15-deoxy-delta(12-14)-prostaglandin-j2 and Inflammation

Lipid-derived electrophilic molecules are endogenously generated and are causally involved in many pathophysiological effects. Prostaglandin D2, a major cyclooxygenase product in a variety of tissues and cells, readily undergoes dehydration to yield the J-series PGs such as 15-deoxy-Δ(12,14)-PGJ2 (15d-PGJ2). Because of the electrophilic α,β-unsaturated ketone moiety present in its cyclopentenone ring, 15d-PGJ2 acts as an endogenous electrophile. 15d-PGJ2 can covalently react via the Michael addition reaction with critical cellular nucleophiles, such as the free cysteine residues of proteins that play a key role in the regulation of the intracellular signaling pathways. Covalent modification of cellular proteins by 15d-PGJ2 may be one of the most important mechanisms by which 15d-PGJ2 induces many biological responses involved in the pathophysiological effects associated with inflammation. This current review is intended to provide a comprehensive summary of 15d-PGJ2 as an endogenous electrophilic mediator of biological activities.

Topics: Animals; Antioxidants; Apoptosis; Humans; Inflammation; Oxidation-Reduction; Oxidative Stress; Prostaglandin D2; Proteins; Signal Transduction

Electrophilic molecules are endogenously generated and are causally involved in many pathophysiological effects. Prostaglandin D (20 (PGD (2)), a major cyclooxygenase product in a variety of tissues, readily undergoes dehydration to yield the cyclopentenone-type PGs of the J (2)-series such as 15-deoxy-Delta (12,14)-PGJ (2) (15d-PGJ (2)). 15d-PGJ (2) is an electrophile, which can covalently react via the Michael addition reaction with nucleophiles, such as the free sulfhydryls of glutathione and cysteine residues in cellular proteins that play an important role in the control of the redox cell-signaling pathways. Covalent binding of 15d-PGJ (2) to cellular proteins may be one of the mechanisms by which 15d-PGJ (2) induces a cellular response involved in most of the pathophysiological effects associated with inflammation. In the present perspective, we provide a comprehensive summary of 15d-PGJ (2) as an electrophilic mediator of cellular responses.

Topics: Animals; Antioxidants; Apoptosis; Cell Differentiation; Cytoskeleton; Humans; Immunologic Factors; Inflammation; Neurons; Oxidation-Reduction; Prostaglandin D2

The cyclopentenone prostaglandin 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is recognized as a potent lipid mediator that is derived from PGD(2), which is produced abundantly in allergic inflammatory sites. It is now established that 15d-PGJ(2) negatively regulates cellular functions through its intracellular targets such as peroxisome proliferator-activated receptor-gamma (PPARgamma). However, recent studies revealed that 15d-PGJ(2) appears to possess not only anti-inflammatory activities but also a proinflammatory potential depending on its concentration and the activation state of the target cell. For instance, at low concentrations, 15d-PGJ(2) enhances eotaxin-induced chemotaxis, shape change, and actin reorganization in eosinophils through its ligation with PPARgamma. Moreover, 15d-PGJ(2) itself is a potent chemoattractant, and it induces calcium mobilization, and up-regulates CD11b expression through its membrane receptor--chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). Conversely, at high concentrations, 15d-PGJ(2) inhibits eosinophil survival by inducing apoptosis in a PPARgamma-independent manner. Here, we discuss the pathophysiological roles of 15d-PGJ(2) that could act as a paracrine, autocrine, and intracrine substance to regulate eosinophil functions.

Topics: Animals; Apoptosis; Autocrine Communication; Calcium Signaling; CD11b Antigen; Cell Shape; Chemotaxis; Cytoskeleton; Dose-Response Relationship, Drug; Eosinophils; Humans; Inflammation; Paracrine Communication; PPAR gamma; Prostaglandin D2; Receptors, Immunologic; Receptors, Prostaglandin

Prostaglandins are potent lipid molecules that affect key aspects of immunity. The original view of prostaglandins was that they were simply immunoinhibitory. This review focuses on recent findings concerning prostaglandin E2 (PGE2) and the PGD2 metabolite 15-deoxy-Delta(12,14)-PGJ2, and their divergent roles in immune regulation. We will highlight how these two seminal prostaglandins regulate immunity and inflammation, and play an emerging role in cancer progression. Understanding the diverse activities of these prostaglandins is crucial for the development of new therapies aimed at immune modulation.

Topics: Adjuvants, Immunologic; Animals; Dinoprostone; Humans; Inflammation; Models, Immunological; Models, Molecular; Neoplasms; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases

Prematurity is the leading cause of childhood death under the age of five. The aetiology of preterm birth is multifactorial; however, inflammation and infection are the most common causal factors, supporting a potential role for immunomodulation as a therapeutic strategy. 15-Deoxy-Delta-12,14-prostaglandin J2 (15dPGJ2) is an anti-inflammatory prostaglandin and has been shown to delay lipopolysaccharide (LPS) induced preterm labour in mice and improve pup survival. This study explores the immunomodulatory effect of 15dPGJ2 on the transcription factors NF-κB and AP-1, pro-inflammatory cytokines, and contraction associated proteins in human cultured myocytes, vaginal epithelial cell line (VECs) and primary amnion epithelial cells (AECs).. Cells were pre-incubated with 32µM of 15dPGJ2 and stimulated with 1ng/mL of IL-1β as an. 15dPGJ2 inhibited IL-1β-induced activation of NF-κB and AP-1, and expression of IL-6, IL-8, TNF-α, COX-2 and PGE2 in myocytes, with no effect on myometrial contractility or cell viability. Despite inhibiting IL-1β-induced activation of NF-κB, expression of IL-6, TNF-α, and COX-2, 15dPGJ2 led to activation of AP-1, increased production of PGE2 and increased cell death in VECs and AECs.. We conclude that 15dPGJ2 has differential effects on inflammatory modulation depending on cell type and is therefore unlikely to be a useful therapeutic agent for the prevention of preterm birth.

Topics: Amnion; Animals; Anti-Inflammatory Agents; Cyclooxygenase 2; Cytokines; Dinoprostone; Epithelial Cells; Female; Humans; Infant, Newborn; Inflammation; Interleukin-6; Interleukin-8; Lipopolysaccharides; Mice; Muscle Cells; NF-kappa B; Premature Birth; Prostaglandin D2; RNA, Messenger; Transcription Factor AP-1; Tumor Necrosis Factor-alpha

Stress granules (SGs) are cytoplasmic RNA-protein aggregates formed in response to inhibition of translation initiation. SGs contribute to the stress response and are implicated in a variety of diseases, including cancer and some forms of neurodegeneration. Neurodegenerative diseases often involve chronic phosphorylation of eukaryotic initiation factor 2α (eIF2α), with deletions of eIF2α kinases or treatment with eIF2α kinase inhibitors being protective in some animal models of disease. However, how and why the integrated stress response (ISR) is activated in different forms of neurodegeneration remains unclear. Because neuroinflammation is common to many neurodegenerative diseases, we hypothesized that inflammatory factors contribute to ISR activation in a cell-nonautonomous manner. Using fluorescence microscopy and immunoblotting, we show here that the endogenously produced product of inflammation, 15-deoxy-Δ

Topics: Cell Line, Tumor; Cytoplasmic Granules; Eukaryotic Initiation Factor-2; Humans; Inflammation; Peptide Chain Initiation, Translational; Phosphorylation; Prostaglandin D2; Stress, Physiological

In the present study, we demonstrate a mechanism through which 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) induces MKP-1 expression in rat primary astrocytes, leading to the regulation of inflammatory responses. We show that 15d-PGJ2 enhances the efficiency of MKP-1 pre-mRNA processing (constitutive splicing and 3'-end processing) and increases the stability of the mature mRNA. We further report that this occurs via the RNA-binding protein, Hu antigen R (HuR). Our experiments show that HuR knockdown abrogates the 15d-PGJ2-induced increases in the pre-mRNA processing and mature mRNA stability of MKP-1, whereas HuR overexpression further enhances the 15d-PGJ2-induced increases in these parameters. Using cysteine (Cys)-mutated HuR proteins, we show that the Cys-245 residue of HuR (but not Cys-13 or Cys-284) is critical for the direct binding of HuR with 15d-PGJ2 and the effects downstream of this interaction. Collectively, our data show that HuR is a novel target of 15d-PGJ2 and reveal HuR-mediated pre-mRNA processing and mature mRNA stabilization as important regulatory steps in the 15d-PGJ2-induced expression of MKP-1. The potential to use a small molecule such as 15d-PGJ2 to regulate the induction of MKP-1 at multiple levels of gene expression could be exploited as a novel therapeutic strategy aimed at combating a diverse range of MKP-1-associated pathologies.

Topics: Animals; Astrocytes; Dual Specificity Phosphatase 1; ELAV Proteins; Gene Expression Regulation; Inflammation; Primary Cell Culture; Prostaglandin D2; Rats; RNA Precursors; RNA Processing, Post-Transcriptional; RNA Stability; RNA, Messenger

Activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) contributes to several beneficial bioactivities of natural products, including induction of an increased cellular stress resistance and prevention or resolution of inflammation. In this study, the potential of a crude leaf extract of Chromolaena odorata, traditionally used against inflammation and skin lesions, was examined for Nrf2 activation. Guided by an Nrf2-dependent luciferase reporter gene assay, the phytoprostane chromomoric acid C-I (1) was identified as a potent Nrf2 activator from C. odorata with a CD (concentration doubling the response of vehicle-treated cells) of 5.2 μM. When tested at 1-10 μM, 1 was able to induce the endogenous Nrf2 target gene heme oxygenase 1 (HO-1) in fibroblasts. Between 2 and 5 μM, compound 1 induced HO-1 in vascular smooth muscle cells (VSMC) and inhibited their proliferation in a HO-1-dependent manner, without eliciting signs of cytotoxicity.

Topics: Cell Culture Techniques; Cell Survival; Chromolaena; Fatty Acids, Unsaturated; Furans; Heme Oxygenase-1; Inflammation; Luciferases; Molecular Structure; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NF-E2-Related Factor 2; Plant Leaves; Vietnam

A well-recognized natural ligand of PPARγ, 15-deoxy-δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) possesses immunomodulatory properties. The aim of this study was to elucidate whether 15d-PGJ(2) was able to attenuate lipopolysaccharide (LPS)-induced inflammatory responses in human retinal pigment epithelial (RPE) cells, which are involved in ocular immune responses. In addition, we examined whether the platelet activating factor (PAF) is associated with the anti-inflammatory activity of 15d-PGJ(2). ARPE19 cells treated with varying concentrations of 15d-PGJ(2) and a PAF antagonist (CV3988) were used in this study. The activity of PAF-acetylhydrolase (PAF-AH) was assayed by treatment with 15d-PGJ(2) and CV3988 in the presence of LPS. 15d-PGJ(2) and CV3988 inhibited the LPS-induced mRNA expression and protein production of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) in ARPE19 cells. These effects resulting from 15d-PGJ(2) were not abrogated by the PPARγ antagonist, indicating that the actions were PPARγ-independent. Furthermore, 15d-PGJ(2) and CV3988 enhanced the PAF-AH activity. Additionally, 15d-PGJ(2) inhibited the phosphorylation of the extracellular signal-regulated kinase (ERK) and the activation of nuclear transcription factor-κB (NF-κB). These results demonstrated that 15d-PGJ(2) reduced LPS-stimulated inflammatory responses in ARPE19 cells by enhancing the PAH-AH activity. These results suggest that 15d-PGJ(2) may have potent anti-inflammatory activity against ocular inflammation.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Anti-Inflammatory Agents; Cell Survival; Chemokine CCL2; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Humans; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-6; Lipopolysaccharides; NF-kappa B; Phosphorylation; PPAR gamma; Prostaglandin D2; Retinal Pigment Epithelium

We have previously demonstrated that the anti-inflammatory prostaglandin 15-deoxy-Δ 12,14-prostaglandin J(2) (15dPGJ(2)) delays inflammation-induced preterm labour in the mouse and improves pup survival through the inhibition of nuclear factor-κB (NF-κB) by a mechanism yet to be elucidated. 15dPGJ(2) is an agonist of the second prostaglandin D(2) receptor, chemoattractant receptor homologous to the T helper 2 cell (CRTH2). In human T helper cells CRTH2 agonists induce the production of the anti-inflammatory interleukins IL-10 and IL-4. We hypothesized that CRTH2 is involved in the protective effect of 15dPGJ(2) in inflammation-induced preterm labour in the murine model. We therefore studied the effects of a specific small molecule CRTH2 agonist on preterm labour and pup survival. An intrauterine injection of lipopolysaccharide (LPS) was administered to CD1 mice at embryonic day 16, ± CRTH2 agonist/vehicle controls. Mice were killed at 4.5 hr to assess fetal wellbeing and to harvest myometrium and pup brain for analysis of NF-κB, and T helper type 1/2 interleukins. To examine the effects of the CRTH2 agonist on LPS-induced preterm labour, mice were allowed to labour spontaneously. Direct effects of the CRTH2 agonist on uterine contractility were examined ex vivo on contracting myometrial strips. The CRTH2 agonist increased fetal survival from 20 to 100% in LPS-treated mice, and inhibited circular muscle contractility ex vivo. However, it augmented LPS-induced labour and significantly increased myometrial NF-κB, IL-1β, KC-GRO, interferon-γ and tumour necrosis factor-α. This suggests that the action of 15dPGJ(2) is not via CRTH2 and therefore small molecule CRTH2 agonists are not likely to be beneficial for the prevention of inflammation-induced preterm labour.

Topics: Animals; Anti-Inflammatory Agents; Brain; Cytokines; Disease Models, Animal; Female; Fetal Death; Humans; Immunologic Factors; Inflammation; Lipopolysaccharides; Mice; Myometrium; Obstetric Labor, Premature; Peptides; Pregnancy; Prostaglandin D2; Receptors, Immunologic; Receptors, Prostaglandin

Cyclopentenone prostaglandins play a modulatory role in inflammation, in part through their ability to covalently modify key proinflammatory proteins. Using mesangial cells as a cellular model of inflammation we have observed that 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) exerts a biphasic effect on cell activation by cytokines, with nanomolar concentrations eliciting an amplification of nitric oxide (NO) production and iNOS and COX-2 levels, and concentrations of 5 μM and higher inhibiting proinflammatory gene expression. An analog of 15d-PGJ(2) lacking the cyclopentenone structure (9,10-dihydro-15d-PGJ(2)) showed reduced ability to elicit both types of effects, suggesting that the electrophilic nature of 15d-PGJ(2) is important for its biphasic action. Interestingly, the switch from stimulatory to inhibitory actions occurred within a narrow concentration range and correlated with the ability of 15d-PGJ(2) to induce heme oxygenase 1 and γ-GCSm expression. These events are highly dependent on the triggering of the antioxidant response, which is considered as a sensor of thiol group modification. Indeed, the levels of the master regulator of the antioxidant response Nrf2 increased upon treatment with concentrations of 15d-PGJ(2) above 5 μM, an effect that could not be mimicked by 9,10-dihydro-15d-PGJ(2). Thus, an interplay of redox and electrophilic signalling mechanisms can be envisaged by which 15d-PGJ(2), as several other redox mediators, could contribute both to the onset and to the resolution of inflammation in a context or concentration-dependent manner.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antioxidants; Cells, Cultured; Dose-Response Relationship, Drug; Immunologic Factors; Inflammation; Mesangial Cells; Prostaglandin D2; Rats

The transcription factor nuclear factor kappaB (NF-kappaB) induces the expression of various inflammatory genes. In the common NF-kappaB signaling pathway, peperomin E and 2,6-didehydropeperomin B inhibited IkappaB degradation upon stimulation with TNF-alpha or interleukin-1. Consistent with these results, peperomin E and 2,6-didehydropeperomin B blocked the TNF-alpha-induced activation of IkappaB kinase, while they had no direct effect on the IkappaB kinase activity. Our present results clearly demonstrate that peperomins inhibit the NF-kappaB signaling pathway by blocking IkappaB kinase activation.

Topics: Anti-Inflammatory Agents; Benzodioxoles; Cell Line, Tumor; Chemistry, Pharmaceutical; Drug Design; Gene Expression Regulation; Humans; Inflammation; Interleukin-1; Models, Chemical; NF-kappa B; Phosphorylation; Signal Transduction; Structure-Activity Relationship; Tumor Necrosis Factor-alpha

Molecular disruption of the lipid carrier AFABP/aP2 in mice results in improved insulin sensitivity and protection from atherosclerosis. Because small molecule inhibitors may be efficacious in defining the mechanism(s) of AFABP/aP2 action, a chemical library was screened and identified 1 (HTS01037) as a pharmacologic ligand capable of displacing the fluorophore 1-anilinonaphthalene 8-sulfonic acid from the lipid binding cavity. The X-ray crystal structure of 1 bound to AFABP/aP2 revealed that the ligand binds at a structurally similar position to a long-chain fatty acid. Similar to AFABP/aP2 knockout mice, 1 inhibits lipolysis in 3T3-L1 adipocytes and reduces LPS-stimulated inflammation in cultured macrophages. 1 acts as an antagonist of the protein-protein interaction between AFABP/aP2 and hormone sensitive lipase but does not activate PPARgamma in macrophage or CV-1 cells. These results identify 1 as an inhibitor of fatty acid binding and a competitive antagonist of protein-protein interactions mediated by AFABP/aP2.

Topics: 3T3-L1 Cells; Animals; Butyric Acid; Crystallography, X-Ray; Drug Evaluation, Preclinical; Fatty Acid-Binding Proteins; Heterocyclic Compounds, 2-Ring; Inflammation; Ligands; Macrophages; Mice; Molecular Structure; Protein Binding; Small Molecule Libraries

Intrauterine infection is a common trigger for preterm birth and is also a risk factor for the subsequent development of neurodevelopmental abnormalities in the neonate. Bacterial lipopolysaccharide (LPS) binds to toll-like receptor-4 (TLR-4) to activate proinflammatory signaling pathways, which are implicated in both preterm delivery and antenatal brain injury. The transcription factor nuclear factor-kappaB (NF-kappaB) is a key player in the orchestration of the inflammatory response and has a central role in parturition. Here we show that intrauterine administration of TLR-4-specific LPS to pregnant mice results in the activation of NF-kappaB in the maternal uterus and the fetal brain, up-regulation of proinflammatory proteins cyclooxygenase-2, chemokine ligand 1, ChemoKine (C-C motif) ligand 2, and cytosolic phospholipase A(2) in myometrium, and induction of preterm delivery. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is an antiinflammatory prostaglandin that plays a role in promoting the resolution of inflammation. We report that coadministration of 15d-PGJ(2) and LPS to pregnant mice delays LPS-induced preterm delivery and confers protection from LPS-induced fetal mortality. This is associated with inhibition of myometrial NF-kappaB, cytosolic phospholipase A(2), and c-Jun N-terminal kinase activation, and of inflammatory protein synthesis. Therefore 15d-PGJ(2) has anti-inflammatory effects via inhibition of multiple aspects of inflammation-driven TRL-4 signaling pathway. Thus, 15d-PGJ(2) or compounds with similar antiinflammatory functions may have potential as therapeutic agents in the management of preterm labor with the added advantage of preventing detrimental effects to the fetus that may result from infection/inflammation.

Topics: Animals; Animals, Newborn; Animals, Outbred Strains; Anti-Inflammatory Agents; Cyclopentanes; Drug Evaluation, Preclinical; Female; Inflammation; Lipopolysaccharides; Mice; Myometrium; NF-kappa B; Obstetric Labor, Premature; Phospholipases A2; Phosphorylation; Pregnancy; Prostaglandin D2; Time Factors; Uterine Diseases; Uterus

Products of lipid peroxidation are generated in a wide range of pathologies associated with oxidative stress and inflammation. Many oxidized lipids contain reactive functional groups that can modify proteins, change their structure and function, and affect cell signaling. However, intracellular localization and protein adducts of reactive lipids have been difficult to detect, and the methods of detection rely largely on antibodies raised against specific lipid-protein adducts. As an alternative approach to monitoring oxidized lipids in cultured cells, we have tagged the lipid peroxidation substrate arachidonic acid and an electrophilic lipid, 15-deoxy-Delta(12,14)-prostaglandin-J2 (15d-PGJ2), with either biotin or the fluorophore BODIPY. Tagged arachidonic acid can be used in combination with conditions of oxidant stress or inflammation to assess the subcellular localization and protein modification by oxidized lipids generated in situ. Furthermore, we show that reactive lipid oxidation products such as 15d-PGJ2 can also be labeled and used in fluorescence and Western blotting applications. This article describes the synthesis, purification, and selected application of these tagged lipids in vitro.

Topics: Arachidonic Acid; Biochemistry; Blotting, Western; Boron Compounds; Diagnostic Imaging; Fluorescent Dyes; Inflammation; Lipids; Oxidative Stress; Phantoms, Imaging; Prostaglandin D2; Protein Binding; Protein Processing, Post-Translational; Protein Transport; Proteins

We previously demonstrated that, in the MC615 cartilage cell line, the p38/NF-kB pathway is activated both during differentiation and in response to an inflammatory stimulus. In both cases, the p38/NF-kB pathway activation leads to the expression of the lipocalin SIP24 and of COX-2. Given the fact that, in the same cells, the COX-2 expression is sustained during the inflammation resolution, at the same time that the SIP24 expression is suppressed, in the present study we tested the hypothesis that COX-2 products play a role in SIP24 repression. Taken together, our results suggest that, during the resolution of inflammation, COX-2 represses the acute phase protein SIP24 and restores physiological conditions, possibly through a pathway involving PPARgamma. Experimental evidences being the following: (1) 15-deoxy-delta 12,14-prostaglandin J(2), but not PGE(2): (i) inhibits the expression of SIP24 in the inflammatory phase and induces COX-2 synthesis; (ii) represses NF-kB activation induced by LPS; (iii) represses the synthesis of microsomal PGE Synthase-1 induced by LPS. (2) PPARgamma and PPARalpha are present in MC615 cells in both proliferating and hyperconfluent cultures. (3) PPARgamma ligand GW7845, but not PPARalpha ligand GW7647: (i) represses the expression of SIP24 induced by LPS; (ii) induces COX-2 expression. (4) p38 is involved in the PPARgamma mediated induction of COX-2. In fact 15-deoxy-delta 12,14-prostaglandin J(2) activates p38 and the cell pretreatment with the p38 specific inhibitor SB203580 represses the expression of COX-2 induced by both the 15-deoxy-delta12,14-prostaglandin J(2) and the PPARgamma ligand GW7845.

Topics: Acute-Phase Proteins; Animals; Anti-Inflammatory Agents, Non-Steroidal; Butyrates; Cartilage; Cells, Cultured; Chondrocytes; Cyclooxygenase 2; Diclofenac; Dinoprostone; Down-Regulation; Enzyme Induction; Enzyme Repression; Imidazoles; Inflammation; Intramolecular Oxidoreductases; Lipocalin-2; Lipocalins; Lipopolysaccharides; Mice; NF-kappa B; Oncogene Proteins; Oxazoles; p38 Mitogen-Activated Protein Kinases; Phenylurea Compounds; PPAR alpha; PPAR gamma; Prostaglandin D2; Prostaglandin-E Synthases; Protein Kinase Inhibitors; Pyridines; Signal Transduction; Time Factors; Tyrosine

We investigated the signaling pathway that leads to the expression of heme oxygenase-1 (HO-1) in murine macrophages in response to 15-deoxy-delta 12,14-prostaglandin J2 (15dPGJ2). 15dPGJ2 caused dose- and time-dependent activation of Rac1, followed by a transient increase in reactive oxygen species (ROS) via NADPH oxidase, which leads to downstream activation of p38 kinase. Inhibition of 15dPGJ2-dependent HO-1 expression significantly attenuated suppression by 15dPGJ2 of LPS-induced iNOS expression and subsequent production of nitric oxide (NO). Our findings strongly suggest that 15dPGJ2 exerts its anti-inflammatory activity through the Rac1-NADPH oxidase-ROS-p38 signaling to the up-regulation of HO-1 in an in vitro inflammation model.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Line; Heme Oxygenase-1; Inflammation; Macrophages; Mice; NADPH Oxidases; Neuropeptides; p38 Mitogen-Activated Protein Kinases; Prostaglandin D2; rac GTP-Binding Proteins; rac1 GTP-Binding Protein; Reactive Oxygen Species; Signal Transduction; Up-Regulation

Hypoxia and inflammation, an unavoidable milieu for renal tubular cells during the development of renal fibrosis, reportedly up-regulate production of plasminogen activator inhibitor-1 (PAI-1), a promoter of tissue fibrosis. Peroxisome proliferator-activated receptor (PPAR)-gamma agonists may modulate renal fibrosis progression via their anti-inflammatory effects in a PPAR-gamma-dependent or -independent manner. However, no information is known about the effects of PPAR-gamma agonists on PAI-1 expression in human proximal renal tubular cells (HPTECs) under hypoxia and/or inflammation.. Confluent HPTECs were exposed to normoxia (18% O(2)), hypoxia (1% O(2)) and/or TNF-alpha at 10 ng/mL for up to 48 h. The cells were incubated with two PPAR-gamma agonists, 15-deoxy-delta 12,14-prostaglandin J2 (15d-PGJ2) and pioglitazone. Precise amounts of PAI-1 mRNA and protein were measured by TaqMan quantitative PCR and immunoassay, respectively. PPAR response element (PPRE) activity induced by 15d-PGJ2 was measured by transfection with PPRE-luciferase construct.. Basal PAI-1 was significantly increased, in a dose-dependent manner, by 15d-PGJ2. It also enhanced hypoxia-, TNF-alpha- and hypoxia plus TNF-alpha-stimulated PAI-1 expression at the mRNA and protein levels. Pioglitazone had no influence on PAI-1 protein production. Although 15d-PGJ2 enhanced PPRE activity significantly in the HPTECs expressing PPAR-gamma, a specific inhibitor for PPAR-gamma, GW9662, did not diminish 15d-PGJ2-induced PAI-1 expression. In contrast, a non-selective tyrosine kinase (TK) inhibitor, genisteine or a MEK1 (MAPK kinase) inhibitor, PD98059, inhibited 15d-PGJ2-induced PAI-1 production completely.. The endogenous PPAR-gamma agonist, 15d-PGJ2, increased PAI-1 expression independently of PPAR-gamma via the activation of TK or MAP kinase in HPTECs and may act as an enhancer of PAI-1 production in the kidney under hypoxic and inflammatory conditions.

Topics: Cell Hypoxia; Cells, Cultured; Flavonoids; Genistein; Humans; Inflammation; Kidney Tubules, Proximal; MAP Kinase Kinase 1; Pioglitazone; Plasminogen Activator Inhibitor 1; PPAR gamma; Prostaglandin D2; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Thiazolidinediones; Tumor Necrosis Factor-alpha

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a nuclear receptor that regulates diverse biological functions including inflammation. The PPARgamma ligands have been reported to exert cardioprotective effects and attenuate myocardial reperfusion injury. Here, we examined the molecular mechanisms of their anti-inflammatory effects. Male Wistar rats were subjected to myocardial ischemia and reperfusion and were treated with the PPAR-gamma ligands, 15-deoxy-Delta-prostaglandin J2 (15d-PGJ2) or ciglitazone, or with vehicle only, in the absence or presence of the selective PPAR-gamma antagonist GW-9662. In vehicle-treated rats, myocardial injury was associated with elevated tissue activity of myeloperoxidase, indicating infiltration of neutrophils, and elevated plasma levels of creatine kinase and tumor necrosis factor-alpha. These events were preceded by activation of the nuclear factor-kappaB pathway. The PPAR-gamma DNA binding was also increased in the heart after reperfusion. Treatment with ciglitazone or 15d-PGJ2 reduced myocardial damage and neutrophil infiltration and blunted creatine kinase levels and cytokine production. The beneficial effects of both ligands were associated with enhancement of PPAR-gamma DNA binding and reduction of nuclear factor-kappaB activation. Treatment with 15d-PGJ2, but not ciglitazone, enhanced DNA binding of heat shock factor 1 and upregulated the expression of the cardioprotective heat shock protein 70. Treatment with 15d-PGJ2, but not ciglitazone, also induced a significant increase in nuclear phosphorylation of the prosurvival kinase Akt. The cardioprotection afforded by ciglitazone was attenuated by the PPAR-gamma antagonist GW-9662. In contrast, GW-9662 did not affect the beneficial effects afforded by 15d-PGJ2. Thus, our data suggest that treatment with these chemically unrelated PPAR-gamma ligands results in diverse anti-inflammatory mechanisms.

Topics: Anilides; Animals; Creatine Kinase; DNA-Binding Proteins; Heat Shock Transcription Factors; HSP70 Heat-Shock Proteins; Hypoglycemic Agents; Immunologic Factors; Inflammation; Ligands; Male; Myocardial Reperfusion Injury; Neutrophil Infiltration; NF-kappa B; Peroxidase; PPAR gamma; Prostaglandin D2; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Signal Transduction; Thiazolidinediones; Transcription Factors; Tumor Necrosis Factor-alpha; Up-Regulation

3,4-dihydroxyacetophenone (DHAP), an active component isolated from leaves of Tumaodongqing (Ilex Pubescens Hook. Et Arn. Var glaber Chang), is initially used to treat cardiovascular diseases. Previously, we found it had anti-inflammatory effect on macrophages by reducing the production of TNF-alpha in vitro. To further determine whether DHAP could influence inflammatory resolution, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ(2)), an arachidonic acid metabolite and also crucial pro-resolving mediator in inflammation, was chosen as the research target. It showed that 10(-5) M DHAP resulted in obvious increase of 15dPGJ(2) in LPS-activated macrophages. Further, inflammation related cytokines and cell apoptosis were also studied. We found DHAP could markedly inhibit LPS-stimulated production of TNF-alpha. However, it could not change the level of IL-10 obviously. At the same time, LPS-triggered apoptosis of macrophage was enhanced by DHAP significantly. After different kinds of cyclooxygenase (COX) inhibitors were administrated, it showed that the effects of DHAP on TNF-alpha and apoptosis were COX-2 dependent. While, inhibition of both COX-1 and COX-2 with indomethacin and administration of 15dPGJ(2) simultaneous reserved the effect of DHAP to inhibit TNF-alpha and enhance apoptosis in LPS-activated macrophages at least partly. The level of COX-2 mRNA and protein were also detected. It was showed that DHAP could increase the expression of COX-2 at both mRNA and protein levels in LPS-activated macrophages. Our results suggest that DHAP could accelerate resolution phase of acute inflammation though enhance the production of 15dPGJ(2), which was also proved to mediate the function of DHAP to inhibit TNF-alpha and enhance apoptosis in vitro. These results are potentially valuable for future use of DHAP.

Topics: Acetophenones; Animals; Apoptosis; Cell Line; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cytokines; Gene Expression Regulation, Enzymologic; Inflammation; Macrophages; Mice; Prostaglandin D2; RNA, Messenger

Hematopoietic prostaglandin D(2) synthase (hPGD(2)S) metabolizes cyclooxygenase (COX)-derived PGH(2) to PGD(2) and 15-deoxyDelta(12-14) PGJ(2) (15d-PGJ(2)). Unlike COX, the role of hPGD(2)S in host defense is ambiguous. PGD(2) can be either pro- or antiinflammatory depending on disease etiology, whereas the existence of 15d-PGJ(2) and its relevance to pathophysiology remain controversial. Herein, studies on hPGD(2)S KO mice reveal that 15d-PGJ(2) is synthesized in a self-resolving peritonitis, detected by using liquid chromatography-tandem MS. Together with PGD(2) working on its DP1 receptor, 15d-PGJ(2) controls the balance of pro- vs. antiinflammatory cytokines that regulate leukocyte influx and monocyte-derived macrophage efflux from the inflamed peritoneal cavity to draining lymph nodes leading to resolution. Specifically, inflammation in hPGD(2)S KOs is more severe during the onset phase arising from a substantial cytokine imbalance resulting in enhanced polymorphonuclear leukocyte and monocyte trafficking. Moreover, resolution is impaired, characterized by macrophage and surprisingly lymphocyte accumulation. Data from this work place hPGD(2)S at the center of controlling the onset and the resolution of acute inflammation where it acts as a crucial checkpoint controller of cytokine/chemokine synthesis as well as leukocyte influx and efflux. Here, we provide definitive proof that 15d-PGJ(2) is synthesized during mammalian inflammatory responses, and we highlight DP1 receptor activation as a potential antiinflammatory strategy.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Drug Design; Eicosanoids; Hematopoietic Stem Cells; Immunity, Innate; Inflammation; Intramolecular Oxidoreductases; Leukocytes; Lipocalins; Mice; Mice, Knockout; Monocytes; Neutrophils; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases

Inflammatory mechanisms may play an important role in the pathogenesis of cisplatin nephrotoxicity. Agonists of the peroxisome proliferator-activated receptor-gamma (PPARgamma), such as rosiglitazone, have been recently demonstrated to regulate inflammation by modulating the production of inflammatory mediators and adhesion molecules. The purpose of this study was to examine the protective effects of rosiglitazone on cisplatin nephrotoxicity and to explore the mechanism of its renoprotection.. Mice were treated with cisplatin with or without pre-treatment with rosiglitazone. Renal functions, histological findings, aquaporin 2 (AQP2) and adhesion molecule expression, macrophage infiltration and tumour necrosis factor-alpha (TNF-alpha) levels were investigated. The effect of rosiglitazone on nuclear factor (NF)-kappaB activity and on viability was examined using cultured human kidney (HK-2) cells.. Rosiglitazone significantly decreased both the damage to renal function and histological pathology after cisplatin injection. Pre-treatment with rosiglitazone reduced the systemic levels of TNF-alpha and down-regulated adhesion molecule expression in addition to the infiltration of inflammatory cells after cisplatin administration. Rosiglitazone restored the decreased AQP2 expression after cisplatin treatment. Pre-treatment with rosiglitazone blocked the phosphorylation of the p65 subunit of NF-kappaB in cultured HK-2 cells. Rosiglitazone had a protective effect via a PPARgamma-dependent pathway in cisplatin-treated HK-2 cells.. These results showed that pre-treatment with rosiglitazone attenuates cisplatin-induced renal damage through the suppression of TNF-alpha overproduction and NF-kappaB activation.

Topics: Anilides; Animals; Apoptosis; C-Peptide; Cell Line; Chromans; Cisplatin; Drug Evaluation, Preclinical; Glioma; Humans; Hypoglycemic Agents; Inflammation; Insulin; Intercellular Adhesion Molecule-1; Kidney; Kidney Diseases; Kidney Function Tests; Kidney Tubules, Proximal; Macrophages; Male; Mice; Mice, Inbred C57BL; Monocytes; PPAR gamma; Prostaglandin D2; Protein Transport; Rosiglitazone; Thiazolidinediones; Transcription Factor RelA; Troglitazone; Tumor Necrosis Factor-alpha

Peroxisome proliferator-activated receptor-gamma is a nuclear receptor transcription factor that regulates cell growth, differentiation and homeostasis. PPARgamma agonists have been used to treat obesity, diabetes, cancer and inflammation and recent studies have shown the protective effects of PPARgamma agonists on experimental allergic encephalomyelitis (EAE), a Th1 cell-mediated autoimmune disease model of multiple sclerosis (MS). Our studies have further demonstrated that the PPARgamma agonists, 15d-PGJ2 and Ciglitazone, inhibit EAE through blocking IL-12 signaling leading to Th1 differentiation and the PPARgamma deficient heterozygous mice (PPARgamma+/-) or those treated with PPARgamma antagonists develop an exacerbated EAE in association with an augmented Th1 response. In this study, we show that the PPARgamma antagonists, Bisphenol A diglycidyl ether (BADGE) and 2-chloro-5-nitro-N-(4-pyridyl)benzamide (T0070907), reverse the inhibition of EAE by the PPARgamma agonists, Ciglitazone and 15-Deoxy-Delta(12,14)-Prostaglandin J2, in C57BL/6 wild-type and PPARgamma+/- mice. The reversal of EAE by BADGE and T0070907 was associated with restoration of neural antigen-induced T cell proliferation, IFNgamma production and Th1 differentiation inhibited by Ciglitazone and 15d-PGJ2. These results suggest that Ciglitazone and 15d-PGJ2 ameliorate EAE through PPARgamma-dependent mechanisms and further confirm a physiological role for PPARgamma in the regulation of CNS inflammation and demyelination in EAE.

Topics: Animals; Antigens; Benzamides; Benzhydryl Compounds; Cell Differentiation; Cell Proliferation; Demyelinating Diseases; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Epoxy Compounds; Female; Immunologic Factors; Inflammation; Interferon-gamma; Mice; Mice, Inbred C57BL; Myelin Proteins; Myelin-Associated Glycoprotein; Myelin-Oligodendrocyte Glycoprotein; PPAR gamma; Prostaglandin D2; Pyridines; Th1 Cells; Thiazolidinediones

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcriptional factor belonging to the nuclear receptor superfamily. PPARgamma, which is predominantly expressed in adipose tissue, plays a major regulatory role in glucose metabolism and adipogenesis. Interestingly, recent studies have demonstrated PPARgamma expression in monocytes/macrophages and its antiinflammatory activities. However, it is unclear whether alveolar macrophages (AMs) express functional PPARgamma. The present study was conducted to investigate the expression of PPARgamma by AMs and to elucidate its functional role. Using reverse transcription-polymerase chain reaction and Western blotting, we demonstrated the strong expression of PPARs messenger RNA and protein in freshly isolated human AMs. Ligands of PPARgamma, 15-deoxy-delta(12,14)prostaglandin J2, and troglitazone significantly decreased LPS-induced tumor necrosis factor-alpha production by AMs. These ligands markedly upregulated the expression of CD36, a scavenger receptor that mediates the phagocytosis of apoptotic neutrophils. Indeed, ligand-treated AMs ingested a significantly higher number of apoptotic neutrophils than untreated AMs. These data indicate that PPARgamma expressed by AMs play an antiinflammatory role through inhibiting cytokine production and increasing their CD36 expression together with the enhanced phagocytosis of apoptotic neutrophils, which is an essential process for the resolution of inflammation. This suggests the potential therapeutic application of PPARgamma ligands in inflammatory disorders of the lung.

Topics: Apoptosis; CD36 Antigens; Chromans; Humans; In Vitro Techniques; Inflammation; Macrophages, Alveolar; Neutrophils; Phagocytosis; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Thiazolidinediones; Transcription Factors; Troglitazone; Tumor Necrosis Factor-alpha

Activated macrophages express high levels of Nrf2, a transcription factor that positively regulates the gene expression of antioxidant and detoxication enzymes. In this study, we examined how Nrf2 contributes to the anti-inflammatory process. As a model system of acute inflammation, we administered carrageenan to induce pleurisy and found that in Nrf2-deficient mice, tissue invasion by neutrophils persisted during inflammation and the recruitment of macrophages was delayed. Using an antibody against 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), it was observed that macrophages from pleural lavage accumulate 15d-PGJ(2). We show that in mouse peritoneal macrophages 15d-PGJ(2) can activate Nrf2 by forming adducts with Keap1, resulting in an Nrf2-dependent induction of heme oxygenase 1 and peroxiredoxin I (PrxI) gene expression. Administration of the cyclooxygenase 2 inhibitor NS-398 to mice with carrageenan-induced pleurisy caused persistence of neutrophil recruitment and, in macrophages, attenuated the 15d-PGJ(2) accumulation and PrxI expression. Administration of 15d-PGJ(2) into the pleural space of NS-398-treated wild-type mice largely counteracted both the decrease in PrxI and persistence of neutrophil recruitment. In contrast, these changes did not occur in the Nrf2-deficient mice. These results demonstrate that Nrf2 regulates the inflammation process downstream of 15d-PGJ(2) by orchestrating the recruitment of inflammatory cells and regulating the gene expression within those cells.

Topics: Animals; Cyclooxygenase 2; DNA-Binding Proteins; Hepatocytes; Inflammation; Isoenzymes; Macrophages; Mice; NF-E2-Related Factor 2; Peritoneum; Pleurisy; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Trans-Activators

Membrane-associated prostaglandin (PG) E(2) synthase-1 (mPGES-1) catalyzes the conversion of PGH(2) to PGE(2), which contributes to many biological processes. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor and plays an important role in growth, differentiation, and inflammation in different tissues. Here, we examined the effect of PPARgamma ligands on interleukin-1beta (IL-1beta)-induced mPGES-1 expression in human synovial fibroblasts. PPARgamma ligands 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)) and the thiazolidinedione troglitazone (TRO), but not PPARalpha ligand Wy14643, dose-dependently suppressed IL-1beta-induced PGE(2) production, as well as mPGES-1 protein and mRNA expression. 15d-PGJ(2) and TRO suppressed IL-1beta-induced activation of the mPGES-1 promoter. Overexpression of wild-type PPARgamma further enhanced, whereas overexpression of a dominant negative PPARgamma alleviated, the suppressive effect of both PPARgamma ligands. Furthermore, pretreatment with an antagonist of PPARgamma, GW9662, relieves the suppressive effect of PPARgamma ligands on mPGES-1 protein expression, suggesting that the inhibition of mPGES-1 expression is mediated by PPARgamma. We demonstrated that PPARgamma ligands suppressed Egr-1-mediated induction of the activities of the mPGES-1 promoter and of a synthetic reporter construct containing three tandem repeats of an Egr-1 binding site. The suppressive effect of PPARgamma ligands was enhanced in the presence of a PPARgamma expression plasmid. Electrophoretic mobility shift and supershift assays for Egr-1 binding sites in the mPGES-1 promoter showed that both 15d-PGJ(2) and TRO suppressed IL-1beta-induced DNA-binding activity of Egr-1. These data define mPGES-1 and Egr-1 as novel targets of PPARgamma and suggest that inhibition of mPGES-1 gene transcription may be one of the mechanisms by which PPARgamma regulates inflammatory responses.

Topics: Amino Acid Motifs; Anilides; Binding Sites; Blotting, Western; Cell Division; Cell Nucleus; Chromans; DNA-Binding Proteins; DNA, Complementary; Dose-Response Relationship, Drug; Early Growth Response Protein 1; Fibroblasts; Genes, Dominant; Genes, Reporter; Humans; Immediate-Early Proteins; Immunologic Factors; Inflammation; Interleukin-1; Intramolecular Oxidoreductases; Ligands; Peroxisome Proliferators; Plasmids; Promoter Regions, Genetic; Prostaglandin D2; Prostaglandin-E Synthases; Protein Binding; Pyrimidines; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; RNA; Synovial Membrane; Thiazolidinediones; Time Factors; Transcription Factors; Transcription, Genetic; Transfection; Troglitazone

The cyclopentenone prostaglandin and PPARgamma agonist 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) displays anti-inflammatory effects in several experimental models. Direct modification of protein thiols is arising as an important mechanism of cyclopentenone prostaglandin action. However, little is known about the extent or specificity of this process. Mesangial cells (MC) play a key role in glomerulonephritis. In this work, we have studied the selectivity of protein modification by 15d-PGJ(2) in MC, and the correlation with the modulation of several proinflammatory genes. MC incubation with biotinylated 15d-PGJ(2) results in the labeling of a distinct set of proteins as evidenced by two-dimensional electrophoresis. 15d-PGJ(2) binds to nuclear and cytosolic targets as detected by fluorescence microscopy and subcellular fractionation. The pattern of biotinylated 15d-PGJ(2)-modified polypeptides is readily distinguishable from that of total protein staining or labeling with biotinylated iodoacetamide. 15d-PGJ(2) addition requires the double bond in the cyclopentane ring. 9,10-Dihydro-15d-PGJ(2), a 15d-PGJ(2) analog that shows the same potency as peroxisome proliferator-activated receptor (PPAR) agonist in MC but lacks the cyclopentenone moiety, displays reduced ability to modify proteins and to block 15d-PGJ(2) binding. Micromolar concentrations of 15d-PGJ(2) inhibit cytokine-elicited levels of inducible nitricoxide synthase, cyclooxygenase-2, and intercellular adhesion molecule-1 in MC. In contrast, 9,10-dihydro-15d-PGJ(2) does not reproduce this inhibition. 15d-PGJ(2) effect is not blocked by the PPARgamma antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662). Moreover, compounds possessing an alpha,beta-unsaturated carbonyl group, like 2-cyclopenten-1-one and 2-cyclohexen-1-one, reduce pro-inflammatory gene expression. These observations indicate that covalent modification of cellular thiols by 15d-PGJ(2) is a selective process that plays an important role in the inhibition of MC responses to pro-inflammatory stimuli.

Topics: Animals; Biotinylation; Cell Nucleus; Cells, Cultured; Cyclooxygenase 2; Cyclopentanes; Cytosol; Gene Silencing; Glomerular Mesangium; Inflammation; Intercellular Adhesion Molecule-1; Isoenzymes; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peroxisome Proliferator-Activated Receptors; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Proteins; Rats; Sulfhydryl Compounds; Transfection

Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) transiently activates protein kinase C zeta (PKC zeta) and Jun N-terminal kinase (JNK) through a phosphoinositide-3-kinase (PI3-kinase)-dependent pathway. Incubation of LPS-treated cells with the cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ(2)) promoted a sustained activation of PKC zeta and JNK and inhibited I kappa B kinase (IKK) and NF-kappa B activity. Accordingly, 15dPGJ(2) induced an imbalance between JNK and IKK activities by increasing the former signaling pathway and inhibiting the latter signaling pathway. Under these conditions, apoptosis was significantly enhanced; this response was very dependent on PKC zeta and JNK activation. The effect of 15dPGJ(2) on PKC zeta activity observed in LPS-activated macrophages was not dependent on a direct action of this prostaglandin on the enzyme but was due to the activation of a step upstream of PI3-kinase. Moreover, LPS promoted the redistribution of activated PKC zeta from the cytosol to the nucleus, a process that was enhanced by treatment of the cells with 15dPGJ(2) that favored a persistent and broader distribution of PKC zeta in the nucleus. These results indicate that 15dPGJ(2) and other cyclopentenone prostaglandins, through the sustained activation of PKC zeta, might contribute significantly to the process of resolution of inflammation by promoting apoptosis of activated macrophages.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Animals; Apoptosis; Cell Nucleus; Cells, Cultured; Enzyme Activation; I-kappa B Kinase; Immunologic Factors; Inflammation; Isoenzymes; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Macrophage Activation; Macrophages; Mice; Mitogen-Activated Protein Kinases; NF-kappa B; Phosphatidylinositol 3-Kinases; Prostaglandin D2; Protein Kinase C; Protein Serine-Threonine Kinases; Signal Transduction

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has been implicated in inhibition of the expression of proinflammatory cytokines and inducible enzymes such as cyclooxygenase-2 (COX-2). Using real-time RT-PCR the present study investigates the impact of two PPAR-gamma agonists, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and ciglitazone, on the expression of several proinflammatory genes in lipopolysaccharide (LPS)-stimulated human blood monocytes. Stimulation of cells with LPS resulted in a profound induction of the expression of COX-2, interleukin (IL)-1, IL-6, tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Treatment of cells with 15d-PGJ(2) (10 microM) was associated with a nearly complete inhibition of the expression of all genes that remained unaltered in the presence of the PPAR-gamma antagonist bisphenol A diglycidyl ether (BADGE; 100 microM). By contrast, treatment of cells with another potent PPAR-gamma agonist, ciglitazone (50 microM), and the PPAR-alpha agonist WY-14,643 (100 microM) did not suppress LPS-induced expression of the investigated genes. Stimulation of monocytes with LPS resulted in an 88% inhibition of PPAR-gamma mRNA expression that was fully restored by 15d-PGJ(2) but only to a partial extent by ciglitazone and WY-14,643. Again, BADGE did not alter the effect of 15d-PGJ(2). Collectively, our results show that alterations of gene expression by 15d-PGJ(2) in LPS-stimulated human blood monocytes are mediated by PPAR-gamma-independent mechanisms. Moreover, it is concluded that both inhibition of proinflammatory gene expression and restoration of LPS-induced decrease of PPAR-gamma expression may contribute to the biological action of 15d-PGJ(2).

Topics: Cyclooxygenase 2; Gene Expression; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunologic Factors; In Vitro Techniques; Inflammation; Interleukin-1; Interleukin-6; Isoenzymes; Lipopolysaccharides; Membrane Proteins; Monocytes; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Thiazoles; Thiazolidinediones; Transcription Factors; Tumor Necrosis Factor-alpha

Failure of acute inflammation to resolve leads to persistence of the inflammatory response and may contribute to the development of chronic inflammation. Thus, an understanding of inflammatory resolution will provide insight into the etiology of chronic inflammation. In an acute pleurisy, polymorphonuclear leukocytes (PMNs) were found to predominate at the onset of the lesion but decreased in number by undergoing apoptosis, the principal mechanism by which PMNs died in this model. PMNs were progressively replaced by monocytes, which differentiated into macrophages. As with PMNs, macrophages also underwent programmed cell death leading to an abatement of the inflammatory response and eventual resolution. It was found that apoptosis of both these inflammatory cell types was mediated by pro-resolving cyclooxygenase 2-derived 15deoxyDelta12-14PGJ2, which is uniquely expressed during active resolution. Although PMN programmed cell death is well understood, the observation that macrophages apoptose during resolution of acute inflammation is less well described. These results provide insight into the mechanisms that switch off acute inflammation and prevent complications of wound healing and potentially the development of immune-mediated chronic inflammation.

Topics: Acute Disease; Animals; Apoptosis; Cyclooxygenase 2; Inflammation; Isoenzymes; Leukocyte Count; Macrophages; Models, Immunological; Neutrophils; Pleurisy; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Rats

Dendritic cells (DC) are professional antigen-presenting cells playing a pivotal role in the induction of immunological responses. There is evidence that DC survival during ongoing immune responses is finite. However, little is known about the mechanisms regulating apoptosis in these cells. Here, we have investigated the effects of the anti-inflammatory cyclopentenone prostaglandins on human monocyte-derived DC.. Phenotype of DC was determined by flow cytometry and their allostimulatory potential in mixed leukocyte reaction. Induction of apoptosis in DC was monitored by staining with annexin-V-FITC and propidium iodide, propidium iodide staining of cell nuclei, and fluorimetric assay of caspase activity. Induction of maturation in DC was obtained by stimulation with TNF-alpha, LPS, IFN-gamma, CD40-ligand, or different combinations of these stimuli. PPAR-gamma expression in DC was determined by RT-PCR.. Exposure of immature DC to cyclopentenone prostaglandins blunted their allostimulatory capacity and skewed their phenotype by downregulating CD1a and costimulatory molecules. These effects were due to activation of caspases and induction of apoptotic cell death in DC by cyclopentenone prostaglandins. Mature DC showed enhanced susceptibility to apoptosis via cyclopentenone prostaglandins as compared with immature DC. Although DC express PPAR-gamma, the corresponding receptor for some of these metabolites, PPAR-gamma activation by a synthetic high-affinity agonist failed to impair DC viability.. Cyclopentenone prostaglandins induce apoptosis of human DC by a PPAR-gamma-independent mechanism. Since these compounds are released during an inflammatory event and show anti-inflammatory properties, they may contribute to the downregulation of DC function through apoptotic cell death.

Topics: Amino Acid Chloromethyl Ketones; Antigens, CD1; Apoptosis; Caspases; CD40 Ligand; Cell Differentiation; Cells, Cultured; Cyclooxygenase 2; Cysteine Proteinase Inhibitors; Dendritic Cells; Dinoprostone; Drug Resistance; Enzyme Activation; Enzyme Inhibitors; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation; Interferon-gamma; Interleukin-4; Isoenzymes; Lipopolysaccharides; Membrane Proteins; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Prostaglandins A; Receptors, Cytoplasmic and Nuclear; T-Lymphocytes; Transcription Factors; Tumor Escape; Tumor Necrosis Factor-alpha

Prostaglandin D(2) (PGD(2)), a major cyclooxygenase product in a variety of tissues, readily undergoes dehydration to yield the cyclopentenone-type PGs of the J(2) series, such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), which have been suggested to exert anti-inflammatory effects in vivo. Meanwhile, the mechanism of these effects is not well understood and the natural site and the extent of its production in vivo remain unclear. In the present study, we raised a monoclonal antibody specific to 15d-PGJ(2) and determined its production in inflammation-related events. The monoclonal antibody (mAb11G2) was raised against the 15d-PGJ(2)-keyhole limpet hemocyanin conjugate and was found to recognize free 15d-PGJ(2) specifically. The presence of 15d-PGJ(2) in vivo was immunohistochemically verified in the cytoplasm of most of the foamy macrophages in human atherosclerotic plaques. In addition, the immunostaining of lipopolysaccharide-stimulated RAW264.7 macrophages with mAb11G2 demonstrated an enhanced intracellular accumulation of 15d-PGJ(2), suggesting that the PGD(2) metabolic pathway, generating the anti-inflammatory PGs, is indeed utilized in the cells during inflammation. The activation of macrophages also resulted in the extracellular production of PGD(2), which was associated with a significant increase in the extracellular 15d-PGJ(2) levels, and the extracellular 15d-PGJ(2) production was reproduced by incubating PGD(2) in a cell-free medium and in phosphate-buffered saline. Moreover, using a chiral high performance liquid chromatography method for separation of PGD(2) metabolites, we established a novel metabolic pathway, in which PGD(2) is converted to 15d-PGJ(2) via an albumin-independent mechanism.

Topics: Animals; Antibodies, Monoclonal; Arteriosclerosis; Cell Line; Cell-Free System; Chromatography, High Pressure Liquid; Cyclooxygenase 2; Cytoplasm; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Humans; Immunoblotting; Immunohistochemistry; Immunologic Factors; Inflammation; Isoenzymes; Macrophages; Membrane Proteins; Mice; Models, Biological; Models, Chemical; Phosphates; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Serum Albumin; Time Factors

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors that are related to retinoid, steroid, and thyroid hormone receptors. The PPAR-gamma receptor subtype seems to play a pivotal role in the regulation of cellular proliferation and inflammation. Recent evidence also suggests that the cyclopentenone prostaglandin (PG) 15-deoxyDelta(12,14)-PGJ(2) (15d-PGJ(2)), which is a metabolite of prostaglandin D(2), functions as an endogenous ligand for PPAR-gamma. We postulated that 15d-PGJ(2) would attenuate inflammation. In the present study, we have investigated the effects of 15d-PGJ(2) of acute and chronic inflammation (carrageenan-induced pleurisy and collagen-induced arthritis, respectively) in animal models. We report for the first time, to our knowledge, that 15d-PGJ(2) (given at 10, 30, or 100 microg/kg i.p. in the pleurisy model or at 30 microg/kg i.p every 48 h in the arthritis model) exerts potent anti-inflammatory effects (e.g., inhibition of pleural exudate formation, mononuclear cell infiltration, delayed development of clinical indicators, and histological injury) in vivo. Furthermore, 15d-PGJ(2) reduced the increase in the staining (immunohistochemistry) for nitrotyrosine and poly (ADP-ribose) polymerase and the expression of inducible nitric-oxide synthase and cyclooxygenase-2 in the lungs of carrageenan-treated mice and in the joints from collagen-treated mice. Thus, 15d-PGJ(2) reduces the development of acute and chronic inflammation. Therefore, the cyclopentenone prostaglandin 15d-PGJ(2) may be useful in the therapy of acute and chronic inflammation.

Topics: Animals; Arthritis, Experimental; Carrageenan; Cyclopentanes; Disease Models, Animal; Immunohistochemistry; Immunologic Factors; Inflammation; Male; Mice; Mice, Inbred BALB C; Pleurisy; Prostaglandin D2

15-Deoxy-delta(12,14)-prostaglandin J2 (dPGJ2) is a bioactive metabolite of the J2 series that has been identified as a ligand for peroxisome proliferator-activated receptor gamma (PPARgamma). Because PPARgamma is highly expressed in macrophages obtained from stimulant-elicited peritonitis, but not in resident peritoneal macrophages, the effect of dPGJ2 was tested on innate functions of macrophages. dPGJ2 inhibited adhesion and phagocytosis of Escherichia coli. Inhibition of these functions by dPGJ2 was not mediated via the adhesion molecule Mac-1. In addition, dPGJ2 inhibited chemotaxis toward zymosan-activated serum and it also inhibited the production of superoxide anion when macrophages were stimulated with phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan (OPZ), but not lipopolysaccharide. Similarly, dPGJ2 inhibited the production of hydrogen peroxide when macrophages were stimulated with either PMA or OPZ. These studies suggest that dPGJ2 may be a negative regulator of macrophage functions.

Topics: Animals; Cell Adhesion; Chemotaxis; Escherichia coli; Hydrogen Peroxide; Inflammation; Ligands; Lipopolysaccharides; Macrophage-1 Antigen; Macrophages, Peritoneal; Male; Oxidative Stress; Peritonitis; Phagocytosis; Prostaglandin D2; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Superoxides; Tetradecanoylphorbol Acetate; Transcription Factors; Zymosan

Cyclooxygenase (COX) has two isoforms. Generally, COX 1 is constitutively expressed in most tissues, where it maintains physiological processes; inducible COX 2 is considered a pro-inflammatory enzyme and a chief target for the treatment of inflammatory diseases. Here we present evidence that COX 2 may have anti-inflammatory properties. In carrageenin-induced pleurisy in rats, the predominant cells at 2 hours are polymorphonuclear leucocytes, whereas mononuclear cells dominate from 24 hours until resolution at 48 hours. In this model, COX 2 protein expression peaked initially at 2 hours, associated with maximal prostaglandin E2 synthesis. However, at 48 hours there was a second increase in COX 2 expression, 350% greater than that at 2 hours. Paradoxically, this coincided with inflammatory resolution and was associated with minimal prostaglandin E2 synthesis. In contrast, levels of prostaglandin D2, and 15deoxy delta(12-14)prostaglandin J2 were high at 2 hours, decreased as inflammation increased, but were increased again at 48 hours. The selective COX 2 inhibitor NS-398 and the dual COX 1/COX 2 inhibitor indomethacin inhibited inflammation at 2 hours but significantly exacerbated inflammation at 48 hours. This exacerbation was associated with reduced exudate prostaglandin D2 and 15deoxy delta(12-14)prostaglandin J2 concentrations, and was reversed by replacement of these prostaglandins. Thus, COX 2 may be pro-inflammatory during the early phase of a carrageenin-induced pleurisy, dominated by polymorphonuclear leucocytes, but may aid resolution at the later, mononuclear cell-dominated phase by generating an alternative set of anti-inflammatory prostaglandins.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Carrageenan; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Indomethacin; Inflammation; Isoenzymes; Male; Membrane Proteins; Monocytes; Neutrophils; Nitrobenzenes; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar; Sulfonamides

Peroxisome proliferator-activated receptor gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily originally shown to play a critical role in adipocyte differentiation and glucose homeostasis, has recently been implicated as a regulator of cellular proliferation and inflammatory responses. Colonic epithelial cells, which express high levels of PPAR-gamma protein, have the ability to produce inflammatory cytokines that may play a role in inflammatory bowel disease (IBD). We report here that PPAR-gamma ligands dramatically attenuate cytokine gene expression in colon cancer cell lines by inhibiting the activation of nuclear factor-kappaB via an IkappaB-alpha-dependent mechanism. Moreover, thiazolidinedione ligands for PPAR-gamma markedly reduce colonic inflammation in a mouse model of IBD. These results suggest that colonic PPAR-gamma may be a therapeutic target in humans suffering from IBD.

Topics: Animals; Caco-2 Cells; Colitis; Cytokines; DNA-Binding Proteins; Epithelium; Gene Expression; HT29 Cells; Humans; I-kappa B Proteins; Inflammation; Inflammatory Bowel Diseases; Interleukin-8; Ligands; Mice; Microbodies; NF-kappa B; NF-KappaB Inhibitor alpha; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Rosiglitazone; Thiazoles; Thiazolidinediones; Transcription Factors