MCP-1 Protein Levels Research Articles

MCP-1-deficient mice show reduced neuroinflammatory responses and increased peripheral inflammatory responses to peripheral endotoxin insult

BackgroundAn endotoxin insult mimics a severe peripheral infection and recent evidence suggests that a single exposure can cause long-term cognitive deficits. A peripheral injection of LPS results in production of pro-inflammatory cytokines, such as IL-1β and TNF-α, in the brain and periphery and these cytokines mediate many effects of the acute phase response including activation of the HPA axis. The chemokine MCP-1 is highly expressed during endotoxemia and although much is known about the importance of MCP-1 in peripheral inflammatory responses to LPS, information about MCP-1 and CNS responses to peripheral LPS is lacking.MethodsC57Bl/6 mice were administered LPS by intraperitoneal (i.p.) injection, serum and brains were collected at several time points, and the time course of MCP-1 protein up-regulation was measured. To examine the role of MCP-1 in activation of the brain during acute systemic inflammation, we injected MCP-1 knockout (MCP-1-/-) or control C57Bl/6 (MCP-1+/+) mice with LPS i.p. and measured the levels of selected cytokines and chemokines in serum and brain extracts 6 hours later. Activated microglia were examined by CD45 immunohistochemistry, and serum corticosterone and ACTH levels were measured by enzyme immunoassay.ResultsWe report that LPS injection induces a robust increase in MCP-1 protein levels in serum and brain, with peak brain levels reached at 6 hrs after LPS administration. MCP-1-/- mice injected with LPS showed higher levels of serum IL-1β and TNF-α compared to LPS-treated MCP-1+/+ mice. In contrast, these MCP-1-/- mice showed significantly lower inductions of brain pro-inflammatory cytokines and chemokines, fewer activated microglia, and a reduction in serum corticosterone levels.ConclusionMCP-1-/- mice have decreased brain inflammation after a peripheral LPS insult, despite an exaggerated peripheral response. These data demonstrate an important role for MCP-1 in regulation of brain inflammation after peripheral endotoxemia.

Genetic Variants in MCP1 and MCP1R are Associated with Markers of Muscle Damage

PURPOSE: Understanding the genetic factors that underlie inflammation and muscle repair could have profound effects on improved therapeutics for pain relief. We hypothesized that single nucleotide polymorphisms (SNPs) in the gene MCP1, which is highly expressed in injured skeletal muscle and involved in the repair and functional recovery of the injured muscle, would be associated with markers of muscle damage. METHODS: We genotyped nine different SNPs in MCP1 and three SNPs in MCP1R in a group of 151 individuals who completed a bout of eccentric exercise (50 maximal eccentric contractions). The SNPs were examined with indicators of muscle damage [strength loss, muscle soreness, and increases in circulating levels of creatine kinase (CK) and myoglobin (Mb)] and maximum baseline isometric strength values. RESULTS: In females, two SNPs in MCP1 were associated with baseline CK levels: rs1026411 (AA: N=42; 73.7 ± 5.5 U/l vs. AG/GG: N=34; 99.7 ± 6.1 U/l, p=0.002), rs13900 (CC: N=42; 73.9 ± 5.4 U/L vs. AG/GG: N=35; 98.9 ± 6.0 U/l, p = 0.003), and a SNP in MCP1R rs1799865 (TT: N=36; 94.7 ± 6.1 U/l vs. CT/CC: N=41; 77.0 ± 5.7 U/l, p = 0.03). In addition, females with a copy of the C allele for rs3918358 in MCP1R had higher myoglobin values four days post-exercise (AA: N=33; 237.8 ± 64.7 ng/ml vs. AC/CC: N=43; 165.3 ± 17.0 ng/ml, p = 0.04) and greater maximum strength loss (AA: N=34; −12.2 ± kg vs. AC/CC: N=43; −24.3 ± kg, p = 0.04). In the MCP1R gene, males with a copy of the rare allele for two SNPs had higher values of myoglobin seven days post-exercise for rs3918358 (AA: N=33; 85.0 ± 19.3 ng/ml vs. AC/CC: N=38; 159.8 ± 18.0 ng/ml, p = 0.007) and rs1799865 (TT: N=32; 76.4 ± 19.0 ng/ml vs. TC/CC: N=40; 165.3 ± 17.0 ng/ml, p = 0.001). In addition, males showed a differences in average baseline strength for rs3918358 (AA: N=33; 119.8 ± 4.8 kg vs. AC/CC: N=41; 106.7 ± 4.2 kg, p = 0.001; p = 0.04. CONCLUSIONS: We have discovered variants in the MCP1 and MCP1R genes that affect measures of muscle damage (muscle soreness, isometric strength, myoglobin, and CK) after a bout of strenuous eccentric exercise. Baseline CK and strength were also affected in a sex-dependent manner. These data suggest that these SNPs affect MCP1 RNA or protein levels, which could modify signaling between macrophages and satellite cells, promoting or hindering repair during the recovery period.

Glucocorticoids reduce pro-inflammatory cytokines and tissue factorin vitroand improve function of transplanted human isletsin vivo

Factors that upregulate the inflammatory status of islets probably contribute to detrimental processes leading to islet loss and impaired post-transplant function. Glucocorticoids have the potential to counteract inflammation and thus improve islet quality and function. However, glucocorticoids have diabetogenic properties and are known to hamper islet function in vivo. We examined the effect of glucocorticoids on human islets in vitro and in vivo after 48 h of exposure to different concentrations of methylprednisolone. Protein and/or mRNA levels of insulin, interleukin (IL)-8, macrophage chemoattractant protein (MCP)-1, tissue factor (TF), and IL-10 were assessed by enzyme immunosorbent assay and real time quantitative reverse transcription-polymerase chain reaction. Viability was assessed with fluorescein diacetate-propidium iodide staining, adenosine triphosphate (ATP) content and caspase activity. Six-hundred islet equivalents (IEQ) were transplanted to severe combined immunodeficiency disease mice and graft function assessed by glucose measurements and intraperitoneal glucose tolerance tests. Glucocorticoids reduce mRNA and protein levels of TF, MCP-1 and IL-8, and enhance ATP content. Insulin secretion was initially inhibited; however, after 7 days in culture, it was superior to controls. Islets exposed to methylprednisolone cured diabetic mice more effectively than control islets. In conclusion, glucocorticoids have potent anti-inflammatory properties on human islets without permanent effects on insulin metabolism. Brief glucocorticoid exposure improves function of transplanted human islets in vivo.

Increased severity of renal ischemia-reperfusion injury with venous clamping compared to arterial clamping in a rat model

Arterial inflow occlusion is a well-known mechanism of renal injury during major vascular surgery. In contrast, renal injury from venous outflow obstruction is poorly understood. The goal of this study was to examine the injury pattern of renal venous outflow obstruction, compare this with the traditional model of arterial occlusion, and examine possible mechanisms. Male Fisher rats were used for the renal warm ischemia model. Twenty-five minutes of renal ischemia was induced by selectively occluding either the renal artery or vein. After 24 h of reperfusion, whole blood and kidney tissue were collected for further analysis. Serum creatinine (SCr) concentrations taken 24 h after reperfusion were significantly greater in the venous occlusion group (V) when compared to the arterial group (A). While histology did not demonstrate significant differences in extent of necrosis between both groups, a stronger inflammatory response resulted from venous occlusion. Specifically, significantly greater MCP-1 mRNA and significantly greater MCP-1, TNF-alpha, and HO-1 protein levels were found in the venous group, while no differences in MIP-2, ICAM-1, and VCAM-1 mRNA expression existed between A and V. Further analysis demonstrated presence of increased cleaved caspase-3 protein in the artery group than in the venous group. Venous renal outflow obstruction results in more severe functional renal injury when compared to arterial inflow occlusion. Macrophage activation and neutrophilic infiltration appear to be exaggerated during venous occlusion.

Increased MCP-1 and microglia in various regions of the human alcoholic brain

Cytokines and microglia have been implicated in anxiety, depression, neurodegeneration as well as the regulation of alcohol drinking and other consumatory behaviors, all of which are associated with alcoholism. Studies using animal models of alcoholism suggest that microglia and proinflammatory cytokines contribute to alcoholic pathologies [Crews, F.T., Bechara, R., Brown, L.A., Guidot, D.M., Mandrekar, P., Oak, S., Qin, L., Szabo, G., Wheeler, M., Zou, J., (2006) Cytokines and alcohol. Alcohol., Clin. Exp. Res. 30:720−730]. In the current study, human postmortem brains from moderate drinking controls and alcoholics obtained from the New South Wales Tissue Resource Center were used to study the cytokine, monocyte chemoattractant protein 1 (MCP-1,CCL2) and microglia markers in various brain regions. Since MCP-1 is a key proinflammatory cytokine induced by chronic alcohol treatment of mice, and known to regulate drinking behavior in mice, MCP-1 protein levels from human brain homogenate were measured using ELISA, and indicated increased MCP-1 concentration in ventral tegmental area (VTA), substantia nigra (SN), hippocampus and amygdala of alcoholic brains as compared with controls. Immunohistochemistry was further performed to visualize human microglia using ionized calcium binding adaptor protein-1 (Iba-1), and Glucose transporter-5 (GluT 5). Alcoholics were found to have brain region-specific increases in microglial markers. In cingulate cortex, both Iba-1 and GluT 5 were increased in alcoholic brains relative to controls. Alternatively, no detectable change was found in amygdala nuclei. In VTA and midbrain, only GluT 5, but not Iba-1 was increased in alcoholic brains. These data suggest that the enhanced expression of MCP-1 and microglia activities in alcoholic brains could contribute to ethanol-induced pathogenesis.

Abstract 977: Mesenchymal Stem Cell Therapy Prevents Ischemia/Reperfusion Injury and Promotes Tissue Regeneration

Ischemic heart disease, especially myocardial infarction, is an important cause of heart failure. However, therapeutic potential of mesenchymal stem cells (MSC) for myocardial or pulmonary ischemia reperfusion (IR) injury is not well studied. Interleukin-10 (IL-10) is an anti-inflammatory cytokine. In this study MSC and IL-10 engineered MSC were tested for their ability to prevent IR injury and promote tissue repair. Methods: Bone marrow cells from Lewis rat were cultured in plastic tissue culture flasks for 72 hours; cells in suspension were discarded and the adherent cells (MSC) were expanded. MSC were transduced with r vIL-10-retrovirus and selected on neomycin (1mg/ml for 7 days). Experimental design: following 120 minutes of left lung ischemia, Group A, rats received vIL10 transduced MSC (∼15 x 10 6 ; i.v.); Group B, rats received empty vector engineered MSC; Group C, received MSC; and Group D, received saline. Group E received no ischemia or MSC. Left lung ischemia was performed by clamping left pulmonary artery/vein/bronchus at end inspiration. At 24h, 72 h, & 7 days following ischemia, blood was collected from left and right pulmonary veins, separately. Results : Mean blood oxygenation (PaO2/FIO2 ratio, mmHg) at day 7 was significantly (P<0.05) reduced following IR injury in Group B (161± 77) and Group D (181± 64), compared to MSC-vIL10 (404± 32; Group A) & MSC (294±103;Group C) treated animals. In Group E, PaO2/FIO2 was 440± 42mm Hg. Significant improvement in lung oxygenation with MSC and vIL-10 therapy was observed as early as 3 days. MSC produced vIL-10 (∼5ng/ml) ex vivo . HP of lungs demonstrated reduced PMN cells with MSC therapy. Inflammatory mediators IL-1a, MCP-1, MIP-1a, and IL1-b protein levels were markedly increased in the injured lung; their analysis in MSC treated groups are underway. I.V. injected Tg GFP+MSC trafficked to the lung & other tissues. VEGF & HGF mRNA in MSC was 66% & 43% of β-actin, respectively. Ex vivo expanded MSC were CD34-, CD45+ (5%), CD29+ (92%), CD80-, CD86+ (10%), CD90+ (∼90%), MHC Class I low & MHC Class II-. Conclusions: Autologous MSC therapy prevented IR injury and served as an excellent vehicle to deliver cytokine. MSC therapy to prevent IR injury and enhance tissue regeneration in cardiopulmonary diseases seems promising.

Inflammatory mediators induced by intratracheal instillation of ultrafine amorphous silica particles

In order to evaluate the pulmonary effects and inflammatory mechanisms of ultrafine amorphous silica particles (UFASs), the UFASs suspension was prepared in PBS and intratracheally administered to A/J mice at doses of 0, 2, 10 and 50 mg/kg ( n = 5 per group). Animals were sacrificed at 24 h, and 1, 4 or 14 weeks following exposures. At each time point, a bronchoalveolar lavage fluid analysis, histopathological examination, quantitative real-time PCR and immunohistochemistry of the lung tissues were assessed. The intratracheal instillation of UFASs significantly increased the lung weights and total BAL cells following exposures. The histopathological examination revealed that UFASs-induced severe inflammation, with neutrophils, at an early stage and chronic granulomatous inflammation at the later stage. The mRNA and protein levels of IL-1β, IL-6, IL-8, TNF-α, MCP-1 and MIP-2 in lung tissues were significantly increased during the early stages, but there were no changes after weeks 1 (TNF-α) or 4 (IL-1β, IL-6, IL-8, MCP-1 and MIP-2). Instillation of UFASs-induced transient, but very severe lung inflammation. Therefore, the cytokines (IL-1β, IL-6, IL-8 and TNF-α) and chemokines (MCP-1 and MIP-2) play important roles in the inflammation induced by the intratracheal instillation of UFASs.

Eicosapentaenoic Acid Is Anti-Inflammatory in Preventing Choroidal Neovascularization in Mice

To investigate the role of eicosapentaenoic acid (EPA), the major omega-3 polyunsaturated fatty acid (PUFA), in the development of choroidal neovascularization (CNV), together with underlying molecular mechanisms. Six-week-old C57BL/6 mice were fed with laboratory chow with 5% EPA or the omega-6 PUFA linoleic acid (LA) for 4 weeks. Laser photocoagulation was performed to induce CNV, and the volume of CNV tissue was evaluated by volumetric measurements. The expression and production of intercellular adhesion molecule (ICAM)-1, monocyte chemotactic protein (MCP)-1, vascular endothelial growth factor (VEGF) and interleukin (IL)-6 in the retinal pigment epithelium (RPE)-choroid in vivo, and stimulated b-End3 endothelial cells and RAW264.7 macrophages in vitro were evaluated by RT-PCR and ELISA. Fatty acid composition in the serum and the RPE-choroid was analyzed by gas chromatography and high-performance liquid chromatography, respectively. Serum levels of C-reactive protein (CRP), IL-6, VEGF, MCP-1, and soluble ICAM-1 were examined by ELISA. The CNV volume in EPA-fed animals was significantly suppressed compared with that in control mice, whereas the LA-rich diet did not affect CNV. The mRNA expression and protein levels of ICAM-1, MCP-1, VEGF, and IL-6 after CNV induction were significantly reduced in EPA-supplemented mice. In vitro, EPA application led to significant inhibition of mRNA and protein levels of ICAM-1 and MCP-1 in endothelial cells and VEGF and IL-6 in macrophages. EPA-fed mice exhibited significantly higher levels of EPA and lower levels of the omega-6 PUFA arachidonic acid in the serum and the RPE-choroid than control animals. EPA supplementation also led to significant reduction of serum levels of IL-6 and CRP after CNV induction. The present study demonstrates for the first time that an EPA-rich diet results in significant suppression of CNV and CNV-related inflammatory molecules in vivo and in vitro. These results suggest that frequent consumption of omega-3 PUFAs may prevent CNV and lower the risk of blindness due to age-related macular degeneration.

Interleukin-6 Receptor-Mediated Activation of Signal Transducer and Activator of Transcription-3 (STAT3) Promotes Choroidal Neovascularization

Interleukin (IL)-6, a potent proinflammatory cytokine, is suggested to be a risk factor for choroidal neovascularization (CNV) because of its increased levels in the serum of patients with age-related macular degeneration; however, the role of IL-6 in CNV has not been defined. The present study reveals the critical contribution of IL-6 signaling and its downstream STAT3 pathway to the murine model of laser-induced CNV. CNV induction by laser treatment stimulated IL-6 expression in the retinal pigment epithelium-choroid complex, and antibody-based blockade of IL-6 receptor or genetic ablation of IL-6 led to significant suppression of CNV. CNV generation was accompanied by STAT3 activation in choroidal endothelial cells and macrophages, and IL-6 receptor blockade resulted in selectively inhibited phosphorylation of STAT3 but not extracellular signal-regulated kinase 1/2. Consistently, pharmacological blockade of STAT3 pathway also suppressed CNV. In addition, IL-6 receptor neutralization led to significant inhibition of the in vivo and in vitro expression of inflammation-related molecules including monocyte chemotactic protein, intercellular adhesion molecule-1, and vascular endothelial growth factor, and of macrophage infiltration into CNV. These results indicate the significant involvement of IL-6 receptor-mediated activation of STAT3 inflammatory pathway in CNV generation, suggesting the possibility of IL-6 receptor blockade as a therapeutic strategy to suppress CNV associated with age-related macular degeneration.

Rosuvastatin Suppresses the Inflammatory Responses Through Inhibition of c-Jun N-terminal Kinase and Nuclear Factor-κB in Endothelial Cells

Rosuvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, has pleiotropic effects that are anti-inflammatory and antiatherothrombotic. It is important to understand the cardioprotective effects of rosuvastatin in order to optimize its additional advantages in the treatment and prevention of cardiovascular diseases. Human umbilical vein endothelial cells (HUVEC) were treated with tumor necrosis factor (TNF)-alpha (10 ng/mL) alone or with rosuvastatin (100 microM). The extent of inflammation was determined by U937 adhesion assay as well as analysis of the expression of intercellular adhesion molecule (ICAM)-1, monocyte chemoattractant protein (MCP)-1, interleukin (IL)-8, IL-6, cyclooxygenase (COX)-2, c-Jun N-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), p38, and signal transducer and activator of transcription (STAT)-3. The activation of nuclear factor kappa B (NF-kappaB) was determined by Western blot. Rosuvastatin decreased the extent of U937 adhesion to TNF-alpha-stimulated HUVEC. Rosuvastatin inhibited the expressions of ICAM-1, MCP-1, IL-8, IL-6, and COX-2 mRNA and protein levels. The activation of JNK and NF-kappaB was also blocked by rosuvastatin. The inhibitors of JNK, NF-kappaB, and STAT-3 produced a statistically significant decrease of the TNF-alpha induced U937 adhesion and IL-6 protein release. This study suggests that the anti-inflammatory activity of rosuvastatin is accompanied by the inhibition of JNK and NF-kappaB.

“Subclinical” gentamicin nephrotoxicity: a potential risk factor for exaggerated endotoxin-driven TNF-α production

This study sought to determine whether gentamicin, a mainstay in treating Gram-negative sepsis, alters endotoxin (lipopolysaccharide; LPS)-driven TNF-alpha increases. CD-1 mice received 1 day of gentamicin treatment. Either 0, 24, or 72 h later, gentamicin-treated and control mice were injected with LPS. Renal cortical and plasma TNF-alpha, as well as MCP-1, protein levels were measured 2 or 24 h post-LPS injection. Renal cortical mRNAs for TNF-alpha, MCP-1, IL-10, and inducible nitric oxide synthase (iNOS) were also determined. Finally, gentamicin's potential impact(s) on TNF-alpha/MCP-1 mRNA levels in nontraditional "target" organs (liver, spleen) was assessed. Gentamicin, when administered alone, slightly increased renal cortical TNF-alpha and MCP-1 mRNAs, but without changing plasma or renal TNF-alpha/MCP-1 protein levels. The gentamicin protocol induced no overt renal damage (assessed by blood urea nitrogen, creatinine, and histology). Nevertheless, gentamicin augmented LPS responsiveness, as manifested, in part, by a doubling of LPS-induced plasma TNF-alpha increases (vs. LPS injection alone). Plasma and renal cortical MCP-1 protein levels were also selectively enhanced. Gentamicin augmented LPS-driven renal mRNA increases (TNF-alpha, MCP-1, IL-10, iNOS). However, this was not an entirely renal-specific response, since gentamicin also enhanced basal and LPS-stimulated hepatic TNF-alpha mRNA levels. Subclinical gentamicin toxicity can potentiate LPS-driven TNF-alpha increases. Alterations in multiple proinflammatory (TNF-alpha; MCP-1; iNOS) and anti-inflammatory (IL-10) genes in the kidney, and possibly in extrarenal organs, may be involved. Thus gentamicin's activity in Gram-negative sepsis may extend beyond its traditional antimicrobial effect.

Upregulation of the chemokine monocyte chemoattractant protein-1 following unilateral nerve injury in the peripheral taste system

Macrophages are recruited to both sides of the tongue following unilateral chorda tympani (CT) nerve injury. The mechanisms responsible for recruiting these macrophages to the peripheral taste system are unknown. Neural degeneration in other systems leads to the upregulation of small molecules that function as chemoattractant cytokines, or chemokines. The chemokines monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-1α are important regulators of macrophage recruitment to sites of infection and injury. We hypothesized that CT nerve sectioning leads to a bilateral upregulation of MCP-1 and MIP-1α. We examined lingual protein levels of MCP-1 and MIP-1α by enzyme-linked immunosorbent assays (ELISA)s at several time points after unilateral CT section in rats. MCP-1 was significantly upregulated on the intact side of the tongue at 12 h after sectioning, and on the injured side at 24–48 h post-injury. However, MIP-1α expression did not significantly change following CT nerve sectioning. These data indicate that chemokines are differentially regulated following neural injury, and that MCP-1 may contribute to the bilateral macrophage response to neural injury. Furthermore, the increase in MCP-1 occurs even in uninjured, distant sites, and may be upstream from the deficits in neural responses from the contralateral CT after sectioning.

Triptolide inhibits CC chemokines expressed in rat adjuvant-induced arthritis

Triptolide, a diterpenoid triepoxide from Tripterygium wilfordii Hook F (TWHF), has been proven to have potent immunosuppressive and anti-inflammatory activities. It has been clinically used to treat patients with rheumatoid arthritis (RA), in which chemokines play an important role in immune and inflammatory responses. To investigate the effect of triptolide on MCP-1, MIP-1α and RANTES, we used complete Freund's adjuvant to induce adjuvant-induced arthritis (AA) in rats. AA in rat is a useful experimental model of human RA. Our data show that the thickness of arthritic ankle decreases with administration of triptolide. Both mRNA and protein levels of MCP-1, MIP-1α and RANTES in synovial tissue of rats with AA are significantly higher than those in normal rats. mRNA levels of MIP-1α and RANTES increase in peripheral blood mononuclear cells of rats with AA in comparison with those in normal rats, whereas no MCP-1 mRNA can be detected. Triptolide can significantly inhibit rat AA induced over-expression of MCP-1, MIP-1α and RANTES at both mRNA and protein levels in a dose-dependent manner. These results may contribute to the therapeutic effects of triptolide in rheumatoid arthritis.

Calcitonin gene-related peptide inhibits interleukin-1β-induced endogenous monocyte chemoattractant protein-1 secretion in type II alveolar epithelial cells

As important multifunctional cells in the lung, alveolar epithelial type II (AEII) cells secrete numerous chemokines on various stimuli. Our previous data showed that AEII cells also express the neuropeptide calcitonin gene-related peptide (CGRP) and the proinflammatory factor interleukin (IL)-1beta induces CGRP secretion in the A549 human AEII cell line. In the present study, the CGRP-1 receptor antagonist human (h)CGRP(8-37) (0.1-1 nM) greatly amplified the production of IL-1beta-induced monocyte chemoattractant protein (MCP)-1. The inhibition of CGRP expression by small interfering RNA significantly increased MCP-1 secretion on IL-1beta stimulation. However, exogenous hCGRP (10-100 nM) suppressed IL-1beta-evoked MCP-1 secretion in MCP-1 promoter activity, and CGRP gene stably transfected cell clones significantly inhibited both the mRNA and protein levels of MCP-1 induced by IL-1beta. These data imply that AEII-derived CGRP suppressed IL-1beta-induced MCP-1 secretion in an autocrine/paracrine mode. Subsequent investigation revealed that CGRP inhibited IL-1beta-evoked NF-kappaB activity by suppressing IkappaBalpha phosphorylation and degradation. Moreover, CGRP attenuated IL-1beta-induced reactive oxygen species (ROS) formation, the early event in proinflammatory factor signaling. We previously showed that the CGRP inhibitory effect was mediated by elevated intracellular cAMP and show here that analogs of cAMP, 8-bromoadenosine 3',5'-cyclic monophosphothioate and the Sp isomer of adenosine 3',5'-cyclic monophosphothioate, mimicked the CGRP suppressive effect on IL-1beta-induced ROS formation, NF-kappaB activation, and MCP-1 secretion. Thus increased endogenous CGRP secretion in lung inflammatory disease might eliminate the excessive response by elevating the cAMP level through inhibiting the ROS-NF-kappaB-MCP-1 pathway.

Lipopolysaccharide-Induced Cytokine and Chemokine Expression in Human Carotid Lesions

The release of cytokines and chemokines from activated immune-competent cells plays a crucial role in determining the pathology of the atherogenic progress. We investigated the effect of bacterial lipopolysaccharide (LPS) on cytokine/chemokine expression in carotid lesions and normal renal arteries. The lesions or renal arteries were incubated for 6 h at 37°C in serum-free media treated with or without LPS. After LPS treatment, increased protein levels of IL-1β, IL-6, IL-8, IL-10, TNF-α and MCP-1 were observed in the culture medium from the lesions measured with cytometric bead array. We were able to detect the induction of IL-1β, IL-6, IL-8, IL-10, TNF-α and MCP-1 mRNA in the lesions after stimulation with LPS using real-time PCR. In renal arteries, LPS also induces mRNA expression of all chemokines and cytokines investigated with the exception of IL-6. However, LPS induces significantly higher levels of TNF-α, IL-1β and IL-10 mRNA in lesions compared to renal arteries. The results suggest that infectious agents are capable of enhancing the production of cytokines/chemokines in an already ongoing inflammatory process such as in atherosclerosis, and that low levels of circulating LPS may affect the levels of pro-inflammatory cytokines much more in atherosclerotic vessels than in normal vessels and may contribute to the development of the atherosclerotic lesion.

Amyloid-β-induced chemokine production in primary human macrophages and astrocytes

In Alzheimer's disease (AD), chemotaxis might be responsible for attracting glial cells towards the neuritic plaque. Using primary monocyte-derived macrophages and primary adult astrocytes as a model, amyloid-beta (Aβ) (1–42) was able to stimulate the production, as measured by RT-PCR, of MIP-1α and MIP-1β mRNA in macrophages and MCP-1 in astrocytes. Cocultures showed in unstimulated as well as in Aβ-stimulated cells an increase in MIP-1α, MIP-1β and MCP-1 mRNA. ELISAs of supernatant samples of stimulated macrophages and astrocytes also showed an increase in MIP-1α and MIP-1β in macrophages and MCP-1 in astrocytes. Stimulated cocultures showed an increase in MIP-1α, MIP-1β and MCP-1 protein levels in contrast to unstimulated cocultures.

Inflammatory chemokine transport and presentation in HEV: a remote control mechanism for monocyte recruitment to lymph nodes in inflamed tissues.

Interstitial fluid is constantly drained into lymph nodes (LNs) via afferent lymph vessels. This conduit enables monocyte-derived macrophages and dendritic cells to access LNs from peripheral tissues. We show that during inflammation in the skin, a second recruitment pathway is evoked that recruits large numbers of blood-borne monocytes to LNs via high endothelial venules (HEVs). Inhibition of monocyte chemoattractant protein (MCP)-1 blocked this inflammation-induced monocyte homing to LNs. MCP-1 mRNA in inflamed skin was over 100-fold upregulated and paralleled MCP-1 protein levels, whereas in draining LNs MCP-1 mRNA induction was much weaker and occurred only after a pronounced rise in MCP-1 protein. Thus, MCP-1 in draining LNs was primarily derived from inflamed skin. In MCP-1(-/-) mice, intracutaneously injected MCP-1 accumulated rapidly in the draining LNs where it enhanced monocyte recruitment. Intravital microscopy showed that skin-derived MCP-1 was transported via the lymph to the luminal surface of HEVs where it triggered integrin-dependent arrest of rolling monocytes. These findings demonstrate that inflamed peripheral tissues project their local chemokine profile to HEVs in draining LNs and thereby exert "remote control" over the composition of leukocyte populations that home to these organs from the blood.

Angiotensin III increases MCP-1 and activates NF-кB and AP-1 in cultured mesangial and mononuclear cells

Monocyte infiltration is a common feature of renal diseases. Angiotensin II (Ang II) participates in inflammatory cell infiltration in the kidney. However, the influence of other peptides of the renin-angiotensin system, such as the N-terminal Ang II degradation product Ang III, has not been addressed. In cultured renal and mononuclear cells, we investigated whether Ang III is involved in monocyte recruitment through the regulation of the chemokine, monocyte chemoattractant protein-1 (MCP-1; Northern blot, Western blot, immunofluorescence, and chemotaxis), and the activation of transcription factors, nuclear factor kappaB (NF-kappaB) and activating protein-1 (AP-1; electrophoretic mobility shift assay). In cultured rat mesangial and mononuclear cells, Ang III increased MCP-1 gene expression and protein levels. Supernatants from Ang III-treated mesangial cells showed increased chemoattractant activity for monocytes, which was partially inhibited by the addition of anti-MCP-1 antibody. Ang III elicited a rapid NF-kappaB activation (8-fold, after 30 min), showing a kinetics and intensity similar to that observed with Ang II and tumor necrosis factor-alpha. The maximal NF-kappaB activation was correlated with nuclear translocation of p50 and p65 subunits and disappearance of cytosolic IkappaB. Ang III also activated AP-1 (5-fold, after 18 h), while SP-1 was unchanged. Two NF-kappaB inhibitors abolished the Ang III-induced MCP-1 mRNA expression, suggesting that overexpression of this chemokine is mediated, at least in part, by NF-kappaB activation. Ang III activates the transcription factors NF-kappaB and AP-1 and increases the expression of related genes, such as MCP-1. Our study describes a novel and potent proinflammatory action of this Ang degradation product, expanding the present view of the renin-angiotensin system.

THE MITOGEN-ACTIVATED PROTEIN KINASE p38 IS NECESSARY FOR INTERLEUKIN-1β-INDUCED MONOCYTE CHEMOATTRACTANT PROTEIN 1 EXPRESSION BY HUMAN MESANGIAL CELLS

Mitogen-activated protein (MAP) kinases have been suggested as potential mediators for interleukin 1β (IL-1β)-induced gene activation. This study investigated the role of the MAP kinases p38 and ERK2 in IL-1β-mediated expression of the chemokine MCP-1 by human mesangial cells. Phosphorylation of p38 kinase, which is necessary for activation, increased significantly after IL-1β treatment. p38 kinase immunoprecipitated from IL-1β-treated cells phosphorylated target substrates to a greater extent than p38 kinase from controls. SB 203580, a selective p38 kinase inhibitor, was used to examine the role of p38 kinase in MCP-1 expression. SB 203580 decreased IL-1β-induced MCP-1 mRNA and protein levels, but did not affect MCP-1 mRNA stability. Because NF-κB is necessary for MCP-1 gene expression, the effect of p38 kinase inhibition on IL-1β induction of NF-κB was measured. SB 203580 (up to 25 μM) had no effect on IL-1β-induced NF-κB nuclear translocation or DNA binding activity. Our previous work showed that IL-1β also activates the MAP kinase ERK2 in human mesangial cells. PD 098059, a selective inhibitor of the ERK activating kinase MEK1, had no effect on IL-1β-induced MCP-1 mRNA or protein levels, or on IL-1β activation of NF-κB. These data indicate that p38 kinase is necessary for the induction of MCP-1 expression by IL-1β, but is not involved at the level of cytoplasmic activation of NF-κB. In contrast, ERK2 does not mediate IL-1β induced MCP-1 gene expression.