Autocrine Regulation of Macrophage Activation via Exocytosis of ATP and Activation of P2Y11 Receptor

It is important to understand the mechanisms that regulate macrophage activation to establish novel therapies for inflammatory diseases, such as sepsis; a systemic inflammatory response syndrome generally caused by bacterial lipopolysaccharide (LPS). In this study, we investigated the involvement of extracellular ATP-mediated autocrine signaling in LPS-induced macrophage activation. We show here that ATP release via exocytosis, followed by activation of P2Y11 receptor, is a major pathway of the macrophage activation, leading to release of cytokines. Treatment of human monocyte THP-1 cells with LPS induced rapid ATP release from cells, and this release was blocked by knockdown of SLC17A9 (vesicular nucleotide transporter, VNUT), which is responsible for exocytosis of ATP. ATP-enriched vesicles were found in cytosol of THP-1 cells. These data suggest the involvement of vesicular exocytosis in the release of ATP. Knockdown of SLC17A9, the P2Y11 antagonist NF157 or knockdown of P2Y11 receptor significantly suppressed both M1-type polarization and IL-6 production in THP-1 cells, indicating an important role of activation of P2Y11 receptor by released ATP in macrophage activation. Next, the effect of NF157 on LPS-induced immune activation was examined in vivo. Administration of LPS to mice caused increase of serum IL-1ß, IL-6, IL-12 and TNF-alpha levels at 3–24 h after the administration. Pre-treatment of LPS-treated mice with NF157 suppressed both elevation of proinflammatory cytokines in serum and M1 polarization of peritoneal/spleen macrophages. Moreover, post-treatment with NF157 at 30 min after administration of LPS also suppressed the elevation of serum cytokines levels. We conclude that vesicular exocytosis of ATP and autocrine, positive feedback through P2Y11 receptors is required for the effective activation of macrophages. Consequently, P2Y11 receptor antagonists may be drug candidates for treatment of inflammatory diseases such as sepsis.     

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.     

A20 Is Critical for the Induction of Pam3CSK4-Tolerance in Monocytic THP-1 Cells

A20 functions to terminate Toll-like receptor (TLR)-induced immune response, and play important roles in the induction of lipopolysacchride (LPS)-tolerance. However, the molecular mechanism for Pam3CSK4-tolerance is uncertain. Here we report that TLR1/2 ligand Pam3CSK4 induced tolerance in monocytic THP-1 cells. The pre-treatment of THP-1 cells with Pam3CSK4 down-regulated the induction of pro-inflammatory cytokines induced by Pam3CSK4 re-stimulation. Pam3CSK4 pre-treatment also down-regulated the signaling transduction of JNK, p38 and NF-κB induced by Pam3CSK4 re-stimulation. The activation of TLR1/2 induced a rapid and robust up-regulation of A20, suggesting that A20 may contribute to the induction of Pam3CSK4-tolerance. This hypothesis was proved by the observation that the over-expression of A20 by gene transfer down-regulated Pam3CSK4-induced inflammatory responses, and the down-regulation of A20 by RNA interference inhibited the induction of tolerance. Moreover, LPS induced a significant up-regulation of A20, which contributed to the induction of cross-tolerance between LPS and Pam3CSK4. A20 was also induced by the treatment of THP-1 cells with TNF-α and IL-1β. The pre-treatment with TNF-α and IL-1β partly down-regulated Pam3CSK4-induced activation of MAPKs. Furthermore, pharmacologic inhibition of GSK3 signaling down-regulated Pam3CSK4-induced A20 expression, up-regulated Pam3CSK4-induced inflammatory responses, and partly reversed Pam3CSK4 pre-treatment-induced tolerance, suggesting that GSK3 is involved in TLR1/2-induced tolerance by up-regulation of A20 expression. Taken together, these results indicated that A20 is a critical regulator for TLR1/2-induced pro-inflammatory responses.     

S-form lipopolysaccharide (LPS), but not lipid A or R-chemo-type LPS, induces interleukin-6 production in vitamin D3-differentiated THP-1 cells

Bacterial lipopolysaccharide (LPS) induces the production of various inflammatory cytokines and the inducibility is considered attributable to the glycolipid part of LPS called lipid A. We report an in vitro model in which lipid A is not necessarily a minimal structure for the LPS activity. Vitamin D3-differentiated THP-1 cells, cultured human monocytic leukemia cells, produced a high level of interleukin-6 (IL-6) by stimulating LPS from Escherichia coli O111:B4, but not by stimulating synthetic E. coli-type lipid A (compound 506), E. coli Re mutant LPS (ReLPS), or alkali-treated LPS. The induction by LPS was inhibited by the anti-CD14 antibodies or by the synthetic lipid A precursor (compound 406). An alkali-treated LPS or compound 506 partially inhibited the LPS-induced IL-6 production. These facts suggest that lipid A alone is not sufficient for the IL-6-inducing activity, but the polysaccharide part in LPS contributes or acts as a co-factor for activation of differentiated THP-1 cells.     

Glycogen synthase kinase-3 is an early determinant in the differentiation of pathogenic Th17 cells

CD4(+) T cells are critical for host defense but are also major drivers of immune-mediated diseases. The classical view of Th1 and Th2 subtypes of CD4(+) T cells was recently revised by the identification of the Th17 lineage of CD4(+) T cells that produce IL-17, which have been found to be critical in the pathogenesis of autoimmune and other diseases. Mechanisms controlling the differentiation of Th17 cells have been well described, but few feasible targets for therapeutically reducing Th17 cells are known. The generation of Th17 cells requires IL-6 and activation of STAT3. During polarization of CD4(+) T cells to Th17 cells, we found that inhibition of glycogen synthase kinase-3 (GSK3) blocked IL-6 production, STAT3 activation, and polarization to Th17 cells. Polarization of CD4(+) T cells to Th17 cells increased by 10-fold the expression of GSK3β protein levels in Th17 cells, whereas GSK3β was unaltered in regulatory T cells. Diminishing GSK3 activity either pharmacologically or molecularly blocked Th17 cell production, and increasing GSK3 activity promoted polarization to Th17 cells. In vivo inhibition of GSK3 in mice depleted constitutive Th17 cells in intestinal mucosa, blocked Th17 cell generation in the lung after Francisella tularensis infection, and inhibited the increase in spinal cord Th17 cells and disease symptoms in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. These findings identify GSK3 as a critical mediator of Th17 cell production and indicate that GSK3 inhibitors provide a potential therapeutic intervention to control Th17-mediated diseases.     

ST2 suppresses IL-6 production via the inhibition of IkappaB degradation induced by the LPS signal in THP-1 cells

LPS induces the production of inflammatory cytokines via the stimulation of Toll-like receptors. In this study, we demonstrated that a soluble secreted form of the ST2 gene product (ST2), a member of the interleukin-1 receptor family, suppressed the production of IL-6 in an LPS-stimulated human monocytic leukemia cell line, THP-1. Immunofluorescence confocal microscopy revealed the binding of ST2 to the surface of the THP-1 cells, in which ST2 led to decreased binding of nuclear factor-kappaB to the IL-6 promoter. Furthermore, the degradation of IkappaB in the cytoplasm after LPS stimulation was reduced by pretreatment with ST2. These results demonstrated that ST2 negatively regulates LPS-induced IL-6 production via the inhibition of IkappaB degradation in THP-1 cells.     

Differential regulation of cytokine and chemokine production in lipopolysaccharide-induced tolerance and priming

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFalpha production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFalpha production. The pro-inflammatory cytokine, IFNgamma, also abolishes suppression of TNFalpha in LPS tolerance. The effect of LPS tolerance on HKSa and IFNgamma-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNgamma differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24 h with LPS (100 ng/ml) or LPS (100 ng/ml) + IFNgamma (1 microg/ml). Cells were subsequently stimulated with LPS or HKSa (10 microg/ml) for 24 h. The production of the cytokines TNFalpha, IL-6, IL-1beta, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFalpha (3070 +/- 711 pg/ml and 217 +/- 9 pg/ml, respectively) and IL-6 (237 +/- 8.9 pg/ml and 56.2 +/- 2.9 pg/ml, p < 0.05, n = 3, respectively) in control cells compared to basal levels (< 25 pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% (p < 0.05, n = 3) reduction in TNFalpha. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFalpha (2.7 fold, from 217 to 580 pg/ml, p < 0.05, n = 3 ). In contrast to suppressed TNFalpha, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076 pg/ml, p < 0.01, n = 3) and also primed to HKSa stimulation (62 fold, from 56 to 3470 pg/ml, p < 0.01, n = 3). LPS induced IL-8 production and to a lesser extent IL-1beta and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1beta, although HKSa stimulation augmented both mediators. In addition, IFNgamma pretreatment reversed LPS tolerance as evidenced by increased TNFalpha levels while IL-6, IL-1beta, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNgamma. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis.     

Molecular and Cellular Mechanisms Responsible for Cellular Stress and Low-grade Inflammation Induced by a Super-low Dose of Endotoxin

Super-low-dose endotoxemia in experimental animals and humans is linked to low-grade chronic inflammatory diseases. However, the underlying molecular and cellular mechanisms are not well understood. In this study, we examined the effects of a super-low dose of LPS on low-grade inflammation in macrophages as well as underlying mechanisms. We observed that a super-low dose of LPS induces mitochondrial fission and cell necroptosis in primary murine macrophages, dependent upon interleukin 1 receptor-associated kinase (IRAK-1). Mechanistically, our study reveals that a super-low dose of LPS causes protein ubiquitination and degradation of mitofusin 1 (Mfn1), a molecule required for maintaining proper mitochondrial fusion. A super-low dose of LPS also leads to dephosphorylation and activation of Drp1, a molecule responsible for mitochondrial fission and cell necroptosis. Furthermore, we demonstrated that a super-low dose of LPS activates receptor interacting protein 3 kinase (RIP3), a key molecule critical for the assembly of the necrosome complex, the initiation of Drp1 dephosphorylation, and necroptosis. The effects of a super-low dose of LPS are abolished in macrophages harvested from IRAK-1-deficient mice. Taken together, our study identified a novel molecular pathway that leads to cellular stress and necroptosis in macrophages challenged with a super-low dose of endotoxin. This may reconcile low-grade inflammation often associated with low-grade endotoxemia.     

EGb761对LPS诱导THP-1细胞释放HMGB1蛋白表达的调节

目的:探讨EGb761对LPS诱导THP-1细胞释放HMGB1蛋白表达的调节,为EGb761的临床运用提供可行的依据。方法:LPS(1μg/m L)诱导不同时间后,western blotting检测THP-1细胞上清液中HMGB1蛋白含量变化及不同浓度EGb761对LPS诱导THP-1细胞释放HMGB1蛋白的表达和NF-κB的活性;酶联免疫吸附法(ELISA)检测细胞中IL-1β、IL-6、TNF-α的含量。共聚焦显微镜观察EGb761对LPS诱导THP-1细胞释放HMGB1蛋白核转位变化。结果:(1)LPS组IL-1β、IL-6、TNF-α的含量在刺激6-12 h后明显高于空白对照组,而EGb761+LPS组IL-1β、IL-6、TNF-α的含量均显著低于LPS组(P0.05)。(2)EGb761处理LPS诱导THP-1细胞6 h后细胞上清液NF-κB活性表达量较空白对照组低,随着处理时间延长至12 h,NF-κB的活性表达量呈明显下降趋势(P0.05)。(3)LPS诱导THP-1细胞18 h后,细胞上清液中HMGB1蛋白含量呈明显升高趋势(P0.05)。(4)不同浓度EGb761对LPS诱导THP-1细胞18 h后,HMGB1蛋白含量较空白对照组有下降趋势,HMGB1蛋白含量随着EGB761浓度增加至100μg/m L呈下降趋势并呈浓度依赖效应(P0.05)。(5)LPS诱导THP-1细胞后,在共聚焦显微镜下可见胞浆中大量HMGB1蛋白标记分布,而EGb761+LPS共同诱导THP-1细胞后胞浆中可见少量HMGB1蛋白分布。结论:LPS可诱导THP-1细胞IL-1β、IL-6、TNF-α表达增多及NF-κB活化,导致HMGB1蛋白表达增多及核转位,而EGB761能抑制THP-1细胞IL-1β、IL-6、TNF-α表达及NF-κB活化,调节HMGB1蛋白的表达及核转位。     

Extracellular Hsp70 induces inflammation and modulates LPS/LTA-stimulated inflammatory response in THP-1 cells

Extracellular Hsp70 (eHsp70) can act as damage-associated molecular pattern (DAMP) via Toll-like receptors TLR2 and TLR4, and stimulate immune and inflammatory responses leading to sterile inflammation and propagation of already existing inflammation. It was found elevated in the blood of patients with chronic obstructive pulmonary disease (COPD), who might suffer occasional bacterial colonizations and infections. We used a monocytic THP-1 cell line as a cellular model of systemic compartment of COPD to assess inflammatory effects of eHsp70 when present alone or together with bacterial products lypopolysaccharide (LPS) and lypoteichoic acid (LTA). THP-1 cells were differentiated into macrophage-like cells and treated with various concentrations of recombinant human Hsp70 protein (rhHsp70), LPS (TLR4 agonist), LTA (TLR2 agonist), and their combinations for 4, 12, 24, and 48 h. Concentrations of IL-1α, IL-6, IL-8, and TNF-α were determined by ELISA. Cell viability was assessed by MTS assay, and mode of cell death by luminometric measurements of caspases-3/7, -8, and -9 activities. rhHsp70 showed cell protecting effect by suppressing caspases-3/7 activation, while LPS provoked cytotoxicity through caspases-8 and -3/7 pathway. Regarding inflammatory processes, rhHsp70 alone induced secretion of IL-1α and IL-8, but had modulatory effects on release of all four cytokines when applied together with LPS or LTA. Combined effect with LPS was mainly synergistic, and with LTA mainly antagonistic, although it was cytokine- and time-dependent. Our results confirmed pro-inflammatory function of extracellular Hsp70, and suggest its possible implication in COPD exacerbations caused by bacterial infection through desensitization or inappropriate activation of TLR2 and TLR4 receptors.     

A heteroglycan from the cyanobacterium Nostoc commune modulates LPS-induced inflammatory cytokine secretion by THP-1 monocytes through phosphorylation of ERK1/2 and Akt

Cyanobacteria (blue-green algae) have been consumed as food and used in folk medicine since ancient times to alleviate a variety of diseases. Cyanobacteria of the genus Nostoc have been shown to produce complex exopolysaccharides with antioxidant and antiviral activity. Furthermore, Nostoc sp. are common in cyanolichen symbiosis and lichen polysaccharides are known to have immunomodulating effects. Nc-5-s is a heteroglycan isolated from free-living colonies of Nostoc commune and its structure has been characterized in detail. The aim of this study was to determine the effects of Nc-5-s on the inflammatory response of lipopolysaccharide (LPS)-stimulated human THP-1 monocytes and how the effects are mediated. THP-1 monocytes primed with interferon-γ and stimulated with LPS in the presence of Nc-5-s secreted less of the pro-inflammatory cytokine interleukin (IL)-6 and more of the anti-inflammatory cytokine IL-10 than THP-1 monocytes stimulated without Nc-5-s. In contrast, Nc-5-s increased LPS-induced secretion of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and IL-8. Nc-5-s decreased LPS-induced phosphorylation of the extracellular regulated kinase (ERK)1/2 and Akt kinase, but did not affect phosphorylation of the p38 kinase, activation of the nuclear factor kappa B pathway, nor DNA binding of c-fos. These results show that Nc-5-s has anti-inflammatory effects on IL-6 and IL-10 secretion by THP-1 monocytes, but its effects are pro-inflammatory when it comes to TNF-α and IL-8. Furthermore, they show that the effects of Nc-5-s may be mediated through the ERK1/2 pathway and/or the Akt/phosphoinositide 3-kinase pathway and their downstream effectors. The ability of Nc-5-s to decrease IL-6 secretion, increase IL-10 secretion and moderate ERK1/2 activation indicates a potential for its development as an anti-inflammatory agent.     

IL-6 trans-signaling modulates TLR4-dependent inflammatory responses via STAT3

Innate immune responses triggered by the prototypical inflammatory stimulus LPS are mediated by TLR4 and involve the coordinated production of a multitude of inflammatory mediators, especially IL-6, which signals via the shared IL-6 cytokine family receptor subunit gp130. However, the exact role of IL-6, which can elicit either proinflammatory or anti-inflammatory responses, in the pathogenesis of TLR4-driven inflammatory disorders, as well as the identity of signaling pathways activated by IL-6 in a proinflammatory state, remain unclear. To define the contribution of gp130 signaling events to TLR4-driven inflammatory responses, we combined genetic and therapeutic approaches based on a series of gp130(F/F) knock-in mutant mice displaying hyperactivated IL-6-dependent JAK/STAT signaling in an experimental model of LPS/TLR4-mediated septic shock. The gp130(F/F) mice were markedly hypersensitive to LPS, which was associated with the specific upregulated production of IL-6, but not TNF-α. In gp130(F/F) mice, either genetic ablation of IL-6, Ab-mediated inhibition of IL-6R signaling or therapeutic blockade of IL-6 trans-signaling completely protected mice from LPS hypersensitivity. Furthermore, genetic reduction of STAT3 activity in gp130(F/F):Stat3(+/-) mice alleviated LPS hypersensitivity and reduced LPS-induced IL-6 production. Additional genetic approaches demonstrated that the TLR4/Mal pathway contributed to LPS hypersensitivity and increased IL-6 production in gp130(F/F) mice. Collectively, these data demonstrate for the first time, to our knowledge, that IL-6 trans-signaling via STAT3 is a critical modulator of LPS-driven proinflammatory responses through cross-talk regulation of the TLR4/Mal signaling pathway, and potentially implicate cross-talk between JAK/STAT and TLR pathways as a broader mechanism that regulates the severity of the host inflammatory response.     

c-Jun N-terminal kinase negatively regulates lipopolysaccharide-induced IL-12 production in human macrophages: role of mitogen-activated protein kinase in glutathione redox regulation of IL-12 production

Although c-Jun N-terminal kinase (JNK) plays an important role in cytokine expression, its function in IL-12 production is obscure. The present study uses human macrophages to examine whether the JNK pathway is required for LPS-induced IL-12 production and defines how JNK is involved in the regulation of IL-12 production by glutathione redox, which is the balance between intracellular reduced (GSH) and oxidized glutathione (GSSG). We found that LPS induced IL-12 p40 protein and mRNA in a time- and concentration-dependent manner in PMA-treated THP-1 macrophages, and that LPS activated JNK and p38 mitogen-activated protein (MAP) kinase, but not extracellular signal-regulated kinase, in PMA-treated THP-1 cells. Inhibition of p38 MAP kinase activation using SB203580 dose dependently repressed LPS-induced IL-12 p40 production, as described. Conversely, inhibition of JNK activation using SP600125 dose dependently enhanced both LPS-induced IL-12 p40 production from THP-1 cells and p70 production from human monocytes. Furthermore, JNK antisense oligonucleotides attenuated cellular levels of JNK protein and LPS-induced JNK activation, but augmented IL-12 p40 protein production and mRNA expression. Finally, the increase in the ratio of GSH/GSSG induced by glutathione reduced form ethyl ester (GSH-OEt) dose dependently enhanced LPS-induced IL-12 p40 production in PMA-treated THP-1 cells. GSH-OEt augmented p38 MAP kinase activation, but suppressed the JNK activation induced by LPS. Our findings indicate that JNK negatively affects LPS-induced IL-12 production from human macrophages, and that glutathione redox regulates LPS-induced IL-12 production through the opposite control of JNK and p38 MAP kinase activation.     

Bacterial lipopolysaccharide and inflammatory mediators augment IL-6 secretion by human endothelial cells

The interaction between human endothelial cells and leukocytes during immunologic and inflammatory responses is in part mediated through the release of soluble mediators. We report that cultured human umbilical vein endothelial cells secrete IL-6 when stimulated with LPS. This effect was inhibited by polymyxin-B. The monokines IL-1 and TNF-alpha were also potent inducers of IL-6, whereas lymphotoxin was only effective at much higher concentrations. Endothelial cell supernatant IL-6 was active as hybridoma-plasmacytoma growth factor and as B-cell stimulating factor. Endothelial IL-6 activity was neutralized by a specific anti-IL-6 antibody and by immunoprecipitation it was shown to be identical in size to human fibroblast-derived IL-6. As IL-6 is possibly an important regulator of host defense responses, production of this cytokine by endothelial cells may contribute to the pathogenesis of various inflammatory and immunologic diseases.