Effect of interleukin-1beta and tumor necrosis factor-alpha on gene expression in human endothelial cells

Interleukin-1(IL-1) and tumor necrosis factor- (TNF-) are two majorcytokines that rise to relatively high levels during systemicinflammation, and the endothelial cell (EC) response to these cytokinesmay explain some of the dysfunction that occurs. To better understandthe cytokine-induced responses of EC at the gene expression level,human umbilical vein EC were exposed to IL-1 or TNF- for varioustimes and subjected to cDNA microarray analyses to study alterations intheir mRNA expression. Of ~4,000 genes on the microarray, expressionlevels of 33 and 58 genes appeared to be affected by treatment withIL-1 and TNF-, respectively; 25 of these genes responded to bothtreatments. These results suggest that the effects of IL-1 andTNF- on EC are redundant and that it may be necessary to suppressboth cytokines simultaneously to ameliorate the systemic response.

     

IkappaBalpha-dependent regulation of low-shear flow-induced NF-kappa B activity: role of nitric oxide

We have investigated the role ofinhibitor B (IB) in the activation of nuclear factor B(NF-B) observed in human aortic endothelial cells (HAEC) undergoinga low shear stress of 2 dynes/cm². Low shear for 6 hresulted in a reduction of IB levels, an activation of NF-B,and an increase in B-dependent vascular cell adhesion molecule 1 (VCAM-1) mRNA expression and endothelial-monocyte adhesion.Overexpression of IB in HAEC attenuated all of these shear-induced responses. These results suggest that downregulation ofIB is the major factor in the low shear-induced activation ofNF-B in HAEC. We then investigated the role of nitric oxide (NO) inthe regulation of IB/NF-B. Overexpression of endothelial nitric oxide synthase (eNOS) inhibited NF-B activation in HAEC exposed to 6 h of low shear stress. Addition of the structurally unrelated NO donors S-nitrosoglutathione (300 µM) orsodium nitroprusside (1 mM) before low shear stress significantlyincreased cytoplasmic IB and concomitantly reduced NF-Bbinding activity and B-dependent VCAM-1 promoter activity. Together,these data suggest that NO may play a major role in the regulation ofIB levels in HAEC and that the application of low shear flowincreases NF-B activity by attenuating NO generation and thusIB levels.

     

Regulation of Fas (CD95)-induced apoptosis by nuclear factor-kappaB and tumor necrosis factor-alpha in macrophages

The APO-1/Fasligand (FasL) and tumor necrosis factor- (TNF-) are twofunctionally related molecules that induce apoptosis ofsusceptible cells. Although the two molecules have been reported toinduce apoptosis via distinct signaling pathways, we have shown that FasL can also upregulate the expression of TNF-, raising thepossibility that TNF- may be involved in FasL-inducedapoptosis. Because TNF- gene expression is under the controlof nuclear factor-B (NF-B), we investigated whether FasL caninduce NF-B activation and whether such activation plays a role inFasL-mediated cell death in macrophages. Gene transfection studiesusing NF-B-dependent reporter plasmid showed that FasL did activateNF-B promoter activity. Gel shift studies also revealed that FasLmobilized the p50/p65 heterodimeric form of NF-B. Inhibition ofNF-B by a specific NF-B inhibitor, caffeic acid phenylethylester, or by dominant expression of the NF-B inhibitory subunitIB caused an increase in FasL-induced apoptosis and areduction in TNF- expression. However, neutralization of TNF- byspecific anti-TNF- antibody had no effect on FasL-inducedapoptosis. These results indicate that FasL-mediated cell deathin macrophages is regulated through NF-B and is independent ofTNF- activation, suggesting the antiapoptotic role of NF-Band a separate death signaling pathway mediated by FasL.

     

p38 MAPK mediates renal tubular cell TNF-{alpha} production and TNF-{alpha}-dependent apoptosis during simulated ischemia

Ischemia causes renal tubular cellloss through apoptosis; however, the mechanisms of this processremain unclear. Using the renal tubular epithelial cell lineLLC-PK₁, we developed a model of simulated ischemia(SI) to investigate the role of p38 MAPK (mitogen-activated proteinkinase) in renal cell tumor necrosis factor- (TNF-) mRNAproduction, protein bioactivity, and apoptosis. Resultsdemonstrate that 60 min of SI induced maximal TNF- mRNA productionand bioactivity. Furthermore, 60 min of ischemia induced renaltubular cell apoptosis at all substrate replacement time pointsexamined, with peak apoptotic cell death occurring after either 24 or 48 h. p38 MAPK inhibition abolished TNF- mRNA production andTNF- bioactivity, and both p38 MAPK inhibition and TNF- neutralization (anti-porcine TNF- antibody) preventedapoptosis after 60 min of SI. These results constitute theinitial demonstration that 1) renal tubular cells produceTNF- mRNA and biologically active TNF- and undergoapoptosis in response to SI, and 2) p38 MAPKmediates renal tubular cell TNF- production and TNF--dependent apoptosis after SI.

     

Role of alpha(v)beta(3)-integrin in TNF-alpha-induced endothelial cell migration

Tumor necrosis factor- (TNF-), oneof the major inflammatory cytokines, is known to influence endothelialcell migration. In this study, we demonstrate that exposure of calfpulmonary artery endothelial cells to TNF- caused an increase in theformation of membrane protrusions and cell migration. Fluorescencemicroscopy revealed an increase in _v3focal contacts but a decrease in ₅₁ focalcontacts in TNF--treated cells. In addition, both cell-surface andtotal cellular expression of _v3-integrinsincreased significantly, whereas the expression of₅₁-integrins was unaltered. Only focalcontacts containing _v3- but not₅₁-integrins were present in membraneprotrusions of cells at the migration front. In contrast, robust focalcontacts containing ₅₁-integrins were present in cells behind the migration front. A blocking antibody to_v3, but not a blocking antibody to₅-integrins, significantly inhibited TNF--inducedcell migration. These results indicate that in response to TNF-,endothelial cells may increase the activation and ligation of_v3 while decreasing the activation andligation of ₅₁-integrins to facilitatecell migration, a process essential for vascular wound healing and angiogenesis.

     

The gamma-subunit of ENaC is more important for channel surface expression than the beta-subunit

The amiloride-sensitiveepithelial sodium channel (ENaC) plays a critical role in fluid andelectrolyte homeostasis and is composed of three homologous subunits:, , and . Only heteromultimeric channels made of ENaCare efficiently expressed at the cell surface, resulting in maximallyamiloride-sensitive currents. To study the relative importance ofvarious regions of the - and -subunits for the expression offunctional ENaC channels at the cell surface, we constructedhemagglutinin (HA)-tagged --chimeric subunits composed of -and -subunit regions and coexpressed them with HA-tagged - and-subunits in Xenopus laevis oocytes. The whole cellamiloride-sensitive sodium current (I_ami) andsurface expression of channels were assessed in parallel using thetwo-electrode voltage-clamp technique and a chemiluminescence assay.Because coexpression of ENaC resulted in largerI_ami and surface expression compared withcoexpression of ENaC, we hypothesized that the -subunit ismore important for ENaC trafficking than the -subunit. Usingchimeras, we demonstrated that channel activity is largely preservedwhen the highly conserved second cysteine rich domains (CRD2) of the- and -subunits are exchanged. In contrast, exchanging the wholeextracellular loops of the - and the -subunits largely reducedENaC currents and ENaC expression in the membrane. This indicates thatthere is limited interchangeability between molecular regions of thetwo subunits. Interestingly, our chimera studies demonstrated that theintracellular termini and the two transmembrane domains of ENaC aremore important for the expression of functional channels at the cellsurface than the corresponding regions of ENaC.

     

Three-dimensional tissue structure affects sensitivity of fibroblasts to TGF-beta 1

Transforming growth factor-(TGF-) is known to induce -smooth muscle actin (-SMA) infibroblasts and is supposed to play a role in myofibroblastdifferentiation and tumor desmoplasia. Our objective was to elucidatethe impact of TGF-1 on -SMA expression in fibroblasts in athree-dimensional (3-D) vs. two-dimensional (2-D) environment. Inmonolayer culture, all fibroblast cultures responded in a similarfashion to TGF-1 with regard to -SMA expression. In fibroblastspheroids, -SMA expression was reduced and induction by TGF-1 washighly variable. This difference correlated with a differentialregulation in the TGF- receptor (TGFR) expression, in particularwith a reduction in TGF-RII in part of the fibroblast types. Ourdata indicate that 1) sensitivity to TGF-1-induced -SMA expression in a 3-D environment is fibroblast-type specific, 2) fibroblast type-independent regulatory mechanisms, suchas a general reduction/loss in TGF-RIII, contribute to an altered TGFR expression profile in spheroid compared with monolayer culture, and 3) fibroblast type-specific alterations in TGFR typesI and II determine the sensitivity to TGF-1-induced -SMAexpression in the 3-D setting. We suggest that fibroblasts that can beinduced by TGF-1 to produce -SMA in spheroid culture reflect a"premyofibroblastic" phenotype.

     

TNF receptor I is required for induction of macrophage heat shock protein 70

Expression of heat shock proteins (HSP) is anadaptive response to cellular stress. Stress induces tumor necrosisfactor (TNF)- production. In turn, TNF- induces HSP70 expression.However, osmotic stress or ultraviolet radiation activates TNF-receptor I (TNFR-I) in the absence of TNF-. We postulated thatTNF- receptors are involved in the induction of HSP70 by cellularstress. Peritoneal M were isolated from wild-type (WT), TNF-knockout (KO), and TNFR (I or II) KO mice. Cells were culturedovernight and then heat stressed at 43 ± 0.5°C for 30 minfollowed by a 4-h recovery at 37°C. Cellular HSP70 expression wasinduced by heat stress or exposure to endotoxin [lipopolysaccharide(LPS)] as determined by immunoblotting. HSP70 expression induced byeither heat or LPS was markedly decreased in TNFR-I KO M, whereasTNFR-II KO M exhibited HSP70 expression comparable to that in WTmice. Expression of HSP70 after heat stress in TNF- KO M was alsosimilar to that in WT mice, suggesting that induction of HSP70 byTNFR-I occurs independently of TNF-. In addition, levels ofsteady-state HSP70 mRNA were similar by RT-PCR in WT and TNFR-I KO Mdespite differences in protein expression. Furthermore, the effect of TNFR-I appears to be cell specific, since HSP70 expression in splenocytes isolated from TNFR-I KO was similar to that in WT splenocytes. These studies demonstrate that TNFR-I is required for thesynthesis of HSP70 in stressed M by a TNF-independent mechanism andsupport an intracellular role for TNFR-I.

     

NF-kappa B inactivation converts a hepatocyte cell line TNF-alpha response from proliferation to apoptosis

Toxins convertthe hepatocellular response to tumor necrosis factor- (TNF-)stimulation from proliferation to cell death, suggesting thathepatotoxins somehow sensitize hepatocytes to TNF- toxicity. Becausenuclear factor-B (NF-B) activation confers resistance to TNF-cytotoxicity in nonhepatic cells, the possibility that toxin-inducedsensitization to TNF- killing results from inhibition ofNF-B-dependent gene expression was examined in the RALA rathepatocyte cell line sensitized to TNF- cytotoxicity by actinomycinD (ActD). ActD did not affect TNF--induced hepatocyte NF-Bactivation but decreased NF-B-dependent gene expression. Expressionof an IB superrepressor rendered RALA hepatocytes sensitive toTNF--induced apoptosis in the absence of ActD. Apoptosis was blockedby caspase inhibitors, and TNF- treatment led to activation ofcaspase-2, caspase-3, and caspase-8 only when NF-B activation wasblocked. Although apoptosis was blocked by the NF-B-dependent factornitric oxide (NO), inhibition of endogenous NO production did notsensitize cells to TNF--induced cytotoxicity. Thus NF-Bactivation is the critical intracellular signal that determines whetherTNF- stimulates hepatocyte proliferation or apoptosis. Althoughexogenous NO blocks RALA hepatocyte TNF- cytotoxicity, endogenousproduction of NO is not the mechanism by which NF-B activationinhibits this death pathway.

     

Differential requirement for NF-kappaB-inducing kinase in the induction of NF-kappaB by IL-1beta,TNF-alpha,and Fas

In this study, weexamined the role of the nuclear factor-B (NF-B)-inducing kinase(NIK) in distinct signaling pathways leading to NF-B activation. Weshow that a dominant-negative form of NIK (dnNIK) delivered byadenoviral (Ad5dnNIK) vector inhibits Fas-induced IBphosphorylation and NF-B-dependent gene expression in HT-29 and HeLacells. Interleukin (IL)-1- and tumor necrosis factor-(TNF-)-induced NF-B activation and B-dependent gene expressionare inhibited in HeLa cells but not in Ad5dnNIK-infected HT-29 cells.Moreover, Ad5dnNIK failed to sensitize HT-29 cells to TNF--inducedapoptosis at an early time point. However, cytokine- andFas-induced signals to NF-B are finally integrated by the IBkinase (IKK) complex, since IB phosphorylation, NF-B DNAbinding activity, and IL-8 gene expression were strongly inhibited inHT-29 and HeLa cells overexpressing dominant-negative IKK(Ad5dnIKK). Our findings support the concept that cytokine signalingto NF-B is redundant at the level of NIK. In addition, this studydemonstrates for the first time the critical role of NIK and IKK inFas-induced NF-B signaling cascade.

     

Induction of TNF-alpha and MnSOD by endotoxin: role of membrane CD14 and Toll-like receptor-4

Endotoxin (LPS) is a potent inducer oftumor necrosis factor- (TNF-) and manganese superoxide dismutase(MnSOD). Recent evidence suggests that LPS induction of TNF- andMnSOD mRNAs is mediated through distinct intracellular signaltransduction pathways. Membrane CD14 (mCD14) and Toll-like receptor-4(TLR4) mediate LPS induction of TNF- in macrophages. In the current study, we evaluated the role of mCD14 and TLR4 in LPS induction ofMnSOD using peritoneal macrophages from CD14 knockout (CD14-KO) miceand mice with the Tlr4 gene point mutation (C3H/HeJ) ordeletion (C57BL/10ScCr). We studied mCD14-dependent (1 and 10 ng/ml)and mCD14-independent (1,000 ng/ml) concentrations of LPS. Compared with control (BALB/c) macrophages, LPS at 1 and 10 ng/ml failed toinduce TNF- or MnSOD mRNA in CD14-KO macrophages. However, LPS at1,000 ng/ml induced TNF- and MnSOD mRNAs equally in macrophages fromCD14-KO and control mice. LPS (1, 10, or 1,000 ng/ml) failed to induceTNF- or MnSOD mRNA and failed to activate nuclear factor-B inC3H/HeJ or C57BL/10ScCr macrophages. Measurements of TNF- and MnSODenzyme activity paralleled TNF- and MnSOD mRNA levels. These datademonstrate that, like TNF-, induction of MnSOD by LPS is mediatedby mCD14 and TLR4 in murine macrophages.

     

Combined modulation of the mesangial machinery for monocyte recruitment by inhibition of NF-kappa B

The activation of nuclear factor-B(NF-B) is required for the induction of many of the adhesionmolecules and chemokines involved in the inflammatory leukocyterecruitment to the kidney. Here we studied the effects of NF-Binhibition on the machinery crucial for monocyte infiltration of theglomerulus during inflammation. In mesangial cells (MC), the proteaseinhibitors MG-132 and N--tosyl-L-lysine chloromethyl ketone or adenoviral overexpression of IB- prevented the complete IB- degradation following tumor necrosis factor- (TNF-) stimulation. This resulted in a marked inhibition ofTNF--induced expression of mRNA and protein for the immunoglobulinmolecules intracellular adhesion molecule-1 and vascular cell adhesionmolecule-1 and the chemokines growth-related oncogene-, monocytechemoattractant protein-1, interleukin-8, or fractalkine in MC.Finally, the inhibition of IB- degradation or IB-overexpression suppressed the chemokine-induced transendothelialmonocyte chemotaxis toward MC and the chemokine-triggered firm adhesionof monocytic cells to MC. The inhibition of NF-B by pharmacologicalintervention or gene transfer may present a multimodal approach tocontrol the machinery propagating inflammatory recruitment of monocytesduring glomerular disease.

     

TNF-alpha and insulin, alone and synergistically, induce plasminogen activator inhibitor-1 expression in adipocytes

Obesity is associated with hyperinsulinemia and elevatedconcentrations of tumor necrosis factor- (TNF-) inadipose tissue. TNF- has been implicated as an inducer of thesynthesis of plasminogen activator inhibitor-1 (PAI-1), the primaryphysiological inhibitor of fibrinolysis, mediated by plasminogenactivators in cultured adipocytes. To identify mechanism(s) throughwhich TNF- induces PAI-1, 3T3-L1 preadipocytes were differentiatedinto adipocytes and exposed to TNF- for 24 h. TNF- selectivelyincreased the synthesis of PAI-1 without increasing activity ofplasminogen activators. Both superoxide (generated by xanthine oxidaseplus hypoxanthine) and hydrogen peroxide were potent inducers of PAI-1, and hydroxyl radical scavengers completely abolished the TNF- induction of PAI-1. Exposure of adipocytes to TNF- or insulin aloneover 5 days increased PAI-1 production. These agonists exert synergistic effects. Results obtained suggest that TNF- stimulates PAI-1 production by adipocytes, an effect potentiated by insulin, andthat adipocyte generation of reactive oxygen centered radicals mediatesthe induction of PAI-1 production by TNF-. Because induction ofPAI-1 by TNF- is potentiated synergistically by insulin, both agonists appear likely to contribute to the impairment of fibrinolytic system capacity typical in obese, hyperinsulinemic patients.

     

Regulation and distribution of MAdCAM-1 in endothelial cells in vitro

Mucosal addressin cell adhesion molecule-1(MAdCAM-1) is a 60-kDa endothelial cell adhesion glycoprotein thatregulates lymphocyte trafficking to Peyer's patches and lymph nodes.Although it is widely agreed that MAdCAM-1 induction is involved inchronic gut inflammation, few studies have investigated regulation ofMAdCAM-1 expression. We used two endothelial lines [bEND.3 (brain) and SVEC (high endothelium)] to study the signal paths that regulate MAdCAM-1 expression in response to tumor necrosis factor (TNF)- using RT-PCR, blotting, adhesion, and immunofluorescence. TNF- induced both MAdCAM-1 mRNA and protein in a dose- and time-dependent manner. This induction was tyrosine kinase (TK), p42/44, p38mitogen-activated protein kinase (MAPK), and nuclear factor(NF)-B/poly-ADP ribose polymerase (PARP) dependent. Because MAdCAM-1is regulated via MAPKs, we examined mitogen/extracellularsignal-regulated kinase (MEK)-1/2 activation in SVEC. We found thatMEK-1/2 is activated by TNF- within minutes and is dependent on TKand p42/44 MAPKs. Similarly, TNF- activated NF-B through TK,p42/44, p38 MAPKs, and PARP pathways in SVEC cells. MAdCAM-1 was alsoshown to be frequently distributed to endothelial junctions both invitro and in vivo. Cytokines like TNF- stimulate MAdCAM-1 inhigh endothelium via TK, p38, p42/22 MAPKs, and NF-B/PARP.MAdCAM-1 expression requires NF-B translocation through both directp42/44 and indirect p38 MAPK pathways in high endothelial cells.

     

PPAR-gamma ligands modulate effects of LPS in stimulated rat synovial fibroblasts

This work demonstrated the constitutive expressionof peroxisome proliferator-activated receptor (PPAR)- and PPAR-in rat synovial fibroblasts at both mRNA and protein levels. A decrease in PPAR- expression induced by 10 µg/ml lipopolysaccharide (LPS) was observed, whereas PPAR- mRNA expression was not modified. 15-Deoxy-^12,14-prostaglandin J₂(15d-PGJ₂) dose-dependently decreased LPS-induced cyclooxygenase (COX)-2 (80%) and inducible nitric oxide synthase (iNOS) mRNA expression (80%), whereas troglitazone (10 µM) only inhibited iNOS mRNA expression (50%). 15d-PGJ₂ decreasedLPS-induced interleukin (IL)-1 (25%) and tumor necrosis factor(TNF)- (40%) expression. Interestingly, troglitazone stronglydecreased TNF- expression (50%) but had no significant effect onIL-1 expression. 15d-PGJ₂ was able to inhibitDNA-binding activity of both nuclear factor (NF)-B and AP-1.Troglitazone had no effect on NF-B activation and was shown toincrease LPS-induced AP-1 activation. 15d-PGJ₂ andtroglitazone modulated the expression of LPS-induced iNOS, COX-2, andproinflammatory cytokines differently. Indeed, troglitazone seems tospecifically target TNF- and iNOS pathways. These results offer newinsights in regard to the anti-inflammatory potential of the PPAR-ligands and underline different mechanisms of action of15d-PGJ₂ and troglitazone in synovial fibroblasts.

     

Adult alveolar epithelial cells express multiple subtypes of voltage-gated K+ channels that are located in apical membrane

Whole cell perforated patch-clampexperiments were performed with adult rat alveolar epithelial cells.The holding potential was 60 mV, and depolarizing voltage stepsactivated voltage-gated K⁺ (Kv) channels. Thevoltage-activated currents exhibited a mean reversal potential of 32mV. Complete activation was achieved at 10 mV. The currents exhibitedslow inactivation, with significant variability in the time coursebetween cells. Tail current analysis revealed cell-to-cell variabilityin K⁺ selectivity, suggesting contributions of multiple Kv-subunits to the whole cell current. The Kv channels also displayedsteady-state inactivation when the membrane potential was held atdepolarized voltages with a window current between 30 and 5 mV.Analysis of RNA isolated from these cells by RT-PCR revealed thepresence of eight Kv -subunits (Kv1.1, Kv1.3, Kv1.4, Kv2.2, Kv4.1,Kv4.2, Kv4.3, and Kv9.3), three -subunits (Kv1.1, Kv2.1, andKv3.1), and two K⁺ channel interacting protein (KChIP)isoforms (KChIP2 and KChIP3). Western blot analysis with available Kv-subunit antibodies (Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3) showedlabeling of 50-kDa proteins from alveolar epithelial cells grown inmonolayer culture. Immunocytochemical analysis of cells from monolayersshowed that Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3 were localized to theapical membrane. We conclude that expression of multiple Kv -, -,and KChIP subunits explains the variability in inactivation gating andK⁺ selectivity observed between cells and that Kv channelsin the apical membrane may contribute to basal K⁺ secretionacross the alveolar epithelium.

     

Polyamines regulate beta-catenin tyrosine phosphorylation via Ca(2+) during intestinal epithelial cell migration

Polyaminesare essential for early mucosal restitution that occurs by epithelialcell migration to reseal superficial wounds after injury. Normalintestinal epithelial cells are tightly bound in sheets, but they needto be rapidly disassembled during restitution. -Catenin is involvedin cell-cell adhesion, and its tyrosine phosphorylation causesdisassembly of adhesion junctions, enhancing the spreading of cells.The current study determined whether polyamines are required for thestimulation of epithelial cell migration by altering -catenintyrosine phosphorylation. Migration of intestinal epithelial cells(IEC-6 line) after wounding was associated with an increase in-catenin tyrosine phosphorylation, which decreased the bindingactivity of -catenin to -catenin. Polyamine depletion by-difluoromethylornithine reduced cytoplasmic free Ca²⁺concentration ([Ca²⁺]_cyt), preventedinduction of -catenin phosphorylation, and decreased cell migration.Elevation of [Ca²⁺]_cyt induced by theCa²⁺ ionophore ionomycin restored -cateninphosphorylation and promoted migration in polyamine-deficient cells.Decreased -catenin phosphorylation through the tyrosine kinaseinhibitor herbimycin-A or genistein blocked cell migration, which wasaccompanied by reorganization of cytoskeletal proteins. These resultsindicate that -catenin tyrosine phosphorylation plays a criticalrole in polyamine-dependent cell migration and that polyamines induce-catenin tyrosine phosphorylation at least partially through[Ca²⁺]_cyt.

     

Resistance of macrophages to the suppressive effect of interleukin-10 following thermal injury

The activation of a macrophage(M)-dependent proinflammatory cascade following thermal injuryplays an important role in the development of immunosuppression andincreased susceptibility to subsequent sepsis in burn patients. Incontrast, although interleukin (IL)-10, an anti-inflammatory cytokinethat can downregulate M activity, has also been implicated inpostburn immune dysfunction, its role in the regulation of Mfunction postburn remains unclear. To study this, C57BL/6 female micewere subjected to a 25% total body surface area third-degree scaldburn, and splenic Ms were isolated 7 days later. Lipopolysaccharide(LPS)-stimulated IL-10, IL-6, tumor necrosis factor (TNF)-, andnitric oxide (NO) production were significantly increased in the burngroup compared with shams. Blockade of endogenous IL-10 activityenhanced IL-6 and TNF- release, but not NO release, in both groups.The addition of exogenous IL-10 to the M cultures dose dependentlysuppressed production of these inflammatory mediators in both groups.The timing of IL-10 addition to the cultures in relation to LPSstimulation, however, was critical. The suppressive effect of exogenousIL-10 was attenuated in both groups when the cells were exposed toIL-10 at 4-6 h after LPS stimulation; however, Ms from injuredmice were significantly better able to maintain inflammatorymediator-productive capacity. The resistance of Ms from injured miceto IL-10-mediated suppression correlated with decreased IL-10 receptor(IL-10R) expression and increased CD11b expression. These findingssuggest that Ms, following thermal injury, display resistance tosuppression by IL-10 due in part to downregulation of IL-10R expression.

     

Arachidonic acid mediates dual effect of TNF-alpha on Ca2+ transients and contraction of adult rat cardiomyocytes

Tumor necrosisfactor (TNF)- has a biphasic effect on heart contractility andstimulates phospholipase A₂ (PLA₂) incardiomyocytes. Because arachidonic acid (AA) exerts a dual effect onintracellular Ca²⁺ concentration([Ca²⁺]_i) transients, we investigated thepossible role of AA as a mediator of TNF- on[Ca²⁺]_i transients and contraction withelectrically stimulated adult rat cardiac myocytes. At a lowconcentration (10 ng/ml) TNF- produced a 40% increase in theamplitude of both [Ca²⁺]_i transients andcontraction within 40 min. At a high concentration (50 ng/ml) TNF-evoked a biphasic effect comprising an initial positive effect peakingat 5 min, followed by a sustained negative effect leading to50-40% decreases in [Ca²⁺]_i transientsand contraction after 30 min. Both the positive and negative effects ofTNF- were reproduced by AA and blocked by arachidonyltrifluoromethylketone (AACOCF3), an inhibitor of cytosolic PLA₂.Lipoxygenase and cyclooxygenase inhibitors reproduced the high-doseeffects of TNF- and AA. The negative effects of TNF- and AA werealso reproduced by sphingosine and were abrogated by the ceramidaseinhibitor n-oleoylethanolamine. These results point out thekey role of the cytosolic PLA₂/AA pathway in mediating thecontractile effects of TNF-.

     

Nitric oxide-dependent downregulation of adipocyte UCP-2 expression by tumor necrosis factor-alpha

Uncoupling protein-2 (UCP-2) is amitochondrial protein expressed in adipocytes and has recently beeninvolved in the control of energy dissipation. Because obesity ischaracterized by an imbalance between energy intake and expenditure andby an enhanced adipocyte-derived secretion of tumor necrosis factor-(TNF-), we asked whether TNF- could directly influence UCP-2expression in adipocytes. Experiments performed in differentiated3T3F442A preadipocytes showed that TNF- (10 ng/ml) induced areduction of UCP-2 trancripts, assessed by Northern blot analysis. Asignificant decrease in UCP-2 expression (40%) was observed after 12 and 24 h of TNF- stimulation of the cells. The characterizationof the mechanisms responsible for the TNF- effect on UCP-2expression demonstrates an involvement of the TNF--induced inducible(i) nitric oxide synthase (NOS) expression. Cell treatment with the NOSinhibitor N^G-nitro-L-arginine methylester (L-NAME; 1 mmol/l) significantly diminished theTNF--mediated sustained downregulation of UCP-2 expression, whereascell treatment with a nitric oxide (NO) donor (10³ mol/lS-nitroso-L-glutathione) mimicked the TNF-effect on UCP-2 expression. Moreover, Western blot analysis clearlyshowed that TNF- alone induces the expression of iNOS after12-24 h treatment of differentiated 3T3F442A cells. Theseexperiments demonstrate that TNF- directly downregulates UCP-2expression via NO-dependent pathways that involve the induction of iNOS expression.