leukotriene-d4 and Inflammation

This mini-review is focused on the enzyme 15-lipoxygenase-1 (15-LO-1) and eoxins in airway inflammatory diseases and Hodgkin lymphoma. Several studies have demonstrated increased expression and activity of 15-LO-1 in the respiratory tissue from asthma patients , indicating a pathophysiological role of this enzyme in airway inflammation. Eoxins were recently identified as pro-inflammatory metabolites of arachidonic acid, formed through the 15-LO-1 pathway, in human eosinophils, mast cells, airway epithelial cells and Hodgkin lymphoma. Mice deficient of 12/15-LO, the ortholog to human 15-LO-1, had an attenuated allergic airway inflammation compared to wild type controls, also indicating a pathophysiological role of this enzyme in respiratory inflammation. The putative therapeutic implications of 15-LO-1 inhibitors in the treatment of asthma, chronic obstructive pulmonary disorder and Hodgkin lymphoma are discussed.

Topics: Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Asthma; Eosinophils; Hodgkin Disease; Humans; Inflammation; Leukotriene D4; Leukotrienes; Lung; Mice; Organ Specificity; Pulmonary Disease, Chronic Obstructive; Reed-Sternberg Cells

Initiation and regulation of allergic inflammation is influenced by many factors, including cell type, membrane receptors, and mediators generated. Furthermore, the altered response of targeted tissues (ie, airway smooth muscle) becomes important to the eventual expression of asthma. Finally, the genetic regulation and association of genetic polymorphisms has enhanced our understanding of host susceptibility. In this review key findings published in 2004 issues of the Journal of Allergy and Clinical Immunology are highlighted to demonstrate recent advances in these areas.

Topics: Animals; Asthma; Basophils; Disease Models, Animal; Eosinophils; Genetic Predisposition to Disease; Humans; Inflammation; Leukotriene D4; Mast Cells; Myocytes, Smooth Muscle; Nitric Oxide; Respiratory System; T-Lymphocytes

The inflammatory mediator LTD4 (leukotriene D4) is present at high levels in many inflammatory conditions, and areas of chronic inflammation have an increased risk for subsequent cancer development. We demonstrate here that following LTD4 stimulation, beta-catenin is translocated to the nucleus, triggering the transcriptional activity of the TCF (T-cell factor)/LEF (lymphoid enhancer factor) family of transcription factors. These events are dependent on phosphoinositide-3 kinase activation and glycogen synthase kinase inhibition. Our data suggest that, similar to Wnt signalling, LTD4 increases free beta-catenin and targets it to the nucleus.

Topics: Gene Expression Regulation; Humans; Inflammation; Inflammatory Bowel Diseases; Leukotriene D4; Oncogenes; Protein Transport; Transcription, Genetic

Exposure to cigarette smoke complicates the treatment and management of asthma through a variety of inflammatory effects. This study aimed to investigate the differences between newly diagnosed cases of asthma in smokers and nonsmokers in terms of localized and systemic biomarkers following treatment with inhaled corticosteroids (ICS) or ICS in combination with a long-acting β2 agonist (LABA).. Specimens of exhaled breath condensate (EBC) from newly diagnosed patients with asthma were used to quantify inflammation in the airways, while blood samples were used to assess systemic inflammation. In both samples, the levels of IL-6, LTB4, LTD4, and 8-isoprostane were measured and these were repeated after 3 months of treatment with ICS or ICS + LABA.. Monitoring the alterations in 8-isoprostane levels in EBC in patients with asthma who smoke may be helpful in deciding on therapeutic management and switching treatments. Asthma control was better in nonsmokers than in smokers.

Topics: Adrenal Cortex Hormones; Asthma; Biomarkers; Breath Tests; Exhalation; Humans; Inflammation; Interleukin-6; Leukotriene B4; Leukotriene D4; Smoking

Cysteinyl leukotrienes (CysLTs), a group of inflammatory lipid mediators, are found elevated in obese-asthmatic patients. Leukotriene D. Primary human small airway epithelial cells (SAECs) were stimulated with different concentrations of LTD

Topics: Airway Remodeling; Allergens; Animals; Asthma; Bronchoalveolar Lavage Fluid; Cells, Cultured; Cytokines; Epithelial Cells; Humans; Inflammasomes; Inflammation; Leukocyte Count; Leukotriene D4; Male; Mice, Inbred BALB C; Mucin 5AC; NLR Family, Pyrin Domain-Containing 3 Protein; Obesity; Ovalbumin; Smad2 Protein; Smad3 Protein; Vimentin

Although arachidonic acid metabolites, cysteinyl leukotrienes (cys-LTs; leukotriene [LT] C4, LTD4, and LTE4), and prostaglandin (PG) E2 are generated at the site of inflammation, it is not known whether crosstalk exists between these 2 classes of inflammatory mediators.. We sought to determine the role of LTD4-PGE2 crosstalk in inducing vascular inflammation in vivo, identify effector cells, and ascertain specific receptors and pathways involved in vitro.. Vascular (ear) inflammation was assessed by injecting agonists into mouse ears, followed by measuring ear thickness and histology, calcium influx with Fura-2, phosphorylation and expression of signaling molecules by means of immunoblotting, PGD2 and macrophage inflammatory protein 1β generation by using ELISA, and expression of transcripts by using RT-PCR. Candidate receptors and signaling molecules were identified by using antagonists and inhibitors and confirmed by using small interfering RNA.. LTD4 plus PGE2 potentiated vascular permeability and edema, gearing the system toward proinflammation in wild-type mice but not in Kit(W-sh) mice. Furthermore, LTD4 plus PGE2, through cysteinyl leukotriene receptor 1 (CysLT1R) and E-prostanoid receptor (EP) 3, enhanced extracellular signal-regulated kinase (Erk) and c-fos phosphorylation, inflammatory gene expression, macrophage inflammatory protein 1β secretion, COX-2 upregulation, and PGD2 generation in mast cells. Additionally, we uncovered that this synergism is mediated through Gi, protein kinase G, and Erk signaling. LTD4 plus PGE2-potentiated effects are partially sensitive to CysLT1R or EP3 antagonists but completely abolished by simultaneous treatment both in vitro and in vivo.. Our results unravel a unique LTD4-PGE2 interaction affecting mast cells through CysLT1R and EP3 involving Gi, protein kinase G, and Erk and contributing to vascular inflammation in vivo. Furthermore, current results also suggest an advantage of targeting both CysLT1R and EP3 in attenuating inflammation.

Topics: Animals; Capillary Permeability; Cell Line; Cell Line, Tumor; Dinoprostone; Edema; Humans; Inflammation; Leukotriene D4; Mast Cells; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Receptors, Leukotriene; Receptors, Prostaglandin E, EP3 Subtype

Inflammation is a major burden in respiratory diseases, resulting in airway hyperresponsiveness. Our hypothesis is that resolution of inflammation may represent a long-term solution in preventing human bronchial dysfunctions. The aim of the present study was to assess the anti-inflammatory effects of RvD2, a member of the D-series resolving family, with concomitant effects on ASM mechanical reactivity. The role and mode of action of RvD2 were assessed in an in vitro model of human bronchi under pro-inflammatory conditions, induced either by 1 μM LTD4 or 10 ng/ml TNF-α pre-treatment for 48h. TNF-α and LTD4 both induced hyperreactivity in response to pharmacological stimuli. Enhanced 5-Lipoxygenase (5-LOX) and cysteinyl leukotriene receptor 1 (CysLTR1) detection was documented in LTD4 or TNF-α pre-treated human bronchi when compared to control (untreated) human bronchi. In contrast, RvD2 treatments reversed 5-LOX/β-actin and CysLTR1/β-actin ratios and decreased the phosphorylation levels of AP-1 subunits (c-Fos, c-Jun) and p38-MAP kinase, while increasing the detection of the ALX/FPR2 receptor. Moreover, various pharmacological agents revealed the blunting effects of RvD2 on LTD4 or TNF-α induced hyper-responsiveness. Combined treatment with 300 nM RvD2 and 1 μM WRW4 (an ALX/FPR2 receptor inhibitor) blunted the pro-resolving and broncho-modulatory effects of RvD2. The present data provide new evidence regarding the role of RvD2 in a human model of airway inflammation and hyperrresponsiveness.

Topics: Actins; Arachidonate 5-Lipoxygenase; Blotting, Western; Bronchi; Bronchoconstriction; Docosahexaenoic Acids; Humans; Inflammation; Leukotriene D4; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Receptors, Leukotriene; Tissue Culture Techniques; Transcription Factor AP-1; Tumor Necrosis Factor-alpha

The role of inflammatory lipid-mediators in tumor progression is well recognized in colorectal cancer; however, if this includes promotion of cancer-initiating cells remains unclear. We show that the inflammatory lipid-mediators leukotriene D4 and prostaglandin E2 increased the Aldehyde dehydrogenase (ALDH(+) ) population, the colony formation capacity, and tumor growth in a xenograft model of colon cancer. The ALDH(+) cells showed significant resistance to irradiation and 5-fluorouracil treatment that could be further augmented by these lipid-mediators, occurring in parallel with increased target gene expression. Our data emphasize a role for tumor microenvironment derived inflammatory lipid-mediators to favor cancer stem cells-like characteristics and thus promote tumor progression.

Topics: Animals; Caco-2 Cells; Cell Line, Tumor; Colonic Neoplasms; Dinoprostone; Disease Progression; Drug Resistance, Neoplasm; Female; Fluorouracil; HCT116 Cells; HT29 Cells; Humans; Inflammation; Leukotriene D4; Lipids; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplastic Stem Cells; Tumor Microenvironment; Xenograft Model Antitumor Assays

Mast cells (MCs) are important effector cells in asthma and pulmonary inflammation, and their proliferation and maturation is maintained by stem cell factor (SCF) via its receptor, c-Kit. Cysteinyl leukotrienes (cys-LTs) are potent inflammatory mediators that signal through CysLT1 R and CysLT2 R located on the MC surface, and they enhance MC inflammatory responses. However, it is not known if SCF and cys-LTs cross-talk and influence MC hyperplasia and activation in inflammation. Here, we report the concerted effort of the growth factor SCF and the inflammatory mediator LTD4 in MC activation. Stimulation of MCs by LTD4 in the presence of SCF enhances c-Kit-mediated proliferative responses. Similarly, SCF synergistically enhances LTD4 -induced calcium, c-fos expression and phosphorylation, as well as MIP1β generation in MCs. These findings suggest that integration of SCF and LTD4 signals may contribute to MC hyperplasia and hyper-reactivity during airway hyper-response and inflammation.

Topics: Calcium; Cell Proliferation; Humans; Inflammation; Leukotriene D4; Mast Cells; Mastocytosis; Phosphorylation; Proto-Oncogene Proteins c-kit; Receptors, Leukotriene; Signal Transduction; Stem Cell Factor

Inflammation and altered immunity are recognized components of severe pulmonary arterial hypertension in human patients and in animal models of PAH. While eicosanoid metabolites of cyclooxygenase and lipoxygenase pathways have been identified in the lungs from pulmonary hypertensive animals their role in the pathogenesis of severe angioobliterative PAH has not been examined. Here we investigated whether a cyclooxygenase-2 (COX-2) inhibitor or diethylcarbamazine (DEC), that is known for its 5-lipoxygenase inhibiting and antioxidant actions, modify the development of PAH in the Sugen 5416/hypoxia (SuHx) rat model. The COX-2 inhibitor SC-58125 had little effect on the right ventricular pressure and did not prevent the development of pulmonary angioobliteration. In contrast, DEC blunted the muscularization of pulmonary arterioles and reduced the number of fully obliterated lung vessels. DEC treatment of SuHx rats, after the lung vascular disease had been established, reduced the degree of PAH, the number of obliterated arterioles and the degree of perivascular inflammation. We conclude that the non-specific anti-inflammatory drug DEC affects developing PAH and is partially effective once angioobliterative PAH has been established.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 5-Lipoxygenase; Arterioles; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Diethylcarbamazine; Dinoprost; Dinoprostone; Humans; Hypertension, Pulmonary; Hypoxia; Inflammation; Leukotriene D4; Lipoxygenase Inhibitors; Lung; Male; Prostaglandins F; Pulmonary Artery; Pyrazoles; Rats; Rats, Sprague-Dawley; Thromboxane B2; Ventricular Function, Right

The cysteinyl leukotrienes (cys-LTs), leukotriene C4 (LTC4), a conjugation product of glutathione and eicosatetraenoic acid, and its metabolites, LTD4 and LTE4, are lipid mediators of smooth muscle constriction and inflammation in asthma. LTD4 is the most potent ligand for the type 1 cys-LT receptor (CysLT1R), and LTC4 and LTD4 have similar lesser potency for CysLT2R, whereas LTE4 has little potency for either receptor. Cysltr1/Cysltr2(-/-) mice, lacking the two defined receptors, exhibited a comparable dose-dependent vascular leak to intradermal injection of LTC4 or LTD4 and an augmented response to LTE4 as compared with WT mice. As LTE4 retains a cysteine residue and might provide recognition via a dicarboxylic acid structure, we screened cDNAs within the P2Y nucleotide receptor family containing CysLTRs and dicarboxylic acid receptors with trans-activator reporter gene assays. GPR99, previously described as an oxoglutarate receptor (Oxgr1), showed both a functional and a binding response to LTE4 in these transfectants. We generated Gpr99(-/-) and Gpr99/Cysltr1/Cysltr2(-/-) mice for comparison with WT and Cysltr1/Cysltr2(-/-) mice. Strikingly, GPR99 deficiency in the Cysltr1/Cysltr2(-/-) mice virtually eliminated the vascular leak in response to the cys-LT ligands, indicating GPR99 as a potential CysLT3R active in the Cysltr1/Cysltr2(-/-) mice. Importantly, the Gpr99(-/-) mice showed a dose-dependent loss of LTE4-mediated vascular permeability, but not to LTC4 or LTD4, revealing a preference of GPR99 for LTE4 even when CysLT1R is present. As LTE4 is the predominant cys-LT species in inflamed tissues, GPR99 may provide a new therapeutic target.

Topics: Animals; Capillary Permeability; Inflammation; Leukotriene C4; Leukotriene D4; Leukotriene E4; Ligands; Mice; Mice, Inbred BALB C; Mice, Knockout; Receptors, Leukotriene

Gastrointestinal eosinophilic (EG) is a rare and heterogeneous condition characterized by patchy or diffuse eosinophilic infiltration of gastrointestinal tissue. Pharmacological study so far has demonstrated that montelukast, an oral leukotriene receptor antagonist, might be considered in patients with this disease. The aim of this study was to evaluate the effect of montelukast on oral ovalbumin (OVA) allergen induced EG inflammation in mice and to suggest some mechanisms underlying this effect. Twenty-four mice were divided into three experimental groups: PBS control, OVA group, and montelukast treated group. The mice were sensitized intraperitoneally and challenged intragastrically with OVA, and were treated with montelukast. Gastrointestinal symptoms were observed after challenged intragastrically with OVA. Eosinophils count in blood, serum OVA specific IgE and gastrointestinal histology were evaluated. Montelukast could significantly reduce the severity of oral allergen-induced eosinophilic inflammation, villous atrophy, and associated symptoms of weight loss associated with diarrhea. Montelukast also could ameliorate OVA-induced gastrointestinal pathological lesions, which was associated with the decrease of IgE and LTD4 levels, and this might be one of the important mechanisms of montelukast that protected gastrointestinal injury from EG. These findings indicated that montelukast therapy may be a novel therapeutic approach for EG and other eosinophil-mediated diseases.

Topics: Acetates; Animals; Body Weight; Cyclopropanes; Enteritis; Eosinophilia; Eosinophils; Female; Gastric Mucosa; Gastritis; Gastroenteritis; Immunoglobulin E; Inflammation; Jejunum; Leukotriene D4; Mice; Mice, Inbred BALB C; Ovalbumin; Quinolines; Stomach; Sulfides

Airway epithelial cells play a central role in the physiopathology of asthma. They release eotaxins when treated with T(H)2 cytokines such as interleukin (IL)-4 or IL-13, and these chemokines attract eosinophils and potentiate the biosynthesis of cysteinyl leukotrienes (cysLTs), which in turn induce bronchoconstriction and mucus secretion. These effects of cysLTs mainly mediated by CysLT(1) and CysLT(2) receptors on epithelial cell functions remain largely undefined. Because the release of inflammatory cytokines, eotaxins, and cysLTs occur relatively at the same time and location in the lung tissue, we hypothesized that they regulate inflammation cooperatively rather than redundantly. We therefore investigated whether cysLTs and the T(H)2 cytokines would act in concert to augment the release of eotaxins by airway epithelial cells.. A549 cells or human primary bronchial epithelial cells were incubated with or without IL-4, IL-13, and/or LTD(4). The release of eotaxin-3 and the expression of cysLT receptors were assessed by ELISA, RT-PCR, and flow cytometry, respectively.. IL-4 and IL-13 induced the release of eotaxin-3 by airway epithelial cells. LTD(4) weakly induced the release of eotaxin-3 but clearly potentiated the IL-13-induced eotaxin-3 release. LTD(4) had no effect on IL-4-stimulated cells. Epithelial cells expressed CysLT(1) but not CysLT(2). CysLT(1) expression was increased by IL-13 but not by IL-4 and/or LTD(4). Importantly, the upregulation of CysLT(1) by IL-13 preceded eotaxin-3 release.. These results demonstrate a stepwise cooperation between IL-13 and LTD(4). IL-13 upregulates CysLT(1) expression and consequently the response to cysLTs This results in an increased release of eotaxin-3 by epithelial cells which at its turn increases the recruitment of leukocytes and their biosynthesis of cysLTs. This positive amplification loop involving epithelial cells and leukocytes could be implicated in the recruitment of eosinophils observed in asthmatics.

Topics: Asthma; Bronchi; Chemokine CCL24; Chemokine CCL26; Chemokines, CC; Cysteine; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Flow Cytometry; Gene Expression Regulation; Humans; Inflammation; Interleukin-13; Interleukin-4; Kinetics; Leukotriene D4; Leukotrienes; Models, Biological; Recombinant Proteins; Th2 Cells

To investigate the sensitizing effects of the cysteinyl leukotrienes (CysLTs) C(4) and D(4) on the proinflammatory responses of chemoattractant-activated human neutrophils in vitro.. Neutrophils were isolated from venous blood taken from healthy, adult, human volunteers.. Cells were exposed to LTC(4) and LTD(4) (50-300 nM) prior to activation with 1 microM of N-formyl-L-methionyl- L-leucyl-L-phenylalanine (fMLF).. A fura-2/AM-based spectrofluorimetric procedure, lucigenin-enhanced chemiluminescence (LECL), a colourimetric method and an ELISA procedure, were used to measure Ca(2+) mobilization, superoxide production, elastase and MMP-8 release respectively following activation of LTC(4)/ D(4)-primed neutrophils with fMLF. Superoxide generation was also measured in the presence and absence of the CysLT receptor 1 antagonist, montelukast (100 nM).. Exposure of neutrophils to either LTC(4) or LTD(4) alone had modest effects on Ca(2+) mobilization, while superoxide generation and elastase release were unaffected. However, relative to the responses of neutrophils activated with fMLF in the absence of the CysLTs, pre-treatment of the cells with either LTC(4)or LTD(4) resulted in significant, augmentation of fMLF-activated elastase and MMP-8 release and superoxide generation, which was attenuated by montelukast.. These previously undocumented sensitizing interactions of LTs C(4) and D(4) with neutrophils may contribute to the activation of these cells in acute and chronic inflammation of both atopic and non-atopic aetiology, while identifying a role for montelukast in regulating neutrophil reactivity.

Topics: Acetates; Adult; Chemotactic Factors; Cyclopropanes; Fluorescent Dyes; Fura-2; Humans; Inflammation; Leukotriene Antagonists; Leukotriene C4; Leukotriene D4; Matrix Metalloproteinase 8; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pancreatic Elastase; Quinolines; Sulfides; Superoxides

The chronicity of bronchial asthma is attributed to persistent airway inflammation and to a variety of structural changes, or remodelling, that includes smooth muscle and goblet cell hyperplasia. To investigate the mechanisms of airway remodelling, the current authors used an established allergen (ovalbumin; OVA)-driven rodent model (the Brown Norway rat). Brown Norway rats were sensitised to OVA and challenged three times at 5-day intervals to evoke airway remodelling. The effects of an epidermal growth factor (EGF) receptor inhibitor, AG1478, and a cysteinyl leukotriene-1 receptor antagonist, montelukast, on epithelial and airway smooth muscle (ASM) cell proliferation in vivo in response to repeated OVA challenge were tested. Three challenges with leukotriene (LT)D(4) were given, to examine their effects on remodelling with and without AG1478 pretreatment. OVA challenges caused ASM hyperplasia, with an increase in mass, epithelial cell proliferation and goblet cell proliferation. AG1478 prevented the changes, as did montelukast. Multiple OVA challenges increased heparin-binding EGF-like growth factor but not EGF expression by airway epithelium. LTD(4) reproduced the changes in remodelling induced by OVA and this was blocked by AG1478. Allergen-induced airway epithelial and airway smooth muscle remodelling is mediated by cysteinyl leukotrienes via the cysteinyl leukotriene-1 receptor with downstream effects on the epidermal growth factor receptor axis.

Topics: Allergens; Animals; Cell Proliferation; Cysteine; ErbB Receptors; Gene Expression Profiling; Goblet Cells; Hyperplasia; Inflammation; Leukotriene D4; Muscle, Smooth; Ovalbumin; Quinazolines; Rats; Receptors, Leukotriene; Tyrphostins

Cysteinyl leukotrienes (cys-LTs) are potent inflammatory lipid mediators, of which leukotriene (LT) E(4) is the most stable and abundant in vivo. Although only a weak agonist of established G protein-coupled receptors (GPCRs) for cys-LTs, LTE(4) potentiates airway hyper-responsiveness (AHR) by a cyclooxygenase (COX)-dependent mechanism and induces bronchial eosinophilia. We now report that LTE(4) activates human mast cells (MCs) by a pathway involving cooperation between an MK571-sensitive GPCR and peroxisome proliferator-activated receptor (PPAR)gamma, a nuclear receptor for dietary lipids. Although LTD(4) is more potent than LTE(4) for inducing calcium flux by the human MC sarcoma line LAD2, LTE(4) is more potent for inducing proliferation and chemokine generation, and is at least as potent for upregulating COX-2 expression and causing prostaglandin D(2) (PGD(2)) generation. LTE(4) caused phosphorylation of extracellular signal-regulated kinase (ERK), p90RSK, and cyclic AMP-regulated-binding protein (CREB). ERK activation in response to LTE(4), but not to LTD(4), was resistant to inhibitors of phosphoinositol 3-kinase. LTE(4)-mediated COX-2 induction, PGD(2) generation, and ERK phosphorylation were all sensitive to interference by the PPARgamma antagonist GW9662 and to targeted knockdown of PPARgamma. Although LTE(4)-mediated PGD(2) production was also sensitive to MK571, an antagonist for the type 1 receptor for cys-LTs (CysLT(1)R), it was resistant to knockdown of this receptor. This LTE(4)-selective receptor-mediated pathway may explain the unique physiologic responses of human airways to LTE(4) in vivo.

Topics: Cell Proliferation; Chemokines; Dose-Response Relationship, Drug; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Inflammation; Leukemia; Leukotriene D4; Leukotriene E4; Lipids; Mast Cells; Models, Biological; PPAR gamma; Prostaglandin D2

Both cysteinyl-leukotrienes and extracellular nucleotides mediate inflammatory responses via specific G-protein-coupled receptors, the CysLT and the P2Y receptors, respectively. Since these mediators accumulate at sites of inflammation, and inflammatory cells express both classes of receptors, their responses are likely to be crossregulated. We investigated the molecular basis of desensitization and trafficking of the CysLT1 receptor constitutively and transiently expressed in the human monocyte/macrophage-like U937 or COS-7 cells in response to LTD4 or nucleotides. Exposure to agonist induced a rapid homologous desensitization of the CysLT1 receptor [as measured by the reduction in the maximal agonist-induced intracellular cytosolic Ca2+ ([Ca2+]i) transient], followed by receptor internalization (as assessed by equilibrium binding and confocal microscopy). Activation of P2Y receptors with ATP or UDP induced heterologous desensitization of the CysLT1 receptor. Conversely, LTD4-induced CysLT1 receptor activation had no effect on P2Y receptor responses, which suggests that the latter have a hierarchy in producing desensitizing signals. Furthermore, ATP/UDP-induced CysLT1 receptor desensitization was unable to cause receptor internalization, induced a faster recovery of CysLT1 functionality and was dependent upon protein kinase C. By contrast, homologous desensitization, which is probably dependent upon G-protein-receptor kinase 2 activation, induced a fast receptor downregulation and, accordingly, a slower recovery of CysLT1 functionality. Hence, CysLT1 receptor desensitization and trafficking are differentially regulated by the CysLT1 cognate ligand or by extracellular nucleotides. This crosstalk may have a profound physiological implication in the regulation of responses at sites of inflammation, and may represent just an example of a feedback mechanism used by cells to fine-tune their responses.

Topics: Adenosine Triphosphate; Animals; Calcium; Chlorocebus aethiops; COS Cells; Dose-Response Relationship, Drug; Extracellular Fluid; Feedback, Physiological; Gene Expression Regulation; Humans; Immunohistochemistry; Inflammation; Leukotriene D4; Membrane Proteins; Nucleotides; Paclitaxel; Peptides, Cyclic; Protein Kinases; Receptors, Leukotriene; Receptors, Purinergic P2; Second Messenger Systems; U937 Cells; Uridine Diphosphate

Inflammatory bowel diseases are associated with increased risk of developing colon cancer. A possible role of the pro-inflammatory leukotriene D4 (LTD4) in this process has been implicated by the findings that LTD4 can signal increased proliferation and survival, both hallmarks of a cancer cell, in non-transformed intestinal epithelial cells. Here we make the novel finding that LTD4 can also signal increased motility in these cells. In parallel, we found that LTD4 induced a simultaneous transient 10-fold increase in Rac but not Cdc42 activity. These data were also supported by the ability of LTD4 to activate the Rac GDP/GTP exchange factor Vav2. Further, LTD4 triggered a 3-fold transient increase in phosphatidylinositol 3-kinase (PI3K) phosphorylation, a possible upstream activator of the Vav2/Rac signaling pathway. The activation of Rac was blocked by the PI3K inhibitors LY294002 and wortmannin and by transfection of a kinase-negative mutant of PI3K or a dominant-negative form of Vav2. Furthermore, Rac was found to co-localize with actin in LTD4-generated membrane ruffles that were formed by a PI3K-dependent mechanism. In accordance, the inhibition of the PI3K and Rac signaling pathway also blocked the LTD4-induced migration of the intestinal cells. The present data reveal that an inflammatory mediator such as LTD4 cannot only increase proliferation and survival of non-transformed intestinal epithelial cells but also, via a PI3K/Rac signaling pathway, trigger a motile response in such cells. These data demonstrate the capacity of inflammatory mediators to participate in the process by which inflammatory bowel conditions increase the risk for colon cancer development.

Topics: Actins; Cell Line; Cell Movement; Cell Surface Extensions; Epithelial Cells; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Inflammation; Intestinal Mucosa; Leukotriene D4; Oncogene Proteins; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein Subunits; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-vav; rac GTP-Binding Proteins; Signal Transduction

A previous study showed that the novel tetrazolephtalimide derivative LASSBio 552 (2-4-[3-(1H-1,2,3,4-tetraazol-5-yl)propoxy]phenethyl-1,3-isoindolinedione) prevents LTD(4)-evoked tracheal contraction. This led us to examine the putative anti-inflammatory effect of LASSBio 552 in comparison with the leukotriene CysLT(1) receptor antagonist zafirlukast using a model of allergic pleurisy in rats. Treatment with either LASSBio 552 (24-96 micromol/kg, i.p.) or zafirlukast (9-72 micromol/kg, i.p.), 1 h before challenge, inhibited eosinophil and mononuclear cell influx into the pleural cavity 24 h post-challenge, but failed to alter the increased levels of eotaxin, plasma leakage, mast cell degranulation and neutrophil infiltration noted 6 h post-challenge. CD4(+) T cell recruitment 24 h post-challenge was also sensitive to LASSBio 552. This treatment failed to alter cysteinyl leukotriene production at 6 h, but clearly inhibited the phenomenon 24 h and 48 h post-challenge. In in vitro settings LASSBio 552 inhibited allergen-evoked cysteinyl leukotriene generation from isolated mast cells, while histamine release remained unchanged. It also slightly inhibited cysteinyl leukotriene production by eosinophils and mononuclear cells triggered by Ca(+2) ionophore A23187. A leukotriene CysLT(1) receptor transfected cell-based assay revealed that LASSBio 552 did not prevent LTD(4)-evoked Ca(+2) influx, indicating that it was not a leukotriene CysLT(1) receptor antagonist. These findings indicate that LASSBio 552 is able to inhibit eosinophil influx triggered by allergen chalenge in a mechanism at least partially associated with suppression of CD4(+) T cell influx and cysteinyl leukotriene production.

Topics: Allergens; Animals; Anti-Asthmatic Agents; Calcium; CD4-Positive T-Lymphocytes; Cell Movement; Chemokine CCL11; Chemokines, CC; CHO Cells; Cricetinae; Cricetulus; Cysteine; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Eosinophils; Female; Indoles; Inflammation; Isoindoles; Leukotriene D4; Leukotrienes; Male; Membrane Proteins; Phenylcarbamates; Pleura; Pleurisy; Rats; Rats, Wistar; Receptors, Leukotriene; Sulfonamides; Tetrazoles; Tosyl Compounds; Transfection

Inhalation of heparin attenuates hyperventilation-induced bronchoconstriction in humans and dogs. The purpose of this study was to determine whether heparin inhibits the late-phase response to hyperventilation, which is characterized by increased peripheral airway resistance (RP), eicosanoid mediator production, neutrophilic/ eosinophilic inflammation, and airway hyperreactivity (AHR) at 5 h after dry air challenge (DAC). Fiberoptic bronchoscopy was used to record RP and airway reactivity (DeltaRP) to aerosol and intravenous histamine before and 5 h after DAC. Bronchoalveolar lavage fluid (BALF) cells and eicosanoid mediators were also measured approximately 5 h after DAC. DAC of vehicle-treated bronchi resulted in late-phase airway obstruction (approximately 120% increase over baseline RP), inflammation, increased BALF concentrations of leukotriene (LT) C(4), LTD(4), and LTE(4) and prostaglandin (PG)D(2), and AHR. Pretreatment with aerosolized heparin attenuated late-phase airway obstruction by approximately 50%, inhibited eosinophil infiltration, reduced BALF concentrations of LTC(4), LTD(4), and LTE(4) and PGD(2), and abolished AHR. We conclude that heparin inhibits hyperventilation-induced late-phase changes in peripheral airway function, and does so in part via the inhibition of eosinophil migration and eicosanoid mediator production and release.

Topics: Administration, Inhalation; Airway Resistance; Animals; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Bronchoscopy; Disease Models, Animal; Dogs; Drug Evaluation, Preclinical; Eicosanoids; Eosinophils; Heparin; Humans; Hyperventilation; Inflammation; Leukotriene C4; Leukotriene D4; Leukotriene E4; Male; Neutrophils; Prostaglandin D2; Time Factors

Aspirin-triggered lipoxin A(4) (ATL, 15-epi-LXA(4)) and leukotriene D(4) (LTD(4)) possess opposing vascular actions mediated via receptors distinct from the LXA(4) receptor (ALX) that is involved in leukocyte trafficking. Here, we identified these receptors by nucleotide sequencing and demonstrate that LTD(4) receptor (CysLT(1)) is induced in human vascular endothelia by interleukin-1beta. Recombinant CysLT(1) receptor gave stereospecific binding with both [(3)H]-LTD(4) and a novel labeled mimetic of ATL ([(3)H]-ATLa) that was displaced with LTD(4) and ATLa ( approximately IC(50) 0.2 to 0.9 nmol/L), but not with a bioinactive ATL isomer. The clinically used CysLT(1) receptor antagonist, Singulair, showed a lower rank order for competition with [(3)H]-ATLa (IC(50) approximately 8.3 nmol/L). In contrast, LTD(4) was an ineffective competitive ligand for recombinant ALX receptor with [(3)H]-ATLa, and ATLa did not compete for [(3)H]-LTB(4) binding with recombinant LTB(4) receptor. Endogenous murine CysLT(1) receptors also gave specific [(3)H]-ATLa binding that was displaced with essentially equal affinity by LTD(4) or ATLa. Systemic ATLa proved to be a potent inhibitor (>50%) of CysLT(1)-mediated vascular leakage in murine skin (200 microg/kg) in addition to its ability to block polymorphonuclear leukocyte recruitment to dorsal air pouch (4 microg/kg). These results indicate that ATL and LTD(4) bind and compete with equal affinity at CysLT(1), providing a molecular basis for aspirin-triggered LXs serving as a local damper of both vascular CysLT(1) signals as well as ALX receptor-regulated polymorphonuclear leukocyte traffic.

Topics: Animals; Aspirin; Binding, Competitive; Capillary Permeability; Cell Line; COS Cells; Dose-Response Relationship, Drug; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Inflammation; Leukotriene D4; Lipoxins; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Neutrophils; Receptors, Cell Surface; Receptors, Formyl Peptide; Receptors, Leukotriene; Receptors, Leukotriene B4; Receptors, Lipoxin; Recombinant Proteins; RNA; Stereoisomerism; Tritium

To study the function of gamma-glutamyl leukotrienase (GGL), a newly identified member of the gamma-glutamyl transpeptidase (GGT) family, we generated null mutations in GGL (GGL(tm1)) and in both GGL and GGT (GGL(tm1)-GGT(tm1)) by a serial targeting strategy using embryonic stem cells. Mice homozygous for GGL(tm1) show no obvious phenotypic changes. Mice deficient in both GGT and GGL have a phenotype similar to the GGT-deficient mice, but approximately 70% of these mice die before 4 weeks of age, at least 2 months earlier than mice deficient only in GGT. These double-mutant mice are unable to cleave leukotriene C(4) (LTC(4)) to LTD(4), indicating that this conversion is completely dependent on the two enzymes, and in some organs (spleen and uterus) deletion of GGL alone abolished more than 90% of this activity. In an experimental model of peritonitis, GGL alone is responsible for the generation of peritoneal LTD(4). Further, during the development of peritonitis, GGL-deficient mice show an attenuation in neutrophil recruitment but not of plasma protein influx. These findings demonstrate an important role for GGL in the inflammatory response and suggest that LTC(4) and LTD(4) have distinctly different functions in the inflammatory process.

Topics: Animals; Dipeptidases; Gene Expression Regulation; Gene Targeting; Inflammation; Leukotriene D4; Mice; Mutation

A method has been developed for the direct quantification of the CD11b integrin on granulocytes by flow cytometric analysis of whole blood specimens following either LTB(4) or lipopolysaccharide (LPS) stimulation. This method has utility in evaluating the pharmacodynamic action of either LTB(4) receptor antagonists or immune cell modulators in effecting CD11b integrin expression and granulocyte activation in human subjects administered such drugs. Previous studies using CD11b as a biomarker of granulocyte activation have faltered because of the difficulty in controlling the activation state of the granulocyte following removal of blood from subjects. The present study has made use of a newly validated method using either LTB(4) or LPS to stimulate CD11b expression on granulocytes and has been used, as one measure, in the evaluation of LPS activity when administered to normal human volunteers.

Topics: Acrylates; Adjuvants, Immunologic; Binding, Competitive; Flow Cytometry; Humans; Inflammation; Integrins; Leukotriene B4; Leukotriene D4; Lipopolysaccharides; Macrophage-1 Antigen; Male; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pyridines; Receptors, Leukotriene B4

Myrtol standardized (Gelomyrtol/Gelomyrtol forte) inhibits the activity of 5-lipoxygenase of human basophil and eosinophil leukocytes and the formation of leukotriene C4 as well as 1.8-cineole (eucalyptol). An increase of prostaglandins (PGE2) in mucous membranes of teat cisterns after topical administration of TPA (tetradecanoylphorbol-13-acetate) was inhibited. In vitro and in vivo studies revealed spasmolytic and broncholytic effects of Myrtol stand. After topical administration into the teat cisterns of the isolated bovine udder Myrtol stand. increased the surface temperature, comparable to effects of menthol.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Basophils; Cattle; Cyclohexanols; Dinoprostone; Drug Combinations; Drug Interactions; Eosinophils; Eucalyptol; Female; Guinea Pigs; Humans; Hyperemia; In Vitro Techniques; Inflammation; Leukemia, Basophilic, Acute; Leukotriene C4; Leukotriene D4; Leukotriene E4; Male; Mammary Glands, Animal; Menthol; Mice; Monoterpenes; Mucous Membrane; Muscle Contraction; Muscle, Smooth; Rats; Terpenes; Tetradecanoylphorbol Acetate; Trachea; Tumor Cells, Cultured

Topics: Animals; Humans; Inflammation; Leukotriene B4; Leukotriene D4; Lipid Metabolism; Lipoxygenase Inhibitors; Tumor Necrosis Factor-alpha

Sickle cell (HbSS) disease is associated with rheological and inflammatory stresses within the microcirculation. In order to determine the role of leukotrienes in the inflammatory processes in HbSS patients, we analysed plasma and urine levels of leukotrienes (LT); LTB4, LTC4, LTD4, and LTE4 as indicators of their in vivo metabolism. Plasma and urine level samples of 15 HbSS patients in steady-state and age-matched healthy, homozygous (HbAA) controls were extracted for leukotrienes and quantitated by HPLC. Control plasma level of leukotrienes (mean +/- SEM, ng ml-1) were: LTB4, 8.95 +/- 0.26; LTC4, 7.24 +/- 0.21; LTD4, 11.42 +/- 0.40; and LTE4, 14.51 +/- 0.50. Corresponding values for HbSS patients were: LTB4, 6.15 +/- 0.42; LTC4, 13.61 +/- 1.45; LTD4, 6.44 +/- 0.51 and LTE4, 4.97 +/- 0.37. The differences were significant at P < 0.05. Urine levels (mean +/- SEM, ng mmol-1 creatinine), for controls were: LTB4, 10.60 +/- 0.35; LTC4, 360.0 +/- 9.82. Values for HbSS urine were: LTB4, 27.50 +/- 3.33; LTC4, 356.0 +/- 17.87; LTD4, 69.90 +/- 14.51. LTD4 was not detected in control urine. These results suggest that sickle cell patients may exhibit impaired ability to catabolize LTC4 in plasma during steady state conditions. This altered metabolism may contribute to the persistent stress of the microcirculation, and is probably related to the abnormal microvascular rheology of sickle blood cells.

Topics: Adult; Anemia, Sickle Cell; Cell Adhesion; Endothelium, Vascular; Female; Humans; Inflammation; Leukocytes; Leukotriene B4; Leukotriene C4; Leukotriene D4; Leukotriene E4; Leukotrienes; Male; Middle Aged

Propolis is a natural product produced by the honey bee. The extract contains amino acids, flavanoids, terpenes and cinnamic acid derivatives. In various in vitro models propolis extract was shown to inhibit platelet aggregation and to inhibit eicosanoid synthesis, suggesting that it might have potent antiinflammatory properties. A 13% aqueous extract was tested orally in three dose levels (1, 5 and 10 ml/kg) on the carrageenan rat paw oedema model and on adjuvant-induced arthritis in rats. In both models, the extract showed potent dose-related antiinflammatory activity, which compared well with that of diclofenac (as a reference standard). The extract was then tested on an isolated sensitized guinea pig lung preparation to study its effect on the release of prostaglandins, leukotrienes and histamine. It is concluded that propolis extract has potent antiinflammatory properties in vivo. Its activity can be well correlated with its effects on the release of various mediators of inflammation.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arthritis, Experimental; Carrageenan; Diclofenac; Dinoprostone; Dose-Response Relationship, Drug; Edema; Guinea Pigs; Histamine Release; In Vitro Techniques; Inflammation; Leukotriene D4; Lung; Male; Propolis; Prostaglandins; Rats