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.     

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.     

Human monocyte chemoattractant protein-1 (MCP-1). Full-length cDNA cloning, expression in mitogen-stimulated blood mononuclear leukocytes, and sequence similarity to mouse competence gene JE

The purpose of this work was to analyze cDNA encoding human monocyte chemoattractant protein-1 (MCP-1), previously isolated from glioma cell line culture fluid. Screening of a cDNA library from total poly(A) RNA of glioma cell line U-105MG yielded a clone that coded for the entire MCP-1. Nucleotide sequence analysis and comparison with the amino acid sequence of purified MCP-1 showed that the cDNA clone comprises a 53-nucleotide 5'-non-coding region, an open reading frame coding for a 99-residue protein of which the last 76 residues correspond exactly to pure MCP-1, and a 389-nucleotide 3'-untranslated region. The hydrophobicity of the first 23 residues is typical of a signal peptide. Southern blot analysis of human and animal genomic DNA showed that there is a single MCP-1 gene, which is conserved in several primates. MCP-1 mRNA was induced in human peripheral blood mononuclear leukocytes (PBMNLs) by PHA, LPS and IL-1, but not by IL-2, TNF, or IFN-gamma. Among proteins with similar sequences, the coding regions of MCP-1 and mouse JE show 68% identity. This suggest that MCP-1 is the human homologue of the mouse competence gene JE.     

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.     

Dexamethasone modulates interleukin-12 production by inducing monocyte chemoattractant protein-1 in human dendritic cells

Glucocorticoids have long been used as first-line immunosuppressants, although their precise mechanism of action has not been fully elucidated yet. This study evaluated the gene and protein expression of monocyte chemoattractant protein-1 (MCP-1), and its relationship with interleukin-12 and interleukin-10 synthesis, in human monocyte-derived dendritic cells exposed to dexamethasone. Dendritic cells were differentiated in the presence or in the absence of dexamethasone and then activated by IFN-gamma+soluble CD40 ligand; the gene and protein expression of target cytokines was measured by real-time PCR and ELISA, respectively. Our results showed that dexamethasone-primed mature dendritic cells expressed low levels of interleukin-12, and, at the opposite, high levels of interleukin-10 and MCP-1. Transfection experiments confirmed the ability of dexamethasone to activate MCP-1 gene promoter. Dexamethasone increased also MCP-2, but not MCP-3 synthesis, and the gene expression of CC chemokine receptor-2 by mature dendritic cells. The addition of anti-MCP-1 blocking antibody depressed MCP-1 release, and increased interleukin-12 production in dexamethasone-treated dendritic cells, thus demonstrating that interleukin-12 downregulation is largely dependent on MCP-1 overexpression. Our findings suggest that the induction of MCP expression in human dendritic cells by dexamethasone, and the amplification of cell response via the upregulation of the chemokine cognate receptor, may be critical to inhibit type 1 T-helper-biased immune response and, possibly, to favor type 2 T-helper-skewed response.     

Production and characterization of mouse monoclonal antibodies against human monocyte chemoattractant protein-1

We developed five different hybridoma cell lines that produced mAb against human monocyte chemoattractant protein-1 (MCP-1). The subclass of all five antibodies was IgG1. All five mAb formed complexes with metabolically labeled MCP-1 that could be demonstrated by immunoprecipitation. The antibodies were specific for MCP-1. They did not cross-react by immunoprecipitation with structurally related host defense cytokines present in metabolically labeled PHA- or LPS-stimulated mononuclear cell culture fluids, nor did they cross-react in a direct ELISA with neutrophil attractant/activation protein-1, with crude platelet lysate proteins, or with pure platelet proteins that have amino acids sequences similar to that of MCP-1. The mAb also reacted with rMCP-1 expressed in Escherichia coli, suggesting that they recognize protein structure rather than the glycosylated portion of human MCP-1. When the mAb were mixed with MCP-1, the monocyte chemotactic response to MCP-1 was inhibited. A sandwich ELISA was developed to detect MCP-1 in biologic fluids containing relatively high concentrations of other proteins. The sensitivity was 300 pg/ml, or 30 pg/ELISA well. An anti-MCP-1 mAb column was used in an improved method of MCP-1 purification. Approximately 240 micrograms of MCP-1 were purified from 5 liters of FCS-containing U-105MG cell culture supernatant. The yield was at least 60%. In addition to two forms of MCP-1 reported previously by us, two more forms of MCP-1 were found in a mixture of culture supernatants of PHA- and LPS-stimulated human PBMC.     

Caveolin-1 knockdown by small interfering RNA suppresses responses to the chemokine monocyte chemoattractant protein-1 by human astrocytes

Astrocytes regulate the integrity of the blood-brain barrier and influence inflammatory processes in the central nervous system. The pro-inflammatory chemokine monocyte chemoattractant protein-1 (MCP-1), which is both released by and stimulates astrocytes, is thought to play a crucial role in both these activities. Because astrocytes have been shown to possess caveolae, vesicular structures that participate in intracellular transport and signal transduction events, we reasoned that expression of the major structural protein of these organelles, caveolin-1, might feature critically in the cellular responses to MCP-1. To test this hypothesis, caveolin-1 level was "knocked down" in human astrocyte cultures by using a small interfering RNA approach. This method resulted in efficient (>90% loss) and specific knockdown of caveolin-1 expression while sparring glial fibrillary acidic protein as well as several other proteins involved in endocytosis. Astrocytes suffering caveolin-1 loss showed diminished ability to down-modulate and internalize the MCP-1 receptor (CCR2) in response to exposure to this chemokine and also demonstrated significantly reduced capacity to undergo chemotaxis and calcium flux when MCP-1-stimulated. The results highlight a potentially prominent role for caveolae and/or caveolin-1 in mediating astrocyte responses to MCP-1, a feature that might significantly dictate the progression of inflammatory events at the blood-brain barrier.     

Phenotypic change regulates monocyte chemoattractant protein-1 (MCP-1) gene expression in human retinal pigment epithelial cells

We investigated the expression profile of monocyte chemoattractant protein-1 (MCP-1) in human retinal pigment epithelial (RPE) cells under different culture conditions and evaluated the molecular mechanism responsible for MCP-1 gene expression in RPE cells. After cellular confluence, total RNA was extracted and used for RT-PCR. Medium conditioned by RPE was used for ELISA and Western blotting. The result showed that RPE cells cultured on plastic expressed MCP-1 constitutively in the absence of any stimuli. On the other hand, growing human RPE on laminin-coated flasks instead of plastic reduced the production of MCP-1. In the RPE cells cultured on plastic, IkappaB was degraded and A20 protein increased concomitantly. MCP-1 upregulation in RPE cells on plastic was attenuated by the addition of MG-132, a proteasome inhibitor. Also, the addition of pyrolidine dithiocarbonate (PDTC) and hypoxic conditions (0.5% O2) decreased MCP-1 production in these cells. These findings suggested that the expression profile of MCP-1 is regulated by phenotypic alterations of the RPE cells. And the increased MCP-1 expression in RPE cells cultured on plastic is caused via spontaneous activation of NFkappaB induced by susceptibility to oxidative stress.     

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.     

Mu-opioid induction of monocyte chemoattractant protein-1, RANTES, and IFN-gamma-inducible protein-10 expression in human peripheral blood mononuclear cells

Strong evidence for the direct modulation of the immune system by opioids is well documented. Mu-opioids have been shown to alter the release of cytokines important for both host defense and the inflammatory response. Proinflammatory chemokines monocyte chemoattractant protein-1 (MCP-1), RANTES, and IFN-gamma-inducible protein-10 (IP-10) play crucial roles in cell-mediated immune responses, proinflammatory reactions, and viral infections. In this report, we show that [D-Ala(2),N:-Me-Phe(4),Gly-ol(5)]enkephalin (DAMGO), a mu-opioid-selective agonist, augments the expression in human PBMCs of MCP-1, RANTES, and IP-10 at both the mRNA and protein levels. Because of the proposed relationship between opioid abuse and HIV-1 infection, we also examined the impact of DAMGO on chemokine expression in HIV-infected cells. Our results show that DAMGO administration induces a significant increase in RANTES and IP-10 expression, while MCP-1 protein levels remain unaffected in PBMCs infected with the HIV-1 strain. In contrast, we show a dichotomous effect of DAMGO treatment on IP-10 protein levels expressed by T- and M-tropic HIV-infected PBMCs. The differential modulation of chemokine expression in T- and M-tropic HIV-1-infected PBMCs by opioids supports a detrimental role for opioids during HIV-1 infection. Modulation of chemokine expression may enhance trafficking of potential noninfected target cells to the site of active infection, thus directly contributing to HIV-1 replication and disease progression to AIDS.     

Transforming growth factor-beta-induced gene expression of monocyte chemoattractant JE in mouse osteoblastic cells, MC3T3-E1

A recent study demonstrated that PDGF-inducible JE is an inflammatory cytokine that directs chemotactic activity of monocytes. Accumulation of monocyte/macrophage lineage cells at site of bone tissue sites is very important for formation of multinucleate osteoclasts, which mediate bone resorption. Since transforming growth factor-beta (TGF-beta) is a potent regulator in bone remodeling, we examined whether TGF-beta induced JE gene expression in mouse osteoblastic cells, MC3T3-E1. TGF-beta induced a maximum JE mRNA expression at 3 hr after initiation of the cytokine treatment. This maximal expression was observed in when TGF-beta was used at a concentration of 1 ng/ml. The chemotactic activity for human monocytes was detected in conditioned medium of TGF-beta-treated cells, and the chemotactic activity was neutralized by anti-JE serum treatment.     

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.     

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.