF-alpha. These findings demonstrate that treatment with BSO in combination with TNF-alpha or CDDP can overcome the resistance of C30 tumor cells to TNF-alpha and CDDP. The depletion of intracellular GSH and downregulation of TNF-alpha mRNA by BSO may play a role in the enhanced cytotoxicity seen with the combination of BSO and TNF-alpha. The synergistic effect obtained with a CDDP selected resistant ovarian cancer cell line suggests that treatment with BSO in conjunction with either TNF-alpha or CDDP, or TNF-alpha and CDDP may have a clinical application in the therapy of TNF-alpha and/or CDDP resistant ovarian tumors'

Tumor necrosis factor alpha inhibits hepatocyte mitochondrial respiration.

Although direct cytotoxicity is a well-established phenomenon of tumor necrosis factor alpha (TNF alpha)-induced tissue damage, the intracellular events leading to cell death are still poorly understood. To study the cytotoxic effects of TNF alpha on normal parenchymal cells, rat hepatocytes were purified and incubated with various concentrations of TNF alpha. Mitochondrial respiration, total protein synthesis, and enzyme release were measured to assess metabolic performance and cell integrity. Treatment with TNF alpha suppressed mitochondrial respiration in a concentration-dependent fashion, resulting in a reduction of the activity of complex I of the respiratory chain to 67.0 +/- 3.5% of that of untreated hepatocytes by 2000 U/mL TNF alpha. Under these conditions protein synthesis and the release of intracellular enzymes were significantly increased. Both hepatocellular enzyme release and inhibition of mitochondrial respiration appear to be associated with the generation of reactive oxygen intermediates by the hepatocyte itself, because oxygen radical scavengers prevented these adverse effects of TNF alpha. Inhibition of protein synthesis by cycloheximide as well as addition of cyclic adenosine monophosphate synergistically enhanced the suppression of mitochondrial respiration by TNF alpha, resulting in complex I activity of 6.9 +/- 1.6% and 24.9 +/- 2.9% of that of untreated cells. These data indicate that inhibition of mitochondrial respiration is one of the mechanisms by which TNF alpha induces tissue injury.

Aging of FRTL-5 rat thyroid cells causes sensitivity to cytotoxicity induced by tumor necrosis factor-alpha.

While investigating the modulation of the growth and function of the FRTL-5 rat thyroid cell line by recombinant human tumor necrosis factor-alpha (TNF alpha), we noticed that pronounced changes in several response parameters occurred with increasing passage number. For young cells (passage less than 20), TNF alpha by itself slightly increased [3H]thymidine incorporation and DNA content, and had a minimal effect on basal 125I uptake. When combined with TSH, TNF alpha had no influence on TSH-stimulated [3H]thymidine incorporation, but significantly inhibited TSH-stimulated 125I uptake. Compared with young cells, aged cells (passage greater than 40), in contrast, developed a high sensitivity to TNF alpha. TNF alpha markedly stimulated [3H]thymidine incorporation into DNA, inhibited TSH-stimulated 125I uptake per micrograms DNA, but dramatically decreased the total DNA content and cell number. TSH augmented the TNF alpha effect in aged cells, resulting in a further reduction of DNA content. Aphidicolin, a specific inhibitor of DNA polymerase-alpha which is associated with DNA replication, dramatically inhibited TNF alpha-induced [3H]thymidine incorporation in both young and aged cells; this suggested that the effect of TNF alpha on FRTL-5 cell growth is related to DNA replication, rather than DNA repair. 51Cr release from FRTL-5 cells, a measure of cytotoxicity, increased 2-fold over baseline in aged cells at a dose of 400 ng/ml TNF alpha and decreased to 70% of baseline in young cells at this same dose. The protein kinase-A (PKA) and protein kinase-C (PKC) signal transduction mechanisms of TNF alpha in aged cells (passage greater than 40) were also studied. TNF alpha increased cAMP and also increased relative PKA and PKC activity in 1-40 min. Phorbol myristate acetate (PMA), an activator of PKC, increased [3H]thymidine incorporation and DNA content. PMA did not affect the TNF alpha-induced increase in [3H]thymidine incorporation or its reduction of DNA content. When the cells were pretreated with a high concentration of PMA (1 microM/24 h) to down-regulate PKC, the TNF alpha dose-dependent increase in [3H]thymidine incorporation and decrease in DNA content were only slightly inhibited, suggesting that the main effects of TNF alpha are independent of PKC. We conclude that the sensitivity of FRTL-5 cells to the cytotoxic effect of TNF alpha increases with aging.

Nitric oxide mediates tumor necrosis factor-α cytotoxicity in endothelial cells

Tumor necrosis factor alpha (TNF-alpha) exerts multiple actions on endothelial cells including among others the expression of pro-coagulant activity and adhesion molecules, and secretion of cytokines. We now show that TNF-alpha induces a time- and dose-dependent cytotoxic effect on cultured bovine aortic endothelial cells. This TNF-induced cytotoxicity, which is preceded by increased production of nitric oxide (NO), is significantly decreased by the NO synthase inhibitor N-iminoethyl-L-ornithine (L-NIO). Dexamethasone, which prevents the expression of cytokine-induced NO synthase in endothelial cells, also inhibits TNF-alpha-dependent cytotoxicity. The results indicate that NO is involved in the cytotoxic effect of TNF-alpha on endothelial cells.

Tumor necrosis factor-alpha alters bovine luteal cell synthetic capacity and viability.

Tumor necrosis factor-alpha (TNF-alpha) is a macrophage-derived cytokine that is also reportedly produced by granulosal cells and is localized in luteal cells. The present study employed serum-free culture of midcycle bovine luteal cells to investigate the effects of TNF-alpha, alone and with other cytokines, on luteal function. TNF-alpha (1-1000 ng/ml) produced a dose-dependent increase in prostaglandin (PG)F2 alpha and 6-keto-PGF1 alpha synthesis on all days of culture, but had no effect on basal progesterone (P4) production. TNF-alpha, in combination with other known stimulators of luteal PG synthesis, interleukin-1 beta (2.5 ng/ml) or interferon-gamma (IFN-gamma, 100 U/ml), had synergistic effects on PGF2 alpha production (greater than 50-fold above control, P less than 0.05) whereas interferon-alpha (1000 U/ml) significantly suppressed TNF-alpha-stimulated PGF2 alpha production. By day 7 of culture, TNF-alpha inhibited LH-stimulated P4 production (P less than 0.05). Luteal cell numbers were significantly reduced by IFN-gamma but not by TNF-alpha alone. However, the combination of TNF-alpha + IFN-gamma was extremely cytotoxic (only 20% of cells maintained as compared to control). Finally, TNF-alpha (100 ng/ml) enhanced the expression of Class I major histocompatibility complex antigens on cultured bovine luteal cells but did not alter IFN-gamma induction of Class II major histocompatibility complex antigens. In light of these findings, it appears that TNF-alpha, in conjunction with other cytokines, is a modulator of luteal cell function in vitro. The stimulation of PG synthesis, as well as cytotoxic effects of TNF-alpha, may suggest a role in luteolysis.