Cytokine-activated human endothelial cells synthesize and secrete a monocyte chemoattractant, MCP-1/JE.

We have demonstrated inducible expression of the mRNA encoding the monocyte chemoattractant MCP-1, the human homolog of the JE gene, in endothelial cells within 3 hours of treatment with IL-1 beta and tumor necrosis factor. IFN-gamma also induced expression of this mRNA after 24 hours, but to a lesser extent. MCP-1/JE protein steadily accumulated in the medium of endothelial cells during a 48-hour exposure to IL-1 beta. Medium conditioned by IL-1 beta-treated endothelial cells contained monocyte chemoattractant activity that was immunoadsorbed by anti-MCP-1 antibodies. These results suggest that endothelial cells secrete a monocyte chemoattractant, MCP-1/JE, in response to inflammatory mediators, and thus may contribute to the accumulation of monocytes at sites of inflammation.     

Production of Monocyte Chemoattractant Protein 1 in Tuberculosis Patients

To investigate the role of monocyte chemoattractant protein 1 (MCP-1) in the immune response to Mycobacterium tuberculosis, we studied MCP-1 production in tuberculosis patients. CD14⁺ blood monocytes from tuberculosis patients spontaneously expressed higher levels of MCP-1 mRNA and protein than CD14⁺ monocytes from healthy tuberculin reactors. MCP-1 production in lymph nodes from tuberculosis patients was also markedly increased. These findings suggest that MCP-1 can contribute to the antimycobacterial inflammatory response by attracting monocytes and T lymphocytes.     

探讨肺纤维化时表达单核细胞趋化蛋白-1的主要效应细胞

目的研究凝血酶对人肺上皮细胞及正常人肺成纤维细胞释放单核细胞趋化蛋白-1（MCP-1）的调节作用。方法人肺支气管上皮细胞（normal human bronchial epithelial cells,NHBE）、人肺癌上皮细胞系A549及正常人肺成纤维细胞（normal human lung fibroblasts,NH-LF）分别培养于96孔或6孔板培养板内,凝血酶诱导4h和24h后,收集上清液及细胞裂解液。ELISA方法检测MCP-1蛋白水平;实时荧光定量RT-PCR技术检测MCP-1mRNA水平。结果凝血酶可诱导NHBE、A549及NH-LF细胞释放MCP-1,但NH-LF细胞的MCP-1释放量远远高于NHBE及A549细胞;凝血酶还可诱导NH-LF细胞mRNA表达。结论人肺成纤维细胞可能是肺内表达MCP-1的主要来源细胞,因此在肺纤维化时可能起重要作用。     

Increased expression of monocyte chemotactic protein-1 during active hepatic fibrogenesis: correlation with monocyte infiltration.

Monocyte chemotactic protein (MCP)-1 is a chemoattractant and activator for circulating monocytes and T lymphocytes. We investigated MCP-1 protein and gene expression during chronic liver disease at different stages, using immunohistochemistry and in situ hybridization, respectively. In normal liver, a modest expression of MCP-1 was confined to few peri-sinusoidal cells and to bile duct epithelial cells. During chronic hepatitis, MCP-1 immunostaining and gene expression were evident in the inflammatory infiltrate of the portal tract. In tissue from patients with active cirrhosis, MCP-1 expression was clearly up-regulated and was present in the portal tract, in the epithelial cells of regenerating bile ducts, and in the active septa surrounding regenerating nodules. A combination of in situ hybridization for MCP-1 and immunohistochemistry showed that activated stellate cells and monocyte/macrophages contribute to MCP-1 expression in vivo together with bile duct epithelial cells. Comparison of serial sections of liver biopsies from patients with various degrees of necro-inflammatory activity showed that infiltration of the portal tracts with monocytes/macrophages is directly correlated with the expression of MCP-1. These data expand previous in vitro studies showing that secretion of MCP-1 may contribute to the formation and maintenance of the inflammatory infiltrate observed during chronic liver disease.     

Cysteinyl leukotrienes induce monocyte chemoattractant protein 1 in human monocytes/macrophages

BACKGROUND: Monocytes/macrophages have a cysteinyl leukotriene 1 (CysLT1) receptor, but its function is poorly understood. Objective To elucidate the biological function of the CysLT1 receptor of human monocytes/macrophages. METHODS: We examined the production of TNF-alpha, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, monocyte chemoattractant protein 1 (MCP-1), macrophage colony-stimulating factor (M-CSF), and eotaxin induced by CysLTs (leukotriene (LT)C4, -D4, and -E4) in THP-1 cells, a human monocytic leukaemia cell line, and peripheral blood CD14+ monocytes/macrophages. Moreover, we examined the effect of CysLTs on the expression of beta-chemokine receptor 2B (CCR2B) as the receptor of MCP-1 by Western blot analysis. RESULTS: ELISA revealed that CysLTs induced MCP-1 in THP-1 cells and peripheral blood CD14+ monocytes/macrophages, but not other cytokines. PCR demonstrated that CysLTs increased MCP-1 mRNA expression in THP-1 cells, and Western blotting showed that CysLTs increased the expression of CCR2B in THP-1 cells. Moreover, we demonstrated that pranlukast, a CysLT1 receptor antagonist, blocked MCP-1 production by CysLTs in THP-1 cells almost completely, and partially inhibited MCP-1 release by CysLTs in peripheral blood CD14+ monocytes/macrophages and CCR2B expression by CysLTs in THP-1 cells. CONCLUSION: CysLTs induce MCP-1 and increase CCR2B expression in human monocytes/macrophages.     

高浓度的糖刺激人肾小球内皮细胞表达单核细胞趋化蛋白-1的影响

目的 :探讨高浓度的糖对培养的人肾小球内皮细胞 (HUGEC)表达单核细胞趋化蛋白 1(MCP 1)的影响 ,以及HUGEC的条件培养基对单核细胞 (MC)的趋化作用及抗MCP 1抗体对MC迁移的影响。方法 :采用原位杂交技术和细胞ELISA法 ,观察MCP 1的表达 ;用改良的Boyden小室微孔滤膜法 ,测定高浓度的糖刺激HUGEC后的条件培养基 ,对MC的趋化作用 ,以及抗MCP 1抗体对MC迁移的影响。结果 :(1)在低浓度的糖(含 5 .5mmol/LD 葡萄糖 )条件下培养的HUGECMCP 1mRNA呈弱表达。高浓度的糖 (含 2 5mmol/LD 葡萄糖 )刺激后 ,8h即出现MCP 1表达增强 ,于 16h达高峰。(2 )高浓度的糖刺激HUGEC后的条件培养基 ,对MC具有明显的趋化作用 ;抗MCP 1抗体可抑制其作用。结论 :在高浓度的糖诱导下 ,人HUGECMCP 1的表达增强 ,其条件培养基对MC具有趋化作用 ,从而可能招引单核细胞迁入内皮下间隙。     

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.     

Monocyte chemoattractant protein 1 and interleukin-8 production in mononuclear cells stimulated by oral microorganisms.

Chemokines are a family of low-molecular-weight proinflammatory cytokines that stimulate recruitment of leukocytes. The chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) are relatively specific chemoattractants for neutrophils and monocytes, respectively. Chemokine expression contributes to the presence of different leukocyte populations observed in normal and pathologic states. In the present studies, peripheral blood mononuclear cells (PBMC) were stimulated by microbes (Candida albicans, Streptococcus mutans, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans) selected based upon their importance as oral pathogens. IL-8 and MCP-1 gene expression and protein release were determined by Northern blot (RNA blot) analysis and enzyme-linked immunosorbent assay. C. albicans, P. gingivalis, and A. actinomycetemcomitans induced high levels of production of both MCP-1 and IL-8. S. mutans was a strong inducer of MCP-1, but it did not stimulate significant production of IL-8. C. albicans, S. mutans, and A. actinomycetemcomitans were 500 to 5,000 times more potent than P. gingivalis in terms of MCP-1 production. In general, the microbe-to-PBMC ratios required for maximum gene expression of MCP-1 were lower than those for IL-8. However, for actual protein release of MCP-1 versus IL-8, differences in the effects of various microbe concentrations were observed only for A. actinomycetemcomitans. These results demonstrate that different oral pathogens induce specific dose-dependent patterns of chemokine gene expression and release. Such patterns may help explain the immunopathology of oral infections, particularly with regard to inflammatory leukocyte recruitment.     

Effect of acute inflammatory lung injury on the expression of monocyte chemoattractant protein-1 (MCP-1) in rat pulmonary alveolar macrophages.

Using a well-characterized rat model of immune complex-mediated acute inflammatory lung injury, we determined that there is a time-dependent elaboration of monocyte chemotactic activity in bronchoalveolar lavage fluid. Monocyte chemotactic activity is also significantly enhanced in culture supernatants from pulmonary alveolar macrophages (PAMs) from injured rat lungs. Northern hybridization analysis revealed markedly increased expression of rat monocyte chemoattractant protein 1 (MCP-1) mRNA in PAMs obtained from rats with immune complex-induced lung injury. The increased expression of MCP-1 mRNA and associated increase in monocyte chemotactic activity present in culture supernatants of PAMs from injured rat lungs suggest that PAMs may participate in the pathogenesis of acute inflammatory lung injury by the secretion of monocyte chemoattractants including MCP-1.     

THE ROLE OF MONOCYTE CHEMOATTRACTANT PROTEIN-1 (MCP-1) IN THE PATHOGENESIS OF COLLAGEN-INDUCED ARTHRITIS IN RATS

Collagen-induced arthritis was produced in rats by intradermal immunization with type II collagen and the expression and production of monocyte chemoattractant protein-1 (MCP-1) were examined by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and Northern blot analysis. Two to three weeks after the immunization, the hindfeet showed swelling and redness, followed by the development of severe arthritis, particularly in the ankle joints. During this period, prominent infiltration of neutrophils and macrophages was observed. Sandwich ELISA and Northern blot analysis revealed that MCP-1 concentrations in the joint lavages and MCP-1 mRNA levels in the joint tissues both peaked at 2 weeks after the immunization. By immunohistochemistry, various types of cells, particularly neutrophils, macrophages, synovial cells, and vascular endothelial cells, stained positively for MCP-1. Finally, injection of a neutralizing monoclonal antibody against rat MCP-1 significantly decreased the number of exudate macrophages in the lesions and reduced the ankle swelling by about 30 per cent compared with controls. These results suggest that MCP-1 plays a critical role in this model in the recruitment of monocytes and in the development of arthritis. © 1997 John Wiley & Sons, Ltd.     

Expression of monocyte chemoattractant protein 1 in human inflamed gingival tissues.

Gingival inflammation is initiated by bacterial colonization on the tooth surface. It is characterized by infiltration of mononuclear cells, a common feature of many forms of chronic inflammation. Monocyte chemoattractant protein 1 (MCP-1) is the predominant monocyte chemoattractant secreted by a variety of different cells in vitro. For this report, we examined MCP-1 expression in bacterially induced gingival inflammation by immunohistochemistry and in situ hybridization. The cell types expressing MCP-1 are identified as vascular endothelial cells and monocytes/macrophages. Correlation analysis shows that the number of cells expressing MCP-1 is related to the degree of inflammation. Our finding that MCP-1 is expressed in inflamed gingival tissue suggests that MCP-1 plays an important role in the recruitment of monocytes and amplification of inflammatory signals in bacterially induced inflammation.     

Expression of monocyte chemotactic protein-1 in human melanoma in vivo.

A common feature of human melanoma is infiltration by monocytes at early stages of tumorigenesis. This infiltration may be highly significant since macrophages have the capacity to alter the behavior of tumor cells. The authors previously demonstrated that the predominant monocyte chemoattractant produced by tumor cells in vitro was monocyte chemotactic protein-1 (MCP-1). The authors identify the expression of MCP-1 in pathologic specimens of both primary and metastatic human melanoma but not in normal skin. The finding that MCP-1 is produced by malignant melanoma suggests that specific genes are expressed in tumor cells that can induce the recruitment of monocytes in vivo.     

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.     

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.     

Morphological Alteration of Cultured Tracheobronchial Epithelial Cells is Accompanied by the Expression of Chemokines, MCP-1 and CINC/gro, in Rats

Morphologically altered epithelial cells are generally observed in fibrotic lung conditions and have been reported to produce several cytokines. To examine the relationship between morphological changes of the tracheobronchial epithelial cells (TBECs) and their chemokine production, we investigated, (1) the mRNA expression and protein secretion of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant/gro (CINC/gro), (2) morphological changes by electron microscopy, and (3) cytokeratin (CK) expression, using a primary culture system of rat TBECs. The constitutive secretion of MCP-1 in the culture supernatant of TBECs increased in a time-dependent manner, whereas the CINC/gro secretion was not changed. These results were consistent with the chemokines'' mRNA expression observed by in situ hybridization. The constitutive secretions of MCP-1 and CINC/gro were inhibited partially but significantly by dexamethasone. With the extension of the culture period, the morphology of the TBECs became flat and spindle in shape, similar to squamous metaplasia, as observed on electron microscopy, and with strong expression of CK 14. Sequential staining using immunocytochemistry and in situ hybridization revealed the coexpression of MCP-1 mRNA and CK 14. These data indicate a significant relationship between the morphological squamoid alteration and the constitutive expression of MCP-1 but not of CINC/gro. It is thought that the squamous metaplasia of TBECs may accompany the alteration of cytokine production and play an important role in chronic lung inflammation.     

ERK 1/2介导结缔组织生长因子刺激系膜细胞产生MCP-1

目的检测结缔组织生长因子（CTGF）是否刺激大鼠肾小球系膜细胞分泌单核细胞趋化蛋白-1（MCP-1），并探讨其作用机制。方法应用CTGF刺激静息的培养的系膜细胞，在刺激后不同时间点应用RT-PCR方法测定MCP-1的mRNA表达，应用酶联免疫吸附试验（ELISA）测定上清液中MCP-1，应用趋化试验测定上清液对单核细胞（THP-1）的趋化作用。应用Western blot测定CTGF对细胞外信号调节激酶（ERK）1／2磷酸化的作用。应用ERK1／2抑制剂PD98059预处理，观察CTGF对上清液中MCP-1分泌的影响。结果应用CTGF刺激后，系膜细胞的MCP-1的mRNA表达上升，上清液中分泌量增加。MCP-1抗体可部分阻止上清液对单核细胞的趋化作用。CTGF诱导ERK1／2磷酸化，而PD98059可抑制这一作用，并部分抑制CTGF诱导的上清液中MCP-1的分泌。结论CTGF可引起系膜细胞分泌MCP-1，其作用机制部分依赖于ERK1／2的磷酸化。     

Increase in circulating levels of monocyte chemoattractant protein-1 with aging.

Monocyte chemoattractant protein-1 (MCP-1) is a member of chemokines with chemoattractant activity for monocytes, T cells, mast cells, and basophils. Precursor mRNA or protein was detected at high levels in the lesions of several diseases, such as pulmonary fibrosis, rheumatoid arthritis, atherosclerosis, and some types of tumors. The regulation of MCP-1 production and the role of this chemokine in pathophysiologic states, however, remain largely unknown. In this study, using an enzyme-linked immunosorbent assay (ELISA), we measured the circulating MCP-1 levels in 405 healthy Japanese subjects of various ages, eliciting a profound age-dependent MCP-1 increase. Multivariate regression analysis revealed that significant predictors of MCP-1 value for males were age (p = 0.033) and serum triglyceride (p = 0.039). For females, age was also a significant predictor (p = 0.00002). One possible explanation is that the plasma MCP-1 concentration might reflect the existence of atherosclerosis, although the plasma MCP-1 concentration from patients with coronary artery disease or cerebrovascular accidents appears not to differ from age-matched, disease-free controls. This is the first report linking an increase in a particular chemokine level with aging.     

Unfractionated heparin suppresses lipopolysaccharide-induced monocyte chemoattractant protein-1 expression in human microvascular endothelial cells by blocking Krüppel-like factor 5 and nuclear factor-κB pathway

Unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH), apart from anticoagulant activities, contain a variety of biological properties such as anti-inflammatory actions possibly affecting sepsis. Chemokines are vital for promoting the movement of circulating leukocytes to the site of infection and are involved in the pathogenesis of sepsis. The purpose of this study was to investigate the effects and potential mechanisms of UFH on lipopolysaccharide (LPS)-induced chemokine production in human pulmonary microvascular endothelial cells (HPMECs). HPMECs were pretreated with UFH (0.1 U/ml and 1 U/ml), 15 min prior to stimulation with LPS (10 μg/ml). Cells were cultured under various experimental conditions for 2 h and 6 h for analysis. UFH markedly decreased LPS-induced interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1) mRNA and protein expression in HPMECs. UFH also attenuated the secretion of these chemokines in culture supernatants. In addition, UFH blocked the chemotactic activities of LPS-stimulated HPMECs supernatants on monocytes migration as expected. UFH inhibited LPS-induced Krüppel-like factor 5 (KLF-5) mRNA and protein levels. Concurrently, UFH reduced nuclear factor (NF)-κB nuclear translocation. Importantly, transfection with siRNA targeting KLF-5 reduced NF-κB activation and chemokines expression. These results demonstrate that interfering with KLF-5 mediated NF-κB activation might contribute to the inhibitory effects of chemokines and monocytes migration by UFH in LPS-stimulated HPMECs.