Prostaglandin D2 Generation Research Articles

Lipid-orchestrated paracrine circuit coordinates mast cell maturation and anaphylaxis through functional interaction with fibroblasts

Interaction of mast cells (MCs) with fibroblasts is essential for MC maturation within tissue microenvironments, although the underlying mechanism is incompletely understood. Through a phenotypic screening of >30 mouse lines deficient in lipid-related genes, we found that deletion of the lysophosphatidic acid (LPA) receptor LPA1, like that of the phospholipase PLA2G3, the prostaglandin D2 (PGD2) synthase L-PGDS, or the PGD2 receptor DP1, impairs MC maturation and thereby anaphylaxis. Mechanistically, MC-secreted PLA2G3 acts on extracellular vesicles (EVs) to supply lysophospholipids, which are converted by fibroblast-derived autotaxin (ATX) to LPA. Fibroblast LPA1 then integrates multiple pathways required for MC maturation by facilitating integrin-mediated MC-fibroblast adhesion, IL-33-ST2 signaling, L-PGDS-driven PGD2 generation, and feedforward ATX-LPA1 amplification. Defective MC maturation resulting from PLA2G3 deficiency is restored by supplementation with LPA1 agonists or PLA2G3-modified EVs. Thus, the lipid-orchestrated paracrine circuit involving PLA2G3-driven lysophospholipid, eicosanoid, integrin, and cytokine signaling fine-tunes MC-fibroblast communication, ensuring MC maturation.

Targeting the DP2 receptor alleviates muscle atrophy and diet-induced obesity in mice through oxidative myofiber transition.

Mammalian skeletal muscles consist of two main fibre types: slow-twitch (type I, oxidative) and fast-twitch (type IIa, fast oxidative; type IIb/IIx, fast glycolytic). Muscle fibre composition switch is closely associated with chronic diseases such as muscle atrophy, obesity, type II diabetes and athletic performance. Prostaglandin D2 (PGD2 ) is a bioactive lipid derived from arachidonic acid that aggravates muscle damage and wasting during muscle atrophy. This study aimed to investigate the precise mechanisms underlying PGD2 -mediated muscle homeostasis and myogenesis. Skeletal muscle-specific PGD2 receptor DP2-deficient mice (DP2fl/fl HSACre ) and their littermate controls (DP2fl/fl ) were subjected to exhaustive exercise and fed a high-fat diet (HFD). X-linked muscular dystrophy (MDX) mice and HFD-challenged mice were treated with the selective DP2 inhibitor CAY10471. Exercise tolerance, body weight, glycometabolism and skeletal muscle fibre composition were measured to determine the role of the skeletal muscle PGD2 /DP2 signalling axis in obesity and muscle disorders. Multiple genetic and pharmacological approaches were also used to investigate the intracellular signalling cascades underlying the PGD2 /DP2-mediated skeletal muscle fibre transition. PGD2 generation and DP2 expression were significantly upregulated in the hindlimb muscles of HFD-fed mice (P<0.05 or P<0.01 vs. normal chow diet). Compared with DP2fl/fl mice, DP2fl/fl HSACre mice exhibited remarkable glycolytic-to-oxidative fibre-type transition in hindlimb muscles and were fatigue resistant during endurance exercise (154.9±6.0 vs. 124.2±8.1min, P<0.05). DP2fl/fl HSACre mice fed an HFD showed less weight gain (P<0.05) and hepatic lipid accumulation (P<0.01), reduced insulin resistance and enhanced energy expenditure (P<0.05) compared with DP2fl/fl mice. Mechanistically, DP2 deletion promoted the nuclear translocation of nuclear factor of activated T cells 1 (NFATc1) by suppressing RhoA/Rho-associated kinase 2 (ROCK2) signalling, which led to enhanced oxidative fibre-specific gene transcription in muscle cells. Treatment with CAY10471 enhanced NFATc1 activity in the skeletal muscles and ameliorated HFD-induced obesity (P<0.05 vs. saline) and insulin resistance in mice. CAY10471 also enhanced exercise tolerance in MDX mice (100.8±8.0 vs. 68.9±11.1min, P<0.05 vs. saline) by increasing the oxidative fibre-type ratio in the muscles (45.1±2.3% vs. 32.3±2.6%, P<0.05 vs. saline). DP2 activation suppresses oxidative fibre transition via RhoA/ROCK2/NFATc1 signalling. The inhibition of DP2 may be a potential therapeutic approach against obesity and muscle disorders.

Niacin Attenuates Pulmonary Hypertension Through H-PGDS in Macrophages.

Pulmonary arterial hypertension (PAH) is characterized by progressive pulmonary vascular remodeling, accompanied by varying degrees of perivascular inflammation. Niacin, a commonly used lipid-lowering drug, possesses vasodilating and proresolution effects by promoting the release of prostaglandin D2 (PGD2). However, whether or not niacin confers protection against PAH pathogenesis is still unknown. This study aimed to determine whether or not niacin attenuates the development of PAH and, if so, to elucidate the molecular mechanisms underlying its effects. Vascular endothelial growth factor receptor inhibitor SU5416 and hypoxic exposure were used to induce pulmonary hypertension (PH) in rodents. We found that niacin attenuated the development of this hypoxia/SU5416-induced PH in mice and suppressed progression of monocrotaline-induced and hypoxia/SU5416-induced PH in rats through the reduction of pulmonary artery remodeling. Niacin boosted PGD2 generation in lung tissue, mainly through H-PGDS (hematopoietic PGD2 synthases). Deletion of H-PGDS, but not lipocalin-type PGDS, exacerbated the hypoxia/SU5416-induced PH in mice and abolished the protective effects of niacin against PAH. Moreover, H-PGDS was expressed dominantly in infiltrated macrophages in lungs of PH mice and patients with idiopathic PAH. Macrophage-specific deletion of H-PGDS markedly decreased PGD2 generation in lungs, aggravated hypoxia/SU5416-induced PH in mice, and attenuated the therapeutic effect of niacin on PAH. Niacin treatment ameliorates the progression of PAH through the suppression of vascular remodeling by stimulating H-PGDS-derived PGD2 release from macrophages.

Nepetin, a natural compound from Inulae flos, suppresses degranulation and eicosanoid generation through PLCγ1 and Akt signaling pathways in mast cells

Nepetin derived from the flowers of Inula japonica, Inulae flos, has been reported to exert several biological activities, including anti-inflammatory responses. In this study, we evaluated the anti-allergic property of nepetin with its molecular mechanisms in bone marrow-derived mast cells (BMMC) and mice. In this in vitro study, we investigated the inhibitory effects of nepetin on degranulation and generation of leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) in IgE/antigen (Ag)-stimulated BMMC. The effect of nepetin on passive cutaneous anaphylaxis (PCA) reaction was also studied in mice. Nepetin reduced degranulation and LTC4 generation in BMMC. The IgE/Ag-mediated signaling pathway demonstrated that nepetin suppressed intracellular Ca2+ level and activation of PLCγ1 and cPLA2. However, MAPKs were not affected by nepetin in BMMC. In addition, nepetin treatment reduced PGD2 production and suppressed cyclooxygenase-2 protein expression via the inhibition of the Akt and nuclear factor-κB signaling pathways. With respect to the local allergic response in vivo, oral administration of nepetin suppressed mast cell-dependent PCA reaction in a dose-dependent manner. The results of this study suggest that nepetin might have an anti-allergic potential related to mast cell-mediated inflammatory diseases.

Prostaglandin D2 generation from human lung mast cells is catalysed exclusively by cyclooxygenase-1

Mast cells are an exceptionally rich source of prostaglandin D2 (PGD2). PGD2 is pro-inflammatory and can cause bronchoconstriction. The enzyme cyclooxygenase (COX) is central to the generation of prostanoids such as PGD2. Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX. COX exists as two isoforms, COX-1 and COX-2. The principal aim of this study was to establish whether COX-1 and/or COX-2 mediates PGD2 generation from human lung mast cells. Mast cells were isolated from human lung tissue and purified by flotation over Percoll and immunomagnetic bead separations. The cells were activated with anti-IgE or Stem Cell Factor (SCF). The generation of PGD2 was determined by ELISA. The effects of NSAIDs (aspirin, ibuprofen, diclofenac, naproxen, indomethacin), COX-1 selective (FR122047), and COX-2 selective (celecoxib) inhibitors on PGD2 generation were determined. The expression of COX-1 and COX-2 in mast cells was determined by Western blotting. All the NSAIDs tested abrogated stimulated PGD2 generation from mast cells except aspirin which was only weakly effective. FR122047 was an effective inhibitor of PGD2 generation (EC50 ~25nM) from mast cells whereas celecoxib was ineffective. Immunoblotting indicated that COX-1 was strongly expressed in all mast cell preparations while COX-2 expression was weak. No induction of COX-2 was observed following activation of mast cells. These findings indicate that COX-1 is the principal isoform involved in generating PGD2 from human lung mast cells. These studies provide insight into the potential behaviour of NSAIDs in the context of respiratory diseases.

Role of TrkA signalling and mast cells in the initiation of osteoarthritis pain in the monoiodoacetate model.

Aiming to delineate novel neuro-immune mechanisms for NGF/TrkA signalling in osteoarthritis (OA) pain, we evaluated inflammatory changes in the knee joints following injection of monoiodoacetate (MIA) in mice carrying a TrkA receptor mutation (P782S; TrkA KI mice). In behavioural studies we monitored mechanical hypersensitivity following intra-articular MIA and oral prostaglandin D2 (PGD2) synthase inhibitor treatments. In immunohistochemical studies we quantified joint mast cell numbers, calcitonin gene-related peptide expression in synovia and dorsal root ganglia, spinal cord neuron activation and microgliosis. We quantified joint leukocyte infiltration by flow cytometry analysis, and PGD2 generation and cyclooxygenase-2 (COX-2) expression in mast cell lines by ELISA and Western blot. In TrkA KI mice we observed rapid development of mechanical hypersensitivity and amplification of dorsal horn neurons and microglia activation 7 days after MIA. In TrkA KI knee joints we detected significant leukocyte infiltration and mast cells located in the vicinity of synovial nociceptive fibres. We demonstrated that mast cells exposure to NGF results in up-regulation of COX-2 and increase of PGD2 production. Finally, we observed that a PGD2 synthase inhibitor prevented MIA-mechanical hypersensitivity in TrkA KI, at doses which were ineffective in wild type (WT) mice. Using the TrkA KI mouse model, we delineated a novel neuro-immune pathway and suggest that NGF-induced production of PGD2 in joint mast cells is critical for referred mechanical hypersensitivity in OA, probably through the activation of PGD2 receptor 1 in nociceptors: TrkA blockade in mast cells constitutes a potential target for OA pain.

Thymic stromal lymphopoietin controls prostaglandin D2 generation in patients with aspirin-exacerbated respiratory disease

Prostaglandin (PG) D2 is the dominant COX product of mast cells and is an effector of aspirin-induced respiratory reactions in patients with aspirin-exacerbated respiratory disease (AERD). We evaluated the role of the innate cytokine thymic stromal lymphopoietin (TSLP) acting on mast cells to generate PGD2 and facilitate tissue eosinophilia and nasal polyposis in patients with AERD. Urinary eicosanoid levels were measured in aspirin-tolerant control subjects and patients with AERD. Nasal polyp specimens from patients with AERD and chronic rhinosinusitis were analyzed by using quantitative PCR, Western blotting, and immunohistochemistry. Human cord blood-and peripheral blood-derived mast cells were stimulated with TSLP invitro to assess PGD2 generation. Urinary levels of a stable PGD2 metabolite (uPGD-M) were 2-fold higher in patients with AERD relative to those in control subjects and increased further during aspirin-induced reactions. Peak uPGD-M levels during aspirin reactions correlated with reductions in blood eosinophil counts and lung function and increases in nasal congestion. Mast cells sorted from nasal polyps expressed PGD2 synthase (hematopoietic PGD2 synthase) mRNA at higher levels than did eosinophils from the same tissue. Whole nasal polyp TSLP mRNA expression correlated strongly with mRNA encoding hematopoietic PGD2 synthase (r=.75), the mast cell-specific marker carboxypeptidase A3 (r=.74), and uPGD-M (r=0.74). Levels of the cleaved active form of TSLP were increased in nasal polyps from patients with AERD relative to those in aspirin-tolerant control subjects. Recombinant TSLP induced PGD2 generation by cultured human mast cells. Our study demonstrates that mast cell-derived PGD2 is a major effector of type 2 immune responses driven by TSLP and suggests that dysregulation of this innate system contributes significantly to the pathophysiology of AERD.

The natural compound nujiangexanthone A suppresses mast cell activation and allergic asthma

Mast cells play an important role in allergic diseases such as asthma, allergic rhinitis and atopic dermatitis. The genus Garcinia of the family Guttiferae is well known as a prolific source of polycyclic polyprenylated acylphloroglucinols and bioactive prenylated xanthones, which exhibit various biological activities including antibacterial, antifungal, anti-inflammatory, antioxidant, and cytotoxic effects. Nujiangexanthone A (N7) is a novel compound isolated from the leaves of Garcinia nujiangensis. In this paper, we sought to determine the anti-allergic and anti-inflammation activity of N7 in vivo and its mechanism in vitro. We found N7 suppressed IgE/Ag induced mast cell activiation, including degranulation and production of cytokines and eicosanoids, through inhibiting Src kinase activity and Syk dependent pathways. N7 inhibited histamine release, prostaglandin D2 and leukotriene C4 generation in mast cell dependent passive cutaneous anaphylaxis animal model. We also found N7 inhibited the IL-4, IL-5, IL-13 and IgE levels in ovalbumin-induced asthma model. Histological studies demonstrated that N7 substantially inhibited OVA-induced cellular infiltration and increased mucus production in the lung tissue. Our study reveals the anti-allergic function of N7, thereby suggesting the utility of this compound as a possible novel agent for preventing mast cell-related immediate and delayed allergic diseases.

Imperatorin Suppresses Degranulation and Eicosanoid Generation in Activated Bone Marrow-Derived Mast Cells.

Imperatorin has been known to exert many biological functions including anti-inflammatory activity. In this study, we investigated the inhibitory effects of imperatorin on the production of inflammatory mediators in mouse bone marrow-derived mast cells (BMMC). Imperatorin inhibited degranulation and the generation of eicosanoids (leukotriene C4 (LTC4) and prostaglandin D2 (PGD2)) in IgE/antigen (Ag)-stimulated BMMC. To elucidate the molecular mechanism involved in this process, we investigated the effect of imperatorin on intracellular signaling in BMMC. Biochemical analyses of the IgE/Ag-mediated signaling pathway demonstrated that imperatorin dramatically attenuated degranulation and the production of 5-lipoxygenase-dependent LTC4 and cyclooxygenase-2-dependent PGD2 through the inhibition of intracellular calcium influx/phospholipase Cγ1, cytosolic phospholipase A2/mitogen-activated protein kinases and/or nuclear factor-κB pathways in BMMC. These results suggest that the effects of imperatorin on inhibition of degranulation and eicosanoid generation through the suppression of multiple steps of IgE/Ag-mediated signaling pathways would be beneficial for the prevention of allergic inflammation.

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.

15,16-Dihydrotanshinone I suppresses IgE-Ag stimulated mouse bone marrow-derived mast cell activation by inhibiting Syk kinase

Ethnopharmacological relevance15,16-Dihydrotanshinone I (DHT-I), isolated from the dried root of Salvia miltiorrhiza Bung, which is traditionally used to treat cardiovascular and inflammatory diseases agent in Chinese medicine. DHT-I has been reported to have a broad range of biological activities, including antibacterial activity, and has been used to treat circulatory disorders, hepatitis, inflammation, cancer, and neurodegenerative diseases. Aim of the studyThe aim of this study was to evaluate the anti-allergic inflammatory effects of DHT-I on degranulation and on the generation of eicosanoids, such as, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4), in IgE/Ag-stimulated bone marrow-derived mast cells (BMMCs). Materials and methodsThe anti-allergic inflammatory activity of DHT-I was evaluated using BMMCs. The effects of DHT-I on mast cell activation were investigated by following degranulation and eicosanoid generation using ELISA and immunoblotting and immunoprecipitation techniques. ResultsDHT-I at a concentration of 20μM markedly inhibited degranulation and the generation of PGD2 and LTC4 in IgE/Ag-stimulated BMMCs (about 90% inhibitions, respectively). Analyses of FcεRI-mediated signaling pathways demonstrated that DHT-I inhibited the phosphorylations of spleen tyrosine kinase (Syk) and linker for activation of T cells (LAT), and inhibited downstream signaling process, including [Ca2+]i mobilization induced by the phosphorylation of phospholipase Cγ1 (PLCγ1), and the activations of mitogen-activated protein kinases (MAPKs) and the Akt-nuclear factor-κB (NF-κB) pathway. ConclusionsDHT-1 inhibits the release of allergic inflammatory mediators from IgE/Ag-stimulated mast cells by suppressing a FcεRI-mediated Syk-dependent signal pathway. This result suggests DHT-I offers a novel developmental basis for drugs targeting allergic inflammatory diseases.

Britanin Suppresses IgE/Ag-Induced Mast Cell Activation by Inhibiting the Syk Pathway.

The aim of this study was to determine whether britanin, isolated from the flowers of Inula japonica (Inulae Flos), modulates the generation of allergic inflammatory mediators in activated mast cells. To understand the biological activity of britanin, the authors investigated its effects on the generation of prostaglandin D2 (PGD2), leukotriene C4 (LTC4), and degranulation in IgE/Ag-induced bone marrow-derived mast cells (BMMCs). Britanin dose dependently inhibited degranulation and the generations of PGD2 and LTC4 in BMMCs. Biochemical analyses of IgE/Ag-mediated signaling pathways demonstrated that britanin suppressed the phosphorylation of Syk kinase and multiple downstream signaling processes, including phospholipase Cγ1 (PLCγ1)-mediated calcium influx, the activation of mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase 1/2, c-Jun NH2-terminal kinase and p38), and the nuclear factor-κB (NF-κB) pathway. Taken together, the findings of this study suggest britanin suppresses degranulation and eicosanoid generation by inhibiting the Syk-dependent pathway and britanin might be useful for the treatment of allergic inflammatory diseases.

Anti-allergic effect of a Korean traditional medicine, Biyeom-Tang on mast cells and allergic rhinitis

BackgroundBiyeom-Tang, a medicine prescribed by oriental clinics, has been used for the treatment of the allergic rhinitis (AR). In the present study, an ethanol extract of Biyeom-Tang (EBT) was investigated for anti-allergic properties on bone-marrow derived mast cells (BMMC) and in vivo models.MethodsThe anti-allergic properties of EBT were evaluated by measuring β-Hex release and the production of prostaglandin D2 (PGD2) and leukotriene C4 (LTC4) on BMMC in vitro and PCA and OVA-induced AR models in vivo.ResultsEBT strongly inhibited a degranulation reaction in a dose dependent manner with an IC50 value of 35.6 μg/ml. In addition, the generation of PGD2 and LTC4 was inhibited in BMMC in a concentration-dependent manner with IC50 values of 7.0 μg/ml and 10.9 μg/ml, respectively. When administrated orally, EBT ameliorated the mast cell-mediated PCA reaction. In the OVA-induced AR model, the increased levels of IgE were reduced by EBT. The levels of cytokines, such as IL-4, IL-5, IL-10, and IL-13 decreased in the splenocytes of EBT-treated mice. The histological analysis shows that the infiltration of inflammatory cells increased by OVA-sensitization was also reduced.ConclusionsTaken together, these results suggested that EBT has anti-allergic and anti-inflammatory effects in vitro and in vivo models.

A Phenotypic Screening Approach in Cord Blood–Derived Mast Cells to Identify Anti-Inflammatory Compounds

Mast cells are unique hematopoietic cells that are richly distributed in the skin and mucosal surfaces of the respiratory and gastrointestinal tract. They play a key role in allergic inflammation by releasing a cocktail of granular constituents, including histamine, serine proteases, and various eicosanoids and cytokines. As such, a number of drugs target either inhibition of mast cell degranulation or the products of degranulation. To identify potential novel drugs and mechanisms in mast cell biology, assays were developed to identify inhibitors of mast cell degranulation and activation in a phenotypic screen. Due to the challenges associated with obtaining primary mast cells, cord blood–derived mononuclear cells were reproducibly differentiated to mast cells and assays developed to monitor tryptase release and prostaglandin D2 generation. The tryptase assay was particularly sensitive, requiring only 500 cells per data point, which permitted a set of approximately 12,000 compounds to be screened robustly and cost-effectively. Active compounds were tested for concomitant inhibition of prostaglandin D2 generation. This study demonstrates the robustness and effectiveness of this approach in the identification of potential novel compounds and mechanisms targeting mast cell–driven inflammation, to enable innovative drug discovery efforts to be prosecuted.

Anti-allergic effect of a Korean folk medicine, KOTMIN5, in vitro and in vivo

A Korean folk medicine, Biyeom Tang (KOTMIN5), composed of Xanthii fructus, Trichosanthis semen, Angelicae dahuricae radix, and Menthae herba, had been used in traditional medicinal clinic for the treatment of inflammatory diseases such as allergic rhinitis. However, the anti-allergic properties of KOTMIN5 and its molecular mechanisms have not been investigated. In the present study, the ethanol extract of KOTMIN5 was investigated for anti-allergic properties in bone-marrow derived mast cells (BMMC) and in vivo model. KOTMIN5 strongly inhibited a degranulation reaction in a dose dependent manner with an IC50 value of 17.6 mg/ml. In addition, the generation of prostaglandin D2 (PGD2) and leukotriene C4 (LTC4) was inhibited in BMMC in a concentration-dependent manner with IC50 values of 33.8 mg/ml and 24.3 mg/ml, respectively. Furthermore, KOTMIN5 reduced compound48/80-induced systemic anaphylactic shock and IgE-mediated passive cutaneous anaphylaxis in mice. These results suggested that KOTMIN5 might be useful in regulating allergic reactions.

212: Innate neutrophil function is regulated locally through IL-6 control of prostaglandins

Interleukin-6 (IL-6) is vital for competent anti-microbial host defense. Mice deficient in IL-6 showed an inability to appropriately control bacterial peritonitis (5 × 108 cfu Staphylococcus epidermidis) and displayed both increased dissemination and reduced bacterial clearance. This response was reversed through local reconstitution of IL-6 trans-signaling using a chimeric sIL-6R-IL-6 fusion protein (termed HDS). Administration of HDS (10–1000 ng/ml) to infected IL-6−/− mice promoted bacterial clearance and reduced bacteremia in a dose-dependent fashion. This response was associated with improved resolution of the acute phase response and a more rapid normalization of body temperature. To define the mechanisms governing IL-6 involvement in anti-microbial immunity, we first developed an ex vivo flow cytometric assay to simultaneously monitor neutrophil respiratory burst and phagocytosis. Bacterial stimulation of circulating blood neutrophils from IL-6−/− and IL-6R−/− mice showed comparable effector function to that displayed by WT controls. Thus, IL-6 deficiency is not associated with an inherent defect in neutrophil function. However, evaluation of infiltrating neutrophils isolated by lavage from the peritoneal cavity of infected IL-6−/− and IL-6R−/− mice showed significantly reduced respiratory burst and phagocytic activity. Interleukin-6 therefore controls responses local to the site of infection and is consistent with the activation of non-hematopoietic stromal cells by IL-6 and sIL-6R. In human peritoneal mesothelial cells and peritoneal membranes from infected mice, IL-6 trans-signaling promoted cyclooxygenase-2 (COX2) expression. Blockade of IL-6 signaling in infected WT mice with the antagonist sgp130 or anti-IL-6R antibodies inhibited peritoneal COX2 expression. Mass spectrometry for the COX2 products, prostaglandin-E2 (PGE2) prostaglandin-D2 (PGD2) in lavage samples from infected mice showed that the generation of PGD2, but not PGE2, was impaired in IL-6 deficiency. Here, PGD2 levels correlated with the number of infiltrating neutrophils undergoing respiratory burst. These studies suggest IL-6 signaling regulates the effector characteristics of infiltrating neutrophils through local control of immuno-regulatory prostaglandin production.

Anti-inflammatory activity of hexane extracts from bones and internal organs of Anguilla japonica suppresses cyclooxygenase-2-dependent prostaglandin D2 generation in mast cells and anaphylaxis in mice

The purpose of this study is to investigate the effects of n-hexane extracts from bones and internal organs of Japanese eel, Anguilla japonica (HEE), on cyclooxygenase-2 (COX-2)-dependent prostaglandin D₂(PGD₂) generation in stem cell factor (SCF), IL-10, plus LPS-induced mouse bone marrow-derived mast cells (BMMCs) and on passive cutaneous anaphylaxis (PCA) in mice. HEE suppressed SCF/IL-10/LPS-induced PGD₂ generation, and concomitantly reduced COX-2 protein expression dose-dependently. To understand the mechanistic basis for the inhibition of PGD₂ generation by HEE, we examined the effects of HEE on upstream signaling pathways essential for COX-2 induction. HEE was found to inhibit the translocation of nuclear factor-κB (NF-κB) p65 subunit to the nucleus and its DNA-binding ability through the inhibition of TAK1, IKK and IκB phosphorylation. Furthermore, HEE also attenuated mitogen-activated protein kinase (MAPK)-mediated regulation of DNA binding of activator protein-1 (AP-1). Moreover, oral administration of HEE inhibited anti-dinitrophenyl (DNP) IgE-induced PCA in a dose dependent manner. Taken together, the present study provides new insights into the anti-inflammatory activity of HEE, which could be a promising candidate to be used for an inflammatory therapy.

Manassantin B Isolated from &lt;i&gt;Saururus chinensis&lt;/i&gt; Inhibits Cyclooxygenase-2-Dependent Prostaglandin D&lt;sub&gt;2&lt;/sub&gt; Generation by Blocking Fyn-Mediated Nuclear Factor-kappaB and Mitogen Activated Protein Kinase Pathways in Bone Marrow Derived-Mast Cells

The authors investigated the effect of manassantin B (Man B) isolated from Saururus chinensis (S. chinensis) on cyclooxygenase-2 (COX-2)-dependent prostaglandin D2 (PGD2) generation in mouse bone marrow derived-mast cells (BMMCs). Man B inhibited the generation of PGD2 dose-dependently by inhibiting COX-2 expression in immunoglobulin E (IgE)/Ag-stimulated BMMCs. To elucidate the mechanism responsible for the inhibition of COX-2 expression by Man B, the effects of Man B on the activation of nuclear factor-kappaB (NF-κB), a transcription factor essential and mitogen-activated protein kinases (MAPKs) for COX-2 induction, were examined. Man B attenuated the nuclear translocation of NF-κB p65 and its DNA-binding activity by inhibiting inhibitors of kappa Bα (IκBα) degradation and concomitantly suppressing IκB kinase (IKK) phosphorylation. In addition, Man B suppressed phosphorylation of MAPKs including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK) and p38. It was also found that Man B suppressed Fyn kinase activation and consequent downstream signaling processes, including those involving Syk, Gab2, and Akt. Taken together, the present results suggest that Man B suppresses COX-2 dependent PGD2 generation by primarily inhibiting Fyn kinase in FcεRI-mediated mast cells.

Heterogeneity in the responses of human lung mast cells to stem cell factor

Stem cell factor (SCF) is a growth factor that is involved in mast cell differentiation and proliferation. SCF primes human lung mast cells for enhanced responses to IgE-directed activation but is not generally recognized as a direct activator. SCF mediates its effects through c-kit. The aim of this study was to reappraise the effects of SCF on human lung mast cells. Mast cells were isolated from human lung. Mast cells were challenged with anti-IgE or SCF and the generation of histamine, cysteinyl-leukotrienes (cys-LTs) and prostaglandin D(2) (PGD(2) ) was assessed as was expression of the activation marker, CD63. The effects of c-kit inhibitors on mediator release were evaluated. Stem cell factor (10 ng/mL) alone was unable to induce mediator release but primed mast cells for enhanced IgE-dependent secretion. At higher concentrations (≥ 30 ng/mL), SCF had more varied effects and even when used alone was able to drive substantial levels of histamine release in about a third of all preparations studied. Similarly, SCF (100 ng/mL) alone was effective in stimulating the generation of cys-LTs in half of the preparations studied. SCF (100 ng/mL) was even more effective at stimulating PGD(2) generation as almost all preparations generated substantial quantities of the prostanoid. Mediator release induced by SCF was accompanied by the up-regulation of the activation marker, CD63. There was a positive correlation between the extent of mediator release induced by SCF and c-kit receptor expression. The effects of SCF on mediator release from mast cells were reversed by the c-kit inhibitor imatinib. These data demonstrate that the responses of mast cells to SCF are heterogeneous. SCF can drive much greater levels of mediator release from mast cells, especially of PGD(2), than hitherto appreciated and this could be important in the context of respiratory diseases.

Impact of IL-33 on PGD2 Generation by Activated Human Cord Blood-Derived Mast Cell: Lack of Effect on Tryptase Release

Mast cells are important not only in allergic reactions, but also in inflammation and are involved in a variety of responses including the immediate release of potent inflammatory mediators after activation by cross-linking of FcεRI molecules. Prostaglandin D2 (PGD2) is a major cyclooxygenase metabolite of arachidonic acid produced by mast cells and it is released following allergen challenge in allergic diseases. IL-33 is an iflammatory cytokine which is critically involved in the regulation of in vitro and in vivo cyclooxygenase production, providing a potential therapeutic target for inflammatory disorders. In this study, using human derived umbelical cord blood mast cells, we show that IL-33 (50 ng/ml), and calcium ionophore A 23187 (0.5 μg/ml), compound 48/80 (10−5 M) or anti-IgE (10 μg/ml), enhaced the production of PGD2 and this effect was inhibited by indomethacin. However, IL-33 was unable to induce tryptase release in these cells. These effects confirm the inflammatory property of IL-33 by stimulating PGD2 but not tryptase in human mast cells. The inhibitory effect of this new cytokine may have a potential therapeutic response in allergic and inflammatory diseases.