Activation of TLR-9 induces IL-8 secretion through peroxynitrite signaling in human neutrophils

Bacterial DNA containing unmethylated CpG motifs is emerging as an important regulator of functions of human neutrophil granulocytes (polymorphonuclear leukocytes (PMN)). These motifs are recognized by TLR-9. Recent studies indicate that peroxynitrite (ONOO-) may function as an intracellular signal for the production of IL-8, one of the key regulators of leukocyte trafficking in inflammation. In this study we investigated whether bacterial DNA (CpG-DNA) could induce ONOO- signaling in human PMN. Human whole blood, isolated PMN (purity, >95%), and high purity (>99%) PMN respond to CpG-DNA, but not to calf thymus DNA, with secretion of IL-8 and, to a lesser extent, IL-6 and TNF. Methylation of cytosines in CpG-DNA resulted in a complete loss of activity. The endosomal acidification inhibitors, bafilomycin A and chloroquine, inhibited CpG-DNA-induced cytokine release from PMN. CpG-DNA-induced IL-8 mRNA expression and release was also blocked by the NO synthase inhibitor Nomega-nitro-L-arginine methyl ester. CpG-DNA evoked concomitant increases in intracellular superoxide and NO levels, leading to enhanced ONOO- formation and, consequently, nuclear accumulation of c-Fos and NF-kappaB. Pharmacological inhibition of NF-kappaB activation attenuated approximately 75% of CpG-DNA-evoked IL-8 release. These results identify ONOO- -dependent activation of NF-kappaB and c-Fos as an important mechanism that mediates PMN responses, including IL-8 gene expression and release, to bacterial DNA and unmethylated CpG motifs in particular. Enhanced ONOO- formation represents a mechanism by which bacterial DNA may contribute to prolongation and amplification of the inflammatory response.     

Lymphotoxin beta receptor induces interleukin 8 gene expression via NF-kappaB and AP-1 activation

The human lymphotoxin beta receptor (LTbetaR), a member of the tumor necrosis factor (TNF) receptor superfamily, is essential for not only the development and organization of secondary lymphoid tissues, but also for chemokine release. Even though LTbetaR was shown to recruit TNF-receptor-associated factor (TRAF) 2, 3, and 5, and to induce cell apoptosis or NF-kappaB activation, however, the downstream signaling leading to chemokine expression is not illustrated yet. In this study, we find that overexpression of LTbetaR in HEK293 cells increases IL-8 promoter activity and leads to IL-8 release. LTbetaR-induced IL-8 gene expression requires NF-kappaB (-80 to -71) and AP-1 (-126 to -12) binding sites located in IL-8 promoter, and NF-kappaB is more crucial than AP-1 for IL-8 gene expression. Reporter assay with dominant-negative mutants of TRAFs reveals that TRAF2, 3, and 5, as well as the downstream signal molecules NIK, IKKalpha, and IKKbeta, are involved in IL-8 gene expression. LTbetaR-mediated IL-8 response was inhibited by the dominant-negative mutants of ASK1, MKK4, MKK7, and JNK, but not by those of MEKK1, TAK1, MEK, ERK, and p38 MAPK. This suggests that IL-8 induction by LTbetaR is via TRAFs-elicited signaling pathways, including NIK/IKK-dependent NF-kappaB activation and ASK/MKK/JNK-dependent AP-1 activation.     

Interleukin-8 production by THP-1 cells stimulated by Salmonella enterica serovar Typhimurium porins is mediated by AP-1, NF-kappaB and MAPK pathways

Interleukin-8 (IL-8) is released in response to inflammatory stimuli, such as bacterial products. Either porins or lipopolysaccharide (LPS) stimulated THP-1 cells to release IL-8 after 24 h. We have previously reported that stimulation of monocytic cells with Salmonella enterica serovar Typhimurium porins led to the activation of mitogen-activated protein kinase cascades and of protein tyrosine kinases (PTKs). In this report, we demonstrate, using two potent and selective inhibitors of MEK activation by Raf-1 (PD-098059) and p38 (SB-203580), that both ERK1/2 and p38 pathways play a key role in the production of IL-8 by porins and LPS. Porin-stimulated expression of activating protein 1 (AP-1) and correlated IL-8 release is also inhibited by PD-098059 or SB-203580 indicating that the Raf-1/MEK1-MEK2/MAPK cascade is required for their activation. Also PTKs modulate the pathway that control IL-8 gene expression, in fact its expression is abolished by tyrphostin. By using N-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN) an inhibitor of nuclear factor-kappaB (NF-kappaB) activity, we also observed IL-8 release modulation. Our results elucidate some of the molecular mechanisms by which AP-1 and NF-kappaB regulate IL-8 release and open new strategies for the design of specific molecules that will modulate IL-8 effects in various infectious diseases.     

Phosphatidylinositol 3-kinase/Akt signaling mediates interleukin-32alpha induction in human pancreatic periacinar myofibroblasts

Interleukin (IL)-32 is a recently described proinflammatory cytokine, characterized by the induction of nuclear factor (NF)-kappaB activation. We studied IL-32alpha expression in human pancreatic periacinar myofibroblasts, which play important roles in the regulation of extracellular matrix metabolism and inflammatory responses in the pancreas. IL-32alpha protein expression was evaluated by Western blot analyses, and IL-32alpha mRNA expression was analyzed by Northern blot and real-time PCR analyses. IL-32alpha mRNA was weakly expressed without a stimulus, and its expression was markedly enhanced by IL-1beta, IFN-gamma, and TNF-alpha. IL-1beta, IFN-gamma, and TNF-alpha enhanced intracellular accumulation of IL-32alpha protein, but IL-32alpha was not detected in supernatants. Each cytokine dose and time dependently induced IL-32alpha mRNA expression. An inhibitor of phosphatidylinositol 3-kinase (LY294002) significantly suppressed IL-1beta-, IFN-gamma-, and TNF-alpha-induced IL-32alpha mRNA expression, although MAPK inhibitors had no effect. Akt activation in response to these cytokines was confirmed by Western blot. Furthermore, LY294002 suppressed both IL-1beta- and TNF-alpha-induced NF-kappaB activation and IL-1beta-, TNF-alpha-, and IFN-gamma-induced activated protein-1 (AP-1) activation. Blockade of NF-kappaB and AP-1 activation by an adenovirus expressing a stable mutant form of IkappaBalpha and a dominant negative mutant of c-Jun markedly suppressed IL-1beta-, IFN-gamma-, and/or TNF-alpha-induced IL-32alpha mRNA expression. Human pancreatic periacinar myofibroblasts expressed IL-32alpha in response to IL-1beta, TNF-alpha, and IFN-gamma. IL-32alpha mRNA expression is dependent on interactions between the phosphatidylinositol 3-kinase/Akt-pathway and the NF-kappaB/AP-1 system.     

Lipoxins and aspirin-triggered lipoxins in neutrophil adhesion and signal transduction

Lipoxin A4 (LXA4) and aspirin-triggered 15-epi-LXA4 (ATL) are emerging as endogenous braking signals for neutrophil-mediated tissue injury. LXA4 and ATL and their metabolically stable analogues display potent inhibitory actions in human isolated cells and blood, including attenuation of expression of adhesion molecules on leukocytes and endothelial cells, neutrophil adhesion to endothelial cells and platelets under shear, and IL-8 production, key events of the acute inflammatory response. The underlying molecular mechanisms include interference with MAPK signaling pathways, modulation of the oxidative chemistry of superoxide, NO and ONOO-, inhibition of activation of NF-kappaB and AP-1, and consequently the expression of interleukin-8 and likely other pro-inflammatory genes. Collectively, these results add to the profile of LXA4/ATL rapid actions that contribute to "stop signaling" involved in regulating neutrophil functions during acute inflammation and suggest that aspirin inhibits neutrophil accumulation through triggering the synthesis of 15-epi-LXA4.     

Oxygen radicals trigger activation of NF-kappaB and AP-1 and upregulation of ICAM-1 in reperfused canine heart

We investigated whether oxygen radicals generated during ischemia-reperfusion trigger postischemic inflammation in the heart. Closed-chest dogs underwent 90-min coronary artery occlusion, followed by 1- or 3-h reperfusion: 10 dogs received the cell-permeant oxygen radical scavenger N-(2-mercaptopropionyl)-glycine (MPG; 8 mg x kg(-1) x h(-1) intracoronary) beginning 5 min before reperfusion, and 9 dogs received vehicle. Blood flow (microspheres), intercellular adhesion molecule (ICAM)-1 protein expression (immunohistochemistry), ICAM-1 gene activation (Northern blotting), nuclear DNA binding activity of nuclear factor (NF)-kappaB and AP-1 (electrophoretic mobility shift assays), and neutrophil (PMN) accumulation (myeloperoxidase activity) were assessed in myocardial tissue samples. ICAM-1 protein expression was high in vascular endothelium after ischemia-reperfusion but was markedly reduced by MPG. MPG treatment also markedly decreased expression of ICAM-1 mRNA and tissue PMN accumulation. Nuclear DNA binding activities of NF-kappaB and AP-1, increased by ischemia-reperfusion, were both markedly decreased by MPG at 1 h of reperfusion. However, by 3 h, AP-1 activity was only modestly reduced by MPG and NF-kappaB activity was not significantly different from ischemic-reperfused controls. These results suggest that oxygen radicals generated in vivo during reperfusion trigger early activation of NF-kappaB and AP-1, resulting in upregulation of the ICAM-1 gene in vascular endothelium and subsequent tissue accumulation of activated PMNs.     

Modulators of inflammation use nuclear factor-kappa B and activator protein-1 sites to induce the caspase-1 and granzyme B inhibitor,proteinase inhibitor 9

Proteinase inhibitor 9 (PI-9) inhibits caspase-1 (interleukin (IL)-1beta-converting enzyme) and granzyme B, thereby regulating production of the pro-inflammatory cytokine IL-1beta and susceptibility to granzyme B-induced apoptosis. We show that cellular PI-9 mRNA and protein are induced by IL-1beta, lipopolysaccharide, and 12-O-tetradecanoylphorbol-13-acetate. We identified functional imperfect nuclear factor-kappaB (NF-kappaB) sites at -135 and -88 and a consensus activator protein-1 (AP-1) site at -308 in the PI-9 promoter region. Using transient transfections in HepG2 cells to assay PI-9 promoter mutations, we find that mutational ablation of the AP-1 site or of either NF-kappaB site reduces IL-1beta-induced expression of PI-9 by approximately 60%. Mutational ablation of the two NF-kappaB sites and of the AP-1 site nearly abolishes both basal and IL-1beta-induced expression of PI-9. Nuclear extracts from IL-1beta-treated HepG2 cells exhibited strong, IL-1beta-inducible binding to the NF-kappaB sites and to the AP-1 site. Electrophoretic mobility shift assays show that after IL-1beta treatment c-Jun/c-Fos and JunD bind to the AP-1 site, whereas the p50/p65 heterodimer binds to the two NF-kappaB sites. Estrogens induce PI-9, but induction of PI-9 by estrogens and IL-1beta is not synergistic. In transiently transfected, estrogen receptor-positive HepG2ER7 cells, estrogens do not interfere with IL-1beta induction, whereas IL-1beta exhibits dose-dependent repression of estrogen-inducible PI-9 expression. Our surprising finding that the pro-inflammatory cytokine IL-1beta strongly induces PI-9 suggests a novel mechanism for regulating inflammation and apoptosis through a negative feedback loop controlling expression of the anti-inflammatory and anti-apoptotic protein, PI-9.     

Interleukin-1 stimulates cytokines, prostaglandin E2 and matrix metalloproteinase-1 production via activation of MAPK/AP-1 and NF-kappaB in human gingival fibroblasts

Interleukin-1 (IL-1) plays a crucial role in the immunopathological responses involved with tissue destruction in chronic inflammatory diseases, such as periodontal disease, as it stimulates host cells including fibroblasts to produce various inflammatory mediators and catabolic factors. We comprehensively investigated the involvement of mitogen-activated protein kinases (MAPKs)/activator protein-1 (AP-1) and IkappaB kinases (IKKs)/IkappaBs/nuclear factor-kappaB (NF-kappaB) in IL-1beta-stimulated IL-6, IL-8, prostaglandin E(2) (PGE(2)) and matrix metalloproteinase-1 (MMP-1) production by human gingival fibroblasts (HGF). Three MAPKs, extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK), which were simultaneously activated by IL-1beta, mediated subsequent c-fos and c-jun mRNA expression and DNA binding of AP-1 at different magnitudes. IKKalpha/beta/IkappaB-alpha/NF-kappaB was also involved in the IL-1 signaling cascade. Further, IL-1beta stimulated HGF to produce IL-6, IL-8, PGE(2) and MMP-1 via activation of the 3 MAPKs and NF-kappaB, as inhibitors of each MAPK and NF-kappaB significantly suppressed the production of IL-1beta-stimulated factors, though these pathways might also play distinct roles in IL-1beta activities. Our results strongly suggest that the MAPKs/AP-1 and IKK/IkappaB/NF-kappaB cascades cooperatively mediate the IL-1beta-stimulated synthesis of IL-6, IL-8, PGE(2) and MMP-1 in HGF.     

Effects of antioxidant and nitric oxide on chemokine production in TNF-alpha-stimulated human dermal microvascular endothelial cells

Chemokines have been implicated convincingly in the driving of leukocyte emigration in different inflammatory reactions. Multiple signaling mechanisms are reported to be involved in intracellular activation of chemokine expression in vascular endothelial cells by various stimuli. Nevertheless, redox-regulated mechanisms of chemokine expression in human dermal microvascular endothelial cells (HDMEC) remain unclear. This study examined the effects of pyrrolidine dithiocarbamate (PDTC, 0.1 mM) and spermine NONOate (Sper-NO, 1 mM) on the secretion and gene expression of chemokines, interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, regulated upon activation normal T cell expressed and secreted (RANTES), and eotaxin. This study also addresses PDTC and Sper-NO effects on activation of nuclear factor kappa B (NF-kappaB) induced by TNF-alpha (10 ng/ml). Treatment with TNF-alpha for 8 h significantly increased secretion of IL-8, MCP-1, and RANTES, but not of eotaxin, in cultured HDMEC. Up-regulation of these chemokines was suppressed significantly by pretreatment with PDTC or Sper-NO for 1 h, but not by 1 mM 8-bromo-cyclic GMP. The mRNA accumulation of IL-8, MCP-1, RANTES, and eotaxin, and activation of NF-kappaB were induced by TNF-alpha for 2 h; all were suppressed significantly by the above two pretreatments. These findings indicate that both secretion and mRNA accumulation of IL-8, MCP-1, and RANTES in HDMEC induced by TNF-alpha are inhibited significantly by pretreatment with PDTC or Sper-NO, possibly via blocking redox-regulated NF-kappaB activation. These results suggest that restoration of the redox balance using antioxidant agents or nitric oxide pathway modulators may offer new opportunities for therapeutic interventions in inflammatory skin diseases.     

Increased AP-1 and NF-kappaB activation and recruitment with the combination of the proinflammatory cytokines IL-1beta, tumor necrosis factor alpha and IL-17 in rheumatoid synoviocytes

To determine the contribution of IL-1beta, tumor necrosis factor alpha (TNF-alpha) and IL-17 to AP-1, NF-kappaB and Egr-1 activation in rheumatoid arthritis, the effect of the cytokines used alone or in combination was measured on TF expression in rheumatoid synoviocytes. Effects on mRNA expression were measured by RT-PCR and effects on nuclear translocation were measured by immunocytochemistry. To assess the functional consequences of cytokine induction, osteoprotegerin levels were measured in synoviocyte supernatants.IL-1beta and TNF-alpha alone at optimal concentration (100 pg/ml) induced the nuclear translocation of NF-kappaB and almost all AP-1 members, except JunB and Egr-1 for IL-1beta and except Fra-2 and Egr-1 for TNF-alpha. IL-17 was clearly less potent since no nuclear translocation was observed, except for a weak activation of Fra-1 and NF-kappaB. More importantly, when these cytokines were used at low concentrations, their combination showed a synergistic effect on almost all the TFs, except for Egr-1, with a particular effect on Fra-1 and NF-kappaB. Increased recruitment of additional factors was induced when the three cytokines were combined. IL-1 and TNF-alpha induced mRNA expression of c-jun while IL-17 had no effect. A synergistic effect was seen with their combination. A similar synergistic effect was observed for osteoprotegerin production when these three cytokines were combined at low concentrations.AP-1 and NF-kappaB pathways were highly sensitive to the combination through synergistic mechanisms. These effects observed in rheumatoid arthritis synoviocytes may reflect the conditions found in the rheumatoid arthritis joint and may contribute to the mode of action of cytokine inhibitors.