Heat shock inhibits IL-12 p40 expression through NF-kappa B signalling pathway in murine macrophages.

We report the effect of heat shock on lipopolysaccharide (LPS)-induced interleukin 12 (IL-12) expression. The augmentation of LPS-induced IL-12 p40 mRNA and p70 protein was significantly suppressed in both peritoneal macrophages and RAW264.7 cells after heat shock at 43 degrees C. The binding activity of nuclear factor kappa B (NF-kappa B) was reduced by prior heat shock. LPS did not induce degradation of the inhibitory protein I-kappa B alpha in the shocked cells, which might be a potential mechanism to block NF-kappa B activation. Furthermore, transient transfection assay in RAW264.7 cells demonstrated that LPS-induced activation of DM703 and DM138 (contains NF-kappa B motif) was highly sensitive to heat shock. These data suggest that heat shock influences expression of IL-12 through the I-kappa B/NF-kappa B pathway.     

Interleukin-10 inhibits tumor necrosis factor-alpha production in lipopolysaccharide-stimulated RAW 264.7 cells through reduced MyD88 expression

The mechanism of interleukin (IL)-10-mediated inhibition of tumor necrosis factor (TNF)-alpha production was studied by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. IL-10 inhibited TNF-alpha production transiently at an early stage after LPS stimulation. IL-10 inhibited the activation of nuclear factor (NF)-kappaB, p38 and stress-activated protein kinase (SAPK) in LPS-stimulated RAW 264.7 cells. Although the level of MyD88 protein increased in response to LPS, IL-10 prevented the LPS-induced MyD88 augmentation. There was no significant difference in the MyD88 mRNA expression between the cells pretreated with or without IL-10 in response to LPS. Therefore, IL-10 was suggested to inhibit LPS-induced TNF-alpha production via reduced MyD88 expression.     

Magnesium isoglycyrrhizinate suppresses LPS-induced inflammation and oxidative stress through inhibiting NF-κB and MAPK pathways in RAW264.7 cells

Magnesium Isoglycyrrhizinate (MgIG), a novel molecular compound extracted from licorice root, has exhibited greater anti-inflammatory activity and hepatic protection than glycyrrhizin and β-glycyrrhizic acid. In this study, we investigated the anti-inflammatory effect and the potential mechanism of MgIG on Lipopolysaccharide (LPS)-treated RAW264.7 cells. MgIG down-regulated LPS-induced pro-inflammatory mediators and enzymes in LPS-treated RAW264.7 cells, including TNF-α, IL-6, IL-1β, IL-8, NO and iNOS. The generation of reactive oxygen species (ROS) in LPS-treated RAW264.7 cells was also reduced. MgIG attenuated NF-κB translocation by inhibiting IKK phosphorylation and IκB-α degradation. Simultaneously, MgIG also inhibited LPS-induced activation of MAPKs, including p38, JNK and ERK1/2. Taken together, these results suggest that MgIG suppresses inflammation by blocking NF-κB and MAPK signaling pathways, and down-regulates ROS generation and inflammatory mediators.     

A pathway through interferon-gamma is the main pathway for induction of nitric oxide upon stimulation with bacterial lipopolysaccharide in mouse peritoneal cells.

Production of nitric oxide (NO) in response to bacterial lipopolysaccharide (LPS) was investigated using cultures of mouse peritoneal exudate cells (PEC) and the macrophage cell line RAW264.7. In the presence of anti-(interferon-gamma) (IFN-gamma), NO production was markedly suppressed in the PEC culture but not in the RAW264.7 culture. In the PEC culture, LPS induced both IFN-gamma production and activation of IFN response factor-1, which leads to the gene expression of inducible NO synthase, but neither was induced in the culture of RAW264.7 cells. In addition to anti-(IFN-gamma), antibodies against interleukin (IL)-12 and IL-18 showed a suppressive effect on LPS-induced NO production in the PEC culture, and these antibodies in synergy showed strong suppression. Stimulation of the PEC culture with IL-12 or IL-18 induced production of IFN-gamma and NO, and these cytokines, in combination, exhibited marked synergism. Stimulation of the culture with IFN-gamma induced production of NO, but not IL-12. The macrophage population in the PEC, prepared as adherent cells, responded well to LPS for IL-12 production, but weakly for production of IFN-gamma and NO. The macrophages also responded well to IFN-gamma for NO production. For production of IFN-gamma by stimulation with LPS or IL-12 + IL-18, nonadherent cells were required in the PEC culture. Considering these results overall, the indirect pathway, through the production of intermediates (such as IFN-gamma-inducing cytokines and IFN-gamma) by the cooperation of macrophages with nonadherent cells, was revealed to play the main role in the LPS-induced NO production pathway, as opposed to the direct pathway requiring only a macrophage population.     

The effect of oxidized low density lipoprotein (oxLDL)-induced heme oxygenase-1 on LPS-induced inflammation in RAW 264.7 macrophage cells

Macrophages take up oxidized low density lipoprotein (oxLDL) after being exposed to it in the blood vessels. oxLDL transforms macrophages into foam cells, which are a hallmark of atherosclerosis. The effects that oxLDL have on the inflammatory responses of foam cells are not clear. Here, we investigated how oxLDL modulates lipopolysaccharide (LPS)-induced inflammatory mediators in RAW 264.7 murine macrophages. Our results showed that oxLDL dramatically induced HO-1 expression, but did not increase pro-inflammatory mediators such as interleukin-1β, tumor necrosis factor-α, iNOS, and monocyte chemoattractant protein (MCP)-1. In RAW 264.7 macrophages, oxLDL markedly inhibited LPS-induced inflammatory mediators such as inducible nitric oxide synthase (iNOS), IL-1β, IL-6, granulocyte macrophage colony-stimulating factor and stromal cell-derived factor-1. Interestingly, however, the down-regulation of HO-1 by siRNA did not recover the inhibition of LPS-induced expression and/or the secretion of inflammatory mediators. oxLDL blocked LPS-induced NF-κB nuclear translocation by inhibiting inhibitory κB (IκB) degradation. Taken together, our results suggest that oxLDL could modulate LPS-induced inflammatory responses by inhibiting NF-κB signaling independently of HO-1 expression.     

Engagement of tumor necrosis factor mRNA by an endotoxin-inducible cytoplasmic protein.

BACKGROUND: Tumor necrosis factor (TNF) production by macrophages plays an important role in the host response to infection. TNF-alpha gene expression in RAW 264.7 macrophages is predominantly regulated at the translational level. A key element in this regulation is an AU-rich (AUR) sequence located in the 3' untranslated region (UTR) of TNF mRNA. In unstimulated macrophages, the translation of TNF mRNA is inhibited via this AUR sequence. Upon stimulation with LPS, this repression is overcome and translation occurs. In this study, we attempted to identify cellular proteins that interact with the AUR sequence and thereby regulate TNF mRNA translation. MATERIALS AND METHODS: RNA probes corresponding to portions of TNF mRNA 3' UTR were synthesized. These labeled RNAs were incubated with cytoplasmic extracts of either unstimulated or lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages. The RNA/protein complexes formed were analyzed by gel retardation. Ultraviolet (UV) cross-linking experiments were performed to determine the molecular weight of the proteins involved in the complexes. RESULTS: TNF mRNA AUR sequence formed two complexes (1 and 2) of distinct electrophoretic mobilities. While the formation of complex 1 was independent of the activation state of the macrophages from which the extracts were obtained, complex 2 was detected only using cytoplasmic extracts from LPS-stimulated macrophages. Upon UV cross-linking, two proteins, of 50 and 80 kD, respectively, were capable of binding the UAR sequence. The 50-kD protein is likely to be part of the LPS-inducible complex 2, since its binding ability was enhanced upon LPS stimulation. Interestingly, complex 2 formation was also triggered by Sendaï virus infection, another potent activator of TNF mRNA translation in RAW 264.7 macrophages. In contrast, complex 2 was not detected with cytoplasmic extracts obtained from B and T cell lines which are unable to produce TNF in response to LPS. Protein tyrosine phosphorylation is required for LPS-induced TNF mRNA translation. Remarkably, the protein tyrosine phosphorylation inhibitor herbimycin A abolished LPS-induced complex 2 formation. Complex 2 was already detectable after 0.5 hr of LPS treatment and was triggered by a minimal LPS dose of 10 pg/ml. CONCLUSIONS: The tight correlation between TNF production and the formation of an LPS-inducible cytoplasmic complex suggests that this complex plays a role in the translational regulation of TNF mRNA.     

Carabrol suppresses LPS-induced nitric oxide synthase expression by inactivation of p38 and JNK via inhibition of I-κBα degradation in RAW 264.7 cells

Carabrol, isolated from Carpesium macrocephalum, showed anti-inflammatory potential in LPS-induced RAW 264.7 murine macrophages. In present study, carabrol demonstrated the inhibitory activity on pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α. In addition, mRNA and protein levels of iNOS and COX-2 were reduced by carabrol. Molecular analysis revealed that these suppressive effects were correlated with the inactivation of p38 and JNK via inhibition of NF-κB activation. Immunoblotting showed that carabrol suppressed LPS-induced degradation of I-κBα and decreased nuclear translocation of p65. Taken together, these results suggest that carabrol can be a modulator of pro-inflammatory signal transduction pathway in RAW 264.7 cells.     

Mitochondrial density contributes to the immune response of macrophages to lipopolysaccharide via the MAPK pathway

We investigated the role of mitochondrial reactive oxygen species (ROS) in the response of macrophages to lipopolysaccharide (LPS) using RAW 264.7 cells and their ρ(o) cells lacking mitochondria. Mitochondrial density, respiratory activity and related proteins in ρ(o) cells were significantly lower than those in RAW cells. LPS rapidly stimulated mitochondrial ROS prior to cytokine secretion, such as TNF-α and IL-6, from RAW 264.7 cells by activating the MAPK pathway, while the response was attenuated in ρ(o) cells. Exposure of ρ(o) cells to H(2)O(2) partially restored the secretion of cytokines induced by LPS. These results suggest that mitochondrial density and/or the respiratory state contribute to intracellular oxidative stress, which is responsible for the stimulation of LPS-induced MAPK signaling to enhance cytokine release from macrophages.     

5-Aminoimidazole-4-carboxamide riboside suppresses lipopolysaccharide-induced TNF-alpha production through inhibition of phosphatidylinositol 3-kinase/Akt activation in RAW 264.7 murine macrophages

5-Aminoimidazole-4-carboxamide riboside (AICAR) is an adenosine analog and a widely used activator of AMP-activated protein kinase (AMPK). We examined the effect of AICAR on LPS-induced TNF-alpha production in RAW 264.7 and peritoneal macrophages and its molecular mechanism in RAW 264.7 macrophages. Treatment with AICAR inhibited LPS-induced increases in TNF-alpha mRNA and protein levels in these cells. AICAR or LPS did not alter the AMPK activity as well as the phosphorylations of AMPK alpha (Thr172) and ACC (Ser79). Moreover, an adenosine kinase inhibitor 5'-iodotubercidin enhanced the suppressive effect of AICAR on TNF-alpha levels. These results suggest that the effect of AICAR on TNF-alpha suppression in RAW 264.7 cells is independent of AMPK activation. In addition, an adenosine receptor antagonist 8-SPT had no effect on AICAR-induced suppression of TNF-alpha levels. Finally, we observed that AICAR inhibited LPS-induced activation of PI 3-kinase and Akt, whereas it had no effect on the activation of p38 and ERK1/2. Taken together, these results suggest that the anti-inflammatory action of AICAR in RAW 264.7 macrophages is independent of AMPK activation and is associated with inhibition of LPS-induced activation of PI 3-kinase/Akt pathway.     

Sphingosine kinase protects lipopolysaccharide-activated macrophages from apoptosis

Lipopolysaccharide (LPS) signaling is critical for the innate immune response to gram-negative bacteria. Here, evidence is presented for LPS stimulation of sphingosine kinase (SPK) in the RAW 264.7 murine macrophage cell line and rat primary hepatic macrophages (HMs). LPS treatment of RAW 264.7 cells resulted in a time- and dose-dependent activation of SPK and membrane translocation of SPK1. Further, LPS-induced SPK activation was blocked by SPK1-specific small interfering RNA (siRNA). Overexpression of Toll-like receptor 4 and MD2, the receptor and coreceptor of LPS, in HEK 293 cells activated SPK activity in the absence of LPS treatment. Inhibition of SPK by the pharmacological inhibitor N,N-dimethylsphingosine (DMS) or SPK1-specific siRNA blocked LPS stimulation of extracellular signal-regulated kinase 1/2 and p38 but enhanced LPS-induced c-Jun N-terminal kinase activation. The SPK inhibitor DMS and dominant-negative SPK1 also blocked LPS activation of Elk-1 and NF-kappaB reporters in RAW 264.7 cells. Inhibition of SPK sensitized RAW 264.7 cells and HMs to LPS-induced apoptosis. These data demonstrate the critical role of SPK1 in LPS signaling in macrophages and suggest that SPK1 is a potential therapeutic target to block hyperimmune responses induced by gram-negative bacteria.     

Anti-inflammatory effect of heme oxygenase 1: glycosylation and nitric oxide inhibition in macrophages

Flavonoids including the aglycones, hesperetin (HT; 5,7,3'-trihydroxy-4'-methoxy-flavanone), and naringenin (NE; 5,7,4'-trihydroxy flavanone) and glycones, hesperidin (HD; 5,7,3'-trihydroxy-4'-methoxy-flavanone 7-rhamnoglucoside) and naringin (NI; 5,7,4'-trihydroxy flavanone 7-rhamno glucoside), were used to examine the importance of rutinose at C7 on the inhibitory effects of flavonoids on lipopolysaccharide (LPS)-induced nitric oxide production in macrophages. Both HT and NE, but not their respective glycosides HD and NI, induced heme oxygenase 1 (HO-1) protein expression in the presence or absence of LPS and showed time and dose-dependent inhibition of LPS-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in RAW264.7, J774A.1, and thioglycolate-elicited peritoneal macrophages. Additive inhibitory effect of an HO-1 inducer hemin and NE or NI on LPS-induced NO production and iNOS expression was identified, and HO enzyme inhibitor tin protoporphyrin (SnPP) attenuated the inhibitory effects of HT, NE, and hemin on LPS-induced NO production. Both NE and HT showed no effect on iNOS mRNA and protein stability in RAW264.7 cells. Removal of rutinose at C7 of HD and NI by enzymatic digestion using hesperidinase (HDase) and naringinase (NIase) produce inhibitory activity on LPS-induced NO production, according to the production of the aglycones, HT and NE, by high-performance liquid chromatography (HPLC) analysis. Furthermore, the amount of NO produced by LPS or lipoteichoic acid (LTA) was significantly reduced in HO-1-overexpressing cells (HO-1/RAW264.7) compared to that in parental cells (RAW264.7). Results of the present study provide scientific evidence to suggest that rutinose at C7 is a negative moiety in flavonoid inhibition of LPS-induced NO production, and that HO-1 is involved in the inhibitory mechanism of flavonoids on LPS-induced iNOS and NO production.     

Inhibition of inducible nitric oxide synthase and cyclooxygenase II by Platycodon grandiflorum saponins via suppression of nuclear factor-kappaB activation in RAW 264.7 cells

Saponins are glycosidic compounds present in many edible and inedible plants. They exhibit potent biological activities in mammalian systems, including several beneficial effects such as anti-inflammation and immunomodulation. In this study, we investigated the effects of seven platycodin saponins on the activities of inducible nitric oxide synthase (iNOS) and cyclooxygenase II (COX-2) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. We found that 2"-O-acetyl polygalacin D (S1), platycodin A (S2), platycodin D (S3), and polygalacin D (S6) inhibited LPS-induced NO production in a concentration-dependent manner. Furthermore, these compounds inhibited the expression of LPS-induced iNOS and COX-2 protein and mRNA without an appreciable cytotoxic effect on RAW 264.7 macrophages, and could suppress induction by LPS of pro-inflammatory cytokines such as prostaglandin E2 (PGE2). Treatment with these compounds of RAW 264.7 cells transfected with a reporter construct indicated a reduced level of LPS-induced nuclear factor-kappaB (NF-kappaB) activity and effectively lowered NF-kappaB binding as measured by electrophoretic mobility shift assay (EMSA). The suppression of NF-kappaB activation appears to occur through the prevention of inhibitor kappaB (IkappaB) degradation. In vivo, platycodin saponin mixture (PS) and S3 protected mice from the lethal effects of LPS. The 89% lethality induced by LPS/galactosamine was reduced to 60% and 50% when PS and S3, respectively, were administered simultaneously with LPS. These results suggest that the main inhibitory mechanism of the platycodin saponins may be the reduction of iNOS and COX-2 gene expression through blocking of NF-kappaB activation.     

Inhibitory effects of 3α-hydroxy-lup-20(29)-en-23, 28-dioic acid on lipopolysaccharide-induced TNF-α, IL-1β, and the high mobility group box 1 release in macrophages

We investigated the anti-inflammatory effects of 3α-hydroxy-lup-20(29)-en-23, 28-dioic acid (HLEDA)—a lupane-type triterpene isolated from leaves of Acanthopanax gracilistylus W. W.Smith (AGS), as well as the underlying molecular mechanisms in lipopolysaccharide (LPS)-induced RAW264.7 cells. Our results demonstrated that HLEDA concentration-dependently reduced the production of nitric oxide (NO), signi?cantly suppressed LPS-induced expression of TNF-α and IL-1β at the mRNA and protein levels in RAW264.7 cells. Further analysis revealed that HLEDA could reduce the secretion of High Mobility Group Box 1 (HMGB1). Additionally, the results showed that HLEDA efficiently decreased nuclear factor-kappaB (NF-κB) activation by inhibiting the degradation and phosphorylation of IκBα. These results suggest that HLEDA exerts anti-inflammatory properties in LPS-induced macrophages, possibly through inhibition of the NF-κB signaling pathway, which mediates the expression of pro-inflammatory cytokines. These results warrant further studies that would concern candidate therapy for diseases, such as fulminant hepatitis and rheumatology of triterpenoids in AGS.