Role of p38 mitogen-activated protein kinase (MAPK) for vacuole formation in lipopolysaccharide (LPS)-stimulated macrophages

The role of p38 mitogen-activated protein kinase (MAPK) on vacuole formation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was examined. LPS definitely induced the formation of vacuoles in RAW 264.7 cells and SB202190 as a p38 specific inhibitor also induced slight vacuole formation. The simultaneous treatment with LPS and SB202190 induced many more vacuoles in RAW 264.7 cells than the treatment with LPS or SB202190 alone, and the vacuoles were extraordinarily large in size. On the other hand, an inactive inhibitor of p38 MAPK did not augment LPS-induced vacuole formation. Further, the inhibitors of other MAPKs and nuclear factor (NF)-kappaB pathways did not affect it. The extraordinarily large vacuoles in RAW 264.7 cells treated with LPS and SB202190 were possibly formed via fusion of small vacuoles. However, SB202190 did not augment vacuole formation in CpG DNA or interferon (IFN)-gamma-stimulated RAW 264.7 cells. The role of p38 MAPK in the vacuole formation in LPS-stimulated macrophages is discussed.     

Enhanced immune response by vacuoles isolated from Saccharomyces cerevisiae in RAW 264.7 macrophages

Vacuoles are membrane vesicles in eukaryotic cells, the digestive system of cells that break down substances absorbed outside the cell and digest the useless components of the cell itself. Researches on anticancer and intractable diseases using vacuoles are being actively conducted. The practical application of the present study to animals requires the determination of the biocompatibility of vacuole. In the present study, we evaluated the effects of vacuoles isolated from Saccharomyces cerevisiae in RAW 264.7 cells. This showed a significant increase in the production of nitric oxide (NO) produced by macrophage activity. Using Reactive Oxygen Species (ROS) assay, we identified that ROS is increased in a manner dependent on vacuole concentration. Western blot analysis showed that vacuole concentration-dependently increased protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2). Therefore, iNOS expression was stimulated to induce NO production. In addition, pro-inflammatory cytokines levels promoted, such as interleukin (IL) 6 (IL-6) and tumor necrosis factor (TNF) α (TNF-α). In summary, vacuoles activate the immune response of macrophages by promoting the production of immune-mediated transporters NO, ROS, and pro-inflammatory cytokines.     

稳定表达鼠源PLEKHA1细胞系的建立及其对细胞迁移的影响

目的：构建稳定表达鼠源PLEKHA1的RAW264.7小鼠巨噬细胞系,探讨PLEKHA1过表达对巨噬细胞迁移的影响。方法：根据小鼠PLEKHA1基因序列设计引物,克隆其编码区序列,酶切后插入pCDH载体,在293FT细胞中进行病毒的包装,用获得的高滴度慢病毒感染RAW264.7细胞,建立能稳定高效表达PLEKHA1的RAW264.7细胞系;在此基础上,观察PLEKHA1对巨噬细胞迁移的影响。结果：经基因克隆、酶切、连接后,构建了鼠源PLEKHA1重组慢病毒表达载体,包装病毒感染RAW264.7细胞,经嘌呤霉素筛选后免疫印迹检测,RAW264.7细胞中PLEKHA1的蛋白表达提高近30倍;同时,发现PLEKHA1的过表达影响RAW264.7细胞的迁移。结论：构建了稳定表达小鼠PLEKHA1的RAW264.7细胞系,PLEKHA1过表达降低RAW264.7细胞的迁移能力。     

蜂蛹多肽对巨噬细胞RAW264.7免疫活性的影响

蜂蛹多肽因具有丰富的营养价值,以及增强免疫、抗肿瘤及抗氧化等生物学活性,而受到了广泛关注,但目前关于蜂蛹多肽纯化组分的体外免疫调节活性的研究尚未见报道。为了探究蜂蛹多肽对巨噬细胞RAW264.7免疫活性的影响,以蜂蛹多肽纯化组分BPP-21为研究对象,研究其在不同浓度（12.5、25、50、100和200 μg·mL-1）下对RAW264.7巨噬细胞的细胞活力、吞噬能力、细胞因子分泌能力、NO分泌能力和氧化应激指标的影响。结果显示,在浓度12.5~200 μg·mL-1范围内,BPP-21对RAW264.7巨噬细胞无明显的细胞毒性作用,可显著提高干扰素-γ（interferon-gamma,IFN-γ）与NO的分泌水平（P<0.05）;在浓度25~200 μg·mL-1范围内,显著增加细胞吞噬能力以及白细胞介素-2（interleukin-2,IL-2）、肿瘤坏死因子α（tumor necrosis factor-alpha,TNF-α）的分泌量（P<0.05）;在浓度50~200 μg·mL-1范围内,显著提高细胞内超氧化物歧化酶（superoxide dismutase,SOD）活力（P<0.05）。研究表明,蜂蛹多肽纯化组分BPP-21可增强RAW264.7巨噬细胞的免疫活性,为蜂蛹多肽免疫调节剂的研究与开发提供了理论依据。     

LOX-1特异性小发夹RNA 表达载体的构建及其对巨噬细胞源性泡沫细胞形成的影响

[摘　要]　目的：靶向血凝素样氧化型低密度脂蛋白受体-1基因的发卡样siRNA(shRNA)表达载体及其对巨噬细胞源性泡沫细胞形成的影响。方法：(1)采用DNA重组技术,将LOX-1 shRNA双链与线性化pGenesil-1质粒表达载体连接,脂质体法转染小鼠单核巨噬细胞（RAW264.7）,半定量逆转录聚合酶链反应法检测LOX-1 mRNA的表达,Western blot法检测LOX-1蛋白的表达。(2) Ox-LDL诱导巨噬细胞建立泡沫细胞模型, LOX-1-shRNA进行干预,干预组使用脂质体法进行细胞转染,转染24小时后,再加入Ox-LDL作用24小时,用油红O染色法及细胞内游离胆固醇及总胆固醇测定法观察对泡沫细胞形成的影响,倒置荧光显微镜观察转染LOX-1 shRNA后RAW264.7细胞对Dil-ox-LDL的摄取率。结果：测序鉴定发现插入的发卡样序列正确,成功合成了发卡样LOX-1基因RNA干扰表达载体;靶向LOX-1基因的发卡样shRNA表达载体转染RAW264.7细胞后,其LOX-1基因和蛋白表达显著下调, 同时可抑制巨噬细胞源性泡沫细胞形成及对Dil-ox-LDL的摄取。结论：成功构建了能有效抑制LOX-1 mRNA表达的发卡样LOX-1基因RNA干扰表达载体,并在一定程度上能抑制巨噬细胞源性泡沫细胞的形成,为进一步利用RNA干扰技术防治动脉粥样硬化提供理论基础。     

人参根提取物增强巨噬细胞RAW264.7的自噬水平并提高其增殖和吞噬能力

研究人参根提取物对巨噬细胞RAW264.7的增殖能力、吞噬能力和自噬水平的影响以及其相关性.用细胞计数试剂(CCK-8)检测不同浓度的人参根以及加入对巨噬细胞RAW264.7增殖的影响;采用中性红吞噬实验检测人参根提取物对巨噬细胞吞噬活性的影响;采用吖啶橙染色法(AO染色法)检测自噬体的形成;采用免疫印迹法(Weste...     

Uptake of histamine by mouse peritoneal macrophages and a macrophage cell line,RAW264.7

We have previously demonstrated that dietary histamine is accumulated in the spleens of L-histidine decarboxylase (HDC)-deficient mice, which lack endogenous histamine synthesis. To characterize the clearance system for dietary histamine in mice, we investigated the cell type and mechanism responsible for histamine uptake in the spleens of HDC-deficient mice. Immunohistochemical analyses using an antihistamine antibody indicated that a portion of the CD14⁺ cells in the spleen is involved in histamine storage. Peritoneal macrophages obtained from Balb/c mice and a mouse macrophage cell line, RAW264.7, had potential for histamine uptake, which was characterized by a low affinity and high capacity for histamine. The histamine uptake by RAW264.7 cells was observed at physiological temperature and was potently inhibited by pyrilamine, chlorpromazine, quinidine, and chloroquine, moderately inhibited by N-methylhistamine, dopamine, and serotonin, and not affected by tetraethylammonium and 1-methyl-4-phenylpyridinium. Intracellular histamine was not metabolized in RAW264.7 cells and was released at physiological temperature in the absence of extracellular histamine. These results suggest that histamine uptake by macrophages may be involved in the clearance of histamine in the local histamine-enriched environment. cation transporter; chlorpromazine; pyrilamine; quinidine     

利用慢病毒载体构建稳定过表达人VEGF_(165)的小鼠巨噬细胞系

目的:通过构建人血管内皮生长因子165(humanVEGF_(165),hVEGF_(165)的慢病毒载体,感染小鼠单核巨噬细胞RAW264.7,建立稳定高表达人VEGF165的小鼠巨噬细胞系。方法:将聚合酶链反应(PCR)扩增得到的hVEGF_(165)和慢病毒载体pLVX-IRES-ZsGreen1双酶切后连接,构建慢病毒表达载体pLVX-hVEGF_(165)-IRES-ZsGreen1。再经双酶切和测序鉴定后,进行病毒包装及浓缩。将该慢病毒载体感染RAW264.7细胞,利用绿色荧光蛋白ZsGreen1进行2次流式分选。用Realtime-PCR、WesternBlot分别检测各组细胞中hVEGF165的mRNA和蛋白表达;ELISA分别检测细胞上清中人VEGF和小鼠VEGF的含量。结果:酶切及测序结果示慢病毒表达载体pLVX-hVEGF165-IRES-ZsGreen1构建正确;流式分选后得到高纯度的ZsGreen1-hVEGF_(165)-RAW264.7细胞。Realtime-PCR、WesternBlot显示该细胞特异高表达hVEGF_(165)基因和蛋白(P均0.01)。ELISA显示该细胞分泌人和小鼠VEGF均显著增加(P均0.01)。结论:成功构建hVEGF_(165)慢病毒表达载体,并建立稳定高表达hVEGF_(165)的小鼠巨噬细胞系。为深入研究该细胞的功能、机制及应用提供充足稳定的细胞来源。     

Identification of TLR2/4-mediated phagocytosis and immune response activation pathways by vacuoles isolated from Saccharomyces cerevisiae

The vacuoles of the yeast Saccharomyces cerevisiae are closely related to mammalian lysosomes and play a role in macromolecular degradation due to the hydrolytic enzymes present inside. The vacuoles also regulate osmotic pressure and control cellular homeostasis. In previous results, vacuoles were shown to activate the immune response of macrophages by promoting the production of immune-mediated transporters nitric oxide (NO), reactive oxygen species (ROS), and pro-inflammatory cytokines. In this study, the effects of vacuoles on the phagocytosis activity of RAW264.7 cells and their potential as immune enhancers were evaluated, and receptors capable of recognizing vacuoles were examined. An investigation using the phagocytes assay showed that phagocytosis activity increased by the vacuole. Besides, after treatment with TLR2/4 inhibitor, the expression of pro-inflammatory cytokines by vacuoles was significantly reduced and the inducible nitric oxide synthase (iNOS) protein was also significantly reduced. However, treatment with a TLR2 inhibitor did not reduce the production of interleukin-6 (IL)-6, a pro-inflammatory cytokine. As a result of confirming the activation of TLR2/4 using Western blot and immunofluorescence (IF), the TLR2/4 protein expression and fluorescence intensity increased depending on the concentration of vacuoles. Yeast vacuoles significantly upregulate protein expression of p-p65/p-p38 MAPKs. In summary, the vacuoles isolated from S. cerevisiae in macrophages have increased phagocytic ability at a concentration of 20 (µg/ml) and can function as immune-enhancing agent suggesting that TLR2/4 mediated the p38 MAPK/nuclear factor kappa B signaling pathway.     

Reactive oxygen species are the major antibacterials against Salmonella Typhimurium purine auxotrophs in the phagosome of RAW 264.7 cells

Intramacrophage survival appears to be a pathogenic trait common to Salmonellae and definition of the metabolic requirements of Salmonella within macrophages might provide opportunities for novel therapeutic interventions. We show that loss of PurG function in Salmonella enterica serovar Typhimurium SL1344 leads to death of the bacterium in RAW264.7 cells, which was due to unavailability of purine nucleotides but not thiamine in the phagosome of RAW264.7 cells. Phagosomal escape of purG mutant restored growth, suggesting that the phagosomal environment, but not the cytosol, is toxic to Salmonella purine auxotrophs. NADPH oxidase inhibition restored the growth of purG mutant in RAW264.7 cells, implying that the Salmonella -containing vacuole acquires reactive oxygen species (ROS) that are lethal to purine auxotrophs. Under purine limiting conditions, purG mutant was unable to repair the damage caused by hydrogen peroxide or UV irradiation, suggesting that ROS-mediated DNA damage may have been responsible for the attenuated phenotype of purG mutant in RAW264.7 cells and in mice. These studies highlight the possibility of utilizing the Salmonella purine nucleotide biosynthetic pathway as a prospective therapeutic target and also underline the importance of metabolic pathways in assembling a comprehensive understanding of the host–pathogen interactions inside phagocytic cells.     

热休克因子1对内毒素所致G-CSF基因表达的影响

为探讨热休克因子1(heatshockfactor 1,HSF1)活化和过表达对内毒素(endotoxin ,ET)所致粒细胞集落刺激因子(granulocyte colonystimulatingfactor,G CSF)基因表达的影响,采用大肠杆菌内毒素即脂多糖(lipopolysaccharide ,LPS)处理RAW2 6 4 7巨噬细胞,并通过热休克预处理诱导HSF1活化,采用Western印迹检测HSP70的表达观察HSF1的活化情况,RT PCR检测热休克反应(heatshockresponse ,HSR)对G CSFmRNA表达的影响;构建HSF1的pcDNA3 1真核表达质粒,采用脂质体转染法建立HSF1过表达RAW 2 6 4 7巨噬细胞株,用免疫细胞化学和Western印迹观察HSF1的表达,RT- PCR及Northern印迹进一步研究HSF1对G CSF基因表达的可能影响.发现LPS诱导巨噬细胞中G- CSFmRNA表达增多,并随时间的延长,表达量逐渐增加;与单纯内毒素处理组相比,热休克预处理后,LPS诱导的巨噬细胞G- CSFmRNA的表达明显被抑制;建立的稳定表达HSF1的RAW 2 6 4 7细胞株中有HSF1蛋白的核移位;HSF1过表达可明显抑制LPS诱导的RAW2 6 4 7巨噬细胞G -CSFmRNA的表达.上述结果表明热休克预处理能抑制LPS诱导的巨噬细胞G- CSFmRNA的表达;HSF1过表达可抑制内毒素诱导的巨噬细胞G CSFmRNA的表达.     

Dental pulp–derived stem cells inhibit osteoclast differentiation by secreting osteoprotegerin and deactivating AKT signalling in myeloid cells

Osteoclasts (OCs) differentiate from the monocyte/macrophage lineage, critically regulate bone resorption and remodelling in both homeostasis and pathology. Various immune and non-immune cells help initiating activation of myeloid cells for differentiation, whereas hyper-activation leads to pathogenesis, and mechanisms are yet to be completely understood. Herein, we show the efficacy of dental pulp–derived stem cells (DPSCs) in limiting RAW 264.7 cell differentiation and underlying molecular mechanism, which has the potential for future therapeutic application in bone-related disorders. We found that DPSCs inhibit induced OC differentiation of RAW 264.7 cells when co-cultured in a contact-free system. DPSCs reduced expression of key OC markers, such as NFATc1, cathepsin K, TRAP, RANK and MMP-9 assessed by quantitative RT-PCR, Western blotting and immunofluorescence detection methods. Furthermore, quantitative RT-PCR analysis revealed that DPSCs mediated M2 polarization of RAW 264.7 cells. To define molecular mechanisms, we found that osteoprotegerin (OPG), an OC inhibitory factor, was up-regulated in RAW 264.7 cells in the presence of DPSCs. Moreover, DPSCs also constitutively secrete OPG that contributed in limiting OC differentiation. Finally, the addition of recombinant OPG inhibited OC differentiation in a dose-dependent manner by reducing the expression of OC differentiation markers, NFATc1, cathepsin K, TRAP, RANK and MMP9 in RAW 264.7 cells. RNAKL and M-CSF phosphorylate AKT and activate PI3K-AKT signalling pathway during osteoclast differentiation. We further confirmed that OPG-mediated inhibition of the downstream activation of PI3K-AKT signalling pathway was similar to the DPSC co-culture–mediated inhibition of OC differentiation. This study provides novel evidence of DPSC-mediated inhibition of osteoclastogenesis mechanisms.     

广叶绣球菌β-D-葡聚糖对巨噬细胞RAW264.7免疫调节作用受体TLR4和TLR2的影响

确定广叶绣球菌β-D-葡聚糖对巨噬细胞RAW264.7的免疫调节作用受体,探索广叶绣球菌β-D-葡聚糖的免疫调节机制。采用MTT法测定不同浓度广叶绣球菌β-D-葡聚糖对巨噬细胞RAW264.7增殖活力的影响,筛选出促进巨噬细胞增殖能力最强的浓度。用筛选出的β-D-葡聚糖浓度作用巨噬细胞RAW264.7;TLR4抗体和TLR2抗体分别作用巨噬细胞RAW264.7 1h,再用含有β-D-葡聚糖的细胞培养液培养。收集细胞培养上清和细胞,检测细胞培养上清中NO、IL-6、TNF-α、IFN-β的生成量;提取细胞内总RNA,采用RT-PCR测定巨噬细胞TLR4 mRNA表达量;提取巨噬细胞总蛋白,采用蛋白免疫印迹western blot测定TLR4的蛋白表达。广叶绣球菌β-D-葡聚糖能够促进巨噬细胞RAW264.7增殖,增加NO、IL-6、TNF-α、IFN-β的生成量,提高TLR4 mRNA表达和蛋白表达,差异极显著（P<0.01）。TLR4抗体作用细胞后,NO、IL-6、TNF-α、IFN-β的生成量明显下降,差异极显著（P<0.01）。TLR2抗体作用细胞后,NO、IL-6、TNF-α、IFN-β的生成量下降,但差异不显著。广叶绣球菌β-D-葡聚糖可以通过细胞表面受体TLR4激活信号转导通路,增强下游细胞因子的释放,从而调节巨噬细胞RAW264.7的免疫功能。TLR2可能不是广叶绣球菌β-D-葡聚糖的免疫受体。     

GM1 Induced the inflammatory response related to the Raf-1/MEK1/2/ERK1/2 pathway in co-culture of pig mesenchymal stem cells with RAW264.7

Pig-human xenotransplantation can trigger cell-mediated immune responses. We explored the role of gangliosides in inflammation related to immune rejection in xenotransplantation. Co-culture of xenogeneic cells (pig-MSCs and RAW264.7) was used to emulate xenotransplantation conditions. MTT assay results indicated that cell viability was significantly decreased in pADMSCs co-cultured with RAW264.7 cells. GM1 and GM3 were highly expressed in pADMSCs co-cultured with RAW264.7 cells. pADMSCs co-cultured with RAW264.7 cells strongly expressed pro-inflammatory proteins such as COX-2, iNOS, p50, p65, pIκBα, and TNF-α. GM1-knockdown pADMSCs co-cultured with RAW 264.7 cells did not show significantly altered cell viability, but pro-inflammatory proteins were markedly inhibited. Co-culture of pADMSCs with RAW264.7 cells induced significant phosphorylation (p) of JNK1/2 and pERK1/2. However, pERK1/2 and pJNK1/2 were decreased and MEK1/2 and Raf1 were suppressed in GM1-knockdown pADMSCs co-cultured with RAW264.7 cells. Thus, the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways were significantly upregulated in response to increases of GM1 in co-cultured xenogeneic cells. However, the inflammatory response was suppressed in co-culture of GM1-knockdown pADMSCs with RAW264.7 cells via down-regulation of the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways. Therefore, the ganglioside GM1 appears to play a major role in the inflammatory response in xenotransplantation via the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways.     

Curcumin enhances non-inflammatory phagocytic activity of RAW264.7 cells

Present study was performed to assess the effect of curcumin treatment on macrophage functions using RAW264.7 cells, a murine macrophage cell line. Phagocytic activity of RAW264.7 cells was enhanced by the treatment with curcumin for 48 h while the nitric oxide synthesis from RAW264.7 cells following lipopolysaccharide exposure was blocked. The incubation of RAW264.7 cells with curcumin dose-dependently inhibited the stimulatory responses of macrophage triggered by lipopolysaccharide; the enhanced secretion of inflammatory cytokines such as TNF-α and IL-1β and the up-regulated expression of surface antigens like CD14 and CD40. Curcumin alone, however, was able to increase the basal level of TNF-α secretion and elevated markedly the expression of CD14 and slightly CD40. The marked enhancement of both phagocytic activity and CD14 was detectable as early as 75 min after curcumin treatment which is the minimum time period required for the phagocytosis and CD14 measurement, suggesting a signaling pathway distinct from that triggered by apoptotic cells. In conclusion, this study elucidates that curcumin treatment enhances the phagocytic activity with blocking nitric oxide synthesis, a scavenger function of macrophages in non-inflammatory condition. In addition, this enhancement of phagocytic activity is triggered directly by the signals from curcumin itself not by apoptotic cells.     

Bromelain activates murine macrophages and natural killer cells in vitro

The innate immune response is critical for effective immunity against most pathogens. In this study, we show that bromelain, a mixture of cysteine proteases, can enhance IFN-gamma-mediated nitric oxide and TNFalpha production by macrophages. Bromelain's effect was independent of endotoxin receptor activation and was not caused by direct modulation of IFN-gamma receptors. Instead, bromelain either enhanced or acted synergistically with IFN-gamma receptor-mediated signals. These effects were seen in both RAW 264.7, a macrophage cell line, and primary macrophage populations. Bromelain also increased IL-2- and IL-12-mediated IFN-gamma production by NK cells. These results indicate a potential role for bromelain in the activation of inflammatory responses in situations where they may be deficient, such as may occur in immunocompromised individuals.     

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.