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.     

Implications of proteasome inhibition: an enhanced macrophage phenotype

The objective of this study was to elucidate the role of the cellular proteasome on endotoxin-mediated activation of the macrophage. To study this role, THP-1 cells were stimulated with lipopolysaccharide (LPS) with selective cells being pretreated with the proteasome inhibitor, lactacystin or MG-132. LPS stimulation led to the phosphorylation and degradation of IRAK, followed by activation of JNK/SAPK, ERK 1/2, and p38. Subsequently, LPS induced the degradation of IkappaB, and the nuclear activation of NF-kappaB and AP-1. Activation of these pathways was associated with the production of IL-6, IL-8, IL-10, and TNF-alpha. Proteasome inhibition with either lactacystin or MG-132 attenuated LPS-induced IRAK degradation, and enhanced activation of JNK/SAPK, ERK 1/2, and p38. Proteasome inhibition, also, led to increased LPS-induced AP-1 activation, and attenuated LPS-induced IkappaB degradation resulting in abolished NF-kappaB activation. Proteasome inhibition led to significant modulation of LPS-induced cytokine production; increased IL-10, no change in IL-6, and decreased IL-8, and TNF-alpha. Thus, this study demonstrates that cellular proteasome is critical to regulation of LPS-induced signaling within the macrophage, and inhibition of the proteasome results in a conversion to an anti-inflammatory phenotype.     

Lipopolysaccharide activated phosphatidylcholine-specific phospholipase C and induced IL-8 and MCP-1 production in vascular endothelial cells

Secretion of proinflammatory cytokines by lipopolysaccharide (LPS) activated vascular endothelial cells (VECs) contributes substantially to the pathogenesis of several inflammatory diseases such as atherosclerosis and septic shock. However, the mechanisms involved in this process are not well understood. Here, we investigated the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in LPS-induced IL-8 and MCP-1 production in VECs. The results showed that LPS elevated the level of PC-PLC and the production of IL-8 and MCP-1 in Human umbilical vein vascular endothelial cells (HUVECs). Blocking the function of PC-PLC by exploiting the neutralization antibody of PC-PLC or tricyclodecan-9-yl-xanthogenate (D609), an inhibitor of PC-PLC, significantly inhibited LPS-induced production of IL-8 and MCP-1 in HUVECs. Furthermore, the in vivo experimental results showed that the levels of PC-PLC, IL-8, and MCP-1 in the aortic endothelium and serum were increased in mice injected with LPS. The increased levels of these molecules were also inhibited by the treatment with D609. The data suggested that blocking PC-PLC function significantly inhibited LPS-induced IL-8 and MCP-1 production in cultured HUVECs and in vivo. PC-PLC might be a potential target for therapy in inflammation associated-diseases such as atherosclerosis.     

GEF-H1/RhoA signalling pathway mediates lipopolysaccharide-induced intercellular adhesion molecular-1 expression in endothelial cells via activation of p38 and NF-κB

The purpose of study is to investigate the effects of GEF-H1/RhoA pathway in regulating intercellular adhesion molecule-1 (ICAM-1) expression in lipopolysaccharide (LPS)-activated endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) to LPS induced GEF-H1 and ICAM-1 expression in dose- and time-dependent up-regulating manners. Pretreatment with Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activity, reduced LPS-related phosphorylation of p65 at Ser 536 in a dose-dependent manner. Inhibition of TLR4 expression significantly blocked LPS-induced RhoA activity, NF-κB transactivation, GEF-H1 and ICAM-1 expression. Coimmunoprecipitation assay indicated that LPS-activated TLR4 and GEF-H1 formed a signalling complex, suggesting that LPS, acting through TLR4, stimulates GEF-H1 expression and RhoA activity, and thereby induces NF-κB transactivation and ICAM-1 gene expression. However, GEF-H1/RhoA regulates LPS-induced NF-κB transactivation and ICAM-1 expression in a MyD88-independent pathway because inhibition of MyD88 expression could not block LPS-induced RhoA activity. Furthermore, pretreatment with Y-27632, an inhibitor of ROCK, significantly reduced LPS-induced p38, ERK1/2 and p65 phosphorylation, indicating that ROCK acts as an upstream effector of p38 and ERK1/2 to promote LPS-induced NF-κB transactivation and ICAM-1 expression. What is more, the p38 inhibitor (SB203580) but not ERK1/2 inhibitor (PD98059) blocked LPS-induce NF-κB transactivation and ICAM-1 expression, which demonstrates that RhoA mediates LPS-induced NF-κB transactivation and ICAM-1 expression dominantly through p38 but not ERK1/2 activation. In summary, our data suggest that LPS-induced ICAM-1 synthesis in HUVECs is regulated by GEF-H1/RhoA-dependent signaling pathway via activation of p38 and NF-κB.     

Inhibition by gangliosides of the specific binding of lipopolysaccharide (LPS) to human monocytes prevents LPS-induced interleukin-1 production

In a previous work we have reported that gangliosides inhibit interleukin 1 (IL-1) release by human monocytes stimulated with lipopolysaccharides (LPS). In the present study we extend this work to IL-1 production and we correlate these observations with the capacity of gangliosides to inhibit the binding of radiolabeled LPS to its specific receptor on human monocytes. Preincubation of 3H-LPS with crude bovine brain gangliosides, as well as purified human brain mono, di, and trisialogangliosides (GM1, GD1a, and GT1b, respectively), led to an inhibition of the specific binding of LPS to the cell surface. Neither ceramide nor N-acetyl neuraminic acid, two constituents of gangliosides, was able by itself to inhibit the specific binding. A strict parallelism was observed with respect to inhibition on LPS-induced IL-1 production and release. Asialoganglioside (asialo-GM1) was inactive in both assays, suggesting that the N-acetyl neuraminic acid plays a role within the ganglioside molecule, with respect to inhibitory activity. We conclude that LPS-induced production and release by human monocytes is not due to a signal triggered by nonspecific absorption and/or intercalation of LPS into cell membrane which occur through hydrophobic interaction mediated by the lipid A region. Addition of exogenous sialogangliosides which blocked LPS-induced IL-1 production and release, did not modify significantly the nonspecific binding of 3H-LPS, whereas it did inhibit the specific binding which is mediated by the polysaccharide moiety of the LPS molecule. These results establish a relationship between the specific endotoxin receptor on monocytes and a LPS-induced cellular function.     

Suppression of osteoprotegerin expression by prostaglandin E2 is crucially involved in lipopolysaccharide-induced osteoclast formation

LPS is a potent stimulator of bone resorption in inflammatory diseases. The mechanism by which LPS induces osteoclastogenesis was studied in cocultures of mouse osteoblasts and bone marrow cells. LPS stimulated osteoclast formation and PGE(2) production in cocultures of mouse osteoblasts and bone marrow cells, and the stimulation was completely inhibited by NS398, a cyclooxygenase-2 inhibitor. Osteoblasts, but not bone marrow cells, produced PGE(2) in response to LPS. LPS-induced osteoclast formation was also inhibited by osteoprotegerin (OPG), a decoy receptor of receptor activator of NF-kappaB ligand (RANKL), but not by anti-mouse TNFR1 Ab or IL-1 receptor antagonist. LPS induced both stimulation of RANKL mRNA expression and inhibition of OPG mRNA expression in osteoblasts. NS398 blocked LPS-induced down-regulation of OPG mRNA expression, but not LPS-induced up-regulation of RANKL mRNA expression, suggesting that down-regulation of OPG expression by PGE(2) is involved in LPS-induced osteoclast formation in the cocultures. NS398 failed to inhibit LPS-induced osteoclastogenesis in cocultures containing OPG knockout mouse-derived osteoblasts. IL-1 also stimulated PGE(2) production in osteoblasts and osteoclast formation in the cocultures, and the stimulation was inhibited by NS398. As seen with LPS, NS398 failed to inhibit IL-1-induced osteoclast formation in cocultures with OPG-deficient osteoblasts. These results suggest that IL-1 as well as LPS stimulates osteoclastogenesis through two parallel events: direct enhancement of RANKL expression and suppression of OPG expression, which is mediated by PGE(2) production.     

Lipopolysaccharide-induced apoptosis of endothelial cells and its inhibition by vascular endothelial growth factor

Endothelial injury is a major manifestation of septic shock induced by LPS. Recently, LPS was shown to induce apoptosis in different types of endothelial cells. In this study, we observed that pretreatment with vascular endothelial growth factor (VEGF), a known cell survival factor, blocked LPS-induced apoptosis in endothelial cells. We then further defined this LPS-induced apoptotic pathway and its inhibition by VEGF. We found that LPS treatment increased caspase-3 and caspase-1 activities and induced the cleavage of focal adhesion kinase. LPS also augmented expression of the pro-apoptotic protein Bax and the tumor suppressor gene p53. The pro-apoptotic Bax was found to translocate to the mitochondria from the cytosol following stimulation with LPS. Pretreatment of endothelial cells with VEGF inhibited the induction of both Bax and p53 as well as the activation of caspase-3. These data suggest that VEGF inhibits LPS-induced endothelial apoptosis by blocking pathways that lead to caspase activation.     

TGF-beta1 represses activation and resultant death of microglia via inhibition of phosphatidylinositol 3-kinase activity

Overactivation of microglial cells may cause severe brain tissue damage in various neurodegenerative diseases. Therefore, the overactivation of microglia should be repressed by any means. The present study investigated the potential mechanism and signaling pathway for the repressive effect of TGF-beta1, a major anti-inflammatory cytokine, on overactivation and resultant death of microglial cells. A bacterial endotoxin LPS stimulated expression of inducible NO synthase (iNOS) and caused death in cultured microglial cells. TGF-beta1 markedly blocked these LPS effects. However, the LPS-evoked death of microglial cells was not solely attributed to excess production of NO. Because phosphatidylinositol 3-kinase (PI3K) was previously shown to play a crucial role in iNOS expression and cell survival signals, we further studied whether PI3K signaling was associated with the suppressive effect of TGF-beta1. Like TGF-beta1, the PI3K inhibitor LY294002 blocked iNOS expression and death in cultured microglial cells. Both TGF-beta1 and LY294002 decreased the activation of caspases 3 and 11 and the mRNA expression of various kinds of inflammatory molecules caused by LPS. TGF-beta1 was further found to decrease LPS-induced activation of PI3K and Akt. TGF-beta1 and LY294002 suppressed LPS-induced p38 mitogen-activated kinase and c-Jun N-terminal kinase activity. In contrast, TGF-beta1 and LY294002 enhanced LPS-induced NF-kappaB activity. Our data indicate that TGF-beta1 protect normal or damaged brain tissue by repressing overactivation of microglial cells via inhibition of PI3K and its downstream signaling molecules.     

A role for neutral sphingomyelinase activation in the inhibition of LPS action by phospholipid oxidation products

Previous studies from our laboratory and others presented evidence that oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylcholine (OxPAPC) and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylethanolamine can inhibit lipopolysaccharide (LPS)-mediated induction of interleukin-8 (IL-8) in endothelial cells. Using synthetic derivatives of phosphatidylethanolamine, we now demonstrate that phospholipid oxidation products containing alpha,beta-unsaturated carboxylic acids are the most active inhibitors we examined. 5-Keto-6-octendioic acid ester of 2-phosphatidylcholine (KOdiA-PC) was 500-fold more inhibitory than OxPAPC, being active in the nanomolar range. Our studies in human aortic endothelial cells identify one important mechanism of the inhibitory response as involving the activation of neutral sphingomyelinase. There is evidence that Toll-like receptor-4 and other members of the LPS receptor complex must be colocalized to the caveolar/lipid raft region of the cell, where sphingomyelin is enriched, for effective LPS signaling. Previous work from our laboratory suggested that OxPAPC could disrupt this caveolar fraction. These studies present evidence that OxPAPC activates sphingomyelinase, increasing the levels of 16:0, 22:0, and 24:0 ceramide and that the neutral sphingomyelinase inhibitor GW4869 reduces the inhibitory effect of OxPAPC and KOdiA-PC. We also show that cell-permeant C6 ceramide, like OxPAPC, causes the inhibition of LPS-induced IL-8 synthesis and alters caveolin distribution similar to OxPAPC. Together, these data identify a new pathway by which oxidized phospholipids inhibit LPS action involving the activation of neutral sphingomyelinase, resulting in a change in caveolin distribution. Furthermore, we identify specific oxidized phospholipids responsible for this inhibition.     

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.     

Novel and detrimental effects of lipopolysaccharide on in vitro generation of immature dendritic cells: involvement of mitogen-activated protein kinase p38

Dendritic cells (DCs) are recognized as major players in the regulation of immune responses to a variety of Ags, including bacterial agents. LPS, a Gram-negative bacterial cell wall component, has been shown to fully activate DCs both in vitro and in vivo. LPS-induced DC maturation involves activation of p38, extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases, and NF-kappaB. Blocking p38 inhibits LPS-induced maturation of DCs. In this study we investigated the role of LPS in the in vitro generation of immature DCs. We report here that in contrast to the observed beneficial effects on DCs, the presence of LPS in monocyte culture retarded the generation of immature DCs. LPS not only impaired the morphology and reduced the yields of the cultured cells, but also inhibited the up-regulation of surface expression of CD1a, costimulatory and adhesion molecules. Furthermore, LPS up-regulated the secretion of IL-1beta, IL-6, IL-8, IL-10, and TNF-alpha; reduced Ag presentation capacity; and inhibited phosphorylation of ERK, but activated p38, leading to a reduced NF-kappaB activity in treated cells. Neutralizing Ab against IL-10, but not other cytokines, partially blocked the effects of LPS. Inhibiting p38 (by inhibitor SB203580) restored the morphology, phenotype, and Ag presentation capacity of LPS-treated cells. SB203580 also inhibited LPS-induced production of IL-1beta, IL-10, and TNF-alpha; enhanced IL-12 production; and recovered the activity of ERK and NF-kappaB. Thus, our study reveals that LPS has dual effects on DCs that are biologically important: activating existing DCs to initiate an immune response, and inhibiting the generation of new DCs to limit such a response.     

Safrole oxide induces human umbilical vein endothelial cell transdifferentiation to 5-hydroxytryptaminergic neuron-like cells through tropomyosin receptor kinase A/cyclooxygenase 2/nuclear factor-kappa B/interleukin 8 signaling

The phenomenon of endothelial-neural transdifferentiation has been observed for a long time, but the mechanism is not clear. We previously found that safrole oxide induced human umbilical vein endothelial cell transdifferentiation into neuron-like cells. In this study, we first validated that these cells induced by safrole oxide were functional 5-hydroxytryptaminergic neuron-like cells. Then, we performed microarray analysis of safrole oxide-treated and -untreated human umbilical vein endothelial cells. Safrole oxide elevated the levels of cyclooxygenase 2 (COX-2), interleukin-8 (IL-8) and reactive oxygen species (ROS), which was accompanied by nuclear factor-kappa B (NF-κB) nuclear translocation during the transdifferentiation. Blockade of tropomyosin receptor kinase A (TrkA) by an inhibitor or short hairpin RNA inhibited the levels of COX-2/IL-8 and the nuclear translocation of NF-κB but did not suppress the increased ROS level. As a result, cells underwent apoptosis. Therefore, via TrkA, safrole oxide may induce endothelial cell transdifferentiation into functional neuron-like cells. During this process, the increased levels of COX-2/IL-8 and the subsequent elevation of ROS production induced NF-κB nuclear translocation and IL-8 secretion. With the activity of TrkA inhibited, the inactive NF-κB regulated the ROS level in a negative feedback manner. Finally, the transdifferentiation pathway was blocked and cells became apoptotic. The TrkA/COX-2/IL-8 signal pathway may have an important role in endothelial-neural transdifferentiation, and safrole oxide may trigger this process by activating TrkA.     

Src tyrosine kinases mediate activations of NF-kappaB and integrin signal during lipopolysaccharide-induced acute lung injury

Src tyrosine kinases (TKs) are signaling proteins involved in cell signaling pathways toward cytoskeletal, membrane and nuclear targets. In the present study, using a selective Src TK inhibitor, PP1, we investigated the roles of Src TKs in the key pulmonary responses, NF-kappaB activation, and integrin signaling during acute lung injury in BALB/C mice intratracheally treated with LPS. LPS resulted in c-Src phosphorylation in lung tissue and the phospho-c-Src was predominantly localized in recruited neutrophils and alveolar macrophages. PP1 inhibited LPS-induced increases in total protein content in bronchoalveolar lavage fluid, neutrophil recruitment, and increases in the production or activity of TNF-alpha and matrix metalloproteinase-9. PP1 also blocked LPS-induced NF-kappaB activation, and phosphorylation and degradation of IkappaB-alpha. The inhibition of NF-kappaB activation by PP1 correlated with a depression of LPS-induced integrin signaling, which included increases in the phosphorylations of integrin beta(3), and of the focal adhesion kinase (FAK) family members, FAK and Pyk2, in lung tissue, and reductions in the fibrinogen-binding activity of alveolar macrophages. Moreover, treatment with anti-alpha(v), anti-beta(3), or Arg-Gly-Asp-Ser (RGDS), inhibited LPS-induced NF-kappaB activation. Taken together, our findings suggest that Src TKs play a critical role in LPS-induced activations of NF-kappaB and integrin (alpha(v)beta(3)) signaling during acute lung injury. Therefore, Src TK inhibition may provide a potential means of ameliorating inflammatory cascade-associated lung injury.     

Mastoparan, a G protein agonist peptide, differentially modulates TLR4- and TLR2-mediated signaling in human endothelial cells and murine macrophages

Previous studies have implicated a role for heterotrimeric G protein-coupled signaling in B cells, monocytes, and macrophages stimulated with LPS and have shown that G proteins coimmunoprecipitate with membrane-bound CD14. In this study, we have extended these observations in human dermal microvessel endothelial cells (HMEC) that lack membrane-bound CD14 and in murine macrophages to define further the role of heterotrimeric G proteins in TLR signaling. Using the wasp venom-derived peptide, mastoparan, to disrupt G protein-coupled signaling, we identified a G protein-dependent signaling pathway in HMEC stimulated with TLR4 agonists that is necessary for the activation of p38 phosphorylation and kinase activity, NF-kappaB and IL-6 transactivation, and IL-6 secretion. In contrast, HMEC activation by TLR2 agonists, TNF-alpha, or IL-1beta was insensitive to mastoparan. In the murine macrophage cell line, RAW 264.7, and in primary murine macrophages, G protein dysregulation by mastoparan resulted in significant inhibition of LPS-induced signaling leading to both MyD88-dependent and MyD88-independent gene expression, while TLR2-mediated gene expression was not significantly inhibited. In addition to inhibition of TLR4-mediated MAPK phosphorylation in macrophages, mastoparan blunted IL-1R-associated kinase-1 kinase activity induced by LPS, but not by TLR2 agonists, yet failed to affect phosphorylation of Akt by phosphoinositol-3-kinase induced by either TLR2- or TLR4-mediated signaling. These data confirm the importance of heterotrimeric G proteins in TLR4-mediated responses in cells that use either soluble or membrane-associated CD14 and reveal a level of TLR and signaling pathway specificity not previously appreciated.     

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.