Astaxanthin inhibits nitric oxide production and inflammatory gene expression by suppressing I(kappa)B kinase-dependent NF-kappaB activation

Astaxanthin, a carotenoid without vitamin A activity, has shown anti-oxidant and anti-inflammatory activities; however, its molecular action and mechanism have not been elucidated. We examined in vitro and in vivo regulatory function of astaxanthin on production of nitric oxide (NO) and prostaglandin E2 (PGE2) as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). Astaxanthin inhibited the expression or formation production of these proinflammatory mediators and cytokines in both lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary macrophages. Astaxanthin also suppressed the serum levels of NO, PGE2, TNF-alpha, and IL-1beta in LPS-administrated mice, and inhibited NF-kappaB activation as well as iNOS promoter activity in RAW264.7 cells stimulated with LPS. This compound directly inhibited the intracellular accumulation of reactive oxygen species in LPS-stimulated RAW264.7 cells as well as H2O2-induced NF-kappaB activation and iNOS expression. Moreover, astaxanthin blocked nuclear translocation of NF-kappaB p65 subunit and I(kappa)B(alpha) degradation, which correlated with its inhibitory effect on I(kappa)B kinase (IKK) activity. These results suggest that astaxanthin, probably due to its antioxidant activity, inhibits the production of inflammatory mediators by blocking NF-kappaB activation and as a consequent suppression of IKK activity and I(kappa)B-alpha degradation.     

Kruppel样因子4对内毒素所致IL-6基因表达的调控及机制研究

探讨Kruppel样因子4(KLF4)对内毒素所致白介素(IL-6)的基因表达以及释放的影响,并对其调控机制做了初步研究．使用RT-PCR和Western blot检测KLF4 mRNA和蛋白质的表达．采用KLF4过表达的RAW264.7巨噬细胞株或反义寡核苷酸技术抑制内源性KLF4的表达,用RT-PCR和ELISA检测内毒素(LPS)刺激后IL-6 mRNA和蛋白质的表达．采用荧光素酶报告基因检测RAW264.7细胞中KLF4过表达对IL-6基因启动子报告基因转录活性的影响．使用EMSA法检测细胞中KLF4与IL-6基因启动子区KLF4元件的结合．结果表明：LPS可以诱导RAW264.7巨噬细胞KLF4的表达以及IL-6蛋白表达．KLF4过表达明显抑制IL-6的mRNA和蛋白质的表达,而KLF4缺失使这种作用消失．荧光素酶报告基因的结果显示,KLF4可以抑制LPS所致的IL-6基因启动子的转录活性．EMSA显示KLF4不能与IL-6启动子区的KLF4结合元件直接结合．结果表明,LPS可以促进RAW264.7小鼠巨噬细胞KLF4的表达和IL-6的释放．KLF4能抑制LPS诱导的IL-6表达和释放,其机制是抑制IL-6启动子的转录活性,但KLF4的抑制作用不是通过直接与IL-6基因的启动子区相结合而实现的．     

Ca2+/calmodulin-dependent protein phosphatase calcineurin mediates the expression of iNOS through IKK and NF-kappaB activity in LPS-stimulated mouse peritoneal macrophages and RAW 264.7 cells

Ca(2+) and Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN) have been known to play crucial roles in immune response and inflammation. Using mouse peritoneal macrophages and RAW 264.7 macrophage cells, we demonstrated that LPS mobilized intracellular free Ca(2+) and induced CN phosphatase activity. iNOS expression and NO secretion in response to LPS were suppressed by Ca(2+) antagonists (TMB-8, BAPTA/AM, and nifedipine) and CN inhibitor (cyclosporin A). Transient expression of constitutively active CN in mouse peritoneal macrophages and RAW 264.7 macrophages strongly activated NF-kappaB, a key mediator of iNOS expression. We also found that CN mediates NF-kappaB activation via IkappaB-alpha hyperphosphorylation and degradation. Overexpression of dominant negative mutant of IKKalpha and -beta demonstrates that only IKKbeta is the target for CN. These results indicate that CN is required for full iNOS expression and the effective activation of NF-kappaB in RAW 264.7 and peritoneal macrophages.     

Lipoteichoic acid-induced nitric oxide synthase expression in RAW 264.7 macrophages is mediated by cyclooxygenase-2, prostaglandin E2, protein kinase A, p38 MAPK, and nuclear factor-kappaB pathways

We recently reported that lipoteichoic acid (LTA), a cell wall component of the gram-positive bacterium Staphylococcus aureus, stimulated inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) release, and cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophages. This study was carried out to further investigate the roles of COX-2 and prostaglandin E2 (PGE2) in LTA-induced iNOS expression and NO release in RAW 264.7 macrophages. Treatment of RAW 264.7 macrophages with LTA caused a time-dependent increase in PGE2 release. LTA-induced iNOS expression and NO release were inhibited by a non-selective COX inhibitor (indomethacin), a selective COX-2 inhibitor (NS-398), an adenylyl cyclase (AC) inhibitor (dideoxyadenosine, DDA), and a protein kinase A (PKA) inhibitor (KT-5720). Furthermore, both PGE2 and the direct PKA activator, dibutyryl-cAMP, also induced iNOS expression in a concentration-dependent manner. Stimulation of RAW 264.7 macrophages with LTA, PGE2, and dibutyryl-cAMP all caused p38 MAPK activation in a time-dependent manner. LTA-mediated p38 MAPK activation was inhibited by indomethacin, NS-398, and SB 203580, but not by PD 98059. The PGE2-mediated p38 MAPK activation was inhibited by DDA, KT-5720, and SB 203580, but not by PD 98059. LTA caused time-dependent activation of the nuclear factor-kappaB (NF-kappaB)-specific DNA-protein complex formation. The LTA-induced increase in kappaB-luciferase activity was inhibited by indomethacin, NS-398, KT-5720, and a dominant negative mutant of p38 alphaMAPK (p38 alphaMAPK DN). These results suggest that LTA-induced iNOS expression and NO release involve COX-2-generated PGE2 production, and AC, PKA, p38 MAPK, and NF-kappaB activation in RAW 264.7 macrophages.     

7,7'-Dihydroxy bursehernin inhibits the expression of inducible nitric oxide synthase through NF-kappaB DNA binding suppression.

This study isolated a lignan, 7,7'-dihydroxy bursehernin, from Geranium thunbergii and investigated whether or not the lignan affects the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS). The gel shift analysis and luciferase reporter gene assays using the iNOS promoter and nuclear factor-kappaB (NF-kappaB) minimal promoter showed that a treatment with 7,7'-dihydroxy bursehernin reduced the reporter activities and binding of NF-kappaB to the NF-kappaB consensus sequence, while it had no effect on the nuclear translocation of p65 and the phosphorylation/degradation of I-kappaBalpha. It was reported that a few natural compounds directly suppressed the binding activity of the NF-kappaB components to DNA. The NF-kappaB binding activity was not reversed by the in vitro exposure of the nuclear extracts to 7,7'-dihydroxy bursehernin, which suggest that a metabolite(s) of 7,7'-dihydroxy bursehernin might target the binding of the NF-kappaB complex to the DNA binding domain region in the promoter region of the iNOS gene. After incubation of RAW264.7 cells with 7,7-dihydroxy bursehernin for 18h, the levels of parent compound were negligible; while a main metabolite, 4-[4-(n-hydroxy-phenyl)-2,3-dimethyl-buta-1,3-dienyl]-benzene-1,2-diol was detected in cell lysates and culture medium.     

Suppression of cytokine production in lipopolysaccharide-stimulated mouse macrophages by novel cationic glucosamine derivative involves down-regulation of NF-kappaB and MAPK expressions

Exposure of macrophages to bacterial lipopolysaccharide (LPS) induces release of proinflammatory cytokines that play crucial roles in chronic inflammation. Glucosamine has reported to possess anti-inflammatory properties and currently is the oral supplement of choice for the management of inflammation related complications including osteoarthritis. In this study, quaternized amino glucosamine (QAGlc), a newly synthesized cationic glucosamine (Glc) derivative was found to inhibit LPS-stimulated production of IL-1beta, IL-6, TNF-alpha, and PGE(2) in RAW264.7, mouse macrophages more potently than its starting material Glc. Since production of cytokines is regulated mainly via activation of NF-kappaB and regulation of mitogen-activated protein kinases (MAPKs), we examined if QAGlc could be responsible for the suppression of NF-kappaB pathway and MAPKs. We used reporter gene assay and Western blotting to examine the effects of QAGlc on activation and translocation of NF-kappaB. Further, QAGlc-mediated inhibition of NF-kappaB was accompanied with a suppression of its translocation. Apparently, QAGlc was shown to attenuate LPS-induced activation of p38 MAPK and JNK in RAW264.7 cells suggesting that inhibition of MAPK-mediated LPS signaling also contribute to suppression of cytokine production following stimulation of macrophages with LPS.     

The release of microparticles by apoptotic cells and their effects on macrophages

Microparticles are small membrane vesicles released from the cell membrane by exogenous budding. To elucidate the interactions of microparticles with macrophages, the effect of microparticles released from Jurkat T cells on RAW 264.7 cells was determined. Microparticles were isolated by differential centrifugation, using FACS analysis with annexin V and cell surface markers for identification. Various inducers of apoptosis increased the release of microparticles from Jurkat cells up to 5-fold. The released microparticles were then cultured with RAW 264.7 cells. As shown by confocal microscopy and FACS analysis, RAW 264.7 macrophages cleared microparticles by phagocytosis. In addition, microparticles induced apoptosis in RAW 264.7 cells in a dose-dependent manner with up to a 5-fold increase of annexin V positive cells and 9-fold increase in caspase 3 activity. Cell proliferation as determined by the MTT test was also reduced. Furthermore, microparticles stimulated the release of microparticles from macrophages. These effects were specific for macrophages, since no apoptosis was observed in NIH 3T3 and L929 cells. These findings indicate that microparticles can induce macrophages to undergo apoptosis, in turn resulting in a further increase of microparticles. The release of microparticles from apoptotic cells may therefore represent a novel amplification loop of cell death.     

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.     

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.     

Differential regulation of NO availability from macrophages and endothelial cells by the garlic component S-allyl cysteine

Garlic has been used as a traditional medicine for prevention and treatment of cardiovascular diseases. However, the molecular mechanism of garlic's pharmacological action has not been clearly elucidated. We examined here the effect of garlic extract and its major component, S-allyl cysteine (SAC), on nitric oxide (NO) production by macrophages and endothelial cells. The present study demonstrates that these reagents inhibited NO production through the suppression of iNOS mRNA and protein expression in the murine macrophage cell line RAW264.7, which had been stimulated with LPS and IFNgamma. The garlic extract also inhibited NO production in peritoneal macrophages, rat hepatocytes, and rat aortic smooth muscle cells stimulated with LPS plus cytokines, but it did not inhibit NO production in iNOS-transfected AKN-1 cells or iNOS enzyme activity. These reagents suppressed NF-kappaB activation and murine iNOS promoter activity in LPS and IFNgamma-stimulated RAW264.7 cells. In contrast, these reagents significantly increased cGMP production by eNOS in HUVEC without changes in activity, protein levels, and cellular distribution of eNOS. Finally, garlic extract and SAC both suppressed the production of hydroxyl radical, confirming their antioxidant activity. These data demonstrate that garlic extract and SAC, due to their antioxidant activity, differentially regulate NO production by inhibiting iNOS expression in macrophages while increasing NO in endothelial cells. Thus, this selective regulation may contribute to the anti-inflammatory effect and prevention of atherosclerosis by these reagents.     

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.     

Contribution of the exosome-associated form of secreted endoplasmic reticulum aminopeptidase 1 to exosome-mediated macrophage activation

Macrophages secrete endoplasmic reticulum aminopeptidase 1 (ERAP1) in response to lipopolysaccharide (LPS) and interferon (IFN)-γ to enhance their phagocytic and nitric oxide (NO) synthetic activities. In this study, we found that a subset of secreted ERAP1 bound to exosomes released from LPS/IFN-γ-treated murine RAW264.7 macrophages compared to untreated cells. ERAP1-bound exosomes enhanced phagocytic and NO synthetic activities of macrophages more efficiently than free ERAP1 and exosomes derived from untreated cells. Deletion of the exon 10 coding sequence in ERAP1 gene resulted in loss of binding to exosomes. By comparing the activities of exosomes derived from wild-type and ERAP1 gene-deficient RAW264.7 cells, we observed that ERAP1 contributed to the exosome-dependent phagocytosis and NO synthesis of the cells. Upon stimulation of RAW264.7 cells with LPS/IFN-γ, TNF-α, IFN-γ, and CCL3 were also associated with the released exosomes. Analyses of cytokine function revealed that while CCL3 in the exosomes was crucial to the phagocytic activity of RAW264.7 cells, TNF-α and IFN-γ primarily contributed to the enhancement of NO synthesis. These results suggest that treatment with LPS/IFN-γ alters the physicochemical properties of exosomes released from macrophages in order to facilitate association with ERAP1 and several cytokines/chemokines. This leads to exosome-mediated enhancement of macrophage functions. It is possible that packaging effector molecules into exosomes upon inflammatory stimuli, facilitates the exertion of effective pathophysiological functions on macrophages. Our data provide the first evidence that ERAP1 associated with exosomes plays important roles in inflammatory processes via activation of macrophages.     

Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways

Lactoferrin (LF) is a component of innate immunity and is known to interact with accessory molecules involved in the TLR4 pathway, including CD14 and LPS binding protein, suggesting that LF may activate components of the TLR4 pathway. In the present study, we have asked whether bovine LF (bLF)-induced macrophage activation is TLR4-dependent. Both bLF and LPS stimulated IL-6 production and CD40 expression in RAW 264.7 macrophages and in BALB/cJ peritoneal exudate macrophages. However, in macrophages from congenic TLR4(-/-) C.C3-Tlr4(lps-d) mice, CD40 was not expressed while IL-6 secretion was increased relative to wild-type cells. The signaling components NF-kappaB, p38, ERK and JNK were activated in RAW 264.7 cells and BALB/cJ macrophages after bLF or LPS stimulation, demonstrating that the TLR4-dependent bLF activation pathway utilizes signaling components common to LPS activation. In TLR4 deficient macrophages, bLF-induced activation of NF-kappaB, p38, ERK and JNK whereas LPS-induced cell signaling was absent. We conclude from these studies that bLF induces limited and defined macrophage activation and cell signaling events via TLR4-dependent and -independent mechanisms. bLF-induced CD40 expression was TLR4-dependent whereas bLF-induced IL-6 secretion was TLR4-independent, indicating potentially separate pathways for bLF mediated macrophage activation events in innate immunity.     

Biological activity of rhBMP-2 released from PLGA microspheres

Human recombinant bone morphogenetic protein-2 (rhBMP-2) has been proven effective in stimulating the regeneration of bone in both skeletal and extraskeletal locations. Through encapsulation within, and release from, biodegradable poly(DL-lactic-co-glycolic acid) (PLGA) microspheres, a proven vehicle for sustained delivery of various proteins, the local concentrations of rhBMP-2 could be maintained at optimal levels to stimulate bone regeneration and remodeling at the site of healing in diverse clinical settings. Thus the purpose of this work was to investigate the encapsulation of rhBMP-2 in PLGA microspheres and its biologic activity upon release. Using in vitro tests in simulated body fluids, the effect of rhBMP-2 released from PLGA microspheres upon osteoblast cell cultures was found to be statistically similar to the effect produced by positive controls consisting of nonencapsulated aqueous rhBMP-2 in simulated body fluids. This clarifies an important step in skeletal tissue engineering strategies aimed at the use of encapsulated rhBMP-2 to stimulate bone regeneration and remodeling.