Identification of high affinity receptors for human monocyte chemoattractant protein-1 on human monocytes

The binding of human monocyte chemoattractant protein-1 (MCP-1) to human monocytes was studied. MCP-1 was radioiodinated with Iodo-beads (Pierce Chemical Co., Rockford, IL) without significant loss of biologic activity. 125I-MCP-1 binding to PBMC occurred within 5 min at 0 degrees C and the binding was inhibited by unlabeled MCP-1 dose dependently but not by neutrophil attractant/activation protein-1 or FMLP. 125I-MCP-1 bound to monocytes; no significant binding to either neutrophils or lymphocytes was observed. Scatchard plot analysis indicated that monocytes had a minimum of 1700 +/- 600 binding sites per cell with a Kd of 1.9 +/- 0.2 x 10(-9) M. For analysis of binding by flow cytometry, MCP-1 was biotinylated. In contrast to radioiodination, biotinylation resulted in loss of activity; potency was 10-fold less, but the efficacy was retained. Detection by flow cytometry of bound biotinylated MCP-1 with avidin-FITC confirmed results obtained with 125I-MCP-1. Biotinylated MCP-1 bound to monocytes but not to lymphocytes; and the binding was inhibited by a 100-fold excess of unlabeled MCP-1.     

Homocysteine stimulates the expression of monocyte chemoattractant protein-1 in endothelial cells leading to enhanced monocyte chemotaxis

The effect of intraperitoneal administration of tocopherol (100 mg/kg wt/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of mitochondria and microsomes isolated from rat liver and testis was studied. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:6 n3 in liver and C20:4 n6 and C22:5 n6 in testis. The lipid peroxidation of liver mitochondria or microsomes produced a significant decrease of C20:4 n6 and C22:6 n3 in the control group, whereas changes in the fatty acid composition of the tocopherol treated group were not observed. The light emission was significantly higher in the control than in the tocopherol treated group. The lipid peroxidation of testis microsomes isolated from the tocopherol group produced a significant decrease of C20:4 n6 , C22:5 n6 and C22:6 n3, these changes were not observed in testis mitochondria. The light emission of both groups was similar. The treatment with tocopherol at the dose and times indicated showed a protector effect on the polyunsaturated fatty acids of liver mitochondria, microsomes and testis mitochondria, whereas those fatty acids situated in testis microsomes were not protected during non enzymatic ascorbateFe²⁺ lipid peroxidation. The protector effect observed by tocopherol treatment in the fatty acid composition of rat testis mitochondria but not in microsomes could be explained if we consider that the sum of C20:4 n6 + C22:5 n6 in testis microsomes is 2-fold than that present in mitochondria.     

Regulatory role of thioredoxin in homocysteine-induced monocyte chemoattractant protein-1 secretion in monocytes/macrophages

We have previously shown that homocysteine (Hcy) can induce monocyte chemoattractant protein-1 (MCP-1) secretion via reactive oxygen species (ROS) in human monocytes. Here, we show that Hcy upregulates expression of an important antioxidative protein, thioredoxin (Trx), via NADPH oxidase in human monocytes in vitro. The increase of Trx expression and activity inhibited Hcy-induced ROS production and MCP-1 secretion. Of note, 2-week hyperhomocysteinemia (HHcy) ApoE^−/− mice showed accelerated lesion formation and parallel lower Trx expression in macrophages than ApoE^−/− mice, suggesting that HHcy-induced sustained oxidative stress in vivo might account for impaired Trx and hence increased ROS production and MCP-1 secretion from macrophages, and subsequently accelerated atherogenesis.     

Low-level monocyte chemoattractant protein-1 stimulation of monocytes leads to tumor formation in nontumorigenic melanoma cells

Tumors commonly produce chemokines for recruitment of host cells, but the biological significance of tumor-infiltrating inflammatory cells, such as monocytes/macrophages, for disease outcome is not clear. Here, we show that all of 30 melanoma cell lines secreted monocyte chemoattractant protein-1 (MCP-1), whereas normal melanocytes did not. When low MCP-1-producing melanoma cells from a biologically early, nontumorigenic stage were transduced to overexpress the MCP-1 gene, tumor formation depended on the level of chemokine secretion and monocyte infiltration; low-level MCP-1 secretion with modest monocyte infiltration resulted in tumor formation, whereas high secretion was associated with massive monocyte/macrophage infiltration into the tumor mass, leading to its destruction within a few days after injection into mice. Tumor growth stimulated by monocytes/macrophages was due to increased angiogenesis. Vessel formation in vitro was inhibited with mAbs against TNF-alpha, which, when secreted by cocultures of melanoma cells with human monocytes, induced endothelial cells under collagen gels to form branching, tubular structures. These studies demonstrate that the biological effects of tumor-derived MCP-1 are biphasic, depending on the level of secretion. This correlates with the degree of monocytic cell infiltration, which results in increased tumor vascularization and TNF-alpha production.     

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.     

Effect of the mitogen-activated kinase inhibitors on monocyte protein-1-stimulated chemotaxis of cells

Monocyte chemoattractant protein (MCP-1) is a major chemoattractant for monocytes and T-lymphocytes although it can cause migration of the HUVECs. We used monocytic cell line THP-1, monocytes of human peripheral blood, and HUVECs to study MCP-1 receptor-mediated cell migration. We showed that THP-1 and the monocytes chemotaxis was decreased in presence of specific inhibitors of p 38 MAP-kinase. Furthermore, it was almost completely diminished by inhibitor of tyrosine kinases. In contrast, MCP-1-stimulated migration of HUVECs was abrogated by specific inhibitor of ERK1/2 MAP-kinases and, to a lesser extent, by blocking tyrosine kinases. These results suggest that intracellular signal pathways activated by MCP-1 in monocytes and HUVECs, are distinct.     

Inhibition of monocyte chemoattractant protein-1 ameliorates rat adjuvant-induced arthritis

Chemokines, including RANTES/CCL5 and MCP-1/CCL2, are highly expressed in the joints of patients with rheumatoid arthritis, and they promote leukocyte migration into the synovial tissue. This study was conducted to determine whether the inhibition of RANTES and MCP-1 therapeutically was capable of ameliorating rat of adjuvant-induced arthritis (AIA). Postonset treatment of AIA using a novel inhibitor for endogenous MCP-1 (P8A-MCP-1) improved clinical signs of arthritis and histological scores measuring joint destruction, synovial lining, macrophage infiltration, and bone erosion. Using immunohistochemistry, ELISA, real-time RT-PCR, and Western blot analysis, we defined joint inflammation, bony erosion, monocyte migration, proinflammatory cytokines, and bone markers, and p-p38 levels were reduced in rat AIA treated with P8A-MCP-1. In contrast, neither the dominant-negative inhibitor for endogenous RANTES (44AANA47-RANTES) nor the CCR1/CCR5 receptor antagonist, methionylated-RANTES, had an effect on clinical signs of arthritis when administered after disease onset. Additionally, therapy with the combination of 44AANA47-RANTES plus P8A-MCP-1 did not ameliorate AIA beyond the effect observed using P8A-MCP-1 alone. Treatment with P8A-MCP-1 reduced joint TNF-alpha, IL-1beta, and vascular endothelial growth factor levels. P8A-MCP-1 also decreased p38 MAPK activation in the joint. Our results indicate that inhibition of MCP-1 with P8A-MCP-1 after the onset of clinically detectable disease ameliorates AIA and decreases macrophage accumulation, cytokine expression, and p38 MAPK activation within the joint.     

Curcumin as a natural regulator of monocyte chemoattractant protein-1

Monocyte chemoattractant/chemotactic protein-1 (MCP-1), a member of the CC chemokine family, is one of the key chemokines that regulate migration and tissue infiltration of monocytes/macrophages. Its role in the pathophysiology of several inflammatory diseases has been widely recognized, thus making MCP-1 a possible target for anti-inflammatory treatments. Curcumin (diferuloylmethane) is a natural polyphenol derived from the rhizomes of Curcuma Longa L. (turmeric). Anti-inflammatory action underlies numerous pharmacological effects of curcumin in the control and prevention of several diseases. The purpose of this review is to evaluate the effects of curcumin on the regulation of MCP-1 as a key mediator of chemotaxis and inflammation, and the biological consequences thereof. In vitro studies have shown that curcumin can decrease MCP-1 production in various cell lines. Animal studies have also revealed that curcumin can attenuate MCP-1 expression and improve a range of inflammatory diseases through multiple molecular targets and mechanisms of action. There is limited data from human clinical trials showing the decreasing effect of curcumin on MCP-1 concentrations and improvement of the course of inflammatory diseases. Most of the in vitro and animal studies confirm that curcumin exert its MCP-1-lowering and anti-inflammatory effects by down-regulating the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathway. As yet, there is limited data from human clinical trials showing the effect of curcumin on MCP-1 levels and improvement of the course of inflammatory diseases. More evidence, especially from human studies, is needed to better assess the effects of curcumin on circulating MCP-1 in different human diseases and the role of this modulatory effect in the putative anti-inflammatory properties of curcumin.     

Circulating monocyte chemoattractant protein-1 in women with gestational diabetes

Monocyte chemoattractant protein 1 (MCP-1) has been implicated as a key factor in the recruitment and activation of peripheral blood leukocytes in atherosclerotic lesions and adipose tissue. Elevated levels of circulating MCP-1 have been found in patients with type 1 and type 2 diabetes, as well as with coronary artery disease. In this study we compared serum MCP-1 concentrations between pregnant women with normal glucose tolerance (NGT), gestational diabetes mellitus (GDM) and non-pregnant healthy women. The group studied consisted of 62 patients with GDM (mean age 30.1 +/- 5.0 years) at 29.0 +/- 3.5 week of gestation, 64 pregnant women with NGT (mean age 30.0 +/- 4.7 years) at 29.2 +/- 2.9 week of gestation and 34 non-pregnant healthy women (mean age 29.8 +/- 4.7 years). Serum MCP-1 concentration was measured using an enzyme - linked immunosorbent assay. Median MCP-1 concentrations did not differ significantly between women with GDM (median 342.3 [interquartile range 267.9-424.4] pg/ml) and NGT (338.0 [274.7-408.2] pg/ml), but were markedly lower than those found in non-pregnant women (485.2 [409.6-642.4] pg/ml, p<0.0001). After adjusting for glucose, the difference between pregnant and non-pregnant women remained highly significant (p<0.0001). In GDM patients MCP-1 levels correlated significantly with fasting glucose (r=0.2665, p=0.0363), insulin (r=0.4330, p=0.0004), HOMA-IR (r=0.4402, p=0.0003), ISQUICKI (r=-0.4402, p=0.0003), HbA1c (r=0.2724, p=0.0322), as well as with prepregnancy and current BMI (r=0.3501, p=0.0057 and r=0.3250, p=0.0106, respectively). Multiple regression analysis revealed that MCP1 concentrations were significantly predicted only by plasma glucose ( beta=0.3489, p=0.00004). Our results suggest that MCP1 levels are decreased in pregnant women, irrespective of their glucose tolerance status.     

Antiphospholipid antibodies induce monocyte chemoattractant protein-1 in endothelial cells

The presence of antiphospholipid Ab is associated with increased risk of thrombosis. The monocyte-endothelial cell interaction has been suggested to play a key role at the site of vascular injury during thrombosis. Therefore, we tested the effect of anticardiolipin Abs (aCL) on the production of monocyte chemoattractant protein-1 (MCP-1) in HUVEC. We found that monoclonal aCL as well as IgG fractions from patients with antiphospholipid syndrome (APS-IgG) could induce the production of MCP-1 in HUVEC. The ability of IgG aCL to induce MCP-1 production could be abrogated by preabsorption with cardiolipin liposomes. Simultaneous addition of either monoclonal aCL or APS-IgG with IL-1beta resulted in synergistic increase in MCP-1 production, whereas the addition of control IgG lacking aCL activity did not alter IL-1beta-induced levels of MCP-1. MCP-1 mRNA expression was also up-regulated when HUVEC were incubated with either APS-IgG or monoclonal aCL, and down-regulated by the treatment of dexamethasone. In addition, we found that serum levels of MCP-1 in 76 systemic lupus erythematosus patients correlated well with the titers of IgG aCL. Collectively, these results indicate that aCL could promote endothelial cell-monocyte cross-talk by enhancing the endothelial production of MCP-1, thereby shifting the hemostatic balance toward the prothrombotic state of APS.     

Cytokine-induced production of monocyte chemoattractant protein-1 by cultured human mesangial cells.

The infiltration of the glomerulus by monocyte-derived macrophages is an important step in the pathogenesis of glomerular injury. The factors regulating glomerular leukocyte traffic remain unknown. We postulated that the glomerular mesangial cell (MC) may participate in the development of glomerular inflammation through the production of the monocyte-specific chemotactic factor, monocyte chemoattractant protein-1 (MCP-1). Using a cell culture system, we found that human MC produced a basal level of monocyte chemotactic activity, which was significantly increased by the inflammatory cytokines IL-1 beta and TNF-alpha. This increase in bioactivity correlated with the increased expression of MCP-1 mRNA by cytokine-conditioned MC. The total chemotactic activity of MC-conditioned supernatants was reduced by more than 80% after immunoadsorption with a specific anti-MCP-1 antibody. Thus, MC could play a role in inflammatory glomerular conditions through the production of MCP-1.     

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.     

Homocysteine induces monocyte chemoattractant protein-1 expression by activating NF-kappaB in THP-1 macrophages

Homocysteinemia is an independent risk factor for cardiovascular disorders. The recruitment of monocytes is an important event in atherogenesis. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that stimulates monocyte migration into the intima of arterial walls. The objective of the present study was to investigate the effect of homocysteine on MCP-1 expression in macrophages and the underlying mechanism of such effect. Human monocytic cell (THP-1)-derived macrophages were incubated with homocysteine. By nuclease protection assay and ELISA, homocysteine (0.05-0.2 mM) was shown to significantly enhance the expression of MCP-1 mRNA (up to 2.6-fold) and protein (up to 4.8-fold) in these cells. Homocysteine-induced MCP-1 expression resulted in increased monocyte chemotaxis. The increase in MCP-1 expression was associated with activation of nuclear factor (NF)-kappaB due to increased phosphorylation of the inhibitory protein (IkappaB-alpha) as well as reduced expression of IkappaB-alpha mRNA in homocysteine-treated cells. In conclusion, our results demonstrate that homocysteine, at pathological concentration, stimulates MCP-1 expression in THP-1 macrophages via NF-kappaB activation.     

Human monocyte chemoattractant protein-1 expressed in a baculovirus system

Human monocyte chemoattractant protein-1 (hMCP-1) was produced using a baculovirus system. The hMCP-1 cDNA was inserted into the genomic DNA of Autographa californica nuclear polyhedrosis virus (AcNPV) using a transfer vector, pJVP10Z. Spodoptera frugiperda insect cells, which were infected with this recombinant virus, secreted recombinant hMCP-1 (re-hMCP-1) at the level of 10–20 μg/ml of culture medium. This product was shown to chemoattract monocytes. Three distinct bands of 11,11.5 and 12 kDa were revealed by immunoblotting analysis, and this heterogeneity was assigned to differences in carbohydrate processing. N-terminal amino-acid sequence analysis of the purified product revealed identity with hMCP-1. Thus, in this system, re-hMCP-1 was produced in large quantities and modified in a manner similar to native hMCP-1.     

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.     

Production of monocyte chemoattractant protein-1 by inflamed synovial tissue and cultured synoviocytes.

This study analyzes the expression of monocyte chemoattractant protein-1 (MCP-1) by inflamed synovial tissue and defines its regulation in cultured synoviocytes. Synoviocytes from patients with rheumatoid arthritis and osteoarthritis express the 0.7-kb MCP-1 mRNA. Stimulation of synoviocytes with IL-1, TNF-alpha, LPS, platelet-derived growth factor, and transforming growth factor-beta-1, but not with basic fibroblast growth factor causes a marked increase in MCP-1 mRNA levels. Expression of the MCP-1 gene is inducible by activators of the protein kinase A (cAMP) and C (PMA) signal transduction pathways and is differentially regulated by the steroids dexamethasone and retinoic acid. Cultured synoviocytes de novo synthesize 12-, 15-, and 15.2-kDa MCP-1 proteins, which increase after stimulation with IL-1. Synovial tissues from donors without joint disease and from patients with rheumatoid or osteoarthritis were analyzed for MCP-1 mRNA expression by in situ hybridization. In these samples MCP-1 mRNA expressing cells were predominantly found in the sublining cell layers, whereas specimens of normal synovial tissue contained only few positive cells. These results identify synoviocytes as a source of MCP-1. Its expression is controlled by peptide regulatory factors that are known to be present in arthritic joints. Detection of cells producing MCP-1 mRNA in synovial tissues from patients with arthritis shows that this gene is expressed in vivo and suggests that MCP-1 can play a role in recruiting monocytes in joint inflammation.     

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.     

HIV-1 Tat induces monocyte chemoattractant protein-1-mediated monocyte transmigration across a model of the human blood-brain barrier and up-regulates CCR5 expression on human monocytes.

AIDS dementia is characterized by neuronal loss in association with synaptic damage. A central predictor for clinical onset of these symptoms is the infiltration of monocytes and macrophages into CNS parenchyma. Chronic HIV-1 infection of monocytes also allows these cells to serve as reservoirs for persistent viral infection. Using a coculture of endothelial cells and astrocytes that models several aspects of the human blood-brain barrier, we examined the mechanism whereby the HIV-derived factor Tat may facilitate monocyte transmigration. We demonstrate that treatment of cocultures on the astrocyte side with HIV-1 Tat induced significant monocyte chemoattractant protein (MCP)-1 protein. Astrocytes, but not endothelial cells, were the source of this MCP-1 expression. Supernatants from Tat-treated cocultures induced significant monocyte transmigration, which was detected by 2.5 h after the addition of PBMC. Pretreatment of the supernatants from Tat-stimulated cocultures with an Ab to MCP-1 completely blocked monocyte transmigration. Flow cytometric analysis of Tat-stimulated PBMC demonstrated that Tat up-regulated expression of the chemokine receptor, CCR5, on monocytes in a time-dependent manner. Taken together, our data indicate that HIV-1 Tat may facilitate the recruitment of monocytes into the CNS by inducing MCP-1 expression in astrocytes. These recruited monocytes may contribute to the pathogenesis of HIV-1-associated AIDS encephalitis and dementia.