Captopril-impaired production of tumor necrosis factor-alpha-induced interleukin-1beta in human monocytes is associated with altered intracellular distribution of nuclear factor-kappaB

We evaluated the effects of the angiotensin-converting enzyme (ACE) inhibitor captopril on pathways for monocyte production of interleukin (IL)-1beta in vitro. Human monocytes were treated with captopril and stimulated with tumor necrosis factor (TNF)-alpha or lipopolysaccharide. Captopril caused a dose-dependent reduction of TNF-alpha induced IL-1beta. LPS-induced IL-1beta generation was not reduced by the ACE inhibitor. Pro-IL-1beta concentrations followed the same pattern as that for mature IL-1beta when monocytes were preincubated with captopril. Also, IL-1beta mRNA concentrations were reduced by captopril pretreatment in parallel with IL-1beta. We sought to determine whether captopril affected the nuclear factor (NF)-kappaB complex in monocytic cells. We found that the translocation of the p-65 component of NF-kappaB to the nucleus was inhibited by captopril. Thus captopril reduced TNF-alpha-induced IL-1beta and IL-1betamRNA synthesis in monocytes, in vitro, probably through interference with NF-kappaB activation of the IL-1beta gene. These results support the hypothesis that captopril has immunomodulating properties.     

Regulation of reactive oxygen species (ROS) production by C18 fatty acids in Jurkat and Raji cells

In the present study, the effects of C18 fatty acids with different numbers of double bonds, SA (stearic acid; C18:0), OA (oleic acid; C18:1), LA (linoleic acid; C18:2) and gamma-LNA (gamma-linolenic acid; C18:3), on ROS (reactive oxygen species) production by Jurkat (a human T-lymphocyte-derived cell line) and Raji (a human B-lymphocyte-derived cell line) cells were investigated. ROS production was determined by NBT (Nitro Blue Tetrazolium) reduction (intracellular and extracellular ROS production) and by dihydroethidium oxidation using flow cytometry (intracellular ROS production). The effectiveness on ROS production was gamma-LNA相似文献   

Ox-LDL-induced LOX-1 expression in vascular smooth muscle cells: role of reactive oxygen species

Oxidized low density lipoprotein (ox-LDL) and lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) have been implicated in the development of atherosclerosis. This study was designed to investigate the expression regulation of LOX-1 by ox-LDL and the potential underlying mechanisms in cultured rat vascular smooth muscle cells (VSMCs). VSMCs were treated with ox-LDL, and the expressions of LOX-1 mRNA and proteins were determined by RT-PCR and western blotting, respectively. The intracellular reactive oxygen species (ROS) production was monitored by flow cytometry with fluorescence probe, DCFH(2) -DA. The effect of several inhibitors including aspirin, NDGA, allopurinol, apocynin, and rotenone on ox-LDL-induced ROS formation and LOX-1 expression was also investigated. The roles of NF-κB p65 and JNK were explored. Ox-LDL significantly induced LOX-1 expression at both mRNA and protein levels in a dose-dependent and time-dependent manner. Aspirin, NDGA, and preconditioned apocynin suppressed ox-LDL-induced intracellular ROS production and LOX-1 expression, while allopurinol and rotenone failed to do so. Vitamin C and N-acetyl-l-cysteine demonstrated similar effect. Furthermore, both NF-κB p65 expression and phosphorylated JNK (p-JNK) to JNK expression ratio were elevated after ox-LDL treatment. In addition, the NF-κB inhibitor PDTC and JNK inhibitor SP600125 pretreatment partly abolished ox-LDL-induced LOX-1 expression. These findings suggested that ROS mediated ox-LDL-induced LOX-1 expression in VSMCs through NF-κB and JNK signaling pathways.     

Dual regulation of tumor necrosis factor-alpha-induced CCL2/monocyte chemoattractant protein-1 expression in vascular smooth muscle cells by nuclear factor-kappaB and activator protein-1: modulation by type III phosphodiesterase inhibition

Monocyte/macrophage infiltration to the subendothelial space of arterial wall is a critical initial step in atherogenesis, in which CC chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1 (MCP-1) is thought to play a key role. This study investigated the effectiveness of phosphodiesterase inhibitors, including the nonselective pentoxifylline (PTX) and the selective type III (cilostamide) and type IV (denbufylline) inhibitors, on cytokine-induced CCL2/MCP-1 production in cultured rat vascular smooth muscle cells (VSMCs), and the signal transduction mechanisms whereby they act. Our results showed that tumor necrosis factor (TNF)-alpha induced a marked increase in CCL2/MCP-1 production in dose- and time-dependent manners. 2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene (U0126) [both inhibitors of p42/44 mitogen-activated protein kinase (MAPK) kinase], and anthra[1hyphen]9-cd]pyrazol-6(2H)-one (SP600125) [an inhibitor of c-Jun NH(2)-terminal kinases (JNKs)] attenuated TNF-alpha-induced CCL2/MCP-1 production, without affecting I-kappaBalpha degradation or p65/nuclear factor-kappaB (NF-kappaB) nuclear translocation. PD98059 abolished TNF-alpha-activated p42/44 MAPK phosphorylation and c-Fos up-regulation, whereas SP600125 inhibited TNF-alpha-activated JNK and c-Jun phosphorylation. The NF-kappaB inhibitor carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132) attenuated TNF-alpha-induced CCL2/MCP-1 production in the presence of increased phospho-JNK and phospho-c-Jun levels. When SP600125 was added simultaneously, MG132 completely inhibited TNF-alpha-induced CCL2/MCP-1 production. Finally, the pretreatment of VSMCs with PTX or cilostamide, but not denbufylline, reduced TNF-alpha-induced CCL2/MCP-1 production, which was preceded by attenuation of p65/NF-kappaB nuclear translocation, p42/44 MAPK, and JNK-c-Jun phosphorylation, and c-Fos up-regulation. These data indicate that TNF-alpha-stimulated CCL2/MCP-1 production in rat VSMCs is dually regulated by activator protein-1 (AP-1) and NF-kappaB pathways, and inhibition of type III phosphodiesterase contributes substantially to the suppressive effect of PTX on CCL2/MCP-1 production via down-regulation of AP-1 and NF-kappaB signals.     

细胞内活性氧在糖基化终末产物促人腹膜间皮细胞分泌单核细胞趋化因子1中的作用

目的观察糖基化终末产物(advanced glycation end products,AGEs)对人腹膜间皮细胞(humanperitoneal mesothelial cell,HPMC)分泌单核细胞趋化蛋白1(monocyte chemoattractant protein-1,MCP-1)的作用及细胞内活性氧族(reactive oxygen species,ROS)在其中的作用。方法分别用不同浓度的糖基化人血清白蛋白(advanced glycation end product s-human serum albumin,AGE HSA)及抗氧化剂N乙酰-L半胱氨酸(N acetyl-L cysteine,NAC)作用于细胞,用逆转录多聚酶链反应(RT PCR)法和酶联免疫吸附法(E L I S A)测定HPMC中MC P 1的表达;再以氧化敏感的荧光染料2,7-二氢二氯荧光素(2,7-dichlorofluorescin,DCFH)染色,流式细胞仪测定ROS强度。结果 AGE HSA能使细胞内ROS水平明显升高,呈现浓度依赖效应;AGE HSA同时以时效和量效方式促进HPMC中MCP-1的表达;而NAC能够明显抑制AGE-HSA所导致的细胞内ROS升高,同时抑制HPMC中MCP-1的分泌。结论 AGE HSA可能部分通过诱导细胞内ROS,促进HPMC表达MCP-1。     

Tumor necrosis factor-alpha-induced apoptosis of human coronary artery endothelial cells: modulation by the peroxisome proliferator-activated receptor-gamma ligand pioglitazone

The cytokine tumor necrosis factor-alpha (TNF-alpha) plays an important role in endothelial injury, which is associated with the release of reactive oxygen species and the induction of apoptosis. We report on our study of TNF-alpha-induced apoptosis in human coronary artery endothelial cells and its modulation by the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligand pioglitazone. Treatment of cells with TNF-alpha (40 ng/mL) resulted in apoptosis as measured by DNA laddering and caspase-3 activation. TNF-alpha treatment decreased the expression of antiapoptotic protein Bcl-2 (P <.05 vs control), but not the expression of Fas or FLIP, in human coronary artery endothelial cells. Treatment of cells with TNF-alpha also enhanced lipid peroxidation (P <.01 vs control). Pretreatment of cells with the PPAR-gamma ligand pioglitazone blocked TNF-alpha-mediated apoptosis, caspase-3 activation, expression of Bcl-2, and lipid peroxidation (P <.01 vs TNF-alpha alone). These results indicate that TNF-alpha induces oxidative stress in human coronary artery endothelial cells, resulting in apoptosis through a reduction in Bcl-2 expression and the subsequent activation of caspase-3. The PPAR-gamma ligand pioglitazone modulates lipid peroxidation, alters Bcl-2 expression and caspase-3 activation, and finally reduces apoptosis. The antioxidant and antiapoptotic effects of pioglitazone may be the mechanism by which this agent reduces endothelial injury.     

4-hydroxynonenal decreases interleukin-6 expression and protein production in primary rat Kupffer cells by inhibiting nuclear factor-kappaB activation

Kupffer cells have been documented to play an important role in the early events of liver injury and regeneration by releasing biologically active mediators such as interleukin-6 (IL-6). 4-Hydroxy-trans-2-nonenal (4-HNE), a major end product of lipid peroxidation, has multiple cytotoxic effects and is implicated in chemical-induced liver injury. Consequently, the purpose of this study was to evaluate the ability of 4-HNE to modulate IL-6 production in isolated primary rat Kupffer cells. 4-HNE (0.1-10 microM) reduced both lipopolysaccharide (LPS)-induced IL-6 protein production and mRNA levels. The role of nuclear factor-kappaB (NF-kappaB) in IL-6 induction was elucidated using Kupffer cells transduced in vitro with a recombinant adenovirus containing a IkappaBalpha super-repressor resistant to phosphorylation and degradation (Ad5IkappaB). Using this system, LPS-induced IL-6 protein production was inhibited by 65% in Ad5IkappaB-infected cells. The treatment of Kupffer cells for 1 h with 4-HNE followed by stimulation for 1 h with LPS (500 ng/ml) resulted in a concentration-dependent decrease in NF-kappaB activation. Similarly, decreased NF-kappaB activity in these cells paralleled a reduction in IkappaBalpha mRNA levels. Furthermore, upon LPS stimulation, 4-HNE stabilized IkappaBalpha, which corresponded to a decrease in phosphorylated IkappaBalpha. At lower 4-HNE concentrations (0-5 microM), interactions between p65 and IkappaBalpha proteins were maintained as detected by immunoprecipitation-immunoblot analyses. In conclusion, these data suggest that 4-HNE inhibits IL-6 production in rat Kupffer cells by preventing activation of the NF-kappaB pathway and suppressing IkappaBalpha phosphorylation. These results have functional implications in that 4-HNE may interfere with the ability of Kupffer cells to produce cytokines proposed to play an important role in liver regeneration.     

Hypoxia-induced endocytosis of Na,K-ATPase in alveolar epithelial cells is mediated by mitochondrial reactive oxygen species and PKC-zeta

During ascent to high altitude and pulmonary edema, the alveolar epithelial cells (AEC) are exposed to hypoxic conditions. Hypoxia inhibits alveolar fluid reabsorption and decreases Na,K-ATPase activity in AEC. We report here that exposure of AEC to hypoxia induced a time-dependent decrease of Na,K-ATPase activity and a parallel decrease in the number of Na,K-ATPase alpha(1) subunits at the basolateral membrane (BLM), without changing its total cell protein abundance. These effects were reversible upon reoxygenation and specific, because the plasma membrane protein GLUT1 did not decrease in response to hypoxia. Hypoxia caused an increase in mitochondrial reactive oxygen species (ROS) levels that was inhibited by antioxidants. Antioxidants prevented the hypoxia-mediated decrease in Na,K-ATPase activity and protein abundance at the BLM. Hypoxia-treated AEC deficient in mitochondrial DNA (rho(0) cells) did not have increased levels of ROS, nor was the Na,K-ATPase activity inhibited. Na,K-ATPase alpha(1) subunit was phosphorylated by PKC in hypoxia-treated AEC. In AEC treated with a PKC-zeta antagonist peptide or with the Na,K-ATPase alpha(1) subunit lacking the PKC phosphorylation site (Ser-18), hypoxia failed to decrease Na,K-ATPase abundance and function. Accordingly, we provide evidence that hypoxia decreases Na,K-ATPase activity in AEC by triggering its endocytosis through mitochondrial ROS and PKC-zeta-mediated phosphorylation of the Na,K-ATPase alpha(1) subunit.     

Bay w 9798, a dihydropyridine structurally related to nifedipine with no calcium channel-blocking properties, inhibits tumour necrosis factor-alpha-induced vascular cell adhesion molecule-1 expression in endothelial cells by suppressing reactive oxygen species generation

Dihydropyridine-based calcium antagonists (DHPs) are widely used to treat hypertension. We have previously shown that nifedipine, one of the most popular DHPs, blocks tumour necrosis factor-alpha (TNF-alpha)-induced monocyte chemoattractant protein-1 as well as vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells by suppressing reactive oxygen species generation (ROS). The molecular mechanism is still to be elucidated, however, because endothelial cells do not possess voltage-operated L-type calcium channels. The aim of this study was to determine in TNF-alpha-exposed human umbilical vein endothelial cells (HUVECs) whether and how Bay w 9798, a dihydropyridine structurally related to nifedipine with no calcium antagonistic properties, may suppress VCAM-1 expression, a key molecule which mediates the adhesion of monocytes to vasculature in the early stages of atherosclerosis. In HUVECs, 10 ng/ml TNF-alpha for 4 h stimulated ROS generation and subsequently upregulated VCAM-1 mRNA levels, both of which were dose-dependently blocked by Bay w 9798. The results demonstrated that Bay w 9798 inhibited VCAM-1 expression in TNF-alpha-exposed cells by suppressing ROS generation. They suggest that the anti-inflammatory and anti-oxidative properties of nifedipine and Bay w 9798 may be ascribed to the dihydropyridine structure, which is common to both molecules and has no calcium antagonistic ability.     

Complement membrane attack complex stimulates production of reactive oxygen metabolites by cultured rat mesangial cells.

To explore possible mechanisms by which complement membrane attack complexes (MAC) that are deposited in the glomerular mesangium might be pathogenic, we stimulated rat glomerular mesangial cells grown in vitro with nascent MACs formed from the purified human complement components C5b6 and normal human serum and measured production of superoxide ion (O2-) and hydrogen peroxide (H2O2). Mesangial cells incubated with C5b6 + serum, which results in cell membrane interaction with the MAC, produce 0.9 +/- 0.15 nmol O2-/10(5) cells per 30 min, which was significantly greater than the amount produced by cells incubated with C5b6 alone, serum alone, or decayed MACs that can no longer interact with the cell membrane (0.3 +/- 0.2, 0.4 +/- 0.1, 0.3 +/- 0.2 nmol O2-/10(5) cells per 30 min, respectively; P less than 0.02). Production of O2- after stimulation with MACs increased during the first 20 min of incubation but then plateaued. Cells exposed to decayed MACs produced small amounts of O2-, which did not increase from 20 to 60 min. Production of H2O2 was also observed after stimulation with MACs, and continued to increase during 60 min of incubation (1.22 +/- 0.16 nmol H2O2/10(5) cells per 60 min), whereas H2O2 production could not be detected after exposure to decayed MACs. Cell viability was not adversely affected by exposure to nascent MACs as determined by trypan blue exclusion or chromium-51 release. These results demonstrate that glomerular mesangial cell membrane interaction with the MAC stimulates the production of the toxic oxygen metabolites O- and H2O2. Activation of the terminal complement pathway by mesangial immune deposits in vivo might lead to tissue injury by stimulation of local production of toxic oxygen-free radicals.     

Enhancement of interleukin-8-induced chemotactic response and reactive oxygen species production in HL-60 cells expressing CXCR1

Neutrophils play a pivotal role in immunity against infection by ingesting and killing invading microbes. Neutrophils isolated from human peripheral blood have been used for a number of studies conducted for evaluation of immunomodulating drugs, cytokines, and microbe products. Human promyelocytic leukemia cells, HL-60, have been extensively studied because they can differentiate into neutrophil-like cells by addition of all-trans retinoic acid or dimethyl sulfoxide. For a system that would always allow experimental use of granulocytic cells in a uniformly activated state, we have established HL-60 cell lines with increased migratory activity by transducing the CXC chemokine receptor 1 (CXCR1) gene. When these cell lines were primed with CXC chemokine ligand 8 (IL-8), a slight increase in reactive oxygen species production induced by phorbol myristate acetate (PMA) or zymosan A stimuli was observed. A significance increase in migratory activity was noticed when the HL-60 cells transduced CXCR1 were stimulated with IL-8 in the Boyden chamber method. The gene-transduced HL-60 cell lines may be used as a substitute for neutrophils in screening the effects of various immunomodulating drugs on the migratory activity induced by IL-8.     

Mitochondrial reactive oxygen species promote production of proinflammatory cytokines and are elevated in TNFR1-associated periodic syndrome (TRAPS)

Reactive oxygen species (ROS) have an established role in inflammation and host defense, as they kill intracellular bacteria and have been shown to activate the NLRP3 inflammasome. Here, we find that ROS generated by mitochondrial respiration are important for normal lipopolysaccharide (LPS)-driven production of several proinflammatory cytokines and for the enhanced responsiveness to LPS seen in cells from patients with tumor necrosis factor receptor-associated periodic syndrome (TRAPS), an autoinflammatory disorder caused by missense mutations in the type 1 TNF receptor (TNFR1). We find elevated baseline ROS in both mouse embryonic fibroblasts and human immune cells harboring TRAPS-associated TNFR1 mutations. A variety of antioxidants dampen LPS-induced MAPK phosphorylation and inflammatory cytokine production. However, gp91(phox) and p22(phox) reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits are dispensable for inflammatory cytokine production, indicating that NADPH oxidases are not the source of proinflammatory ROS. TNFR1 mutant cells exhibit altered mitochondrial function with enhanced oxidative capacity and mitochondrial ROS generation, and pharmacological blockade of mitochondrial ROS efficiently reduces inflammatory cytokine production after LPS stimulation in cells from TRAPS patients and healthy controls. These findings suggest that mitochondrial ROS may be a novel therapeutic target for TRAPS and other inflammatory diseases.     

Vascular dysfunction of venous bypass conduits is mediated by reactive oxygen species in diabetes: role of endothelin-1

Diabetes is associated with increased risk for complications following coronary bypass grafting (CABG) surgery. Augmented superoxide (*O2*) production plays an important role in diabetic complications by causing vascular dysfunction. The potent vasoconstrictor endothelin-1 (ET-1) is also elevated in diabetes and following CABG; however, the effect of ET-1 on *O2* generation and/or vascular dysfunction in bypass conduits remain unknown. Accordingly, this study investigated basal and ET-1-stimulated *O2* production in bypass conduits and determined the effect of *O2* on conduit reactivity. Saphenous vein specimens were obtained from nondiabetic (n = 24) and diabetic (n = 24) patients undergoing CABG. Dihydroethidium staining and NAD(P)H oxidase activity assays (5380 +/- 940 versus 16,362 +/- 2550 relative light units/microg) demonstrated increased basal *O2* levels in the diabetes group (p < 0.05). Plasma ET-1 levels were associated with elevated basal *O2* levels, and treatment of conduits with exogenous ET-1 further increased *O2* production and augmented vasoconstriction. Furthermore, vascular relaxation was impaired in the diabetic group (75 versus 40%), which was restored by *O2* scavenger superoxide dismutase. These findings suggest that ET-1 causes bypass conduits dysfunction via stimulation of *O2* production in diabetes. Novel therapies that attenuate *O2* generation in bypass conduits may improve acute and late outcome of CABG in diabetic patients.     

Dependence of reactive oxygen species and FLICE inhibitory protein on lipofectamine-induced apoptosis in human lung epithelial cells

Cationic liposomes such as lipofectamine (LF) are widely used as nonviral gene delivery vectors; however, their clinical application is limited by their cytotoxicity. These agents have been shown to induce apoptosis as the primary mode of cell death, but their mechanism of action is not well understood. The present study investigated the mechanism of LF-induced apoptosis and examined the role of reactive oxygen species (ROS) in this process. We found that LF induced apoptosis of human epithelial H460 cells through a mechanism that involves caspase activation and ROS generation. Inhibition of caspase activity by pan-caspase inhibitor (z-VAD-fmk) or by specific caspase-8 inhibitor (z-IETD-fmk) or caspase-9 inhibitor (z-LEHD-fmk) inhibited the apoptotic effect of LF. Overexpression of FLICE-inhibitory protein (FLIP) or B-cell lymphoma-2, which are known inhibitors of the extrinsic and intrinsic death pathways, respectively, similarly inhibited apoptosis by LF. Induction of apoptosis by LF was shown to require ROS generation because its inhibition by ROS scavengers or by ectopic expression of antioxidant enzyme superoxide dismutase and glutathione peroxidase strongly inhibited the apoptotic effect of LF. Electron spin resonance studies showed that LF induced multiple ROS; however, superoxide was found to be the primary ROS responsible for LF-induced apoptosis. The mechanism by which ROS mediate the apoptotic effect of LF involves down-regulation of FLIP through the ubiquitination pathway. In demonstrating the role of FLIP and ROS in LF death signaling, we document a novel mechanism of apoptosis regulation that may be exploited to decrease cytotoxicity and increase gene transfection efficiency of cationic liposomes.     

Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin 1

Recombinant human tumor necrosis factor (rTNF alpha) injected intravenously into rabbits produces a rapid-onset, monophasic fever indistinguishable from the fever produced by rIL-1. On a weight basis (1 microgram/kg) rTNF alpha and rIL-1 produce the same amount of fever and induce comparable levels of PGE2 in rabbit hypothalamic cells in vitro; like IL-1, TNF fever is blocked by drugs that inhibit cyclooxygenase. At higher doses (10 micrograms/kg) rTNF alpha produces biphasic fevers. The first fever reaches peak elevation 45-55 min after bolus injection and likely represents a direct action on the thermoregulatory center. During the second fever peak (3 h later), a circulating endogenous pyrogen can be shown present using passive transfer of plasma into fresh rabbits. This likely represents the in vivo induction of IL-1. In vitro, rTNF alpha induces the release of IL-1 activity from human mononuclear cells with maximal production observed at 50-100 ng/ml of rTNF alpha. In addition, rTNF alpha and rIFN-gamma have a synergistic effect on IL-1 production. The biological activity of rTNF alpha could be distinguished from IL-1 in three ways: the monophasic pyrogenic activity of rIL-1 was destroyed at 70 degrees C, whereas rTNF alpha remained active; anti-IL-1 neutralized IL-1 but did recognize rTNF alpha or natural cachectin nor neutralize its cytotoxic effect; and unlike IL-1, rTNF alpha was not active in the mitogen-stimulated T cell proliferation assay. The possibility that endotoxin was responsible for rTNF alpha fever and/or the induction of IL-1 was ruled-out in several studies: rTNF alpha produced fever in the endotoxin-resistant C3H/HeJ mice; the IL-1-inducing property of rTNF alpha was destroyed either by heat (70 degrees C) or trypsinization, and was unaffected by polymyxin B; pyrogenic tolerance to daily injections of rTNF alpha did not occur; levels of endotoxin, as determined in the Limulus amebocyte lysate, were below the minimum rabbit pyrogen dose; and these levels of endotoxin were confirmed by gas chromatography/mass spectrometry analysis for the presence of beta-hydroxymyristic acid. Although rTNF alpha is not active in T cell proliferation assays, it may mimic IL-1 in a T cell assay, since high concentrations of rTNF alpha induced IL-1 from epithelial or macrophagic cells in the thymocyte preparations. These studies show that TNF (cachectin) is another endogenous pyrogen which, like IL-1 and IFN-alpha, directly stimulate hypothalamic PGE2 synthesis. In addition, rTNF alpha is an endogenous inducer of IL-1.(ABSTRACT TRUNCATED AT 400 WORDS)