Differential expression of the isoforms for the monocyte chemoattractant protein-1 receptor, CCR2, in monocytes.

Two isoforms of human CCR2, the receptor for monocyte chemoattractant protein-1 (MCP-1), have been identified but their relative expression in monocytes and contribution to inflammatory responses mediated by MCP-1 remain uncertain. All available information on CCR2 expression is based on mRNA data because isoform-specific antibodies were not available until now. To analyze the relative expression of each isoform, we made two antibodies that specifically recognized CCR2A and CCR2B. Examination of receptor protein with these isoform-specific antibodies showed that the total expression of CCR2B in monocytes was about 10-fold higher than that of CCR2A with an equal distribution between the cell surface and intracellular pools. A detailed analysis using purified plasma membranes demonstrated that about 90% of all CCR2 on the cell surface were composed of CCR2B. The relatively abundant expression of CCR2B on the cell surface suggests a principal role of this isoform as a mediator of monocyte responses to MCP-1 in inflammation.     

单核细胞趋化蛋白-1在糖尿病肾病中的作用

糖尿病肾病是多因素引起的复杂性疾病,近年研究发现炎症反应参与了该病的发生与发展.单核细胞趋化蛋白-1是趋化因子CC亚家族的一员,在募集巨噬细胞等炎性细胞参与炎症反应中扮演着重要的角色.其趋化单核巨噬细胞于糖尿病肾组织中,可介导溶酶体释放,产生氧自由基,促进单核巨噬细胞表达β1-转化生长因子(transforming growth factor β1,TGF-β1),而广泛浸润臣噬细胞加剧了肾小球基底膜增厚、细胞外基质堆积,进而发展为肾小球硬化和间质纤维化.深入研究单核细胞趋化蛋白-1在糖尿病肾病中的作用,可望为糖尿病肾病的预防和治疗提供新的思路和途径.     

Association of monocyte chemoattractant protein-1 2518G/A gene polymorphism with diabetic nephropathy risk

Abstract

Results from the published studies on the association between monocyte chemoattractant protein-1 (MCP-1) ?2518 A/G gene polymorphism and diabetic nephropathy (DN) risk are still conflicting. This meta-analysis was performed to evaluate the relationship between MCP-1 A/G gene polymorphism and DN risk and to explore whether MCP-1 A allele, AA genotype or GG genotype could become a predictive marker for DN risk. Association studies were identified from the databases of PubMed, Embase, Cochrane Library and CBM-disc (China Biological Medicine Database) as of 1 March 2014, and eligible investigations were synthesized using meta-analysis method. Four studies were identified for the analysis of association between MCP-1 A/G gene polymorphism and DN risk, and all the included studies were form Asian population. The association between MCP-1 A/G gene polymorphism and DN susceptibility was not found (A allele: OR?=?1.19; 95% CI: 0.97–1.45; p?=?0.10; AA genotype: OR?=?1.27; 95% CI: 0.95–1.70; p?=?0.11; GG genotype: OR?=?0.77; 95% CI: 0.57–1.05; p?=?0.10). In the sensitive analysis, according to the control source from hospital, we found that AA genotype was associated with the DN risk (OR?=?1.45; 95% CI: 1.05–2.00; p?=?0.02). However, other associations were not found in the sensitive analysis according to the control source from hospital or population. Our results indicate that AA homozygous might be a significant genetic molecular marker to predict the diabetes mellitus patients developing into DN. However, more investigations are required to further clarify this association.     

Induction of monocyte chemoattractant protein-1 release from A549 cells by agonists of protease-activated receptor-1 and -2

Hypersecretion of cytokines and serine proteases has been observed in asthma. However, the influence of proteases and protease-activated receptors (PARs) on monocyte chemoattractant protein-1 (MCP-1) release from airway epithelial cells remains largely unknown. In the present study, A549 cells were challenged with agonists of PARs, and levels of MCP-1 released in the supernatant and mRNA expression were examined by ELISA and real time polymerase chain reaction (PCR), respectively. The results show that thrombin, tryptase, elastase and trypsin induced an up to 6.5-, 1.8-, 1.6-, and 3.1-fold increase in MCP-1 release from A549 cells, respectively, following a 16-h incubation period. The protease-induced secretion of MCP-1 can be abolished by specific protease inhibitors. Agonist peptides of PAR-1 and PAR-2 stimulate MCP-1 secretion up to 15- and 12.7-fold, respectively. Real-time PCR showed that MCP-1 mRNA is up-regulated by the serine proteases tested and by agonist peptides of PAR-1 and PAR-2. In conclusion, serine proteases can stimulate MCP-1 release from A549 cells possibly through a PARs-related mechanism, suggesting that they are likely to contribute to MCP-1-related airway inflammatory disorders in man.     

Reactive oxygen species and ERK 1/2 mediate monocyte chemotactic protein-1-stimulated smooth muscle cell migration

Summary Monocyte chemotactic protein-1 (MCP-1), a potent chemoattractant for monocytes, is thought to play a major role in atherosclerosis, but whether its atherogenic effects involve the direct modulation of vascular smooth muscle cell (SMC) functions remains unclear. This study examined the effects of MCP-1 on the migration of cultured A7r5 SMCs and the signaling pathways involved. Addition of recombinant MCP-1 stimulated SMC migration in modified Boyden chambers coated with type I collagen in a concentration-dependent manner, with 10^–9 M being maximally effective. Using untreated A7r5 cells, two MCP-1 receptors, CCR2 and CCR4, were detected and MCP-1 secretion was significantly increased by stimulation with platelet-derived growth factor. MCP-1-stimulated A7r5 migration was completely blocked by the NAD(P)H oxidase inhibitor, diphenylene iodonium (DPI), and dose-dependently inhibited by polyethylene glycol-conjugated superoxide dismutase (PEG-SOD), suggesting a role for reactive oxygen species (ROS) in this process. During MCP-1 stimulation, ROS production increased rapidly, then gradually decayed over 60 min, and this effect was markedly decreased by pretreatment with DPI or PEG-SOD. Interestingly, U0126 and PD98059, which inhibit activation of extracellular signal-regulated kinases 1/2 (ERK 1/2), significantly inhibited MCP-1-activated ROS generation. Furthermore, transfection of an active mutant of MEK1 (ERK 1/2 kinase) markedly increased superoxide production in rat aortic smooth muscle cells, as detected by dihydroethydium staining, suggesting that ERK 1/2 activation stimulates ROS generation. ERK 1/2 activation was increased for at least 30 min in cells incubated with MCP-1, and this effect was abolished by U0126 or DPI pretreatment. These results demonstrate that MCP-1 is a chemoattractant for SMCs and that MCP-1-stimulated migration requires both ROS production and ERK 1/2 activation in a positive activation loop, which may contribute to the atherogenic effects of MCP-1.These authors contributed equally to this work.     

Induction of monocyte chemoattractant protein-1 by Porphyromonas gingivalis in human endothelial cells

The association between periodontal and cardiovascular diseases could be mediated by direct interaction of periodontal pathogens with cardiac tissue. In order to explore this possibility, the effect of the periodontal pathogen Porphyromonas gingivalis on monocyte chemoattractant protein-1 (MCP-1) production by endothelial cells was investigated. When incubated with live P. gingivalis 381, MCP-1 production by human umbilical vein endothelial cells (HUVEC) was potently increased. Compared to the type strain 381, non-adhesive/invasive strains (W50 and DPG3) did not increase MCP-1 production, which was also demonstrated at the mRNA level. Killed P. gingivalis 381 was much less effective than live bacteria for MCP-1 induction. Treatment of HUVEC with cytochalasin D, an inhibitor of endocytosis, prevented MCP-1 mRNA up-regulation by P. gingivalis 381, suggesting that internalization of P. gingivalis is necessary for MCP-1 induction. In conclusion, the secretion of high levels of MCP-1 resulting from interactions of P. gingivalis with endothelial cells could enhance atherosclerosis progression by contributing to the recruitment of monocytes.     

N-cadherin mediates angiogenesis by regulating monocyte chemoattractant protein-1 expression via PI3K/Akt signaling in prostate cancer cells

Over the past decade, evidence continues to mount showing that N-cadherin is a critical protein in cancer progression and metastasis. In the present study, we evaluated the expression of N-cadherin in human prostate cancer tissue specimens and cell lines. Enhanced expression of N-cadherin was observed in both the malignant and bone-metastasized prostate tissue specimens compared to the healthy prostate tissues. Consistent with the tissue array data, N-cadherin was highly expressed in PC3, but not in Du145 and LNCaP human prostate cell lines. Based on cell to cell binding assay, we found that N-cadherin expression facilitates homotypic interaction between human prostate cancer cells and human microvascular endothelial cells (HMEC). Human angiogenesis antibody array and in vitro angiogenesis assay showed that siRNA-mediated knockdown of N-cadherin reduced the secretion of monocyte chemoattractant protein-1 (MCP-1), which played a potential role in stimulating capillary network formation of HMEC. Additionally, culture supernatant of Du145 cells transfected with full-length N-cadherin expressing plasmid showed increased MCP-1 expression and chemoattractant ability compared to normal Du145 cells. Further, we noticed that blocking PI3K activity inhibited N-cadherin mediated MCP-1 expression. Our data demonstrated that N-cadherin in prostate cancer cell mediates cell–cell adhesion and regulates MCP-1 expression via the PI3K/Akt signaling pathway.     

Borrelia burgdorferi-induced monocyte chemoattractant protein-1 production in vivo and in vitro

Matrix metalloproteinase 9 (MMP-9) is selectively upregulated in erythema migrans (EM) lesions with acute Lyme disease. This study explored whether upregulation of MMP-9 was associated with monocyte chemoattractant protein 1 (MCP-1) production, and Borrelia burgdorferi (B. burgdorferi) could induce MCP-1 production in vivo and in vitro. The results indicated that expression of MCP-1 was significantly increased in U937 cells by B. burgdorferi. The activity of MMP-9 could be elevated by recombinant MCP-1 (rMCP-1) in U937 cells. MMP-9 was not upregulated by B. burgdorferi in fibroblasts. However, the expression of MCP-1 was significantly increased in the presence of B. burgdorferi in fibroblasts. The level of MCP-1 in EM lesions and in serum of patients with acute Lyme disease was also significantly elevated compared to that for healthy controls. The secreted MCP-1 may affect the production of MMP-9 in fibroblasts and/or macrophages.     

Inhibition of the angiogenesis by the MCP-1 (monocyte chemoattractant protein-1) binding peptide

The CC chemokine, monocyte chemoattractant protein-1 (MCP-1), plays a crucial role in the initiation of atherosclerosis and has direct effects that promote angiogenesis. To develop a specific inhibitor for MCP-1-induced angiogenesis, we performed in vitro selection employing phage display random peptide libraries. Most of the selected peptides were found to be homologous to the second extracellular loops of CCR2 and CCR3. We synthesized the peptide encoding the homologous sequences of the receptors and tested its effect on the MCP-1 induced angiogenesis. Surface plasmon resonance measurements demonstrated specific binding of the peptide to MCP-1 but not to the other homologous protein, MCP-3. Flow cytometry revealed that the peptide inhibited the MCP-1 binding to THP-1 monocytes. Moreover, CAM and rat aortic ring assays showed that the peptide inhibited MCP-1 induced angiogenesis. Our observations indicate that the MCP-1-binding peptide exerts its anti-angiogenic effect by interfering with the interaction between MCP-1 and its receptor.     

Dexamethasone suppresses monocyte chemoattractant protein-1 production via mitogen activated protein kinase phosphatase-1 dependent inhibition of Jun N-terminal kinase and p38 mitogen-activated protein kinase in activated rat microglia

Microglial cells release monocyte chemoattractant protein-1 (MCP-1) which amplifies the inflammation process by promoting recruitment of macrophages and microglia to inflammatory sites in several neurological diseases. In the present study, dexamethasone (Dex), an anti-inflammatory and immunosuppressive drug has been shown to suppress the mRNA and protein expression of MCP-1 in activated microglia resulting in inhibition of microglial migration. This has been further confirmed by the chemotaxis assay which showed that Dex or MCP-1 neutralization with its antibody inhibits the microglial recruitment towards the conditioned medium of lipopolysaccharide (LPS)-treated microglial culture. This study also revealed that the down-regulation of the MCP-1 mRNA expression by Dex in activated microglial cells was mediated via mitogen-activated protein kinase (MAPK) pathways. It has been demonstrated that Dex inhibited the phosphorylation of Jun N-terminal kinase (JNK) and p38 MAP kinases as well as c-jun, the JNK substrate in microglia treated with LPS. The involvement of JNK and p38 MAPK pathways in induction of MCP-1 production in activated microglial cells was confirmed as there was an attenuation of MCP-1 protein release when microglial cells were treated with inhibitors of JNK and p38. In addition, Dex induced the expression of MAP kinase phosphatase-1 (MKP-1), the negative regulator of JNK and p38 MAP kinases in microglial cells exposed to LPS. Blockade of MKP-1 expression by triptolide enhanced the phosphorylation of JNK and p38 MAPK pathways and the mRNA expression of MCP-1 in activated microglial cells treated with Dex. In summary, Dex inhibits the MCP-1 production and subsequent microglial cells migration to the inflammatory site by regulating MKP-1 expression and the p38 and JNK MAPK pathways. This study reveals that the MKP-1 and MCP-1 as novel mediators of biological effects of Dex may help developing better therapeutic strategies for the treatment of patients with neuroinflammatory diseases.     

单核细胞趋化蛋白-1、高迁移率族蛋白B1与溃疡性结肠炎患者肠道菌群变化的相关性

目的 探讨单核细胞趋化蛋白-1（MCP-1）、高迁移率族蛋白B1（HMGB1）与溃疡性结肠炎（UC）患者肠道菌群变化的相关性。方法 选取2016年9月‒2018年1月遂宁市中心医院收治的98例UC患者资料,根据Mayo评分系统将UC患者分为活动期组（n=50）和缓解期组（n=48）。选取同期进行体检的健康人50例作为对照组。比较各组间肠道菌群,血清MCP-1、HMGB1水平,并进行Pearson相关分析。结果 活动期组患者肠道乳杆菌、双歧杆菌含量[（5.34±0.87）、（5.81±0.83）CFU/g]显著低于缓解期组和对照组[（8.07±0.86）、（8.35±0.88）CFU/g;（8.13±0.91）、（8.46±0.95）CFU/g]（F=12.035,P0.05）。活动期组和缓解期组患者血清MCP-1、HMGB1水平[（267.42±23.51）、（21.35±2.26）ng/mL;（188.15±20.73）、（6.28±1.38）ng/mL]显著高于对照组[（106.38±15.92）、（2.13±0.41）ng/mL]（F=84.163,P<0.001;F=25.386,P<0.001）;活动期组患者血清MCP-1、HMGB1水平[（267.42±23.51）、（21.35±2.26）ng/mL]显著高于缓解期组[（188.15±20.73）、（6.28±1.38）ng/mL]（t=17.676、39.641,均P<0.05）。经过Pearson相关性分析,MCP-1、HMGB1与UC患者乳杆菌、双歧杆菌含量呈负相关（r=‒0.715、‒0.659,r=‒0.703、‒0.614,均P<0.001）,与大肠埃希菌、肠球菌、拟杆菌含量呈正相关（r=0.783、0.702,r=0.762、0.735,r=0.653、0.612,均P<0.001）。结论 MCP-1、HMGB1作为促炎因子可介导肠黏膜炎性反应,引起UC患者肠道菌群的紊乱。     

Interplay of CCR2 signaling and local shear force determines vein graft neointimal hyperplasia in vivo

Leukocytes play a central role in vein graft neointimal hyperplasia, which is significantly augmented under low shear conditions. The current concept is that shear force regulates leukocyte adhesion predominately through up-regulation of chemokines and growth factors within the graft wall. Using rabbit and murine vein graft models, we demonstrate that CC chemokine receptor 2/monocyte chemoattractant protein-1 mediated monocyte recruitment and a low shear environment act synergistically to augment neointimal hyperplasia development and removal of either of the conditions leads to a significant reduction in neointimal thickening. We propose a novel concept that the shear stress response element phenotypically stems from the complex interplay of the biological and physical microenvironments.     

Increased expression levels of monocyte CCR2 and monocyte chemoattractant protein-1 in patients with diabetes mellitus

Increased monocyte recruitment into subendothelial space in atherosclerotic lesions is one of the hallmarks of diabetic angiopathy. The aim of this study was to determine the state of peripheral blood monocytes in diabetes associated with atherosclerosis. Diabetic patients treated with/without an oral hypoglycemic agent and/or insulin for at least 1 year were recruited (n=106). We also included 24 non-diabetic control subjects. We measured serum levels of monocyte chemoattractant protein (MCP)-1, fasting plasma glucose (FPG), HbA1c, total cholesterol, triglyceride, body mass index (BMI), high sensitivity CRP (hs-CRP) and evaluated CCR2, CD36, CD68 expression on the surface of monocytes. Serum MCP-1 levels were significantly (p<0.05) higher in diabetic patients than in normal subjects. In diabetic patients, serum MCP-1 levels correlated significantly with FPG, HbA1c, triglyceride, BMI, and hs-CRP. The expression levels of CCR2, CD36, and CD68 on monocytes were significantly increased in diabetic patients and were more upregulated by MCP-1 stimulation. Our data suggest that elevated serum MCP-1 levels and increased monocyte CCR2, CD36, CD68 expression correlate with poor blood glucose control and potentially contribute to increased recruitment of monocytes to the vessel wall in diabetes mellitus.     

Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes

Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-κB activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.     

Stress defense mechanisms of NADPH-dependent thioredoxin reductases (NTRs) in plants

Plants establish highly and systemically organized stress defense mechanisms against unfavorable living conditions. To interpret these environmental stimuli, plants possess communication tools, referred as secondary messengers, such as Ca²⁺ signature and reactive oxygen species (ROS) wave. Maintenance of ROS is an important event for whole lifespan of plants, however, in special cases, toxic ROS molecules are largely accumulated under excess stresses and diverse enzymes played as ROS scavengers. Arabidopsis and rice contain 3 NADPH-dependent thioredoxin reductases (NTRs) which transfer reducing power to Thioredoxin/Peroxiredoxin (Trx/Prx) system for scavenging ROS. However, due to functional redundancy between cytosolic and mitochondrial NTRs (NTRA and NTRB, respectively), their functional involvements under stress conditions have not been well characterized. Recently, we reported that cytosolic NTRA confers the stress tolerance against oxidative and drought stresses via regulation of ROS amounts using NTRA-overexpressing plants. With these findings, mitochondrial NTRB needs to be further elucidated.     

Development of a microplate bioassay for monocyte chemoattractant protein-1 based on activation of p44/42 mitogen-activated protein kinase

Monocyte chemoattractant protein-1 (MCP-1) is a potential therapeutic target for the treatment of several inflammatory conditions, including rheumatoid arthritis and chronic obstructive pulmonary disease. Current cell-based assays for MCP-1 use monocyte chemotaxis or calcium flux as a readout. Here, we describe an alternative bioassay based on MCP-1-induced phosphorylation of the mitogen-activated protein kinases (MAPK) p44 (ERK1) and p42 (ERK2). Adherent cells expressing the MCP-1 receptor CCR2B are treated with MCP-1 in 96-well plates in the presence or absence of inhibitors, fixed and permeabilized with methanol, and then probed with a monoclonal antibody that selectively recognizes the doubly phosphorylated form of p44/42 MAPK. Bound antibody is detected with a secondary antibody-peroxidase conjugate and a chromogenic substrate. The phosphorylation of p44/42 MAPK as detected in this assay peaks after 3-5 min of MCP-1 treatment, and the concentration of MCP-1 required for half-maximal p44/42 MAPK phosphorylation is 1-3 nM. MCP-1-induced phosphorylation of p44/42 MAPK is dependent upon the expression of CCR2B. The assay can be used for screening and characterization of small molecule inhibitors and antibodies blocking the binding of MCP-1 to its receptor. Since the assay is rapid and simple, it may represent a useful alternative to chemotaxis or calcium mobilization assays for the analysis of MCP-1 inhibitors.     

Effects of losartan on expression of monocyte chemoattractant protein-1 (MCP-1) in hyperuricemic nephropathy rats

Abstract

The monocyte chemoattractant protein-1 (MCP-1) plays an important role in the pathogenesis of progression of renal failure. This is based on the observations done both in various animal models of renal damage and in different types of human renal disease. During the development of non-infectious kidney stones, crystals are formed and deposited on the kidneys and the kidneys are surrounded by monocytes/macrophages. We have proposed that in response to crystal exposure, renal epithelial cells produce chemokines, which attract the monocytes/macrophages to the sites of crystal deposition. In this study, we investigated the expression of MCP-1 protein by SD rats exposed to oxonic acid (OA). Our study showed that hyperuricemia accelerates renal progression via a mechanism linked to high MCP-1 which may mediate the inflammation reaction of renal diseases induced by hyperuricemia. Losartan may retard the progression of advanced renal dysfunction, and the mechanism was partly due to blocking of renal inflammation induced by the uric acid. Because the number of experiments performed here is very few, results must be confirmed by more extensive studies with a larger sample size.