Tissue Tumor Necrosis Factor Research Articles

Human Interleukin 10 Gene Therapy Decreases the Severity and Mortality of Lethal Pancreatitis in Rats

Background. Studies have proven the validity of interleukin-10 (IL-10) in the treatment of experimental pancreatitis. Prophylactic human IL-10 (hIL-10) gene treatment attenuated the severity in cerulein models. Our research aims to study whether the therapeutic hIL-10 gene could decrease both severity and mortality in a lethal pancreatic model.Methods. Severe acute pancreatitis (SAP) was induced by sodium taurocholate. A plasmid–hIL-10 construct (pcDNA3–hIL-10) complexed with cationic liposomes was administered to SAP rats by a single intraperitoneal injection. Levels of hIL-10 in the pancreas, liver, and lungs were determined by ELISA kits. The severity of pancreatitis was assessed in terms of serum amylase, histology, and tissue tumor necrosis factor α (TNF-α). Mortality, observed for 7 days, was evaluated for gene therapy or control groups.Results. After hIL-10 gene therapy, hIL-10 levels in the pancreas, liver, and lungs increased significantly and the serum amylase, tissue TNF-α, and histological changes in pancreas, liver, and lungs decreased markedly. Therefore, mortality was significantly reduced in the hIL-10 gene therapy group, in which 70% of rats survived in the 7-day observation, while only 10% survived in untreated groups (P < 0.05).Conclusion. We found that liposome/hIL-10 gene therapy decreased severity and mortality in SAP, even carried out after SAP establishment, predicting a more convenient shift to clinical applications.

Lipopolysaccharide activation of the MEK-ERK1/2 pathway in human monocytic cells mediates tissue factor and tumor necrosis factor α expression by inducing Elk-1 phosphorylation and Egr-1 expression: Presented in abstract form at the 42nd annual meeting of the American Society of Hematology, December 1-5, 2000, San Francisco, CA.

Lipopolysaccharide (LPS) induces human monocytes to express many proinflammatory mediators, including the procoagulant molecule tissue factor (TF) and the cytokine tumor necrosis factor alpha (TNF-alpha). The TF and TNF-alpha genes are regulated by various transcription factors, including nuclear factor (NF)-kappaB/Rel proteins and Egr-1. In this study, the role of the MEK-ERK1/2 mitogen-activated protein kinase (MAPK) pathway in LPS induction of TF and TNF-alpha gene expression in human monocytic cells was investigated. The MAPK kinase (MEK)1 inhibitor PD98059 reduced LPS induction of TF and TNF-alpha expression in a dose-dependent manner. PD98059 did not affect LPS-induced nuclear translocation of NF-kappaB/Rel proteins and minimally affected LPS induction of kappaB-dependent transcription. In contrast, PD98059 and dominant-negative mutants of the Ras-Raf1-MEK-ERK (extacellular signal-regulated kinase) pathway strongly inhibited LPS induction of Egr-1 expression. In kinetic experiments LPS induction of Egr-1 expression preceded induction of TF expression. In addition, mutation of the Egr-1 sites in the TF and TNF-alpha promoters reduced expression of these proinflammatory genes. It was demonstrated that LPS induction of the Egr-1 promoter was mediated by 3 SRE sites, which bound an LPS-inducible complex containing serum response factor and Elk-1. LPS stimulation transiently induced phosphorylation of Elk-1 and increased the functional activity of a GAL4-Elk-1TA chimeric protein via the MEK-ERK1/2 pathway. The data indicate that LPS induction of Egr-1 gene expression is required for maximal induction of the TNF-alpha and TF genes in human monocytic cells.

Adipose tissue tumor necrosis factor and interleukin-6 expression in human obesity and insulin resistance.

Adipose tissue expresses tumor necrosis factor (TNF) and interleukin (IL)-6, which may cause obesity-related insulin resistance. We measured TNF and IL-6 expression in the adipose tissue of 50 lean and obese subjects without diabetes. Insulin sensitivity (S(I)) was determined by an intravenous glucose tolerance test with minimal-model analysis. When lean [body mass index (BMI) <25 kg/m(2)] and obese (BMI 30-40 kg/m(2)) subjects were compared, there was a 7.5-fold increase in TNF secretion (P < 0.05) from adipose tissue, and the TNF secretion was inversely related to S(I) (r = -0.42, P < 0.02). IL-6 was abundantly expressed by adipose tissue. In contrast to TNF, plasma (rather than adipose) IL-6 demonstrated the strongest relationship with obesity and insulin resistance. Plasma IL-6 was significantly higher in obese subjects and demonstrated a highly significant inverse relationship with S(I) (r = -0.71, P < 0.001). To separate the effects of BMI from S(I), subjects who were discordant for S(I) were matched for BMI, age, and gender. By use of this approach, subjects with low S(I) demonstrated a 3.0-fold increased level of TNF secretion from adipose tissue and a 2.3-fold higher plasma IL-6 level (P < 0.05) compared with matched subjects with a high S(I). Plasma IL-6 was significantly associated with plasma nonesterified fatty acid levels (r = 0.49, P < 0.002). Thus the local expression of TNF and plasma IL-6 are higher in subjects with obesity-related insulin resistance.

Regulation of interleukin 8 production and gene expression in human adipose tissue in vitro.

A variety of cytokines and other compounds are produced in the human adipose tissue and may have autocrine functions in the adipose tissue as well as be involved in the complications seen in association with obesity. Because it recently has been reported that interleukin 8 (IL-8), through its effects on the macrophage and endothelial cell, may be involved in the pathogenesis of atherosclerosis, we found it of interest to investigate whether IL-8 is produced in human adipose tissue in vitro. Human sc adipose tissue was investigated both in incubations with whole adipose tissue fragments as well as with isolated mature adipocytes. In adipose tissue fragments, IL-1beta (3 nM) and tumor necrosis factor alpha (0.6 nM) were able to stimulate IL-8 production by 12-fold and 5-fold, respectively (P < 0.001), when incubated for 48 h. Incubations with isolated adipocytes were performed up to 6 h, and IL-1beta and tumor necrosis factor alpha significantly increased IL-8 production by 50-60% (P < 0.05). Dexamethasone (50 nM) decreased IL-8 production from adipose tissue fragments by 57% (P < 0.01) and from adipocytes by 37% (P < 0.05). IL-8 messenger RNA expression in adipocytes incubated with IL-1beta was increased already after 2 h (P < 0.05). Thus, the effect of proinflammatory cytokines and dexamethasone on IL-8 production in adipose tissue seems to be mediated at the transcriptional level. In conclusion, it is demonstrated for the first time that IL-8 is produced and released from human adipose tissue and from isolated adipocytes in vitro, which may indicate that IL-8 from adipose tissue could be involved in some of the obesity-related complications.

Endogenous PPARγ mediates anti-inflammatory activity in murine ischemia-reperfusion injury

Background & Aims: Peroxisome proliferator–activated receptor γ (PPARγ) is a nuclear receptor whose activation has been linked to several physiologic pathways including those related to the regulation of intestinal inflammation. We sought to determine whether PPARγ could function as an endogenous anti-inflammatory pathway in a murine model of intestinal ischemia-reperfusion (I/R) injury. Methods: PPARγ-deficient and wild-type mice were examined for their response to I/R procedure. Treatment with a PPARγ-specific ligand was also performed. Results: In a murine model of intestinal I/R injury, we observed more severe injury in PPARγ-deficient mice and protection against local and remote tissue injury in mice treated with a PPARγ-activating ligand, BRL-49653. Activation of PPARγ resulted in down-regulation of intercellular adhesion molecule 1 expression by intestinal endothelium and tissue tumor necrosis factor α messenger RNA levels most likely by inhibition of the NF-κB pathway. Conclusions: These data strongly suggest that an endogenous PPARγ pathway exists in tissues that may be amenable to therapeutic manipulation in I/R-related injuries.GASTROENTEROLOGY 2001;120:460-469

Effects of lipopolysaccharide on TNF-α production, hepatic NOS2 activity, and hepatic toxicity in rats with cirrhosis

Background/Aims: Septic shock results in high mortality in patients with cirrhosis. Nitric oxide synthase 2 (NOS2) is induced by bacterial lipopolysaccharides (LPS) and plays a major role in the inflammatory response to bacterial infections. Little is known about the regulation of NOS2 in cirrhosis under septic conditions. Thus, the aim of this study was to determine tissue NOS2 activity, serum nitrate and tumor necrosis factor (TNF-α) levels and hepatic toxicity in cirrhotic rats after LPS administration. Methods: Serum nitrates, TNF-α and transaminases were determined after LPS-administration in rats with secondary biliary cirrhosis and in sham-operated rats. Liver, lung, aortic and peritoneal macrophage NOS2 activities were determined by converting L[ 14C] arginine into L[ 14C] citrulline in a calcium free medium. Nitrate and TNF-α production were determined in a culture medium of peritoneal macrophages after in vivo LPS administration. Results: LPS (1.5 mg/kg) induced 50% mortality in cirrhotic rats and no mortality in sham-operated rats. After LPS, TNF-α, nitrate and transaminase levels were significantly higher in cirrhotic rats compared to sham-operated rats. After LPS administration, there were no differences in NOS2 activity in the aorta, lungs, or peritoneal macrophages of the two groups, whereas NOS2 activity was significantly higher in the cirrhotic liver compared to the normal liver. Conclusions: In rats with cirrhosis, LPS administration induces higher mortality, hepatic toxicity, hepatic NOS2 activation and TNF-α release than in sham-operated rats. These results confirm the harmful role of septic shock in liver disease.

Effects of alpha 1-acid glycoprotein on tissue factor expression and tumor necrosis factor secretion in human monocytes

Activated monocytes express tissue factor (TF) and secrete tumor necrosis factor α (TNFα), which are important in the initiation of blood coagulation and inflammation. We investigated the effect of α 1-acid glycoprotein (α 1-AGP), an acute phase protein, on the induction of the expression of TF and the secretion of TNFα in human monocytes in vitro. The TF activity of both fresh human monocytes and human monocytic cell line U937 significantly increased in a dose-dependent manner after a 6 h incubation with human or bovine α I-AGP. The activity of TF gradually tailed off after 24 h. RT-PCR and southern blot analysis revealed that TF mRNAα synthesis was induced in monocytes. Inhibition of α 1-AGP induced TF expression by actinomycin D (ActD) further support that de novo TF mRNA synthesis was required. The specificity of the α1-AGP-induced TF activity was demonstrated by anti-α 1-AGP antibody inhibition. TNFca secretion in α 1-AGP stimulated monocytes was also increased; this could be blocked by pentoxifylline (PTX). The possible contamination of lipopolysaccharide (LPS) in the α 1-AGP was excluded by limulus amoebocyte lysate. Therefore, these results indicate that α 1-AGP may contribute to the cellular initiation of coagulation and inflammation by increasing TF expression and TNFα secretion of monocytes.

Acetylsalicylic Acid and Sodium Salicylate Inhibit LPS-lnduced NF-kB/c-Rel Nuclear Translocation, and Synthesis of Tissue Factor (TF) and Tumor Necrosis Factor alfa (TNF-a) in Human Monocytes

We have investigated the effects of acetylsalicylic acid and sodium salicylate on the LPS-induced synthesis of the pro-coagulant protein tissue factor (TF) and the pro-inflammatory protein tumor necrosis factor-alpha (TNF-alpha), as well as the prostaglandin PGE2 in human monocytes. Both drugs dose-dependently inhibited LPS-induced TF and TNF-alpha synthesis at the mRNA and the protein level, and reduced PGE2 production. As evidenced by electro mobility shift assay (EMSA) and the use of a NF-kappa B prototypic probe, these drugs probably exert their inhibitory effects by interference with the nuclear translocation of NF-kappa B/c-Rel proteins. These data may expand the understanding of the anti-thrombotic and anti-inflammatory effects of these drugs when activation of monocytes occurs.

Tissue Tumor Necrosis Factor mRNA Expression Following Cecal Ligation and Puncture or Intraperitoneal Injection of Endotoxin

Tumor necrosis factor (TNF) has been implicated as a key mediator of the septic response. Although very high serum levels of TNF are detected in animal models of endotoxemia or gram-negative bacteremia, human patients with sepsis rarely have greatly elevated TNF serum levels. It has therefore been postulated that TNF may act in a paracrine fashion to cause local injury. In order to examine the role of locally produced TNF in sepsis, we compared serum TNF levels and TNF messenger RNA (mRNA) expression in various tissues following cecal ligation and puncture (CLP) or intraperitoneal injection of a sublethal dose of endotoxin. TNF mRNA expression was determined by the reverse transcription differential polymerase chain reaction using β-actin as an internal standard. Serum levels of TNF were threefold higher after endotoxin administration compared to CLP. TNF mRNA in peritoneal macrophages rose fourfold after both endotoxin injection and CLP, with rapid return to normal in endotoxin-treated animals. There was a significant increase in TNF mRNA in the liver and lung, but not the spleen, during the first 24 hr after CLP. An increase in TNF mRNA was seen in all three tissues after injection of endotoxin. These results support the concept of locally produced TNF as a contributing factor in tissue damage and multiple organ failure during sepsis.

Serum components enhance bacterial lipopolysaccharide-induced tissue factor expression and tumor necrosis factor-alpha secretion by bovine alveolar macrophages in vitro.

We have compared the effect of bacterial lipopolysaccharide (LPS) in combination with normal adult bovine serum (NBS), fetal bovine serum (FBS), or a bovine serum fraction on tissue factor expression and tumor necrosis factor alpha (TNF-alpha) secretion by bovine alveolar macrophages. At a concentration of 1 ng/ml, bacterial LPS alone failed to induce measurable tissue factor expression by the macrophages, but the presence of FBS, NBS, or a fraction of normal pooled bovine serum isolated by ion-exchange chromatography (fraction 2) markedly potentiated the effect of LPS. A protein concentration of 64 micrograms/ml NBS, 192 micrograms/ml FBS, and only 640 ng/ml fraction 2 was required to induce maximal tissue factor expression on the macrophages in combination with 1 ng/ml LPS. Comparison of quantities of added serum protein required to induce maximal potentiating effects indicated that fraction 2 was 100 times more potent than whole NBS and 300 times more potent than whole FBS. We similarly found that TNF-alpha secretion by macrophages exposed to LPS was responsive to serum and was highly responsive to fraction 2. LPS alone (1 ng/ml) induced a relatively low level of TNF-alpha secretion by the macrophages, and the presence of FBS, NBS, or fraction 2 potentiated the effect of LPS. A concentration of 64.0 micrograms/ml NBS, 320.0 micrograms/ml FBS, and 3.2 micrograms/ml fraction 2 serum protein induced near-maximal TNF-alpha secretion by the macrophages. Comparison of the concentration of serum protein required to induce these potentiating effects indicated that fraction 2 was approximately 20 times more potent than whole NBS and 100 times more potent than whole FBS. The stimulatory effect of LPS plus fraction 2 serum proteins was dependent on the CD14 receptor, as monoclonal antibodies directed against CD14 (My4, 60bd; 10 micrograms/ml) inhibited tissue factor expression and TNF-alpha secretion by the macrophages.

Lipoproteins do not modulate the tissue factor activity, plasminogen activator or tumour necrosis factor production induced by lipopolysaccharide stimulation of human monocytes.

Upon stimulation with lipopolysaccharides (LPS), monocytes are able to produce tissue factor (TF), the most powerful physiological procoagulant substance known. In several assay systems LPS bound to lipoprotein has been reported to be less active than unbound LPS in stimulating monocytes. In the present study the LPS-induced TF activity was, however, not prevented by lipoproteins (VLDL, LDL, HDL). In fact, the very low density (VLDL) fraction further increased the TF inducing capacity of LPS. The lipoproteins per se mediated reduced plasminogen activator (PA) production in monocytes. LPS had an even more and significant depressing effect on PA production, which was not further decreased in the presence of lipoproteins. Furthermore, LPS-induced release of tumour necrosis factor (TNF), a marker of monocyte activity, was not inhibited by lipoproteins. Our experiments suggest that lipoproteins do not render LPS less effective in stimulating TNF release, procoagulant and fibrinolytic activities in human monocytes.

Increased lipopolysaccharide-induced tissue factor activity and tumour necrosis factor production in monocytes after intake of aspirin: possible role of prostaglandin E2.

To determine how aspirin intake might influence lipopolysaccharide (LPS)-induced tissue factor (TF) activity and tumour necrosis factor (TNF) in human blood monocytes, we collected blood before and at various times after intake of 300 mg aspirin in 25 healthy volunteers. Aspirin intake reduced LPS-induced thromboxane B2 and PGE2 production in whole blood by 50% and 65% respectively, measured 1 h after aspirin intake. Subsequently, a 95% rise in LPS-induced TF activity in monocytes was seen as compared to a 26% rise in TNF. The rise in TF activity was maximal within 1 h after aspirin intake and no further rise was observed 3, 4 or 24 h after aspirin intake. In contrast, TF activity induced by incubating whole blood in the absence of LPS fell rapidly after the intake of aspirin. In separate experiments, a dose-dependent inhibition by PGE2 was observed in LPS-induced TF activity in monocytes. It is proposed that the increased LPS-induced TF activity and TNF production following aspirin intake may be due to suppressed PGE2 formation. The more pronounced rise in TF activity compared to TNF production may be due to an enhancement of the platelet lipoxygenase pathway that has been shown to be important for LPS-induced TF activity in monocytes.