Bacterial lipopolysaccharide activates nuclear factor-kappaB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes

Bacterial lipopolysaccharide (LPS)-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells, play an important role in the pathogenesis of Gram-negative bacteria-induced sepsis syndrome. Activation of NF-kappaB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. Here we investigated the role and involvement of interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) signal transducer molecules in LPS signaling in human dermal microvessel endothelial cells (HDMEC) and THP-1 monocytic cells. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-kappaB signaling cascade. In transient cotransfection experiments, dominant negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-kappaB-luciferase activity. LPS-induced NF-kappaB activation was not inhibited by a dominant negative mutant of TRAF2 that is involved in TNF signaling. LPS-induced activation of NF-kappaB-responsive reporter gene was not inhibited by IL-1 receptor antagonist. TLR2 and TLR4 were expressed on the cell surface of HDMEC and THP-1 cells. These findings suggest that a signal transduction molecule in the LPS receptor complex may belong to the IL-1 receptor/toll-like receptor (TLR) super family, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in mononuclear phagocytes and endothelial cells.     

Lipopolysaccharide and interleukin 1 inhibit interferon-gamma-induced Fc receptor expression on human monocytes

The objectives of these studies were to study the effects of bacterial lipopolysaccharide (LPS) on interferon-gamma (IFN-gamma)-induced Fc receptor expression on human monocytes and to examine whether these effects were mediated through stimulation of interleukin 1 (IL-1) production. Fc receptor expression was determined by binding of monomeric monoclonal murine immunoglobulin (Ig)G2a and cytofluorographic analysis. IL-1 activity in monocyte supernatants and lysates was assayed by augmentation of mitogen-induced murine thymocyte proliferation. IFN-gamma induced the expression of Fc receptors on human monocytes that were specific for murine IgG2a. This induction was inhibited by the addition of LPS in amounts as low as 2 to 8 pg/ml. LPS inhibition of IFN-gamma-induced Fc receptor expression was paralleled by the appearance of IL-1 in monocyte lysates and supernatants. The addition of purified human or recombinant IL-1 beta at the initiation of culture similarly inhibited the expression of IFN-gamma-induced Fc receptors on the monocytes. LPS also inhibited Fc receptor expression on the human myelomonocytic cell line THP-1 after induction with IFN-gamma or phorbol myristate acetate alone or with both agents together. This inhibition also was paralleled by the production of IL-1 but the addition of exogenous IL-1 to the THP-1 cells had no effect on IFN-gamma-induced Fc receptor expression. Tumor necrosis factor (TNF) inhibited IFN-gamma-induced Fc receptor expression on human monocytes but was much less potent than comparable amounts of IL-1. TNF also did not inhibit Fc receptor expression on THP-1 cells. In fact, IL-1 or TNF led to an enhancement in IFN-gamma-induced Fc receptor expression on THP-1 cells. These results indicate that LPS can inhibit IFN-gamma-induced Fc receptor expression on human monocytes and that IL-1 and TNF may mediate these effects of LPS. Thus, an autocrine or paracrine role is suggested for these cytokines. The possibility exists that intracellular IL-1 resulting from LPS stimulation may be at least in part responsible for inhibition of Fc receptor expression.     

A glycosylation-deficient endothelial cell mutant with modified responses to transforming growth factor-beta and other growth inhibitory cytokines: evidence for multiple growth inhibitory signal transduction pathways.

An endothelial cell line (M40) resistant to growth inhibition by transforming growth factor-beta type 1 (TGF beta 1) was isolated by chemical mutagenesis and growth in the presence of TGF beta 1. Like normal endothelial cells, this mutant is characterized by high expression of type II TGF beta receptor and low expression of type I TGF beta receptor. However, the mutant cells display a type II TGF beta receptor of reduced molecular weight as a result of a general defect in N-glycosylation of proteins. The alteration does not impair TGF beta 1 binding to cell surface receptors or the ability of TGF beta 1 to induce fibronectin or plasminogen activator inhibitor-type I production. M40 cells were also resistant to growth inhibition by tumor necrosis factor alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) but were inhibited by interferon-gamma (IFN gamma) and heparin. These results imply that TGF beta 1, TNF alpha, and IL-1 alpha act through signal transducing pathways that are separate from pathways for IFN gamma and heparin. Basic fibroblast growth factor was still mitogenic for M40, further suggesting that TGF beta 1, TNF alpha, and IL-1 alpha act by direct inhibition of cell growth rather than by interfering with growth stimulatory pathways.     

TNFα and IL-1β are mediated by both TLR4 and Nod1 pathways in the cultured HAPI cells stimulated by LPS

A growing body of evidence recently suggests that glial cell activation plays an important role in several neurodegenerative diseases and neuropathic pain. Microglia in the central nervous system express toll-like receptor 4 (TLR4) that is traditionally accepted as the primary receptor of lipopolysaccharide (LPS). LPS activates TLR4 signaling pathways to induce the production of proinflammatory molecules. In the present studies, we verified the LPS signaling pathways using cultured highly aggressively proliferating immortalized (HAPI) microglial cells. We found that HAPI cells treated with LPS upregulated the expression of TLR4, phospho-JNK (pJNK) and phospho-NF-κB (pNF-κB), TNFα and IL-1β. Silencing TLR4 with siRNA reduced the expression of pJNK, TNFα and IL-1β, but not pNF-κB in the cells. Inhibition of JNK with SP600125 (a JNK inhibitor) decreased the expression of TNFα and IL-1β. Unexpectedly, we found that inhibition of Nod1 with ML130 significantly reduced the expression of pNF-κB. Inhibition of NF-κB also reduced the expression of TNFα and IL-1β. Nod1 ligand, DAP induced the upregulation of pNF-κB which was blocked by Nod1 inhibitor. These data indicate that LPS-induced pJNK is TLR4-dependent, and that pNF-κB is Nod1-dependent in HAPI cells treated with LPS. Either TLR4-JNK or Nod1-NF-κB pathways is involved in the expression of TNFα and IL-1β.     

Synergistic induction of interleukin 2 receptor (TAC) expression on YT cells by simultaneous activation of distinct signal transduction pathways

The human NK-like leukemic cell line YT was used to study interleukin 2 receptor (IL-2R; Tac) expression induced by activators of distinct signal transduction pathways. Tac expression was induced by active phorbol esters (12-O-tetradecanoylphorbol 13-acetate [TPA] and 4 beta-phorbol 12,13-didecanoate), which directly activate protein kinase C (PKC), as well as forskolin (FK), a stimulator of adenylate cyclase. A synergistic effect on Tac expression was obtained by simultaneous stimulation with optimal concentrations of phorbol esters and FK. Inactive phorbol esters (4 beta-phorbol, 4 alpha-phorbol 12,13-didecanoate) and the inactive analog of FK (1,9-dideoxyforskolin) had no effect on Tac expression. The active phorbol esters synergized also with interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF alpha) in Tac expression. Staurosporine, a potent inhibitor of PKC in vitro, inhibited Tac expression marginally in YT cells stimulated with FK, and enhanced Tac expression in cultures treated with TPA, TNF alpha, or IL-1. Based on the assumption that synergistic effects are observed when two agonists use different signaling pathways, these findings provide evidence that IL-1, TNF, and TPA use different pathways/regulatory elements to regulate Tac expression on the cell surface. Synergistic upregulation of Tac expression by simultaneous activation of distinct pathways may be an important mechanism to modulate the immune response.     

Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of the RID, E3-14.7K, and E1B-19K genes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus infections. The ability of adenovirus to inhibit killing through these receptors may prolong acute and persistent infections.     

Adenovirus RIDbeta subunit contains a tyrosine residue that is critical for RID-mediated receptor internalization and inhibition of Fas- and TRAIL-induced apoptosis

The adenovirus-encoded receptor internalization and degradation (RID) protein (previously named E3-10.4K/14.5K), which is composed of RIDalpha and RIDbeta subunits, down-regulates a number of cell surface receptors in the tumor necrosis factor (TNF) receptor superfamily, namely Fas, TRAIL receptor 1, and TRAIL receptor 2. Down-regulation of these "death" receptors protects adenovirus-infected cells from apoptosis induced by the death receptor ligands Fas ligand and TRAIL. RID also down-regulates certain tyrosine kinase cell surface receptors, especially the epidermal growth factor receptor (EGFR). RID-mediated Fas and EGFR down-regulation occurs via endocytosis of the receptors into endosomes followed by transport to and degradation within lysosomes. However, the molecular interactions underlying this function of RID are unknown. To investigate the molecular determinants of RIDbeta that are involved in receptor down-regulation, mutations within the cytoplasmic tail of RIDbeta were constructed and the mutant proteins were analyzed for their capacity to internalize and degrade Fas and EGFR and to protect cells from death receptor ligand-induced apoptosis. The results demonstrated the critical nature of a tyrosine residue near the RIDbeta C terminus; mutation of this residue to alanine abolished RID function. Mutating the tyrosine to phenylalanine did not abolish the function of RID, arguing that phosphorylation of the tyrosine is not required for function. These data suggest that this tyrosine residue forms part of a tyrosine-based sorting signal (Yxxphi). Additional mutations that target another potential sorting motif and several possible protein-protein interaction motifs had no discernible effect on RID function. It was also demonstrated that mutation of serine 116 to alanine eliminated phosphorylation of RIDbeta but did not affect any of the functions of RID that were examined. These results suggest a model in which the tyrosine-based sorting signal in RID plays a role in RID's ability to down-regulate receptors.     

Effects of soluble TNF receptor treatment on lipopolysaccharide-induced myocardial cytokine expression

Tumor necrosis factor (TNF)-alpha plays a key role in the pathogenesis of septic shock syndrome, and myocardial TNF-alpha expression may contribute to this pathophysiology. We examined the myocardial expression of TNF-alpha-related cytokines and chemokines in mice exposed to lipopolysaccharide (LPS) and tested the effects of anti-TNF therapy on myocardial cytokine expression. Cytokine mRNA levels were measured by RNase protection assay, and protein levels in the plasma and myocardium were assessed by enzyme-linked immunosorbent assays. LPS (4 microg/g body wt ip) induced marked cytokine expression, including TNF-alpha, interleukin (IL)-1beta, IL-6, and monocyte chemotactic protein (MCP)-1, in both the plasma and myocardium. Pretreatment with adenovirus-mediated TNF receptor fusion protein (AdTNFR1; 10(9) plaque-forming units iv) decreased plasma cytokine levels. In contrast, whereas myocardial IL-1beta expression was also suppressed, expression of IL-6 and MCP-1 was not inhibited by AdTNFR1. In summary, anti-TNF treatment differentially altered the cytokine expression in the plasma and myocardium during endotoxemia. Inability to block myocardial expression of IL-6 and MCP-1 suggests a possible mechanism for the failure of anti-TNF therapies in the treatment of endotoxin shock.     

Ganglioside GD1a impedes lipopolysaccharide-induced maturation of human dendritic cells

Immunosuppressive membrane gangliosides are released by tumor cells and inhibit normal antigen presenting cell (APC) function. To better understand this process, we have studied the effect of gangliosides on lipopolysaccharide (LPS)-induced maturation of human dendritic cells (DCs). Immature DCs were generated in vitro from human peripheral blood monocytes and were exposed for 72 h to a highly purified ganglioside, G(D1a). During the last 24 h, LPS was added to effect maturation. As assessed by fluorescence activated cell sorting (FACS) analysis, incubation in 50 microM G(D1a) significantly blunted the LPS-induced maturation of the dendritic cells. The expected up-regulation of expression of the co-stimulatory molecules CD80 and CD86 was ablated and that of CD40 was reduced, as were surface CD83 expression and intracellular CD208 production. In addition, ganglioside pretreatment of DC markedly inhibited the allostimulatory capacity and partially prevented the down-regulation of FITC-dextran uptake characteristic of LPS-activated DC. Furthermore, ganglioside-exposed DC also evidenced a broad down-regulation of the cytokine release that is normally initiated by LPS exposure, i.e., there was no increase in IL-1 beta, IL-6, IL-10, IL-12, or tumor necrosis factor (TNF)-alpha release. That a common mechanism may underlie these defects was suggested by the finding that G(D1a) exposure of DC also inhibited the nuclear binding of NF-kappa B that is normally induced by LPS. These results suggest that tumor gangliosides may blunt the anti-tumor immune response in vivo by binding and interfering with dendritic cell maturation.     

The lysosomal transmembrane protein 9B regulates the activity of inflammatory signaling pathways

The intracellular signaling pathway by which tumor necrosis factor (TNF) induces its pleiotropic actions is well characterized and includes unique components as well as modules shared with other signaling pathways. In addition to the currently known key effectors, further molecules may however modulate the biological response to TNF. In our attempt to characterize novel regulators of the TNF signaling cascade, we have identified transmembrane protein 9B (TMEM9B, c11orf15) as an important component of TNF signaling and a module shared with the interleukin 1beta (IL-1beta) and Toll-like receptor (TLR) pathways. TMEM9B is a glycosylated protein localized in membranes of the lysosome and partially in early endosomes. The expression of TMEM9B is required for the production of proinflammatory cytokines induced by TNF, IL-1beta, and TLR ligands but not for apoptotic cell death triggered by TNF or Fas ligand. TMEM9B is essential in TNF activation of both the NF-kappaB and MAPK pathways. It acts downstream of RIP1 and upstream of the MAPK and IkappaB kinases at the level of the TAK1 complex. These findings indicate that TMEM9B is a key component of inflammatory signaling pathways and suggest that endosomal or lysosomal compartments regulate these pathways.     

Involvement of protein tyrosine kinase in Toll-like receptor 4-mediated NF-kappa B activation in human peripheral blood monocytes

Bacterial lipopolysaccharide (LPS) is a powerful activator of the innate immune system. Exposure to LPS induces an inflammatory reaction in the lung mediated primarily by human blood monocytes and alveolar macrophages, which release an array of inflammatory chemokines and cytokines including IL-8, TNF-alpha, IL-1beta, and IL-6. The signaling mechanisms utilized by LPS to stimulate the release of cytokines and chemokines are still incompletely understood. Pretreatment with the protein tyrosine kinase-specific inhibitors genistein and herbimycin A effectively blocked LPS-induced NF-kappaB activation as well as IL-8 gene expression in human peripheral blood monocytes. However, when genistein was added 2 min after the addition of LPS, no inhibition was observed. Utilizing a coimmunoprecipitation assay, we further showed that LPS-stimulated tyrosine phosphorylation of Toll-like receptor 4 (TLR4) may be involved in downstream signaling events induced by LPS. These findings provide evidence that LPS-induced NF-kappaB activation and IL-8 gene expression use a signaling pathway requiring protein tyrosine kinase and that such regulation may occur through tyrosine phosphorylation of TLR4.     

Lipopolysaccharide-mediated reactive oxygen species and signal transduction in the regulation of interleukin-1 gene expression

Lipopolysaccharide (LPS) stimulates macrophages to release inflammatory cytokines, interleukin-1 beta (IL-1), and tumor necrosis factor (TNF). LPS-induced TNF suppresses scavenger receptor functions in macrophages (van Lenten, B. J., and Fogelman, A. M. (1992) J. Immunol. 148, 112-116), which is regulated by TNF-mediated protein kinases (Hsu, H. Y., and Twu, Y. C. (2000) J. Biol. Chem. 275, 41035-41048). To examine the molecular mechanism for LPS induction of IL-1 in macrophages, we demonstrated that LPS quickly stimulated reactive oxygen species (ROS), and 3 h later induced prointerleukin-1 beta (pro-IL-1, precursor of IL-1) production and IL-1 secretion. LPS stimulated pro-IL-1 message/protein between 3 and 10 h; however, there was a 40% reduction of pro-IL-1 in preincubation of the antioxidant, N-acetylcysteine (NAC). Moreover, NAC moderated LPS-induced IL-1 secretion partially via interleukin 1-converting enzyme. The maximal activity of LPS-induced ERK, JNK, and p38 was 12- (30 min), 5- (30 min), and 16-fold (15 min), respectively. In contrast, NAC reduced ERK activity to 60% and decreased p38 activity to the basal level, but JNK activity was induced 2-fold. Furthermore, the pharmacological antagonists LY294002, SB203580, curcumin, calphostin C, and PD98059 revealed the diverse roles of LPS-mediated protein kinases in pro-IL-1. On the other hand, NAC and diphenyleneiodonium chloride partially inhibited LPS-induced Rac activity and protein-tyrosine kinase (PTK), indicating that LPS-mediated ROS and NADPH oxidase correspond to Rac activation and IL-1 expression. Our findings establish for the first time that LPS-mediated PTK/phosphatidylinositol 3-kinase/Rac/p38 pathways play a more important role than pathways of PTK/PKC/MEK/ERK and of PTK/phosphatidylinositol 3-kinase/Rac/JNK in the regulation of pro-IL-1/IL-1. The findings also further elucidate the critical role of LPS-mediated ROS in signal transduction pathways. Our results suggest that understanding LPS-transduced signals in IL-1 induction upon the antibacterial action of macrophages should provide a therapeutic strategy for aberrant inflammatory responses leading to severe cellular injury or concurrent multiorgan septic damage.     

CXC chemokine receptor 4 expression and function in human astroglioma cells

Chemokines constitute a superfamily of proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the CNS, are a source of chemokines within the diseased brain. Specifically, we have shown that primary human astrocytes and human astroglioma cell lines produce the CXC chemokines IFN-gamma-inducible protein-10 and IL-8 and the CC chemokines monocyte chemoattractant protein-1 and RANTES in response to stimuli such as TNF-alpha, IL-1beta, and IFN-gamma. In this study, we investigated chemokine receptor expression and function on human astroglioma cells. Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was observed upon treatment with the cytokines TNF-alpha and IL-1beta. The peak of CXCR4 expression in response to TNF-alpha and IL-1beta was 8 and 4 h, respectively. CXCR4 protein expression was also enhanced upon treatment with TNF-alpha and IL-1beta (2- to 3-fold). To study the functional relevance of CXCR4 expression, stable astroglioma transfectants expressing high levels of CXCR4 were generated. Stimulation of cells with the ligand for CXCR4, stromal cell-derived factor-1alpha (SDF-1alpha), resulted in an elevation in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase cascade, specifically, extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest, SDF-1alpha treatment induced expression of the chemokines monocyte chemoattractant protein-1, IL-8, and IFN-gamma-inducible protein-10. SDF-1alpha-induced chemokine expression was abrogated upon inclusion of U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK signaling cascade is involved in this response. Collectively, these data suggest that CXCR4-mediated signaling pathways in astroglioma cells may be another mechanism for these cells to express chemokines involved in angiogenesis and inflammation.     

Human cytomegalovirus blocks tumor necrosis factor alpha- and interleukin-1beta-mediated NF-kappaB signaling

NF-kappaB plays an important role in the early cellular response to pathogens by activating genes involved in inflammation, immune response, and cell proliferation and survival. NF-kappaB is also utilized by many viral pathogens, like human cytomegalovirus (HCMV), to activate their own gene expression programs, reflecting intricate roles for NF-kappaB in both antiviral defense mechanisms and viral physiology. Here we show that the NF-kappaB signaling pathway stimulated by proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) becomes inhibited in HCMV-infected cells. The block to NF-kappaB signaling is first noticeable during the early phase of infection but is fully established only at later times. Biochemical and genetic evidence demonstrates that the viral inhibition of proinflammatory signaling by distinct cytokines occurs upstream of the convergence point of NF-kappaB-activating pathways, i.e., the IkappaB kinase complex, and that it is mediated via different mechanisms. Consistent with this, we further show that an HCMV variant that has lost the ability to downregulate TNF-alpha-induced NF-kappaB signaling also fails to downregulate surface expression of TNF receptor 1, thereby mechanistically linking the inhibition of TNF-alpha-induced NF-kappaB signaling by HCMV to TNF receptor targeting. Our data support a model whereby HCMV inhibits cytokine-induced NF-kappaB signaling at later times during infection, and we suggest that this contributes to the inhibition of the cell's antiviral defense program.