Suppressive effect of tamarixetin, isolated from Inula japonica, on degranulation and eicosanoid production in bone marrow-derived mast cells.

BACKGROUND: The aim of this study was to evaluate the inhibitory effect of tamarixetin on the production of inflammatory mediators in IgE/antigen-induced mouse bone marrow-derived mast cells (BMMCs). MATERIALS AND METHODS: The effects of tamarixetin on mast cell activation were investigated with regard to degranulation, eicosanoid generation, Ca2+ influx, and immunoblotting of various signaling molecules. RESULTS: Tamarixetin effectively decreased degranulation and the eicosanoid generation such as leukotriene C4 and prostaglandin D2 in BMMCs. To elucidate the mechanism involved, we investigated the effect of tamarixetin on the phosphorylation of signal molecules. Tamarixetin inhibited the phosphorylation of Akt and its downstream signal molecules including IKK and nuclear factor κB. In addition, tamarixetin downregulated the phosphorylation of cytosolic phospholipase A2 (cPLA2) and p38 mitogen-activated protein kinase. CONCLUSIONS: Taken together, this study suggests that tamarixetin inhibits degranulation and eicosanoid generation through the PLCγ1 as well as Akt pathways in BMMCs, which would be potential for the prevention of allergic inflammatory diseases.

Roles of cysteinyl leukotrienes and their receptors in immune cell-related functions.

The cysteinyl leukotrienes (cys-LTs), leukotriene C4, (LTC4), LTD4, and LTE4, are lipid mediators of inflammation. LTC4 is the only intracellularly synthesized cys-LT through the 5-lipoxygenase and LTC4 synthase pathway and after transport is metabolized to LTD4 and LTE4 by specific extracellular peptidases. Each cys-LT has a preferred functional receptor in vivo; LTD4 to the type 1 cys-LT receptor (CysLT1R), LTC4 to CysLT2R, and LTE4 to CysLT3R (OXGR1 or GPR99). Recent studies in mouse models revealed that there are multiple regulatory mechanisms for these receptor functions and each receptor plays a distinct role as observed in different mouse models of inflammation and immune responses. This review focuses on the integrated host responses to the cys-LT/CysLTR pathway composed of sequential ligands with preferred receptors as seen from mouse models. It also discusses potential therapeutic targets for LTC4 synthase, CysLT2R, and CysLT3R.

The organization of leukotriene biosynthesis on the nuclear envelope revealed by single molecule localization microscopy and computational analyses.

The initial steps in the synthesis of leukotrienes are the translocation of 5-lipoxygenase (5-LO) to the nuclear envelope and its subsequent association with its scaffold protein 5-lipoxygenase-activating protein (FLAP). A major gap in our understanding of this process is the knowledge of how the organization of 5-LO and FLAP on the nuclear envelope regulates leukotriene synthesis. We combined single molecule localization microscopy with Clus-DoC cluster analysis, and also a novel unbiased cluster analysis to analyze changes in the relationships between 5-LO and FLAP in response to activation of RBL-2H3 cells to generate leukotriene C4. We identified the time-dependent reorganization of both 5-LO and FLAP into higher-order assemblies or clusters in response to cell activation via the IgE receptor. Clus-DoC analysis identified a subset of these clusters with a high degree of interaction between 5-LO and FLAP that specifically correlates with the time course of LTC4 synthesis, strongly suggesting their role in the initiation of leukotriene biosynthesis.

Alisol B 23-Acetate Inhibits IgE/Ag-Mediated Mast Cell Activation and Allergic Reaction.

Alisol B 23-acetate (AB23A), a natural triterpenoid, has been reported to exert hepatoprotective and antitumor activities. Aiming to investigate the anti-inflammatory activity, this study examined the effect of AB23A on mast cells and allergic reaction. AB23A inhibited the degranulation of mast cells stimulated by immunoglobulin E/antigen (IgE/Ag), and also decreased the synthesis of leukotriene C4 (LTC4), production of interlukin-6 (IL-6), and expression of cyclooxygenase-2 (COX-2) in a concentration-dependent manner with no significant cytotoxicity in bone marrow-derived mast cells (BMMCs). AB23A inhibited spleen tyrosine kinase (Syk) and the downstream signaling molecules including phospholipase Cγ (PLCγ), serine-threonine protein kinase/inhibitor of nuclear factor kappa-B kinase/nuclear factor kappa-B (Akt/IKK/NF-κB), and mitogen-activated protein kinases/cytosolic phospholipase A2 (MAPK/cPLA2). Furthermore, AB23A blocked mobilization of Ca2+. Similar results were obtained in other mast cell lines Rat basophilic leukemia (RBL)-2H3 cells and a human mast cell line (HMC-1). In addition, AB23A attenuated allergic responses in an acute allergy animal model, passive cutaneous anaphylaxis (PCA). Taken together, this study suggests that AB23A inhibits the activation of mast cells and ameliorates allergic reaction, and may become a lead compound for the treatment of mast cell-mediated allergic diseases.

Seongsanamides A-D: Antiallergic Bicyclic Peptides from Bacillus safensis KCTC 12796BP.

Six seongsanamides were isolated from the culture broth of Bacillus safensis KCTC 12796BP, and their structures were elucidated by spectroscopic data analysis combined with Marfey's method, electronic circular dichroism calculations, and biosynthetic gene cluster analysis. Compounds 1-4 were bicyclic peptides with isodityrosine residues; 5 and 6 were monocyclic peptides. Only the bicyclic seongsanamides inhibited degranulation and LTC4/PGD2 generation in IgE/Ag-stimulated bone marrow-derived mast cells. Oral administration of 1 suppressed mast cell-dependent passive cutaneous anaphylaxis reaction.

Leptin Elicits LTC4 Synthesis by Eosinophils Mediated by Sequential Two-Step Autocrine Activation of CCR3 and PGD2 Receptors.

Leptin is a cytokine, produced mainly by mature adipocytes, that regulates the central nervous system, mainly to suppress appetite and stimulate energy expenditure. Leptin also regulates the immune response by controlling activation of immunomodulatory cells, including eosinophils. While emerging as immune regulatory cells with roles in adipose tissue homeostasis, eosinophils have a well-established ability to synthesize pro-inflammatory molecules such as lipid mediators, a key event in several inflammatory pathologies. Here, we investigated the impact and mechanisms involved in leptin-driven activation of eicosanoid-synthesizing machinery within eosinophils. Direct in vitro activation of human or mouse eosinophils with leptin elicited synthesis of lipoxygenase as well as cyclooxygenase products. Displaying selectivity, leptin triggered synthesis of LTC4 and PGD2, but not PGE2, in parallel to dose-dependent induction of lipid body/lipid droplets biogenesis. While dependent on PI3K activation, leptin-driven eosinophil activation was also sensitive to pertussis toxin, indicating the involvement of G-protein coupled receptors on leptin effects. Leptin-induced lipid body-driven LTC4 synthesis appeared to be mediated through autocrine activation of G-coupled CCR3 receptors by eosinophil-derived CCL5, inasmuch as leptin was able to trigger rapid CCL5 secretion, and neutralizing anti-RANTES or anti-CCR3 antibodies blocked lipid body assembly and LTC4 synthesis induced by leptin. Remarkably, autocrine activation of PGD2 G-coupled receptors DP1 and DP2 also contributes to leptin-elicited lipid body-driven LTC4 synthesis by eosinophils in a PGD2-dependent fashion. Blockade of leptin-induced PGD2 autocrine/paracrine activity by a specific synthesis inhibitor or DP1 and DP2 receptor antagonists, inhibited both lipid body biogenesis and LTC4 synthesis induced by leptin stimulation within eosinophils. In addition, CCL5-driven CCR3 activation appears to precede PGD2 receptor activation within eosinophils, since neutralizing anti-CCL5 or anti-CCR3 antibodies inhibited leptin-induced PGD2 secretion, while it failed to alter PGD2-induced LTC4 synthesis. Altogether, sequential activation of CCR3 and then PGD2 receptors by autocrine ligands in response to leptin stimulation of eosinophils culminates with eosinophil activation, characterized here by assembly of lipidic cytoplasmic platforms synthesis and secretion of the pleiotropic lipid mediators, PGD2, and LTC4.

Leukotrienes and kidney diseases.

PURPOSE OF REVIEW: This review will critically highlight the role of leukotrienes as mediators of renal diseases and drug nephrotoxicity. It will also discuss the recently identified mechanism of cysteinyl leukotrienes induction and action, and will propose clinical implementation of these findings. RECENT FINDINGS: Since last reviewed in 1994, leukotrienes were shown to mediate drug-associated nephrotoxicity, transplant rejection and morbidity in several models of renal diseases. Although leukotrienes may be released by various infiltrating leukocytes, a recent study demonstrated that cytotoxic agents trigger production of leukotriene C4 (LTC4) in mouse kidney cells by activating a biosynthetic pathway based on microsomal glutathione-S-transferase 2 (MGST2). LTC4 then elicits nuclear accumulation of hydrogen peroxide-generating NADPH oxidase 4, leading to oxidative DNA damage and cell death. LTC4 inhibitors, commonly used as systemic asthma drugs, alleviated drug-associated damage to proximal tubular cells and attenuated mouse morbidity. SUMMARY: Cysteinyl leukotrienes released by mast cells trigger the symptoms of asthma, including bronchoconstriction and vasoconstriction. Therefore, effective leukotriene inhibitors were approved as orally administered asthma drugs. The findings that leukotrienes mediate the cytotoxicity of nephrotoxic drugs, and are involved in numerous renal diseases, suggest that such asthma drugs may ameliorate drug-induced nephrotoxicity, as well as some renal diseases.

Schistosomal Lipids Activate Human Eosinophils via Toll-Like Receptor 2 and PGD2 Receptors: 15-LO Role in Cytokine Secretion.

Parasite-derived lipids may play important roles in host-pathogen interactions and immune evasion mechanisms. Remarkable accumulation of eosinophils is a characteristic feature of inflammation associated with parasitic disease, especially caused by helminthes. Infiltrating eosinophils are implicated in the pathogenesis of helminth infection by virtue of their capacity to release an array of tissue-damaging and immunoregulatory mediators. However, the mechanisms involved in the activation of human eosinophils by parasite-derived molecules are not clear. Here we investigated the effects and mechanisms of schistosomal lipids-induced activation of human eosinophils. Our results showed that stimulation of human eosinophils in vitro with total lipid extracts from adult worms of S. mansoni induced direct activation of human eosinophils, eliciting lipid droplet biogenesis, synthesis of leukotriene (LT) C4 and eoxin (EX) C4 (14,15 LTC4) and secretion of eosinophil pre-formed TGFß. We demonstrated that main eosinophil activating components within S. mansoni lipid extract are schistosomal-derived lysophosphatidylcholine (LPC) and prostaglandin (PG)D2. Moreover, TLR2 is up-regulated in human eosinophils upon stimulation with schistosomal lipids and pre-treatment with anti-TLR2 inhibited both schistosomal lipids- and LPC-, but not PGD2-, induced lipid droplet biogenesis and EXC4 synthesis within eosinophils, indicating that TLR2 mediates LPC-driven human eosinophil activation. By employing PGD2 receptor antagonists, we demonstrated that DP1 receptors are also involved in various parameters of human eosinophil activation induced by schistosomal lipids, but not by schistosomal LPC. In addition, schistosomal lipids and their active components PGD2 and LPC, triggered 15-LO dependent production of EXC4 and secretion of TGFß. Taken together, our results showed that schistosomal lipids contain at least two components-LPC and PGD2-that are capable of direct activation of human eosinophils acting on distinct eosinophil-expressed receptors, noticeably TLR2 as well as DP1, trigger human eosinophil activation characterized by production/secretion of pro-inflammatory and immunoregulatory mediators.

Troglitazone, a PPAR-γ agonist, decreases LTC4 concentration in mononuclear cells in patients with asthma.

BACKGROUND: Asthma is an inflammatory disorder with multiple mediators involved in the inflammatory response. Despite several attempts, no new anti-inflammatory drugs have been registered for asthma treatment for several years. However, thiazolidinediones, peroxisome proliferator-activated receptor agonists, have demonstrated some anti-inflammatory properties in various experimental settings. The aim of this study was to assess the influence of troglitazone on LTC4 and 15-HETE concentrations. It also evaluates TNF-induced eotaxin synthesis in peripheral blood mononuclear cells from 14 patients with mild asthma and 13 healthy controls. METHODS: PBMCs were isolated from the whole blood of the asthmatics and healthy subjects and pretreated with 0.1, 1 or 10µM of Troglitazone. The cells were then exposed to 10-6M calcium jonophore or 10ng/ml TNF. The production and release of LTC4, 15-HETE and eotaxin were then assessed. RESULTS: Troglitazone caused a dose-dependent inhibition in LTC4 synthesis in both asthmatics and healthy subjects. Troglitazone did not influence 15-HETE or eotaxin production in either asthmatic patients or in healthy individuals. CONCLUSION: Due to its inhibition of LTC4 synthesis, troglitazone therapy is an interesting potential therapeutic approach in asthma and other LTC4 related inflammatory disorders.

Pulmonary epithelial cancer cells and their exosomes metabolize myeloid cell-derived leukotriene C4 to leukotriene D4.

Leukotrienes (LTs) play major roles in lung immune responses, and LTD4 is the most potent agonist for cysteinyl LT1, leading to bronchoconstriction and tissue remodeling. Here, we studied LT crosstalk between myeloid cells and pulmonary epithelial cells. Monocytic cells (Mono Mac 6 cell line, primary dendritic cells) and eosinophils produced primarily LTC4 In coincubations of these myeloid cells and epithelial cells, LTD4 became a prominent product. LTC4 released from the myeloid cells was further transformed by the epithelial cells in a transcellular manner. Formation of LTD4 was rapid when catalyzed by γ-glutamyl transpeptidase (GGT)1 in the A549 epithelial lung cancer cell line, but considerably slower when catalyzed by GGT5 in primary bronchial epithelial cells. When A549 cells were cultured in the presence of IL-1ß, GGT1 expression increased about 2-fold. Also exosomes from A549 cells contained GGT1 and augmented LTD4 formation. Serine-borate complex (SBC), an inhibitor of GGT, inhibited conversion of LTC4 to LTD4 Unexpectedly, SBC also upregulated translocation of 5-lipoxygenase (LO) to the nucleus in Mono Mac 6 cells, and 5-LO activity. Our results demonstrate an active role for epithelial cells in biosynthesis of LTD4, which may be of particular relevance in the lung.

Protective Effect of an Antibody against Specific Extracellular Domain of TLR2 on Agonists-Driven Inflammatory and Allergic Response.

Specific blocking strategies of TLR2-mediated inflammatory signaling and hypersensitivity reactions may offer novel therapeutic strategies to prevent a variety of diseases. In this study, we investigated the blocking effects of a new anti-TLR2 antibody anti-T20 against a 20 mer peptide T20 located in the extracellular specific domain of mouse TLR2. In addition, the effects of the anti-T20 in vitro, measuring the inhibition of the IL-6 and TNF-α production in response to PGN, LTA, and Pam3CSK4-stimulated RAW264.7 cells, were determined. In vivo, the effects of anti-T20 on a lethal anaphylaxis model using PGN-challenged OVA allergic mice, including the rectal temperature and mortality, and serum levels of TNF-α, IL-6, and LTC4 were assayed. The results showed that anti-T20 specifically bound to TLR2 and significantly inhibited PGN, LTA, and Pam3CSK4-driven TNF-α and IL-6 production by RAW264.7 cells. Also, anti-T20 protected OVA allergic mice from PGN-induced lethal anaphylaxis, and the serum levels of TNF-α, IL-6, and LTC4 of anti-T20 treated PGN-challenged OVA allergic mice were decreased as compared to isotype control of anti-T20 treated mice. In summary, this study produced a new antibody against the specific extracellular domain of TLR2 which has protective effect on TLR2 agonists-driven inflammatory and allergic response.

Effect of lipopolysaccharide and cytokines on synthesis and secretion of leukotrienes from endometrial epithelial cells of pigs.

In the present study, effects were studied of lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-4 and IL-10 on the mRNA and protein expression of 5-lipooxygenase (5-LO), leukotriene (LT)A4 hydrolase (LTAH) and LTC4 synthase (LTCS), and secretion of LTB4 and LTC4 from endometrial epithelial cells of pigs, as well as on viability of these cells. Cells were incubated for 24h with LPS (10 or 100ng/ml of medium), TNF-α, IL-1ß, IL-4 or IL-10 (each cytokine: 1 or 10ng/ml of medium). Larger doses of TNF-α and IL-10 and both doses of IL-1ß increased the relative abundance of mRNA/protein of 5-LO in the cells. A similar effect was exerted by the smaller dose of LPS on 5-LO mRNA content. Smaller doses of LPS and IL-4, and the larger dose of IL-10 increased the relative abundance of mRNA/protein LTAH, while both doses of TNF-α and the larger dose of IL-1ß increased the protein content of this enzyme. Relative abundance of the mRNA/protein of LTCS was greater with the smaller dose of LPS, both doses of TNF-α and greater doses of IL-1ß and IL-10, while relative abundance of LTCS mRNA was greater in response to the larger dose of LPS and both doses of IL-4. The LTB4 and LTC4 release was increased by the smaller dose of LPS, both doses of TNF-α and larger doses of IL-1ß and IL-10. The IL-4 at the smaller dose exerted a stimulatory effect on LTB4 release. Larger doses of TNF-α and IL-4 enhanced cell viability. Interactions with LPS and cytokines revealed in this study may represent mechanisms important for the regulation of endometrium functions of pigs under physiological or pathological conditions.

NecroX-5 suppresses IgE/Ag-stimulated anaphylaxis and mast cell activation by regulating the SHP-1-Syk signaling module.

BACKGROUND: IgE/Ag-stimulated mast cells release various pro-allergic inflammatory mediators, including histamine, eicosanoids, and pro-inflammatory cytokines. NecroX-5, a cell permeable necrosis inhibitor, showed cytoprotective effects in both in vitro and in vivo models. However, the anti-allergic effect of NecroX-5 has not yet been investigated. The aims of this study were to evaluate the anti-allergic activity of NecroX-5 in vivo and to investigate the underlying mechanism in vitro. METHODS: The anti-allergic activity of NecroX-5 was evaluated in vitro using bone marrow-derived mast cells (BMMCs) and IgE receptor-bearing RBL-2H3 or KU812 cells and in vivo using a mouse model of passive anaphylaxis. The levels of histamine, eicosanoids (PGD2 and LTC4 ), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were measured using enzyme immunoassay kits. The mechanism underlying the action of NecroX-5 was investigated using immunoblotting, immunoprecipitation, and gene knockdown techniques. RESULTS: NecroX-5 markedly inhibited mast cell degranulation and the synthesis of eicosanoids, TNF-α, and IL-6 by suppressing the activation of Syk, LAT, phospholipase Cγ1, MAP kinases, the Akt/NF-κB pathway, and intracellular Ca(2+) mobilization via the activation of phosphatase SHP-1. Oral administration of NecroX-5 effectively suppressed mast cell-dependent passive cutaneous and systemic anaphylactic reactions in a dose-dependent manner. CONCLUSIONS: NecroX-5 might be a potential candidate for the development of a novel anti-allergic agent that suppresses IgE-dependent mast cells signaling.

Lipoxin Inhibits Fungal Uptake by Macrophages and Reduces the Severity of Acute Pulmonary Infection Caused by Paracoccidioides brasiliensis.

Cysteinyl leukotrienes (CysLTs) and lipoxins (LXs) are lipid mediators that control inflammation, with the former inducing and the latter inhibiting this process. Because the role played by these mediators in paracoccidioidomycosis was not investigated, we aimed to characterize the role of CysLT in the pulmonary infection developed by resistant (A/J) and susceptible (B10.A) mice. 48 h after infection, elevated levels of pulmonary LTC4 and LXA4 were produced by both mouse strains, but higher levels were found in the lungs of susceptible mice. Blocking the CysLTs receptor by MTL reduced fungal loads in B10.A, but not in A/J mice. In susceptible mice, MLT treatment led to reduced influx of PMN leukocytes, increased recruitment of monocytes, predominant synthesis of anti-inflammatory cytokines, and augmented expression of 5- and 15-lipoxygenase mRNA, suggesting a prevalent LXA4 activity. In agreement, MTL-treated macrophages showed reduced fungal burdens associated with decreased ingestion of fungal cells. Furthermore, the addition of exogenous LX reduced, and the specific blockade of the LX receptor increased the fungal loads of B10.A macrophages. This study showed for the first time that inhibition of CysLTs signaling results in less severe pulmonary paracoccidioidomycosis that occurs in parallel with elevated LX activity and reduced infection of macrophages.

Inhibitory effect of oblongifolin C on allergic inflammation through the suppression of mast cell activation.

Oblongifolin C (OC), a natural small molecule compound extracted from Garcinia yunnanensis Hu, has been previously shown to have anti-cancer effect, but the anti-allergic effect of OC has not yet been investigated. The aim of the present study is to determine the anti-allergic effect of OC on IgE/Ag-induced mouse bone marrow-derived mast cells (BMMCs) and on the passive systemic anaphylaxis (PSA) reaction in mice. OC clearly suppressed cyclooxygenase-2 (COX-2)-dependent prostaglandin D2 (PGD2) generation as well as leukotriene C4 (LTC4) generation and the degranulation reaction in IgE/Ag-stimulated BMMCs. Biochemical analyses of the IgE/Ag-mediated signaling pathways showed that OC suppressed the phosphorylation of phospholipase Cγ1 (PLCγ1)-mediated intracellular Ca(2+) influx and the nuclear factor-κB (NF-κB) pathway, as well as the phosphorylation of mitogen-activated protein (MAP) kinases. Although OC did not inhibit the phosphorylation of Fyn, Lyn, and Syk, it directly inhibited the tyrosine kinase activity in vitro. Moreover, oral administration of OC inhibited the IgE-induced PSA reaction in a dose-dependent manner. Taken together, the present study provides new insights into the anti-allergic activity of OC, which could be a promising candidate for allergic therapy.

Assay of mast cell mediators.

Mediator release from activated mast cells is a major initiator of the symptomology associated with allergic disorders such as anaphylaxis and asthma. Thus, methods to monitor the generation and release of such mediators have widespread applicability in studies designed to understand the processes regulating mast cell activation and for the identification of therapeutic approaches to block mast cell-driven disease. In this chapter, we discuss approaches used for the determination of mast cell degranulation, lipid-derived inflammatory mediator production, and cytokine/chemokine gene expression as well as cytokine release.

Roles of coactosin-like protein (CLP) and 5-lipoxygenase-activating protein (FLAP) in cellular leukotriene biosynthesis.

5-Lipoxygenase (5LO) is a key enzyme in leukotriene (LT) biosynthesis. Two accessory proteins, coactosin-like protein (CLP) and 5-lipoxygenase-activating protein (FLAP), can support 5LO activity. To study the roles of CLP and FLAP, we knocked down these proteins in the human monocytic cell line Mono Mac 6 (MM6). Expression of CLP increased MM6 cellular 5LO activity for all stimuli tested. CLP is not absolutely crucial, however; some 5LO activity remained in all incubations of CLP knockdown cells. FLAP knockdown had minor effects in the presence of exogenous arachidonic acid, but led to prominent reductions in 5LO product formation from endogenous substrate. Similar effects were observed after CLP and FLAP knockdown in human primary macrophages as well. In addition, FLAP knockdown reduced conversion of leukotriene A4 to leukotriene C4 (LTC4), suggesting a role for the activity of LTC4 synthase. After stimulation of MM6 cells by phorbol myristate acetate and ionophore A23187, a perinuclear ring pattern was observed for 5LO. This redistribution from cytosolic to perinuclear was clearly compromised in both CLP- and FLAP-deficient cells. In addition, association of CLP with the nucleus was almost absent after 5LO knockdown, and was clearly reduced in FLAP knockdown cells. Coimmunoprecipitation experiments indicated that 5LO-CLP complex formation in MM6 cells was increased by stimulation with ionophore, and that this complex was formed to the same extent in FLAP knockdown cells. A possible interpretation of our findings is that on cell stimulation, formation of the 5LO-CLP complex augments the translocation from cytosol to nucleus, whereas FLAP stabilizes association of this complex with the perinuclear membrane.

The catalytic formation of leukotriene C4: a critical step in inflammatory processes.

Leukotrienes (LT) are a family of drug-like molecules involved in the pathobiology of bronchial asthma and are responsible for smooth muscle contraction. Leukotriene C4 synthase (LTC4S) is a nuclear-membrane enzyme responsible for the conjugation of leukotriene A4 (LTA4) to glutathione to form LTC4, a cysteinyl leukotriene. In this study, the mechanism of LTA4 binding by LTC4S has been computationally examined. More specifically, docking and molecular dynamics simulations were used to gain insight into the substrate-bound active site. These studies identified two possible orientations for bound LTA4: 'tail-to-head' and 'head-to-tail'. An ONIOM(QM/MM) approach was then used to elucidate the mechanism by which glutathione may add to LTA4. In particular, the thiolate of glutathione acts as a nucleophile attacking C6 of LTA4 forming a S-C6 bond. Concomitantly, a proton is transferred from the guanidinium of Arg31 to the epoxide ring oxygen. This results in opening of the epoxide ring and stabilization of the LTC4 product complex. Within the present computational methodology the 'tail-to-head' orientation appears to be the most likely substrate orientation.

In vivo sex differences in leukotriene biosynthesis in zymosan-induced peritonitis.

Leukotrienes (LTs) are 5-lipoxygenase (5-LO) metabolites which are implicated in sex-dependent inflammatory diseases (asthma, autoimmune diseases, etc.). We have recently reported sex differences in LT biosynthesis in in vitro models such as human whole blood, neutrophils and monocytes, due to down-regulation of 5-LO product formation by androgens. Here we present evidences for sex differences in LT synthesis and related inflammatory reactions in an in vivo model of inflammation (mouse zymosan-induced peritonitis). On the cellular level, differential 5-LO subcellular compartmentalization in peritoneal macrophages (PM) from male and female mice might be the basis for these differences. Sex differences in vascular permeability and neutrophil recruitment (cell number and myeloperoxidase activity) into peritoneal cavity were evident upon intraperitoneal zymosan injection, with more prominent responses in female mice. This was accompanied by higher levels of LTC4 and LTB4 in peritoneal exudates of female compared to male mice. Interestingly, LT peritoneal levels in orchidectomized mice were higher than in sham male mice. In accordance with the in vivo results, LT formation in stimulated PM from female mice was higher than in male PM, accompanied by alterations in 5-LO subcellular localization. The increased formation of LTC4 in incubations of PM from orchidectomized mice confirms a role of sex hormones. In conclusion, sex differences observed in LT biosynthesis during peritonitis in vivo may be related, at least in part, to a variant 5-LO localization in PM from male and female mice.

Modulation of basophils' degranulation and allergy-related enzymes by monomeric and dimeric naphthoquinones.

Allergic disorders are characterized by an abnormal immune response towards non-infectious substances, being associated with life quality reduction and potential life-threatening reactions. The increasing prevalence of allergic disorders demands for new and effective anti-allergic treatments. Here we test the anti-allergic potential of monomeric (juglone, menadione, naphthazarin, plumbagin) and dimeric (diospyrin and diosquinone) naphthoquinones. Inhibition of RBL-2H3 rat basophils' degranulation by naphthoquinones was assessed using two complementary stimuli: IgE/antigen and calcium ionophore A23187. Additionally, we tested for the inhibition of leukotrienes production in IgE/antigen-stimulated cells, and studied hyaluronidase and lipoxidase inhibition by naphthoquinones in cell-free assays. Naphthazarin (0.1 µM) decreased degranulation induced by IgE/antigen but not A23187, suggesting a mechanism upstream of the calcium increase, unlike diospyrin (10 µM) that reduced degranulation in A23187-stimulated cells. Naphthoquinones were weak hyaluronidase inhibitors, but all inhibited soybean lipoxidase with the most lipophilic diospyrin, diosquinone and menadione being the most potent, thus suggesting a mechanism of competition with natural lipophilic substrates. Menadione was the only naphthoquinone reducing leukotriene C4 production, with a maximal effect at 5 µM. This work expands the current knowledge on the biological properties of naphthoquinones, highlighting naphthazarin, diospyrin and menadione as potential lead compounds for structural modification in the process of improving and developing novel anti-allergic drugs.