cys-LT Synthesis Research Articles

CysLT1 Receptor Is Protective against Oxidative Stress in a Model of Irritant-Induced Asthma.

The bronchoconstrictive and proinflammatory properties of cysteinyl leukotrienes (cysLTs) in allergic asthma mediate their effects predominantly through the cysLT1 receptor (cysLT1R). However, the role of cysLTs and cysLT1R in innate immune-triggered asthma is largely unexplored. We explored the synthesis of cysLTs and cysLT1R as determinants of airway responses in an oxidative stress-induced model of irritant asthma. Wild-type (WT) mice exposed to 100 ppm Cl2 for 5 min had airway neutrophilia, increased cysLT production, and pulmonary expression of cysLT-related biosynthetic genes. CysLT1R-deficient (CysLTr1(-/-)) mice that were exposed to Cl2 demonstrated airway hyperresponsiveness to inhaled methacholine significantly greater than in WT BALB/c mice. Compared to WT mice, airway neutrophilia and keratinocyte chemoattractant production levels were higher in CysLTr1(-/-) mice and airway hyperresponsiveness was ameliorated using a granulocyte depletion Ab. CysLTr1(-/-) mice also demonstrated prolonged bronchial epithelial cell apoptosis following Cl2 WT mice showed increased antioxidant and NF erythroid 2-related factor 2 (Nrf2) gene expression, Nrf2 nuclear translocation in bronchial epithelial cells, and increased reduced glutathione/oxidized glutathione following Cl2 exposure whereas CysLTr1(-/-) mice did not. Furthermore, CysLTr1(-/-) mice demonstrated increased pulmonary E-cadherin expression and soluble E-cadherin shedding compared with WT mice. Loss of a functional cysLT1R results in aberrant antioxidant response and increased susceptibility to oxidative injury, apparently via a cysLT1R-dependent impairment of Nrf2 function.

Tandem Benzophenone Amino Pyridines, Potent and Selective Inhibitors of Human Leukotriene C4 Synthase.

Cysteinyl leukotrienes (cys-LTs) are lipid mediators of inflammation. The enzyme catalyzing synthesis of cys-LTs, leukotriene C4 synthase (LTC4S), is considered an important drug target. Here we report the synthesis and characterization of three tandem benzophenone amino pyridines as inhibitors of LTC4S in vitro and in vivo. The inhibitors were characterized in vitro using recombinant human LTC4S, MonoMac 6 cells, and a panel of peripheral human immune cells. In vivo, the compounds were tested in the Zymosan A-induced peritonitis mouse model. The molecules, denoted TK04, TK04a, and TK05, were potent and selective inhibitors of LTC4S with IC50 values of 116, 124, and 95 nM, respectively. Molecular docking revealed binding in a hydrophobic crevice between two enzyme monomers and interaction with two catalytic residues, Arg104 and Arg31. The TK compounds potently inhibited cys-LT biosynthesis in immune cells. In coincubations of platelets and polymorphonuclear leukocytes, inhibition of LTC4S led to shunting of LTA4 toward anti-inflammatory lipoxin A4, which was significantly enhanced by simultaneous inhibition of LTA4H. Finally, we found that TK05 (6 mg⋅kg(-1)⋅body weight) reduces LTE4 levels in peritoneal lavage fluid by 88% and significantly decreases vascular permeability in vivo. Our findings indicate that the TK compounds are valuable experimental tools in eicosanoid research in vitro and in vivo. Their chemical structures may serve as leads for further inhibitor design. Novel drugs depleting cys-LT production could be beneficial for treatment of inflammatory diseases associated with overexpression of LTC4S.

Endogenous secreted phospholipase A2 group X regulates cysteinyl leukotrienes synthesis by human eosinophils

Phospholipase A2s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA2α (cPLA2α) is thought to be the dominant PLA2 in eosinophils; however, eosinophils also have secreted PLA2 (sPLA2) activity that has not been fully defined. To examine the expression of sPLA2 group X (sPLA2-X) in eosinophils, the participation of sPLA2-X in the formation of CysLTs, and the mechanism by which sPLA2-X initiates the synthesis of CysLTs in eosinophils. Peripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. Arabbit polyclonal anti-sPLA2-X antibody identified sPLA2-X by Western blot. Weused confocal microscopy to colocalize the sPLA2-X to intracellular structures. An inhibitor of sPLA2-X (ROC-0929) that does not inhibit other mammalian sPLA2s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA2α, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT. Eosinophils express the mammalian sPLA2-X gene (PLA2G10). The sPLA2-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA2α also attenuated the fMLP-mediated formation of CysLT. The sPLA2-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA2α during cellular activation, indicating that sPLA2-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA2α. We further demonstrate that sPLA2-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1β increased the expression of PLA2G10 by eosinophils. These results demonstrate that sPLA2-X plays a significant role in the formation of CysLTs by human eosinophils. The predominant role of the enzyme is the regulation of MAPK activation that leads to the phosphorylation of cPLA2α. The sPLA2-X protein is regulatedby proteolytic cleavage, suggesting that an inflammatory environment may promote the formation of CysLTs through this mechanism. These results have important implications forthe treatment of eosinophilic disorders such as asthma.

Cathelicidin rCRAMP stimulates rat mast cells to generate cysteinyl leukotrienes, synthesize TNF and migrate: involvement of PLC/A2, PI3K and MAPK signaling pathways.

Cathelicidins represent a family of cationic peptides involved in host defense systems. Apart from exerting direct anti-microbial effects, cathelicidins can regulate immune responses by affecting the activity of cells playing a role in antibacterial defense. Taking into account that mast cells are critical components of host defense, the aim of this study was to determine whether rat cathelicidin-related anti-microbial peptide (rCRAMP) can influence mast cell activity. We have demonstrated that activation of fully mature rat mast cells with rCRAMP resulted in generation and release of cysteinyl leukotrienes (cysLTs). However, rCRAMP failed to induce mast cell degranulation and histamine release. We also found that rCRAMP stimulated rat mast cells to synthesize TNF, but not CXCL8. What is more, this peptide induced GM-CSF, IL-1β, CCL2 and CCL3 but not IL-33 mRNA expression in mast cells. Finally, we showed that this cathelicidin serves as potent chemoattractant for rat mast cells. rCRAMP-mediated cysLT synthesis and mast cell migration were strongly inhibited by IL-10 pre-treatment. With the use of specific inhibitors, we established that activation of PLC/A2 and ERK1/2, but not p38, was required for rCRAMP-induced mast cell stimulation, while PI3K-dependent pathway is involved in both TNF synthesis and mast cell migration. Our results suggest that cathelicidins can amplify inflammatory responses by causing mast cells accumulation and by stimulating these cells to release potent pro-inflammatory mediators.

Fatty acids, inflammation, and asthma

Fatty acids and consequently diet play an essential role in the formation of inflammatory mediators involved in the pathogenesis of asthma. Because intake variations of omega-6 (n-6) and omega-3 (n-3) fatty acids ultimately determine cell membrane incorporation, changes in diet have the potential to modify downstream production of inflammatory mediators derived from these compounds. It has long been hypothesized that decreasing the n-6/n-3 ratio could reduce the production of more proinflammatory mediators while increasing the formation of downstream metabolites that can serve to limit or resolve inflammation. In turn, these changes would result in improved asthma outcomes or would lower the risk for asthma incidence. This review will focus on the role of fatty acid inflammatory and resolving mediators and will summarize the clinical and epidemiologic data on how diet and obesity alter fatty acid profiles that can contribute to asthma.

LEUKOTRIENE C4 SYNTHASE AND LEUKOTRIENE RECEPTOR-1 GENES POLYMORPHISM AMONG ATOPIC ASTHMATIC PATIENTS

Asthma is a complex polygenic disease in which Cysteinyl Leukotrienes (Cys-LTs) are a potential risk factors causing airway inflammation and remodeling, which are characteristics of asthma. The polymorphisms in the leukotriene C4 synthase -444A/C and cysteinyl leukotriene receptor1 927 T/C genes has been implicated in susceptibility to asth ma. The objective of this study was to analyse two different polymorphisms, LTC4S-444 A/C and Cys-LTR1 927 T/C single nucleotide polymorphism and to determine whether there is an association betwee n these polymorphisms and asthma development. The study included two groups (30 asthmatics and 30 hea lthy controls). They were genotyped for the LTC4S444 A/C and CysLTR1 927 T/C polymorphisms by PCR-RFLP. Their total serum IgE levels and urinary LTE4 levels were measured by Enzyme Linked Immunosorbent Assay (ELISA). IgE levels and urinary leukotriene E4 levels were higher in patient group than control group. The genotype and allele frequencies of both LTC4S-444 A/C and CysLTR1 927 T/C polymorphism were not significantly different between asthmatic patients and control gr oup. While urinary leukotriene E4 levels were significantly higher in variant types of LTC4 synth ase (AC and CC) compared to wild type (AA). This study does not support the role of these polymorphi sms in genetic susceptibility to asthma but provide an evidence for a functional role of LTC4 synthase-444 A/C polymorphism on Cys-LT synthesis.

Cysteinyl leukotrienes and their receptors; emerging concepts.

Cysteinyl leukotrienes (cys-LTs) are potent mediators of inflammation derived from arachidonic acid through the 5-lipoxygenase/leukotriene C4 synthase pathway. The derivation of their chemical structures and identification of their pharmacologic properties predated the cloning of their classical receptors and the development of drugs that modify their synthesis and actions. Recent studies have revealed unanticipated insights into the regulation of cys-LT synthesis, the function of the cys-LTs in innate and adaptive immunity and human disease, and the identification of a new receptor for the cys-LTs. This review highlights these studies and summarizes their potential pathobiologic and therapeutic implications.

A Phase I Trial Of Zileuton In Sickle Cell Disease

Sickle cell disease (SCD) is associated with a high prevalence of airway hyperreactivity (AHR) and chronic inflammation at a steady state in the absence of infection or acute sickle events; SCD patients have also been reported to have increased circulating leukotrienes (LT) (Setty et al, 2002; Jennings et al, 2008): both the pro-inflammatory LT-B4 and cysteinyl leukotrienes (cys-LTs; LT-C4, -D4 and -E4), known to promote AHR. We sought to explore the mechanisms of increased AHR in SCD. Five-lipoxygenase (5LO) is a key enzyme in LT-B4 and cysLT synthesis, we have recently reported that expression of 5LO is significantly increased in blood mononuclear cells (MNCs) in patients with SCD (Patel et al 2009). Zileuton is a specific inhibitor of 5LO that decreases LT production, is licensed for asthma and is now available in BID dosing (Zyflo CR, Cornerstone Therapeutics), but has never been tested in patients with SCD. We performed a phase 1 clinical trial to determine the safety and pharmacodynamics (PK) of twice daily administration of ZL (Zyflo CR, the study drug was provided by Cornerstone Therapeutics) in patients with SCD after obtaining an IND from the FDA. The primary objective of this study was to determine a safe and tolerable dose of ZL using a 3x3 dose escalation design of twice daily ZL for 6 weeks, with monitoring of blood counts, serum and urine chemistries, echo, EKG and clinical events. Patients 7-30 years of age with SCD at steady state that were not on hydroxyurea or chronic transfusions were enrolled. A total of 14 subjects were consented and screened for the ZL trial; 4 failed screening and 11 subjects received the study drug; 2 subjects were withdrawn due to acute sickle events. The 1200 mg BID dose that is approved for asthma was well tolerated by 3 patients; the dose was then escalated to 3000 mg/day. We found that 1800 mg alternating with 1200 mg of ZL was a safe and clinically well tolerated dose in 6 patients. No clinically significant changes of any of the clinical/laboratory safety parameters were observed from baseline values that were attributable to ZL. PK, another primary objective of this study was performed using a D-optimal sampling strategy (day 1 through week 6) and data analyzed by nonlinear mixed effects modeling (NONMEM). ZL plasma concentrations were determined by validated LC-MS/MS assay. The Cmax of ZL were 2445 ± 795 ng/ml and 2510 ± 489 ng/ml at 2400 mg/d and 3000 mg/d and the minimum zileuton exposure as measured by predose troughs were 445.0 ± 137.7 ng/ml and 703.7 ± 172.2 ng/ml at 2400 mg/d and 3000 mg/d respectively. PK details will be presented. The secondary objective was to determine the pharmacodynamics (PD) of ZL in patients with SCD. PD end points were 1. CysLT levels before and after ZL administration for 6 weeks, 2. circulating levels of inflammatory biomarkers: sVCAM, sICAM, IL-6, IL-13 and macrophage chemoattractant protein-1 (MCP-1), 3. Methacholine challenge test (MCT) for AHR and 4. fetal hemoglobin. MCT was negative in 5 of 9 of patients; It is to be noted that exclusion of patients on hydroxyurea and chronic transfusions excluded the majority of patients with moderate to severe SCD that predominantly have AHR; 4 patients had a positive MCT, 2 of them became negative after receiving ZL while there was no change in the other 2 patients. LT and inflammatory markers are under analysis. There was no increase in HbF, F cells/F-retics in any of the patients, unlike results reported in vitro in erythroid cultures (Haynes et al, Blood 2004). Our study shows the safety and PK of ZL in SCD and that a higher dose of ZL (than used for asthma) was safely tolerated by SCD patients with good compliance. This phase I trial may form the basis of a phase II/III trial of zileuton in SCD. Clinicaltrial.gov # NCT01136941. Disclosures:No relevant conflicts of interest to declare.

SAT0024 Increased production of leukotrienes by peripheral blood mononuclear cells is associated with more severe disease and worse prognosis in patients with systemic sclerosis

BackgroundLeukotrienes (LTs) are a group of pro-inflammatory and pro-fibrotic, arachidonic acid-derived lipid mediators. We have shown that LTs are increased in the lungs of patients with interstitial lung disease (ILD)...

Different potency of bacterial antigens TLR2 and TLR4 ligands in stimulating mature mast cells to cysteinyl leukotriene synthesis

The aim of study was to compare the potency of different bacterial antigens to induce rat mature mast cell to cysteinyl leukotriene (cysLT) generation. We examined Toll-like receptor (TLR)2 agonists, i.e. lipoteichoic acid (LTA) Staphylococcus faecalis, Streptococcus pyogenes, Bacillus subtilis and Staphylococcus aureus, lipoarabinomannan (LAM) Mycobacterium smegmatis, peptydoglican (PGN) Staphylococcus aureus, as well as TLR4 agonists, i.e. lipopolysaccharide (LPS) Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis, Pophyromonas gingivalis and Escherichia coli. We also estimated the effect of tumor necrosis factor (TNF)-, interleukin (IL)-6-, CCL5-, and IL-10-priming on mast cell cysLT synthesis following bacterial antigen activation. We found that all bacterial antigens activated mast cells to cysLT generation; however, the extent of cysLT release in response to stimulation varied. Out of the examined antigens LPS P. gingivalis exhibited the highest potency, as it induced cysLT generation acting at a very low concentration (10(-4) ng/mL). Other LPSs affected mast cells at higher (up to 10(5) -fold) concentrations. LTAs were the most effective at concentrations of 5 × 10(2) ng/mL, while LAM and PGN stimulated mast cells to maximal cysLT generation at concentrations as high as 10(5) ng/mL. Anti-TLR2 and anti-TLR4 antibodies, as well as nuclear factor κB (NF-κB) inhibitor significantly diminished cysLT generation in response to bacterial antigen stimulation. Priming with TNF, IL-6 and CCL5 did not affect bacterial antigen-induced cysLT generation, while IL-10-pretreatment caused significant decrease in cysLT synthesis by mast cells. These observations might have a great pathophysiological importance; inasmuch cysLTs strongly influence the development and intensity of inflammation during bacterial infection.

Synthesis of cysteinyl leukotrienes in human endothelial cells: subcellular localization and autocrine signaling through the CysLT2receptor

The purpose of this study was to characterize enzyme, receptor, and signaling involved in the synthesis and the activity of cysteinyl leukotrienes (cys-LTs) in human umbilical vein endothelial cells (HUVECs). We used primary cultures of HUVECs and evaluated the formation of cys-LTs by RP-HPLC. Suicide inactivation and subcellular localization of the enzyme responsible for the conversion of leukotriene (LT) A(4) into LTC(4) were studied by repeated incubations with LTA(4) and immunogold electron microscopy. The CysLT(2) receptor in HUVECs was characterized by equilibrium binding studies, Western blot analysis, and immunohistochemistry. Concentration-response curves in HUVECs and in transfected COS-7 cells were used to characterize a novel specific CysLT(2) receptor antagonist (pA(2) of 8.33 and 6.79 against CysLT(2) and CysLT(1) receptors, respectively). The results obtained provide evidence that the mGST-II synthesizing LTC(4) in HUVECs is pharmacologically distinguishable from the LTC(4)-synthase (IC(50) of MK886 <5 μM for LTC(4)-synthase and >30 μM for mGST-II), is not suicide-inactivated and is strategically located on endothelial transport vesicles. The CysLT(2) receptor is responsible for the increase in intracellular Ca(2+) following exposure of HUVECs to cys-LTs and is coupled to a pertussis toxin-insensitive G(q) protein. The synthesis of cys-LTs from LTA(4) by endothelial cells is directly associated with the activation of the CysLT(2) receptor (EC(50) 0.64 μM) in a typical autocrine fashion.

Eosinophil Cysteinyl Leukotriene Synthesis Mediated by Exogenous Secreted Phospholipase A2 Group X

Secreted phospholipase A(2) group X (sPLA(2)-X) has recently been identified in the airways of patients with asthma and may participate in cysteinyl leukotriene (CysLT; C(4), D(4), and E(4)) synthesis. We examined CysLT synthesis and arachidonic acid (AA) and lysophospholipid release by eosinophils mediated by recombinant human sPLA(2)-X. We found that recombinant sPLA(2)-X caused marked AA release and a rapid onset of CysLT synthesis in human eosinophils that was blocked by a selective sPLA(2)-X inhibitor. Exogenous sPLA(2)-X released lysophospholipid species that arise from phospholipids enriched in AA in eosinophils, including phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine as well as plasmenyl phosphatidylcholine and phosphatidylethanolamine. CysLT synthesis mediated by sPLA(2)-X but not AA release could be suppressed by inhibition of cPLA(2)α. Exogenous sPLA(2)-X initiated Ser(505) phosphorylation of cPLA(2)α, an intracellular Ca(2+) flux, and translocation of cPLA(2)α and 5-lipoxygenase in eosinophils. Synthesis of CysLTs in response to sPLA(2)-X or lysophosphatidylcholine was inhibited by p38 or JNK inhibitors but not by a MEK 1/2 inhibitor. A further increase in CysLT synthesis was induced by the addition of sPLA(2)-X to eosinophils under conditions of N-formyl-methionyl-leucyl-phenylalanine-mediated cPLA(2)α activation. These results indicate that sPLA(2)-X participates in AA and lysophospholipid release, resulting in CysLT synthesis in eosinophils through a mechanism involving p38 and JNK MAPK, cPLA(2)α, and 5-lipoxygenase activation and resulting in the amplification of CysLT synthesis during cPLA(2)α activation. Transactivation of eosinophils by sPLA(2)-X may be an important mechanism leading to CysLT formation in the airways of patients with asthma.

A Novel 5-Lipoxygenase-Activating Protein Inhibitor, AM679, Reduces Inflammation in the Respiratory Syncytial Virus-Infected Mouse Eye

Respiratory syncytial virus (RSV) is an important cause of viral respiratory disease in children, and RSV bronchiolitis has been associated with the development of asthma in childhood. RSV spreads from the eye and nose to the human respiratory tract. Correlative studies of humans and direct infection studies of BALB/c mice have established the eye as a significant pathway of entry of RSV to the lung. At the same time, RSV infection of the eye produces symptoms resembling allergic conjunctivitis. Cysteinyl leukotrienes (CysLTs) are known promoters of allergy and inflammation, and the first step in their biogenesis from arachidonic acid is catalyzed by 5-lipoxygenase (5-LO) in concert with the 5-LO-activating protein (FLAP). We have recently developed a novel compound, AM679, which is a topically applied and potent inhibitor of FLAP. Here we show with the BALB/c mouse eye RSV infection model that AM679 markedly reduced the RSV-driven ocular pathology as well as the synthesis of CysLTs in the eye. In addition, AM679 decreased the production of the Th2 cell cytokine interleukin-4 but did not increase the viral load in the eye or the lung. These results suggest that FLAP inhibitors may be therapeutic for RSV-driven eye disease and possibly other inflammatory eye indications.

Transcellular biosynthesis of cysteinyl leukotrienes in vivo during mouse peritoneal inflammation

Leukotrienes (LTs) are lipid mediators of inflammation formed by enzymatic oxidation of arachidonic acid. One intriguing aspect of LT production is transcellular biosynthesis: cells expressing 5-lipoxygenase (5LO) form LTA(4) and transfer it to cells expressing LTA(4) hydrolase (LTA(4)H) or LTC(4) synthase (LTC(4)S) to produce LTB(4) or LTC(4). This process has been demonstrated in vivo for LTB(4), but not for cysteinyl LTs (cysLTs). We examined transcellular cysLT synthesis during zymosan-induced peritonitis, using bone marrow transplants with transgenic mice deficient in key enzymes of LT synthesis and analyzing all eicosanoids by liquid chromatography/tandem mass spectrometry. WT mice time-dependently produced LTB(4) and cysLTs (LTC(4), LTD(4), and LTE(4)). 5LO(-/-) mice were incapable of producing LTs. WT bone marrow cells restored this biosynthetic ability, but 5LO(-/-) bone marrow did not rescue LT synthesis in irradiated WT mice, demonstrating that bone marrow-derived cells are the ultimate source of all LTs in this model. Total levels of 5LO-derived products were comparable in LTA(4)H(-/-) and WT mice, but were reduced in LTC(4)S(-/-) animals. No differences in prostaglandin production were observed between these transgenic or chimeric mice. Bone marrow cells from LTA(4)H(-/-) or LTC(4)S(-/-) mice injected into 5LO(-/-) mice restored the ability to synthesize LTB(4) and cysLTs, providing unequivocal evidence of efficient transcellular biosynthesis of cysLTs. These results highlight the potential relevance of transcellular exchange of LTA(4) for the synthesis of LTs mediating biological activities during inflammatory events in vivo.

Role of cysteinyl-leukotrienes in the pathogenesis of nasal polyposis in patients with asthma

Cysteinyl leukotrienes (CysLTs: leukotrienes C4, D4, and E4) have long been implicated in the pathogenesis of allergic diseases such as asthma. CysLTs are potent bronchoconstrictors that have the additional effects of edema, mucous secretion, and eosinophilic accumulation, and airway remodeling. LTE4 has been identified as a major metabolite of LTC4, and urinary LTE4 (U-LTE4) is considered as the most reliable analytic parameter for monitoring the endogenous synthesis of CysLTs. From our extensive study of U-LTE4 in adult asthma, we identified four factors for hyperleukotrienuria. These factors were aspirin-intolerance, eosinophilic nasal polyposis (ENP), vasculitis, and severe asthma. In ENP there is prominent infiltration of eosinophils in the sinus and polyp tissues, which is linked to adult asthma and aspirin-sensitivity, and ENP is the most important factor for overproduction of CysLTs in asthmatics. We demonstrated that there is a significant decrease in the U-LTE4 concentration after the sinus surgery of asthmatics with and without aspirin intolerance. Recent studies reported that ENP tissue contain and produce a large amount of CysLTs, and there is a close relationship between CysLT production and eosinophil accumulation in ENP. These observations suggest that ENP is not only a local allergic disease, but also a systemic inflammatory disease with CysLT overproduction.

Interactions Between Leukotriene C4 and Interleukin 13 Signaling Pathways in a Mouse Model of Airway Disease

During an asthmatic episode, leukotriene C4 (LTC4) and interleukin 13 (IL-13) are released into the airways and are thought to be central mediators of the asthmatic response. However, little is known about how these molecules interact or affect each other's signaling pathway. To determine if the LTC4 and IL-13 signaling pathways interact with each other's pathways. We examined airway responsiveness, cysteinyl LTs (Cys-LTs), and Cys-LT and IL-13 receptor transcript levels in wild-type mice and in mice that were deficient in gamma-glutamyl leukotrienase (an enzyme that converts LTC4 to LTD4), STAT6 (signal transducer and activator of transcription 6 [a critical molecule in IL-13 signaling]), and IL-4Ralpha (a subunit of the IL-13 receptor). Wild-type (C57BL/129SvEv) and gamma-glutamyl leukotrienase-deficient mice showed increased airway responsiveness after intranasal instillation of IL-13; similar results were observed after intranasal instillation of IL-13 or LTC4 in a second wild-type strain (BALB/c). Interleukin 13 treatment reduced levels of Cys-LTs in bronchoalveolar lavage fluid. This change was unaccompanied by changes in other arachidonic acid metabolites or in RNA transcript levels of enzymes associated with Cys-LT synthesis. Interleukin 13 treatment also increased transcript levels of the Cys-LT 1 and Cys-LT 2 receptors, while LTC4 increased transcript levels of the alpha1 chain of the IL-13 receptor. Furthermore, IL-4Ralpha-deficient mice had increased airway responsiveness to LTC4 but not to IL-13, whereas STAT6-deficient mice failed to respond to either agonist. These findings indicate that LTC4 and IL-13 are dependent on or signal through STAT6 to increase airway responsiveness and that both agonists regulate expression of each other's receptors.

Targeted Gene Disruption Reveals the Role of the Cysteinyl Leukotriene 2 Receptor in Increased Vascular Permeability and in Bleomycin-induced Pulmonary Fibrosis in Mice

The cysteinyl leukotrienes (cys-LTs) mediate both acute and chronic inflammatory responses in mice, as demonstrated by the attenuation of the IgE/antigen-mediated increase in microvascular permeability and of bleomycin-induced pulmonary fibrosis, respectively, in a strain with targeted disruption of leukotriene C(4) synthase to prevent cys-LT synthesis. Our earlier finding that the acute, but not the chronic, injury was attenuated in a strain with targeted disruption of the cysteinyl leukotriene 1 (CysLT(1)) receptor suggested that the chronic injury might be mediated through the CysLT(2) receptor. Thus, we generated CysLT(2) receptor-deficient mice by targeted gene disruption. These mice developed normally and were fertile. The increased vascular permeability associated with IgE-dependent passive cutaneous anaphylaxis was significantly reduced in CysLT(2) receptor-null mice as compared with wild-type mice, whereas plasma protein extravasation in response to zymosan A-induced peritoneal inflammation was not altered. Alveolar septal thickening after intratracheal injection of bleomycin, characterized by interstitial infiltration with macrophages and fibroblasts and the accumulation of collagen fibers, was significantly reduced in CysLT(2) receptor-null mice as compared with the wild-type mice. The amounts of cys-LTs in bronchoalveolar lavage fluid after bleomycin injection were similar in the CysLT(2) receptor-null mice and the wild-type mice. Thus, in response to a particular pathobiologic event the CysLT(2) receptor can mediate an increase in vascular permeability in some tissues or promote chronic pulmonary inflammation with fibrosis.

Rac and protein kinase C-delta regulate ERKs and cytosolic phospholipase A2 in FcepsilonRI signaling to cysteinyl leukotriene synthesis in mast cells.

Although cysteinyl leukotrienes (cysLTs) are known to be principal inflammatory lipid mediators released from IgE-stimulated mast cells, the signaling mechanisms involved in the synthesis of cysLTs remain largely unknown. In the present study, therefore, we investigated the signaling pathway by which IgE induces cysLTs synthesis after binding to its high affinity receptor (FcepsilonRI) in RBL-2H3 mast cells. We found that IgE-induced cysLT synthesis is completely abolished in RBL-2H3(Rac-N17) cells, a stable cell line expressing Rac(N17), a dominant negative Rac1 mutant; conversely, synthesis was enhanced in cells expressing Rac(V12), a constitutively active Rac1 mutant, suggesting that Rac1 is a key mediator of IgE signaling to cysLT synthesis. Further analysis aimed at identifying mediators downstream of Rac1 revealed that pretreating cells with a protein kinase C-delta (PKC-delta) inhibitor or infection with an adenoviral vector harboring a dominant negative PKC-delta mutant significantly attenuates IgE-induced ERKs phosphorylation, cytosolic phospholipase A(2) phosphorylation/translocation, and cysLT synthesis. In addition, the expression of Rac(N17) blocked PKC-delta translocation and impaired the phosphorylation of ERKs and cytosolic phospholipase A(2) otherwise elicited by IgE stimulation. Taken together these results suggest that PKC-delta also plays a critical mediatory role in the IgE signaling pathway leading to cysLT synthesis, acting downstream of Rac1. Finally, the physiological significance of PKC-delta in the IgE signaling pathway was demonstrated in an Ag (OVA)-challenged in vivo mouse model, in which induced levels of cysLTs and airway responsiveness in lung airways were significantly diminished by prior i.p. injection of a PKC-delta inhibitor.

Leukotriene receptors in rhinitis and sinusitis.

Cysteinyl leukotrienes (CysLTs) mediate their biologic activities through interactions with the CysLT1 and CysLT2 receptors. CysLT1 receptors are prominently expressed on smooth muscle cells and lung fibroblasts, whereas CysLT2 receptors are expressed on heart Purkinje fiber cells, adrenal chromaffin cells, and endothelial cells. Both receptors are expressed on eosinophils and mast cells, but CysLT1 receptors alone are on neutrophils. Antigen-presenting cells more prominently express the type 2 receptor. CysLT1 receptors are uniquely important for bronchospasm, whereas CysLT2 receptors can stimulate endothelial cell adherence, myofibroblast proliferation, and chemokine production by mast cells. Comprehensive inhibition of the proinflammatory activities of CysLTs might require either combination CysLT1 and CysLT2 receptor antagonists or inhibitors of the CysLT synthesis pathway.

Disodium cromoglycate suppresses the induction of cysteinyl leukotriene synthesis during granulocytic differentiation in HL-60 cells

Objectives: Disodium cromoglycate (DSCG) and nedocromil sodium are anti-asthma drugs that have a variety of physiological and biological effects. We examined whether DSCG affects the induction of cysteinyl leukotriene (cysLT) synthesis during dimethyl sulfoxide (DMSO)-induced granulocytic differentiation in HL-60 cells. Methods: HL-60 cells were differentiated to mature granulocyte-like cells by DMSO in the presence or absence of DSCG for 5 days. Then, we measured A23187-stimulated production of LTC 4, an initial product of cysLTs. We also examined the mRNA expression and enzyme activity of LTC 4 synthase and other LT-synthetic enzymes. Results: The amount of LTC 4 production was 732.0±19.0 pg/10 6 cells in DMSO-differentiated HL-60 cells. The value was significantly decreased to 420.7±22.7 pg/10 6 cells in the presence of DSCG at 100 μg/ml. The DMSO-induced mRNA expression and enzyme activity of LTC 4 synthase was also suppressed by DSCG. Conclusions: Our results indicate that DSCG suppresses the DMSO-induced LTC 4 synthase-activity by inhibiting mRNA expression of LTC 4 synthase, which might be a novel anti-allergic action of DSCG.