Leukotriene D4 paradoxically limits LTC4-driven platelet activation and lung immunopathology.

BACKGROUND: The 3 cysteinyl leukotrienes (cysLTs), leukotriene (LT) C4 (LTC4), LTD4, and LTE4, have different biologic half-lives, cellular targets, and receptor specificities. CysLT2R binds LTC4 and LTD4in vitro with similar affinities, but it displays a marked selectivity for LTC4in vivo. LTC4, but not LTD4, strongly potentiates allergen-induced pulmonary eosinophilia in mice through a CysLT2R-mediated, platelet- and IL-33-dependent pathway. OBJECTIVE: We sought to determine whether LTD4 functionally antagonizes LTC4 signaling at CysLT2R. METHODS: We used 2 different in vivo models of CysLT2R-dependent immunopathology, as well as ex vivo activation of mouse and human platelets. RESULTS: LTC4-induced CD62P expression; HMGB1 release; and secretions of thromboxane A2, CXCL7, and IL-33 by mouse platelets were all were blocked by a selective CysLT2R antagonist and inhibited by LTD4. These effects did not depend on CysLT1R. Inhaled LTD4 blocked LTC4-mediated potentiation of ovalbumin-induced eosinophilic inflammation; recruitment of platelet-adherent eosinophils; and increases in IL-33, IL-4, IL-5, and IL-13 levels in lung tissue. In contrast, the effect of administration of LTE4, the preferred ligand for CysLT3R, was additive with LTC4. The administration of LTD4 to Ptges-/- mice, which display enhanced LTC4 synthesis similar to that in aspirin-exacerbated respiratory disease, completely blocked the physiologic response to subsequent lysine-aspirin inhalation challenges, as well as increases in levels of IL-33, type 2 cytokines, and biochemical markers of mast cell and platelet activation. CONCLUSION: The conversion of LTC4 to LTD4 may limit the duration and extent of potentially deleterious signaling through CysLT2R, and it may contribute to the therapeutic properties of desensitization to aspirin in aspirin-exacerbated respiratory disease.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Decrease Lung Injury in Mice.

Human mesenchymal stem cell (MSC) extracellular vesicles (EV) can reduce the severity of bacterial pneumonia, but little is known about the mechanisms underlying their antimicrobial activity. In the current study, we found that bacterial clearance induced by MSC EV in Escherichia coli pneumonia in C57BL/6 mice was associated with high levels of leukotriene (LT) B4 in the injured alveolus. More importantly, the antimicrobial effect of MSC EV was abrogated by cotreatment with a LTB4 BLT1 antagonist. To determine the role of MSC EV on LT metabolism, we measured the effect of MSC EV on a known ATP-binding cassette transporter, multidrug resistance-associated protein 1 (MRP1), and found that MSC EV suppressed MRP1 mRNA, protein, and pump function in LPS-stimulated Raw264.7 cells in vitro. The synthesis of LTB4 and LTC4 from LTA4 are competitive, and MRP1 is the efflux pump for LTC4 Inhibition of MRP1 will increase LTB4 production. In addition, administration of a nonspecific MRP1 inhibitor (MK-571) reduced LTC4 and subsequently increased LTB4 levels in C57BL/6 mice with acute lung injury, increasing overall antimicrobial activity. We previously found that the biological effects of MSC EV were through the transfer of its content, such as mRNA, microRNA, and proteins, to target cells. In the current study, miR-145 knockdown abolished the effect of MSC EV on the inhibition of MRP1 in vitro and the antimicrobial effect in vivo. In summary, MSC EV suppressed MRP1 activity through transfer of miR-145, thereby resulting in enhanced LTB4 production and antimicrobial activity through LTB4/BLT1 signaling.

Identification of the minimal region of peptide derived from ADP-ribosylation factor1 (ARF1) that inhibits IgE-mediated mast cell activation.

Mast cells play a pivotal role in allergic reactions and inflammations. Aggregation of the high affinity IgE receptor (FcεRI) eventually leads to the release of granule components such as histamine, as well as the de novo synthesis of inflammatory cytokines and lipid mediators. These substances are involved in the development of allergy and inflammation. Therefore, efficient inhibitors of mast cell activation would be therapeutically beneficial. We previously demonstrated that the synthetic peptide derived from the NH2-terminal region (2-17: GNIFANLFKGLFGKKE) of a small GTPase ARF1 (ADP-ribosylation factor1) inhibited FcεRI-induced mast cell degranulation. However, detailed structure-activity relationship study of NH2-terminal portion of ARF1 peptide has not been done. In addition, it is still unclear whether the NH2-terminal peptide of ARF1 suppresses FcεRI-induced production of cytokines and lipid mediators such as leukotriene C4 (LTC4) from mast cells. Here we show that amino acid residues K10-K16 are necessary for ARF1 peptide to efficiently inhibit FcεRI-induced activation of bone marrow-derived mast cells (BMMCs), indicated by decreased mast cell degranulation, cytokine secretion and leukotriene release. Furthermore, we show that ARF1 peptide inhibits IgE-mediated passive cutaneous anaphylaxis reaction. Our results suggest that the peptide derived from ARF1 could be developed into a novel anti-allergic agent for therapeutic intervention in allergy and mast cell-related pathologies.

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.

Elevated Systemic Levels of Eosinophil, Neutrophil, and Mast Cell Granular Proteins in Strongyloides Stercoralis Infection that Diminish following Treatment.

Infection with the helminth parasite Strongyloides stercoralis (Ss) is commonly clinically asymptomatic that is often accompanied by peripheral eosinophilia. Granulocytes are activated during helminth infection and can act as immune effector cells. Plasma levels of eosinophil and neutrophil granular proteins convey an indirect measure of granulocyte degranulation and are prominently augmented in numerous helminth-infected patients. In this study, we sought to examine the levels of eosinophil, neutrophil, and mast cell activation-associated granule proteins in asymptomatic Ss infection and to understand their kinetics following anthelmintic therapy. To this end, we measured the plasma levels of eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, eosinophil major basic protein, neutrophil elastase, myeloperoxidase, neutrophil proteinase-3, mast cell tryptase, leukotriene C4, and mast cell carboxypeptidase-A3 in individuals with asymptomatic Ss infection or without Ss infection [uninfected (UN)]. We also estimated the levels of all of these analytes in infected individuals following definitive treatment of Ss infection. We demonstrated that those infected individuals have significantly enhanced plasma levels of eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, eosinophil major basic protein, elastase, myeloperoxidase, mast cell tryptase, leukotriene C4, and carboxypeptidase-A3 compared to UN individuals. Following the treatment of Ss infection, each of these granulocyte-associated proteins drops significantly. Our data suggest that eosinophil, neutrophil, and mast cell activation may play a role in the response to Ss infection.

Microsomal glutathione transferase 2 modulates LTC4 synthesis and ROS production in Apostichopus japonicus.

Microsomal glutathione transferase 2 (mGST2) is an integral membrane protein involved in detoxication of xenobiotics, and has also been suggested to catalyze the biosynthesis of pro-inflammatory mediator leukotriene C4 (LTC4) as homologous to LTC4 synthase (LTC4S) in mammals. In the present study, a novel mGST2 homology was identified from Apostichopus japonicus (designated as AjmGST2) by RACE approaches. The full-length cDNA of AjmGST2 was of 1917bp encoding a polypeptide of 161 amino acids residues. Multiple sequences alignment and phylogenetic analysis together supported that AjmGST2 belonged to a new member in invertebrate mGSTs family and close to mammalian LTC4S. Spatial expression analysis revealed that AjmGST2 was ubiquitously expressed in all examined tissues with the larger magnitude in intestine. AjmGST2 transcripts in coelomocytes were slightly induced post 6h challenge of pathogenic Vibrio splendidus and reached the peak expression at 48h. The increased expression profiles of AjmGST2 were also detected in lipopolysaccharide (LPS) exposed primary coelomocytes. Consistently, LTC4 contents were also induced by a 1.56-fold increase in the same condition. Functional assay further revealed that AjmGST2 might be functioned as LTC4S to promote LTC4 synthesis. AjmGST2 knock-down by specific siRNA significantly depressed LTC4 contents with 27.0% decrease at 24h. Meantime, ROS levels were elevated by 40.1% in vitro. All of these results indicated that AjmGST2 performed dual functions roles as LTC4S and ROS eliminator in sea cucumber immune response.

Leukotriene C4 Potentiates IL-33-Induced Group 2 Innate Lymphoid Cell Activation and Lung Inflammation.

Asthma is a complex disease that is promoted by dysregulated immunity and the presence of many cytokine and lipid mediators. Despite this, there is a paucity of data demonstrating the combined effects of multiple mediators in asthma pathogenesis. Group 2 innate lymphoid cells (ILC2s) have recently been shown to play important roles in the initiation of allergic inflammation; however, it is unclear whether lipid mediators, such as cysteinyl leukotrienes (CysLTs), which are present in asthma, could further amplify the effects of IL-33 on ILC2 activation and lung inflammation. In this article, we show that airway challenges with the parent CysLT, leukotriene C4 (LTC4), given in combination with low-dose IL-33 to naive wild-type mice, led to synergistic increases in airway Th2 cytokines, eosinophilia, and peribronchial inflammation compared with IL-33 alone. Further, the numbers of proliferating and cytokine-producing lung ILC2s were increased after challenge with both LTC4 and IL-33. Levels of CysLT1R, CysLT2R, and candidate leukotriene E4 receptor P2Y12 mRNAs were increased in ILC2s. The synergistic effect of LTC4 with IL-33 was completely dependent upon CysLT1R, because CysLT1R-/- mice, but not CysLT2R-/- mice, had abrogated responses. Further, CysLTs directly potentiated IL-5 and IL-13 production from purified ILC2s stimulated with IL-33 and resulted in NFAT1 nuclear translocation. Finally, CysLT1R-/- mice had reduced lung eosinophils and ILC2 responses after exposure to the fungal allergen Alternaria alternata Thus, CysLT1R promotes LTC4- and Alternaria-induced ILC2 activation and lung inflammation. These findings suggest that multiple pathways likely exist in asthma to activate ILC2s and propagate inflammatory responses.

Airway eosinophil migration into lymph nodes in mice depends on leukotriene C4.

BACKGROUND: We previously demonstrated in mice that airway eosinophils traffic from the airway lumen into lung-draining paratracheal lymph nodes. However, mechanisms whereby eosinophils traverse from the lungs and home to paratracheal lymph nodes remain unclear. We investigated roles of cysteinyl leukotrienes in mediating eosinophil trafficking from lungs to paratracheal lymph nodes. METHODS: The expression of CCR7 was determined by flow cytometry. Transwell assays were used to test chemotactic responses of leukotriene C4 synthase-deficient and control airway eosinophils to the chemokine CCL19 ex vivo. Eosinophils from the spleens of IL-5 transgenic mice, fluorescently labeled ex vivo, were intratracheally injected into ovalbumin-sensitized and ovalbumin aerosol-challenged leukotriene C4 synthase-deficient and control mice. Eosinophils were identified by microscopy and flow cytometry in the lungs and paratracheal lymph nodes. RESULTS: Mouse eosinophils expressed CCR7, the receptor for CCL19, and responded chemotactically to CCL19. Leukotriene C4 synthase-deficient eosinophils exhibited impaired chemotaxis to CCL19 that was restored by exogenous leukotriene C4 . The migration of intratracheally injected eosinophils into paratracheal lymph nodes from distal alveolar lung was diminished in leukotriene C4 synthase-deficient mice compared with wild-type mice, with increased retention of eosinophils in the lungs of leukotriene C4 synthase-deficient mice. Exogenous administration of leukotriene C4 restored trafficking of eosinophils to paratracheal lymph nodes in leukotriene C4 synthase-deficient mice. CONCLUSIONS: Our findings that cysteinyl leukotrienes are involved in regulating airway and lung eosinophil migration into paratracheal lymph nodes identify previously unrecognized roles for the cysteinyl leukotrienes in regulating the pulmonary trafficking of eosinophils in experimental allergic asthma.

Elm Tree (Ulmus parvifolia) Bark Bioprocessed with Mycelia of Shiitake (Lentinus edodes) Mushrooms in Liquid Culture: Composition and Mechanism of Protection against Allergic Asthma in Mice.

Mushrooms can break down complex plant materials into smaller, more digestible and bioactive compounds. The present study investigated the antiasthma effect of an Ulmus parvifolia bark extract bioprocessed in Lentinus edodes liquid mycelium culture (BPUBE) against allergic asthma in chicken egg ovalbumin (OVA)-sensitized/challenged mice. BPUBE suppressed total IgE release from U266B1 cells in a dose-dependent manner without cytotoxicity. Inhibitory activity of BPUBE against OVA-specific IgE secretion in bronchoalveolar lavage fluid (BALF) was observed in OVA-sensitized/challenged asthmatic mice. BPUBE also inhibited OVA-specific IgG and IgG1 secretion into serum from the allergic mice, suggesting the restoration of a Th2-biased immune reaction to a Th1/Th2-balanced status, as indicated by the Th1/Th2 as well as regulatory T cell (Treg) cytokine profile changes caused by BPUBE in serum or BALF. Inflammatory cell counts in BALF and lung histology showed that leukocytosis and eosinophilia induced by OVA-sensitization/challenge were inhibited by the oral administration of BPUBE. Amelioration of eosinophil infiltration near the trachea was associated with reduced eotaxin and vascular cell adhesion molecule-1 (VCAM-1) levels. Changes in proinflammatory mediator levels in BALF suggest that BPUBE decreased OVA-sensitization-induced elevation of leukotriene C4 (LTC4) and prostaglandin D2 (PGD2). The finding that asthma-associated biomarker levels of OVA-sensitized/challenged mice were much more inhibited with BPUBE treatment than NPUBE (not-bioprocessed Ulmus parvifolia extract) treatment suggested the production of new bioactive compounds by the mushroom mycelia that may be involved in enhancing the observed antiasthmatic properties. The possible relation of the composition determined by proximate analysis and GC/MS to observed bioactivity is discussed. The results suggest that the elm tree (Ulmus parvifolia) bark bioprocessed with mycelia of shiitake (Lentinus edodes) mushrooms has the potential to prevent and/or treat allergic asthma.

Transglutaminase 2 expressed in mast cells recruited into skin or bone marrow induces the development of pediatric mastocytosis.

BACKGROUND: Mastocytosis is characterized by a pathological increase in mast cells in organs such as skin and bone marrow. Transglutaminase 2 (TG2) expressed in mast cells contributes to allergic diseases, but its role in mastocytosis has not been investigated. This study aimed to investigate whether TG2 contributes to pediatric mastocytosis. METHODS: Serum, various skin tissues or bone marrow (BM) biopsy and aspirates were obtained from pediatric normal control or patients with indolent systemic mastocytosis (SM), mastocytoma, and urticaria pigmentosa (UP). Tryptase, individual cytokines, leukotriene C4 (LTC4 ), and TG2 activity in the serum were determined by enzyme-linked immunosorbent assay, mast cell population by May-Grünwald-Giemsa, CD 117 by immunofluorescence, cell surface molecules by Western blot, and colocalization of c-kit and TG2 or IL-10-expressing cells, CD25, and FOXP3 by immunohistochemistry. RESULTS: Infiltration of CD25(+) CD117(+) CD2(-) mast cells into BM and scalp/trunk/ear dermis; expression of FcεRI, tryptase, c-kit, FOXP3, CCL2/CCR2, and vascular cell adhesion molecule-1; and colocalization of c-kit and TG2 were enhanced in patient's skin tissues or BM, particularly SM, but colocalization of c-kit and IL-10-expressing cells was decreased vs. normal tissues. Amounts of LTC4 and inflammatory cytokines, expression of tryptase or TG2 activity were increased in patient's serum, BM aspirates, or ear/scalp skin tissues, respectively, vs. normal persons, but IL-10 level was decreased. CONCLUSION: The data suggest that mast cells, recruited in the skin and BM by CCL2/CCR, may induce the development of pediatric mastocytosis through reducing IL-10 due to upregulating TG2 activity via transcription factor nuclear factor-κB. Thus, TG2 may be used in diagnosis of pediatric mastocytosis, particularly SM.

Platelet-driven leukotriene C4-mediated airway inflammation in mice is aspirin-sensitive and depends on T prostanoid receptors.

Cysteinyl leukotrienes (cysLTs) are bronchoconstricting lipid mediators that amplify eosinophilic airway inflammation by incompletely understood mechanisms. We recently found that LTC4, the parent cysLT, potently activates platelets in vitro and induces airway eosinophilia in allergen-sensitized and -challenged mice by a platelet- and type 2 cysLT receptor-dependent pathway. We now demonstrate that this pathway requires production of thromboxane A2 and signaling through both hematopoietic and lung tissue-associated T prostanoid (TP) receptors. Intranasal administration of LTC4 to OVA-sensitized C57BL/6 mice markedly increased the numbers of eosinophils in the bronchoalveolar lavage fluid, while simultaneously decreasing the percentages of eosinophils in the blood by a TP receptor-dependent mechanism. LTC4 upregulated the expressions of ICAM-1 and VCAM-1 in an aspirin-sensitive and TP receptor-dependent manner. Both hematopoietic and nonhematopoietic TP receptors were essential for LTC4 to induce eosinophil recruitment. Thus, the autocrine and paracrine functions of thromboxane A2 act downstream of LTC4/type 2 cysLT receptor signaling on platelets to markedly amplify eosinophil recruitment through pulmonary vascular adhesion pathways. The findings suggest applications for TP receptor antagonists in cases of asthma with high levels of cysLT production.

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies.

BACKGROUND: Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC4 (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC). METHODS: We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC4 and LTD4 by competing their binding to their receptors. RESULTS: mAbLTC and scFvLTC inhibited their binding of LTC4 or LTD4 to CysLT1 receptor (CysLT1R) and CysLT2 receptor (CysLT2R) overexpressed in Chinese hamster ovary cells. The induction by LTD4 of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT1R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC4- and LTD4-induced aggregation of mouse platelets expressing CysLT2R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT2R antagonists but not to CysLT1R antagonists. CONCLUSIONS: These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT1R and CysLT2R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors. GENERAL SIGNIFICANCE: mAbLTC can be used in the treatment of inflammatory diseases such as asthma.

Fatty acids induce leukotriene C4 synthesis in macrophages in a fatty acid binding protein-dependent manner.

Obesity results in increased macrophage recruitment to adipose tissue that promotes a chronic low-grade inflammatory state linked to increased fatty acid efflux from adipocytes. Activated macrophages produce a variety of pro-inflammatory lipids such as leukotriene C4 (LTC4) and 5-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) suggesting the hypothesis that fatty acids may stimulate eicosanoid synthesis. To assess if eicosanoid production increases with obesity, adipose tissue of leptin deficient ob/ob mice was analyzed. In ob/ob mice, LTC4 and 12-HETE levels increased in the visceral (but not subcutaneous) adipose depot while the 5-HETE levels decreased and 15-HETE abundance was unchanged. Since macrophages produce the majority of inflammatory molecules in adipose tissue, treatment of RAW264.7 or primary peritoneal macrophages with free fatty acids led to increased secretion of LTC4 and 5-HETE, but not 12- or 15-HETE. Fatty acid binding proteins (FABPs) facilitate the intracellular trafficking of fatty acids and other hydrophobic ligands and in vitro stabilize the LTC4 precursor leukotriene A4 (LTA4) from non-enzymatic hydrolysis. Consistent with a role for FABPs in LTC4 synthesis, treatment of macrophages with HTS01037, a specific FABP inhibitor, resulted in a marked decrease in both basal and fatty acid-stimulated LTC4 secretion but no change in 5-HETE production or 5-lipoxygenase expression. These results indicate that the products of adipocyte lipolysis may stimulate the 5-lipoxygenase pathway leading to FABP-dependent production of LTC4 and contribute to the insulin resistant state.

Oxidative stress potentially enhances FcεRI-mediated leukotriene C4 release dependent on the late-phase increase of intracellular glutathione in mast cells.

Cysteinyl leukotrienes (cysLTs), which include leukotriene C4 (LTC4), are the predominant class of LTs synthesized by mast cells. CysLTs can induce many of the abnormalities seen in asthma. LTC4 is generated by the conjugation of LTA4 with reduced glutathione (GSH) by LTC4 synthase. During screening of the effects of prostanoids on high-affinity IgE receptor (FcεRI)-mediated LTC4 release from mast cells, we realized that some prostanoids, including ONO-AE1-259-01 and ONO-AE-248, inhibited LTC4 release, which was associated with a decrease in the amount of intracellular total GSH. We ascertained that l-buthionine-S,R-sulfoximine (BSO), a selective inhibitor of glutamate-cysteine ligase, inhibited LTC4 release. In addition, cell-permeable GSH, the glutathione reduced form ethyl ester (GSH-OEt), enhanced LTC4 release in accordance with the change in intracellular total GSH. Depletion of intracellular total GSH induced by ONO-AE-248 or BSO enhanced FcεRI-mediated LTB4 release in contrast to LTC4. Oxidative stress contributes to many pathological conditions including asthma. GSH is a major soluble antioxidant and a cofactor for several detoxifying enzymes including GSH peroxidase. Exposure of mast cells to hydrogen peroxide (H2O2) or diamide to mimic oxidative stress unexpectedly increased rather than decreased the intracellular reduced GSH content as well as total GSH in the late phase (i.e., 24 or 48 h after exposure), which was accompanied by an increase in LTC4 release. In conclusion, FcεRI-mediated LTC4 release from mast cells is mainly regulated by the amount of intracellular GSH. In some cases, oxidative stress may induce a late-phase increase in intracellular GSH, resulting in enhanced LTC4 release from mast cells.

Anti-allergic flavones from Arthraxon hispidus.

Bioactivity-guided fractionation for an EtOAc-soluble fraction of methanolic extract of Arthraxon hispidus, using primary cell assay with bone marrow-derived mast cells (BMMC), led to an isolation of six new flavones and nine known compounds. The structures of the new compounds were established by one dimensional (1D)- and 2D-NMR spectroscopic data, as luteolin 8-C-ß-kerriopyranoside (1), luteolin 8-acetic acid methyl ester (2), 7-methyl-luteolin 8-C-ß-(6-deoxyxylo-3-uloside) (3), apigenin 8-C-α-fucopyranoside (4), apigenin 8-C-ß-fucopyranoside (5) and luteolin 8-C-ß-fucopyranoside (6). All the isolates were evaluated for inhibitory activities on interleukin-6 release in the primary cultures using BMMC. Of the tested compounds, compounds 2, 3 and 10 were found to inhibit interleukin-6 release. Furthermore, compound 2 displayed inhibitory activity against prostaglandin D2, leukotriene C4, and ß-hexosaminidase releases.

Wasp venom allergy: effect of anti-IgE antibody on wasp venom anaphylaxis in a mouse model.

BACKGROUND: Although anti-IgE antibody (Ab) therapy was recently shown to be effective in patients with bronchial asthma, no study has reported the effect of IgE therapy in the prevention of wasp venom anaphylaxis. In this study, we used a mouse model of wasp venom allergy to investigate the effect of anti-IgE Ab on wasp venom anaphylaxis. METHODS: We developed a mouse model of wasp venom allergy by intraperitoneally (i.p.) injecting wasp venom into BALB/c mice twice on experimental day (day) 0 and 7. On day 20, a group of mice received an i.p. injection of mouse anti-IgE Ab as a pretreatment, and another group received rat anti-IgG1 Ab. On day 21, the animals were challenged by i.p. injection of wasp venom, and 30 min later, body temperature was measured and serum levels of leukotriene (LT) B4 and LTC4 were determined using enzyme immunoassay. RESULTS: The body temperature of mice treated with anti-IgE Ab and controls before and after wasp venom challenge was 37.8±0.2 vs 37.7± 0.3°C before challenge and 37.8±0.2 vs 37.1± 0.3°C after challenge, respectively, showing that anti-IgE Ab treatment significantly prevented body temperature from falling (p <0.05). Furthermore, anti-IgE Ab treatment reduced total serum IgE levels in the treated mice (42.2±15.9 pg/ml), compared with controls (105.9±23.1 pg/ml, p <0.05), and inhibited the secretion of LTC4 in the treated mice (32.0±18.8 pg/ml), but not in the controls (162.4±12.4 pg/ml, p <0.05), following challenge with wasp venom. CONCLUSION: The results of the present study indicate that anti-IgE Ab treatment is an effective preventive measure against wasp venom-induced anaphylaxis.

In vivo intranasal anti-CD23 treatment inhibits allergic responses in a murine model of allergic rhinitis.

Although CD23-dependent transcytosis of IgE and IgE-derived immune complexes across respiratory epithelial cells is likely to play a pivotal role in the initiation and development of airway allergic inflammation, there is currently a lack of physiological support for this phenomena to suggest that the targeting of CD23 could be used as a means of therapeutic intervention. The present study was designed to detect the CD23 expression in the nasal mucosa of allergic rhinitis (AR) murine model by immunohistochemistry and western blotting, and to investigate whether intranasal anti-CD23 treatment could inhibit allergen-induced upper airway inflammation in the AR model. This is the first report to show that CD23 was constitutively expressed in murine nasal epithelial cells, and its expression was significantly up-regulated in the AR murine model. In vivo, the up-regulation of CD23 expression was correlated with increased serum IL-4 levels. Following intranasal anti-CD23 treatment, nasal symptoms were alleviated and histopathologic examination showed a significant decrease in eosinophilic infiltration. Meanwhile, ELISA analysis showed levels of serum leukotriene C4 (LTC4), eosinophil cation protein (ECP), ovalbumin (OVA)-specific IgE and IL-4 also significantly decreased, as were LTC4 and OVA-specific IgE in the nasal lavage fluid. Furthermore, Western blotting analysis showed that ECP expression in the nasal mucosa was down-regulated. Finally, flow cytometric analysis revealed anti-CD23 treatment inhibited Th2 cell responses. These results indicate that intranasal anti-CD23 treatment can reduce allergic responses in a murine model of allergic rhinitis.

Inhibitory effect of N-Acyl dopamines on IgE-mediated allergic response in RBL-2H3 cells.

Recently, endogenous N-acyl dopamines have been found to show anti-inflammatory and immunomodulatory activities. However, the effect of the N-acyl dopamines on allergic responses was not reported. In this study, we investigated whether N-acyl dopamines might inhibit immunoglobulin E-mediated degranulation in RBL-2H3 cells. When RBL-2H3 cells were exposed to palmitoyl dopamine (NP-DA), oleoyl dopamine (NO-DA) or arachidonoyl dopamine (NA-DA) at micromolar levels, all these compounds significantly inhibited the release of ß-hexosaminidase, a marker of degranulation, as well as tumor necrosis factor (TNF)-α. In comparison, NP-DA, potently suppressing the release of ß-hexosaminidase (IC50, 3.5 µM) and TNF-α (IC50, 2.2 µM), was more potent than NO-DA or NA-DA. Additionally, NP-DA markedly suppressed the formation of prostaglandin E2, prostaglandin D2 and leukotriene C4, corresponding to pro-inflammatory lipid mediators in asthma. In the mechanistic analyses, where the effect of NP-DA on the FcεRI cascade was examined, NP-DA significantly inhibited the phosphorylation and expression of Syk, but not Lyn. And, NP-DA also suppressed phosphorylation of ERK1/2 and Akt. Further, NP-DA decreased the phosphorylation of cPLA2 and 5-lipoxygenase (5-LO), but not cyclooxygenase-2 (COX-2). Based on these results, it is suggested that NP-DA exert anti-allergic effect on allergic response through suppressing the activation of Syk, ERK1/2, Akt, cPLA2 and 5-LO. Besides, a strong inhibition of COX-2 activity by NP-DA may be additional mechanism for its anti-allergic action. Such an anti-allergic action of N-acyl dopamines may contribute to further information about biological functions of N-acyl dopamines.

Montelukast inhibits leukotriene stimulation of human dendritic cells in vitro.

BACKGROUND: Leukotrienes are potent inflammatory mediators which modulate immune responses and induce bronchoconstriction in susceptible individuals. Montelukast (MK) is a leukotriene receptor (CysLT1) antagonist that has been shown to prevent exacerbation of asthma. Considering the plethora of potential cellular targets for MK, specific mechanisms for its therapeutic action are still not fully understood. In vitro, we determined whether human dendritic cell function could be affected by leukotriene C(4) (LTC(4)) treatment and whether MK had potential in modulating this response. We also studied the effect of LTC(4) in the context of response to an airway virus (respiratory syncytial virus, RSV). METHODS: Human monocyte-derived dendritic cells (moDCs) exposed to LTC(4), MK, or both, were cocultured with autologous T cells, with or without RSV. The effects of LTC(4) and MK on cell function were determined by ELISA and proliferation assays. RESULTS: Both moDCs and their precursors--monocytes--express LTC(4) receptor CysLT1, making them potential targets for MK. moDCs cultured with LTC(4) release the eosinophil chemoattractant RANTES (CCL5) and induce greater T cell proliferation. Both were blocked by the presence of MK. MK treatment, albeit anti-inflammatory, did not interfere with the moDC-dependent T cell-proliferative responses induced by RSV. CONCLUSIONS: LTC(4), chronically present in the airways of asthma patients, could induce an exaggerated inflammatory response to airway infection via dendritic cell activation, which would be prevented by MK. Our study provides additional insight into the mechanisms of action of this leukotriene receptor antagonist.