Processing of newly synthesized cachectin/tumor necrosis factor in endotoxin-stimulated macrophages

The biosynthesis and processing of cachetin/tumor necrosis-factor (TNF) were examined in the murine macrophage-like cell line RAW 264.7. Lipopolysaccharide-stimulated cells secreted both glycosylated and nonglycosylated 17-kilodalton (kDa) mature cachectin/TNF into the culture medium. Secreted cachectin/TNF was derived from membrane-associated precursors that were precipitated by polyclonal antisera raised against either the mature protein or synthetic peptide fragments of the 79 amino acid cachectin/TNF prohormone sequence. About half of the precursors were N-glycosylated, apparently cotranslationally. The cachectin/TNF precursors were then proteolytically cleaved to release soluble mature cytokine into the medium, while the membrane-bound 14-kDa presequence remained cell associated. During the period of LPS stimulation, the amount of macrophage cell surface cachectin/TNF remained at a low level, suggesting that both nonglycosylated and glycosylated precursors of cachectin/TNF are efficiently cleaved by these cells. These findings suggest the presence of a unique mechanism for the secretion of cachectin/TNF.     

Additive effects of IL-1 and TNF on induction of prostacyclin synthesis in human vascular endothelial cells

The effect of interleukin 1 (IL-1) and tumor necrosis factor (TNF) on the induction of prostacyclin (PGI2) synthesis in human vascular endothelial cells (EC) was investigated. Both IL-1 and TNF increased PGI2 production by EC in both a time- and dose-dependent manner, and a combination of the two cytokines additively enhanced PGI2 production. Metabolic inhibitors including indomethacin and cycloheximide abolished cytokine induced PGI2 synthesis. Since, the effect of TNF was not inhibited by neutralization with antibody to IL-1 alpha and IL-1 beta, it seems that the mechanism is not mediated by endogenous IL-1 induced by TNF.     

Tumors secreting human TNF/cachectin induce cachexia in mice

Anorexia and weight loss are serious complications that adversely effect the prognosis of cancer patients. It has been suggested that TNF/cachectin may cause cachexia. To determine if TNF/cachectin can induce progressive weight loss in tumor-bearing animals, a clone of the human TNF/cachectin gene was isolated and inserted into a mammalian expression vector. This construct was transfected into CHO cells, and a cell line (CHO/TNF-20) that secretes TNF/cachectin was isolated. A cell line (CHO/CMV-Neo) that contains the same expression vector without the TNF/cachectin gene was also isolated. Nude mice injected intraperitoneally with CHO/TNF-20 cells died more quickly than mice injected with CHO/CMV-Neo cells. Eighty-seven percent of mice inoculated intramuscularly with CHO/TNF-20 cells developed severe cachexia and weight loss. All mice bearing CHO/CMV-Neo tumors maintained or increased their body weight. We conclude that mice bearing tumors that secrete TNF/cachectin develop progressive wasting and die more quickly than mice bearing control tumors.     

Cachectin/tumor necrosis factor-alpha alters red blood cell kinetics and induces anemia in vivo

Chronic inflammatory diseases are often associated with decreased red blood cell (RBC) mass. The cytokines cachectin/tumor necrosis factor-alpha (TNF) and interleukin 1 (IL 1) are produced by monocytes/macrophages in response to many inflammatory stimuli and have been implicated in the anemia of chronic disease. This study was undertaken to evaluate the mechanisms by which cachectin/TNF, IL 1, or endotoxin induce anemia. Hematologic parameters and RBC kinetics were quantitated in rats given chronic sublethal quantities of either recombinant human cachectin/TNF, recombinant human IL 1 alpha, or Salmonella endotoxin for 7 days. Cachectin/TNF or endotoxin treatment resulted in a 25 or 31% decrease, respectively, in total RBC mass, whereas RBC mass was unchanged by IL 1 administration. Anemia associated with either chronic cachectin or endotoxin administration was characterized by normal mean corpuscular volume, mean corpuscular hemoglobin content, and reticulocyte numbers. [59Fe]RBC survival was significantly shortened in animals given cachectin, IL 1 or endotoxin, but the magnitude of the response was greatest in cachectin/TNF-or endotoxin-treated rats. Although cachectin/TNF-IL 1-, or endotoxin treatment resulted in similar hypoferremia and shortened plasma iron half-life, endotoxin or cachectin/TNF treatment (but not IL 1) significantly reduced the incorporation of plasma 59Fe into newly synthesized RBCs. We conclude that chronic cachectin/TNF administration produces anemia by decreasing RBC synthesis and reducing the life span of circulating RBCs. An endogenous cachectin/TNF response during inflammatory disease may contribute to an associated anemic state, whereas the modestly reduced red cell life span induced by IL 1 does not lead to a net reduction in RBC mass, presumably owing to a preserved RBC synthetic rate.     

Nitric oxide enhances PGI(2)production by human pulmonary artery smooth muscle cells

To evaluate the effect of exogenous nitric oxide (NO) and endogenous NO on the production of prostacyclin (PGI(2)) by cultured human pulmonary artery smooth muscle cells (HPASMC) treated with lipopolysaccharide (LPS), interleukin-1(beta)(IL-1(beta)), tumor necrosis factor alpha (TNF(alpha)) or interferon gamma (IFN(gamma)), HPASMC were treated with LPS and cytokines together with or without sodium nitroprusside (SNP), NO donor, N(G)-monomethyl-L-arginine (L-NMMA), NO synthetase inhibitor, and methylene blue (MeB), an inhibitor of the soluble guanylate cyclase. After incubation for 24 h, the postculture media were collected for the assay of nitrite by chemiluminescence method and the assay of PGI(2)by radioimmunoassay. The incubation of HPASMC with various concentrations of LPS, IL-1(beta)or TNF(alpha)for 24 h caused a significant increase in nitrite release and PGI(2)production. However, IFN(gamma)slightly increased the release of nitrite and had little effect on PGI(2)production. Although the incubation of these cells for 24 h with SNP did not cause a significant increase in PGI(2)production, the incubation of HPASMC with SNP and 10 microg/ml LPS, or with SNP and 100 U/ml IL-1(beta)further increase PGI(2)production and this enhancement was closely related to the concentration of SNP. However, stimulatory effect of SNP on PGI(2)production was not found in TNF(alpha)- and IFN(gamma)- treated HPASMC. Addition of L-NMMA to a medium containing LPS or IL-1(beta)reduced nitrite release and attenuated the stimulatory effect of those agents on PGI(2)production. MeB significantly suppressed the production of PGI(2)by HPASMC treated with or without LPS or IL-1(beta). The addition of SNP partly reversed the inhibitory effect of MeB on PGI(2)production by HPASMC. These experimental results suggest that NO might stimulate PGI(2)production by HPASMC. Exogenous NO together with endogenous NO induced by LPS or cytokines from smooth muscle cells might synergetically enhance PGI(2)production by these cells, possibly in clinical disorders such as sepsis and acute respiratory distress syndrome.     

Human recombinant TNF suppresses lipoprotein lipase activity and stimulates lipolysis in 3T3-L1 cells

Tumor necrosis factor (TNF) has been reported to be identical to "cachectin," a monokine which we have previously proposed as a mediator of the enhanced catabolism observed in patients or animals responding to various invasive stimuli such as infections. Detailed quantitative studies were conducted on the effects of TNF on fatty acid metabolism in 3T3-L1 cells in order to explore the extent of the catabolic effects exerted by TNF compared with those by the crude cachectin. 3T3-L1 adipocytes responded to recombinant human TNF, showing a decrease in LPL activity and an increase in intracellular lipolysis. When TNF in the crude cachectin preparation was completely neutralized with anti-TNF antibody, about 75% of LPL suppression activity in the crude cachectin was absorbed, indicating that most of the mediator responsible for LPL suppression in the crude preparation is TNF. In contrast to the above effect on LPL, TNF markedly increased the lipolysis of stored fat in the cells. The effect on LPL was observed as early as 2 h after the addition of TNF, but enhancement of lipolysis required a time lag of at least 3 h before any increase of glycerol release became apparent. The effective concentrations of TNF for the stimulation of lipolysis were much higher (2.5 to 49 nM) than those for LPL suppression (50 pM to 50 nM), but both were in the same range as the concentration required for tumoricidal effect. These results demonstrate that cachectin is synonymous with TNF and that it plays an important role in the pathophysiology of deranged lipid metabolism through both suppression of LPL and enhancement of lipolysis in patients coping with invasive conditions such as infections.     

Cachectin/tumor necrosis factor: production, distribution, and metabolic fate in vivo

A highly specific radioreceptor assay for cachectin/tumor necrosis factor (TNF) was utilized to measure the time course of lipopolysaccharide (LPS)-induced hormone production in rabbits. Cachectin/TNF bioactivity was monitored in the same serum samples by measuring lipoprotein lipase (LPL) suppression in 3T3-L1 cells. Cachectin/TNF is produced in large quantities by LPS-treated rabbits without priming by bacillus Calmette Guérin, C. parvum, or other agents. Nanomolar concentrations of the hormone are achieved, with peak levels occurring at 2 hr postinjection; the hormone is rapidly cleared thereafter. In separate studies, mice were used to assess the distribution and metabolic fate of cachectin/TNF. Radioiodinated hormone is cleared from the plasma with a half-life of 6 to 7 min. Studies of the tissue distribution of label after injection demonstrate that liver, kidneys, skin, and gastrointestinal tract take up most of the hormone. Electrophoretic analysis of tissues recovered from injected animals suggests that the hormone is very rapidly degraded after binding.     

Cachectin/tumor necrosis factor and interleukin-1 show different modes of combined effect on lipoprotein lipase activity and intracellular lipolysis in 3T3-L1 cells

Cachectin/tumor necrosis factor (cachectin/TNF) and interleukin-1 (IL-1) share many effects in various tissues and cells, including suppression of lipoprotein lipase (LPL) activity and enhancement of intracellular lipolysis. A possible interaction between cachectin/TNF and IL-1 in these lipase systems was studied in 3T3-L1 adipocytes. The two cytokines showed marked synergy in their suppression of LPL activity in these adipocytes. The least effective dose of cachectin/TNF or IL-1 was at around 5 x 10(-11) or 2.5 x 10(-12) M, respectively. However, when present in combination in amounts as small as 1/20 or 1/100 of the minimum effective dose for either cytokine alone (2.5 x 10(-12) M cachectin/TNF and 2.5 x 10(-14) M IL-1), the cytokines showed marked suppression of LPL activity. In marked contrast, such synergism was not seen for enhancement of intracellular lipolysis. This discrepancy in the combined effects of the two monokines on the two different enzyme systems in the same cells suggests that synergism between cachectin/TNF and IL-1 is unlikely at the level of their receptors on the surface of 3T3-L1 cells. Because the two monokines are considered to be secreted from macrophages under similar conditions, their effect on LPL suppression in many pathophysiological situations would be much greater than that of either monokine alone.     

Suppression of lipoprotein lipase in 3T3-L1 cells by a mediator produced by SEKI melanoma, a cachexia-inducing human melanoma cell line

Production of a cachexia-inducing factor(s) by the SEKI melanoma cell line, established from a human melanoma, has been well documented. Conditioned medium from cultures of this melanoma cell line contains a factor(s) that inhibits the activity of lipoprotein lipase (LPL) in fully differentiated 3T3-L1 adipocytes. The mode of inhibition of this enzyme by the factor, i.e. its dose-dependency and time course, is very similar to that of LPL-inhibition by a macrophage-derived cachexia-inducing factor, cachectin/tumor necrosis factor (cachectin/TNF). However, the conditioned medium of SEKI melanoma cells does not contain any immuno-reactive substances reactive in enzyme-linked immunosorbent assay (ELISA) with anti-cachectin/TNF antibody, or with anti-interleukin 1 alpha or beta antibodies. This LPL-suppression factor present in the conditioned medium seems to be a peptide because of its heat-lability and apparent molecular weight of more than 25,000. The conditioned media from cultures of four other different cell lines were found to show no significant suppression of LPL activity. These results imply that SEKI melanoma cells produce a cachexia-inducing factor(s) similar to cachectin/TNF but that the molecule involved is different.     

IL-1 and related cytokines enhance thrombin-stimulated PGI2 production in cultured endothelial cells without affecting thrombin-stimulated von Willebrand factor secretion or platelet-activating factor biosynthesis

We examined the effects of various cytokines on alpha-thrombin-stimulated prostaglandin (PG) I2 production, von Willebrand factor (vWF) secretion, and platelet-activating factor (PAF) synthesis in cultured human umbilical vein endothelial cells (HUVEC). A 24-h pretreatment with IL-1 beta doubled the low level of constitutive PGI2 production. In contrast, alpha-thrombin increased PGI2 production fivefold in untreated HUVEC. The most striking increase in PGI2 production was observed in IL-1 beta-treated HUVEC that were subsequently stimulated with thrombin. PGI2 production was two to three times greater than in untreated, thrombin-stimulated HUVEC and nearly eightfold greater than in IL-1 beta-treated but unstimulated HUVEC. Enhanced thrombin-stimulated PGI2 production was also observed in HUVEC pretreated with the related cytokines IL-1 alpha, TNF, or lymphotoxin. This cytokine effect was selective for PGI2 production because none of these cytokines altered either constitutive or thrombin-stimulated vWF secretion or PAF biosynthesis. IL-1 beta enhancement of thrombin-stimulated PGI2 production was concentration and time dependent and required protein synthesis. IL-1 beta pretreatment also enhanced PGI2 production in response to another agonist, histamine, and to exogenously added substrates, arachidonic acid or PGH2. Our results indicate that activation by IL-1 and related cytokines selectively primes endothelial cells for enhanced PGI2 production, but not vWF secretion or PAF synthesis, in response to thrombin and histamine. The evidence suggests that this effect is mediated through specific induction of biosynthetic enzymes for PGI2.     

The effect of tumor necrosis factor/cachectin on follicle-stimulating hormone-induced aromatase activity in cultured rat granulosa cells

We investigated the effects of tumor necrosis factor (TNF)/cachectin on follicle-stimulating hormone (FSH)-induced aromatase activity in cultured rat granulosa cells using the stereospecific transfer of 3H from [1 beta-3H] androstenedione into 3H2O. TNF (10 pg/ml-10 ng/ml) inhibited FSH (250 ng/ml)-induced aromatase activity in a concentration-dependent manner, and 10 ng/ml of TNF completely abolished the FSH-induced aromatase activity. A time course analysis of the effects of TNF showed that TNF had no effect on induced aromatase activity, but inhibited the further induction of the enzyme by FSH. TNF (10 ng/ml) also inhibited the ability of TGF beta (1 ng/ml) to enhance aromatase activity and increase progesterone synthesis. Thus, TNF is a component of the complex array of proteins that modulate ovarian function and, as such, may play a physiological role in the regulation of the granulosa cell. In view of its association with cachexia, it may also play a pathophysiological role in the suppression of reproductive function during chronic illness.     

Transforming growth factor alpha stimulates prostacyclin production by cultured human vascular endothelial cells more potently than epidermal growth factor

Transforming growth factor alpha (TGF alpha) induces dose- and time-dependent stimulation of prostacyclin (PGI2) production by cultured human umbilical vein endothelial cells. The lowest stimulatory concentration of TGF alpha was 0.1 ng/ml and the maximal response, a 2.7-fold rise, was obtained with 10 ng/ml. The stimulation, which lasted at least 24 h, was blocked by cycloheximide and by indomethacin. TGF alpha induced PGI2 production at 10-100 times lower concentrations than did epidermal growth factor (EGF), although in stimulating endothelial cell growth the two factors were equipotent. This is the first demonstration that TGF alpha enhances PGI2 production by human cells. Moreover, this is the first evidence that it acts as both an agonist (growth) and a superagonist (PGI2 production) of EGF in the same cell type. I suggest that this phenomenon may be involved with the angiogenic activity of TGF alpha.     

Tumor necrosis factor as a pharmacological target

As indicated by its name, tumor necrosis factor (TNF), cloned in 1985, was originally described as a macrophage-derived endogenous mediator that can induce hemorrhagic necrosis of solid tumors and kill some tumor cell lines in vitro. Unfortunately, its promising use as an anticancer agent was biased by its toxicity, which was clear soon from the first clinical trials with TNF in cancer. Almost at the same time TNF was being developed as an anticancer drug, it became clear that TNF was identical to a mediator responsible for cachexia associated with sepsis, which was termed cachectin. This research led to the finding that TNF is, in fact, the main lethal mediator of sepsis and to the publication of a huge number of articles showing that TNF inhibits the toxic effects of bacterial endotoxins, which are now described as systemic inflammatory response. Although the clinical trials with anti-TNF in sepsis have not been successful thus far, undoubtedly as a result of the complexity of this clinical setting, these studies ultimately led to the identification of TNF as a key inflammatory mediator and to the development of anti-TNF molecules (soluble receptors and antibodies) for important diseases including rheumatoid arthritis and Crohn’s disease. On the other side, the mechanisms by which TNF and related molecules induce cell death have been studied in depth, and their knowledge might, in the future, suggest means of improve the therapeutic index of TNF in cancer.     

Metabolic effects of tumour necrosis factor-α on rat brown adipose tissue

Intravenous administration of a single dose (100 g/kg bw) of recombinant tumour necrosis factor- (TNF, cachectin) to rats increased the rate ofin vitro fatty acid synthesis in interscapular brown adipose tissue (IBAT) from both glucose and alanine, without changes in the oxidation of these substrates to¹⁴CO₂. Lactate production and glycerol release were also unaffected by treatment with the cytokine. Additionally, the presence of TNF in the incubation media did not affect fatty acid synthesis, suggesting an indirect effect of the cytokine. The activities of different enzymes of glucose and alanine metabolism such as hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase and alanine transaminase, did not suffer changes as a consequence of TNF administration. The same applied to the enzymatic activities involved in fatty acid synthesis such as fatty acid synthase, acetyl-CoA carboxylase and ATP-citrate lyase. Conversely, citrate levels in IBAT were increased in animals treated with TNF, suggesting that it could be the cause for the increased fatty acid synthesis in this tissue.     

Cytokine production in a model of wound healing: the appearance of MIP-1, MIP-2, cachectin/TNF and IL-1

Macrophages are essential for normal wound repair and many of their effects on healing wounds are likely to be mediated by the secretion of cytokines. This study examines the appearance of messenger RNA (mRNA) for cachectin/tumor necrosis factor (TNF), IL 1, and macrophage inflammatory proteins 1 and 2 (MIP-1 and MIP-2), as well as the mature peptides, in a model of wound healing using wound chambers. RNA for all four cytokines can be detected in wound inflammatory cells by polymerase chain reaction amplification throughout the first 7 days. Cachectin/TNF and IL 1 protein levels peaked on the first day after wound chamber implantation, and MIP-1 and MIP-2 were detected only on day 3. The data suggest that these cytokines participate in the early inflammatory response to wounding.     

Cocaine use and its effect on umbilical artery prostacyclin production

Cocaine's association with adverse perinatal outcome has been attributed to its inhibition of norepinephrine uptake. This study examined the effect of cocaine on umbilical artery prostacyclin (PGI2) production. Umbilical arteries from pregnant cocaine users and controls were incubated in vitro and PGI2 levels in the media determined by measuring its stable metabolite, 6-keto-PGF1 alpha, by RIA. Cocaine users showed a significant decrease (p less than .05) in PGI2 production from their umbilical arteries when compared to controls. This appears to be through a direct effect of cocaine, as it decreases PGI2 production when added in vitro to umbilical arteries from controls. In addition, in vitro phospholipase A2 activity is inhibited by cocaine in a dose-dependent manner. These results suggest that the adverse perinatal outcome associated with cocaine use may be due in part to reduced vascular PGI2 production in the fetus.     

Effect of tumor necrosis factor/cachectin on the activity of the low density lipoprotein receptor on human skin fibroblasts

We have investigated the effects of recombinant human tumor necrosis factor (TNF)/cachectin on the cellular binding of human low density lipoprotein (LDL) to human skin fibroblasts. When recombinant TNF was added to cultured cells, LDL binding doubled after 24 h of incubation. The effect of TNF was dose-dependent and its maximal effect was observed at concentrations of 1-10 ng/ml. TNF also stimulated the growth of human skin fibroblasts 1.6-fold. These results indicate that TNF increases LDL-receptor activity, which might be related to its stimulatory effect on cell growth.     

Ca2+ redistribution from bound to free form is required for tumor necrosis factor actions in 30A5 preadipocytes

Tumor necrosis factor/cachectin (TNF) inhibits differentiation of 30A5 preadipocytes into adipocytes. In this process, TNF inhibits the expression of the gene for acetyl-coenzyme-A carboxylase, the rate-limiting enzyme for biogenesis of long chain fatty acids. One of the early reactions caused by TNF is the Ca2+ redistribution of Ca2+ from the bound form to the free form. This Ca2+ redistribution results in a transient Ca2+ efflux. High concentrations of Mg2+ inhibit Ca2+ redistribution and efflux. This inhibition reverses the repression of acetyl-coenzyme-A carboxylase and reverses the TNF inhibition of the differentiation of 30A5 preadipocytes into adipocytes. This indicates that Ca2+ redistribution between the bound and the free form is an obligatory event in the sequence of actions caused by TNF in 30A5 cells.     

Arachidonic acid strongly stimulates prostaglandin I3 (PGI3) production from eicosapentaenoic acid in human endothelial cells

Eicosapentaenoic acid (EPA) is a prominent polyunsaturated fatty acid in fish oil which inhibits blood platelet aggregation and thromboxane A2 formation but not prostacyclin-like material generation from vascular endothelium. In this study we investigated interaction between EPA and arachidonic acid (AA) during their oxygenation by cultured endothelial cells. As measured by gas chromatography-mass spectrometry (GC-MS), AA increased markedly prostaglandin I3 (PGI3) production from EPA while that of PGI2 from AA was decreased by EPA. However, increasing the ratio AA/EPA over one almost suppressed the inhibition of PGI2 formation by EPA, and the stimulation of PGI3 production by AA was even higher. The effect of AA on EPA conversion to minor prostaglandins like PGE3 and PGF3 alpha was similar then confirming the stimulating effect and suggesting it is occurring at the cyclooxygenase instead of the prostacyclin synthase level. Altogether these data indicate that, in certain nutritional states where the liberation of EPA from endothelial cells will be accompanied with that of endogenous AA, substantial amounts of PGI3 could contribute to the prostacyclin-like activity of the vessel wall in addition to PGI2.     

Prostaglandin I2 stimulation of granulosa cell cyclic AMP production.

The ability of prostaglandin I2 (PGI2) to stimulate cyclic AMP production by granulosa cells, isolated from intact immature rats, has been demonstrated in vitro. The minimal effective dose was 15 ng/ml, which was comparable to the minimal effective dose for PGE2. However, a concentration of 15 microgram/ml PGI2 was required to stimulate cyclic AMP production maximally, compared to a concentration of 1 microgram/ml PGE2, which produced the maximum response. It therefore appears that PGI2 is not more effective than PGE2 in stimulating cyclic AMP production in granulosa cells, and is possibly less effective. Submaximal concentrations of PGI2 appeared to be able to modify the stimulation of cyclic AMP production by follicle-stimulating hormone (FSH), but whether or not PGI2 plays any role in follicular function remains to be established.