MCP-1 Gene Expression Research Articles

Elevated expression of S100B and RAGE positively correlates with pro‐inflammatory cytokine production that may contributie to rapid neurodegeneration in INCL

Infantile neuronal ceroid lipofuscinosis (INCL) is caused by mutations in the palmitoyl-protein thioesterase-1 (PPT1) gene. PPT1 cleaves thioester linkages in S-acylated (palmitoylated) proteins and its mutation causes abnormal accumulation of fatty-acylated polypeptides leading to INCL pathogenesis. We recently reported that neuronal apoptosis in INCL is at least in part caused by endoplasmic reticulum (ER) stress activating unfolded protein response (UPR) and caspase activation. It has been reported that ER stress disrupts Ca2+ homeostasis and stimulates the production of reactive oxygen species (ROS). Here, we report that the levels of Ca2+ binding protein, S100B, in human INCL as well as in the PPT1-KO mice that mimic INCL, are markedly elevated. We also demonstrate that the expression of a receptor for S-100B, RAGE (advanced glycation end products), is also significantly increased. We further demonstrate that elevated levels of S-100B and RAGE expression is positively correlated with increased level of Src-phosphorylation and IL-6 as well as MCP-1 gene expression in cultured neurospheres from PPT1-KO mice. We propose that: PPT1-deficiency-mediated ER stress increases ROS production and disrupts calcium homeostasis, which in turn stimulates S100B over-expression and S-100B interaction with RAGE activates signaling pathway(s) stimulating pro-inflammatory mediator production and apoptosis contributing to rapid neurodegeneration in INCL.

Vanin‐1 controls granuloma formation and macrophage polarization in Coxiella burnetii infection

Q fever is caused by Coxiella burnetii, a bacterium that survives in MPhi. Vanin-1 is a membrane-anchored pantetheinase that controls tissue inflammation. Consequently, Vanin-1-deficient mice represent a unique mouse model in which stress-induced inflammation is limited by the reaction of resident tissue cells. To investigate the contribution of host tissues in the control of a bacterial infection, we infected Vanin-1-deficient mice with C. burnetii. Mortality and morbidity of mice were not affected. The lack of Vanin-1 had no effect on C. burnetii clearance but decreased the formation of granulomas in spleen and liver. Granuloma formation depends upon MPhi recruitment and activation in these tissues. Whereas the former was slightly impaired in mutant mice, the lack of Vanin-1 significantly affected the activation pattern of BM-derived MPhi stimulated by C. burnetii. While their microbicidal activity against C. burnetii was moderately impaired, they exhibited decreased inducible nitric oxide synthase (iNOS) and MCP-1 gene expression, and increased IL-10 and arginase expression. In liver from mutant mice, increased arginase expression and decreased expression of MCP-1 and iNOS were reminiscent of MPhi data. These results suggest a role of Vanin-1 in granuloma formation in response to C. burnetii by skewing MPhi activation toward an M2 program.

Apolipoprotein E3- and Nitric Oxide–Dependent Modulation of Endothelial Cell Inflammatory Responses

Although apolipoprotein E3 (apoE3) is known to be atheroprotective, its mechanisms of protection in endothelial cells remain unclear. Cultured human aortic endothelial cells were stimulated with tumor necrosis factor (TNF)-alpha in the presence of human recombinant apoE3 solubilized in dimyristoyl phosphatidylcholine liposomes. Using flow cytometry and real-time polymerase chain reaction, a significant increase of inflammatory cell adhesion proteins (vascular cell adhesion molecule-1 and E-Selectin), and MCP-1, interleukin-8, and intercellular adhesion molecule-1 gene expression was observed within 5 hours of TNF-alpha exposure, which was markedly attenuated in cells coincubated with apoE3. Treatment with apoE4 resulted in increased inflammatory gene expression relative to either TNF treatment alone or TNF + apoE3 treatment. NO synthase inhibition experiments demonstrated NO to be an active participant in the actions of both TNF and apoE. To clarify the role of NO, dose-response experiments were performed with 0.03 to 300 micromol/L DEA-NONOate. Using flow cytometry and real-time polymerase chain reaction, a modulatory role of NO in TNF-induced endothelial cell activation was observed. These data suggest a role of vascular wall apoE3 to balance the intracellular redox state in injured endothelial cells via NO-dependent pathways.

Prevention of abdominal aortic aneurysms by simultaneous inhibition of NFκB and ets using chimeric decoy oligonucleotides in a rabbit model

Abdominal aortic aneurysm (AAA) is one of the major vascular diseases caused by atherosclerosis. Because treatment for AAA mainly consists of surgery to prevent deaths from AAA rupture and there is a conspicuous absence of alternative therapeutic strategies, the development of minimally invasive treatment is needed. To develop a novel therapeutic approach, we examined the simultaneous inhibition of the transcription factors NFkappaB and ets, which regulate inflammation and matrix degradation, in a rabbit AAA model. In this study, we employed chimeric decoy oligodeoxynucleotides (ODN), containing the consensus sequences of both the NFkappaB- and ets-binding sites, to inhibit both the transcription factors simultaneously. Using a delivery sheet, we examined the inhibitory effect of chimeric decoy ODN on aortic dilatation. Ultrasound and angiographic analysis demonstrated that treatment with chimeric decoy ODN significantly prevented the progression of elastase-induced aortic dilatation. The inhibitory effect of chimeric decoy ODN on aortic dilatation was also confirmed by histological studies. Treatment with chimeric decoy ODN reduced the activities of matrix metalloproteinase (MMP)-2 and MMP-9 and markedly inhibited the proteolysis of elastin as compared to scrambled decoy ODN. Interestingly, treatment with chimeric decoy ODN also suppressed VCAM-1 and MCP-1 gene expression, leading to inhibition of macrophage infiltration in the adventitia and media. The present study in a rabbit model provides a novel strategy to treat AAA by the simultaneous inhibition of both NFkappaB and ets using chimeric decoy ODN. Further modification of chimeric decoy ODN would be useful to treat AAA as a decoy-based therapy.

Dietary cholesterol and differential monocyte chemoattractant protein-1 gene expression in aorta and liver of apo E-deficient mice

In humans, hypercholesterolemia, steatohepatitis, and risk for arteriosclerosis are associated. Apolipoprotein E-deficient mice, a widely used animal model, show both arteriosclerosis and steatohepatitis in response to high-fat and cholesterol diets. We have found a relationship between these conditions and a higher mRNA aortic and hepatic monocyte chemoattractant protein-1 ( mcp-1) gene expression. Both tissues respond in a similar way when dietary cholesterol is provided for a few weeks but differently if the conditions persist for a protracted period of time. After 8 months of treatment, the mcp-1 gene expression in the aorta continues increasing but in the liver decreases. This coincides with a significant increase in hepatic ppar- δ anti-inflammatory gene expression. Apparently, the arterial wall cannot prevent the deleterious effects of higher mcp-1 expression by increasing ppar- δ gene expression and the lesion progress. However, in the liver, the activation of anti-inflammatory genes may reduce the hepatic mcp-1 expression which significantly decreases the inflammatory response. This differential inflammatory gene expression in aorta and liver may support the idea that anti-inflammatory transcription factors are involved in the response to diet and inflammation. Therefore, the use of cholesterol-enriched diets should be carefully considered in the apolipoprotein E-deficient mice because they may trigger different stimuli and seriously hinder the interpretation of possible findings.

Activation of Nrf2/ARE pathway protects endothelial cells from oxidant injury and inhibits inflammatory gene expression

The antioxidant response element (ARE) is a transcriptional control element that mediates expression of a set of antioxidant proteins. NF-E2-related factor 2 (Nrf2) is a transcription factor that activates ARE-containing genes. In endothelial cells, the ARE-mediated genes are upregulated by atheroprotective laminar flow through a Nrf2-dependent mechanism. We tested the hypothesis that activation of ARE-regulated genes via adenovirus-mediated expression of Nrf2 may suppress redox-sensitive inflammatory gene expression. Expression of Nrf2 in human aortic endothelial cells (HAECs) resulted in a marked increase in ARE-driven transcriptional activity and protected HAECs from H2O2-mediated cytotoxicity. Nrf2 suppressed TNF-alpha-induced monocyte chemoattractant protein (MCP)-1 and VCAM-1 mRNA and protein expression in a dose-dependent manner and inhibited TNF-alpha-induced monocytic U937 cell adhesion to HAECs. Nrf2 also inhibited IL-1beta-induced MCP-1 gene expression in human mesangial cells. Expression of Nrf2 inhibited TNF-alpha-induced activation of p38 MAP kinase. Furthermore, expression of a constitutively active form of MKK6 (an upstream kinase for p38 MAP kinase) partially reversed Nrf2-mediated inhibition of VCAM-1 expression, suggesting that p38 MAP kinase, at least in part, mediates Nrf2's anti-inflammatory action. In contrast, Nrf2 did not inhibit TNF-alpha-induced NF-kappaB activation. These data identify the Nrf2/ARE pathway as an endogenous atheroprotective system for antioxidant protection and suppression of redox-sensitive inflammatory genes, suggesting that targeting the Nrf2/ARE pathway may represent a novel therapeutic approach for the treatment of inflammatory diseases such as atherosclerosis.

The host response to the probiotic Escherichia coli strain Nissle 1917: Specific up-regulation of the proinflammatory chemokine MCP-1

BackgroundThe use of live microorganisms to influence positively the course of intestinal disorders such as infectious diarrhea or chronic inflammatory conditions has recently gained increasing interest as a therapeutic alternative. In vitro and in vivo investigations have demonstrated that probiotic-host eukaryotic cell interactions evoke a large number of responses potentially responsible for the effects of probiotics. The aim of this study was to improve our understanding of the E. coli Nissle 1917-host interaction by analyzing the gene expression pattern initiated by this probiotic in human intestinal epithelial cells.MethodsGene expression profiles of Caco-2 cells treated with E. coli Nissle 1917 were analyzed with microarrays. A second human intestinal cell line and also pieces of small intestine from BALB/c mice were used to confirm regulatory data of selected genes by real-time RT-PCR and cytometric bead array (CBA) to detect secretion of corresponding proteins.ResultsWhole genome expression analysis revealed 126 genes specifically regulated after treatment of confluent Caco-2 cells with E. coli Nissle 1917. Among others, expression of genes encoding the proinflammatory molecules monocyte chemoattractant protein-1 ligand 2 (MCP-1), macrophage inflammatory protein-2 alpha (MIP-2α) and macrophage inflammatory protein-2 beta (MIP-2β) was increased up to 10 fold. Caco-2 cells cocultured with E. coli Nissle 1917 also secreted high amounts of MCP-1 protein. Elevated levels of MCP-1 and MIP-2α mRNA could be confirmed with Lovo cells. MCP-1 gene expression was also up-regulated in mouse intestinal tissue.ConclusionThus, probiotic E. coli Nissle 1917 specifically upregulates expression of proinflammatory genes and proteins in human and mouse intestinal epithelial cells.

Modulation of intra-pulmonary TGF-b expression by mycophenolate mofetil in lupus prone MRL/lpr mice

We investigated the expression profile of inflammatory cytokines in the lung of lupus-prone MRL/lpr mice and evaluated the therapeutic potential of mycophenolate mofetil (MMF) in reducing pulmonary cytokines in active lupus. Eight-week old female MRL/lpr mice (n = 20) were treated with MMF in vehicle by oral gavage. Disease control MRL/lpr mice (n = 30) or normal control MRL mice (n = 20) received vehicle alone. The mice were sacrificed after eight or 12 weeks of treatment. Gene expression and protein synthesis of IL-1beta, MCP-1 and TGF-beta1 in lung tissues were determined. We found an increase in the gene expression of IL-1beta, MCP-1 and TGF-beta1 in lung tissues of untreated MRL/lpr mice compared with MRL mice at either 16 weeks or 20 weeks of age. MMF treatment significantly prolonged the survival of MRL/lpr mice, down-regulated the gene expression of IL-1beta, MCP-1 and TGF-beta1 in lung tissues at the end of eight or 12 weeks of treatment. Protein synthesis of TGF-beta1 was decreased following eight weeks of MMF treatment. We conclude that MMF treatment can reduce the TGF-beta1 gene expression and protein synthesis in lung tissues of lupus-prone mice. Our findings provide experimental data suggesting a beneficial potential of MMF therapy in pulmonary involvement of lupus.

Tumor necrosis factor‐α increases chemokine gene expression and production in synovial fibroblasts from human temporomandibular joint

Synovitis, which is characterized by infiltration of inflammatory cells, often accompanies progression of clinical symptoms of the temporomandibular joint (TMJ). Synovial fibroblasts of the TMJ are believed to play important roles in progression of synovitis. The purpose of this study was to examine production and gene expression of chemokines by synovial fibroblasts stimulated by tumor necrosis factor-alpha (TNF-alpha). Protein levels of chemokines were measured by enzyme-linked immunosorbent assay (ELISA). Gene expression of chemokines was analyzed by real-time polymerase chain reaction (PCR). Production of interleukin (IL)-8, growth-related oncogene (GRO)-alpha, monocyte chemoattractant protein (MCP)-1, and regulated upon activation normal T-cell expressed and secreted (RANTES) protein by synovial fibroblasts was increased by TNF-alpha. In contrast, stromal cell-derived factor (SDF)-1alpha, macrophage inflammatory protein (MIP)-1alpha and -1beta were not detectable in conditioned media of synovial fibroblasts, with or without TNF-alpha treatment. Increases in gene expression of IL-8, GRO-alpha, MCP-1, and RANTES in response to TNF-alpha treatment were detected. Increased protein production and gene expression of chemokines by synovial fibroblasts in response to TNF-alpha treatment appears to play an important role in recruitment of inflammatory cells into synovium and the progression of synovitis in the TMJ.

Effects of the NF-κB Inhibitor Pyrrolidine Dithiocarbamate on Experimentally Induced Autoimmune Anterior Uveitis

To determine the effect of pyrrolidine dithiocarbamate (PDTC), a nuclear factor (NF)-kappaB inhibitor, on chemokine and chemokine receptor expression and thus elucidate the role of NF-kappaB in the pathogenesis of experimental autoimmune anterior uveitis (EAAU). Uveitis was induced in Lewis rats with the injection of melanin-associated antigen into the footpad. PDTC (200 mg/kg and 100 mg/kg) was administered intraperitoneally daily, beginning 1 day after the immunization. The clinical inflammatory activity of the anterior chamber was recorded daily and scored. Immunohistochemical staining and an electrophoretic mobility shift assay assessed the effect of PDTC on NF-kappaB activation in the iris/ciliary body tissues. Gene expression profiles of chemokine and chemokine receptors were semiquantitatively examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Aqueous chemokine levels were measured by enzyme-linked immunosorbent assay (ELISA). PDTC significantly attenuated the clinical scores and monocyte/lymphocyte infiltration in rats with EAAU. PDTC effectively inhibited NF-kappaB activation in the iris and ciliary body, and markedly inhibited the expression of chemokine genes, including monocyte chemoattractant protein (MCP)-1, regulated-on-activation normal T-cell expressed and secreted (RANTES), and interleukin (IL)-8 and chemokine receptors genes including CCR2, CCR5, and CXCR3. Activation of NF-kappaB appears to play an important role in the pathogenesis of EAAU, through transcriptional control of MCP-1, RANTES, and IL-8 gene expression. Blocking NF-kappaB reduces ocular inflammation and may be an effective strategy in the treatment of acute anterior uveitis.

Interference with MCP-1 gene expression by vector generated triple helix-forming RNA oligonucleotides

Triple helix-forming oligonucleotides (TFOs) that specifically bind to double-stranded DNA sequences can be rationally designed, while intracellular delivery of single stranded RNA TFOs has not yet been studied in detail. In this report, we demonstrate gene and sequence-specific inhibition of MCP-1 gene expression due to interference of intracellular-generated single-stranded RNA (CU-TFO) with an overlapping SP-1/AP-1 target. Binding of synthetic 19-nucleotide (19-nt) CU-TFO to the MCP-1 promoter duplex was verified by triplex blotting. Furthermore, we confirmed binding of a 1.1-kb fusion transcript containing the 19-nt pyrimidine CU sequence to a plasmid-encoded MCP-1 promoter target duplex at pH 7.0. In tumour necrosis factor-alpha-stimulated HEK cells, CU-TFOs inhibited MCP-1 protein release by 76 +/- 10.2% compared to intracellular-generated control oligonucleotides. Interleukin-8 as a control target gene was not affected by CU-TFO, confirming both highly specific and effective chemokine gene repression by transfectable TFO-shuttle vectors.

Differential expression of cytokines and chemokines during secondary neuron death following brain injury in old and young mice

Adverse effects of age on the outcome of brain injury are well documented, but the mechanism is not well understood. Enhanced expression of proinflammatory cytokines and chemokines has long been linked to neuronal and glial responses to brain injury. In the present study, we used retrograde degeneration of thalamus as a model for secondary neuron death after cortical injury. We investigated the inflammatory component of glial responses to injury by determining mRNA expression of cytokines (TNF-α, IL-6, IL-1β and IFN-γ), chemokines (MCP-1 and RANTES) and iNOS in thalamus at day 0, 1, 3 and 7 after visual cortex ablation in mice aged 24 (old) and 4 months (young). Old mice demonstrated higher basal expression of TNF-α, IL-6, IL-1β, MCP-1, RANTES and iNOS than young mice, while basal IFN-γ expression was lower in the brains of older mice. Following injury, thalami of old mice demonstrated higher gene expression of TNF-α, IL-1β, MCP-1, RANTES, and iNOS than young mice. Maximal expression of these five genes was observed 1 day following injury. IL-6 showed maximal gene expression 3 days after injury. By contrast, the increases in IFN-γ expression after injury were smaller in old than in young mice. Our data demonstrate that both the baseline levels and the responses to injury of specific inflammatory molecules increase during aging. The increased expression of these critical molecules in inflammatory cascades may be responsible for increased secondary neuron death after injury in aging brain, suggesting that strategies to reduce these age-specific differences may help in the development of specific targets for pharmacologic intervention for the aging population.

Effect of two nitrogenous diphenyl ether pesticides on mast cell activation

We examined the effect of two nitrogenous diphenyl ether pesticides, nitrofen (NIP) and chlornitrofen (CNP), on mast cell activation. RBL-2H3 (rat basophilic leukemia) cells were exposed to NIP or CNP for 30 min to investigate their effect on degranulation, and for 3 h to investigate their effect on cytokine production and gene expression. NIP and CNP increased IgE receptor-mediated β-hexosaminidase release, MCP-1 release, and TNF-α release in a dose-dependent manner. The increasing effect of CNP on their release was greater than that of NIP. In the gene expression experiment, 30 μg/ml CNP significantly upregulated Egr-1, MCP-1 and GADD45a gene expression. These results suggest that at higher concentrations (more than 30 μg/ml) the nitrogenous diphenyl ether pesticides had both a degranulation-enhancing effect and proinflammatory cytokine-production enhancing effect through the expression of some transcription factors in RBL-2H3 cells.

Modulation of natural killer cell activity by restraint stress during an influenza A/PR8 infection in mice

These experiments were designed to examine the influences of restraint stress (RST) on natural killer (NK) activity and to determine its consequences on influenza A/PR8 (A/PR8) viral replication in mice. The data showed that RST delayed the recruitment of NK1.1 + cells into the lung parenchyma during infection. Quantification of MIP-1α and MCP-1 gene expression by real-time PCR revealed that RST suppressed the chemokines responsible for NK cell recruitment into the infected tissue. Additionally, RST suppressed the expression of several macrophage-derived cytokines involved in the effector response of NK cells. IL-15, which is the main cytokine involved in NK cell development and homeostasis, and IL-12, which is important for NK cytotoxicity, were both suppressed. As the NK cell response is an important innate response to control viral replication, we hypothesized that the RST-mediated reduction in NK cell numbers and function would enable viral replication to continue unchecked. In fact, there was enhanced viral replication in the lungs of RST animals. Interestingly, expression of the anti-viral type I interferons (IFN-α and IFN-β) was elevated presumably in response to the elevated viral load in the stressed mice. Together, these data show that RST suppressed expression of the cytokine genes involved in the recruitment and activation of NK cells during an experimental influenza viral infections. The consequence of this effect was diminished NK cell function and enhanced viral replication.

Pharmacologic prevention of both restenosis and atherosclerosis progression: AGI-1067, probucol, statins, folic acid and other therapies.

In this article, the authors intend to provide an update on clinical trials of pharmacologic prevention of restenosis after percutaneous coronary interventions, placed in the perspective of the use of orally administered therapy for the prevention of atherosclerosis progression and clinical events. AGI-1067, the mono-succinic acid ester of probucol, is a phenolic antioxidant member of a novel class of agents termed v-protectants. It has strong antioxidant properties equipotent to those of probucol and antiinflammatory properties. It inhibits gene expression of VCAM-1 and MCP-1 and has been effective at preventing atherosclerosis in all tested animal models including the non-human primate. In the Canadian Antioxidant Restenosis Trial (CART) 1, AGI-1067 and probucol improved lumen dimensions at the site of percutaneous coronary intervention. AGI-1067 also improved luminal dimensions of non-intervened coronary reference segments in the Canadian Antioxidant Restenosis Trial, which suggests a direct antiatherosclerosis effect. Probucol reduced post-percutaneous coronary intervention restenosis and progression of carotid atherosclerosis in other clinical trials. Although statins reduce atherosclerotic events, they do not appear to have a significant effect on restenosis. The failure of folate therapy to protect against restenosis in the Folate After Coronary Intervention Trial (FACIT) occurred despite significant reductions in homocysteine levels. Prevention of both post-percutaneous coronary intervention restenosis and atherosclerosis progression with a pharmacologic agent such as AGI-1067 may be an attractive treatment paradigm. Two important trials that test the antioxidant/antiinflammatory hypothesis are ongoing with AGI-1067: the Canadian Atherosclerosis and Restenosis Trial 2, which assesses its value for the reduction of both atherosclerosis progression and post-percutaneous coronary interventions restenosis, and the Aggressive Reduction of Inflammation Stops Events (ARISE) trial which is evaluating its effects on cardiovascular events.

Superoxide, H2O2, and iron are required for TNF-alpha-induced MCP-1 gene expression in endothelial cells: role of Rac1 and NADPH oxidase.

Reactive oxygen species (ROS) play an important but not yet fully defined role in the expression of inflammatory genes such as monocyte chemoattractant protein (MCP)-1. We used complementary molecular and biochemical approaches to explore the roles of specific ROS and their molecular linkage to inflammatory signaling in endothelial cells. Adenovirus-mediated expression of superoxide dismutase and catalase inhibited TNF-alpha-induced MCP-1 gene expression, suggesting important roles of superoxide (O(2)(-).) and H(2)O(2) in MCP-1 gene activation. In addition, the iron chelator 1,2-dimethyl-3-hydroxypyridin-4-one and the hydroxyl radical scavengers dimethylthiourea and dimethyl sulfoxide inhibited TNF-alpha-induced MCP-1 expression, suggesting important roles of iron and hydroxyl radicals in inflammatory signal activation. In contrast, scavenging of peroxynitrite with 5,10,15,20-tetrakis-(4-sulfonatophenyl)prophyrinato iron (III) chloride had no effect on TNF-alpha-induced MCP-1 expression. Inhibition of NADPH oxidase, the major oxidase responsible for O(2)(-). generation, with diphenylene iodonium suppressed TNF-alpha-induced MCP-1 mRNA accumulation. Rac1 is an upstream signaling molecule for the activation of NADPH oxidase and O(2)(-). generation. Expression of dominant negative N17Rac1 by adenovirus suppressed TNF-alpha-induced MCP-1 mRNA levels and MCP-1 protein secretion. Expression of N17Rac1 inhibited TNF-alpha-induced MCP-1 and NF-kappaB transcriptional activity. These data suggest that ROS such as superoxide and H(2)O(2) derived from Rac1-activated NADPH oxidase mediate TNF-alpha-induced MCP-1 expression in endothelial cells.

Microglia from mice transgenic for a provirus encoding a monocyte-tropic HIV type 1 isolate produce infectious virus and display in vitro and in vivo upregulation of lipopolysaccharide-induced chemokine gene expression.

A large body of evidence has indicated that microglia are the predominant cellular location for HIV-1 in the brains of HIV-1-infected individuals and play a direct role in the development of HIV-1-associated dementia (HAD). Therefore, investigation of the mechanism by which HIV-1-infected microglia contribute to the development of HIV-associated dementia should be facilitated by the creation of a mouse model wherein microglia carry replication-competent HIV-1. To circumvent the inability of HIV-1 to infect mouse cells, we developed a mouse line that is transgenic for a full-length proviral clone of a monocyte-tropic HIV-1 isolate, HIV-1(JR-CSF) (JR-CSF mice), whose T cells and monocytes produce infectious HIV-1. We detected expression of the long terminal repeat-regulated proviral transgene in the microglia of these transgenic mice and demonstrated that it was increased by in vitro and in vivo stimulation with lipopolysaccharide. Furthermore, microglia isolated from JR-CSF mouse brains produced HIV-1 that was infectious in vitro and in vivo. We examined the effect that carriage of the HIV-1 provirus had on chemokine gene regulation in the brains of these mice and demonstrated that MCP-1 gene expression by JR-CSF mouse microglia and brains was more responsive to in vitro and in vivo stimulation with lipopolysaccharide than were microglia and brains from control mice. Thus, this study indicates that the JR-CSF mice may represent a new mouse model to study the effect of HIV-1 replication on microglia function and its contribution to HIV-1-associated neurological disease.

Antioxidants inhibit the generation of MCP-1

Objective: Endometriosis has been referred to an inflammatory disease in the peritoneal cavity and a number of inflammatory genes have been recognized to be induced by oxidative stress and attenuated by antioxidants. Our previous study has shown that co-culture of endometrial cells with mesothelial cells resulted in significant high-level productions of MCP-1 and other cytokines. Therefore, it is important to determine whether the induction of MCP-1 by endometrial cells when added to the mesothelial cells involves an oxidative stress and whether the addition of antioxidants would decrease MCP-1 gene expression. The object of this study is to determine whether antioxidants have effects on the production of MCP-1 in the inflammatory reaction induced by co-culturing of endometrial cells with mesothelial cells.

Toll-Like receptor 4 mediates inflammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cells

Bacterial lipopolysaccharide (LPS) stimulates Kupffer cells and participates in the pathogenesis of alcohol-induced liver injury. However, it is unknown whether LPS directly affects hepatic stellate cells (HSCs), the main fibrogenic cell type in the injured liver. This study characterizes LPS-induced signal transduction and proinflammatory gene expression in activated human HSCs. Culture-activated HSCs and HSCs isolated from patients with hepatitis C virus-induced cirrhosis express LPS-associated signaling molecules, including CD14, toll-like receptor (TLR) 4, and MD2. Stimulation of culture-activated HSCs with LPS results in a rapid and marked activation of NF-κB, as assessed by in vitro kinase assays for IκB kinase (IKK), IκBα steady-state levels, p65 nuclear translocation, NF-κB-dependent luciferase reporter gene assays, and electrophoretic mobility shift assays. Lipid A induces NF-κB activation in a similar manner. Both LPS- and lipid A-induced NF-κB activation is blocked by preincubation with either anti-TLR4 blocking antibody (HTA125) or Polymyxin B. Lipid A induces NF-κB activation in HSCs from TLR4-sufficient (C3H/OuJ) mice but not from TLR4-deficient (C3H/HeJ) mice. LPS also activates c-Jun N-terminal kinase (JNK), as assessed by in vitro kinase assays. LPS up-regulates IL-8 and MCP-1 gene expression and secretion. LPS-induced IL-8 secretion is completely inhibited by the IκB super repressor (Ad5IκB) and partially inhibited by a specific JNK inhibitor, SP600125. LPS also up-regulates cell surface expression of ICAM-1 and VCAM-1. In conclusion, human activated HSCs utilize components of TLR4 signal transduction cascade to stimulate NF-κB and JNK and up-regulate chemokines and adhesion molecules. Thus, HSCs are a potential mediator of LPS-induced liver injury. (H epatology 2003;37:1043-1055.)

PDGF BB and bFGF stimulate DNA synthesis and upregulate CSF-1 and MCP-1 gene expression in dental follicle cells

CSF-1 and MCP-1, released by dental follicle cells, stimulate the influx of monocytes into the follicle sac and enhance the formation of osteoclasts that, in turn, resorb alveolar bone for the eruption pathway. PDGF and bFGF, released by cells adjacent to the follicle or by activated monocytes, are prime candidates that may regulate CSF-1 and MCP-1 gene expression. The present study demonstrates that PDGF and bFGF are mitogens for dental follicle cells and stimulate CSF-1 and MCP-1 mRNA, but with different time course kinetics. Peak induction of CSF-1 mRNA was observed at 6–8 h, while maximal MCP-1 induction was observed at 2 h. These findings suggest that MCP-1 is an early chemotactic signal for monocytes and that subsequent release of CSF-1 may act synergistically with MCP-1 to enhance monocyte influx. Further understanding of the molecular mechanisms by which cytokines regulate CSF-1 and MCP-1 may lead to more effective treatment regimens for disorders associated with abnormal tooth eruption.