Immunostimulatory Activities of the Sulfated Polysaccharide Ascophyllan from Ascophyllum nodosum in in Vivo and in Vitro Systems

Splenic natural killer (NK) cell activity against YAC-1 cells increased in mice intraperitoneally injected with ascophyllan. Ascophyllan enhanced the cytotoxicity of RAW264.7 cells toward YAC-1 cells in a concentration-dependent manner. The cytotoxicity of ascophyllan-stimulated RAW264.7 cells as to YAC-1 cells was suppressed with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of nitric oxide (NO) synthase, suggesting the involvement of NO in the cytotoxicity of ascophyllan-stimulated RAW264.7 cells.     

Dioscorealide B suppresses LPS‐induced nitric oxide production and inflammatory cytokine expression in RAW 264.7 macrophages: The inhibition of NF‐κB and ERK1/2 activation

Dioscorealide B (DB), a naphthofuranoxepin has been purified from an ethanolic extract of the rhizome of Dioscorea membranacea Pierre ex Prain & Burkill which has been used to treat inflammation and cancer in Thai Traditional Medicine. Previously, DB has been reported to have anti‐inflammatory activities through reducing nitric oxide (NO) and tumor necrosis factor‐α (TNF‐α) production in lipopolysaccharides (LPS)‐induced RAW 264.7 macrophage cells. In this study, the mechanisms of DB on LPS‐induced NO production and cytokine expression through the activation of nuclear factor‐κB (NF‐κB) and ERK1/2 are demonstrated in RAW 264.7 cells. Through measurement with Griess's reagent, DB reduced NO level with an IC₅₀ value of 2.85 ± 0.62 µM that was due to the significant suppression of LPS‐induced iNOS mRNA expression as well as IL‐1β, IL‐6, and IL‐10 mRNA at a concentration of 6 µM. At the signal transduction level, DB significantly inhibited NF‐κB binding activity, as determined using pNFκB‐Luciferase reporter system, which action resulted from the prevention of IκBα degradation. In addition, DB in the range of 1.5–6 µM significantly suppressed the activation of the ERK1/2 protein. In conclusion, the molecular mechanisms of DB on the inhibition of NO production and mRNA expression of iNOS, IL‐1β, IL‐6, and IL‐10 were due to the inhibition of the upstream kinases activation, which further alleviated the NF‐κB and MAPK/ERK signaling pathway in LPS‐induced RAW264.7 macrophage cells. J. Cell. Biochem. 109: 1057–1063, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of irradiation on cytokine secretion and nitric oxide production by inflammatory macrophages

This study explored the effects of low-dose and high-dose irradiation on inflammatory macrophage cells, specifically inflammatory cytokine secretion and nitric oxide (NO) production after irradiation. To elucidate the effect of irradiation on active and inactive macrophages, we exposed LPS-treated or untreated murine monocyte/macrophage RAW 264.7 cell lines to low-dose to high-dose radiation (0.01–10 Gy). We analyzed the effects of irradiation on RAW 264.7 cell proliferation by MTT assays and analyzed cytokine secretion and NO production related to inflammation by ELISA assays. Low-to-high doses of radiation did not significantly affect the proliferation of LPS-treated or untreated RAW 264.7 cells. Pro-inflammatory cytokine IL-1ß was generally increased in RAW 264.7 cells at 3 days after radiation. Especially, IL-1ß was significantly increased in only high dose-irradiation (2 and 10 Gy irradiation) groups in LPS-untreated RAW 264.7 cells but increased in both low and high dose-irradiation groups (0.01–10 Gy) in LPS-treated RAW 264.7 cells at 3 days after irradiation. Whereas, the expression of IL-1ß was prolonged in high-dose irradiation group at 5 days after irradiation. The production of anti-inflammatory cytokine IL-10 did not change significantly at 3 days after radiation but was significantly reduced at 5 days after 10 Gy radiation. The effect of irradiation on the secretion of IL-1ß and IL-10 was not significantly different between RAW 264.7 cells treated or not treated with LPS. The effect of irradiation on NO secretion by RAW 264.7 cells showed a specific pattern. NO was produced after low-dose irradiation but reduced in a high-dose irradiation group at 3 days after irradiation. However, NO production was not changed after low-dose irradiation and reduced at 5 days after high-dose irradiation. These results showed that irradiation affected the inflammatory system and regulated NO production in both activated and inactivated macrophages through different regulation mechanisms, depending on irradiation dose.     

Sentulic acid isolated from Sandoricum koetjape Merr attenuates lipopolysaccharide and interferon gamma co-stimulated nitric oxide production in murine macrophage RAW264 cells

A seco-triterpenoid, sentulic acid (SA) isolated from Sandoricum koetjape Merr attenuated nitric oxide (NO) production following co-stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFNγ) in RAW264.7 macrophage cells. The mRNA expression levels of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), IFNγ, interleukin (IL)-6, and IL-12 in LPS/IFNγ co-stimulated RAW264.7 cells also decreased upon SA treatment. To determine the molecular mechanisms underlying the inhibitory effect of SA on LPS/IFNγ-induced NO production in RAW264.7 cells, we further analyzed Toll-like receptor (TLR) signaling by western blotting. The expression of TLR4 and IFN signaling molecules in cells treated with SA was significantly suppressed compared to that in cells not treated with SA. Additionally, SA inhibited the binding of LPS to the TLR4 receptor in RAW264.7 cells stimulated with Alexa Fluor 488-conjugated LPS. These results demonstrate that SA attenuates NO production after LPS/IFNγ co-stimulation in RAW264.7 cells by inhibiting the binding of LPS to TLR4. Our findings suggest that SA is beneficial for the treatment of inflammatory diseases.     

The enhancing action of D-galactosamine on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells

The effect of D-galactosamine (D-GalN) on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was examined. D-GalN augmented the production of NO, but not tumor necrosis factor (TNF)-alpha in LPS-stimulated RAW 264.7 cells. Pretreatment of D-GalN augmented the NO production whereas its post-treatment did not. D-GalN augmented the NO production in RAW 264.7 cells stimulated with either TNF-alpha and interferon-gamma. The augmentation of LPS-induced NO production by D-GalN was due to enhanced expressions of an inducible type of NO synthase mRNA and proteins. Intracellular reactive oxygen species (ROS) were exclusively generated in RAW 264.7 cells stimulated with D-GalN and LPS. Scavenging of intracellular ROS abrogated the augmentation of NO production. It was therefore suggested that D-GalN might augment LPS-induced NO production through the generation of intracellular ROS.     

Degradation of IkappaBalpha in activated RAW264.7 cells is blocked by the phosphatidylinositol 3-kinase inhibitor LY294002

The mechanism by which lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) induces production of proinflammatory cytokines in murine macrophages, and the role of phosphatidylinositol 3-kinase (PI3-kinase) have not been well investigated. Activation of nuclear factor κB (NF-κB) is initiated by the phosphorylation of the inhibitory subunit, IκB, which targets IκB for degradation and leads to the release of active NF-κB. In this study we demonstrate that 2- (4-morpholinyl)-8-phenylchromone (LY294002), which inhibits PI3-kinase, specifically inhibited degradation of IκBα in RAW264.7 cells stimulated with interferon-γ (IFN-γ) plus LPS or IFN-γ plus PMA. To elucidate the importance of this activity in RAW264.7 cells, we examined tumor necrosis factor-α (TNF-α) and interleukin IL)-6 production in the activated cells. Pretreatment of the cells with LY294002 resulted in the inhibition of TNF-α and IL-6 production in RAW264.7 cells stimulated with IFN-γ plus LPS or IFN-γ plus PMA. Furthermore, LY294002 inhibited the production of nitric oxide NO) in RAW264.7 cells stimulated with IFN-γ plus LPS or IFN-γ plus PMA. LY294002 also inhibited inducible NO synthase (iNOS) mRNA expression in the activated RAW264.7 cells. In conclusion, the present results suggest that PI3-kinase is involved in the signal transduction pathway responsible for LPS- or PMA-mediated TNF-α and IL-6 production, and that LY294002 inhibits NO generation through blocking the degradation of IκBα in activated RAW264.7 cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ganglioside GM3 suppresses lipopolysaccharide‐induced inflammatory responses in rAW 264.7 macrophage cells through NF‐κB,AP‐1, and MAPKs signaling

Gangliosides are known to specifically inhibit vascular leukocyte recruitment and consequent interaction with the injured endothelium, the basic inflammatory process. In this study, we have found that the production of nitric oxide (NO), a main regulator of inflammation, is suppressed by GM3 on murine macrophage RAW 264.7 cells, when induced by LPS. In addition, GM3 attenuated the increase in cyclooxyenase‐2 (COX‐2) protein and mRNA levels in lipopolysaccharide (LPS)‐activated RAW 264.7 cells in a dose‐dependent manner. Moreover, GM3 inhibited the expression and release of pro‐inflammatory cytokines of tumor necrosis factor‐alpha (TNF‐α), interleukin‐6 (IL‐6), and interleukin‐1β (IL‐1β) in RAW 264.7 macrophages. At the intracellular level, GM3 inhibited LPS‐induced nuclear translocation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and activator protein (AP)‐1 in RAW 264.7 macrophages. We, therefore, investigated whether GM3 affects mitogen‐activated protein kinase (MAPK) phosphorylation, a process known as the upstream signaling regulator. GM3 dramatically reduced the expression levels of the phosphorylated forms of ERK, JNK, and p38 in LPS‐activated RAW 264.7 cells. These results indicate that GM3 is a promising suppressor of the vascular inflammatory responses and ganglioside GM3 suppresses the LPS‐induced inflammatory response in RAW 264.7 macrophages by suppression of NF‐κB, AP‐1, and MAPKs signaling. Accordingly, GM3 is suggested as a beneficial agent for the treatment of diseases that are associated with inflammation.     

Effects of alpha-melanotropin C-terminal tripeptide analogues on macrophage NO production

The C-terminal tripeptide of melanocyte-stimulating hormone, MSH (11-13) (Lys-Pro-Val), possesses strong anti-inflammatory actions, which are mediated via mechanisms that are not fully understood. To shed more light into these mechanisms we have here synthesised and evaluated the activities of L- and D-Val substituted cyclic modifications of MSH (11-13) on nitric oxide (NO) in macrophage RAW 264.7 cells, as well as on binding to melanocortin receptors (MCRs) in B16-F1 and MCR expressing insect cells, and for effects on cAMP. MSH (11-13) and its analogues did neither bind to MCRs nor stimulate cAMP in RAW 264.7 and B16-F1 cells, except H-, which showed a tendency to increase cAMP at high (10-100 microM) concentrations. However, all investigated peptides dose dependently inhibited NO in LPS/IFN-gamma-stimulated RAW 264.7, cells with a structure activity relationship suggesting the existence of a distinct receptive site. This site appears to be distinct from the MCRs and not linked with cAMP.     

Mechanisms of activation of the RAW264.7 macrophage cell line by transfected mammalian DNA

Bacterial DNA can stimulate the production of cytokines and nitric oxide (NO), while mammalian DNA can block these responses. If mammalian DNA is transfected into macrophages, however, it can stimulate NO production, without inducing IL-12. To define further this activity, signaling pathways induced by transfected calf thymus (CT) DNA were studied. Using RAW264.7 cells as a model, CT DNA in the transfection agent FuGENE 6 activated cells through the NF-kappaB and MAPKs pathways, similar to bacterial DNA and LPS. The role of these pathways was further investigated using specific inhibitors, with studies indicating that NO production is blocked by inhibitors of NF-kappaB and p38 but not other MAPKs. These data indicate that the immune activity of DNA is influenced by context or intracellular location and that, when transfected into cells, mammalian DNA can activate cells through signaling pathways similar to those of bacterial DNA.     

Bacillus alcalophilus peptidoglycan induces IFN-alpha-mediated inhibition of vaccinia virus replication

Bacterial products such as cell walls (CW) and peptidoglycan (PGN) are known to activate macrophages and NK cells during microbial infections. In this report, we demonstrated that whole CW and PGN of four Gram-positive bacteria are capable of enhancing the anti-poxviral activity of murine macrophage RAW 264.7 cells. Among the major Bacillus alcalophilus CW components, PGN contributes the most to antiviral activity and induces remarkably higher levels of IFN-alpha. Anti-IFN-alpha/beta antibody, but not anti-IFN-gamma, anti-IFN-gamma receptor, or anti-IL-12, reversed the PGN-induced inhibition of vaccinia virus replication and reduced nitric oxide (NO) production. Our data thus suggest that PGN induce antiviral activity through IFN-alpha and to a lesser extent, through NO production.     

Silicate modulates the cross-talk between osteoblasts (SaOS-2) and osteoclasts (RAW 264.7 cells): Inhibition of osteoclast growth and differentiation

It has been shown that inorganic monomeric and polymeric silica/silicate, in the presence of the biomineralization cocktail, increases the expression of osteoprotegerin (OPG) in osteogenic SaOS‐2 sarcoma cells in vitro. In contrast, silicate does not affect the steady‐state gene expression level of the osteoclastogenic ligand receptor activator of NF‐κB ligand (RANKL). In turn it can be expected that the concentration ratio of the mediators OPG/RANKL increases in the presence of silicate. In addition, silicate enhances the growth potential of SaOS‐2 cells in vitro, while it causes no effect on RAW 264.7 cells within a concentration range of 10–100 µM. Applying a co‐cultivation assay system, using SaOS‐2 cells and RAW 264.7 cells, it is shown that in the presence of 10 µM silicate the number of RAW 264.7 cells in general, and the number of TRAP⁺ RAW 264.7 cells in particular markedly decreases. The SaOS‐2 cells retain their capacity of differential gene expression of OPG and RANKL in favor of OPG after exposure to silicate. It is concluded that after exposure of the cells to silicate a factor(s) is released from SaOS‐2 cells that causes a significant inhibition of osteoclastogenesis of RAW 264.7 cells. It is assumed that it is an increased secretion of the cytokine OPG that is primarily involved in the reduction of the osteoclastogenesis of the RAW 264.7 cells. It is proposed that silicate might have the potential to stimulate osteogenesis in vivo and perhaps to ameliorate osteoporotic disorders. J. Cell. Biochem. 113: 3197–3206, 2012. © 2012 Wiley Periodicals, Inc.     

Effect of bismuth subgallate on nitric oxide and prostaglandin E2 production by macrophages

Bismuth subgallate (BSG) is used widely in clinics, including Vincent's angina, syphilis, and adenotonsillectomy. This study examined the effects of BSG on nitric oxide (NO) and prostaglandin E2 (PGE2) production in activated RAW 264.7 cells. BSG suppressed production of NO and PGE2 in a dose-dependent manner. BSG could increase TGF-beta1 production, which in turn might promote degradation of iNOS mRNA, thus inhibiting NO production. Additionally, BSG inhibited mPGES protein expression and COX-2 activity in activated RAW 264.7 cells. Exogenous addition of SNP reversed the inhibition effect of PGE2 production by BSG. This behavior indicates that PGE2 inhibition by BSG exerts an indirect effect through NO inhibition.     

Cellular non-heme iron content is a determinant of nitric oxide-mediated apoptosis, necrosis, and caspase inhibition

In this report, we tested the hypothesis that cellular content of non-heme iron determined whether cytotoxic levels of nitric oxide (NO) resulted in apoptosis versus necrosis. The consequences of NO exposure on cell viability were tested in RAW264.7 cells (a cell type with low non-heme iron levels) and hepatocytes (cells with high non-heme iron content). Whereas micromolar concentrations of the NO donor S-nitroso-N-acetyl-DL-penicillamine induced apoptosis in RAW264.7 cells, millimolar concentrations were required to induce necrosis in hepatocytes. Caspase-3 activation and cytochrome c release were evident in RAW264.7 cells, but only cytochrome c release was detectable in hepatocytes following high dose S-nitroso-N-acetyl-DL-penicillamine exposure. Pretreating RAW264.7 cells with FeSO(4) increased intracellular non-heme iron to levels similar to those measured in hepatocytes and delayed NO-induced cell death, which then occurred in the absence of caspase-3 activation. Iron loading was also associated with the formation of intracellular dinitrosyl-iron complexes (DNIC) upon NO exposure. Cytosolic preparations containing DNIC as well as pure preparations of DNIC suppressed caspase activity. These data suggest that non-heme iron content is a key factor in determining the consequence of NO on cell viability by regulating the chemical fate of NO.     

EphB4/ephrinB2逆向信号对RAW264．7破骨细胞分化中PDZ结构域蛋白表达变化的研究

Eph受体是酪氨酸蛋白激酶受体家族中最大的亚家族,ephrin(Eph受体相互作用蛋白)是其配体,它们是膜结合蛋白,相互依赖进行信号转导.内居蛋白(syntenin)与Pick1属于PDZ结构域(PSD-95/Dlg-/Zo-1 domain)蛋白,报道称能与ephrinB配体结合,但是否受Eph受体调控尚未见报道.以RAW264.7细胞株为研究对象,通过蛋白质印迹及/或免疫荧光分析显示RAW264.7细胞经RANKL诱导的破骨细胞表达ephrinB2、内居蛋白(syntenin)和Pick1三个蛋白质.将提前成簇的可溶性EphB4蛋白加入培养液,与ephrinB2配体结合,用来研究EphB4/ephrinB2逆向信号对syntenin和Pick1表达水平变化的影响.免疫印迹及Real-time RT-PCR分析结果显示,在EphB4-Fc实验组中Pick1的蛋白质及mRNA水平都有明显增加,然而在EphB4-Fc实验组与Fc对照组别间syntenin的蛋白质及mRNA水平未见明显变化.免疫共沉淀结果显示,syntenin和Pick1不能与ephrinB2共沉淀.以上结果初步探索了体外破骨细胞分化过程中,EphB4/ephrinB2逆向信号对PDZ结构域蛋白(ephrinB2配体潜在的下游信号分子)表达变化的调控.     

自然杀伤细胞参与肝脏恶性肿瘤免疫逃逸研究进展

自然杀伤细胞是机体固有免疫系统重要组成部分,在肝脏等免疫器官中含量丰富,而且免疫表型、功能等表现出器官特异性。在正常情况下,靶细胞表面的配体与自然杀伤细胞表面的活化性受体直接结合并释放细胞毒性物质,诱导活化靶细胞凋亡程序,从而发挥抗感染、抗肿瘤作用。然而肿瘤细胞仍能够通过多种途径逃逸机体的免疫监视功能,研究认为肿瘤细胞抗原异常表达、肿瘤微环境中细胞因子及其他免疫细胞相互作用等因素所引起的自然杀伤细胞活性降低对于诱导肿瘤免疫逃逸起重要作用。本文综述了自然杀伤细胞在肝脏恶性肿瘤发生过程中参与免疫逃逸的机制及研究进展,以期为临床抗肿瘤免疫治疗的研究提供参考。