Signal pathway involved in inhibition by lipoxin A4 of production of interleukins induced in endothelial cells by lipopolysaccharide

Objective and design:  We examine whether lipoxin A₄4 (LXA₄) inhibits production of interleukins (ILs) in endothelial cells and what signal pathway might participate in the actions of LXA₄. Methods:  Cultured pulmonary microvascular endothelial cells (PMVEC) were treated with lipopolysaccharide (LPS), with or without preincubation with LXA₄. Results:  The results showed that LPS induced production of IL-1β, IL-6 and IL-8 in rat PMVEC, upregulated the expressions of myeloid differentiation factor 88 (MyD88), phosphorylated p38 and p42/44 mitogen-activated protein kinase (MAPK), phosphorylated phosphoinositide 3-kinase (PI3-K), DNA-binding activities of nuclear factor-κ B (NF-κB) and activator protein-1(AP-1). The blockade of p38 MAPK, p42/44 MAPK, PI3-K, NF-κB or AP-1 partially inhibited production of IL-1β, IL-6 and IL-8 stimulated by LPS, respectively. LXA₄ significantly inhibited LPS-stimulated secretion of protein and expressions of mRNA of IL-1β, IL-6 and IL-8, activation of p38 MAPK, p42/44 MAPK, PI3-K, NF-κB and AP-1 but not MyD88 in PMVEC. Conclusions:  LXA₄ inhibits synthesis of IL-1β, IL-6 and IL-8 in PMVEC and this antagonism is related to PI3-K, p38 and p42/44 MAPK, NF-κB and AP-1 pathway-dependent signal transduction. Received 7 August 2007; returned for revision 21 December 2007; received from final revision 17 February 2008; accepted by M. Katori 11 March 2008     

Clostridium difficile toxin A promotes dendritic cell maturation and chemokine CXCL2 expression through p38, IKK, and the NF-κB signaling pathway

Clostridium difficile toxin A causes acute colitis associated with intense infiltrating neutrophils. Although dendritic cells (DCs) play an important role in the regulation of inflammation, little is known about the effects of toxin A on the maturation and neutrophil-attracting chemokine expression in DCs. This study investigated whether C. difficile toxin A could influence the maturation of mouse bone-marrow-derived DCs and chemokine CXCL2 expression. Toxin A increased the DC maturation which was closely related to CXCL2 upregulation. Concurrently, toxin A activated the signals of p65/p50 nuclear factor kappa B (NF-κB) heterodimers and phospho-IκB kinase (IKK) in DCs. The increased DC maturation, CXCL2 expression, and neutrophil chemoattraction were significantly downregulated in the NF-κB knockout mice. In addition, toxin A activated the phosphorylated signals of mitogen-activated protein kinases (MAPKs), such as ERK, p38, and JNK. Of all three MAPK signals, p38 MAPK was significantly related to DC maturation. Thus, suppression of p38 activity using SB203580 and siRNA transfection resulted in the significant reduction of IKK activity, DC maturation, and CXCL2 upregulation by toxin A. These results suggest that p38 MAPK may lead to the activation of IKK and NF-κB signaling, resulting in enhanced DC maturation and CXCL2 expression in response to C. difficile toxin A stimulation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cryptotanshinone Suppressed Inflammatory Cytokines Secretion in RAW264.7 Macrophages through Inhibition of the NF-κB and MAPK Signaling Pathways

Cryptotanshinone (CTS), a major constituent extracted from the medicinal herb Salvia miltiorrhiza Bunge, has well-documented antioxidative and anti-inflammatory effects. In the present study, the pharmacological effects and underlying molecular mechanisms of CTS on lipopolysaccharide (LPS)-induced inflammatory responses were investigated. By enzyme-linked immunosorbent assay, we observed that CTS reduced significantly the production of proinflammatory mediators (tumor necrosis factor-α and interleukin-6) induced by LPS in murine macrophage-like RAW264.7 cells. Mechanistically, CTS inhibited markedly the phosphorylation of mitogen-activated protein kinases (MAPKs), including ERK1/2, p38MAPK, and JNK, which are crucially involved in regulation of proinflammatory mediator secretion. Moreover, immunofluorescence and western blot analysis indicated that CTS abolished completely LPS-triggered nuclear factor-κB (NF-κB) activation. Taken together, these data implied that NF-κB and MAPKs might be the potential molecular targets for clarifying the protective effects of CTS on LPS-induced inflammatory cytokine production in macrophages.     

In situ glomerular expression of activated NF-κB in human lupus nephritis and other non-proliferative proteinuric glomerulopathy

Nuclear Factor-κB (NF-κB) has been suggested to play a role in the cellular and molecular mechanisms underlying glomerular injury. We investigated the potential role of NF-κB activation in the pathogenesis of glomerular injury in 31 patients with class III–V lupus nephritis (LN), 14 patients with non-proliferative proteinuric glomerulopathy and six normal controls. The expression of NF-κB subunits p65 and p50, and the NF-κB regulated proinflammatory mediators tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) as well as CD68 and synaptopodin was examined by Southwestern histochemistry (SWH) or immunohistochemistry. In contrast to non-proliferative glomerulopathy and normal controls, NF-κB activation (both p65 and p50) was enhanced in glomerular endothelial, mesangial cells or infiltrating cells in class IV LN, along with upregulation of TNF-α, IL-1β, IL-6 and ICAM-1 expression. Glomerular endothelial and mesangial activation of NF-κB and mesangial ICAM-1 expression correlated with disease activity and the level of glomerular macrophage infiltration. Podocyte NF-κB overactivation (predominantly p65) paralleled podocyte expression of TNF-α and IL-1β in patients with LN and non-proliferative glomerulopathy. Podocyte staining scores of NF-κB and p65 were positively correlated with the severity of proteinuria in LN and non-proliferative glomerulopathy. These results suggest a pathogenic role for NF-κB in glomerular injury by multiple mechanisms.     

p38MAPK在重症急性胰腺炎大鼠枯否细胞分泌促炎细胞因子中的作用

目的:探讨p38MAPK信号转导通路在重症急性胰腺炎(SAP)大鼠枯否细胞(KCs)分泌促炎细胞因子TNF-α和IL-1β中的作用。方法:30只SD大鼠随机分为:①假手术对照(SO)组;②SAP组;③SAP+CNI-1493(p38MAPK抑制剂)组。SAP模型通过胰胆管逆行注射5%牛磺胆酸钠诱导。假手术或造模后12h处死动物分离出KCs,采用实时定量PCR方法检测KCs内TNF-α和IL-1βmRNA的表达,采用Westernblot法检测p38MAPK活化情况,并用ELISA法检测血浆的TNF-α和IL-1β含量。结果:SAP大鼠KCs内TNF-α和IL-1βmRNA的表达明显强于假手术组,p38MAPK活性显著高于SO组,同时血浆TNF-α和IL-1β含量明显高于SO组,使用CNI-1493的SAP大鼠上述指标均显著低于SAP组。结论:p38MAPK信号转导通路介导了SAP大鼠的KCs促炎细胞因子TNF-α和IL-1β的分泌,阻断p38MAPK信号转导通路对于SAP防治可能具有重要意义。     

Glutamine decreases intestinal nuclear factor kappa B activity and pro-inflammatory cytokine expression after traumatic brain injury in rats

Objective: To investigate whether glutamine supplementation modulates intestinal nuclear factor kappa B (NF-κB) activity and pro-inflammatory cytokine expression after traumatic brain injury (TBI) in rats. Materials and methods: Right parietal cortical contusion in male rats was made by the weight-dropping method. After trauma, the rats were randomly given chow alone or glutamine mixed chow for 5 d. Gut samples were extracted at 5 d postinjury. We measured NF-κB binding activity by electrophoretic mobility shift assay; NF-κB subunits p50 and p65 expression by immunohistochemistry; the concentrations of interleukin-1β, tumor necrosis factor-α and interleukin-6 by enzyme-linked immunosorbent assay; intestinal mucosal morphological changes by histopathological study and electron microscopy; and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. Results: Administration of glutamine following TBI could decrease NF-κB binding activity, NF-κB p65 protein expression and concentrations of pro-inflammatory cytokines in the gut. TBI-induced damage of gut structure was ameliorated after glutamine supplementation. Conclusion: The results of the present study suggest that the therapeutic benefit of post-TBI glutamine supplementation might be due to its inhibitory effects on intestinal NF-κB activation and pro-inflammatory cytokine expression. Received 14 May 2007; returned for revision 9 July 2007; accepted by I. Ahnfeld-R?nne 16 August 2007     

IKK-2 is required for TNF-α-induced invasion and proliferation of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) can contribute to tissue repair by actively migrating to sites of tissue injury. However, the cellular and molecular mechanisms of MSC recruitment are largely unknown. The nuclear factor (NF)-κB pathway plays a pivotal role in regulating genes that influence cell migration, cell differentiation, inflammation, and proliferation. One of the major cytokines released at sites of injury is tumor necrosis factor-α (TNF-α), which is known to be a key regulator of the NF-κB pathway. Therefore, we hypothesized that TNF-α may lead to MSC invasion and proliferation by activation of the NF-κB pathway. TNF-receptor 1 and 2, NF-κB (p65), and IκB kinase 2 (IKK-2) are expressed in human MSCs (hMSCs). Stimulation of hMSCs with TNF-α caused a p65 translocation from the cytoplasm to nucleoplasm but did not change the expression profile of MSC markers. TNF-α strongly augmented the migration of hMSCs through the human extracellular matrix. Using lentiviral gene transfer, overexpressing a dominant-negative mutant of IKK-2 (dn-IKK-2) significantly blocked this effect. NF-κB target genes associated with migration (vascular cell adhesion molecule-1, CD44, and matrix metalloproteinase 9) were upregulated by TNF-α stimulation and blocked by dn-IKK-2. Moreover, using the bromodeoxyuridine assay, we showed that the inhibition of the NF-κB pathway caused a significant reduction in the basal proliferation rate. TNF-α stimulated the proliferation of hMSCs, whereas overexpression of dn-IKK-2 significantly blocked this effect. TNF-α led to the upregulated expression of the proliferation-associated gene cyclin D1. In conclusion, we demonstrated that the NF-κB pathway components, p65 and IKK-2, are expressed in hMSCs. Our data provide evidence that this signal transduction pathway is implicated in TNF-α-mediated invasion and proliferation of hMSCs. Therefore, hMSC recruitment to sites of tissue injury may, at least in part, be regulated by the NF-κB signal transduction pathway.     

Gamma-Linolenic Acid Inhibits Inflammatory Responses by Regulating NF-κB and AP-1 Activation in Lipopolysaccharide-Induced RAW 264.7 Macrophages

Gamma linolenic acid (GLA) is a member of the n-6 family of polyunsaturated fatty acids and can be synthesized from linoleic acid (LA) by the enzyme delta-6-desaturase. The therapeutic values of GLA supplementation have been documented, but the molecular mechanism behind the action of GLA in health benefits is not clear. In this study, we assessed the effect of GLA with that of LA on lipopolysaccharide (LPS)-induced inflammatory responses and further explored the molecular mechanism underlying the pharmacological properties of GLA in mouse RAW 264.7 macrophages. GLA significantly inhibited LPS-induced protein expression of inducible nitric oxide synthase, pro-interleukin-1β, and cyclooxygenase-2 as well as nitric oxide production and the intracellular glutathione level. LA was less potent than GLA in inhibiting LPS-induced inflammatory mediators. Both GLA and LA treatments dramatically inhibited LPS-induced IκB-α degradation, IκB-α phosphorylation, and nuclear p65 protein expression. Moreover, LPS-induced nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) nuclear protein–DNA binding affinity and reporter gene activity were significantly decreased by LA and GLA. Exogenous addition of GLA but not LA significantly reduced LPS-induced expression of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK)-1. Our data suggest that GLA inhibits inflammatory responses through inactivation of NF-κB and AP-1 by suppressed oxidative stress and signal transduction pathway of ERK and JNK in LPS-induced RAW 264.7 macrophages.     

Verapamil modulates LPS-induced cytokine production via inhibition of NF-kappa B activation in the liver

Objective: To investigate the effect of verapamil on Lipopolysaccharide (LPS)-induced cytokines [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10)] and nuclear factor kappa B (NF-κ B) in the liver. Methods and Materials: Adult male Sprague-Dawley rats were randomly divided into seven groups of eight rats each: control rats treated with saline (0.9 % NaCl); rats treated with saline and then challenged intraperitoneally with LPS (10 mg/kg); rats treated intraperitoneally with different levels of verapamil (1, 2.5, 5, 10 mg/kg) and then challenged with LPS (10 mg/kg); and rats treated only with verapamil (10 mg/kg). TNF-α, IL-6, IL-10 and NF-κ B in the liver tissues were investigated as well as the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) one hour after LPS injection. Results: LPS alone stimulated production of TNF-α, IL-6 and IL-10, and activated NF-κ B in the liver. Pretreatment with verapamil before LPS challenge reduced acute liver injury, down-regulated production of LPS-induced pro-inflammatory cytokines (TNF-α and IL-6), up-regulated production of anti-inflammatory cytokines (IL-10) and inhibited NF-κ B activation in the liver in a dose-dependent manner. Conclusion: Verapamil can attenuate acute liver injury by down-regulating the production of TNF-α and IL-6 and up-regulating IL-10 in the liver, possibly via inhibition of NF-κ B. Received 8 August 2005; returned for revision 25 September 2005; returned for final revision 18 November 2005; accepted by M. Katori 6 December 2005     

Hyaluronan inhibits cytokine production by lipopolysaccharide-stimulated U937 macrophages through down-regulation of NF-κB via ICAM-1

Objective: This study investigated the inhibitory mechanism of hyaluronan (HA) on lipopolysaccharide (LPS)-stimulated production of proinflammatory cytokines in U937 macrophages. Methods: HA was added to U937 macrophage cultures in the presence of LPS, with or without pretreatment with anti-intercellular adhesion molecule-1 (ICAM-1) antibody. Secreted levels of tumor necrosis factor α (TNFα), interleukin (IL)-1β, and IL-6 were determined by enzyme-linked immunosorbent assay. The phosphorylation of nuclear factor (NF)-κB, IκBα, and mitogen-activated protein kinases (MAPKs) was analyzed by immunoblotting. Results: LPS stimulated production of TNFα, IL-1β, and IL-6. In contrast to 800 kDa HA, 2700 kDa HA at 1 mg/ml inhibited LPS-induced cytokine production. Anti-ICAM-1 antibody blocked the effects of HA on the LPS actions on U937 cells. LPS activated NF-κB and MAPK pathways, whereas HA down-regulated p65 NF-κB and IκBα phosphorylation by LPS without affecting MAPKs. Inhibition studies revealed the requirement of NF-κB for LPS-stimulated cytokine production. Anti-ICAM-1 antibody reversed the inhibitory effects of HA on phosphorylation of p65 NF-κB and IκBα. Conclusion: HA of intrinsic molecular weight suppresses LPS-stimulated production of proinflammatory cytokines via ICAM-1 through down-regulation of NF-κB and IκB. Exogenous HA injected into arthritic joints could act as an anti-NF-κB agent by the mechanism demonstrated in the present study. Received 23 September 2006; returned for revision 12 October 2006; accepted J. Di Battista 18 December 2006     

Enteropathogenic <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> Outer Membrane Proteins Induce iNOS by Activation of NF-κB and MAP Kinases

Enteropathogenic Escherichia coli (EPEC) infects the human intestinal epithelium and is a major cause of infantile diarrhea in developing countries. Nitric oxide (NO) is an important modulator of intestinal inflammatory response. The aim of the present study was to investigate whether EPEC outer membrane proteins (OMPs) up regulate epithelial cell expression of inducible nitric oxide synthase (iNOS) and to examine the role of NF-κB and MAP kinases (MAPK) on nitrite production. iNOS mRNA expression was assessed by RT-PCR. Nitrite levels were measured by Griess reaction. NF-κB activation by OMPs was evaluated by EMSA and immunoblotting was done to detect MAPK activation. EPEC OMP up regulated iNOS, induced nitrite production and NF-κB and MAPK were activated in caco-2 cells. The nitrite levels decreased when NF-κB and MAPK inhibitors were used. Thus, EPEC OMPs induce iNOS expression and NO production through activation of NF-κB and MAPK. These authors contributed equally to this work.     

Involvement of adenylate cyclase and p70S6-kinase activation in IL-10 up-regulation in human monocytes by gp41 envelope protein of human immunodeficiency virus type 1

Our previous results show that recombinant gp41 (aa565–647), the extracellular domain of HIV-1 transmembrane glycoprotein, stimulates interleukin-10 (IL-10) production in human monocytes. The signal cascade transducing this effect is not yet clear. In this study, we examined whether gp41-induced IL-10 up-regulation is mediated by the previously described synergistic activation of cAMP and NF-κB pathways. gp41 induced cAMP accumulation in monocytes in a time- and concentration-dependent manner and the adenylate cyclase inhibitor SQ 22536 suppressed gp41-induced IL-10 production in monocytes. In contrast, gp41 failed to stimulate NF-κB binding activity in as much as no NF-κB bound to the main NF-κB-binding site 2 of the IL-10 promoter after addition of gp41. We also examined the involvement of other signal transduction pathways. Specific inhibitors of p70^S6-kinase (rapamycin), and G_i protein (pertussis toxin), prevented induction of IL-10 production by gp41 in monocytes, while inhibitors of the phosphatidylinositol 3-kinase (PI 3-kinase) (wortmannin) and mitogen-activated protein kinase (MAPK) pathway (PD 98059) did not. Thus HIV-1 gp41-induced IL-10 up-regulation in monocytes may not involve NF-κB, MAPK, or PI 3-kinase activation, but rather may operate through activation of adenylate cyclase and pertussis-toxin-sensitive G_i/G_o protein to effect p70^S6-kinase activation. Received: 28 September 1998 / Received after revision: 27 October 1998 / Accepted: 3 November 1998     

Wortmannin,PI3K/Akt signaling pathway inhibitor,attenuates thyroid injury associated with severe acute pancreatitis in rats

Increasing evidences suggest that PI3K/AKT pathway plays an important role in the pathogenesis of inflammatory diseases such as acute pancreatitis. However, the exact effect of PI3K/AKT on thyroid injury associated with acute pancreatitis has not been investigated. This study aimed to investigate the protective effects of wortmannin, PI3K/AKT inhibitor, on thyroid injury in a rat model of severe acute pancreatitis (SAP). Sixty male SD rats were randomly divided into four groups: sham operating group (SO), SAP group, wortmannin treatment (WOR) group and drug control (WOR-CON) group. Serum amylase (AMY), lipase (LIP) and thyroid hormone levels were evaluated. The morphological change of thyroid tissue was analyzed under the light and transmission electron microscopy. AKT, P38MAPK and NF-κB expression in the thyroid tissue was evaluated by immunohistochemical staining. Oxidative stress and inflammatory cytokines were detected. Results showed that wortmannin attenuated the following: (1) serum AMY, LIP and thyroid hormone (2) pancreatic and thyroid pathological injuries (3) thyroid MDA, (4) thyroid ultrastructural change, (5) serum TNF-α, IL-6 and IL-1β (6) AKT, MAPKP38 and NF-κB expression in thyroid tissues. These results suggested that wortmannin attenuates thyroid injury in SAP rats, presumably because of its role on prevent ROS generation and inhibits the activation of P38MAPK, NF-κB pathway. Our findings provide new therapeutic targets for thyroid injury associated with SAP.