PGD2 Generation Research Articles

Anti-allergic actions of a Chinese patent medicine, huoxiangzhengqi oral liquid, in RBL-2H3 cells and in mice

Context Huoxiangzhengqi oral liquid (HXZQ-OL), a traditional Chinese medicine formula, has antibacterial, anti-inflammation and gastrointestinal motility regulation effects. Objective The study investigates the anti-allergic activity and underlying mechanism of HXZQ-OL. Materials and methods IgE/Ag-mediated RBL-2H3 cells were used to evaluate the anti-allergic activity of HXZQ-OL (43.97, 439.7 and 4397 μg/mL) in vitro. The release of cytokines and eicosanoids were quantified using ELISA. RT-qPCR was used to measure the gene expression of cytokines. The level of intracellular Ca2+ was measured with Fluo 3/AM. Immunoblotting analysis was performed to investigate the mechanism of HXZQ-OL. In the passive cutaneous anaphylaxis (PCA), BALB/c mice (5 mice/group) were orally administrated with HXZQ-OL (263.8, 527.6 and 1055 mg/kg/d) or dexamethasone (5 mg/kg/d, positive control) for seven consecutive days. Results HXZQ-OL not only inhibited degranulation of mast cells (IC50, 123 μg/mL), but also inhibited the generation and secretion of IL-4 (IC50, 171.4 μg/mL), TNF-α (IC50, 88.4 μg/mL), LTC4 (IC50, 52.9 μg/mL) and PGD2 (IC50, 195.8 μg/mL). Moreover, HXZQ-OL suppressed the expression of IL-4 and TNF-α mRNA, as well as the phosphorylation of Fyn, Lyn and multiple downstream signalling proteins including MAPK and PI3K/NF-κB pathways. In addition, HXZQ-OL (527.5 mg/kg) attenuated the IgE-mediated PCA with 55% suppression of Evans blue exudation in mice. Conclusions HXZQ-OL attenuated the activation of mast cell and PCA. Therefore, HXZQ-OL might be used as an alternative treatment for allergic diseases.

DMSO-induced drastic changes in cellular processes and epigenetic landscape in vitro

Classical FcεRI-induced mast cell (MC) activation causes synthesis of arachidonic acid (AA)–derived eicosanoids (leukotriene [LT] C4, prostaglandin [PG] D2, and thromboxane A2), which mediate vascular leak, bronchoconstriction, and effector cell chemotaxis. Little is known about the significance and regulation of eicosanoid generation in response to nonclassical MC activation mechanisms.We sought to determine the regulation and significance of MC-derived eicosanoids synthesized in response to IL-33, a cytokine critical to innate type 2 immunity.We used an ex vivo model of mouse bone marrow–derived mast cells and an IL-33–dependent in vivo model of aspirin-exacerbated respiratory disease (AERD).IL-33 potently liberates AA and elicits LTC4, PGD2, and thromboxane A2 production by bone marrow–derived mast cells. Unexpectedly, the constitutive function of COX-1 is required for IL-33 to activate group IVa cytosolic phospholipase A2 with consequent AA release for synthesis of all eicosanoids, including CysLTs. In contrast, COX-1 was dispensable for FcεRI-driven CysLT production. Inhibition of COX-1 prevented IL-33–induced phosphorylation of extracellular signal-related kinase, an upstream effector of cytosolic phospholipase A2, which was restored by exogenous PGH2, implying that the effects of COX-1 required its catalytic function. Administration of a COX-1–selective antagonist to mice completely prevented the generation of both PGD2 and LTC4 in a model of AERD in which MC activation is IL-33 driven.MC-intrinsic COX-1 amplifies IL-33–induced activation in the setting of innate type 2 immunity and might help explain the phenomenon of therapeutic desensitization to aspirin by nonselective COX inhibitors in patients with AERD.

Aspirin desensitization in aspirin-exacerbated respiratory disease: New insights into the molecular mechanisms.

Aspirin desensitization (AD) has been the only available modifying treatment in aspirin-exacerbated respiratory disease (AERD). The mechanisms of AD are nonetheless poorly understood. Though very effective, AD is limited by its risks and side-effects. Moving forward to the targeted biologicals era, the aim of this study was to characterize the airway inflammatory response to long-term AD, including TSLP dynamics, in order to assess potential new targets in AERD. Adult patients with aspirin challenge-confirmed AERD underwent an oral AD followed by daily ingestion of aspirin for at least 6 months. Clinical data and inflammatory biomarkers were measured and compared, before and after AD. Induced sputum analyses were performed at baseline, one and six months after AD (differential cell count and levels of sputum supernatant leukotriene C4, prostaglandin D2 and E2, and TSLP). AD was followed by significant clinical improvement, as quantified by all monitored parameters. The good clinical outcomes of AD in our study are supported by overall changes observed in the arachidonic acid metabolites (decreased PGD2 over a constant LTC4/PGE2). TSLP increased (mean baseline 0.1 ± 0.03; 1 month 3.68 ± 7; 6 months 212.2 ± 44 pg/ml; p < 0.01). Our findings suggest that new biologicals blocking TSLP might have a clinical benefit in AERD, by cutting down the TSLP-induced PGD2 generation.

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.

Leukotriene D4 and prostaglandin E2 signals synergize and potentiate vascular inflammation in a mast cell–dependent manner through cysteinyl leukotriene receptor 1 and E-prostanoid receptor 3

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

Piperine inhibits the activities of platelet cytosolic phospholipase A2 and thromboxane A2 synthase without affecting cyclooxygenase-1 activity: different mechanisms of action are involved in the inhibition of platelet aggregation and macrophage inflammatory response.

PURPOSE: Piperine, a major alkaloid of black pepper (Piper nigrum) and long pepper (Piper longum), was shown to have anti-inflammatory activity through the suppression of cyclooxygenase (COX)-2 gene expression and enzyme activity. It is also reported to exhibit anti-platelet activity, but the mechanism underlying this action remains unknown. In this study, we investigated a putative anti-platelet aggregation mechanism involving arachidonic acid (AA) metabolism and how this compares with the mechanism by which it inhibits macrophage inflammatory responses; METHODS: Rabbit platelets and murine macrophage RAW264.7 cells were treated with piperine, and the effect of piperine on the activity of AA-metabolizing enzymes, including cytosolic phospholipase A2 (cPLA2), COX-1, COX-2, and thromboxane A2 (TXA2) synthase, as well as its effect on AA liberation from the plasma membrane components, were assessed using isotopic labeling methods and enzyme immunoassay kit; RESULTS: Piperine significantly suppressed AA liberation by attenuating cPLA2 activity in collagen-stimulated platelets. It also significantly inhibited the activity of TXA2 synthase, but not of COX-1, in platelets. These results suggest that piperine inhibits platelet aggregation by attenuating cPLA2 and TXA2 synthase activities, rather than through the inhibition of COX-1 activity. On the other hand, piperine significantly inhibited lipopolysaccharide-induced generation of prostaglandin (PG)E2 and PGD2 in RAW264.7 cells by suppressing the activity of COX-2, without effect on cPLA2; CONCLUSION: Our findings indicate that piperine inhibits platelet aggregation and macrophage inflammatory response by different mechanisms.

Identification of orally bioavailable small-molecule inhibitors of hematopoietic prostaglandin D2 synthase using X-ray fragment based drug discovery

Using X-ray crystallographic screening, fragments 4 and 6 were identified as inhibitors of hematopoietic prostaglandin D2 synthase (H-PGDS). Both fragments induced a small protein movement in the X-ray crystal structure relative to the apo structure, where the highly polar nature of the ligand complemented the induced protein conformation. The manuscript describes the fragment optimisation of 4 and 6 followed by fragment growth to lead molecule 10. This showed favourable physicochemical properties and evidence of oral activity in blocking PGD2 generation in vivo.

Regulation of Prostaglandin E2 signaling by cysteinyl leukotrienes in human mast cells

Cysteinyl leukotrienes (cys‐LTs), LTC4, LTD4 and LTE4, are critical mediators of airway hyper‐responsiveness and asthma. We have previously shown that LTD4 regulates mast cell (MCs) functions through the activation of G‐protein‐coupled receptors, CysLT1R and CysLT2R. The role of Prostaglandin E2 (PGE2), the other arachidonic acid metabolite in asthma is controversial as it act as both pro‐ and anti‐inflammatory, depending on the receptor through which it exerts its effect. Although cysLTs were shown to induce PGD2 generation in mast cells, it is not known whether there is cross‐talk between cysLTs and PGE2. Here, we found that PGE2 by itself does not activate calcium and MIP‐1β in mast cells, but cys‐LT priming induced robust PGE2‐dependent calcium flux and MIP‐1β generation. Further, using immunoprecipitation assays, we found that PGE2 receptor EP1R interacts with CysLT1R and this interaction was enhanced upon LTD4 stimulation. Importantly, we found that LTD4 and PGE2 synergistically increased the production of pro‐inflammatory cytokine, MIP1β in mast cells which was substantially inhibited by blocking protein kinase C (PKC)s. Taken together, our results suggest that cysLTs augment inflammation by activating PGE2 signaling through the priming of PGE2 receptors. This unravels a novel phenomenon by which inflammatory mediators generated from the same precursor can cooperate to enhance inflammation.

Attenuation of niacin‐induced prostaglandin D2 generation by omega‐3 fatty acids in THP‐1 macrophages

The present investigation examined the ability of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), omega‐3 polyunsaturated fatty acids (PUFAs), to attenuate niacin‐induced PGs in THP‐1 macrophages. Niacin induced both PGD2 and PGE2 generation in a dose dependent manner. Niacin also caused an increase in cytosolic calcium and activation of cytosolic phospholipase A2 (cPLA2). The increase in PGD2 and PGE2 was reduced by both DHA and EPA, but not by oleic acid (OLA). Omega‐3 PUFAs efficiently incorporated into cellular phospholipids at the expense of AA, whereas OLA incorporated to a higher extent but had no effect on AA levels. Omega‐3 PUFAs also reduced surface expression of HM74A, a human niacin receptor. Furthermore, omega‐3 PUFAs also inhibited the niacin‐induced increase in cytosolic calcium. Niacin and/or omega‐3 PUFAs minimally affected cyclooxygenase‐1 (COX‐1) activity and had no effect on Cox‐2 activity. The effects of niacin on PGD2 generation were further confirmed using Langerhans dendritic cells. Results of the present study indicate that omega‐3 PUFAs reduced niacin‐induced PG formation by diminishing the availability of their substrate and as well as reducing the surface expression of niacin receptors. In conclusion, our study suggests that the regular use of omega‐3 PUFAs along with niacin can potentially reduce the niacin‐induced flushing response in sensitive patients.

Citreorosein, a naturally occurring anthraquinone derivative isolated from Polygoni cuspidati radix, attenuates cyclooxygenase-2-dependent prostaglandin D2 generation by blocking Akt and JNK pathways in mouse bone marrow-derived mast cells

In this study, we examined the effects of citreorosein (CIT), an anthraquinone component of Polygoni cuspidati radix (P. cuspidati, Polygonaceae), on cyclooxygenase (COX)-2 dependent prostaglandin (PG)D2 generation in mast cells, central effector cells of allergy and other inflammatory diseases. CIT strongly inhibited COX-2-dependent PGD2 generation in a concentration-dependent manner in mouse bone marrow-derived mast cells (BMMCs) stimulated with stem cell factor (SCF)/IL-10/LPS. In an effort to identify the mechanisms underlying the inhibition of COX-2-dependent PGD2 generation by CIT, we examined the effects of this compound on MAP kinases, Akt and NF-κB signaling pathways, which are essential for COX-2 induction. CIT inhibited nuclear translocation of the nuclear factor (NF)-κB p65 subunit and its cognate DNA-binding activity, which correlated with its inhibitory effects on the phosphorylation of Akt and IKK and subsequent phosphorylation and degradation of IκB. Furthermore, CIT significantly attenuated the DNA binding of activator protein (AP)-1 that regulates COX-2 expression through the reduction of the phosphorylation of c-Jun. Moreover, inhibition of PGD2 generation by CIT was accompanied by a decrease in phosphorylation of cytosolic phospholipase A2α. Taken together, the present study suggests that CIT represents a potential therapeutic approach for the treatment of inflammatory diseases.

PGD2, IL-1-Family Members and Mast Cells

Cytokines are immunomodulatory and inflammatory compounds produced by many different cell types. The IL-1 family consists of at least eleven cytokines including IL-18 and IL-13 and are essential to the host defence against severe infections and mediate inflammation. IL-18 also enhances tumour rejection and has high capacity to augment the cytotoxicity of NK cells and T cells. IL-33 stimulates basophils and mast cells to produce cytokines and histamine independently of IgE. Mast cells play a crucial role in the development of allergy through the cross-linking of their surface receptors for IgE leading to degranulation and inflammation. Activated mast cells induce the generation of PGD2, detectable in 2–15 minutes after challenge, and LTC4. Here we review the interrelationship between PGD2, IL-1 family members and mast cells.

Anti-inflammatory Compounds from the Leaves of Ailanthus altissima Meihua JIN

In our ongoing search for biological components from the Korea endemic plants, the MeOH extract of Ailanthus altissima leaves (Simaroubaceae) showed cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) dual inhibitory activity by assessing their effects on the production of prostaglandin () and leukotriene () in mouse bone marrow-derived mast cells (BMMCs). In further study, eight compounds, squalene (1), -sitosterol (2), scopoletin (3), quercetin (4), luteolin (5), astragalin (6), scopolin (7), and daucosterol (8) were isolated, the chemical structures were elucidated on the basis of physicochemical and spectroscopic data and by comparison with those of published literatures. Among the compounds, 2, 4, and 5 strongly inhibited both the COX-2-dependent PGD2 generation with values of 2.6, 7.3 and 2.5 , respectively and the generation of in the 5-LOX dependent phase with values of 2.0, 5.1 and 1.8 , respectively, which suggest that the anti-inflammatory activity of A. altissima might occur in part via the inhibition of both and generation by 2, 4 and 5.

The Phosphoinositide 3-Kinase-Dependent Activation of Btk Is Required for Optimal Eicosanoid Production and Generation of Reactive Oxygen Species in Antigen-Stimulated Mast Cells

Activated mast cells are a major source of the eicosanoids PGD(2) and leukotriene C(4) (LTC(4)), which contribute to allergic responses. These eicosanoids are produced following the ERK1/2-dependent activation of cytosolic phospholipase A(2), thus liberating arachidonic acid, which is subsequently metabolized by the actions of 5-lipoxygenase and cyclooxygenase to form LTC(4) and PGD(2), respectively. These pathways also generate reactive oxygen species (ROS), which have been proposed to contribute to FcepsilonRI-mediated signaling in mast cells. In this study, we demonstrate that, in addition to ERK1/2-dependent pathways, ERK1/2-independent pathways also regulate FcepsilonRI-mediated eicosanoid and ROS production in mast cells. A role for the Tec kinase Btk in the ERK1/2-independent regulatory pathway was revealed by the significantly attenuated FcepsilonRI-dependent PGD(2), LTC(4), and ROS production in bone marrow-derived mast cells of Btk(-/-) mice. The FcepsilonRI-dependent activation of Btk and eicosanoid and ROS generation in bone marrow-derived mast cells and human mast cells were similarly blocked by the PI3K inhibitors, Wortmannin and LY294002, indicating that Btk-regulated eicosanoid and ROS production occurs downstream of PI3K. In contrast to ERK1/2, the PI3K/Btk pathway does not regulate cytosolic phospholipase A(2) phosphorylation but rather appears to regulate the generation of ROS, LTC(4), and PGD(2) by contributing to the necessary Ca(2+) signal for the production of these molecules. These data demonstrate that strategies to decrease mast cell production of ROS and eicosanoids would have to target both ERK1/2- and PI3K/Btk-dependent pathways.

NF-κB-Inducing Kinase Regulates Cyclooxygenase 2 Gene Expression in Macrophages by Phosphorylation of PU.1

Selective expression of cyclooxygenase 2 (COX-2) by macrophages could have an important role in the pathobiology of inflammation. We reported a functional synergism between PU.1 and other transcription factors that contributes to COX-2 gene expression in macrophages. PU.1 resides in the nuclear compartment and is activated by phosphorylation to bind to cognate DNA elements containing a 5'-GGAA/T-3' motif, but the involved kinase has not been discovered. We tested the hypothesis that NF-kappaB-inducing kinase (NIK) regulates COX-2 gene expression in macrophages through inducible phosphorylation of PU.1. Our initial experiments showed an in vitro protein-protein binding interaction between myc-NIK and GST-PU.1. Purified myc-NIK had a strong in vitro kinase activity for purified GST-PU.1, and this activity and production of COX-2 protein is blocked by treatment with a nonspecific kinase inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. We used short interfering RNA to develop a stable NIK knockdown macrophage cell line that had an approximately 50% decrease in COX-2 protein production and decreased generation of PGD(2), and this was correlated with decreased binding of activated PU.1 to the COX-2 promoter in response to treatment with endotoxin. These findings suggest a novel role for NIK in mediating COX-2 gene expression in endotoxin-treated macrophages by a mechanism that involves phosphorylation of PU.1.

Essential role for hematopoietic prostaglandin D2 synthase in the control of delayed type hypersensitivity

Hematopoietic prostaglandin D(2) synthase (hPGD(2)S) metabolizes cyclooxygenase-derived prostaglandin (PG) H(2) to PGD(2), which is dehydrated to cyclopentenone PGs, including 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). PGD(2) acts through two receptors (DP1 and DP2/CRTH2), whereas 15d-PGJ(2) can activate peroxisome proliferator-activated receptors or inhibit a range of proinflammatory signaling pathways, including NF-kappaB. Despite eliciting asthmatic and allergic reactions through the generation of PGD(2), it is not known what role hPGD(2)S plays in T helper (Th)1-driven adaptive immunity. To investigate this question, the severity and duration of a delayed type hypersensitivity reaction was examined in hPGD(2)S knockout and transgenic mice. Compared with their respective controls, knockouts displayed a more severe inflammatory response that failed to resolve, characterized histologically as persistent acute inflammation, whereas transgenic mice had little detectable inflammation. Lymphocytes isolated from inguinal lymph nodes of hPGD(2)S(-/-) animals showed hyperproliferation and increased IL-2 synthesis effects that were rescued by 15d-PGJ(2), but not PGD(2), working through either of its receptors. Crucially, 15d-PGJ(2) exerted its suppressive effects through the inhibition of NF-kappaB activation and not through peroxisome proliferator-activated receptor signaling. In contrast, lymph node cultures from transgenics proliferated more slowly and synthesized significantly less IL-2 than controls. Therefore, contrary to its role in driving Th2-like responses, this report shows that hPGD(2)S may act as an internal braking signal essential for bringing about the resolution of Th1-driven delayed type hypersensitivity reactions. Consequently, hPGD(2)S-derived cyclopentenone PGs may protect against inflammatory diseases, where T lymphocytes play a pathogenic role, as in rheumatoid arthritis, atopic eczema, and chronic rejection.

MITF Is Necessary for Generation of Prostaglandin D2 in Mouse Mast Cells

Mast cells generate eicosanoids that are linked to asthma and other inflammatory diseases. A basic-helix-loop-helix leucine zipper transcription factor termed MITF is essential for the development of mast cells. Although other substances also linked to inflammatory reactions (such as various proteases and serotonin) require MITF for their expression, the role of MITF in eicosanoid generation has not been studied. We examined eicosanoid generation in bone marrow-derived mast cells (BMMCs) of tg/tg mice that lack MITF. Most eicosanoids generated by BMMCs are either prostaglandin (PG) D2 or leukotriene C4. The former is synthesized via the cyclooxygenase pathway, whereas the latter is synthesized via the 5-lipoxygenase pathway. In response to stimulation with IgE and antigens, BMMCs of tg/tg mice synthesized leukotriene C4 normally. However, neither immediate nor delayed PGD2 production was detected in these BMMCs. This indicates that MITF is a transcription factor that specifically activates the cyclooxygenase pathway, but not the 5-lipoxygenase pathway. Significant decreases in expression of hematopoietic PGD2 synthase (hPGDS, a terminal synthase for PGD2) were observed at both mRNA and protein levels in tg/tg BMMCs. MITF transactivated the hPGDS gene via a CACCTG motif located in the promoter region. MITF appeared to be essential for generation of PGD2 by enhancing expression of the hPGDS gene in BMMCs.

Human mast cells express inducible PGE synthase and generate PGE2 in response to TLR2-dependent activation

Rationale Mast cells (MCs) are effectors of both innate and adaptive immunity. MC activation through Fc epsilon RI results in their generation of leukotriene (LT)C4 through the 5-lipoxygenase/leukotriene C4 synthase (5-LO/LTC4S) pathway, and of prostaglandin (PG)D2 through the cyclooxygenase (COX)/PGD2 synthase pathway. PGE2 is generated by both constitutive and inducible enzymatic systems and serves a key role in cell activation and gene induction; its generation by MCs has not been recognized. Methods Cultured human MCs (hMCs) derived from cord blood were stimulated by either Fc epsilon RI cross-linkage, or by staphylococcal peptidoglycan (PGN), a ligand for Toll-like receptor (TLR)2. Eicosanoids were measured by ELISA, and relevant enzymes and proteins were assessed by western blots. Results Unexpectedly, both ligands elicited the generation of PGE2, as well as LTC4 and PGD2. Whereas Fc epsilon RI-dependent activation was far more potent for LTC4 and PGD2 generation, PGN was more potent for PGE2 generation, which occurred with a strikingly delayed kinetics (maximal at 24 h). Both activation stimuli induced the expression of COX-2 and two terminal synthases, mPGES-1 and mPGES-2. Delayed-phase PGE2 generation was completely prevented by NS-398, a selective PGHS-2 inhibitor. Conclusions MCs express an inducible system for PGE2 generation, and likely produce different respective profiles of eicosanoids in innate and adaptive immune responses. The inducible COX-2/mPGES system may form a pathogenetic link between MCs and non-allergic diseases in which PGE2 plays a major role.

Cysteinyl leukotrienes and uridine diphosphate induce cytokine generation by human mast cells through an interleukin 4-regulated pathway that is inhibited by leukotriene receptor antagonists.

We previously reported that interleukin (IL)-4 upregulates the expression of leukotriene C4 synthase (LTC4S) by human cord blood–derived mast cells (hMCs), augments their high-affinity Fc receptor for IgE (FcεRI)-dependent generation of eicosanoids and cytokines, and induces a calcium flux in response to cysteinyl leukotrienes (cys-LTs) and uridine diphosphate (UDP) that is blocked by cys-LT receptor antagonists. We speculated that this IL-4–dependent, receptor-mediated response to the cys-LTs and UDP might induce cytokine generation by hMCs without concomitant exocytosis. Unlike hMCs maintained in cytoprotective stem cell factor (SCF) alone, hMCs primed for 5 d with IL-4 responded to UDP (1 μM), LTC4 (100 nM), and LTD4 (100 nM) by producing IL-5, tumor necrosis factor (TNF)-α, and especially large quantities of macrophage inflammatory protein (MIP)-1β de novo at 6 h, preceded by the induced expression of the corresponding mRNAs. Cys-LT– and UDP-mediated cytokine production by the primed hMCs occurred without histamine release or PGD2 generation and was inhibited by the CysLT1 receptor antagonist MK571. Additionally, pretreatment of hMCs with MK571 or with the cys-LT biosynthetic inhibitor MK886 decreased IL-5 and TNF-α production in response to IgE receptor cross-linkage, implying a positive feedback by endogenously produced cys-LTs. Cys-LTs and UDP thus orchestrate a novel, IL-4–regulated, non-IgE–dependent hMC activation for cytokine gene induction that could be initiated by microbes, cellular injury, or neurogenic or inflammatory signals; and this pathobiologic event would not be recognized in tissue studies where hMC activation is classically defined by exocytosis.

T helper cell type 2 cytokines coordinately regulate immunoglobulin E-dependent cysteinyl leukotriene production by human cord blood-derived mast cells: profound induction of leukotriene C(4) synthase expression by interleukin 4.

Human mast cells (hMCs) derived in vitro from cord blood mononuclear cells exhibit stem cell factor (SCF)-dependent comitogenic responses to T helper cell type 2 (Th2) cytokines. As cysteinyl leukotriene (cys-LT) biosynthesis is a characteristic of immunoglobulin (Ig)E-activated mucosal hMCs, we speculated that Th2 cytokines might regulate eicosanoid generation by hMCs. After passive sensitization for 5 d with IgE in the presence of SCF, anti-IgE-stimulated hMCs elaborated minimal cys-LT (0.1 +/- 0.1 ng/10(6) hMCs) and abundant prostaglandin (PG)D(2) (16.2 +/- 10.3 ng/10(6) hMCs). Priming of hMCs by interleukin (IL)-4 with SCF during passive sensitization enhanced their anti-IgE-dependent histamine exocytosis and increased their generation of both cys-LT (by 27-fold) and PGD(2) (by 2. 5-fold). Although priming with IL-3 or IL-5 alone for 5 d with SCF minimally enhanced anti-IgE-mediated cys-LT generation, these cytokines induced further six- and fourfold increases, respectively, in IgE-dependent cys-LT generation when provided with IL-4 and SCF; this occurred without changes in PGD(2) generation or histamine exocytosis relative to hMCs primed with IL-4 alone. None of these cytokines, either alone or in combination, substantially altered the levels of cytosolic phospholipase A(2) (cPLA(2)), 5-lipoxygenase (5-LO), or 5-LO activating protein (FLAP) protein expression by hMCs. In contrast, IL-4 priming dramatically induced the steady-state expression of leukotriene C(4) synthase (LTC(4)S) mRNA within 6 h, and increased the expression of LTC(4)S protein and functional activity in a dose- and time-dependent manner, with plateaus at 10 ng/ml and 5 d, respectively. Priming by either IL-3 or IL-5, with or without IL-4, supported the localization of 5-LO to the nucleus of hMCs. Thus, different Th2-derived cytokines target distinct steps in the 5-LO/LTC(4)S biosynthetic pathway (induction of LTC(4)S expression and nuclear import of 5-LO, respectively), each of which is necessary for a full integrated functional response to IgE-dependent activation, thus modulating the effector phenotype of mature hMCs.

A new class of COX-2 inhibitor, rutaecarpine from Evodia rutaecarpa.

We investigated the effect of a new class of COX-2 inhibitor, rutaecarpine, on the production of PGD2 in bone marrow derived mast cells (BMMC) and PGE2 in COX-2 transfected HEK293 cells. Inflammation was induced by lambda-carrageenan in male Splague-Dawley (SD) rats. Rutaecarpine (8,13-Dihydroindolo[2',3':3,4]pyridol[2,1-b]quinazolin -5(7H)-one) was isolated from the fruits of Evodia rutaecarpa. BMMC were cultured with WEHI-3 conditioned medium. c-Kit ligand and IL-10 were obtained by their expression in baculovirus. The generation of PGD2 and PGE2 were determined by their assay kit. COX-1 and COX-2 protein and mRNA expression was determined by BMMC in the presence of KL, LPS and IL-10. Rutaecarpine and indomethacin dissolved in 0.1% carboxymethyl cellulose was administered intraperitoneally and, 1 h later, lambda-carrageenan solution was injected to right hind paw of rats. Paw volumes were measured using plethysmometer 5 h after lambda-carrageenan injection. Rutaecarpine inhibited COX-2 and COX-1 dependent phases of PGD2 generation in BMMC in a concentration-dependent manner with an IC50 of 0.28 microM and 8.7 microM, respectively. It inhibited COX-2-dependent conversion of exogenous arachidonic acid to PGE2 in a dose-dependent manner by the COX-2-transfected HEK293 cells. However, rutaecarpine inhibited neither PLA2 and COX-1 activity nor COX-2 protein and mRNA expression up to the concentration of 30 microM in BMMC, indicating that rutaecarpine directly inhibited COX-2 activity. Furthermore, rutaecarpine showed in vivo anti-inflammatory activity on rat lambda-carrageenan induced paw edema by intraperitoneal administration. Anti-inflammatory activity of Evodia rutaecarpa could be attributed at least in part by inhibition of COS-2.