Discovery and optimization of oxadiazole-based FLAP inhibitors.

Structure activity relationship (SAR) investigation of an oxadiazole based series led to the discovery of several potent FLAP inhibitors. Lead optimization focused on achieving functional activity while improving physiochemical properties and reducing hERG inhibition. Several compounds with favorable in vitro and in vivo properties were identified that were suitable for advanced profiling.

Androgen-mediated sex bias impairs efficiency of leukotriene biosynthesis inhibitors in males.

Proinflammatory leukotrienes (LTs) are produced by 5-lipoxygenase (5-LO) aided by 5-LO-activating protein (FLAP). LT biosynthesis inhibitors are currently under clinical investigation as treatments for respiratory and cardiovascular diseases. Here, we have revealed a sex bias in the efficiency of clinically relevant LT biosynthesis inhibitors, showing that their effects are superior in females. We found that androgens cause these sex differences by impeding the LT-biosynthetic 5-LO/FLAP complex assembly. Lower doses of the FLAP inhibitor MK886 were required to reduce LTB4 levels in exudates of female versus male mice and rats. Following platelet-activating factor-induced shock, MK886 increased survival exclusively in female mice, and this effect was abolished by testosterone administration. FLAP inhibitors and the novel-type 5-LO inhibitors licofelone and sulindac sulfide exhibited higher potencies in human blood from females, and bioactive 5-LO/FLAP complexes were formed in female, but not male, human and murine leukocytes. Supplementation of female blood or leukocytes with 5α-dihydrotestosterone abolished the observed sex differences. Our data suggest that females may benefit from anti-LT therapy to a greater extent than males, prompting consideration of sex issues in LT modifier development.

Discovery of the first dual inhibitor of the 5-lipoxygenase-activating protein and soluble epoxide hydrolase using pharmacophore-based virtual screening.

Leukotrienes (LTs) are pro-inflammatory lipid mediators derived from arachidonic acid (AA) with roles in inflammatory and allergic diseases. The biosynthesis of LTs is initiated by transfer of AA via the 5-lipoxygenase-activating protein (FLAP) to 5-lipoxygenase (5-LO). FLAP inhibition abolishes LT formation exerting anti-inflammatory effects. The soluble epoxide hydrolase (sEH) converts AA-derived anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatetraenoic acids (di-HETEs). Its inhibition consequently also counteracts inflammation. Targeting both LT biosynthesis and the conversion of EETs with a dual inhibitor of FLAP and sEH may represent a novel, powerful anti-inflammatory strategy. We present a pharmacophore-based virtual screening campaign that led to 20 hit compounds of which 4 targeted FLAP and 4 were sEH inhibitors. Among them, the first dual inhibitor for sEH and FLAP was identified, N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N'-(3,4-dichlorophenyl)urea with IC50 values of 200 nM in a cell-based FLAP test system and 20 nM for sEH activity in a cell-free assay.

Down-regulation of NF-κB expression by n-3 fatty acid-rich linseed oil is modulated by PPARγ activation, eicosanoid cascade and secretion of cytokines by macrophages in rats fed partially hydrogenated vegetable fat.

PURPOSE: The industrially produced partially hydrogenated vegetable fat (PHVF) contains trans fatty acid mostly comprising of elaidic acid (18:1 ∆9t). PHVF is used as a cooking medium in Southeast Asian countries. The purpose of this study is to evaluate the effects of dietary PHVF on inflammatory mediators and possible ameliorative effects of n-3 fatty acid (α-linolenic acid, ALA)-rich linseed oil (LSO) on the inflammatory mediators. METHODS: Male Wistar weaning rats were fed AIN-93-purified diet supplemented with one of the following lipids for 60 days, groundnut oil (GNO, 10 wt%), PHVF (10 wt%), LSO (10 wt%), PHVF blended with LSO at 2.5, 5.0 and 7.5 wt% levels. The final fat level in the diet was maintained at 10 wt%. RESULTS: The macrophages from rats fed PHVF showed higher levels of total cholesterol and free cholesterol as compared to those from rats fed GNO and LSO. Macrophages from rats fed PHVF down-regulated the expression of PPARγ and up-regulated the expressions of cytosolic phospholipase A2, cyclooxygenase-2, 5-lipoxygenase and nuclear factor-kappa B p65. The macrophages from rats fed PHVF secreted higher levels of pro-inflammatory eicosanoids and cytokines. The rats fed PHVF blended with LSO at incremental amounts showed a significant reduction in the expressions of pro-inflammatory markers in dose-dependent manner. CONCLUSION: Detrimental effects of dietary PHVF in enhancing pro-inflammatory agents in rats could be significantly reduced by providing ALA (n-3 PUFA)-rich LSO.

Increased PUFA Content and 5-Lipoxygenase Pathway Expression Are Associated with Subcutaneous Adipose Tissue Inflammation in Obese Women with Type 2 Diabetes.

Obese women with type 2 diabetes mellitus (T2DM) have more inflammation in their subcutaneous white adipose tissue (sWAT) than age-and-BMI similar obese women with normal glucose tolerance (NGT). We aimed to investigate whether WAT fatty acids and/or oxylipins are associated with the enhanced inflammatory state in WAT of the T2DM women. Fatty acid profiles were measured in both subcutaneous and visceral adipose tissue (vWAT) of 19 obese women with NGT and 16 age-and-BMI similar women with T2DM. Oxylipin levels were measured in sWAT of all women. Arachidonic acid (AA) and docosahexaenoic acid (DHA) percentages were higher in sWAT, but not vWAT of the T2DM women, and AA correlated positively to the gene expression of macrophage marker CD68. We found tendencies for higher oxylipin concentrations of the 5-LOX leukotrienes in sWAT of T2DM women. Gene expression of the 5-LOX leukotriene biosynthesis pathway was significantly higher in sWAT of T2DM women. In conclusion, AA and DHA content were higher in sWAT of T2DM women and AA correlated to the increased inflammatory state in sWAT. Increased AA content was accompanied by an upregulation of the 5-LOX pathway and seems to have led to an increase in the conversion of AA into proinflammatory leukotrienes in sWAT.

Identification of novel benzimidazole derivatives as inhibitors of leukotriene biosynthesis by virtual screening targeting 5-lipoxygenase-activating protein (FLAP).

Pharmacological suppression of leukotriene biosynthesis by 5-lipoxygenase (5-LO)-activating protein (FLAP) inhibitors is a promising strategy to intervene with inflammatory, allergic and cardiovascular diseases. Virtual screening targeting FLAP based on a combined ligand- and structure-based pharmacophore model led to the identification of 1-(2-chlorobenzyl)-2-(1-(4-isobutylphenyl)ethyl)-1H-benzimidazole (7) as developable candidate. Compound 7 potently suppressed leukotriene formation in intact neutrophils (IC(50)=0.31 µM) but essentially failed to directly inhibit 5-LO suggesting that interaction with FLAP causes inhibition of leukotriene synthesis. For structural optimization, a series of 46 benzimidazole-based derivatives of 7 were synthesized leading to more potent analogues (70-72, 82) with IC(50)=0.12-0.19 µM in intact neutrophils. Together, our results disclose the benzimidazole scaffold bearing an ibuprofen fingerprint as a new chemotype for further development of anti-leukotriene agents.

Endogenous n-3 polyunsaturated fatty acids delay progression of pancreatic ductal adenocarcinoma in Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice.

Preclinical studies suggest that diets rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs) may be beneficial for prevention of pancreatic cancer. Nutritional intervention studies are often complex, and there is no clear evidence, without potential confounding factors, on whether conversion of n-6 PUFAs to n-3 PUFAs in pancreatic tissues would provide protection. Experiments were designed using n-3 fatty acid desaturase (Fat-1) transgenic mice, which can convert n-6 PUFA to n-3 FAs endogenously, to determine the impact of n-3 PUFAs on pancreatic intraepithelial neoplasms (PanINs) and their progression to pancreatic ductal adenocarcinoma (PDAC). Six-week-old female p48(Cre/+)-LSL-Kras(G12D/+) and compound Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice were fed (AIN-76A) diets containing 10% safflower oil for 35 weeks. Pancreata were evaluated histopathologically for PanINs and PDAC. Results showed a dramatic reduction in incidence of PDAC (84%; P < .02) in Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice compared to p48(Cre/+)-LSL-Kras(G12D/+) mice. Importantly, significant reductions of pancreatic ducts with carcinoma (90%; P < .0001) and PanIN 3 (~50%; P < .001) lesions were observed in the compound transgenic mice. The levels of n-3 PUFA were much higher (>85%; P < .05-0.01) in pancreas of compound transgenic mice than in those of p48(Cre/+)-LSL-Kras(G12D/+) mice. Molecular analysis of the pancreas showed a significant down-regulation of proliferating cell nuclear antigen, cyclooxygenase-2, 5-lipoxygenase (5-LOX), 5-LOX-activating protein, Bcl-2, and cyclin D1 expression levels in Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice compared to p48(Cre/+)-LSL-Kras(G12D/+) mice. These data highlight the promise of dietary n-3 FAs for chemoprevention of pancreatic cancer in high-risk individuals.

Decreasing dietary linoleic acid promotes long chain omega-3 fatty acid incorporation into rat retina and modifies gene expression.

Age-related macular degeneration (AMD) may be partially prevented by dietary habits privileging the consumption of ω3 long chain polyunsaturated fatty acids (ω3s) while lowering linoleic acid (LA) intake. The present study aimed to document whether following these epidemiological guidelines would enrich the neurosensory retina and RPE with ω3s and modulate gene expression in the neurosensory retina. Rat progenitors and pups were fed with diets containing low or high LA, and low or high ω3s. After scotopic single flash and 8-Hz-Flicker electroretinography, rat pups were euthanized at adulthood. The fatty acid profile of the neurosensory retina, RPE, liver, adipose tissue and plasma was analyzed using gas chromatography. Gene expression was analyzed with real-time PCR in the neurosensory retina. Diets rich in ω3s efficiently improved the incorporation of ω3s into the organs and tissues. This raising effect was magnified by lowering LA intake. Compared to a diet with high LA and low ω3s, low LA diets significantly upregulated LDL-receptor gene expression. Similar but not significant upregulation of CD36, ABCA1, ALOX5 and ALOX12 gene expression was observed in rats fed with low LA. No effect was observed on retinal function. Increasing the intake in ω3s and lowering LA improved the enrichment with ω3s of the tissues, including the neurosensory retina and RPE, and upregulated genes involved in lipid trafficking in the neurosensory retina. Those results consistently reinforced the beneficial role of ω3s in the prevention of AMD, especially when the diet contained low levels of LA, as suggested from epidemiological data.

Traditional Chinese medicine, a solution for reducing dual stroke risk factors at once?

Based on genome wide association studies (GWAS), the activities of phosphodiesterase 4D (PDE4D) and 5-Lipoxygenase activating protein (ALOX5AP) were suggested as two of the major factors involved in ischemic stroke risks. Uncontrolled PDE4D activities often lead to cAMP-induced stroke and cardiovascular diseases. Overexpression of ALOX5AP, on the other hand, had been shown to play a major role in inflammation pathway that could induce the development of atherosclerosis and stroke. To eliminate the risk factors that lead to stroke, we reported the identification and analysis of dual-targeting compounds that could reduce PDE4D and ALOX5AP activities from traditional Chinese medicine (TCM). We employed world's largest TCM database, TCM Database@Taiwan, for in silico drug identification. We also introduced machine learning predictive models, as well as pharmacophore model, for characterizing the drug-like candidates. Both myristic acid and pentadecanoic acid were identified. The follow-up analysis on molecular dynamics simulation further determined the major roles of the carboxyl group for forming stable molecular interactions. Intriguingly, the carboxyl group demonstrated different bonding patterns with PDE4D and ALOX5AP, through electrostatic interaction and hydrogen bonds, respectively. In addition, the large volume occupied by the ligand hydrophobic regions could achieve inhibition through occupying the vacant spaces in the binding site. These pharmacophores held true for both candidates against each protein targets. Hence, we proposed the presence of the carboxyl group and hydrophobic regions as potent dual targeting features that inhibit both PDE4D and ALOX5AP activities.

Sulindac sulfide suppresses 5-lipoxygenase at clinically relevant concentrations.

Sulindac is a non-selective inhibitor of cyclooxygenases (COX) used to treat inflammation and pain. Additionally, non-COX targets may account for the drug's chemo-preventive efficacy against colorectal cancer and reduced gastrointestinal toxicity. Here, we demonstrate that the pharmacologically active metabolite of sulindac, sulindac sulfide (SSi), targets 5-lipoxygenase (5-LO), the key enzyme in the biosynthesis of proinflammatory leukotrienes (LTs). SSi inhibited 5-LO in ionophore A23187- and LPS/fMLP-stimulated human polymorphonuclear leukocytes (IC(50) approximately 8-10 microM). Importantly, SSi efficiently suppressed 5-LO in human whole blood at clinically relevant plasma levels (IC(50) = 18.7 microM). SSi was 5-LO-selective as no inhibition of related lipoxygenases (12-LO, 15-LO) was observed. The sulindac prodrug and the other metabolite, sulindac sulfone (SSo), failed to inhibit 5-LO. Mechanistic analysis demonstrated that SSi directly suppresses 5-LO with an IC(50) of 20 muM. Together, these findings may provide a novel molecular basis to explain the COX-independent pharmacological effects of sulindac under therapy.

FLAP inhibitors for the treatment of inflammatory diseases.

Leukotrienes are lipid inflammatory mediators that are implicated in asthma, COPD, arthritis, cardiovascular disease and cancer. Leukotriene synthesis requires 5-lipoxygenase activating protein (FLAP), which acts as a scaffolding protein for the assembly of other enzymes involved in the leukotriene synthetic pathway occurring at the nuclear envelope of leukocytes. By blocking the formation of both leukotriene B4 and the cysteinyl leukotrienes (ie, LTC4 , LTD4 and LTE4), FLAP inhibitors act as broad-spectrum leukotriene-modifier drugs that may have a wide range of therapeutic applications. FLAP inhibitors such as MK-886, MK-0591 and veliflapon (BAY-X-1005, DG-031) demonstrated promise in clinical trials with patients with inflammatory diseases in the mid 1990 s, but, unlike the 'lukast' class of cysteinyl-leukotriene receptor antagonists, these compounds were not brought to market. The elucidation of the 3D structure of FLAP has enabled novel compound development, and several FLAP inhibitors including 2190914 (AM-103) and GSK-2190915 (both under development by GlaxoSmithKline plc) have entered phase II trials for the treatment of inflammatory disease, including asthma.

The nuclear membrane organization of leukotriene synthesis.

Leukotrienes (LTs) are signaling molecules derived from arachidonic acid that initiate and amplify innate and adaptive immunity. In turn, how their synthesis is organized on the nuclear envelope of myeloid cells in response to extracellular signals is not understood. We define the supramolecular architecture of LT synthesis by identifying the activation-dependent assembly of novel multiprotein complexes on the outer and inner nuclear membranes of mast cells. These complexes are centered on the integral membrane protein 5-Lipoxygenase-Activating Protein, which we identify as a scaffold protein for 5-Lipoxygenase, the initial enzyme of LT synthesis. We also identify these complexes in mouse neutrophils isolated from inflamed joints. Our studies reveal the macromolecular organization of LT synthesis.

Expression of enzymes and receptors of the leukotriene pathway in human neuroblastoma promotes tumor survival and provides a target for therapy.

The metabolism of arachidonic acid by the cyclooxygenase (COX) or lipoxygenase (LO) pathways generates eicosanoids that have been implicated in the pathogenesis of a variety of human diseases, including cancer. In this study, we examined the expression and significance of components within the 5-LO pathway in human neuroblastoma, an embryonal tumor of the sympathetic nervous system. High expression of 5-LO, 5-LO-activating protein (FLAP), leukotriene A(4) hydrolase, leukotriene C(4) synthase, and leukotriene receptors was detected in a majority of primary neuroblastoma tumors and all cell lines investigated. Expression of 5-LO and FLAP was evident in tumor cells but not in nonmalignant adrenal medulla where neuroblastomas typically arise. Moreover, neuroblastoma cells produce leukotrienes, and stimulation of neuroblastoma cells with leukotrienes increased neuroblastoma cell viability. Inhibitors of 5-LO (AA-861), FLAP (MK-886), or the leukotriene receptor antagonist montelukast inhibited neuroblastoma cell growth by induction of G(1)-cell cycle arrest and apoptosis. Similarly, specific 5-LO and leukotriene receptor silencing by small interfering RNA decreased neuroblastoma cell growth. These findings provide new insights into the pathobiology of neuroblastoma, and the use of leukotriene pathway inhibitors as a novel adjuvant therapy for children with neuroblastoma warrants further consideration.

What's all the FLAP about?: 5-lipoxygenase-activating protein inhibitors for inflammatory diseases.

Leukotrienes have physiological roles in innate immune responses and pathological roles in inflammatory diseases, such as asthma, allergic rhinitis and atherosclerosis. Anti-leukotriene therapy has proven benefits in the treatment of respiratory disease, either through the inhibition of leukotriene synthesis or the selective antagonism of leukotriene receptors. The first committed step in the synthesis of leukotrienes is the oxidation of arachidonic acid (AA) by 5-lipoxygenase (5-LO), and the integral membrane protein 5-lipoxygenase-activating protein (FLAP) is an essential partner of 5-LO for this process. FLAP was molecularly identified via a photoaffinity probe and an affinity gel based on MK-886, a selective leukotriene inhibitor that has no activity against broken-cell preparations of 5-LO. Several FLAP inhibitors showed efficacy in early clinical trials in asthma but were not developed commercially for unpublished reasons. Recently, the FLAP (ALOX5AP) gene has been linked to risk for myocardial infarction, stroke and restenosis, reigniting pharmaceutical interest in this target. In addition, the recent determination of the crystal structure of inhibitor-bound FLAP offers exciting potential for novel FLAP inhibitor design.

[Effects of among compositions of Herba Ephedrae decoction on genic xpression of 5-lipoxygenase activating protein, IL-4 and leukotriene C4 in asthmatic mice].

OBJECTIVE: To explore the regularity of recipe composition by observing inhibitory effects on the genic expression of 5-lipoxygenase activating protein, IL-4 and the leukotriene C4 in asthmatic mice. METHOD: The mice were challenged with OVA and administered ig with the Herba Ephedrae decoction (HED), separated compositions (2500 mg x kg(-1), calculated by Herba Ephedrae) and dexamethasone (2 mg x kg(-1)) respectively once daily for seven days. The real-time fluorescence quantitative PCR method was employed to measure the contents of FLAP mRNA and IL-4 mRNA expressions in lung and the ELISA method was used to determine the content of LTC4 in the washing solution of pulmonary alveolus and bronchi. RESULT: In the lung of asthma mice, the expressions of FLAP and IL-4 and the content of LTC4 were significantly augmented compared with the control group. The HED and the separated compositions could suppress the expressions of FLAP and IL-4 and LTC4 release to a great extent in mice. CONCLUSION: The HED had the remarkable effects of antianaphylaxis asthma and the original formula HED worked best. These results confirmed the rationality and scientific level of HED.

Effects of among compositions of Herba Ephedrae decoction on genic xpression of 5-lipoxygenase activating protein, IL-4 and leukotriene C4 in asthmatic mice / 中国中药杂志

<p><b>OBJECTIVE</b>To explore the regularity of recipe composition by observing inhibitory effects on the genic expression of 5-lipoxygenase activating protein, IL-4 and the leukotriene C4 in asthmatic mice.</p><p><b>METHOD</b>The mice were challenged with OVA and administered ig with the Herba Ephedrae decoction (HED), separated compositions (2500 mg x kg(-1), calculated by Herba Ephedrae) and dexamethasone (2 mg x kg(-1)) respectively once daily for seven days. The real-time fluorescence quantitative PCR method was employed to measure the contents of FLAP mRNA and IL-4 mRNA expressions in lung and the ELISA method was used to determine the content of LTC4 in the washing solution of pulmonary alveolus and bronchi.</p><p><b>RESULT</b>In the lung of asthma mice, the expressions of FLAP and IL-4 and the content of LTC4 were significantly augmented compared with the control group. The HED and the separated compositions could suppress the expressions of FLAP and IL-4 and LTC4 release to a great extent in mice.</p><p><b>CONCLUSION</b>The HED had the remarkable effects of antianaphylaxis asthma and the original formula HED worked best. These results confirmed the rationality and scientific level of HED.</p>

Increased expression of lipoxygenase enzymes during pollen season in nasal biopsies of pollen-allergic patients.

BACKGROUND: Exposure of patients sensitized to pollen triggers development of seasonal allergic rhinitis symptoms (SAR). Eicosanoids are a group of arachidonic acid metabolites contributing to the symptoms of SAR. The aim of this study was to investigate seasonal changes in the expression of enzymes of the eicosanoid pathway in the nasal mucosa of patients with SAR. METHODS: Twenty SAR patients allergic to birch or grass and eight healthy subjects were included in the study. Patients registered rhinoconjunctivitis symptoms and use of rescue medication before and during the pollen season. Nasal biopsies were obtained before and around the peak of the season, sectioned and stained using markers for eosinophils, mast cells, T cells and neutrophils. Antibodies against the following enzymes were also used: cyclo-oxygenase (COX-1, COX-2), 5-lipoxygenase (5-LO), 5-lipoxygenase-activating factor (FLAP), LTA4 hydrolase (LTA4h) and LTC4 synthase (LTC4s). RESULTS: During the pollen season symptoms of rhinoconjunctivitis and medication score increased significantly (P=0.001; P=0.001 respectively). During the pollen season numbers of eosinophils (P=0.02) and cell positive 5-LO (P=0.02), LTC4s (P=0.04) and LTA4h (P=0.02) increased significantly. During season number of mast cells and cells expressing 5-LO and LTA4h were higher in SAR than in healthy controls group (P=0.02; P=0.01; P=0.03 respectively). CONCLUSION: In sensitized patients exposure to pollen allergen results in increased expression of enzymes of the eicosanoid pathway.

5-Lipoxygenase as a putative link between cardiovascular and psychiatric disorders.

There is evidence of an association between depression and anxiety and cardio- cerebro-vascular conditions, but the mechanisms of this association are unknown. Here we review a possible role for the 5-lipoxygenase (5-LOX) pathway. 5-LOX is an enzyme that, in association with 5-LOX-activating protein (FLAP), leads to the synthesis of leukotrienes from omega-6 arachidonic acid. Production of active leukotrienes can be reduced by dietary omega-3 fatty acids, which also are beneficial in cardiac and psychiatric (e.g., depression) pathologies. Human 5-LOX and FLAP gene polymorphisms are a risk factor in atherosclerosis and cardio-cerebro-vascular pathologies; an overactive 5-LOX pathway is found in these diseases. Studies with 5-LOX-deficient transgenic mice suggest that 5-LOX activity may contribute to anxiety- and depression-like behaviors. Future research should characterize the role of the 5-LOX pathway in comorbid cardio-cerebro-vascular and psychiatric disorders and in the therapeutic actions of dietary omega-3 fatty acids.

Prostaglandins inhibit 5-lipoxygenase-activating protein expression and leukotriene B4 production from dendritic cells via an IL-10-dependent mechanism.

PGs produced from arachidonic acid by the action of cyclooxygenase enzymes play a pivotal role in the regulation of both inflammatory and immune responses. Because leukotriene B4 (LTB4), a product of 5-lipoxygenase (5-LO) pathway, can exert numerous immunoregulatory and proinflammatory activities, we examined the effects of PGs on LTB4 release from dendritic cells (DC) and from peritoneal macrophages. In concentration-dependent manner, PGE1 and PGE2 inhibited the production of LTB4 from DC, but not from peritoneal macrophage, with an IC50 of 0.04 microM. The same effect was observed with MK-886, a 5-LO-activating protein (FLAP)-specific inhibitor. The decreased release of LTB4 was associated with an enhanced level of IL-10. Furthermore, the inhibition of LTB4 synthesis by PGs was significantly reversed by anti-IL-10, suggesting the involvement of an IL-10-dependent mechanism. Hence, we examined the effects of exogenous IL-10 on the 5-LO pathway. We demonstrate that IL-10 suppresses the production of LTB4 from DC by inhibiting FLAP protein expression without any effect on 5-LO and cytosolic phospholipase A2. Taken together, our results suggest links between DC cyclooxygenase and 5-LO pathways during the inflammatory response, and FLAP is a key target for the PG-induced IL-10-suppressive effects.

Studies of differential gene expression in clinically derived eosinophil populations.

BACKGROUND: Influx of eosinophils into the post-capillary bronchial epithelium and the subsequent release of inflammatory mediators is characteristic of the late phase of asthmatic attacks. The genes that serve to predispose the peripheral blood eosinophils of asthmatics to undergo this process are poorly defined. The aim of this report is to describe the differential gene expression of both the known pro-inflammatory genes 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) and novel cDNA sequences in eosinophils derived from clinical samples. METHODS: Novel cDNA sequences representing genes upregulated in peripheral blood eosinophils of asthmatic as compared with nonasthmatic patients were identified by differential display polymerase chain reaction (DDPCR). The differential expression of these sequences, in addition to known pro-inflammatory genes, were then studied by reverse dot blotting of amplified RNA generated from the eosinophils of nonasthmatic donors, asthmatic donors, asthmatic donors taking steroids, interleukin (IL) -3, IL-5, granulocyte-macrophage colony stimulating factor- (GM-CSF) treated eosinophils from asthmatic donors and the eosinophilic cell line AML14. RESULTS: Four unique DDPCR-generated 3'UTR DNA fragments were identified that showed differing patterns of expression between the eosinophil populations of interest. Expression of each of the novel clones was increased in the peripheral blood eosinophils of asthmatics and downregulated in those donors taking steroids. Expression of 5-lipoxygenase was not found to vary between the different eosinophil populations, whereas FLAP was induced by treatment with the cytokine cocktail in both primary eosinophils and the eosinophilic cell line AML14. CONCLUSION: The differential regulation of the novel cDNA sequences and FLAP in the range of eosinophil populations studied suggest that they may provide clinically relevant therapeutic targets. Moreover, the procedures used in these studies may provide a general approach to the study of differential gene expression in small numbers of cells such as those obtained from clinical samples.