Cytokine-stimulated secretion of group II phospholipase A2 by rat mesangial cells. Its contribution to arachidonic acid release and prostaglandin synthesis by cultured rat glomerular cells.

Potent pro-inflammatory cytokines, such as interleukin 1 (IL-1) or tumor necrosis factor (TNF) alpha have been found to increase group II phospholipase A2 (PLA2) synthesis and secretion by mesangial cells. In all cases 85-90% of the enzyme is secreted from the cells and a parallel increase in prostaglandin (PG)E2 synthesis is observed. We report here that co-incubation with a monoclonal antibody that specifically binds and neutralizes rat group II PLA2 attenuates IL-1 beta and TNF alpha-stimulated PGE2 production by 45% and 52%, respectively. CGP43182, a specific inhibitor of group II PLA2, potently blocks mesangial cell group II PLA2 in vitro with a half-maximal inhibitory concentration (IC50) of 1.5 microM, while only slightly affecting mesangial cell high molecular weight PLA2. CGP 43182 markedly attenuates IL-1 beta- and TNF alpha-stimulated PGE2 synthesis in intact mesangial cells with IC50's of 1.3 and 1.0 microM, respectively. PLA2 secreted from cytokine-stimulated mesangial cells was purified to homogeneity. Addition of the purified enzyme to unstimulated mesangial cells causes a marked release of arachidonic acid and a subsequent increased synthesis of PGE2. Moreover, addition of purified PLA2 to a cloned rat glomerular epithelial cell line and cultured bovine glomerular endothelial cells augmented both arachidonic acid release and PGE2 synthesis, with the endothelial cells being especially sensitive. Thus, cytokine-triggered synthesis and secretion of group II PLA2 by mesangial cells contributes, at least in part, to the observed synthesis of PGE2 that occurs in parallel to the enzyme secretion. Furthermore, extracellular PLA2 secreted by mesangial cells is able to stimulate arachidonic acid release and PGE2 synthesis by the adjacent endothelial and epithelial cells. These data suggest that expression and secretion of group II PLA2 triggered by pro-inflammatory cytokines may crucially participate in the pathogenesis of inflammatory processes within the glomerulus.     

Inhibition of NF-kappaB by IkappaB prevents cytokine-induced NO production and promotes enterocyte apoptosis in vitro

Nuclear factor-kappaB (N-kappaB) plays a key role in gut inflammation. NF-kappaB up-regulates proinflammatory genes encoding cytokines, adhesion molecules, and inducible nitric oxide synthase (iNOS). However, NF-kappaB has also been shown to up-regulate protective or anti-apoptotic factors. We utilized an adenoviral vector carrying a super-repressor form of the inhibitor of NF-kappaB, IkappaB, to examine the effects of NF-kappaB inhibition on cytokine-induced nitric oxide production and apoptosis in rat small intestinal epithelial cells (IEC-6). Chemical inhibitors of NF-kappaB, including pyrrolidine dithiocarbamate (PDTC), tosyl-lysine-chloromethylketone (TLCK), genistein, and N-acetyl-leu-leu-norleucinal (n-LLnL) were also utilized. Treatment of AdIkappaB-transfected cells with cytomix [1000 U/mL IFN-gamma, 1 nM IL-1beta, and 10 ng/mL tumor necrosis factor alpha (TNFalpha)] or TNFalpha-containing cytokine combinations resulted in inhibition of cytokine-induced nitrite production and a marked increase in apoptosis compared to control cells. Apoptosis occurred independently of nitric oxide (NO) production since exogenous sources of NO did not inhibit apoptosis. Inducible NOS and clAP were down-regulated in AdIkappaB-transfected cells treated with cytomix. TLCK and LLnL treatment also induced apoptosis in cytomix-treated cells, while PDTC and genistein did not. Thus, although NF-kappaB up-regulates various pro-inflammatory genes, it may also have protective or anti-apoptotic effects in enterocytes. NF-kappaB appears necessary for upregulating cIAP in IEC-6 cells upon cytokine exposure.     

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.     

L-Alanine induces changes in metabolic and signal transduction gene expression in a clonal rat pancreatic beta-cell line and protects from pro-inflammatory cytokine-induced apoptosis

Acute effects of nutrient stimuli on pancreatic beta-cell function are widely reported; however, the chronic effects of insulinotropic amino acids, such as L-alanine, on pancreatic beta-cell function and integrity are unknown. In the present study, the effects of prolonged exposure (24 h) to the amino acid L-alanine on insulin secretory function, gene expression and pro-inflammatory cytokine-induced apoptosis were studied using clonal BRIN-BD11 cells. Expression profiling of BRIN-BD11 cells chronically exposed to L-alanine was performed using oligonucleotide microarray analysis. The effect of alanine, the iNOS (inducible nitric oxide synthase) inhibitor NMA (N(G)-methyl-L-arginine acetate) or the iNOS and NADPH oxidase inhibitor DPI (diphenylene iodonium) on apoptosis induced by a pro-inflammatory cytokine mix [IL-1beta (interleukin-1beta), TNF-alpha (tumour necrosis factor-alpha) and IFN-gamma (interferon-gamma)] was additionally assessed by flow cytometry. Culture for 24 h with 10 mM L-alanine resulted in desensitization to the subsequent acute insulin stimulatory effects of L-alanine. This was accompanied by substantial changes in gene expression of BRIN-BD11 cells. Sixty-six genes were up-regulated >1.8-fold, including many involved in cellular signalling, metabolism, gene regulation, protein synthesis, apoptosis and the cellular stress response. Subsequent functional experiments confirmed that L-alanine provided protection of BRIN-BD11 cells from pro-inflammatory cytokine-induced apoptosis. Protection from apoptosis was mimicked by NMA or DPI suggesting L-alanine enhances intracellular antioxidant generation. These observations indicate important long-term effects of L-alanine in regulating gene expression, secretory function and the integrity of insulin-secreting cells. Specific amino acids may therefore play a key role in beta-cell function in vivo.     

Neuroprotective effects of glyceryl nonivamide against microglia-like cells and 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y human dopaminergic neuroblastoma cells

Glyceryl nonivamide (GLNVA), a vanilloid receptor (VR) agonist, has been reported to have calcitonin gene-related peptide-associated vasodilatation and to prevent subarachnoid hemorrhage-induced cerebral vasospasm. In this study, we investigated the neuroprotective effects of GLNVA on activated microglia-like cell mediated- and proparkinsonian neurotoxin 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in human dopaminergic neuroblastoma SH-SY5Y cells. In coculture conditions, we used lipopolysaccharide (LPS)-stimulated BV-2 cells as a model of activated microglia. LPS-induced neuronal death was significantly inhibited by diphenylene iodonium (DPI), an inhibitor of NADPH oxidase. However, capsazepine, the selective VR1 antagonist, did not block the neuroprotective effects of GLNVA. GLNVA reduced LPS-activated microglia-mediated neuronal death, but it lacked protection in DPI-pretreated cultures. GLNVA also decreased LPS activated microglia induced overexpression of neuronal nitric-oxide synthase (nNOS) and glycoprotein 91 phagocyte oxidase (gp91(phox)) on SH-SY5Y cells. Pretreatment of BV-2 cells with GLNVA diminished LPS-induced nitric oxide production, overexpression of inducible nitric-oxide synthase (iNOS), and gp91(phox) and intracellular reactive oxygen species (iROS). GLNVA also reduced cyclooxygenase (COX)-2 expression, inhibitor of nuclear factor (NF)-kappaB (IkappaB)alpha/IkappaBbeta degradation, NF-kappaB activation, and the overproduction of tumor necrosis factor-alpha, interleukin (IL)-1beta, and prostaglandin E2 in BV-2 cells. However, GLNVA augmented anti-inflammatory cytokine IL-10 production on LPS-stimulated BV-2 cells. Furthermore, in 6-OHDA-treated SH-SY5Y cells, GLNVA rescued the changes in condensed nuclear and apoptotic bodies, prevented the decrease in mitochondrial membrane potential, and reduced cells death. GLNVA also suppressed accumulation of iROS and up-regulated heme oxygenase-1 expression. 6-OHDA-induced overexpression of nNOS, iNOS, COX-2, and gp91(phox) was also reduced by GLNVA. In summary, the neuroprotective effects of GLNVA are mediated, at least in part, by decreasing the inflammation- and oxidative stress-associated factors induced by microglia and 6-OHDA.     

Sympathoadrenal modulation of stress-activated signaling in burn trauma

Burn injury stimulates stress-responsive components, p38 mitogen-activated protein kinase (MAPK)/c-Jun N-terminal kinases (JNK)/nuclear factor (NF)-kappaB. p38 MAPK plays a role in postburn cardiomyocyte tumor necrosis factor-alpha secretion and cardiac dysfunction. Since burn trauma increases circulating catecholamine levels, which in turn modulate inflammatory cytokine production, we hypothesized that increased sympathetic activity after major burn trauma may trigger postburn cardiac p38 MAPK activation via an adrenergic receptor-mediated phenomenon. We examined adrenergic receptor populations involved in burn-activated cardiac stress signaling. Sprague Dawley rats were divided into six groups: 1) control, 2) control plus alpha1-adrenergic agonist phenylephrine (2 microg/kg, intravenous), 3) control plus beta-adrenergic agonist isoproterenol (1 microg/kg, intravenous), 4) burn (fluid resuscitation with lactated Ringer's 4 ml/kg/% burn), 5) burn plus alpha1-adrenergic antagonist prazosin (1 mg/kg, by mouth), and 6) burn plus beta-adrenergic antagonist propranolol (3.3 mg/kg, by mouth). Phenylephrine, but not isoproterenol, increased cardiac p38 MAPK/JNK/NF-kappaB activation. Burn trauma activated p38 MAPK, JNK, and NF-kappaB, and this stress response was blocked by either prazosin or propranolol. Thus, stimulation of the adrenergic pathway may constitute one upstream activator of stress response in burn.     

Cytokines down-regulate alpha1-adrenergic receptor expression during endotoxemia

OBJECTIVE: The reduced pressure response to norepinephrine in septic patients has directed our interest to the regulation of alpha1-adrenergic receptors in vitro and in vivo during conditions mimicking acute sepsis. DESIGN: Prospective animal trial followed by a controlled cell culture study. SETTING: Laboratory of the Department of Anesthesiology. SUBJECTS: Male Sprague-Dawley rats weighing 200 to 250 g and a mesangial cell line. INTERVENTIONS: Experimental endotoxemia was induced in rats with lipopolysaccharide, and blood pressure dose-response studies with norepinephrine were performed. Alpha1-receptor gene expression was determined in various organs by a specific RNase protection assay, and tissue concentrations of the proinflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha were measured. Rat renal mesangial cells were incubated with these cytokines or with nitric oxide donors to investigate the regulation of alpha1-adrenergic receptors during severe inflammation on a cellular level. MEASUREMENTS AND MAIN RESULTS: The pressor effect of norepinephrine was markedly diminished during endotoxemia. The animals showed down-regulated mRNA levels of alpha1A-, alpha1B- and alpha1D-receptors in all organs investigated, and the tissue concentrations of interleukin-1beta and tumor necrosis factor-alpha were highly increased during experimental endotoxemia. Incubation of cultured rat renal mesangial cells with the cytokines resulted in diminished alpha -receptor gene expression and [3H]prazosin binding capacity, whereas incubation of the cells with nitric oxide donors did not affect alpha1B-receptor expression. In line, blocking of cytokine-induced nitric oxide synthesis by coincubation of mesangial cells with N(G)-nitro-L-arginine methyl ester did not influence cytokine-induced down-regulation of alpha1B-receptors. CONCLUSIONS: Our data show that endotoxemia causes a systemic down-regulation of alpha1-receptors on the level of gene expression and suggest that this effect is likely mediated by proinflammatory cytokines in a synergistic but nitric oxide-independent fashion. We propose that this down-regulation of alpha1-adrenergic receptors contributes to the attenuated blood pressure response to norepinephrine and, therefore, to septic circulatory failure in patients.     

Antioxidants attenuate nuclear factor-kappa B activation and tumor necrosis factor-alpha production in alcoholic hepatitis patient monocytes and rat Kupffer cells, in vitro

There is increased tumor necrosis factor-alpha (TNF) activity in alcoholic hepatitis (AH). OBJECTIVES: To examine the effects of antioxidants and glutathione enhancing agents on NF-kappaB activation and TNF production in Kupffer cells and monocytes. DESIGN AND METHODS: Isolated rat Kupffer cells and peripheral blood monocytes from AH patients were treated in vitro. NF-kappaB activation was assessed by electrophoretic mobility shift assay and TNF was measured in cell culture supernatants. RESULTS: Monocytes from AH patients had greater TNF production compared to normal volunteers. Pretreatment with antioxidants or gluathione enhancing agents inhibited TNF production and NF-kappaB activation in both monocytes from normal and AH patients as well as in rat Kupffer cells. CONCLUSIONS: There may be a therapeutic role for antioxidants or glutathione enhancing agents in disease states with increased TNF activity such as AH.     

Inhibition of NADPH oxidases prevents chronic ethanol-induced bone loss in female rats

Previous in vitro data suggest that ethanol (EtOH) activates NADPH oxidase (Nox) in osteoblasts leading to accumulation of reactive oxygen species (ROS). This might be a mechanism underlying inhibition of bone formation and increased bone resorption observed in vivo after EtOH exposure. In a rat model in which cycling females were infused intragastrically with EtOH-containing liquid diets, EtOH significantly decreased bone formation and stimulated osteoblast-dependent osteoclast differentiation. These effects were reversed by exogenous 17-β-estradiol coadministration. Moreover, coadministration of N-acetyl cysteine (NAC), an antioxidant, or diphenylene iodonium (DPI), a specific Nox inhibitor, also abolished chronic EtOH-associated bone loss. EtOH treatment up-regulated mRNA levels of Nox1, 2, 4, and the receptor activator of nuclear factor-κB ligand (RANKL), an essential factor for differentiation of osteoclasts in bone. Protein levels of Nox4, a major Nox isoform expressed in nonphagocytic cells, was also up-regulated by EtOH in bone. 17-β-Estradiol, NAC, and DPI were able to normalize EtOH-induced up-regulation of Nox and RANKL. In vitro experiments demonstrated that EtOH directly up-regulated Nox expression in osteoblasts. Pretreatment of osteoblasts with DPI eliminated EtOH-induced RANKL promoter activity. Furthermore, EtOH induced RANKL gene expression, and RANKL promoter activation in osteoblasts was ROS-dependent. These data suggest that inhibition of Nox expression and activity may be critical for prevention of chronic EtOH-induced osteoblast-dependent bone loss.     

山莨菪碱对失血性休克肝枯否细胞IκB激酶-β的影响及意义

目的 :探讨 IκB激酶 β( IKKβ)在失血性休克肝脏损伤中可能的病理机制以及山莨菪碱 ( 6 5 42 )的治疗作用。方法 :通过失血性休克和内毒素双项打击制备家兔休克模型 ,采用原位杂交方法 ( ISH)结合原位定量分析检测肝枯否细胞 ( KC)中 IKKβ的 m RNA表达 ;用凝胶电泳迁移率改变分析法 ( EMSA)和酶联免疫吸附实验 ( EL ISA)分别检测 KC中 NFκB活性和细胞培养液上清中 TNFα含量 ,并进行肝脏组织的病理学光镜检查。结果 :休克组 KC中 IKKβ的 m RNA表达 ( 0 .2 1± 0 .0 3)、NFкB活性 ( 2 .2 9± 0 .2 5 )和培养液上清中TNFα含量〔( 5 6 0 .2 1± 31.0 4) ng/ L〕均较正常对照组明显增高 ( P均 <0 .0 1)。 6 5 42能明显降低 IKKβm RNA的表达 ( 0 .14± 0 .0 3)、NFκB的活性 ( 1.35± 0 .17)以及 TNFα的含量〔( 30 0 .79± 2 3.47) ng/ L〕,P均 <0 .0 1,并能减轻肝脏组织的损伤程度。结论 :IKKβ激活在失血性休克和内毒素导致的肝脏损伤中起关键作用 ;6 5 42通过抑制 IKKβ表达 ,影响 IKKβ、NFκB和 TNFα信号转导通路 ,发挥对失血性休克继发肝脏损害的保护作用。     

Interleukin 1 alpha causes rapid activation of cytosolic phospholipase A2 by phosphorylation in rat mesangial cells.

We have shown previously that interleukin 1 (IL-1) stimulates eicosanoid production in glomerular mesangial cells (MC) by de novo synthesis of a 14-kD, group II phospholipase A2 (PLA2). IL-1-stimulated prostaglandin E2 synthesis precedes expression of this enzyme, suggesting that another PLA2 isoform must be more rapidly activated. In the presence but not absence of calcium inophore, [3H]arachidonate release is increased significantly as early as 5 min after addition of IL-1, and IL-1 concurrently stimulates a Ca(2+)-dependent phospholipase activity, which was characterized as the cytosolic form of PLA2 (cPLA2). IL-1 does not alter either cPLA2 mRNA expression or mass in serum-stimulated MC, suggesting that cPLA2 activity is increased by a posttranslational modification. IL-1 treatment for 30 min doubles 32P incorporation into immunoprecipitable cPLA2 protein, concordant with the increase in enzyme activity. Immunoblot analysis of extracts derived from IL-1-treated (30 min) cells demonstrates a decreased mobility of cPLA2, and treatment of MC lysates with acid phosphatase significantly reduces cytokine-activated cPLA2 activity, further indicating that IL-1 stimulates phosphorylation of the enzyme. IL-1 treatment (24 h) of serum-deprived MC doubled cPLA2 mRNA, protein, and activity. In summary, IL-1 increases cPLA2 activity in a biphasic, time-dependent manner both by posttranslational modification and de novo synthesis. We consider cPLA2 activation a key step in IL-1-stimulated synthesis of pro-inflammatory, lipid mediators, and an integral event in the phenotypic responses induced in target cells by this cytokine.     

Selective decontamination of the digestive tract: impact on cytokine release and mucosal damage after hemorrhagic shock

OBJECTIVE: To determine if selective decontamination of the digestive tract (SDD) influences the proinflammatory immune response of the gut after hemorrhage. DESIGN: Random assignment to either unmanipulated control after 7 days of SDD or conventional rat chow, or hemorrhagic shock group after the same time of conventional rat chow or SDD. SETTING: University animal laboratory. SUBJECTS: Male Wistar rats, weighing between 300 and 350 g. INTERVENTION: Animals of the control group were not manipulated until organ harvesting, whereas animals of the hemorrhagic shock group were bled to 30 +/- 5 mm Hg for 90 mins by withdrawal/reinfusion of shed blood and were resuscitated by Ringer's lactate equivalent to the shed blood volume. MEASUREMENTS AND MAIN RESULTS: Rats were killed after resuscitation (hemorrhagic shock group) or completed feeding (control group). Whole portal and caval blood was obtained, and splenic macrophages and gut mononuclear cells were harvested to measure supernatant tumor necrosis factor-alpha and IL-6 by bioassay. Mesenteric lymph nodes were obtained to determine bacterial translocation, and a histologic specimen was taken from the distal ileum. Feces were harvested to examine the effect of SDD. SDD eliminated Gram-negative enteric bacteria and had no influence on mucosal damage or on bacterial translocation in control animals and animals after hemorrhage. In animals receiving conventional rat chow, hemorrhagic shock led to significantly (p <.05) elevated lipopolysaccharide-stimulated proinflammatory cytokine (tumor necrosis factor-alpha and interleukin-6) release in whole portal blood, splenic macrophages, and gut mononuclear cells compared with the control group without shock. In contrast, hemorrhagic shock after SDD led to suppressed or unchanged cytokine release compared with unmanipulated animals receiving SDD. However, SDD itself induced significant (p <.05) cytokine release in these organs. Furthermore, plasma concentrations of tumor necrosis factor-alpha and interleukin-6 were significantly (p <.05) elevated in animals after hemorrhage and SDD compared with animals after hemorrhage alone. CONCLUSIONS: Hemorrhagic shock led to significant cytokine release. In contrast, cytokine release after hemorrhage and SDD was unchanged or suppressed. Furthermore, in control animals without hemorrhagic shock, SDD induced significant cytokine release. Therefore, selective decontamination of the gut, as practiced in some patients, may induce additional proinflammatory cytokine release, which can add to the proinflammatory burst in case of a complication such as hemorrhagic shock.