Overexpression of tumor necrosis factor-alpha increases production of hydroxyl radical in murine myocardium

Transgenic (TG) mice with cardiac-specific overexpression of tumor necrosis factor-alpha develop congestive heart failure with myocardial inflammation. The purpose of this study was to investigate the effects of tumor necrosis factor-alpha on reactive oxygen species (ROS) in this mouse model of cardiomyopathy. Myocardial production of hydroxyl radical detected by electron spin resonance spectroscopy was significantly increased in TG. Myocardial expression of Mn-SOD was significantly decreased in TG, whereas that of Cu,Zn-SOD was unaltered. Myocardial expression of catalase was unchanged, whereas that of glutathione peroxidase was significantly increased, in TG. Histological analysis revealed that macrophages and CD4-positive lymphocytes were increased in TG myocardium. To investigate whether these infiltrating inflammatory cells were the source of ROS, we treated TG mice with cyclophosphamide for 7 days. Although cyclophosphamide significantly suppressed the infiltration of inflammatory cells, it did not diminish the production of hydroxyl radical in TG myocardium. Damaged myocytes, but not infiltrating inflammatory cells, may be the source of ROS in TG.     

Fcgamma-receptor signaling augments the LPS-stimulated increase in serum tumor necrosis factor-alpha levels

The phagocytosis of IgG-coated erythrocytes (EIgG) has been shown to augment the bacterial lipopolysaccharide (LPS)-stimulated increase in serum tumor necrosis factor-alpha (TNF-alpha) levels. The present study evaluated the role of Fcgamma-receptor (FcgammaR) signaling and complement activation in the effect of EIgG on the TNF-alpha response to LPS. The role of FcgammaR was determined using FcR gamma-chain knockout mice that lack functional FcgammaRI and FcgammaRIII. In wild-type animals, EIgG caused a 16-fold augmentation of the serum TNF-alpha response to LPS, whereas there was no augmentation in the FcgammaR-deficient animals. Heat-damaged erythrocytes also augmented the TNF-alpha response to LPS. This effect was absent in FcgammaR-deficient animals. An IgG antibody against heated erythrocytes was detected in mouse serum. The complement activation caused by EIgG had little effect on the LPS-stimulated increase in serum TNF-alpha levels as indicated by activation of complement with cobra venom factor or IgM-coated erythrocytes as well as studies with C5-deficient mice. These results indicate that FcgammaR signaling primarily mediates the augmented serum TNF-alpha response to LPS caused by EIgG.     

Ozone-induced lung inflammation and hyperreactivity are mediated via tumor necrosis factor-alpha receptors

This study was designed to investigate the mechanisms through which tumor necrosis factor (Tnf) modulates ozone (O(3))-induced pulmonary injury in susceptible C57BL/6J (B6) mice. B6 [wild-type (wt)] mice and B6 mice with targeted disruption (knockout) of the genes for the p55 TNF receptor [TNFR1(-/-)], the p75 TNF receptor [TNFR2(-/-)], or both receptors [TNFR1/TNFR2(-/-)] were exposed to 0.3 parts/million O(3) for 48 h (subacute), and lung responses were determined by bronchoalveolar lavage. All TNFR(-/-) mice had significantly less O(3)-induced inflammation and epithelial damage but not lung hyperpermeability than wt mice. Compared with air-exposed control mice, O(3) elicited upregulation of lung TNFR1 and TNFR2 mRNAs in wt mice and downregulated TNFR1 and TNFR2 mRNAs in TNFR2(-/-) and TNFR1(-/-) mice, respectively. Airway hyperreactivity induced by acute O(3) exposure (2 parts/million for 3 h) was diminished in knockout mice compared with that in wt mice, although lung inflammation and permeability remained elevated. Results suggested a critical role for TNFR signaling in subacute O(3)-induced pulmonary epithelial injury and inflammation and in acute O(3)-induced airway hyperreactivity.     

Cytotoxic mechanism of tumor necrosis factor-alpha

Many intracellular pathways are set in motion by the binding of tumor necrosis factor (TNF) to its cell surface receptor. Major steps in the TNF-mediated cytotoxicity cascade include G protein-coupled activation of phospholipases, generation of free radicals, and damage to nuclear DNA by endonucleases. Ultimately the cells undergo apoptosis and die. Understanding how TNF initiates these pathways will facilitate the rational design of pharmaceuticals that can attenuate or potentiate the action of this important cytokine.     

Lung volumes after an increase in lung distension in pneumonectomized ferrets

To investigate the role of lung distension in compensatory lung growth, the right lung of each of 21 adult male ferrets was replaced with a silicone rubber balloon filled with mineral oil. Three to thirteen weeks after surgery, the oil was removed through a subcutaneous port. Lung volumes were measured serially until 3-6 wk after balloon deflation. With pneumonectomy the total lung capacity (TLC) decreased to less than 50% of the preoperative value and remained essentially unchanged while the balloon was inflated. At balloon deflation, TLC and vital capacity did not change immediately, whereas functional residual capacity increased by 44%, indicating a change of 2-3 cmH2O in end-expiratory transpulmonary pressure. TLC increased by 10% within 3 days and continued to increase over the subsequent 3-5 wk by a total of 25% over TLC at balloon deflation. There was little difference in this response between animals whose balloons were deflated 3 wk after surgery and those in which deflation was delayed up to 13 wk. After pneumonectomy in the adult ferret, the remaining lung increases in volume in response to an increase in lung distension even weeks or months after surgery. The extent to which this volume increase involves lung tissue growth or depends on previous lung resection is at present unknown. This model may be useful for studies of the mechanisms by which lung distension influences lung volume and compensatory lung growth.     

Eicosanoids and tumor necrosis factor-alpha in the kidney

The thick ascending limb of Henle's loop (TAL) is capable of metabolizing arachidonic acid (AA) by cytochrome P450 (CYP450) and cyclooxygenase (COX) pathways and has been identified as a nephron segment that contributes to salt-sensitive hypertension. Previous studies demonstrated a prominent role for CYP450-dependent metabolism of AA to products that inhibited ion transport pathways in the TAL. However, COX-2 is constitutively expressed along all segments of the TAL and is increased in response to diverse stimuli. The ability of Tamm-Horsfall glycoprotein, a selective marker of cortical TAL (cTAL) and medullary (mTAL), to bind TNF and localize it to this nephron segment prompted studies to determine the capacity of mTAL cells to produce TNF and determine its effects on mTAL function. The colocalization of calcium-sensing receptor (CaR) and COX-2 in the TAL supports the notion that activation of CaR induces TNF-dependent COX-2 expression and PGE? synthesis in mTAL cells. Additional studies showed that TNF produced by mTAL cells inhibits ??Rb uptake, an in vitro correlate of natriuresis, in an autocrine- and COX-2-dependent manner. The molecular mechanism for these effects likely includes inhibition of Na?-K?-2Cl? cotransporter (NKCC2) expression and trafficking.     

Molecular design of conjugated tumor necrosis factor-alpha: synthesis and characteristics of polyvinyl pyrrolidone modified tumor necrosis factor-alpha

We conjugated tumor necrosis factor-alpha (TNF-alpha) with the synthetic polymeric modifier polyvinyl pyrrolidone (PVP) to facilitate its clinical use for anti-tumor therapy. TNF-alpha was chemically conjugated with the terminal carboxyl-bearing PVP at one end of its main chain, which was radically polymerized via the formation of an amide bond between the lysine amino groups of TNF-alpha and carboxyl group of PVP. In vitro specific bioactivity of PVP-conjugated TNF-alpha (PVP-TNF-alpha) relative to that of native TNF-alpha gradually decreased with increases in the degree of PVP attachment. In contrast, PVP-TNF-alpha in which 40% of TNF-alpha lysine residues were coupled with PVP (MPVP-TNF-alpha) exhibited the highest anti-tumor activity among the conjugated derivatives examined. MPVP-TNF-alpha had more than 200-fold higher anti-tumor efficacy than native TNF-alpha, and the anti-tumor activity of MPVP- TNF-alpha was more than 5-fold stronger than that MPEG- TNF-alpha which had the highest anti-tumor activity among PEG-conjugated TNF-alphas examined. Additionally, a high dose of native TNF-alpha induced toxic side-effects such as body weight reduction, piloerection and tissue inflammation, while no side effects were observed following i.v. administration of MPVP-TNF-alpha. The plasma half-life of MPVP-TNF-alpha (360 min) was about 80 and 3-fold longer than those of native TNF-alpha (4.6 min) and MPEG-TNF-alpha (122 min), respectively. These results suggested that PVP is a useful polymeric modifier for increasing the anti-tumor activity of PVP.     

Formation of an alpha-helix in human tumor necrosis factor-alpha by guanidine hydrochloride-induced unfolding

Human tumor necrosis factor-alpha (TNF-alpha) is a trimeric protein consisting primarily of beta-sheet. GdnHCl-induced unfolding of TNF-alpha was investigated at room temperature by circular dichroism (CD) and size exclusion chromatography. The secondary and tertiary structure of TNF-alpha persisted up to 0.9N GdnHCl regardless of incubation time, but, in the range of 1.2 N to 2.1 N GdnHCl, there was loss of tertiary structure accompanied by the formation of an alpha-helix, as revealed by far- and near-UV CD spectra. The structural changes occurred gradually in 1.2 and 2.1 N GdnHCl, but were rapid in 1.5 and 1.8 N GdnHCl. The GdnHCl-induced state of TNF-alpha is an unfolded, alpha-helical aggregate of about 130 monomers, as shown by size exclusion chromatography. We suggest the most likely pathway for the transition from beta-sheet to alpha-helix.     

Structure and functions of tumor necrosis factor-alpha converting enzyme

Tumor necrosis factor-alpha converting enzyme (TACE) is the first described and best characterized secretase. In this review the structure and the possible roles for TACE are summarized. The substrate specificity and the regulation of TACE activity as well as redundancy and possible cooperations of distinct secretases are also discussed.     

Structure of tumor necrosis factor-alpha haploblocks in European populations

DNA variants in the tumor necrosis factor-α (TNF) and linked lymphotoxin-α genes, and specific alleles of the highly polymorphic human leukocyte antigen B (HLA-B) gene have been implicated in a plethora of immune and infectious diseases. However, the tight linkage disequilibrium characterizing the central region of the human major histocompatibility complex (MHC) containing these gene loci has made difficult the unequivocal interpretation of genetic association data. To alleviate these difficulties and facilitate the design of more focused follow-up studies, we investigated the structure and distribution of HLA-B-specific MHC haplotypes reconstructed in a European population from unphased genotypes at a set of 25 single nucleotide polymorphism sites spanning a 66-kilobase long region across TNF. Consistent with the published data, we found limited genetic diversity across the so-called TNF block, with the emergence of seven common MHC haplotypes, termed TNF block super-haplotypes. We also found that the ancestral haplotype 8.1 shares a TNF block haplotype with HLA-B*4402. HLA-B*5701, a known protective allele in HIV-1 pathogenesis, occurred in a unique TNF block haplotype.     

Critical roles of mucin-1 in sensitivity of lung cancer cells to tumor necrosis factor-alpha and dexamethasone

Lung cancer is the leading cause of death from cancer. Mucins are glycoproteins with high molecular weight, responsible for cell growth, differentiation, and signaling, and were proposed to be correlated with gene heterogeneity of lung cancer. Here, we report aberrant expression of mucin genes and tumor necrosis factor receptors in lung adenocarcinoma tissues compared with normal tissues in GEO datasets. Mucin-1 (MUC1) gene was selected and considered as the target gene; furthermore, the expression pattern of adenocarcinomic cells (A549, H1650, or H1299 cells) was validated under the stimulation with tumor necrosis factor-alpha (TNFα) or dexamethasone (DEX), separately. MUC1 gene interference was done to A549 cells to show its role in sensitivity of lung cancer cells to TNFα and DEX. Results of our experiments indicate that MUC1 may regulate the influence of inflammatory mediators in effects of glucocorticoids (GCs), as a regulatory target to improve therapeutics. It shows the potential effect of MUC1 and GCs in lung adenocarcinoma (LADC), which may help in LADC treatment in the future.     

Molecular weight of recombinant human tumor necrosis factor-alpha

Recombinant DNA-derived human tumor necrosis factor-alpha from Escherichia coli was examined by equilibrium ultracentrifugation under conditions similar to those where gel filtration experiments suggested an oligomeric structure. Short-column equilibrium experiments at concentrations in the range 0.015-0.12% at pH 8.5 in 0.04 M Tris/Tris-HCl gave molecular weights corresponding to 3 times the sequence molecular weight both in the presence and absence of 0.1 M NaCl. Long (2.6 mm)-column experiments under the same solvent conditions indicated molecular weights of 51,900 +/- 900 in the absence of added NaCl and 52,600 +/- 700 in the presence of added 0.1 M NaCl. No evidence of any species other than the trimer was found.     

Stimulation-induced down-regulation of tumor necrosis factor-alpha converting enzyme

The extracellular domains of many proteins, including growth factors, cytokines, receptors, and adhesion molecules, are proteolytically released from cells, a process termed "shedding." Tumor necrosis factor-alpha converting enzyme (TACE/ADAM-17) is a metalloprotease-disintegrin that sheds tumor necrosis factor-alpha and other proteins. To study the regulation of TACE-mediated shedding, we examined the effects of stimulation of cells on TACE localization and expression. Immunofluorescence microscopy revealed a punctate distribution of TACE on the surface of untreated cells, and stimulation of monocytic cells with lipopolysaccharide did not affect TACE staining. Phorbol 12-myristate 13-acetate (PMA), a potent inducer of shedding, decreased cell-surface staining for TACE. Surface biotinylation experiments confirmed and extended this observation; PMA decreased the half-life of surface-biotinylated TACE without increasing the turnover of total cell-surface proteins. Soluble fragments of TACE were not detected in the medium of cells that had down-regulated TACE, and TACE was not down-regulated when endocytosis was inhibited. Antibody uptake experiments suggested that cell-surface TACE was internalized in response to PMA. Surprisingly, a metalloprotease inhibitor prevented the PMA-induced turnover of TACE. Thus, PMA activates shedding and causes the down-regulation of a major "sheddase," suggesting that induced shedding may be regulated by a mechanism that decreases the amount of active TACE on the cell surface.     

The molecular action of tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is a polypeptide hormone newly synthesized by different cell types upon stimulation with endotoxin, inflammatory mediators (C5a anaphylatoxin), or cytokines such as interleukin-1 and, in an autocrine manner, TNF itself. The net biological effect of TNF-alpha may vary depending on relative concentration, duration of cell exposure and presence of other mediators which may act in synergism with this cytokine. TNF-alpha may be relevant either in pathological events occurring in cachexia and endotoxic shock and inflammation or in beneficial processes such as host defense, immunity and tissue homeostasis. The biological effects of TNF-alpha are triggered by the binding to specific cell surface receptors. The formation of TNF-alpha-receptor complex activates a variety of biochemical pathways that include the transduction of the signal at least in part controlled by guanine-nucleotide-binding regulatory proteins (G proteins), its amplification through activation of adenyl cyclase, phospholipases and protein kinases with the generation of second messenger pathways. The transduction of selected genes in different cell types determines the characteristics of the cell response to TNF-alpha. The full understanding of the molecular mechanisms of TNF-alpha will provide the basis for a pharmacological approach intended to inhibit or potentiate selected biological actions of this cytokine.     

八肽胆囊收缩素减轻肿瘤坏死因子诱导的离体兔肺动脉反应性变化及内皮细胞损伤

为探讨八肽胆囊收缩素（ＣＣＫ－８）缓解内毒素休克时肺动脉高压的作用机制,应用离体血管环张力测定技术及扫描电镜方法,观察了ＣＣＫ－８对肿瘤坏死因子α（ＴＮＦ－α）引起的肺动脉反应性及肺动脉内皮细胞超微结构变化的影响。结果显示：离体脑动脉经ＴＮＦ－α（４０００Ｕ／ｍｌ）孵育２ｈ对苯肾上腺素（ＰＥ）的收缩反应、对乙酰胆碱（ＡＣｈ）及硝普钠（ＳＮＰ）的内皮依赖性及非内皮依赖性舒张反应均无明显影响。ＴＮＦ－     

Reduced testicular steroidogenesis in tumor necrosis factor-alpha knockout mice

We previously demonstrated that the expression of Mullerian inhibiting substance (MIS) in Sertoli cells is downregulated by tumor necrosis factor alpha (TNF-alpha), which is secreted by meiotic germ cells, in mouse testes. Several studies have reported that MIS that is secreted by Sertoli cells inhibits steroidogenesis and, thus, the synthesis of testosterone in testicular Leydig cells. Here, we demonstrate that in TNF-alpha knockout testes, which show high levels of MIS, steroidogenesis is decreased compared to that in wild-type testes. The levels of testosterone and the mRNA levels of steroidogenesis-related genes were significantly lower after puberty in TNF-alpha knockout testes than in wild-type testes. Furthermore, the number of sperm was reduced in TNF-alpha knockout mice. Histological analysis revealed that spermatogenesis is also delayed in TNF-alpha knockout testes. In conclusion, TNF-alpha knockout mice show reduced testicular steroidogenesis, which is likely due to the high level of testicular MIS compared to that seen in wild-type mice.     

Effects of tumor necrosis factor-alpha and interleukin-6 in elderly populations

Aging is associated with low-grade increases in circulating levels of TNF-alpha and IL-6. A wide range of factors, including smoking, obesity, infections, the decline in sex hormones, and the genotype, induce and modify this age-related inflammatory activity, which on the other hand may cause age-related pathology. Several classical risk factors are indeed controlled by TNF-alpha and IL-6. TNF-alpha induces insulin resistance and endothelial dysfunction, IL-6 promotes procoagulant changes and both cytokines cause dyslipidaemia. Moreover, systemic low-grade elevations in both cytokines have been related to cardiovascular diseases and TNF-alpha has been associated with Alzheimer's disease and type 2 diabetes mellitus. TNF-alpha and IL-6 are also differently and independently of each other associated with mortality in elderly populations, indicating points of distinction in the biological effects of the two cytokines. Moreover, the association between cytokines and mortality is independent of co-morbidity, suggesting that low-grade increases in circulating cytokines are strong, independent risk factors of morbidity and mortality in old populations, although life style factors and co-morbidity may modulate levels.