Interleukin-8 induces nuclear transcription factor-kappaB through a TRAF6-dependent pathway

Considering the potential role of interleukin-8 (IL-8) in inflammation, angiogenesis, tumorigenesis, and metastasis, we investigated the molecular mechanism involved in IL-8-mediated signaling. In this report we provide evidence that like TNF, an inducer of NF-kappaB and also a NF-kappaB-dependent gene product, IL-8 induces NF-kappaB in a unique pathway. IL-8 induces NF-kappaB activation in a dose-dependent manner in different cell types as detected by a DNA-protein binding assay. IL-8 induces NF-kappaB-dependent reporter gene expression as well as ICAM-1, VCAM-1, and Cox-2 expression. IL-8 also induces IkappaBalpha phosphorylation followed by degradation and p65 translocation. IL-8 induces c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinase (MAPK) in a dose- and time-dependent manner. IL-8-induced NF-kappaB activation is for the most part unaltered when cells are transfected with dominant-negative TRADD, FADD, or TRAF2, but is inhibited with dominant-negative TRAF6-, NIK-, IKK-, or IkappaBalpha-transfected cells. The data suggest that IL-8-induced NF-kappaB activation proceeds through a TRAF2-independent but TRAF6-dependent pathway, followed by recruitment of IRAK and activation of IKK. IL-8-induced NF-kappaB activation is not observed in a cell-permeable peptide that has TRAF6 binding motif-treated cells or IRAK-deficient cells. IL-8-induced NF-kappaB activation proceeds mostly through interaction with TRAF6 and partially through the Rho-GTPase pathways. This is the first report that IL-8 induces NF-kappaB in a distinct pathway, and activation of NF-kappaB and its dependent genes may be one of the pathways of IL-8-induced inflammation and angiogenesis.     

IL-18 receptors, their role in ligand binding and function: anti-IL-1RAcPL antibody, a potent antagonist of IL-18

IL-18 is critical in eliciting IFN-gamma production from Th1 cells both in vitro and in vivo. Th1 cells have been implicated in the pathogenesis of autoimmune disorders, making antagonists of IL-18 promising therapeutics. However, specificity and binding characteristics of IL-18R components have only been superficially explored. In this study, we show that IL-1R related protein 1 (IL-1Rrp1) and IL-1R accessory protein-like (IL-1RAcPL) confer responsiveness to IL-18 in a highly specific (no response to other IL-1 ligands) and unique manner (no functional pairing with other IL-1Rs and IL-1R-like molecules). Cotransfection with both receptor components resulted in expression of both low and high affinity binding sites for IL-18 (K:(d) of 11 and 0.4 nM, respectively). We prepared anti-IL-1RAcPL mAb TC30-28E3, which, in contrast to soluble R proteins, effectively inhibited the IL-18-induced activation of NF-kappaB. Quantitative PCR showed that Th1 but not Th2 cells are unique in that they coexpress IL-1Rrp1 and IL-1RAcPL. mAb TC30-28E3 inhibited IL-18-induced production of IFN-gamma by Th1 cells, being at least 10-fold more potent than anti-IL-18 ligand mAb. This study shows that IL-1RAcPL is highly specific to IL-18, is required for high affinity binding of IL-18, and that the anti-IL-1RAcPL mAb TC30-28E3 potently antagonizes IL-18 responses in vitro, providing a rationale for the use of anti-IL-1RAcPL Abs to inhibit Th1-mediated inflammatory pathologies.     

Melanoma differentiation-associated gene-7/IL-24 gene enhances NF-kappa B activation and suppresses apoptosis induced by TNF

Melanoma differentiation-associated gene-7 (mda-7), also referred to as IL-24, is a novel growth regulatory cytokine that has been shown to regulate the immune system by inducing the expression of inflammatory cytokines, such as TNF, IL-1, and IL-6. Whether the induction of these cytokines by MDA-7 is mediated through activation of NF-kappaB or whether it regulates cytokine signaling is not known. In the present report we investigated the effect of MDA-7 on NF-kappaB activation and on TNF-induced NF-kappaB activation and apoptosis in human embryonic kidney 293 cells. Stable or transient transfection with mda-7 into 293 cells failed to activate NF-kappaB. However, TNF-induced NF-kappaB activation was significantly enhanced in mda-7-transfected cells, as indicated by DNA binding, p65 translocation, and NF-kappaB-dependent reporter gene expression. Mda-7 transfection also potentiated NF-kappaB reporter activation induced by TNF receptor-associated death domain and TNF receptor-associated factor-2. Cytoplasmic MDA-7 with deleted signal sequence was as effective as full-length MDA-7 in potentiating TNF-induced NF-kappaB reporter activity. Secretion of MDA-7 was not required for the potentiation of TNF-induced NF-kappaB activation. TNF-induced expression of the NF-kappaB-regulated gene products cyclin D1 and cyclooxygenase-2, were significantly up-regulated by stable expression of MDA-7. Furthermore, MDA-7 expression abolished TNF-induced apoptosis, and suppression of NF-kappaB by IkappaBalpha kinase inhibitors enhanced apoptosis. Overall, our results indicate that stable or transient MDA-7 expression alone does not substantially activate NF-kappaB, but potentiates TNF-induced NF-kappaB activation and NF-kappaB-regulated gene expression. Potentiation of NF-kappaB survival signaling by MDA-7 inhibits TNF-mediated apoptosis.     

The zinc finger protein A20 inhibits TNF-induced NF-kappaB-dependent gene expression by interfering with an RIP- or TRAF2-mediated transactivation signal and directly binds to a novel NF-kappaB-inhibiting protein ABIN.

The zinc finger protein A20 is a tumor necrosis factor (TNF)- and interleukin 1 (IL-1)-inducible protein that negatively regulates nuclear factor-kappa B (NF-kappaB)-dependent gene expression. However, the molecular mechanism by which A20 exerts this effect is still unclear. We show that A20 does not inhibit TNF- induced nuclear translocation and DNA binding of NF-kappaB, although it completely prevents the TNF- induced activation of an NF-kappaB-dependent reporter gene, as well as TNF-induced IL-6 and granulocyte macrophage-colony stimulating factor gene expression. Moreover, NF-kappaB activation induced by overexpression of the TNF receptor-associated proteins TNF receptor-associated death domain protein (TRADD), receptor interacting protein (RIP), and TNF recep- tor-associated factor 2 (TRAF2) was also inhibited by expression of A20, whereas NF-kappaB activation induced by overexpression of NF-kappaB-inducing kinase (NIK) or the human T cell leukemia virus type 1 (HTLV-1) Tax was unaffected. These results demonstrate that A20 inhibits NF-kappaB-dependent gene expression by interfering with a novel TNF-induced and RIP- or TRAF2-mediated pathway that is different from the NIK-IkappaB kinase pathway and that is specifically involved in the transactivation of NF-kappaB. Via yeast two-hybrid screening, we found that A20 binds to a novel protein, ABIN, which mimics the NF-kappaB inhibiting effects of A20 upon overexpression, suggesting that the effect of A20 is mediated by its interaction with this NF-kappaB inhibiting protein, ABIN.     

IL-17 reduces TNF-induced Rantes and VCAM-1 expression

Functional diversity of the memory T-cell-derived cytokine IL-17 was explored at the receptor level. IL-17 inhibited TNF-induced chemokine Rantes expression in human synovial fibroblasts and mouse lung fibroblasts. This inhibitory activity of IL-17 (IC50=0.2 ng/ml) was 6-fold more potent than its stimulatory activity on TNF-alpha-induced IL-6 secretion (ED50=1.2 ng/ml), measured in the same cells. IL-17 also inhibited the TNF-mediated expression of adhesion molecule VCAM-1, and the NF-kappaB binding to the VCAM-1 promoter-specific site, along with the inhibitor of NF-kappaB, IkappaB-beta. Neutralization of the human IL-17 receptor (IL-17R) by M202 antibody competitively reverses the IL-17-induced IL-6 upregulation. However, M202 only partially affected the inhibitions by IL-17. Yet, IL-17R was essential for the Rantes inhibition, as assessed in lung-derived fibroblasts from IL-17R gene deficient mice. Therefore, inhibitory and stimulatory functions of IL-17 involve receptor IL-17R but show distinct dose-responses and in turn different sensitivities to an IL-17R antagonizing antibody.     

Saccharomyces boulardii produces a soluble anti-inflammatory factor that inhibits NF-kappaB-mediated IL-8 gene expression

Saccharomyces boulardii (Sb) is a non-pathogenic yeast that ameliorates intestinal injury and inflammation caused by a wide variety of enteric pathogens. We hypothesized that Sb may exert its probiotic effects by modulation of host cell signaling and pro-inflammatory gene expression. Human HT-29 colonocytes and THP-1 monocytes were stimulated with IL-1beta, TNFalpha or LPS in the presence or absence of Sb culture supernatant (SbS). IL-8 protein and mRNA levels were measured by ELISA and RT-PCR, respectively. The effect of SbS on IkappaB alpha degradation was studied by Western blotting and on NF-kappaB-DNA binding by EMSA. NF-kappaB-regulated gene expression was evaluated by transient transfection of THP-1 cells with a NF-kappaB-responsive luciferase reporter gene. SbS inhibited IL-8 protein production in IL-1beta or TNFalpha stimulated HT-29 cells (by 75% and 85%, respectively; P<0.001) and prevented IL-1beta-induced up-regulation of IL-8 mRNA. SbS also inhibited IL-8 production, prevented IkappaB alpha degradation, and reduced both NF-kappaB-DNA binding and NF-kappaB reporter gene up-regulation in IL-1beta or LPS-stimulated THP-1 cells. Purification and characterization studies indicate that the S. boulardii anti-inflammatory factor (SAIF) is small (<1 kDa), heat stable, and water soluble. The probiotic yeast Saccharomyces boulardii exerts an anti-inflammatory effect by producing a low molecular weight soluble factor that blocks NF-kappaB activation and NF-kappaB-mediated IL-8 gene expression in intestinal epithelial cells and monocytes. SAIF may mediate, at least in part, the beneficial effects of Saccharomyces boulardii in infectious and non-infectious human intestinal disease.     

IL-8 released constitutively by primary bronchial epithelial cells in culture forms an inactive complex with secretory component

The bronchial epithelium is a source of both alpha and beta chemokines and, uniquely, of secretory component (SC), the extracellular ligand-binding domain of the polymeric IgA receptor. Ig superfamily relatives of SC, such as IgG and alpha(2)-macroglobulin, bind IL-8. Therefore, we tested the hypothesis that SC binds IL-8, modifying its activity as a neutrophil chemoattractant. Primary bronchial epithelial cells were cultured under conditions to optimize SC synthesis. The chemokines IL-8, epithelial neutrophil-activating peptide-78, growth-related oncogene alpha, and RANTES were released constitutively by epithelial cells from both normal and asthmatic donors and detected in high m.w. complexes with SC. There were no qualitative differences in the production of SC-chemokine complexes by epithelial cells from normal or asthmatic donors, and in all cases this was the only form of chemokine detected. SC contains 15% N-linked carbohydrate, and complete deglycosylation with peptide N-glycosidase F abolished IL-8 binding. In micro-Boyden chamber assays, no IL-8-dependent neutrophil chemotactic responses to epithelial culture supernatants could be demonstrated. SC dose-dependently (IC(50) approximately 0.3 nM) inhibited the neutrophil chemotactic response to rIL-8 (10 nM) in micro-Boyden chamber assays and also inhibited IL-8-mediated neutrophil transendothelial migration. SC inhibited the binding of IL-8 to nonspecific binding sites on polycarbonate filters and endothelial cell monolayers, and therefore the formation of haptotactic gradients, without effects on IL-8 binding to specific receptors on neutrophils. The data indicate that in the airways IL-8 may be solubilized and inactivated by binding to SC.     

IL-33 enhances Siglec-8 mediated apoptosis of human eosinophils

IL-33 activates eosinophils directly via the ST2 receptor. Like IL-5, IL-33 induces eosinophilia and eosinophilic airway inflammation in mouse models and primes human eosinophil responses. Previously, we reported that IL-5 priming enhances Siglec-8 mediated mitochondrial and reactive oxygen species (ROS)-dependent eosinophilic apoptosis and eliminates caspase dependence of this cell death process. Whether IL-33, like IL-5, augments pro-apoptotic pathways involving receptors such as Siglec-8 and in a similar manner has not been explored. Annexin-V labeling was performed to detect apoptosis in human eosinophils pre-incubated with or without a range of concentrations of IL-33 and/or IL-5 in the presence or absence of Siglec-8 monoclonal antibody (mAb) 2C4 and inhibitors of caspases. Tetramethyl-rhodamine staining was used as a marker of mitochondrial membrane potential loss and injury. ROS production was determined by measuring the superoxide dismutase-inhibitable reduction of cytochrome c. Cleavage of poly(ADP-ribose) polymerase (PARP) was assessed using Western blotting. Eosinophils cultured alone or with mAb 2C4 underwent low levels of apoptosis at 24 h. 2C4-induced eosinophil apoptosis was markedly and equally enhanced after culture for 24 h with either IL-33 or IL-5, although IL-5 was more potent. Effects on apoptosis with IL-33 and IL-5 were synergistic. In contrast, percentages of cells exhibiting reduced mitochondrial membrane potential were greater with IL-33 than IL-5 and effects of these cytokines were also synergistic. Antimycin, an inhibitor of mitochondrial electron transport, almost completely inhibited 2C4-induced apoptosis with either IL-33 or IL-5. Surprisingly, 2C4-induced eosinophil ROS production was significantly enhanced with IL-5 but not IL-33. Siglec-8-mediated apoptosis in the presence of IL-33 was more sensitive in magnitude than IL-5 to inhibition by the pan-caspase inhibitor Z-VAD-FMK, yet both cytokine conditions were associated with PARP cleavage. These data demonstrate that IL-33 is as effective but less potent than IL-5 in enhancing Siglec-8-mediated eosinophil apoptosis, and can synergize with IL-5. Eosinophils primed by IL-33 and/or IL-5 in vivo would be expected to display enhanced susceptibility to undergoing Siglec-8-induced apoptosis.     

TLR8-mediated NF-kappaB and JNK activation are TAK1-independent and MEKK3-dependent

TLR8-mediated NF-kappaB and IRF7 activation are abolished in human IRAK-deficient 293 cells and IRAK4-deficient fibroblast cells. Both wild-type and kinase-inactive mutants of IRAK and IRAK4, respectively, restored TLR8-mediated NF-kappaB and IRF7 activation in the IRAK- and IRAK4-deficient cells, indicating that the kinase activity of IRAK and IRAK4 is probably redundant for TLR8-mediated signaling. We recently found that TLR8 mediates a unique NF-kappaB activation pathway in human 293 cells and mouse embryonic fibroblasts, accompanied only by IkappaBalpha phosphorylation and not IkappaBalpha degradation, whereas interleukin (IL)-1 stimulation causes both IkappaBalpha phosphorylation and degradation. The intermediate signaling events mediated by IL-1 (including IRAK modifications and degradation and TAK1 activation) were not detected in cells stimulated by TLR8 ligands. TLR8 ligands trigger similar levels of IkappaBalpha phosphorylation and NF-kappaB and JNK activation in TAK1(-/-) mouse embryo fibroblasts (MEFs) as compared with wild-type MEFs, whereas lack of TAK1 results in reduced IL-1-mediated NF-kappaB activation and abolished IL-1-induced JNK activation. The above results indicate that although TLR8-mediated NF-kappaB and JNK activation are IRAK-dependent, they do not require IRAK modification and are TAK1-independent. On the other hand, TLR8-mediated IkappaBalpha phosphorylation, NF-kappaB, and JNK activation are completely abolished in MEKK3(-/-) MEFs, whereas IL-1-mediated signaling was only moderately reduced in these deficient MEFs as compared with wild-type cells. The differences between IL-1R- and TLR8-mediated NF-kappaB activation are also reflected at the level of IkappaB kinase (IKK) complex. TLR8 ligands induced IKKgamma phosphorylation, whereas IKKalpha/beta phosphorylation and IKKgamma ubiquitination that can be induced by IL-1 were not detected in cells treated with TLR8 ligands. We postulate that TLR8-mediated MEKK3-dependent IKKgamma phosphorylation might play an important role in the activation of IKK complex, leading to IkappaBalpha phosphorylation.