alpha-Melanocyte-stimulating hormone inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production in leukocytes by modulating protein kinase A,p38 kinase,and nuclear factor kappa B signaling pathways

The neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) inhibits inflammation by down-regulating the expression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) in leukocytes via stimulation of alpha-MSH cell surface receptors. However, the signaling mechanism of alpha-MSH action has not yet been clearly elucidated. Here, we have investigated signaling pathways by which alpha-MSH inhibits lipopolysaccharide (LPS)-induced TNF-alpha production in leukocytes such as THP-1 cells. We focused on the possible roles of protein kinase A (PKA), p38 kinase, and nuclear factor kappa B (NF kappa B) signaling. In THP-1 cells, LPS is known to activate p38 kinase, which in turn activates NF kappa B to induce TNF-alpha production. We found that pretreatment of cells with alpha-MSH blocked LPS-induced p38 kinase and NF kappa B activation as well as TNF-alpha production. This response was proportional to alpha-MSH receptor expression levels, and addition of an alpha-MSH receptor antagonist abolished the inhibitory effects. In addition, alpha-MSH treatment activated PKA, and PKA inhibition abrogated the inhibitory effects of alpha-MSH on p38 kinase activation, NF kappa B activation, and TNF-alpha production. Taken together, our results indicate that stimulation of PKA by alpha-MSH causes inhibition of LPS-induced activation of p38 kinase and NF kappa B to block TNF-alpha production.     

Activation of c-Jun-N-terminal kinase is critical in mediating lipopolysaccharide-induced changes in the rat hippocampus

Lipopolysaccharide (LPS) exerts a myriad of effects in rat hippocampus; it increases the concentration of the proinflammatory cytokine, interleukin-1beta (IL-1beta), and signalling via the IL-1 type I receptor (IL-1RI) resulting in phosphorylation of the stress-activated protein kinase, c-jun-N-terminal kinase (JNK) and impairment in long-term potentiation (LTP). This study was designed to establish whether activation of JNK is a pivotal event in mediating the effects of LPS in hippocampus and therefore LPS-treated rats were injected intracerebroventricularly with saline, the JNK inhibitor D-JNKI1, or with the anti-inflammatory cytokine IL-4, which antagonizes the effects of IL-1beta upstream of JNK activation. We report that IL-4 blocked the LPS-induced increase in IL-1RI expression and associated increases in phosphorylation of JNK and c-jun, whereas D-JNKI1 inhibited the LPS-induced phosphorylation of c-jun. Both IL-4 and D-JNKI1 inhibited the increase in caspase-3 staining which was associated with LPS treatment, and both abrogated the LPS-induced inhibition of LTP in perforant path-granule cell synapses. The data presented are consistent with the proposal that JNK activation, probably as a result of increased IL-1RI activation, is a critical step in mediating the detrimental effects of LPS in hippocampus.     

Lipopolysaccharide‐induced increase in signalling in hippocampus is abrogated by IL‐10 – a role for IL‐1β?

Parenterally administered lipopolysaccharide (LPS) increases the concentration of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) in the rat hippocampus and evidence suggests that this effect plays a significant role in inhibiting long-term potentiation (LTP). The anti-inflammatory cytokine IL-10, antagonizes certain effects of IL-1beta, so if the effects of LPS are mediated through an increase in IL-1beta, it might be predicted that IL-10 would also abrogate the effect of LPS. Here, we report that IL-10 reversed the inhibitory effect of LPS on LTP and the data couple this with an inhibitory effect on the LPS-induced increase in IL-1beta. LPS treatment increased hippocampal expression of IL-1 receptor Type I protein. Consistent with the LPS-induced increases in IL-1beta concentration and receptor expression, were downstream changes which included enhanced phosphorylation of IRAK and the stress-activated kinases, JNK and p38; these LPS-induced changes were reversed by IL-10, which concurs with the idea that these events are triggered by increased activation of IL-1RI by IL-1beta. We provide evidence which indicates that LPS treatment leads to evidence of cell death and this was reversed in hippocampus prepared from LPS-treated rats which received IL-10. The evidence is therefore consistent with the idea that IL-10 acts to protect neuronal tissue from the detrimental effects induced by LPS.     

NF-kappa B regulates the LPS-induced expression of interleukin 12 p40 in murine peritoneal macrophages: roles of PKC, PKA, ERK, p38 MAPK, and proteasome

NF-kappa B plays a critical role in coordinating the control of gene expression during monocyte/macrophage activation. In this report we describe our investigation of the mechanisms of LPS-induced NF-kappa B activation and IL-12 expression in murine peritoneal suppressor macrophages. Treatment of these macrophages with LPS induced I kappa B alpha degradation and NF-kappa B activation. EMSAs demonstrated that NF-kappa B bound to a cis-acting element located in the murine IL-12 p40 promoter. LPS signal transduction has been shown to involve a variety of signal pathways. The results in this paper indicate that LPS-induced NF-kappa B binding activity was independent of PKC, PKA, ERK, and p38 MAPK, but was regulated by proteasome. Furthermore, Proteasome Inhibitor I abolished the LPS-induced mRNA expression of IL-12 p35 and p40, and SB203580 reduced these mRNA levels, whereas the blockade of PKC, PKA, and ERK had little effect. These data demonstrate that the LPS-induced activation of proteasome. I kappa B. NF-kappa B and p38 MAPK signal pathways regulate the IL-12 expression in murine peritoneal suppressor macrophages.     

A pivotal role for interleukin-4 in atorvastatin-associated neuroprotection in rat brain

Inflammatory changes, characterized by an increase in pro-inflammatory cytokine production and up-regulation of the corresponding signaling pathways, have been described in the brains of aged rats and rats treated with the potent immune modulatory molecule lipopolysaccharide (LPS). These changes have been coupled with a deficit in long-term potentiation (LTP) in hippocampus. The evidence suggests that anti-inflammatory agents, which attenuate the LPS-induced and age-associated increase in hippocampal interleukin-1beta (IL-1beta) concentration, lead to restoration of LTP. Here we report that atorvastatin, a member of the family of agents that act as inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, exerts powerful anti-inflammatory effects in brain and that these effects are mediated by IL-4 and independent of its cholesterol-lowering actions. Treatment of rats with atorvastatin increased IL-4 concentration in hippocampal tissue prepared from LPS-treated and aged rats and abrogated the age-related and LPS-induced increases in pro-inflammatory cytokines, interferon-gamma (IFNgamma) and IL-1beta, and the accompanying deficit in LTP. The effect of atorvastatin on the LPS-induced increases in IFNgamma and IL-1beta was absent in tissue prepared from IL-4(-/-) mice. The increase in IL-1beta in LPS-treated and aged rats is associated with increased microglial activation, assessed by analysis of major histocompatibility complex II expression, and the evidence suggests that IFNgamma may trigger this activation. We propose that the primary effect of atorvastatin is to increase IL-4, which antagonizes the effects of IFNgamma, the associated increase in microglial activation, and the subsequent cascade of events.     

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.     

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.     

Distinct role of p38 and c-Jun N-terminal kinases in IL-10-dependent and IL-10-independent regulation of the costimulatory molecule B7.2 in lipopolysaccharide-stimulated human monocytic cells

The costimulatory molecule B7.2 (CD86) plays a vital role in immune activation and development of Th responses. The molecular mechanisms by which B7.2 expression is regulated are not understood. We investigated the role of mitogen-activated protein kinases (MAPK) in the regulation of B7.2 expression in LPS-stimulated human monocytic cells. LPS stimulation of human monocytes resulted in the down-regulation of B7.2 expression that could be abrogated by anti-IL-10 Abs. Furthermore, SB202190, a specific inhibitor of p38 MAPK, inhibited LPS-induced IL-10 production and reversed B7.2 down-regulation, suggesting that LPS-induced B7.2 down-regulation may be mediated, at least in part, via regulation of IL-10 production by p38 MAPK. In contrast to human promonocytic THP-1 cells that are refractory to the inhibitory effects of IL-10, LPS stimulation enhanced B7.2 expression. This IL-10-independent B7.2 induction was not influenced by specific inhibitors of either p38 or p42/44 MAPK. To ascertain the role of the c-Jun N-terminal kinase (JNK) MAPK, dexamethasone, an inhibitor of JNK activation, was used, which inhibited LPS-induced B7.2 expression. Transfection of THP-1 cells with a plasmid expressing a dominant-negative stress-activated protein/extracellular signal-regulated kinase kinase 1 significantly reduced LPS-induced B7.2 expression, thus confirming the involvement of JNK. To study the signaling events downstream of JNK activation, we show that dexamethasone did not inhibit LPS-induced NF-kappaB activation in THP-1 cells, suggesting that JNK may not be involved in NF-kappaB activation leading to B7.2 expression. Taken together, our results reveal the distinct involvement of p38 in IL-10-dependent, and JNK in IL-10-independent regulation of B7.2 expression in LPS-stimulated monocytic cells.     

NF-kappaB- and C/EBPbeta-driven interleukin-1beta gene expression and PAK1-mediated caspase-1 activation play essential roles in interleukin-1beta release from Helicobacter pylori lipopolysaccharide-stimulated macrophages

Helicobacter pylori is a Gram-negative microaerophilic bacterium that causes chronic gastritis, peptic ulcer, and gastric carcinoma. Interleukin-1beta (IL-1beta) is one of the potent proinflammatory cytokines elicited by H. pylori infection. We have evaluated the role of H. pylori lipopolysaccharide (LPS) as one of the mediators of IL-1beta release and dissected the signaling pathways leading to LPS-induced IL-1beta secretion. We demonstrate that both the NF-kappaB and the C/EBPbeta-binding elements of the IL-1beta promoter drive LPS-induced IL-1beta gene expression. NF-kappaB activation requires the classical TLR4-initiated signaling cascade leading to IkappaB phosphorylation as well as PI-3K/Rac1/p21-activated kinase (PAK) 1 signaling, whereas C/EBPbeta activation requires PI-3K/Akt/p38 mitogen-activated protein (MAP) kinase signaling. We observed a direct interaction between activated p38 MAP kinase and C/EBPbeta, suggesting that p38 MAPK is the immediate upstream kinase responsible for activating C/EBPbeta. Most important, we observed a role of Rac1/PAK1 signaling in activation of caspase-1, which is necessary for maturation of pro-IL-1beta. H. pylori LPS induced direct interaction between PAK1 and caspase-1, which was inhibited in cells transfected with dominant-negative Rac1. PAK1 immunoprecipitated from lysates of H. pylori LPS-challenged cells was able to phosphorylate recombinant caspase-1, but not its S376A mutant. LPS-induced caspase-1 activation was abrogated in cells transfected with caspase-1(S376A). Taken together, these results suggested a role of PAK1-induced phosphorylation of caspase-1 at Ser376 in activation of caspase-1. To the best of our knowledge our studies show for the first time that LPS-induced Rac1/PAK1 signaling leading to caspase-1 phosphorylation is crucial for caspase-1 activation. These studies also provide detailed insight into the regulation of IL-1beta gene expression by H. pylori LPS and are particularly important in the light of the observations that IL-1beta gene polymorphisms are associated with increased risk of H. pylori-associated gastric cancer.     

Induction of heme oxygenase-1 expression in murine macrophages is essential for the anti-inflammatory effect of low dose 15-deoxy-Delta 12,14-prostaglandin J2

15-Deoxy-Delta 12,14-prostaglandin J2 (15d-PGJ2), a cyclopentenone prostaglandin, displays a potent anti-inflammatory effect at micromolar concentrations (>2 microM) through direct inhibition of nuclear factor (NF)-kappa B activation. Here we show that at submicromolar concentrations (0.1-0.5 microM) 15d-PGJ2 retains the ability to suppress the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in lipopolysaccharide (LPS)-activated murine J774 macrophages under the conditions of a prolonged incubation (>12 h). Western blot analysis revealed that the expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1), was induced and coincident with the anti-inflammatory action of 15d-PGJ2. Inhibition of HO-1 activity or scavenging carbon monoxide (CO), a byproduct derived from heme degradation, significantly attenuated the suppressive activity of 15d-PGJ2. Furthermore, LPS-induced NF-kappa B activation assessed by the inhibitory protein of NF-kappa B(I kappa B) degradation and p50 nuclear translocation was diminished in cells subjected to prolonged treatment with the low concentration of 15d-PGJ2. Treatment of cells with the protein synthesis inhibitor, cycloheximide, or the specific p38 MAP kinase inhibitor, SB203580, blocked the induction of HO-1 and suppression of LPS-induced I kappa B degradation mediated by 15d-PGJ2. Likewise, HO inhibitor and CO scavenger were effective in abolishing the inhibitory effects of 15d-PGJ2 on NF-kappa B activation induced by LPS. The functional role of CO was further demonstrated by the use of a CO releasing molecule, tricarbonyldichlororuthenium(II) dimer, which significantly suppressed LPS-induced nuclear translocation of p50 as assessed by confocal immunofluorescence. Collectively, these data suggest that even at submicromolar concentrations 15d-PGJ2 can exert an anti-inflammatory effect in macrophages through a mechanism that involves the action of HO/CO.     

CCK-8对内毒素休克大鼠肺脏细胞因子的抑制效应

观察八肽胆囊收缩素(cholecystokinin-octapeptide,CCK-8）改善脂多糖(lipopolysaccharide,LPS）引起的大鼠内毒素性休克（endotoxic shock,ES）过程中血清及肺脏细胞因子的变化,探讨p38比裂素活化蛋白激酶（p38 mito-gen-activated protein kinase,p38 MAPK）的信号转导作用。用生理多道记录仪观察尾静脉注入LPS(p38 mito-gen-activated protein kinase,p38 MAPK）的信号转导作用。用生理多道记录仪观察尾静脉注入 LPS（8mg/kg i.v.）复制的SD大鼠ES模型、LPS注入前10min尾静脉注入CCK-8(40ug/kg i.v.）、单独注入CCK-8(40Uug/kg i.v.）或生理盐水（对照）的四组大鼠平均动脉血压（MAP）的改变,应用ELISA试剂盒检测血清和肺脏中炎性细胞因子（TNF-a、IL-1β和IL-6）的变化。用Western blot检测肺脏p38 MAPK的表达。结果显示：CCK-8可改善LPS引起的大鼠MAP的下降。与对照组相比,LPS可显著增加血清和肺脏TNF-a、IL-1β和IL-6含量;CCK-8可显著抑制LPS诱导的血清和肺脏TNF-a、IL-1β和IL-6的增加。CCK-8可增加ES大鼠肺脏磷酸化p38 MAPK的表达。结果提示CCK-8可改善ES大鼠MAP的降低,并对肺脏促炎性细胞因子过量产生有抑制作用,p38MAPK可能参与了其信号转导机制。     

Enhanced induction of LPS-induced fibroblast MCP-1 by interferon-gamma: involvement of JNK and MAPK phosphatase-1

IFN-gamma has significant immunoregulatory activity and plays an important role in both innate and adaptive immunity. Additive effects of IFN-gamma and the Toll-like receptor ligand LPS has been investigated in macrophages, but in fibroblasts is incompletely understood. IFN-gamma and LPS synergistically induced MCP-1 and NO release in primary murine dermal fibroblasts. IFN-gamma enhanced LPS-induced JNK and p38 MAPK phosphorylation but had no effect on NF-kappaB activity. The induction of both MCP-1 and NO was attenuated by inhibition of JNK but not p38 MAPK. Serine 727 STAT1 phosphorylation by IFN-gamma was increased by LPS, and this was also attenuated by inhibition of JNK but not p38 MAPK. IFN-gamma inhibited the basal expression of MAPK phosphatase-1, a negative regulator of MAPK signaling pathway. These results suggest that enhancement of LPS-induced JNK activation by IFN-gamma associated with inhibition of MAPK phosphatase-1 may be one of the mechanisms of additive effects between IFN-gamma and LPS in fibroblasts.     

Nuclear factor (NF)-kappa B blockade attenuates but does not abrogate LPS-mediated interleukin (IL)-1 beta biosynthesis in alveolar epithelial cells

The role that the nuclear factor (NF)-kappa B plays in regulating the biosynthesis of interleukin (IL)-1 beta, an inflammatory cytokine, has been investigated in vitro. Irreversible inhibition of the proteasome complex by carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132; 1-50 microM) had no inhibitory effect on lipopolysaccharide (LPS)-mediated IL-1 beta biosynthesis. Furthermore, selective inhibition of NF-kappa B by the action of caffeic acid phenylethyl ester (CAPE; 1-100 microM) and sulfasalazine (SSA; 0.1-10 mM), a potent and irreversible inhibitor of NF-kappa B, partially attenuated but did not abolish LPS-dependent IL-1 beta secretion. Incorporation of a selectively permeant inhibitor of NF-kappa B, SN-50 (1-20 microM), a peptide which contains the nuclear localization sequence (NLS) for the p50 NF-kappa B subunit and the amino-terminal sequence of Kaposi fibroblast growth factor to promote cell permeability, attenuated in a dose-dependent manner LPS-mediated release of IL-1 beta. It is concluded that the NF-kapp B pathway is partially implicated and its blockade attenuates but does not abrogate LPS-dependent IL-1 beta biosynthesis in alveolar epithelial cells.