Monocyte Chemoattractant Protein-1 Protein Secretion Research Articles

Inhibitory Effects of Eicosapentaenoic Acid on Vascular Endothelial Growth Factor-Induced Monocyte Chemoattractant Protein-1, Interleukin-6, and Interleukin-8 in Human Vascular Endothelial Cells.

Vascular endothelial growth factor (VEGF) induces monocyte chemoattractant protein-1 (MCP-1) and plays an important role in vascular inflammation and atherosclerosis. We investigated the mechanisms of VEGF-induced MCP-1 expression and the effects of eicosapentaenoic acid (EPA) in human umbilical vein endothelial cells (HUVECs). Real-time reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that VEGF enhanced MCP-1 gene expression and protein secretion in HUVECs. Western immunoblot analysis revealed that VEGF induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and inhibitor of nuclear factor (NF)-κB (IκB). Treatment with pharmacological inhibitors of p38 MAPK (SB203580) or NF-κB (BAY11-7085) significantly suppressed VEGF-induced MCP-1 in HUVECs. EPA inhibited VEGF-induced MCP-1 mRNA, protein secretion, phosphorylation of p38 MAPK, and the translocation of phospho-p65 to the nucleus. Additionally, VEGF also stimulated gene expressions of interleukin (IL)-6 and IL-8, which were suppressed by SB203580, BAY11-7085, and EPA. The present study has demonstrated that VEGF-induced activation of MCP-1, IL-6, and IL-8 involves the p38 MAPK and NF-κB signaling pathways and that EPA inhibits VEGF-induced MCP-1, IL-6, and IL-8 via suppressing these signaling pathways. This study supports EPA as a beneficial anti-inflammatory and anti-atherogenic drug to reduce the VEGF-induced activation of proinflammatory cytokine and chemokines.

The role of Krüppel-like factor 4 in transforming growth factor-β-induced inflammatory and fibrotic responses in human proximal tubule cells.

Krüppel-like factor 4 (KLF4) is known to mitigate inflammation in several cell types. Using human proximal tubule cells, the present study aimed to investigate the role of KLF4 in regulating transforming growth factor (TGF)-β₁ induced inflammatory and fibrotic responses. Human kidney proximal tubular cells were exposed to high glucose, or TGF-β₁ and KLF4 expressions were determined. Cells were then transfected with empty vector or KLF4 and exposed to 2-ng/mL TGF-β₁ for up to 72 h. Inflammatory proteins (macrophage migration inhibitory factor and monocyte chemoattractant protein-1) and pro-fibrotic proteins (fibronectin and collagen IV) were measured after 72 h by enzyme-linked immunosorbent assay and western blot, respectively. To determine the relevance to in vivo models of chronic kidney disease, KLF4 protein expression in streptozotocin-induced diabetic mice was determined. Krüppel-like factor 4 messenger RNA (mRNA) levels were significantly reduced in high glucose-treated human kidney proximal tubular cells. High glucose increased TGF-β₁ mRNA expression, which significantly increased migration inhibitory factor and monocyte chemoattractant protein-1 protein secretion. Transforming growth factor-β₁ significantly increased fibronectin and collagen IV protein expression. The overexpression of KLF4 significantly reduced TGF-β-mediated increases in migration inhibitory factor and monocyte chemoattractant protein-1 but had no effect on TGF-β-mediated fibronectin and collagen IV mRNA and protein expression. The levels of KLF4 mRNA were significantly reduced in the diabetic kidney, and diabetic animals had a significant reduction in renal tubular expression of KLF4 proteins. This data suggest that KLF4 reduces inflammation induced by TGF-β₁, suggesting a therapeutic role for KLF4 in diabetic nephropathy.

Effects of LPS and dietary free fatty acids on MCP-1 in 3T3-L1 adipocytes and macrophages in vitro

Background:High levels of free fatty acids (FFA) have been suggested to be one of the underlying mechanisms for adipose tissue (AT) inflammation and dysfunction in obesity. Human AT produces several adipokines including monocyte chemoattractant protein-1 (MCP-1), which are involved in the pathogenesis of obesity-mediated inflammation.Objective:In this study, we investigated the effects of lipopolysaccharide (LPS) and a panel of dietary FFA on MCP-1 gene and protein expression in adipocytes and macrophages. Furthermore, we investigated whether the effect of LPS and FFA were mediated through the toll-like receptor 4 (TLR4).Methods:3T3-L1 adipocytes and THP-1 macrophages were incubated for 24 h with the following FFA: monounsaturated fatty acid (oleic acid), saturated fatty acid (palmitic acid) and trans fatty acid (elaidic acid; 500 μM) with and without LPS (2 ng ml−1), and MCP-1 and TLR4 mRNA expression and MCP-1 protein secretion was determined.Results:The results showed that LPS significantly increased MCP-1 and TLR4 expression and MCP-1 secretion in 3T3-L1 adipocytes, and that the MCP-1 expression was blocked by a TLR4 inhibitor (CLI095). The effects of the various FFA on MCP-1 mRNA expression and protein secretion in the adipocytes showed no significant changes either alone or in combination with LPS. In macrophages, palmitic acid increased MCP-1 mRNA expression by 1.8-fold (P<0.05), but oleic acid and elaidic acid had no effects.Conclusions:In conclusion, in 3T3-L1 adipocyte, the TLR4-agonist, LPS, stimulates the proinflammatory chemokine MCP-1. The different classes of FFA did not induce MCP-1 mRNA expression or protein secretion in the adipocytes, but the saturated FFA, palmitic acid, induced MCP-1 mRNA expression in macrophages, possibly because of the higher expression level of TLR4 in the macrophages than the adipocytes. Our results indicate that FFA may induce AT inflammation through proinflammatory stimulation of macrophages.

Zinc Suppressed the Airway Inflammation in Asthmatic Rats: Effects of Zinc on Generation of Eotaxin, MCP-1, IL-8, IL-4, and IFN-γ

Airway epithelium is rich in labile zinc (Zn), which may have an important protective role in the airway epithelium. The aim of this study is to investigate the effects of Zn on the airway inflammation and the generation of eotaxin, monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), interleukin-4 (IL-4), and interferon-γ (IFN-γ) in rat models of ovalbumin (OVA)-induced allergic airway inflammation. For this purpose, animal model of asthma was established by OVA challenge and zinc-deficient and zinc-supplemented diets were given. Thirty-two Sprague-Dawley rats were divided into four groups: zinc-deficient diet with OVA treatment group, zinc-supplemented diet with OVA treatment group, zinc-normal diet with OVA treatment group, and zinc-normal diet with saline treatment group. Twenty-four hours after asthma was induced, lung histomorphological changes, cells in bronchoalveolar lavage fluid (BALF), contents of eotaxin, MCP-1, and IL-8 in BALF, and the expression of IFN-γ and IL-4 mRNAs were observed. Compared with the group of zinc-normal diet with OVA challenge rats, the group of zinc-deficient rats had higher numbers of eosinophils, neutrophils, and monocytes in BALF, as well as higher contents of eotaxin and MCP-1 in BALF and lower expression of lung IFN-γ mRNA. Conversely, Zn supplementation would decrease the numbers of eosinophils, neutrophils, and monocytes in BALF; suppress eotaxin and MCP-1 protein secretion; and increase lung IFN-γ mRNA expression. No significant difference was observed in IL-8 and IL-4 among OVA-challenged rats with different zinc diets. These studies suggested that Zn may be an important anti-inflammatory mediator of airway inflammation.

Effect of irreversibly glycated LDL in human vascular smooth muscle cells: lipid loading, oxidative and inflammatory stress

The major complication of diabetes is accelerated atherosclerosis, the progression of which entails complex interactions between the modified low-density lipoproteins (LDL) and the cells of the arterial wall. Advanced glycation end product-modified-LDL (AGE-LDL) that occurs at high rate in diabetes contributes to diabetic atherosclerosis, but the underlying mechanisms are not fully understood. The aim of this study was to assess the direct effect of AGE-LDL on human vascular smooth muscle cells (hSMC) dysfunction. Cultured hSMC incubated (24 hrs) with human AGE-LDL, native LDL (nLDL) or oxidized LDL (oxLDL) were subjected to: (i) quantification of the expression of the receptors for modified LDL and AGE proteins (LRP1, CD36, RAGE) and estimation of lipid loading, (ii) determination of NADPH oxidase activity and reactive oxygen species (ROS) production and (iii) evaluation of the expression of monocyte chemoattractant protein-1 (MCP-1). The results show that exposure of hSMC to AGE-LDL (compared to nLDL) induced: (a) increased NADPH oxidase activity (30%) and ROS production (28%) by up-regulation of NOX1, NOX4, p22phox and p67phox expression, (b) accumulation of intracellular cholesteryl esters, (c) enhanced gene expression of LRP1 (160%) and CD36 (35%), and protein expression of LRP1, CD36 and RAGE, (d) increased MCP-1 gene expression (160%) and protein secretion (300%) and (e) augmented cell proliferation (30%). In conclusion, AGE-LDL activates hSMC (increasing CD36, LRP1, RAGE), inducing a pro-oxidant state (activation of NADPHox), lipid accumulation and a pro-inflammatory state (expression of MCP-1). These results may partly explain the contribution of AGE-LDL and hSMC to the accelerated atherosclerosis in diabetes.

Fucoxanthin regulates adipocytokine mRNA expression in white adipose tissue of diabetic/obese KK- A y mice

Fucoxanthin, a marine carotenoid found in edible brown seaweeds, attenuates white adipose tissue (WAT) weight gain and hyperglycemia in diabetic/obese KK- A y mice, although it does not affect these parameters in lean C57BL/6J mice. In perigonadal and mesenteric WATs of KK- A y mice fed fucoxanthin, mRNA expression levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α), which are considered to induce insulin resistance, were markedly reduced compared to control mice. In contrast to KK- A y mice, fucoxanthin did not alter MCP-1 and TNF-α mRNA expression levels in the WAT of lean C57BL/6J mice. Interleukin-6 (IL-6) and plasminogen activator inhibitor-1 mRNA expression levels in WAT were also decreased by fucoxanthin in KK- A y mice. In differentiating 3T3-F442A adipocytes, fucoxanthinol, which is a fucoxanthin metabolite found in WAT, attenuated TNF-α-induced MCP-1 and IL-6 mRNA overexpression and protein secretion into the culture medium. In addition, fucoxanthinol decreased TNF-α, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) mRNA expression in RAW264.7 macrophage-like cells stimulated by palmitic acid. These findings indicate that fucoxanthin regulates mRNA expression of inflammatory adipocytokines involved in insulin resistance, iNOS, and COX-2 in WAT and has specific effects on diabetic/obese KK- A y mice, but not on lean C57BL/6J mice.

Curcumin Attenuates Glucose-Induced Monocyte Chemoattractant Protein-1 Synthesis in Aortic Endothelial Cells by Modulating the Nuclear Factor-ĸB Pathway

Background/Aims: High glucose (HG) induces monocyte chemoattractant protein-1 (MCP-1) synthesis in endothelial cells through nuclear factor ĸB (NFĸB). We investigated whether curcumin, losartan and sodium salicylate (NaSal) attenuate HG-induced MCP-1 synthesis in rat aortic endothelial cells (RAECs) and explored the mechanism of action. Methods: RAECs were stimulated with HG (25 mmol/l) for 24 h in the presence or absence of curcumin, losartan, NaSal or NFĸB inhibitor, Bay 11-0782. The MCP-1 protein and mRNA levels were determined by enzyme-linked immunosorbent assay and real-time reverse transcriptase-polymerase chain reaction, respectively. Nuclear translocation of NFĸB subunit p65 and NFĸB DNA-binding activity was studied using confocal microscopy and electrophoretic mobility shift assay, respectively. Results: A significant increase in the synthesis of MCP-1 protein and mRNA (2-fold) was observed in HG-primed RAECs compared to control glucose (5.5 mmol/l). Curcumin (30 µmol/l) significantly decreased HG-induced MCP-1 protein (74%) and mRNA (53%) synthesis. There was no inhibition of HG-induced MCP-1 protein secretion by losartan and NaSal. In HG-stimulated RAECs, curcumin attenuated the nuclear translocation of p65 and decreased the NFĸB DNA-binding activity. Conclusion: Curcumin blocks HG-induced MCP-1 synthesis in RAECs partly via the NFĸB pathway.

Mitomycin C Upregulates IL-8 and MCP-1 Chemokine Expression via Mitogen-Activated Protein Kinases in Corneal Fibroblasts

To investigate the expression of chemokines and their signaling pathways after application of mitomycin C (MMC) to corneal fibroblasts. Primary porcine and human corneal fibroblasts from passages 3 to 6 were treated with MMC at concentrations of 0.05, 0.1, or 0.2 mg/mL for 1, 2, 5, or 10 minutes. The relative expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) were investigated with reverse transcription, and quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA). The effects of MMC on the activation of kinases were analyzed by Western blot analysis with specific antiphosphokinase antibodies. The signaling pathways by which MMC regulates the expression of IL-8 and MCP-1 were evaluated by pharmacological kinase-specific inhibitors. The expression of IL-8 and MCP-1 were upregulated after MMC treatment in a time- and concentration-dependent manner. Furthermore, the upregulated expression of IL-8 and MCP-1 increased with longer incubation time. MMC treatment enhanced the phosphorylation of p38, JNK, and ERK at different time points. The MMC-related IL-8 and MCP-1 expression was inhibited by both a p38 inhibitor (SB203580) and an ERK inhibitor (PD98059). A JNK inhibitor (SP600125) reduced the expression of MMC-induced MCP-1 but not of IL-8. MMC treatment upregulated the expression of IL-8 and MCP-1 mRNA and protein secretion by the activation of mitogen-activated protein kinases (MAPKs) in corneal fibroblasts.

Interleukin (IL)-4 and IL-13 up-regulate monocyte chemoattractant protein-1 expression in human bronchial epithelial cells: involvement of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 and Janus kinase-2 but not c-Jun NH2-terminal kinase 1/2 signalling pathways

The Th2 cytokines interleukin (IL)-4 and IL-13 and chemokine monocyte chemoattractant protein-1 (MCP-1) are significantly involved in bronchial hyperreactivity (BHR) and remodelling in allergic asthma. Although IL-4 and IL-13 can regulate a number of chemokines from bronchial epithelium, their regulatory effect on the expression of MCP-1 is as yet unproved. We aim to investigate the intracellular signalling mechanisms of IL-4 and IL-13 regulating the expression and secretion of MCP-1 from human bronchial epithelial cells. BEAS-2B cells, derived from a human bronchial epithelial cell line, were activated with or without IL-4 and/or IL-13 for different time intervals. MCP-1 gene expression and protein secretion were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Activation of signalling molecules p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and Janus kinase-2 (JAK-2) was accessed by Western blotting. IL-4 and IL-13 were found to up-regulate gene expression and significantly increase the release of MCP-1 from BEAS-2B cells. Both cytokines could activate p38 MAPK, ERK and JAK-2, but not JNK activity. Inhibition of p38 MAPK, ERK and JAK-2 activities by pretreating the cells with their corresponding inhibitors SB203580, PD98059 and AG490, respectively, significantly suppressed IL-4- and IL-13-induced MCP-1 production in BEAS-2B cells. Together, the above results illustrate that the activation of p38 MAPK, ERK and JAK-2 but not JNK is crucial for IL-4- and IL-13-induced MCP-1 release in human bronchial epithelial cells. Our findings may provide insight into the future development of more effective therapeutic agents for treating allergic asthma.

Chemokine gene activation in human bone marrow‐derived osteoblasts following exposure to particulate wear debris

Particulate wear debris induces the expression of pro-inflammatory cytokine and chemokine genes in various cell types of the periprosthetic region. We have previously reported that titanium particles stimulate the selective induction of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) chemokines in human osteoblast-like osteosarcoma cells. In this study, we characterize the human bone marrow-derived osteoblast chemokine response to titanium particles. We demonstrate that titanium particles result in enhanced IL-8 and MCP-1 protein secretion as well as differential chemokine gene activation. Osteoblast chemokine expression was regulated at the level of gene transcription, with a time-dependent induction of NF-kappaB activation. Inhibition studies with N-acetyl-L-cysteine (Nac) and MG-132 suggest that titanium particle activation of NF-kappaB activity and IL-8 chemokine expression involves oxidant signaling and IkappaBalpha-proteasomal degradation. Activation of the NF-kappaB transcription factor, as well as the IL-8 gene, are redox-regulated. We also demonstrate that while cytochalasin D, a potent inhibitor of phagocytosis, suppressed the titanium particle effect on IL-8 protein release in human bone marrow-derived osteoblasts, the inhibitor had no effect on IL-8 expression in MG-63 osteoblast-like cells. Collectively, these results provide insight into the potential mechanisms responsible for the particulate activation of osteoblast chemokine expression and suggest an important role for the osteoblast in the pathogenesis of periprosthetic osteolysis.

The Effect of Anti-inflammatory Properties of Mycophenolate Mofetil on the Development of Lung Reperfusion Injury

Lung ischemia-reperfusion injury (LIRI) is associated with an increased incidence of both primary graft failure and obliterative bronchiolitis. The immunosuppressant mycophenolate mofetil (MMF) has recently been shown to attenuate inflammatory injury in acute ischemia-reperfusion models via a mechanism that is presently unclear. These experiments studied the effects of MMF in a warm, in situ LIRI model, focusing on transcriptional regulation of pro-inflammatory mediators. Left lungs of rats were rendered ischemic for 90 minutes and reperfused for up to 4 hours. Treated animals received 10 mg/kg of intravenous MMF at 2 hours before ischemia. Left lung injury was quantitated by myeloperoxidase (MPO) content, permeability indices and bronchoalveolar lavage (BAL) inflammatory cell counts. Lungs were analyzed by electrophoretic mobility shift assay (EMSA) for transcription factor transactivation and by enzyme-linked immunoassay for BAL chemokine protein content. MMF significantly reduced lung vascular permeability indices, MPO content and alveolar leukocyte counts at 4 hours of reperfusion. There was significant attenuation of activator protein 1 (AP-1) and early growth response 1 (EGR-1) transactivation, whereas nuclear factor-kappaB (NF-kappaB) was unaffected. Reductions in bronchoalveolar lavage monocyte chemoattractant protein 1 (MCP-1) and cytokine-induced neutrophil chemoattractant (CINC) protein content were found at 4 hours of reperfusion. MMF limits lung ischemia-reperfusion-induced increases in vascular permeability and inflammatory cell sequestration in lung parenchyma and alveolar spaces. The protection is mediated at the transcriptional level via an attenuation of early EGR-1 and AP-1 transactivation, which was found to be associated with reduced late MCP-1 and CINC protein secretion. The use of MMF in concert with an agent that affects NF-kappaB activation may provide even further protection against lung reperfusion injury as multiple inflammatory pathways are inhibited.

Sphingosine kinase-1 mediates TNF-alpha-induced MCP-1 gene expression in endothelial cells: upregulation by oscillatory flow.

Atherosclerosis is a focal inflammatory disease and preferentially occurs in areas of low fluid shear stress and oscillatory flow, whereas the risk of atherosclerosis is decreased in regions of high fluid shear stress and steady laminar flow. Sphingosine kinase-1 (SphK1) catalyzes the conversion of sphingosine to sphingosine-1 phosphate (S1P), a sphingolipid metabolite that plays important roles in angiogenesis, inflammation, and cell growth. In the present study, we demonstrated that exposure of human aortic endothelial cells to oscillatory flow (shear stress, +/-5 dyn/cm(2) for 48 h) resulted in a marked increase in SphK1 mRNA levels compared with endothelial cells kept in static culture. In contrast, laminar flow (shear stress, 20 dyn/cm(2) for 48 h) decreased SphK1 mRNA levels. We further investigated the role of SphK1 in TNF-alpha-induced expression of inflammatory genes, such as monocyte chemoattractant protein-1 (MCP-1) and VCAM-1 by using small interfering RNA (siRNA) specifically for SphK1. Treatment of endothelial cells with SphK1 siRNA suppressed TNF-alpha-induced increase in MCP-1 mRNA levels, MCP-1 protein secretion, and activation of p38 MAPK. SphK1 siRNA also inhibited TNF-alpha-induced cell surface expression of VCAM-1, but not ICAM-1, protein. Exposure of endothelial cells to S1P led to an increase in MCP-1 protein secretion and MCP-1 mRNA levels and activation of NF-kappaB-mediated transcriptional activity. Treatment of endothelial cells with the p38 MAPK inhibitor SB-203580 suppressed S1P-induced MCP-1 protein secretion. These data suggest that SphK1 mediates TNF-alpha-induced MCP-1 gene expression through a p38 MAPK-dependent pathway and may participate in oscillatory flow-mediated proinflammatory signaling pathway in the vasculature.

Hypoxia selectively inhibits monocyte chemoattractant protein-1 production by macrophages.

Hypoxia, a local decrease in oxygen tension occurring in inflammatory and tumor lesions, modulates gene expression in macrophages. Because macrophages are important chemokine producers, we investigated the regulatory effects of hypoxia on macrophage-derived chemokines. We demonstrated that hypoxia inhibits the production of the macrophage and T lymphocyte chemotactic and activating factor, monocyte chemoattractant protein-1 (MCP-1). Exposure of mouse macrophages to low oxygen tension resulted in the down-regulation of constitutive MCP-1 mRNA expression and protein secretion. Hypoxia inhibitory effects were selective for MCP-1 because the chemokines macrophage inflammatory protein-1beta (MIP-1beta), RANTES, IFN-gamma-inducible protein-10, and MIP-2 were not affected, and MIP-1alpha was induced. Hypoxia also inhibited, in a time-dependent fashion, MCP-1 up-regulation by IFN-gamma and LPS. Moreover, the inhibitory action of hypoxia was exerted on human monocytic cells. MCP-1 down-regulation was associated with inhibition of gene transcription and mRNA destabilization, suggesting a dual molecular mechanism of control. Finally, we found that the triptophan catabolite picolinic acid and the iron chelator desferrioxamine, which mimic hypoxia in the induction of gene expression, differentially regulated the expression of MCP-1. This study characterizes a novel property of hypoxia as a selective inhibitor of MCP-1 production induced by different stimuli in macrophages and demonstrates that down-regulation of gene expression by hypoxia can be controlled at both transcriptional and posttranscriptional levels. Inhibition of MCP-1 may represent a negative regulatory mechanism to control macrophage-mediated leukocyte recruitment in pathological tissues.

Glucose-mediated induction of TGF-β1 and MCP-1 in mesothelial cells in vitro is osmolality and polyol pathway dependent

Glucose is converted to sorbitol and then to fructose via the polyol pathway that has been implicated in the pathogenesis of organ damage. The contribution of the polyol pathway to mesothelial cell activation has, however, not been fully determined. The effect of increasing glucose concentrations on transforming growth factor-beta 1 (TGF-beta 1) and monocyte chemoattractant protein-1 (MCP-1) secretion by human peritoneal mesothelial cells (HPMC) was examined. The importance of the polyol pathway was identified by its specific inhibition with an aldose reductase inhibitor. Incubation of HPMC with 5 to 100 mmol/L glucose resulted in an induction of aldose reductase mRNA and intracellular sorbitol accumulation accompanied by the induction of TGF-beta 1 and MCP-1 mRNA expression and protein secretion. Mannitol at the same concentrations also induced aldose reductase, TGF-beta 1 and MCP-1 mRNA and protein expression but at a lower level than glucose. Sorbinil dose-dependently reduced both intracellular sorbitol levels (79.8% reduction of 60 mmol/L D-glucose induced intracellular sorbitol with 100 micromol/L sorbinil (N = 3, P < 0.01) and glucose-induced TGF-beta 1 and MCP-1 secretion. Mannitol induced TGF-beta 1 and MCP-1 secretion was not reduced by sorbinil. The addition of 15 to 40 mmol/L sodium lactate, either alone or in the presence of D-glucose enhanced TGF-beta 1 and MCP-1 secretion, which was inhibited by sorbinil. In contrast, sodium pyruvate appeared to antagonize D-glucose-induced TGF-beta 1 and MCP-1 secretion. These data suggest that the polyol pathway and osmolality contribute to the regulation of HPMC function by glucose. Control of polyol pathway activation might reduce glucose-mediated damage to the peritoneal membrane and promote its long-term survival.

Role of high glucose-induced nuclear factor-kappaB activation in monocyte chemoattractant protein-1 expression by mesangial cells.

Although high glucose (HG) has been shown to induce nuclear factor-kappaB (NF-kappaB) activation in vascular cells, the upstream regulation and the biologic significance of NF-kappaB activation in diabetic renal injury are not clear. It was, therefore, examined if HG-induced generation of reactive oxygen species (ROS) and protein kinase C (PKC) activation are involved in NF-kappaB activation in mesangial cells (MC), and the role of NF-kappaB activation in HG-induced monocyte chemoattractant protein-1 (MCP-1) expression by MC was further investigated. Recent observations suggest that MCP-1 may play a role in the development and progression of diabetic nephropathy. HG rapidly induced NF-kappaB activation in MC as estimated by electrophoretic mobility shift assay. Supershift assay suggests that most of the binding activity arose from p50/p50 and p50/p65 dimers. Antioxidants, pyrrolidine dithiocarbamate, N-acetyl-L-cystein, and trolox effectively inhibited HG-induced NF-kappaB activation in MC. HG rapidly generated dichlorofluorescin-sensitive intracellular ROS in MC as measured by laser-scanning confocal microscopy. HG also activated PKC rapidly in MC. Inhibition of PKC effectively blocked HG-induced intracellular ROS generation and NF-kappaB activation in MC. HG increased MCP-1 mRNA expression by 1.9-fold and protein secretion by 1.6-fold that of control glucose in MC transfected with control vector but not in MC transfected with dominant negative mutant inhibitor of NF-kappaB (IkappaBalphaM). Inhibition of either PKC or ROS effectively blocked HG-induced, but not basal, MCP-1 protein secretion by MC transfected with control vector. Thus this study demonstrates that HG rapidly activates NF-kappaB in MC through PKC and ROS and suggests that HG-induced NF-kappaB activation in MC may play a role in diabetic renal injury through upregulation of MCP-1 mRNA and protein expression.

Effect of High Glucose Concentration on the Synthesis of Monocyte Chemoattractant Protein-1 in Human Peritoneal Mesothelial Cells: Involvement of Protein Kinase C

Human peritoneal mesothelial cells (HMC) contribute to the activation and control of inflammatory processes in the peritoneum by their potential to produce various inflammatory mediators. The present study was designed to assess the effect of glucose, the osmotic active compound in most commercially available peritoneal dialysis fluids, on the synthesis of the C-C chemokine monocyte chemoattractant protein-1 (MCP-1) in cultured HMC. The MCP-1 concentration in the cell supernatants was determined by enzyme-linked immunosorbent assay and the MCP-1 mRNA expression was examined using Northern blot analysis. Incubation of HMC with glucose (30–120 mM) resulted in a time- and concentration-dependent increase in MCP-1 protein secretion and mRNA expression. After 24 h the MCP-1 synthesis was increased from 2.8 ± 0.46 to 4.2 ± 0.32 ng/10⁵ cells (n = 5, p < 0.05) in HMC treated with 60 mM glucose. In contrast, osmotic control media containing either the metabolically inert monosaccharide mannitol or NaCl did not influence MCP-1 production. The stimulating effect of high glucose on MCP-1 expression in HMC was mimicked by activation of protein kinase C (PKC) with the phorbol ester PMA (20 nM). Coincubation of the cells with glucose and the specific PKC inhibitor Ro 31–8220 completely blunted glucose-mediated MCP-1 expression. In summary, our results indicate that glucose induces MCP-1 synthesis by a PKC-dependent pathway. Since osmotic control media did not increase MCP-1 release, it is suggested that the effect of glucose is mainly related to metabolism and not to hyperosmolarity. These data may in part explain elevated steady-state levels of MCP-1 found in the dialysis effluent of continuous ambulatory peritoneal dialysis patients.

ASBESTOS EXPOSURE INDUCES MCP-1 SECRETION BY PLEURAL MESOTHELIAL CELLS

We showed previously that both crocidolite and chrysotile asbestos inhalation induced a persistent macrophage in ammatory response within the pleural space of the rat. We postulated that the stimulus for pleural macrophage recruitment after asbestos exposure was the induction of monocyte chemoattractant protein-1 (MCP-1) synthesis by pleural mesothelial cells. To test this hypothesis, rat pleural mesothelial cells (RPMC) were cultured with or without chrysotile or crocidolite asbestos fibers (8mug/cm2) in the presence (50ng/mL) or absence of either tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta). MCP-1 mRNA expression was assessed by RT-PCR in RPMC cultured for 2 to 24 hours, and MCP-1 protein secretion was measured by ELISA in conditioned medium from 24-hour and 48-hour cultures. Crocidolite and chrysotile fibers induced MCP-1 mRNA expression in RPMC which was maximal after 12 hours in the absence of cytokines, but which peaked after 2 hours when RPMC were challenged with asbestos+TNF-alpha or IL-1beta. Both types of asbestos also significantly increased MCP-1 protein secretion after 24 and 48 hours (P <. 0001), an effect that was potentiated by cytokine stimulation. Rats exposed by inhalation to either chrysotile or crocidolite asbestos fibers also had greater amounts of MCP-1 protein in their pleural lavage fluid than did sham-exposed rats. These findings suggest that MCP-1 secretion by RPMC may have a role in the initiation and/or potentiation of asbestos-induced pleural injury.

Thiazolidinedione inhibits the production of monocyte chemoattractant protein-1 in cytokine-treated human vascular endothelial cells

The chemokine monocyte chemoattractant protein-1 is a potent chemoattractant for monocytes. Monocyte chemoattractant protein-1 is produced by vascular endothelial cells during inflammatory diseases such as atherosclerosis. In this study, we examined the effects of a thiazolidinedione on monocyte chemoattractant protein-1 expression in human vascular endothelial cells. In human vascular endothelial cells, interleukin-1β and tumor necrosis factor-α induced endogenous monocyte chemoattractant protein-1 protein secretion, mRNA expression and promoter activity. The thiazolidinedione inhibited these effects. In summary, our results indicated that the suppression of the expression of monocyte chemoattractant protein-1 can be accomplished by thiazolidinedione treatment, raising the possibility that thiazolidinedione may be of therapeutic value in the treatment of diseases such as atherosclerosis.

Endogenous monocyte chemoattractant protein-1 recruits monocytes in the zymosan peritonitis model.

The role of monocyte chemoattractant protein-1 (MCP-1) in the recruitment of blood-derived monocytes in a model of zymosan peritoneal inflammation was investigated. After zymosan injection (1 mg) a rapid influx of polymorphonuclear leukocytes (PMN) and monocytes into the peritoneal cavity associated with mouse MCP-1 (JE) gene activation and protein secretion in the exudates occurred. MCP-1 production (maximal at 4 h) preceded the accumulation of monocytes (F4/80-positive cells, maximally recovered between 16 and 24 h). Treatment of mice with a single injection of anti-mouse MCP-1 antibody inhibited 16-h monocyte accumulation by approximately 40%, however, a significant decrease in the number of PMN was also measured. Finally, intraperitoneal injection of murine recombinant MCP-1 (1 microg) produced a selective accumulation of monocytes (F4/80-positive cells) into the peritoneal cavity. In conclusion, we show the novel existence of a strict relationship between MCP-1 production and leukocyte accumulation in this model of acute inflammation.

Nitric Oxide Modulates the Expression of Monocyte Chemoattractant Protein 1 in Cultured Human Endothelial Cells

The recruitment of monocytes into the arterial wall is one of the earliest events in the pathogenesis of atherosclerosis. Since monocyte chemoattractant protein 1 (MCP-1) plays a key role in the subendothelial recruitment of monocytes, we tested whether nitric oxide (NO) modulates the expression of MCP-1 in cultured human endothelial cells. Inhibition of basal NO production by NG-nitro-L-arginine (L-NAG) upregulates endothelial MCP-1 mRNA expression (250 +/- 20%) and protein secretion. Exogenous addition of NO dose-dependently decreased MCP-1 mRNA expression and secretion. Changes in MCP-1 mRNA expression and protein secretion were paralleled by corresponding changes in chemotactic activity of cell-conditioned media for monocytes. An MCP-1 antibody reduced monocyte chemotactic activity by 85% and completely abolished the increased monocyte chemotactic activity induced by the inhibition of NO production. Elevation of endothelial cGMP levels had no significant effect on MCP-1 mRNA expression. Inhibition of basal endothelial NO production by L-NAG increased binding activity of a nuclear factor kappa B (NF-kappa B)-like transcriptional regulatory factor, whereas exogenous addition of NO decreased NF-kappa B-like binding activity during stimulation with tumor necrosis factor-alpha. Thus, NO modulates MCP-1 expression and monocyte chemotactic activity secreted by human umbilical vein endothelial cells (HUVECs) in culture. The activation of NF-kappa B-like transcriptional regulatory proteins by inhibition of NO suggests a molecular link between an oxidant-sensitive transcriptional regulatory mechanism and NO synthesis in HUVECs.