Characterization of oak and birch dust-induced expression of cytokines and chemokines in mouse macrophage RAW 264.7 cells

Occupational exposure to wood dust is related to several respiratory diseases, such as allergic rhinitis, chronic bronchitis, and asthma. However, virtually nothing is known about molecular mechanisms behind wood dust-induced pulmonary inflammation. To elucidate the effects of wood dust exposure on cytokine and chemokine expression in murine macrophage cell line cells, mouse RAW 264.7 cells were exposed to two selected hardwood dusts, oak and birch. TiO2 and LPS were used as controls. Expression patterns of several cytokines, chemokines, and chemokine receptors were assessed by real-time quantitative PCR system and by ELISA. Exposure to birch dust caused a major increase in TNF-alpha and IL-6 protein levels whereas a weaker induction of TNF-alpha protein was found after exposure to oak dust. Inorganic TiO2 dust did not induce significant cytokine expression. With respect to the chemokines, a dose-dependent, about 10-fold induction of CCL2 mRNA and protein was found after exposure to birch dust. Oak dust induced weakly CCL2 protein. Similarly, birch dust induced a strong expression of CCL3, CCL4, and CXCL2/3 mRNA whereas only moderate levels of these chemokine mRNAs were detected after oak dust exposure. In contrast, expression of CCL24 mRNA was inhibited by more than 40-fold by both oak and birch dusts. TiO2 dust induced about five-fold expression of CCL3 and CCL4 mRNA but did not affect significantly other chemokines. These results suggest that exposure to birch or oak dusts may influence the development of the inflammatory process in the airways by modulating the expression of macrophage-derived cytokines and chemokines.     

The effects of wood dusts on the redox status and cell death in mouse macrophages (RAW 264.7) and human leukocytes in vitro

Wood dusts are classified as carcinogenic to humans and also produce other toxic, allergic, and acute effects in woodworkers. However, little is known about causative agents in wood dusts and their mechanisms of action. The effects of different tree species and particle size for biological activity were studied. The differences in the production of reactive oxygen species (ROS) and cell death (necrotic and apoptotic) between mouse macrophage (RAW 264.7) cells and human polymorphonuclear leukocytes (PMNL) for pine, birch, and beech dust exposures were investigated in vitro. The pine and birch dust exposure (1-100 microg/ml) produced concentration-dependent ROS production in both the cells, which was one order of magnitude higher with pine dust. The ROS production was faster in human PNML than murine RAW cells. The higher concentrations (500 and/or 1000 microg/ml) decreased ROS formation. With pine and birch dust exposure, this was probably due to the necrotic cell death. The pine dust concentrations of 500 and 1000 microg/ml were cytotoxic to human PMNL. The beech dust exposure activated the ROS production and decreased the cell viability only at the highest concentrations, being least potent of the three dusts. A sign of the apoptotic cell death in the murine RAW cells was observed at the pine dust concentration of 100 microg/ml. The exposure to the birch and beech dusts with a smaller particle size (<5 microm) produced greater ROS production than exposure to the corresponding dust with a wide range of particle sizes. However, changing the particle size did not affect the cell viability. The results indicate that the type of wood dust (tree species and possibly particle size) has a significant impact on the function and viability of phagocytic cells.     

八肽胆囊收缩素抑制LPS诱导的RAW264.7细胞AP-1活性及IL-6表达

目的研究八肽胆囊收缩素(CCK-8)对LPS诱导RAW264.7细胞IL-6表达的影响及相关机制。方法用ELISA及RT-PCR法检测RAW264.7细胞IL-6蛋白及mR-NA表达;用EMSA方法检测RAW264.7细胞AP-1 DNA结合活性。结果①LPS可时间依赖性的诱导RAW264.7细胞IL-6蛋白及mRNA表达;②10-10 mol.L-1 CCK-8对LPS诱导的RAW264.7细胞IL-6表达无明显影响;10-8、10-6 mol.L-1 CCK-8浓度依赖性地抑制了LPS诱导的RAW264.7细胞IL-6表达;③10-10 mol.L-1 CCK-8未影响LPS诱导的AP-1活性,10-8、10-6 mol.L-1 CCK-8浓度依赖性地抑制了LPS诱导的AP-1活性。结论 CCK-8通过抑制AP-1 DNA结合活性而抑制了LPS诱导的RAW264.7细胞IL-6表达,这可能是CCK-8发挥抗炎作用的信号转导机制之一。     

芦荟大黄素对LPS诱导的RAW264.7细胞NO生成及iNOS表达的影响

目的观察芦荟大黄素(aloe-emodin)对脂多糖(LPS)诱导的RAW264.7细胞一氧化氮(NO)生成及诱生型一氧化氮合酶(iNOS)mRNA表达的作用。方法采用LPS诱导的RAW264.7细胞株建立细胞炎症反应模型。采用Griess试剂法测定NO释放量;采用硝普钠释放NO法测定NO自由基含量的变化;采用反转录聚合酶链反应(RT-PCR)分析iNOS mRNA表达改变。结果芦荟大黄素在0.69～2.50mg·L-1剂量范围内可抑制LPS诱导的RAW264.7细胞NO的释放,并呈剂量和时间依赖关系;芦荟大黄素在0.63～5.00mg·L-1剂量范围内可下调LPS诱导的RAW264.7细胞iNOS mRNA含量;而此范围内芦荟大黄素无直接清除NO自由基作用,不影响iNOS活性。结论芦荟大黄素可明显降低LPS诱导的RAW264.7细胞NO释放,呈时间和剂量依赖关系,此作用并非通过捕捉NO或抑制iNOS活性来实现,而是通过抑制iNOS mRNA表达发挥作用的。     

Effect of PPARalpha activation of macrophages on the secretion of inflammatory cytokines in cultured adipocytes

The relationship between adipocytes and infiltrated macrophages in fat tissue is important for the pathogenesis of insulin resistance through the activation of cytokines. Peroxisome proliferator-activated receptors (PPARs) play a role in the regulation of cytokine secretion in these cells. We studied the effect of the PPARalpha activation of macrophages on the modulation of the tumor necrosis factor alpha (TNFalpha) expression in adipocytes using a cell culture system. A conditioned medium of lipopolysaccharide (LPS)-stimulated RAW264.7 cells, a macrophage cell line, induced the level of TNFalpha mRNA in 3T3-L1 adipocytes. This effect was inhibited by the addition of neutralizing antibody against interleukin 6 (IL-6) in the conditioned medium or the preincubation of RAW264.7 cells with a specific PPARalpha agonist, K-111 (2,2-dichloro-12-(4-chlorophenyl)dodecanoic acid). K-111 reduced both the IL-6 production and mRNA expression in RAW264.7 cells, and its effect was stronger than that of rosiglitazone, a PPARgamma agonist. The activation of the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) pathway and nuclear factor kappa B (NF-kappaB) subunits of p65 was significantly inhibited by K-111. The blocking of IL-6 production through the SAPK/JNK pathway or by transfection with siRNA specific for IL-6 abolished the inhibitory effect of K-111 on the TNFalpha expression in the 3T3-L1 adipocytes. As a result, the IL-6 produced by RAW264.7 cells is an inducer of TNFalpha expression in 3T3-L1 adipocytes, and the IL-6 secretion is inhibited by the activation of PPARalpha. The PPARalpha activators may suppress the pathogenetical secretion of TNFalpha in the adipocytes through the functional modulation of the infiltrated macrophages.     

Mechanisms of Particle-Induced Pulmonary Inflammation in a Mouse Model: Exposure to Wood Dust

Repeated airway exposure to wood dust has long been known tocause adverse respiratory effects such as asthma and chronicbronchitis and impairment of lung function. However, the mechanismsunderlying the inflammatory responses of the airways after wooddust exposure are poorly known. We used a mouse model to elucidatethe mechanisms of particle-induced inflammatory responses tofine wood dust particles. BALB/c mice were exposed to intranasallyadministered fine (more than 99% of the particles had a particlesize of 5 µm, with virtually identical size distribution)birch or oak dusts twice a week for 3 weeks. PBS, LPS, and titaniumdioxide were used as controls. Intranasal instillation of birchor oak dusts elicited influx of inflammatory cells to the lungsin mice. Enhancement of lymphocytes and neutrophils was seenafter oak dust exposure, whereas eosinophil infiltration washigher after birch dust exposure. Infiltration of inflammatorycells was associated with an increase in the mRNA levels ofseveral cytokines, chemokines, and chemokine receptors in lungtissue. Oak dust appeared to be a more potent inducer of theseinflammatory mediators than birch dust. The results from ourin vivo mouse model show that repeated airway exposure to wooddust can elicit lung inflammation, which is accompanied by inductionof several proinflammatory cytokines and chemokines. Oak andbirch dusts exhibited quantitative and qualitative differencesin the elicitation of pulmonary inflammation, suggesting thatthe inflammatory responses induced by the wood species may risevia different cellular mechanisms.     

Interrelationship between hemolysis and lipid peroxidation of human erythrocytes induced by silicic acid and silicate dusts

Silicic acid and silicate dusts (slate dust and chrysotile asbestos) cause hemolysis of erythrocytes in vitro. The peroxidation of polyunsaturated fatty acids (PUFA) of erythrocyte membrane lipids is also enhanced by incubating the erythrocytes with silicic acid and silicate dusts in in vitro. Hemolysis of erythrocytes elicited by silicic acid and silicate dusts is inhibited significantly by polyvinyl-pyrrolidone and dipalmitoyl lecithin (DPL). These agents, however, have no effect on silicic acid and silicate dust induced peroxidation of erythrocyte membrane lipids. On the other hand, peroxidation of erythrocyte membrane lipids, induced by silicic acid and silicate dusts, is inhibited almost completely by adding superoxide dismutase and catalase to the incubation system, whilst the hemolysis of erythrocytes induced by silicic acid and silicate dusts is unaffected by these agents. Similarly the lysis of erythrocytes, induced by silicic acid and silicate dusts, proceeds at a much faster rate than silicic acid and silicate dust induced lipid peroxidation. These results indicate that silicic acid and silicate dust induced hemolysis and lipid peroxidation represent two independent processes.     

蟛蜞菊内酯对脂多糖诱导RAW264.7细胞环氧化酶2、NO及TNF-α的影响

目的:研究蟛蜞菊内酯对脂多糖(lipopo-lysaccharide,LPS)诱导RAW264.7巨噬细胞环氧化酶2(COX-2)、NO及TNF-α的作用。方法:ELISA方法检测0.2、2、20μmol/L不同浓度蟛蜞菊内酯对终浓度为10μg/mL LPS诱导RAW264.7细胞产生TNF-α、NO及前列腺素E2(PGE2)的影响,Western blot方法检测蟛蜞菊内酯对LPS诱导COX-2酶蛋白表达的影响。结果:LPS能够明显诱导小鼠RAW264.7细胞产生的COX-2酶蛋白,蟛蜞菊内酯低中高3个浓度均能抑制LPS诱导产生的COX-2酶蛋白表达。PGE2可以被LPS诱导增加,与空白组比有显著差异。蟛蜞菊内酯低中高3个浓度均能抑制LPS诱导产生的PGE2、NO和TNF-α,呈现剂量依赖性。结论:蟛蜞菊内酯抗炎的作用机制可能为抑制COX-2的蛋白表达,进而抑制PGE2的生成,也可能与抑制NO和TNF-α生成有关。     

Lipopolysaccharide neutralization by the antibacterial peptide CM4

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria. Binding of LPS to the CD14+ murine macrophage cell line RAW264.7 results in pro-inflammatory cytokine secretion. In extreme cases, it leads to septic shock in vivo. Therefore, the pursuit for molecules with antiendotoxin properties is urgent. In this study, we investigated the efficacy of antibacterial peptide CM4 in binding Escherichia coli LPS in vitro. CM4 avidly bound to E. coli LPS, as proven by the limulus amoebocyte lysate assay. Furthermore, the killing activity of CM4 against E. coli was progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptide's affinity for endotoxin. Flow cytometric analysis revealed that CM4 inhibited the binding of FITC-conjugated LPS to RAW264.7 cells. Likewise, the inhibition of peptide to LPS-dependent cytokine induction was analyzed. CM4 suppressed LPS-induced TNF-alpha and IL-6 mRNA expression and blocked release of TNF-alpha and NO following LPS challenge in RAW264.7 cells. Together these observations indicate that antibacterial peptide CM4 probably exerts protective actions against endotoxin shock by blocking the binding of LPS to CD14+ cells.     

Isotrifoliol inhibits pro-inflammatory mediators by suppression of TLR/NF-κB and TLR/MAPK signaling in LPS-induced RAW264.7 cells

Soybeans, produced by Glycine max (L.) Merr., contain high levels of isoflavones, such as genistein and daidzein. However, soy leaves contain more diverse and abundant flavonol glycosides and coumestans, as compared to the soybean. This study investigated the anti-inflammatory effects of the major coumestans present in soy leaf (coumestrol, isotrifoliol, and phaseol) in lipopolysaccharide (LPS)-induced RAW264.7 cells. Coumestans significantly reduced LPS-induced nitric oxide (NO), prostaglandin E2 (PGE₂), and reactive oxygen species (ROS) production; isotrifoliol had the most potent anti-inflammatory activity. Isotrifoliol reduced LPS-mediated induction of mRNA expression of inducible nitric-oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-1β, IL-6, tumor necrosis factor alpha (TNFα), and chemokines, such as chemokine (C-C motif) ligand (CCL) 2, CCL3, and CCL4. Isotrifoliol prevented NF-κB p65 subunit activation by reducing the phosphorylation and degradation of the inhibitor of NF-κB. And isotrifoliol significantly suppressed phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). Furthermore, isotrifoliol suppressed LPS-induced Toll-like Receptor (TLR) signaling pathway, including mRNA expression of TNF receptor associated factor 6, transforming growth factor beta-activated kinase 1 (TAK1), TAK1 binding protein 2 (TAB2), and TAB3. These results demonstrate that isotrifoliol exerts an anti-inflammatory effect by suppressing the expression of inflammatory mediators via inhibition of TLR/NF-κB and TLR/MAPK signaling in LPS-induced RAW264.7 macrophages. Therefore, isotrifoliol can be used as an anti-inflammatory agent, and coumestan-rich soy leaf extracts may provide a useful dietary supplement.     

Involvement of cytokines in the hepatic expression of metallothionein by ursolic acid

Ursolic acid (UA), a pentacyclic triterpene acid, is reported to have inducing activity of hepatic metallothionein (MT) which responsible for the detoxification of heavy metals; however, the mechanism underlying its effects is poorly understood. To further determine the underlying mechanism of UA, this study investigated the effects of UA on the induction of hepatic MT expression in an in vitro model, using murine hepatoma cell line Hepa-1c1c7 and murine macrophage cell line RAW 264.7 cell cultures. The UA added directly to Hepa-1c1c7 cells had no effect on MT induction. However, MT and its mRNA levels were markedly increased when Hepa-1c1c7 cells were cultured with UA-treated conditioned media from RAW 264.7. Concomitant treatment with UA and pentoxifylline, a TNF-alpha synthesis inhibitor, to RAW 264.7 cells decreased the effects of UA on the MT induction. In UA-exposed RAW 264.7 cell cultures, TNF-alpha and IL-6 production and TNF-alpha and IL-6 mRNA levels increased. When antibodies to TNF-alpha or/and IL-6 were added to UA-treated conditioned media from RAW 264.7, the MT induction activity was inhibited. These results demonstrate that UA induces hepatic MT expression through TNF-alpha and IL-6 released from UA-activated macrophages, which may be the mechanism, whereby UA elicits its biological effects.     

(1→3)-β-d-Glucan stimulates nitric oxide generation and cytokine mRNA expression in macrophages

Beta-glucans are known for their potent ability to induce nonspecific inflammatory reactions and are believed to play a role in bioaerosol-induced respiratory symptoms seen in both occupational and residential environments. Here, the ability of a (1→3)-β-d-glucan (Curdlan) to stimulate nitric oxide generation and cytokine mRNA expression in rat alveolar macrophages (AMs) and the murine monocyte/macrophage cell line, RAW 264.7 was investigated. Exposure to (1→3)-β-d-glucan (20, 100 and 500 μg/ml) induced a dose-dependent increase in the expression of inducible nitric oxide synthase mRNA and a release of nitric oxide into the culture medium in both rat AMs and RAW 264.7 cells. The mRNA expression of a number of other inflammatory mediators such as interleukin-1β, interleukin-6, tumor necrosis factor-α and cyclooxygenase-2 was also increased by the exposure to β-glucan. The capability of (1→3)-β-d-glucan (500 μg/ml) to induce mRNA synthesis of these various mediators were comparable to that of endotoxin (1 μg/ml). These results imply that (1→3)-β-d-glucan stimulates the generation of nitric oxide, cytokines and prostaglandins in macrophages and suggest the possibility that this may contribute to bioaerosol-induced respiratory symptoms seen in exposed individuals.     

Cytotoxicity of hydroxyapatite nanoparticles is shape and cell dependent

Nanosized hydroxyapatite (nHA) has been proposed as drug delivery vehicles because of its biocompatibility. While the possible risks of nHA inducing inflammation have been highlighted, the specific influence of varying nHA particle morphology is still unclear. In order to establish this understanding, nHA of four different shapes—needle (nHA-ND), plate (nHA-PL), sphere (nHA-SP) and rod (nHA-RD)—were synthesized. The particle effects with the concentration of 10–300 μg/mL on cytotoxicity, oxygen species generation, production of inflammatory cytokines (TNF-α and IL-6), particle–cell association and cellular uptake were evaluated on BEAS-2B and RAW264.7 cells. Results show that nHA-ND and nHA-PL induced the most significant cell death in BEAS-2B cultures compared to nHA-SP and nHA-RD. Necrosis–apoptosis assay by FITC Annexin V and propidium iodide (PI) staining revealed loss of the majority of BEAS-2B by necrosis. No significant cell death was recorded in RAW264.7 cultures exposed to any of the nHA groups. Correspondingly, no significant differences were observed in TNF-α level for RAW264.7 cells upon incubation with nHA of different shapes. In addition, nHA-RD exhibited a higher degree of particle–cell association and internalization in both BEAS-2B and RAW264.7 cells, compared to nHA-ND. The phenomena suggested that higher particle–cell association and increased cellular uptake of nHA need not result in increased cytotoxicity, indicating the importance of particle shape on cytotoxicity. Specifically, needle- and plate-shaped nHA induced the most significant cell-specific cytotoxicity and IL-6 expression but showed the least particle–cell association. Taken collectively, we demonstrated the shape-dependent effects of nHA on cytotoxicity, inflammatory cytokine expression and particle–cell association.     

Dermatophagoides farinae extract induces severe atopic dermatitis in NC/Nga mice,which is effectively suppressed by the administration of tacrolimus ointment

Atopic dermatitis (AD) is a chronic inflammatory skin disease, which is accompanied by marked increases in the levels of inflammatory cells, including mast cells and eosinophils as well as T cells and macrophages. To investigate the expression pattern of chemokines in AD, a house dust mite, Dermatophagoides farinae extracts (DfE)-induced NC/Nga AD model was developed in mice, and this model was used to determine the expression levels of chemokines in atopic lesions using DNA microarrays and RT-PCR. When NC/Nga mice were repeatedly treated with DfE for 4 to 7 weeks on the back skin, the mRNA expression levels of CCL20/LARC, CCL24/eotaxin-2, CCL17/TARC, and CCL11/eotaxin-1 were markedly induced and lesser of CCL2/MCP-1, within the inflammatory lesion of the back skin. Immunohistochemical staining revealed the expression of these chemokines in the epidermis and dermis of DfE-treated NC/Nga mice. Interestingly, repeated application of tacrolimus ointment potently inhibited DfE-induced atopic dermatitis in NC/Nga mice concomitant with the inhibition of these changes in chemokine gene and protein expression levels particularly of CCL20/LARC, CCL17/TARC, and CCL11/eotaxin-1. These data indicate that severe atopic dermatitis induced by DfE accompanies elevated chemokine levels, and it was proposed that tacrolimus ointment is beneficial for the treatment of severe AD.     

Ethyl pyruvate inhibits the acetylation and release of HMGB1 via effects on SIRT1/STAT signaling in LPS-activated RAW264.7 cells and peritoneal macrophages

High mobility group box 1 (HMGB1), a cytokine present in the late phase of sepsis, may be a potential target for the treatment of sepsis. For HMGB1 to be actively secreted from macrophages during infections, it must be post-translationally modified. Although ethyl pyruvate (EP), a simple aliphatic ester derived from pyruvic acid, has been shown to inhibit the release of HMGB1 in lipopolysaccharide (LPS)-treated RAW 264.7 cells, the underlying mechanism(s) are not yet clear. We investigated the hypothesis that the upregulation of SIRT1 by EP might promote the deacetylation of HMGB1, which reduces HMGB1 release in LPS-activated macrophages. Our results show that EP induced the expression of the SIRT1 protein in RAW264.7 cells and that it significantly inhibited the LPS-induced acetylation of HMGB1. Transfection with a SIRT1-overexpressing vector resulted in a significant decrease in the acetylation of HMGB1 in LPS-activated RAW264.7 cells relative to control cells. The genetic ablation or the pharmacological inhibition of SIRT1 by sirtinol increased LPS-induced HMGB1 acetylation. Moreover, EP inhibited the acetylation of HMGB1 in peritoneal macrophages treated with LPS. Interestingly, EP significantly reduced the LPS-induced phosphorylation of STAT1, which was significantly reversed by siSIRT1 transfection in RAW264.7 cells, indicating that SIRT1 negatively regulates the phosphorylation of STAT1. Overall, the results show that EP promotes the deacetylation of HMGB1 via the inhibition of STAT1 phosphorylation through the upregulation of SIRT1, which reduces HMGB1 release in LPS-activated RAW264.7 cells. In conclusion, EP might be useful in the treatment of diseases that target HMGB1, such as sepsis.     

非诺贝特对血管紧张素II诱导RAW264.7细胞Toll样受体4表达, 髓过氧化物酶活性及表达的抑制作用

探讨非诺贝特对血管紧张素Ⅱ (AngⅡ) 诱导的小鼠巨噬细胞株RAW264.7细胞Toll样受体4 (TLR4) mRNA、蛋白表达及髓过氧化物酶 (MPO) 活性、mRNA及蛋白表达的影响及其抗炎机制。采用RT-PCR检测TLR4和MPO mRNA水平, Western blotting检测TLR4和MPO的蛋白表达,比色法测定细胞培养上清液中的MPO活性。结果显示,非诺贝特浓度依赖性地减少AngⅡ诱导的RAW264.7细胞TLR4 mRNA及蛋白表达,抑制AngⅡ诱导的RAW264.7细胞MPO活性、mRNA及蛋白表达。此外,TLR4阻断剂对AngⅡ诱导的RAW264.7细胞MPO活性有部分的抑制作用,而非诺贝特可增强这一抑制效应。同时非诺贝特明显拮抗TLR4特异性配体脂多糖的促MPO分泌效应。以上结果表明,非诺贝特可下调AngⅡ诱导的RAW264. 7细胞TLR4表达,并通过干预TLR4,影响胞内信号转导途径,抑制MPO分泌,减轻炎症反应,这可能是其新的抗炎机制之一。


     

双环醇对脂多糖诱导巨噬细胞iNOS蛋白的表达和NF-κB活化的抑制作用

目的炎症是肝炎病毒或其它因素所致的肝损伤的一个共同特征,该文目的系研究抗肝炎新药双环醇对与炎症反应相关分子的调节作用。方法以无毒性浓度双环醇预先与巨噬细胞株RAW264.7和小鼠腹腔巨噬细胞温孵1h后,加入一定量脂多糖(LPS)共同培养适当时间以诱导上述细胞的活化,培养上清中NO2-的含量和TNF-α的水平分别用Griess试剂及L929细胞株生物法测定,用Westernblot方法测定iNOS蛋白的表达和NF-κB的活化。结果双环醇在0.1～0.5mmol.L-1剂量依赖性降低1mg.L-1LPS引起的RAW264.7和小鼠腹腔巨噬细胞NO的释放以及TNF-α的分泌,双环醇0.5mmol.L-1能够明显抑制1mg.L-1LPS引起的iNOS蛋白的表达和NF-κB的活化。结论双环醇对与炎症相关的调控因子iNOS蛋白的表达和NF-