Anthony P. Sampson

Variant LTC 4 synthase allele modifies cysteinyl leukotriene synthesis in eosinophils and predicts clinical response to zafirlukast

Clinical trials with leukotriene synthesis inhibitors and cysteinyl leukotriene (cys-LT) receptor antagonists have shown that cys-LTs (LTC4, LTD4, and LTE4) are the predominant mediators of bronchoconstriction in response to diverse asthma triggers including allergen, PAF, sulphur dioxide, and non-steroidal anti-inflammatory drugs (NSAIDs).1 In addition, inhalation of a single dose of a cys-LT causes persistent eosinophilia in the bronchial mucosa and sputum of asthmatic patients,2 3 and LT modifier drugs significantly reduce airway and blood eosinophilia in clinical asthma.4 We have shown that, in the asthmatic airway, eosinophils represent the majority of cells that express the terminal enzyme in the cys-LT pathway, LTC4 synthase, and hence have the capacity to generate cys-LT upon stimulation.5 6 Inflammation and bronchoconstriction in the asthmatic airway may be sustained by a vicious cycle of cys-LT synthesis and eosinophil recruitment (fig 1). Anomalies in the genetic and cytokine factors that regulate cys-LT pathway enzymes in eosinophils may thus be central to the pathophysiology of asthma. Figure 1 Schematic diagram illustrating that genetic and cytokine factors modulate the vicious cycle of cysteinyl leukotriene synthesis and eosinophil recruitment in the asthmatic airway. Cys-LTs are synthesised from membrane derived arachidonic acid. During stimulus-specific cell activation, arachidonic acid released by phospholipases including cytosolic phospholipase A2(PLA2)is translocated to the 5-lipoxygenase activating protein (FLAP) and converted in two steps to leukotriene (LT)A4 by 5-lipoxygenase (5- LO). LTA4 is converted to LTB4 by cells expressing LTA4hydrolase, and/or to LTC4 by cells expressing LTC4 synthase, which conjugates LTA4 to reduced glutathione. After carrier mediated cellular export of LTC4, the sequential cleavage of glutamate and glycine residues provides the extracellular receptor active metabolites LTD4 and LTE4, respectively. Cys-LTs act at specific …

Leukotriene antagonists and synthesis inhibitors: new directions in asthma therapy.

This article briefly reviews the advances in our understanding of asthma with a particular focus on the role of inflammation, before providing a concise overview of current knowledge of leukotrienes in the pathophysiology of the disease. The development of leukotriene receptor antagonists and synthesis inhibitors in briefly described; and acute exercise, allergen, and aspirin challenge studies with these agents are reviewed. Clinical studies with leukotriene antagonists and inhibitors have confirmed the central role of leukotrienes in asthma pathophysiology. In conclusion, we suggest that the new generation of leukotriene receptor antagonists may be suitable as first-line therapy in patients with mild to moderate asthma. Further studies are required to determine whether the leukotriene synthesis inhibitors will be equally effective or provide any additional antiinflammatory benefit.

Sputum tumour necrosis factor-alpha and leukotriene concentrations in cystic fibrosis.

It is postulated that a vigorous host inflammatory response in the cystic fibrosis lung contributes to lung injury. Tumour necrosis factor-alpha (TNF-alpha) may play a part in that process and in the generation of leukotrienes. Therefore, the relationships between sputum TNF-alpha, leukotriene concentration, and lung function abnormalities in 16 children with cystic fibrosis were investigated. Each subject provided sputum samples and performed spirometry. TNF-alpha was measured by enzyme linked immunosorbent assay; individual leukotrienes were separated using high performance liquid chromatography and quantified by radioimmunoassay. The geometric mean concentration of TNF-alpha was 129.7 pg/ml and 95% confidence interval 48.2 to 348.3. Mean (SEM) leukotriene B4 (LTB4) was 97.8 (22.9) pmol/g and total cysteinyl leukotrienes were 60.9 (14.8) pmol/g. Mean (SD) forced expiratory volume in one second (FEV1) of the group was 53 (15)% of predicted and forced vital capacity (FVC) was 65 (14)% of predicted. There was a significant positive correlation between TNF-alpha and both LTB4 and the total cysteinyl leukotriene sputum content. An inverse relationship existed between TNF-alpha and FEV1 and FVC. Moreover, a negative correlation was observed between sputum LTB4 and FEV1 and FVC. These results suggest that TNF-alpha and the leukotrienes may participate in the airways inflammation and airflow obstruction observed in cystic fibrosis subjects and support the hypothesis that TNF-alpha upregulates the 5-lipoxygenase pathway in vivo.

Allelic association and functional studies of promoter polymorphism in the leukotriene C4 synthase gene (LTC4S) in asthma

Background: LTC4 synthase is essential for the production of cysteinyl leukotrienes (Cys-LT), critical mediators in asthma. We have identified a novel promoter polymorphism at position −1072 (G/A) and a −444 (A/C) polymorphism has previously been reported. The role of these polymorphisms in the genetic susceptibility to asthma was examined. Methods: To test for genetic association with asthma phenotypes, 341 white families (two asthmatic siblings) and 184 non-asthmatic control subjects were genotyped. Genetic association was assessed using case control and transmission disequilibrium test (TDT) analyses. LTC4S promoter luciferase constructs and transiently transfected human HeLa and KU812F cells were generated to determine the functional role of these polymorphisms on basal transcription. Results: No associations were observed in case control analyses (–1072 A, q=0.09; −444 C, q=0.29); the TDT identified a borderline association between the −444 C allele and bronchial responsiveness to methacholine (p=0.065). Asthmatic children with the −444 C allele had a lower mean basal forced expiratory volume in 1 second (97.4 v 92.7% predicted, p=0.005). LTC4S promoter luciferase analyses provided no evidence for a functional role of either polymorphism in determining basal transcription. Conclusion: This study does not support a role for these polymorphisms in genetic susceptibility to asthma but provides evidence to suggest a role in determining lung function parameters.

Expression of 5-lipoxygenase and cyclooxygenase pathway enzymes in nasal polyps of patients with aspirin-intolerant asthma.

In aspirin‐intolerant subjects, adverse bronchial and nasal reactions to cyclooxygenase (COX) inhibitors are associated with over‐production of cysteinyl‐leukotrienes (cys‐LTs) generated by the 5‐lipoxygenase (5‐LO) pathway. In the bronchi of patients with aspirin‐intolerant asthma, we previously linked cys‐LT over‐production and aspirin hyper‐reactivity with elevated immunoexpression in eosinophils of the terminal enzyme for cys‐LT production, LTC4 synthase. We investigated whether this anomaly also occurs in the nasal airways of these patients. Immunohistochemical expression of 5‐LO and COX pathway proteins was quantified in nasal polyps from 12 patients with aspirin‐intolerant asthma and 13 with aspirin‐tolerant asthma. In the mucosa of polyps from aspirin‐intolerant asthmatic patients, cells immunopositive for LTC4 synthase were four‐fold more numerous than in aspirin‐tolerant asthmatic patients (p = 0.04). There were also three‐fold more cells expressing 5‐LO (p = 0.037), with no differences in 5‐LO activating protein (FLAP), COX‐1 or COX‐2. LTC4 synthase‐positive cell counts correlated exclusively with mucosal eosinophils (r = 0.94, p < 0.001, n = 25). Co‐localisation confirmed that five‐fold higher eosinophil counts (p = 0.007) accounted for the increased LTC4 synthase expression in polyps from aspirin‐intolerant asthmatic patients, with no alterations in mast cells or macrophages. Within the epithelium, increased counts of eosinophils (p = 0.006), macrophages (p = 0.097), and mast cells (p = 0.034) in aspirin‐intolerant asthmatic polyps were associated only with 2.5‐fold increased 5‐LO‐positive cells (p < 0.05), while the other enzymes were not different. Our results indicate that a marked over‐representation of LTC4 synthase in mucosal eosinophils is closely linked to aspirin intolerance in the nasal airway, as in the bronchial airways. Copyright

Specific immunoglobulin E for staphylococcal enterotoxins in nasal polyps from patients with aspirin‐intolerant asthma

Background Nasal polyps infiltrated with eosinophils are commonly found in chronic asthmatic patients, more frequently in those with aspirin‐intolerant asthma (AIA) than aspirin‐tolerant asthma (ATA). Some studies have suggested a contribution of superantigens derived from Staphylococcus sp to nasal polyposis and eosinophilia, but their relative importance in AIA and ATA subjects is unknown.

Role of cysteinyl leukotrienes in adenosine 5`-monophosphate induced bronchoconstriction in asthma

Background: Adenosine induced bronchoconstriction in patients with asthma is thought to be mediated by the synthesis and release of autacoids from airway mast cells. In vitro, adenosine induced constriction of asthmatic bronchi is blocked by a combination of specific histamine and cysteinyl leukotriene receptor antagonists, but the relative contribution of these mediators in vivo is unclear. We hypothesised that adenosine induced bronchoconstriction in asthmatic patients may be blocked by pretreatment with the orally active selective cysteinyl leukotriene-1 (CysLT1) receptor antagonist, montelukast. Methods: In a randomised, double blind, crossover study, oral montelukast (10 mg) or placebo was administered once daily on two consecutive days to 18 patients with mild to moderate persistent atopic asthma. Incremental doses of adenosine 5`-monophosphate (AMP) from 0.39 to 400 mg/ml were inhaled by dosimeter and the dose producing a 20% fall in FEV1 (PC20AMP) after AMP inhalation was recorded. Leukotriene E4 (LTE4) urinary concentrations were measured by enzyme immunoassay 4 hours after AMP challenge. Results: Montelukast pretreatment provided highly significant protection against adenosine induced bronchoconstriction, with geometric mean PC20AMP values of 52.6 mg/ml (95% CI 35.2 to 78.7) after placebo and 123.9 mg/ml (95% CI 83.0 to 185.0) after montelukast (p=0.006). The geometric mean of the montelukast/placebo PC20AMP ratio was 2.4 (95% CI 1.3 to 4.2). Montelukast had no significant effect on 4 hour urinary excretion of LTE4 compared with placebo. Conclusions: Selective CysLT1 receptor antagonism with montelukast provides highly significant protection against AMP induced bronchoconstriction in patients with atopic asthma, implying that cysteinyl leukotrienes are generated from airway mast cells through preferential activation of their A2B receptors.

Rhinovirus infection increases 5-lipoxygenase and cyclooxygenase-2 in bronchial biopsy specimens from nonatopic subjects.

Rhinovirus infections cause wheeze, cough, and bronchial hyperresponsiveness. To investigate the involvement of cysteinyl-leukotrienes and prostanoids in these symptoms, bronchial biopsy specimens from 9 normal subjects (nonatopic and with no history of chronic lung disease) were immunostained for 5-lipoxygenase (5-LO) and cyclooxygenase (COX) pathway enzymes 2 weeks before and 4 days after experimental infection with human rhinovirus serotype 16. 5-LO-positive cell counts increased 9-fold (from 0.48 to 4.4 cells/mm(2); P <.05), and 5-LO-activating protein (FLAP)-positive cell counts increased 3.6-fold (from 1.8 to 6.5 cells/mm(2); P =.09). Levels of leukotriene A(4) hydrolase and leukotriene C(4) synthase were unchanged. COX-2--positive cell counts increased from 0 to 2.6 cells/mm(2) (P =.009), with no change in COX-1 levels. Increases of 3-4-fold were seen in levels of macrophages (P =.02) and mast cells (P =.07) but not of eosinophils (P >.4), and bronchoalveolar lavage fluid cysteinyl-leukotriene levels doubled (from 11.2 to 20.4 pg/mL; P =.13). Cold symptom scores correlated with bronchial immunostaining for FLAP (rho = 0.93; P =.001). In normal subjects, rhinovirus colds induce bronchial inflammation with markedly enhanced expression of 5-LO pathway proteins and COX-2.

Enhanced excretion of urinary leukotriene E4 in coronary artery disease and after coronary artery bypass surgery.

Background:Excretion of leukotriene (LT) E4, the major urinary metabolite of cysteinyl leukotrienes in humans, is increased in patients with unstable angina and myocardial infarction, suggesting that cysteinyl leukotrienes are released into the circulation during episodes of myocardial ischaemia. Furthermore, leukotrienes are known to induce potent vasoconstrictive effects in human atherosclerotic coronary arteries and the saphenous vein. Accordingly, we measured urinary excretion of LTE4 in patients with stable coronary artery disease both before and after coronary artery bypass surgery, and in age-matched healthy controls, to study the relation between the systemic synthesis of cysteinyl leukotrienes and stable coronary artery disease, as well as the possible changes after bypass surgery. Methods:LTE4 was isolated from urine samples by solid-phase extraction, purified by reverse-phase high-performance liquid chromatography, and subsequently quantified by radioimmunoassay. Results:In patients with coronary artery disease, preoperative urinary LTE4 levels were normally distributed on a log10 scale, with a geometric mean of 115 pmol/mmol creatinine (95% confidence interval 67–196) compared with 63.0 pmol/mmol creatinine (95% confidence interval 47.9–82.7) in healthy subjects (P<0.05). Urinary LTE4 levels increased further in patients after coronary artery bypass surgery with levels peaking on the second day after surgery (266.2 pmol/mmol creatinine, 95% confidence interval 167.2–423.9) at significantly higher than preoperative levels (P<0.02), and then decreasing by day 3. Conclusions:Levels of cysteinyl leukotrienes are raised in coronary artery disease patients both before and after coronary artery bypass surgery. As these mediators are capable of inducing potent vasoconstrictive effects on atherosclerotic coronary arteries and the saphenous vein, our results could have important clinical and possibly therapeutic implications.

Colonic expression of leukotriene-pathway enzymes in inflammatory bowel diseases

Background Leukotrienes derived from the 5‐lipoxygenase pathway are proinflammatory lipid mediators that possibly play a role in inflammatory bowel diseases. The expression of 5‐lipoxygenase pathway proteins has not previously been examined in colonic mucosa in inflammatory bowel disease. Results Quantitative immunohistochemical analyses showed that, compared to those of the control subjects (n = 9), colonic biopsies from patients with active inflammatory bowel disease (n = 17) had 3‐ to 7‐fold higher mean counts of cells expressing 5‐lipoxygenase (P = 0.03), 5‐lipoxygenase‐activating protein (P = 0.005), and the leukotriene A4 hydrolase (P = 0.004), which make up the biosynthetic pathway of the potent neutrophil chemotaxin leukotriene B4. Immunoexpression of the leukotriene C4 synthase was unaltered (P > 0.2). The increased representation of leukotriene B4–pathway enzymes was associated with higher counts of neutrophils (P = 0.0001), macrophages (P = 0.03), eosinophils (P = 0.0004), CD8+ T cells (P < 0.001), activated T cells (P < 0.05), and B cells (P < 0.05) but not of mast cells (P > 0.9). These eicosanoid and cellular changes were most marked in the subgroup of patients with ulcerative colitis (n = 9), and were absent in patients with quiescent disease (n = 6). The anomalies in the 5‐lipoxygenase pathway were accompanied as expected by more cells immunostaining for cytokine‐inducible COX‐2 (P = 0.004, n = 17), but this study also revealed a greater number of cells expressing COX‐1 in the samples from the patients in the ulcerative colitis subgroup (P = 0.03, n = 9). Conclusions The 5‐lipoxygenase data provide a cellular basis for increased tissue synthesis of the leukotriene B4, as reflected in the colonic mucosa and rectal dialysates of patients with active inflammatory bowel disease, which contributes to neutrophil influx and colonic injury. The COX‐1/COX‐2 data highlight the ambiguous functional role of prostanoid pathways in inflammatory bowel diseases. (Inflamm Bowel Dis 2007)