PGD2 Levels Research Articles

Malva sylvestris L. extract suppresses desferrioxamine‐induced PGE2 and PGD2 release in differentiated U937 cells: the development and validation of an LC‐MS/MS method for prostaglandin quantification

Malva sylvestris is a species used worldwide as an alternative to anti-inflammatory therapies; however, its mechanism of action remains unknown. In this paper, the anti-inflammatory effects of M. sylvestris alcoholic extracts were evaluated by measuring the pro-inflammatory mediators PGE₂ and PGD₂ in desferrioxamine-stimulated phorbol 12-myristate 13-acetate-differentiated U937 cells. An HPLC-DAD fingerprint of the M. sylvestris extract was performed and caffeic acid, ferulic acid and scopoletin were identified and quantified. An HPLC-MS/MS method was developed and validated to separate and measure the prostaglandins. The lower limits of detection (~0.5 ng/mL for PGE₂ and PGD₂) and quantification (1.0 ng/mL for PGE₂ and PGD₂) indicated that the method is highly sensitive. The calibration curves showed excellent coefficients of correlation (r > 0.99) over the range of 1.0-500.0 ng/mL, and at different levels, the accuracy ranged from 96.4 to 106.4% with an RSD < 10.0% for the precision study. This method was successfully applied using U937-d cells. A significant dose-dependent reduction of PGE2 and PGD2 levels occurred using 10 µg/mL (10.74 ± 2.86 and 9.60 ± 6.89%) and 50 µg/mL of extract (48.37 ± 3.24 and 53.06 ± 6.15%), suggesting that the anti-inflammatory mechanisms evoked by M. sylvestris may be related to modulation of these mediators.

Pharmacological significance of prostaglandin E2 and D2 transport at the brain barriers.

Prostaglandin (PG) E2 and PGD2, which are biosynthesized from arachidonic acid generated by enzymatic cleavage of membrane phospholipid in response to various stimuli, play key roles in multiple brain pathophysiological processes, including modulation of synaptic plasticity, neuroinflammation, and sleep promotion. Concentrations of PGE2 and PGD2 in brain interstitial fluid (ISF) and cerebrospinal fluid (CSF) are maintained at appropriate levels for normal brain function by regulatory systems. The blood-brain barrier (BBB) and the blood-CSF barrier (BCSFB) possess ISF/CSF-to-blood efflux transport systems that are the primary cerebral clearance pathways for PGE2 and PGD2. However, regulatory dysfunction at the brain barriers may seriously affect brain function. In a mouse inflammation model, significant reduction of PGE2 efflux transport at the BBB has been observed. Several kinds of cephalosporin antibiotics and nonsteroidal anti-inflammatory drugs inhibit the BBB- and BCSFB-mediated efflux transport of PGE2 and PGD2. Especially, drugs that inhibit multidrug resistance-associated protein 4 (MRP4)-mediated PGE2 transport are capable of reducing PGE2 efflux at the BBB. Thus, it might be important in the treatment of inflammatory and infectious diseases to use drugs that do not inhibit clearance of PGE2 at the brain barriers, in order to avoid unexpected adverse CNS effects. Further, considering that PGD2 in CSF is a natural sleep-promoting factor, changes in the activity of the PGD2 efflux transport system at the BCSFB may modify the PGD2 level in CSF, thus affecting physiological sleep. These findings indicate that the efflux transport systems at the brain barriers play key roles in the pathophysiology and pharmacology of PGE2 and PGD2.

S19 Increased CRTH2 expression in asthmatic bronchial epithelium

Introduction and ObjectivesProstaglandin-D2 (PGD2) mediates chemotaxis of Th2 cells, basophils and eosinophils through the chemoattractant receptor homologous-molecule expressed on T-helper-type-2 cells (CRTh2). Pulmonary PGD2 levels are increased in patients with...

Eicosanoid post-mortem induction in kidney tissue is prevented by microwave irradiation

Previously, we, and others, have demonstrated a rapid and significant post-mortem increase in brain prostanoid (PG) levels analyzed without microwave fixation, and this is not the result of PG trapping or destruction in microwave-irradiated brain tissue. In the present study, we demonstrate a dramatic increase in kidney eicosanoid levels when analyzed without microwave fixation which was mainly accounted for by the 142-, 81-, and 62-fold increase in medullary 6-ketoPGF1α, PGE2, and PGF2α, levels, respectively, while PGD2 and TXB2 levels were increased ~7-fold. Whole kidney and cortex PG were also significantly increased in non-microwaved tissue, but at lesser extent. Arachidonic acid and the lipoxygenase products hydroxyeicosatetraenoic acids (HETE) were also induced in whole kidney, cortex, and medulla 1.5- to 5.5-fold depending upon tissue and metabolite. Cyclooxygenase inhibition with indomethacin decreased PG mass in non-microwaved tissue to basal levels, however HETE and arachidonic acid were not decreased. These data demonstrate the critical importance of kidney tissue fixation to limiting artifacts during kidney eicosanoid analysis.

A selective antagonist of histamine H4 receptors prevents antigen‐induced airway inflammation and bronchoconstriction in guinea pigs: involvement of lipocortin‐1

Among the pathogenic mechanisms of asthma, a role for oxidative/nitrosative stress has been well documented. Recent evidence suggests that histamine H₄ receptors play a modulatory role in allergic inflammation. Here we report the effects of compound JNJ 7777120 (JNJ), a selective H4 receptor antagonist, on antigen-induced airway inflammation, paying special attention to its effects on lipocortin-1 (LC-1/annexin-A1), a 37 kDA anti-inflammatory protein that plays a key role in the production of inflammatory mediators. Ovalbumin (OA)-sensitized guinea pigs placed in a respiratory chamber were challenged with antigen. JNJ (5, 7.5 and 10 mg.kg⁻¹) was given i.p. for 4 days before antigen challenge. Respiratory parameters were recorded. Bronchoalveolar lavage (BAL) fluid was collected and lung specimens taken for further analyses 1 h after antigen challenge. In BAL fluid, levels of LC-1, PGD2 , LTB4 and TNF-α were measured. In lung tissue samples, myeloperoxidase, caspase-3 and Mn-superoxide dismutase activities and 8-hydroxy-2-deoxyguanosine levels were measured. OA challenge decreased LC-1 levels in BAL fluid, induced cough, dyspnoea and bronchoconstriction and increased PGD2 , LTB4 and TNF-α levels in lung tissue. Treatment with JNJ dose-dependently increased levels of LC-1, reduced respiratory abnormalities and lowered levels of PGD2 , LTB4 and TNF-α in BAL fluid. Antigen-induced asthma-like reactions in guinea pigs decreased levels of LC-1 and increased TNF-α and eicosanoid production. JNJ pretreatment reduced allergic asthmatic responses and airway inflammation, an effect associated with LC-1 up-regulation.

Effects of Long-Term Oral Administration of Arachidonic Acid and Docosahexaenoic Acid on the Immune Functions of Young Rats

Natural killer (NK) cells have many functional activities, including cytotoxicity and the capacity to produce cytokines and chemokines. NK cell activity is regulated partly by eicosanoids, which are produced from arachidonic acid (ARA) and eicosapentaenoic (EPA) acid. In this study, we investigated the effects of long-term therapy with ARA or docosahexaenoic acid (DHA) on the cytotoxic effects of the NK cells of young rats, which were fed on a nonfish oil diet for two generations. Control oil, ARA (240 mg/kg BW/day) or DHA (240 mg/kg BW/day) were orally administrated to the rats for 13 weeks before determining the cytotoxic activity of NK cells from the spleen against YAC-1 mouse lymphoma cell line, as well as the plasma levels of docosanoids or eicosanoids and inflammatory cytokines. Long-term ARA administration significantly suppressed the cytotoxic activity of NK cells. Moreover, ARA administration significantly increased the plasma levels of ARA, prostaglandin (PG) E2, and PGD2. However, DHA administration did not produce any different effects compared with those in the control rats. Furthermore, the inflammatory cytokine levels were not affected by the administration of ARA or DHA. These results suggest that long-term ARA administration has an inhibitory effect on the tumor cytotoxicity of NK cells in rat spleen lymphocytes owing to the enhanced synthesis of PGE2 and PGD2 from ARA because of the elevated plasma ARA levels in young rats.

Cysteinyl leukotriene-receptor-1 antagonists interfere with PGE2 synthesis by inhibiting mPGES-1 activity

Because of their favourable safety profile and beneficial anti-inflammatory properties, the CysLT1 receptor antagonists (LTRA), montelukast, zafirlukast and pranlukast are approved for the treatment of asthma and are frequently prescribed as add-on therapeutics to reduce the amount of inhaled glucocorticoids and β2-agonists. There is evidence that some of these anti-inflammatory properties might be of a secondary nature and therefore, unrelated to the CysLT1 antagonism. Here, we show that LTRA inhibit PGE2 formation in cytokine-stimulated Hela and A549 carcinoma cells and in lipopolysaccharide (LPS)-stimulated human leukocyte preparations (IC50∼20μM). Neither expression of enzymes involved in PGE2 synthesis nor arachidonic acid release and COX activities were inhibited by the compounds. In contrast, mPGES-1 activity was suppressed at low micromolar levels (IC50 between 2 and 4μM). This suppression was specific for PGE2 synthesis, since PGD2 and PGI2 levels in LPS-stimulated leukocyte preparations were not negatively affected. PGF2α levels were concomitantly inhibited, probably due to its direct synthesis from PGE2. Several major conclusions can be drawn from this study: (A) clinical trials investigating elevated doses of the compounds are helpful to confirm suppression of PGE2 synthesis in vivo; (B) studies investigating the role of CysLTs in cell culture or animal models of inflammation and cancer have to be reassessed carefully, if higher doses of LTRA were applied or serum levels in cell culture assays were low; and (C) LTRA may serve as new scaffolds for the development of potent, selective and well tolerated mPGES-1 inhibitors.

Prostaglandin D2 Inhibits Wound-Induced Hair Follicle Neogenesis through the Receptor, Gpr44

Prostaglandins (PGs) are key inflammatory mediators involved in wound healing and regulating hair growth; however, their role in skin regeneration after injury is unknown. Using wound-induced hair follicle neogenesis (WIHN) as a marker of skin regeneration, we hypothesized that PGD2 decreases follicle neogenesis. PGE2 and PGD2 were elevated early and late respectively during wound healing. The levels of WIHN, lipocalin-type prostaglandin D2 synthase (Ptgds) and its product PGD2 each varied significantly among background strains of mice after wounding and all correlated such that the highest Ptgds and PGD2 levels were associated with the lowest amount of regeneration. Additionally, an alternatively spliced transcript variant of Ptgds missing exon 3 correlated with high regeneration in mice. Exogenous application of PGD2 decreased WIHN in wild type mice and PGD2 receptor Gpr44 null mice showed increased WIHN compared to strain-matched control mice. Furthermore, Gpr44 null mice were resistant to PGD2-induced inhibition of follicle neogenesis. In all, these findings demonstrate that PGD2 inhibits hair follicle regeneration through the Gpr44 receptor and imply that inhibition of PGD2 production or Gpr44 signaling will promote skin regeneration.

Change in Prostaglandin Expression Levels and Synthesizing Activities in Dry Eye Disease

To investigate the expression level of prostaglandins (PGs) and their de novo synthesis in dry eye (DE) disease. Cross-sectional case-control study and in vivo mouse experimental study. Forty-six eyes from 23 DE patients and 33 eyes from 17 age- and sex-matched controls were studied. Also, DE-induced murine eyes were compared with control eyes. Patients completed a symptom questionnaire using a 100-mm visual analog scale (VAS). Nano-liquid chromatography tandem mass spectrometry was used for the quantification of PGE2 and PGD2. A DE disease environmental chamber was used to induce DE in mice. One week after induction, enzyme expressions of cyclooxygenase-1, cyclooxygenase-2 (COX-2), PG E synthase (PGES), and PG D synthase (PGDS) in the lacrimal glands, meibomian glands, and corneas were examined using immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). The mean PGE2 and PGD2 levels in the tears of DE patients were measured and compared with symptom severity scores. Immunohistochemistry staining patterns and qRT-PCR data of DE mice were quantified. The mean PGE2 level in the tears of DE patients (2.72 ±3 .42 ng/ml) was significantly higher than that in the control group (0.88 ± 0.83 ng/ml; P = 0.003). However, the mean PGD2 level in the tears of DE patients (0.11 ± 0.22 ng/ml) was significantly lower (0.91 ± 3.28 ng/ml; P = 0.028). The mean PGE2-to-PGD2 ratio correlated strongly with VAS scoring (P = 0.008). In DE mice, COX-2 mRNA was significantly higher in ocular surface tissue and lacrimal glands. Furthermore, PGES mRNA was significantly higher in ocular surface tissue, whereas PGDS mRNA was decreased. Immunohistochemistry staining showed elevated COX-2 expression in the lacrimal glands, meibomian glands, corneas, and conjunctivas. Furthermore, PGES expression was found in periductal infiltrated cells of the lacrimal glands and conjunctival epithelium. Also, PGDS expression was decreased in meibomian glands and increased focally in the conjunctival epithelium. A reciprocal change in PGE2 and PGD2 levels was found in the tears of DE patients, which correlated with patients' symptom scores. These clinical results were supported by increased COX-2 and PGES expression levels found in tear-producing tissues of DE mice.

Exhaled eicosanoid profiles in children with atopic asthma and healthy controls

Chronic endobronchial inflammation is a hallmark of pediatric asthma and involves the arachidonic acid pathway. Its non-volatile metabolites can be quantified in the exhaled breath condensate (EBC), and single substances have been studied as non-invasive biomarkers for the diagnosis and monitoring of children with asthma. The aim of this study was to compare the content and profile of a wider range of eicosanoids in the EBC between patients and a control group. EBC was sampled from 33 children (aged 12.4 ± 3.1 years) with stable atopic asthma (26 on inhaled steroid treatment) and 25 healthy controls (11.8 ± 3.2 years). Validated high performance liquid chromatography coupled with a tandem mass spectrometry platform (HPLC-MS2 ) was used to measure 13 different compounds. In addition, exhaled nitric oxide levels (FeNO) were measured and bronchial hyperresponsiveness (BHR) was assessed by an exercise challenge test in all subjects. An analytical approach was used for multivariate regression modeling of disease status using the most relevant variables. The levels of PGEM (P < 0.001), PGD2 (P < 0.001), 6keto-PGF1α (P = 0.03), LTC4 (P < 0.001), trans-LTC4 (P = 0.04), and 5HETE (P = 0.02) were significantly higher in asthmatics compared to healthy children, while 11-dehydro TXB2 was significantly less abundant (P = 0.02). The eicosanoids asthma classification ratio (EACR) was computed as the logistic regression function using four variables: PGEM, PGD2, LTC4, and 5HETE. This composite parameter discriminated asthmatic from healthy children better than FEV1, FeNO, or BHR. Complementary measurements of PGEM, PGD2, LTC4, and 5HETE in small-volume EBC samples are feasible by HPLC-MS2 and showed a specific profile in our study population. EACR should be evaluated further in the context of diagnosing and monitoring childhood asthma.

Pharmacological inhibition of Akt and downstream pathways modulates the expression of COX-2 and mPGES-1 in activated microglia

BackgroundMicroglia are considered a major target for modulating neuroinflammatory and neurodegenerative disease processes. Upon activation, microglia secrete inflammatory mediators that contribute to the resolution or to further enhancement of damage in the central nervous system (CNS). Therefore, it is important to study the intracellular pathways that are involved in the expression of the inflammatory mediators. Particularly, the role of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and glycogen synthase kinase-3 (GSK-3) pathways in activated microglia is unclear. Thus, in the present study we investigated the role of Akt and its downstream pathways, GSK-3 and mTOR, in lipopolysaccharide (LPS)-activated primary rat microglia by pharmacological inhibition of these pathways in regard to the expression of cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES-1) and to the production of prostaglandin (PG) E2 and PGD2.FindingsWe show that inhibition of Akt by the Akt inhibitor X enhanced the production of PGE2 and PGD2 without affecting the expression of COX-2, mPGES-1, mPGES-2 and cytosolic prostaglandin E synthase (cPGES). Moreover, inhibition of GSK-3 reduced the expression of both COX-2 and mPGES-1. In contrast, the mTOR inhibitor rapamycin enhanced both COX-2 and mPGES-1 immunoreactivity and the release of PGE2 and PGD2. Interestingly, NVP-BEZ235, a dual PI3K/mTOR inhibitor, enhanced COX-2 and reduced mPGES-1 immunoreactivity, albeit PGE2 and PGD2 levels were enhanced in LPS-stimulated microglia. However, this compound also increased PGE2 in non-stimulated microglia.ConclusionTaken together, we demonstrate that blockade of mTOR and/or PI3K/Akt enhances prostanoid production and that PI3K/Akt, GSK-3 and mTOR differently regulate the expression of mPGES-1 and COX-2 in activated primary microglia. Therefore, these pathways are potential targets for the development of novel strategies to modulate neuroinflammation.

P4-05-04: Arachidonic Acid-Induced Elevated Expression of 5-Lipoxygenase Is Linked to Metastatic Migration of Breast Cancer Cells.

Abstract Background: In invasive breast cancers, cancerous cells spread outside the ducts of the breast and metastasize to lung and other tissues. Although the rapid arachidonic acid (AA) metabolism and concomitant increase of eicosanoid molecules are involved in the proliferation and invasion of breast cancer cells, the exact mechanism by which AA metabolites regulate these phenomena are not well understood. Here, we show that leukotriene B4 (LTB4), one of the metabolites of AA, which is produced by the action of 5-lipoxygenase (5-LOX), causes the proliferation and metastatic migration of breast cancer cells. Material and Methods: MDA-MB-231 (invasive breast cancer cells) and MCF7 (non-invasive breast cancer cells) were used in this study. Briefly, cells were treated with AA (100 μM) and nordihydroguaiarectin acid (NDGA, 10 μM), and the secreted eicosanoids were characterized by HPLC. Immunofluorescence microscopy was performed to elucidate the expression and intracellular localization of 5-LOX. The metastatic migration was analyzed by wound-healing assays. Results: Our results suggest that while MDA-MB-231 cells produce high levels of PGE2 and PGD2, MCF7 cells synthesize excess HETE compounds (HETE5 and HETE8). Interestingly, MDA-MB-231 cells, when stimulated with AA, show the increased syntheses of LTB4 (∼3 fold) and decreased PGE2 and PGD2 (∼2 fold). In MCF7, on the contrary, AA treatment reduced the syntheses of all eicosanoids. Furthermore, the expression of 5-LOX in MDA-MB-231 cells was also increased by ∼2 fold. We observed that AA promotes cell migration in MDA-MB-231 cells, which could be blocked by NDGA, a generic inhibitor of LOX enzymes. Because LTB4 production and its binding with BLT receptors are linked to IL-8 secretion, we measured the level of IL-8 synthesized by both MCF7 and MDA-MB-231 cells. The results suggest that MDA-MB-231 cells secrete excess IL-8 (∼2 fold) when stimulated with AA, suggesting that LTB4 and IL-8 interactive pathways are important for cancer metastasis. Discussion: Our results demonstrate that LTB4 synthesis is linked to the metastatic migration of MDA-MB-231 cells, and that it therefore should be considered as a target for developing new drugs for the treatment of invasive breast cancers. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-05-04.

Specificity of eicosanoid production depends on the TLR-4-stimulated macrophage phenotype

Eicosanoid metabolism differs in profile and quantity between macrophages of different tissue origin and method of elicitation, as well as between primary and immortalized macrophages after activation with inflammatory stimuli. Using a lipidomic approach, we comprehensively analyzed the eicosanoids made by murine RPMs, TGEMs, BMDM, and the macrophage-like cell line RAW after stimulation with the TLR-4-specific agonist KLA. Direct correlation among total COX metabolites, COX side-products (11-HETE, 15-HETE), COX-2 mRNA, and protein at 8 h was found when comparing each cell type. Comprehensive qPCR analysis was used to compare relative transcript levels between the terminal prostanoid synthases themselves as well as between each cell type. Levels of PGE(2), PGD(2), and TxB(2) generally correlated with enzyme transcript expression of PGES, PGDS, and TBXS, providing evidence of comparable enzyme activities. PGIS transcript was expressed only in RPM and TGEM macrophages and at an exceptionally low level, despite high metabolite production compared with other synthases. Presence of PGIS in RPM and TGEM also lowered the production of PGE(2) versus PGD(2) by approximately tenfold relative to BMDM and RAW cells, which lacked this enzyme. Our results demonstrate that delayed PG production depends on the maximal level of COX-2 expression in different macrophages after TLR-4 stimulation. Also, the same enzymes in each cell largely dictate the profile of eicosanoids produced depending on the ratios of expression between them, with the exception of PGIS, which appears to have much greater synthetic capacity and competes selectively with mPGES-1.

Interactions of estradiol and NSAIDS on carrageenan-induced hyperalgesia

How exogenous estrogen affects inflammatory responses is poorly understood despite the large numbers of women receiving estrogen-alone hormone therapy. The aim of this study was to determine if estradiol alters injury- or inflammation-induced nociceptive responses after carrageenan administration in females and whether its effects are mediated through cyclo-oxygenase (COX) and prostaglandins (PG). To this end, paw withdrawal latencies and serum levels of PGE2 and PGD2 were measured in rats treated with estradiol (0, 10, 20, and 30%) and/or SC560 (COX-1 inhibitor) or NS398 (COX-2 inhibitor) after intraplantar carrageenan administration. Estradiol significantly increased withdrawal latencies before (baseline condition) and after carrageenan administration to one hindpaw. NS398 was anti-nociceptive only in carrageenan treated animals. SC560 increased withdrawal latencies in both paws at 1 and 5hours after carrageenan administration. Co-administration of estradiol and NS398, but not SC560, was additive except for a prolonged anti-nociceptive effects of estradiol combined with NS398. The anti-nociceptive effect extended beyond that observed with either drug or estradiol alone at the 5-hour time point. Estradiol had no significant effect on PGE2 serum levels, but both COX antagonists decreased them. Although neither estradiol nor the COX inhibitors alone had an effect on PGD2 serum levels, co-administration of NS398 and estradiol significantly elevated PGD2 levels. Taken together, our results suggest that estradiol is anti-nociceptive in the thermal test and reduces carrageenan-induced hyperalgesia. These effects are minimally altered through PG-mediated mechanisms.

Angiogenic effect of laminin involves modulation of cyclooxygenase-2 and prostaglandin levels

The molecular mechanism of the angiogenic effect of laminin (Ln) was studied using human umbilical vein endothelial cells (HUVECs) maintained in culture on Ln-1 substratum. High-pressure liquid chromatography analysis showed that in cells maintained on Ln, the levels of proangiogenic prostaglandin E(2) (PGE(2)) increased and that of antiangiogenic PGD(2) decreased. The angiogenic effect of PGE(2) and PGD(2) was confirmed by assessing the expression of CD31 and E-selectin in HUVECs. Immunoblot analysis, reverse transcription-polymerase chain reaction and cyclooxygenase (COX) assay showed increase in the expression and activity of COX-2 in cells maintained on Ln. Use of pharmacological inhibitors suggested that the modulation in the expression of COX-2 and thereby the levels of PGE(2) and PGD(2) in endothelial cells by Ln is mediated through the α(6)β(4) integrin-p38MAPK (mitogen-activated protein kinase)-NF-κB signaling pathway.

Cyclooxygenase-2 Deficiency Attenuates Adipose Tissue Differentiation and Inflammation in Mice

Obesity is associated with a variety of disorders and is a significant health problem in developed countries. One factor controlling the level of adiposity is the differentiation of cells into adipocytes. Adipocyte differentiation requires expression of peroxisome proliferator-activated receptor γ (PPARγ), which is activated by ligands to regulate expression of genes involved in adipocyte differentiation. Although 15-deoxy-Δ(12,14)-prostaglandin (PG) J(2) (15d-PGJ(2)) has long been known to be a potent activator of PPARγ, the importance of its synthesis in adipose tissue in vivo is not clear. The current study utilized mice deficient in cyclooxygenase-2 (COX-2) to examine the role of COX-2-derived PGs as in vivo modulators of adiposity. As compared with strain- and age-matched wild-type controls, the genetic deficiency of COX-2 resulted in a significant reduction in total body weight and percent body fat. Although there were no significant differences in food consumption between groups, COX-2-deficient mice showed increased metabolic activity. Epididymal adipose tissue from wild-type mice produced a significantly greater level of 15d-PGJ(2), as compared with adipose tissue isolated from mice deficient in COX-2. Furthermore, production of the precursor required for 15d-PGJ(2) formation, PGD(2), was also significantly reduced in COX-2-deficient adipose tissue. The expression of markers for differentiated adipocytes was significantly reduced in adipose tissue from COX-2-deficient mice, whereas preadipocyte marker expression was increased. Macrophage-dependent inflammation was also significantly reduced in adipose tissue of COX-2-deficient mice. These findings suggest that reduced adiposity in COX-2-deficient mice results from attenuated PPARγ ligand production and adipocyte differentiation.

The Macrophage-Specific Transcription Factor Can Be Modified Posttranslationally by Ubiquitination in the Lipopolysaccharide-Treated Macrophages

Background: Macrophages are one of the most important inflammatory cells in innate immunity. PU.1 is a macrophage-specific transcription factor. Ubiquitins are the ultimate regulator of eukaryotic transcription. The ubiquitination process for PU.1 is unknown. This study investigated the lipopolysaccharide (LPS)-induced activation of PU.1 and its relation to ubiquitins in the macrophages. Methods: Raw264.7 cells, the primary cultured alveolar, pulmonary, and bone marrow derived macrophages were used. The Raw264.7 cells were treated with MG-132, NH4Cl, lactacytin and LPS. Nitric oxide and prostaglandin D2 and E2 were measured. Immunoprecipitation and Western blots were used to check ubiquitination of PU.1. Results: The PU.1 ubiquitination increased after LPS (1ug/mL) treatment for 4 hours on Raw264.7 cells. The ubiquitination of PU.1 by LPS was increased by MG-132 or NH4Cl pretreatment. Two hours of LPS treatment on macrophages, PU.1 activation was not induced nor increased with the inhibition of proteasomes and/or lysosomes. The ubiquitination of PU.1 was increased in LPS-treated Raw264.7 cells at 12- and at 24 hours. LPS-treated cells increased nitric oxide production, which was diminished by MG-132 or NH4Cl. LPS increased the production of PGE2 in the alveolar and peritoneal macrophages of wild type mice; however, PGE2 was blocked or diminished in Rac2 null mice. Pretreatment of lactacystin increased PGE2, however it decreased the PGD2 level in the macrophages derived from the bone marrow of B57/BL6 mice. Conclusion: LPS treatment in the macrophages ubiquitinates PU.1. Ubiquitination of PU.1 may be involved in synthesis of nitric oxide and prostaglandins.

Up-Regulation of the Inflammatory Response by Ovariectomy in Collagen-Induced Arthritis. Effects of Tin Protoporphyrin IX

We have studied the influence of ovariectomy on the inflammatory response and bone metabolism on CIA as a model of postmenopausal arthritis as well as the effects of tin protoporphyrin IX (SnPP), a heme oxygenase inhibitor. Ovariectomy in non-arthritic mice produced increased serum PGD2 levels and up-regulated the expression of COX-2, h-PGDS, l-PGDS, and HO-1 in the joints. In CIA, ovariectomy potentiated the inflammatory response with higher levels of serum IL-6 and MMP-3, local PGD2 and MMP-3 as well as trabecular bone erosion. In OVX-CIA, SnPP decreased the serum levels of IL-6, MMP-3, and PGD2; down-regulated TNFα, COX-2, hPGDS, PGD2, PGE2, and MMP-3 in joint tissues; and also decreased focal bone loss in the inflamed joint. Ovariectomy up-regulates inflammatory mediators in non-arthritic and in arthritic animals. In the OVX-CIA model, SnPP exerts anti-inflammatory effects which are not associated with the prevention of systemic bone loss.

The prostaglandin D 2 receptor CRTH2 is important for allergic skin inflammation after epicutaneous antigen challenge

Cutaneous prostaglandin (PG) D₂ levels increase after scratching. Chemoattractant receptor-homologous molecule expressed on receptor on T(H)2 cells (CRTH2) mediates chemotaxis to PGD₂ and is expressed on T(H)2 cells and eosinophils, which infiltrate skin lesions in patients with atopic dermatitis. We sought to examine the role of CRTH2 in a murine model of atopic dermatitis. CRTH2(-/-) mice and wild-type control animals were epicutaneously sensitized by means of repeated application of ovalbumin (OVA) to tape-stripped skin for 7 weeks and then challenged by means of OVA application to tape-stripped previously unsensitized skin for 1 week. Skin histology was assessed by means of hematoxylin and eosin staining and immunohistochemistry. Cytokine mRNA expression was examined by means of quantitative RT-PCR. Levels of PGD₂, antibody, and cytokines were measured by means of ELISA. PGD₂ levels significantly increased in skin 24 hours after tape stripping, although not in skin subjected to repeated sensitization with OVA. Allergic skin inflammation developed normally at sites of chronic epicutaneous sensitization with OVA in CRTH2(-/-) mice but was severely impaired in previously unsensitized skin challenged with OVA, as evidenced by significantly decreased skin infiltration with eosinophils and CD4(+) cells and impaired T(H)2 cytokine mRNA expression. Impaired skin inflammation at sites of acute OVA challenge in CRTH2(-/-) mice was not due to an impaired systemic response to epicutaneous sensitization because OVA-specific IgG1 and IgE antibody levels and OVA-driven splenocyte secretion of cytokines in these mice were comparable with those seen in wild-type control animals. CRTH2 promotes allergic skin inflammation in response to cutaneous exposure to antigen in previously sensitized mice.

Changes in Salivary Prostaglandin Levels During Menstrual Migraine With Associated Dysmenorrhea

To measure prostaglandin levels in the saliva of individuals during menstrual migraine associated with dysmenorrhea (MMaD) and in response to treatment with a single tablet combination of sumatriptan succinate and naproxen sodium. Prostaglandins are thought to play a role in MMaD as elevated serum prostaglandin levels have been reported during attacks of menstrual migraine and are increased in the menstrual fluid of women with dysmenorrhea. While triptans are the primary line of migraine treatment, nonsteriodal anti-inflammatory drugs are the most commonly prescribed therapy for dysmenorrhea symptoms. Data from recent clinical studies have provided evidence that treatment with a single tablet combination of sumatriptan and naproxen sodium is an effective abortive therapy for attacks of MMaD. Women diagnosed with MMaD were treated with a sumatriptan succinate and naproxen sodium single tablet combination or placebo at time of migraine attack. Saliva samples were collected at time of attack as well as 2 and 4 hours after treatment. PGD(2), PGE(2), PGF(2), PGI(2), and TXA(2) levels were determined by enzyme-linked immunosorbent assay. Elevated levels of PGD(2), PGF(2), and TXA(2) at 2 and 4 hours and PGE(2) at 4 hours were found in saliva obtained from placebo subjects when compared with onset of attack levels. However, in subjects treated with a single tablet combination of sumatriptan and naproxen sodium, the levels of PGD(2), PGF(2), and PGE(2) were not elevated at either time point while TXA(2) levels were still elevated at 4 hours. Data from this pilot study provide evidence that saliva levels of several prostaglandins increase during attacks of MMaD and that treatment with a single tablet combination of sumatriptan and naproxen sodium prevents elevation of prostaglandin levels.