Toll like receptor 5 ligand induces monocyte chemoattractant protein-1 in mouse osteoblastic cells

Flagellin, the ligand of Toll like receptor 5, is the major subunit of bacterial flagella. Flagellin stimulates various cells to release chemokines. Monocyte chemoattractant protein-1 (MCP-1) is a member of the CC chemokine family that is involved in monocyte infiltration in inflammatory diseases. It has been reported that serum MCP-1 levels increase proportionally with the severity of periodontal disease. Inflammatory mediators induce MCP-1 production in various cells, including osteoblasts. However, it remains unclear whether MCP-1 is released from osteoblasts in response to flagellin. In the present study, we investigated the effects of flagellin on the expression of MCP-1 in the mouse osteoblastic cell line, MC3T3-E1 (E1) cells. Flagellin markedly increased MCP-1 mRNA level in a dose-dependent manner. The effect of flagellin on MCP-1 mRNA expression in E1 cells was transient, with a peak at 1 h. Concomitant with MCP-1 mRNA expression, MCP-1 protein levels were clearly elevated at 3 h after flagellin exposure. In addition, we revealed that JNK and MEK-ERK1/2 are involved in flagellin-induced MCP-1 expression in E1 cells. These results indicated that bacterial flagellin may play an important role in the progression of periodontitis. Results of further studies will provide more clues to the prevention of periodontal diseases.     

Dramatic decrease of circulating levels of monocyte chemoattractant protein-1 in Kawasaki disease after gamma globulin treatment.

Kawasaki disease (KD) is a systemic vasculitis preferentially affecting coronary arteries. Extensive monocytes/macrophages infiltrate in the vascular lesions, implying the involvement of a chemotactic cytokine in their recruitment. We investigated the role of monocyte chemoattractant protein-1 (MCP-1, also termed monocyte chemotactic and activating factor) in KD. In the immunohistochemical studies using the cardiac tissues of patients with fatal KD, MCP-1 but not interleukin (IL) -8 or macrophage inflammatory protein-1alpha was localized at the extracellular matrix associated with mononuclear cellular infiltration. The sites of MCP-1 expression correlated with the distribution of the acute inflammation, including early coronary vasculitis. In prospectively studied patients with KD, circulating levels of MCP-1, IL-8, tumor necrosis factor alpha (TNF-alpha), and IL-1alpha were elevated in 73, 77, 57, and 0% of samples before gamma globulin (GG) treatment (400 mg/kg x 5 days = total 2 g/kg), respectively, compared with respective control values. GG treatment correlated with a rapid decrease in the circulating levels of MCP-1 (P = 0.001) but not IL-8 (P = 0.19) or TNF-alpha (P = 0.33). In the sensitive Western blotting, MCP-1 bound to GG. Furthermore, GG inhibited the MCP-1-induced Ca2+ influx in a human monocytic cell line in vitro. These findings suggest a role of MCP-1 in KD, and indicate that GG treatment may block MCP-1 activity, thus alleviating KD vasculitis.     

c-Jun氨基末端激酶-激活蛋白1信号通路调控血管紧张素Ⅱ诱导的单核细胞趋化蛋白1表达

目的探讨c-Jun氨基末端激酶-激活蛋白1信号通路在肾小球肾炎单核细胞趋化蛋白1表达中的作用.方法实验性肾炎应用兔抗鼠肾小球基底膜肾毒血清制备.应用凝胶电泳迁移率、超迁移实验和非放射性激酶活性检测法检测实验性肾炎肾组织和体外培养的肾小球系膜细胞内激活蛋白1活化及其组成以及c-Jun氨基末端激酶活性,应用核酸酶保护法检测体外培养的肾小球系膜细胞中单核细胞趋化蛋白1表达.结果实验性肾炎大鼠肾组织中c-Jun氨基末端激酶和激活蛋白1活性明显增强,分别是正常对照组的(3.82±0.58)倍和(5.36±0.61)倍,活化的激活蛋白1主要含有c-Jun和c-Fos亚基;c-Jun氨基末端激酶和激活蛋白1活化与单核细胞趋化蛋白1表达呈显著正相关.血管紧张素Ⅱ可诱导体外培养的肾小球系膜细胞单核细胞趋化蛋白1表达、c-Jun氨基末端激酶和激活蛋白1活化,其作用呈剂量依赖性增加,100 nmol/L血管紧张素Ⅱ诱导单核细胞趋化蛋白1表达、c-Jun氨基末端激酶和激活蛋白1活性增加分别是对照组的(20.99±4.71)倍、(6.91±1.65)倍和(7.82±1.32)倍;应用c-Jun氨基末端激酶特异性抑制剂SP600125显著抑制血管紧张素Ⅱ诱导激活蛋白1活化和单核细胞趋化蛋白1表达.结论血管紧张素Ⅱ可通过诱导c-Jun氨基末端激酶-激活蛋白1信号通路促进单核细胞趋化蛋白1表达,从而在肾小球肾炎中发挥一定的作用.     

Expression of monocyte chemoattractant protein-1 in Kawasaki disease: the anti-inflammatory effect of gamma globulin therapy

We investigated the expression of monocyte chemoattractant protein-1 (MCP-1) mRNA, protein, and its bioactivity in plasma, mononuclear cells (MNC) and polymorphonuclear leukocytes (PML) in patients with Kawasaki disease, including those who were treated with intravenous gamma globulin. MCP-1 mRNA expression was increased in the MNC and the plasma level of MCP-1 and monocyte chemotactic activity in plasma in the acute phase as compared with healthy control levels and decreased after the gamma globulin therapy. The infused gamma globulin contained MCP-1 protein with monocyte chemotactic activity and did not show a neutralising effect against MCP-1 protein in vitro . Our results suggest that the infusion of gamma globulin may reduce the production of MCP-1 in MNC in patients with Kawasaki disease, subsequently reducing its level in plasma. The changes in MCP-1 level after gamma globulin therapy may serve to alleviate the symptoms in the acute phase in patients with Kawasaki disease.     

Nitric oxide suppresses EPO-induced monocyte chemoattractant protein-1 in endothelial cells: implications for atherogenesis in chronic renal disease

Patients with advanced chronic renal disease (CRD) suffer from excessive morbidity and mortality due to complications of accelerated atherosclerosis. Approximately 90% of dialysis-dependent end stage renal disease patients suffer from anemia. Recombinant human erythropoietin (EPO) in combination with iron has become widely used to treat anemic CRD patients. While treatment with EPO results in improved quality of life it may also contribute to the development of atherosclerosis. Recent studies suggest that a reduction in nitric oxide (NO) availability may be linked to EPO-induced vascular dysfunction. Furthermore, CRD per se is thought to result in a state of NO deficiency. The present study suggests that EPO may exert proatherogenic activity by augmenting the cytokine-induced expression of monocyte-chemoattractant protein-1 (MCP-1) in human umbilical vein endothelial cells (HUVECs) and by stimulating the proliferation of HUVECs and human vascular smooth muscle cells (HVSMCs). Augmentation of MCP-1 expression appears to be linked to EPO-induced downregulation of endothelial NO synthase (ecNOS). NO released from a series of synthetic donor compounds suppressed the EPO-mediated augmentation of cytokine-induced MCP-1 expression. In vitro studies revealed that EPO reduces ecNOS expression at both the protein and mRNA levels and that EPO also mediates a reduction in ecNOS enzymatic activity. These observations suggest potential mechanisms through which EPO may contribute to the development of accelerated atherosclerosis, particularly in the setting of CRD where NO availability may already be compromised.     

Interleukin-15 strongly inhibits interleukin-8 and monocyte chemoattractant protein-1 production in human colonic epithelial cells

Interleukin-15 (IL-15) is a novel cytokine with actions similar to IL-2 because of common receptor components. Although IL-15 is expressed in colonic epithelial cells and may regulate epithelial cell function, its effects on these cells are not fully defined. We explored the regulatory effects of IL-15 on IL-8 and monocyte-chemoattractant protein-1 (MCP-1) production in the colonic epithelial cell line Caco-2 as well as in freshly isolated human colonic epithelial cells. IL-15 was added to intestinal epithelial cells under various culture conditions. Levels of chemokines were determined by enzyme-linked immunosorbent assay. To determine the elements of the IL-2/IL-15R complex involved we used neutralizing antibodies specific for individual receptor chains. IL-15 down-regulates IL-8 and MCP-1 production in Caco-2 cells as well as in freshly isolated human colonic epithelial cells in a dose-dependent manner. Intestinal epithelial cells became more responsive to IL-15-induced suppression when activated with greater IL-1 doses. Strong chemokine suppression was seen when IL-15 was given prior to, simultaneous with, or after stimulatory agent. Anti-IL-2Rgamma antibodies efficiently blocked (82% inhibition) the suppression induced by IL-15, while anti-IL-2Rbeta antibodies were less effective. The involvement of beta-chain was further suggested by the finding that a mixture of both monoclonal antibodies (mAb) at a suboptimal concentration (1 microgram/ml of each mAb) produced a synergistic inhibitory effect on down-regulation of epithelial chemokine production. These results show that IL-15 can suppress IL-8 and MCP-1 secretion by intestinal epithelial cells. A microenvironment containing high concentrations of IL-15 may alter the recruitment of neutrophils to enterocytes at least partly by inhibiting IL-8 and MCP-1 production.     

Mechanoregulation of monocyte chemoattractant protein-1 expression in rat vascular smooth muscle cells

The authors have previously shown that arterial wall strain mediates the development of vessel wall inflammation in experimental hypertension. The current studies explore the mechanoregulation of monocyte chemoattractant protein-1 (MCP-1), a potent pro-inflammatory chemokine, by mitogen-activated protein kinases (MAPK) and oxidative stress. Rat aortic smooth muscle (RASM) cells were subjected to cyclic strain on a uniform biaxial strain device. Strain rapidly activated both ERK1/2(MAPK) and p38(MAPK), with peak activation at 5 min. Strain induced a twofold increase in MCP-1 mRNA, which was attenuated by PD 98059, a specific ERK1/2(MAPK) inhibitor, and SB 203580, a specific p38(MAPK) inhibitor. Cyclic strain also increased production of superoxide anion via an NADPH oxidase-dependent mechanism. To assess the potential role of reactive oxygen species in MAPK activation, cells were stretched in the presence of N-acetylcysteine, which had no effect on p38(MAPK) activation, but significantly inhibited ERK1/2(MAPK) activation and MCP-1 expression. In conclusion, redox-sensitive activation of ERK1/2(MAPK) and redox-insensitive activation of p38(MAPK) regulate straininduced MCP-1 expression in RASM cells. These findings define a role for MAPK signal transduction in establishing a pro-inflammatory state in the arterial wall, and thus implicate a potential molecular link between arterial wall strain and atherosclerosis.     

Induction of monocyte chemoattractant protein-1 (MCP-1) production by Pseudomonas nitrite reductase in human pulmonary type II epithelial-like cells

Monocyte chemoattractant protein-1 (MCP-1), a chemoattractant for monocytes, is presumed to play a pivotal role in the recruitment and accumulation of monocytes in various diseases including pulmonary infections. We examined here whether or not Pseudomonas nitrite reductase (PNR), a recently identified IL-8 inducer in various respiratory cells, could stimulate human pulmonary type II epithelial-like cells (A549) to induce MCP-1 production. A time- and dose-dependent induction of MCP-1 protein synthesis associated with an increase of MCP-1 mRNA expression by A549 cells was observed in response to PNR. New protein translation was not required for PNR-mediated MCP-1 mRNA expression in the same cells. When anti-human MCP-1 monoclonal antibody was used for neutralizing of monocyte chemotactic factor (MCF) activities in the culture supernatants of these cells stimulated with PNR, significant reductions of MCF activities (the mean reduction rate; 49-59%, P<0. 05) were observed. These data suggest that PNR may contribute to monocyte migration, through inducing pulmonary epithelial cell-derived MCP-1 production in the airway of patients with pneumonia due to P. aeruginosa.     

IL-17对心肌细胞单核细胞趋化蛋白-1表达的影响

目的:研究IL-17对原代培养心肌细胞中单核细胞趋化蛋白-1(MCP-1)表达的影响。方法:差速贴壁法分离乳鼠心肌细胞进行原代培养,采用逆转录聚合酶链反应(RT-PCR)检测心肌细胞IL-17R和MCP-1基因表达情况,并采用酶联免疫吸附法(ELISA)检测心肌细胞培养上清中MCP-1蛋白的表达。结果:心肌细胞存在IL-17R的基因表达。IL-17呈浓度依赖式上调心肌细胞MCP-1基因和蛋白的表达水平,与不加刺激因子的对照组相比差异有统计学意义(P<0.05)。心肌细胞MCP-1 mRNA的表达量在IL-17作用4 h时达到高峰,随后开始下降,而IL-17作用下心肌细胞MCP-1蛋白的表达呈时间依赖式升高,与对照组相比有显著性差异(P<0.05)。结论:心肌细胞表达IL-17R。IL-17可上调心肌细胞MCP-1的表达,其作用与IL-17的浓度和作用时间有关。     

Production of monocyte chemoattractant protein-1 by malignant fibrous histiocytoma: relation to the origin of histiocyte-like cells.

Human malignant fibrous histiocytoma (MFH) comprise both fibroblast-like cells and histiocyte-like cells. We previously showed that the latter are not neoplastic cells, but are infiltrating macrophages. Since migration of blood monocytes into the tumor might be a response to a locally elaborated monocyte chemoattractant, we designed experiments to determine if the fibroblast-like tumor cells produced a chemoattractant for human monocytes. Malignant fibrous histiocytoma from three patients was put into culture. Cells of all three lines had a spindle shape, and showed no reactivity with antibodies against macrophages (MAC387), HLA-DR (LN3), or leukocyte common antigen. Immunohistochemically, they stained with antibody against human monocyte chemoattractant protein-1 (MCP-1). Culture supernatants of the three cell lines had chemotactic activity for monocytes. This activity was due to MCP-1, since it was absorbed by an anti-MCP-1 column. The production of MCP-1 by MFH tumor lines was confirmed by immunoprecipitation of metabolically labeled MCP-1. These results suggest that the histiocyte-like cells are the infiltrated macrophages that originate from blood monocytes attracted by tumor-derived MCP-1.     

Thromboxane A2 (TXA2) receptor blockade suppresses monocyte chemoattractant protein-1 (MCP-1) expression by stimulated vascular endothelial cells

In a previous study, it was reported that stimulation with a TXA2 receptor agonist, U46619, augments the expression of adhesion molecules by human umbilical vein endothelial cells (HUVEC). In the present study we showed that U46619 augments the expression of MCP-1 in HUVEC, both at the protein and mRNA levels. Pretreatment with TXA2 receptor antagonists greatly diminishes the extent of tumour necrosis factor-alpha (TNF-alpha)-, platelet-activating factor (PAF)-, or U46619-induced mRNA accumulation and production of MCP-1. Protein kinase C (PKC) inhibitors diminish U46619-induced mRNA accumulation and production of MCP-1. NAC, which inhibits nuclear factor kappaB (NF-kappaB) activation and activating protein 1 (AP-1) binding activity, inhibits the expression of MCP-1 at the protein and mRNA levels. These results indicate that in HUVEC stimulation via the TXA2 receptors augments MCP-1 production by induction of the NF-kappaB and AP-1 binding activity through the PKC system.     

Transforming growth factor-β stimulates IL-1β-induced monocyte chemoattractant protein-1 expression in human synovial cells via the ERK/AP-1 pathway

Objective and Design: Transforming growth factor- β (TGF-β) has not only a fibrogenic role, but also monocyte/ macrophage chemotactic properties in a synovial joint. However, little is known about the effects of TGF-β on monocyte chemoattractant protein-1 (MCP-1) expression in human synovial cells under inflammatory status. The aim of this study was to determine whether TGF-modulates MCP-1 production under the chronic inflammation, and to elucidate the cell signaling mechanism involved. Materials and methods: Human synovial cells were exposed to IL-1β, which mimics the environment of chronic inflammation. Production of MCP-1 protein and expression of MCP-1 mRNA were determined by ELISA and real-time PCR. Results: TGF-β upregulated the expression of MCP-1 mRNA and protein with or without IL-1β. TGF-β and IL-1β synergistically enhanced MCP-1 gene expression, and an AP-1 binding site was involved in the signal transduction. In addition, MEK inhibitor completely suppressed TGF-β-induced MCP-1 production. Conclusions: TGF-β and IL-1β synergistically enhance MCP-1 gene expression through the activation of the MEK/ERK1/2 pathways, which leads to AP-1 activation. The impairment of MCP-1 regulation by TGF-β in resident synovial cells might represent an important mechanism of chronic inflammation and tissue fibrosis in a synovial joint. MCP-1 should be considered a valid target for therapeutic intervention. Received 22 September 2005; returned for revision 3 February 2006; accepted by J. Skotnicki 29 May 2006     

Monocyte chemoattractant protein-1 production by intestinal epithelial cells in vitro: a role for p38 in epithelial chemokine expression.

The intestinal epithelial cell (IEC) represents the first cellular barrier to infection. Consistent with this sentinel role, IEC are known to produce a variety of chemokines in response to bacterial infection or proinflammatory cytokines. These chemokines act as potent leukocyte activators and chemoattractants in vivo. In this report, we begin to characterize the regulation of expression of the chemokine monocyte chemoattractant protein-1 (MCP-1) in the rat small intestinal IEC-18 line. Following stimulation with either interleukin-1beta (IL-1beta) or lipopolysaccharide (LPS), IEC-18 cells produced MCP-1, with IL-1 proving a more effective stimulus than LPS at both the mRNA and protein levels. Expression of MCP-1 due to either stimulus was inhibited by tyrosine kinase inhibitors, prompting us to investigate potential phosphotyrosine-dependent targets responsible for MCP-1 expression. We detected activation of p38, a member of the mitogen-activated protein kinase family, following either IL-1 or LPS treatment. Specific inhibition of this kinase using the compound SB203580 caused a destabilization of MCP-1 mRNA. These data point to a role for p38 in the regulation of MCP-1 mRNA expression by the IEC.     

两步法细菌内同源重组制备重组单核细胞趋化因子-1腺病毒

目的两步法提高细菌内同源重组效率制备重组人单核细胞趋化蛋白1（MCP-1）复制缺陷型腺病毒。方法制备含有腺病毒骨架质粒pAdEasy—1的大肠杆菌BJ5183，HindⅢ酶切筛选未发生细菌内重组的菌落，并将其制备成感受态（BJ5183pAdEasy—1）RT—PCR法克隆得纠MCP—1 cDNA片段，连接到pMD18-T载体后亚克隆至腺病毒穿梭载体pAdTrack上陶建重组穿俊载体pAdTrackMCP—1.PmeⅠ酶切线性化并碱性磷酸酶处理pAdTrack—MCP—1，然后电穿孔转化感受态BJ5183pAdEasy-1。PacⅠ酶切筛选得到正确重组的腺病毒载体pad—MCP—1，将pAd—MCP—1转染入HEK293细胞中包装病毒颗粒，以PCR法鉴定？结果成功构建pad—MCP-1，效率町达50％以上，pad—MCP—1能有效转染HEK293细胞并在细胞内包装PCR鉴定病毒携带有MCP—1基因片段、结论两步法细菌内同源重组构建再组腺病毒载体较常规电穿孔共转化效率高．成功制备重组腺病毒颗粒为进一步研究MCP-1的作用提供了重要的方法？     

Hypoxia induces expression of the chemokines monocyte chemoattractant protein-1 (MCP-1) and IL-8 in human dermal fibroblasts

Hypoxia is an important factor in the pathophysiology of vascular and inflammatory diseases. Leucocyte infiltration, as a consequence of adhesion molecule up-regulation and chemokine release, is a prominent feature of these diseases. The objective of our study was to investigate the potential role of resident fibroblasts in hypoxia-induced chemotactic responses. We show that MCP-1 and IL-8 mRNA are specifically induced by hypoxia in dermal fibroblasts. This response is paralleled by increased NF-kappaB p65/p50 binding activity, and it is inhibited by pretreatment with N-acetyl-L-cysteine. MCP-1 secreted by fibroblasts is chemotactic for monocytic cells and this activity is significantly increased by hypoxia. Chemotactic index correlates with MCP-1 protein levels and is significantly decreased by neutralizing anti-MCP-1 MoAb. These findings demonstrate the ability of resident fibroblasts to mediate chemotaxis of leucocytes through the release of chemokines in response to hypoxia. Our data point to MCP-1 as an important component in this response, and therefore it may be a potential target in inflammatory responses associated with hypoxia.     

睾酮双向影响脐静脉内皮细胞分泌单核细胞趋化蛋白1

雄激素与动脉硬化(arteriosc lerosis,AS)的关系日益受到关注,单核细胞趋化蛋白1(monocyte chemoattractant prote in-1,MCP-1)是促进AS的重要炎症介质。本研究以人脐静脉内皮细胞(hum an umb ilical ve in endothelial cell,HUVEC)为对象,观察不同浓度睾酮对MCP-1蛋白表达的影     

Virulence of Mycobacterium tuberculosis affects interleukin-8, monocyte chemoattractant protein-1 and interleukin-10 production by human mononuclear phagocytes.

Microbial virulence and cytokine-mediated immune responses to Mycobacterium tuberculosis infection are important determinants of the pathogenesis of human tuberculosis. To determine the interrelationship between mycobacterial virulence and cytokine induction, human monocytes and monocyte-derived macrophages were infected with attenuated (H37Ra) and virulent (H37Rv and CH306) strains of M. tuberculosis and the amount of proinflammatory [interleukin (IL)-8 and monocyte chemoattractant protein (MCP)- 1] and inhibitory (IL- 10) cytokines was measured in the culture supernatants by enzyme-linked immunosorbent assay (ELISA). Infection with live bacilli induced de novo synthesis of IL-8, MCP-1 and IL-10, since cytokine release was abolished when cells were preincubated with the protein synthesis inhibitor cycloheximide. A differential production of antiinflammatory and inhibitory cytokines was observed. The amount of IL-8 and MCP-1 release was inversely related to strain virulence, the attenuated H37Ra strain being more prone than virulent strains to induce secretion of chemokines. In contrast, virulent strains induced greater amounts of the inhibitory cytokine IL-10. Efficient upregulation of IL-10 synthesis, but not of chemokines, required infection of cells with live bacilli, since heat killing of organisms or challenge with soluble mycobacterial products completely abrogated the effect. Moreover, cells infected with virulent strains produced IL-10 even at a very low bacillus-to-cell ratio and secreted IL-10 continuously during the 96 h that followed infection. The results suggest that the degree of virulence affects host cell responses to M. tuberculosis infection. Continued production of IL-10 may be one of the means by which M. tuberculosis downregulates acute local inflammatory reactions, favoring the development of tuberculosis.