Prostaglandin E2 and cysteinyl leukotriene concentrations in sputum: association with asthma severity and eosinophilic inflammation.

BACKGROUND: Inflammation of the airways in asthma is associated with the production of cysteinyl leukotrienes (cysLT), prostaglandin (PG)E(2), 8-isoprostane, nitric oxide and other mediators. However, the relationship between asthma severity or eosinophilic inflammation and the concentrations of mediators in sputum is unclear. OBJECTIVE: To assess sputum PGE(2), cysLT, 8-isoprostane and nitrate concentrations, as well as urinary leukotriene (LT)E(4) and 9alpha,11beta-prostaglandin (PG)F(2) concentrations, in patients with differing severities of asthma and eosinophilic or non-eosinophilic airway inflammation. METHODS: Inflammatory cells in sputum were assessed in 12 patients with mild, 14 with moderate and 12 with severe persistent asthma, as well as in 13 control subjects. Asthmatic patients were categorized into those with eosinophilic or non-eosinophilic airway inflammation. Sputum PGE(2), cysLT and 8-isoprostane, and urinary LTE(4) were extracted on immunoaffinity sorbents, and the concentrations of all mediators were measured using enzyme immunoassays. Sputum nitrate concentrations were measured on a chemiluminescence analyzer. RESULTS: Sputum PGE(2) concentrations were higher in both moderate (1710 pg/mL) and severe asthmatic (1590 pg/mL) compared with control subjects (827 pg/mL) (P<0.05). CysLT concentrations were higher in moderate asthmatic compared with control or severe asthmatic subjects (P<0.05). Sputum PGE(2) concentrations were lower in patients with eosinophilic (1180 pg/mL) compared with non-eosinophilic airway inflammation (2520 pg/mL) (P=0.02). In contrast, sputum cysLT and urinary LTE(4) concentrations were higher in those with eosinophilic airway inflammation (P<0.05). Forced expiratory volume in 1 s was inversely correlated with sputum eosinophils in all asthmatic patients (r(s)=-0.5, P=0.002). There were no significant differences in sputum 8-isoprostane or nitrate concentrations. CONCLUSIONS: Increased airway concentrations of PGE(2) are consistent with the hypothesis that PGE(2) has a bronchoprotective and anti-inflammatory role in patients with more severe asthma. A reduced PGE(2) to cysLT ratio in the airways may adversely affect lung function and contribute to persistence of symptoms and airway remodelling in patients with eosinophilic airway inflammation.

Clinical effects of sulphite additives.

Sulphites are widely used as preservative and antioxidant additives in the food and pharmaceutical industries. Topical, oral or parenteral exposure to sulphites has been reported to induce a range of adverse clinical effects in sensitive individuals, ranging from dermatitis, urticaria, flushing, hypotension, abdominal pain and diarrhoea to life-threatening anaphylactic and asthmatic reactions. Exposure to the sulphites arises mainly from the consumption of foods and drinks that contain these additives; however, exposure may also occur through the use of pharmaceutical products, as well as in occupational settings. While contact sensitivity to sulphite additives in topical medications is increasingly being recognized, skin reactions also occur after ingestion of or parenteral exposure to sulphites. Most studies report a 3-10% prevalence of sulphite sensitivity among asthmatic subjects following ingestion of these additives. However, the severity of these reactions varies, and steroid-dependent asthmatics, those with marked airway hyperresponsiveness, and children with chronic asthma, appear to be at greater risk. In addition to episodic and acute symptoms, sulphites may also contribute to chronic skin and respiratory symptoms. To date, the mechanisms underlying sulphite sensitivity remain unclear, although a number of potential mechanisms have been proposed. Physicians should be aware of the range of clinical manifestations of sulphite sensitivity, as well as the potential sources of exposure. Minor modifications to diet or behaviour lead to excellent clinical outcomes for sulphite-sensitive individuals.

Cyclooxygenase-2 gene polymorphisms in an Australian population: association of the -1195G > A promoter polymorphism with mild asthma.

BACKGROUND: Cyclooxygenase (COX)-2 is an inducible enzyme responsible for catalysing the formation of prostaglandins (PGs) in settings of inflammation. Single nucleotide polymorphisms (SNPs) of the COX-2 gene may influence gene transcription and PG production in the asthmatic airway. OBJECTIVE: To evaluate the frequencies of COX-2 SNPs in an Australian Caucasian population, and determine potential associations between common COX-2 promoter SNPs and asthma, asthma severity and aspirin-intolerant asthma (AIA). METHODS: The frequencies of 25 COX-2 SNPs were determined in a random population (n=176). The SNPs with a minor allele frequency of >10% were then studied in asthmatic (n=663), non-asthmatic controls (n=513) and AIA subjects (n=58). Genotype, allele and haplotype associations were assessed. Functional assessment of SNPs was performed by transfection into HeLa cells measured using the luciferase dual-reporter assay system. RESULTS: Eighteen COX-2 SNPs were not detected, five were rare and two promoter SNPs, -1195G>A (rs689465), and -1290A>G (rs689466), were further studied. The A allele of the -1195 SNP was present at a significantly higher frequency among all asthmatic subjects (P=0.012). Over 60% of the asthmatic individuals were -1195A homozygotes compared with 54.6% of the control subjects (odds ratio, 1.35; 95% CI, 1.06-1.72, P=0.03). After classifying for severity, the mild asthmatics represented 64.6% of -1195AA individuals, the highest of all the asthma groups compared with 54.6% of the control subjects (odds ratio, 1.5; 95% CI, 1.12-2.02, P=0.02). The -1290A/-1195G/-765G haplotype was associated with a reduced incidence of asthma (odds ratio, 0.76; 95% CI, 0.61-0.95, P=0.017). CONCLUSION: The -1195G>A polymorphism appears to be associated with asthma, and in particular with mild asthma.

Plasma phospholipase A2 activity in patients with asthma: association with body mass index and cholesterol concentration.

BACKGROUND: Secretory phospholipases A2 (sPLA2) have functions relevant to asthmatic inflammation, including eicosanoid synthesis and effects on dendritic cells and T cells. The aim of this study was to measure sPLA2 activity in patients with stable and acute asthma and to assess potential associations with body mass index (BMI), and plasma cholesterol and vitamin C concentrations. METHODS: Plasma sPLA2 activity and concentrations of cholesterol and vitamin C were measured in 23 control subjects and 61 subjects with stable asthma (42 mild to moderate, 19 severe). In addition, sPLA2 activity was measured in 36 patients experiencing acute asthma and in 22 of these patients after recovery from the acute attack. RESULTS: sPLA2 activity was not significantly greater in severe (499.9 U; 95% confidence interval (CI) 439.4 to 560.4) compared with mild to moderate asthmatic subjects (464.8; 95% CI 425.3 to 504.3) or control subjects (445.7; 95% CI 392.1 to 499.4), although it was higher in patients with acute asthma (581.6; 95% CI 541.2 to 622.0; p<0.001). Male gender, high plasma cholesterol, increased BMI and atopy were associated with increased sPLA2 activity, while plasma vitamin C was inversely correlated with sPLA2 activity in patients with stable asthma and in control subjects. There were significant interactions between gender and plasma cholesterol and between gender and vitamin C in relation to sPLA2 activity. CONCLUSIONS: Plasma sPLA2 may provide a biological link between asthma, inflammation, increased BMI, lipid metabolism and antioxidants. Interactions among these factors may be pertinent to the pathophysiology and increasing prevalence of both asthma and obesity.

Molecular genetics and mechanisms of apoptosis in carcinomas of the lung and pleura: therapeutic targets.

Cancers of the lung and pleura remain a major cause of cancer deaths, both in men and women, with strong causal relationships between cigarette smoking and asbestos fibres, and deaths from lung cancer and mesothelioma, respectively. The poor survival rates for small cell lung cancer and mesotheliomas argue powerfully for greater understanding of mechanisms of carcinogenesis, genetic abnormalities and the role of tumour suppressor genes and proteins in carcinomas of the lung and pleura. Despite progress in the development of newer cytotoxic drugs, lung cancer remains a lethal disease. Chemotherapy and radiotherapy produce only a modest improvement in survival of patients with advanced disease. Increased knowledge of molecular mechanisms of lung cancer and apoptosis are providing opportunities for treating lung cancer with new classes of molecularly targeted drugs. These novel therapies should target the abnormalities in lung cancer by maximizing the effects of anti-tumour molecules, with minimal side effects on normal tissues. Of the several molecular targets, those receiving attention are p53 gene replacement, Bcl-2 downregulation, apoptosis by induced by TNF, the FAS/CD95 receptor system and TRAIL, and inhibition of NF-kappaB. Although several studies have shown benefits, there is a need for well planned clinical trials of drugs that target the apoptotic cascade. Stem cell therapy and gene replacement offer the prospect of novel approaches that are likely in the near future to play a definitive role in the treatment of advanced lung cancer. Furthermore, with their apparent minimal toxicity to normal tissues, the newer molecular targets represent attractive investigational directions for innovative cancer therapies.

Changes in bronchial hyperresponsiveness following high- and low-sulphite wine challenges in wine-sensitive asthmatic patients.

BACKGROUND: Previous studies suggest that challenge of most wine-sensitive asthmatic patients may not result in a reduction in forced expiratory volume in 1 s (FEV(1)). OBJECTIVE: The aim of this study was to assess whether changes in bronchial hyperresponsiveness (BHR) occur following wine challenge of asthmatic patients who report sensitivity to wine, and whether such changes could help clarify the role of sulphite additives in wine-induced asthmatic responses. METHODS: Eight self-reporting wine-sensitive asthmatic patients completed double-blind challenges with high- and low-sulphite wines on separate days. FEV(1) and histamine PC(20) were measured before and after consumption of 150 mL of wine. RESULTS: None of the eight subjects demonstrated a clinically significant >or=15%) reduction in FEV(1) following challenge with either high- or low-sulphite wine. In contrast, one patient demonstrated clinically significant increase in BHR following challenge with both high- and low-sulphite wines, and a smaller increase in BHR following placebo challenge. A second patient showed a significant increase, while another showed a significant decrease in BHR following challenge with low-sulphite wine. A fourth patient showed borderline increases in BHR following challenge with both high- and low-sulphite wines. CONCLUSIONS: Although changes in BHR, in the absence of reductions in FEV(1), were observed in some asthmatic patients following wine challenge, these changes were not consistent with a single aetiology. Consequently, this study did not support a major role for the sulphite additives in wine-induced asthmatic responses in the patients studied. The aetiology of wine-induced asthma is likely to be complex and appears to vary among individuals who are sensitive to these drinks.

Cyclooxygenase-1 gene polymorphisms in patients with different asthma phenotypes and atopy.

Cyclooxygenase-1 (COX-1) regulates the biosynthesis of prostaglandins, which are important mediators in asthma. The possible association of COX-1 gene polymorphisms with asthma has not been investigated. The allele frequencies of 20 COX-1 polymorphisms were determined in a random Australian Caucasian population using MassARRAY technology. Informative and potentially functional promoter (c.8592C > T, c.1676C > T) and coding region (c.22C > T, c.50C > T) polymorphisms were investigated in carefully phenotyped patients with mild (n = 316), moderate (n = 241), severe (n = 86) or aspirin-intolerant asthma (AIA) (n = 58), and in nonasthmatic subjects (n = 477). There were no allelic, genotypic or haplotypic associations between these four polymorphisms and asthma or asthma severity. Over-representation of the c.50TT genotype among AIA patients (3.4%) compared with aspirin-tolerant patients (0.8%), and a global haplotype association with AIA did not reach statistical significance. The c.22TT genotype was less frequent among atopic (0.1%) rather than nonatopic individuals (1.2%; odds ratio = 9.05, 95% confidence interval 1.01-81.29). In conclusion, the present investigation of cyclooxygenase-1 polymorphisms in asthma indicates that they do not appear to play a substantial role in genetic pre-disposition for asthma or asthma severity. However, the c.22TT genotype confers a small protective effect against atopy. Potential associations with aspirin-intolerant asthma were identified and warrant further investigation in a larger population of aspirin-intolerant asthma patients.

Plasma concentrations of dietary and nondietary antioxidants are low in severe asthma.

Low antioxidant levels and oxidative stress due to airway inflammation may be important determinants of asthma severity. The objective of the present study was to determine whether lower antioxidant intake and plasma antioxidant concentrations are associated with more severe asthma. Dietary antioxidant intakes and asthma severity were assessed using questionnaires, and plasma concentrations of ascorbic acid, vitamin E, carotenoids, bilirubin, albumin, uric acid and total antioxidant status were measured in 53 mild-to-moderate and 28 severe asthmatic patients and in 43 nonasthmatic subjects. Vitamin C and carotene intakes were lower in males than females and were particularly low in males with severe asthma. Plasma ascorbic acid was lower in severe (31.9+/-3.6 microM) compared with mild-to-moderate asthmatic (52.3+/-2.6) or control subjects (52.7+/-2.9). Low plasma ascorbic acid (odds ratio (OR) 0.93; 95% confidence interval (CI) 0.9-0.97), bilirubin (OR 0.69; 95% CI 0.51-0.93) and increased plasma cholesterol (OR 1.98; 95% CI 1.05-3.73) were independently associated with severe asthma. Albumin was positively and cholesterol negatively correlated with lung function. Low plasma concentrations of specific antioxidants are associated with more severe asthma. Increased antioxidant intake may help reduce the burden of severe asthma, particularly in males.

A functional polymorphism in the promoter region of the cyclooxygenase-2 gene is not associated with asthma and atopy in an Australian population.

BACKGROUND: Cyclooxygenase (COX)-2 is a key inducible enzyme that regulates the production of anti-inflammatory prostaglandin E(2). A single-nucleotide polymorphism, -765G>C, located within a stimulatory protein-1 binding site in the COX-2 promoter region, has been shown to have significantly lower promoter activity in vitro compared with the wild-type and was associated with decreased plasma levels of C-reactive protein after coronary artery bypass surgery. We hypothesized that this polymorphism, which may result in decreased COX-2 transcription, could be associated with more severe asthma, and/or aspirin-intolerant asthma (AIA). OBJECTIVE: To determine the association between the -765G>C COX-2 polymorphism and asthma, disease severity and AIA in a large, well-phenotyped Australian population. METHODS: PCR and restriction fragment length polymorphism analysis was used to characterize the polymorphism in an Australian Caucasian population of patients with mild (n=322), moderate (n=254) or severe (n=88) asthma and in non-asthmatic control subjects (n=512), as well as in patients with AIA (n=58). Genotype and allele association analyses were performed using chi(2) tests. RESULTS: The polymorphic -765C allele was present in approximately 30% of asthmatic patients and non-asthmatic controls. There was no association between the -765G>C polymorphism and asthma (P=0.920), disease severity (P=0.840), atopy (P=0.655) or AIA (P=0.841) in this population. CONCLUSION: Although the -765G>C polymorphism may have lower promoter activity and result in decreased COX-2 expression, it is not associated with asthma, disease severity, AIA or atopy in this Australian population.

Urinary leukotriene E4 and 9 alpha, 11 beta-prostaglandin F concentrations in mild, moderate and severe asthma, and in healthy subjects.

BACKGROUND: Airway inflammation in asthma is associated with cysteinyl leukotriene and prostaglandin D(2) production. Measurement of urinary metabolites of these eicosanoids may be useful for monitoring asthma patients. However, the influence of asthma phenotype and severity on basal urinary excretion of these metabolites is unknown. OBJECTIVE: To compare urinary leukotriene (LT)E(4) and 9 alpha, 11 beta-prostaglandin (PG)F(2) concentrations in large groups of mild, moderate and severe asthmatic patients and healthy control subjects. METHODS: Asthma severity, treatment and aspirin sensitivity were assessed by questionnaire in 168 asthmatic patients. Basal LTE(4) and 9 alpha, 11 beta-PGF(2) concentrations were measured in urine samples from these patients and from 175 control subjects using enzyme immunoassays. RESULTS: Urinary LTE(4) was correlated with 9 alpha, 11 beta-PGF(2) in both control subjects and asthmatic patients (P<0.002). Median LTE(4) and 9 alpha, 11 beta-PGF(2) concentrations in patients with severe asthma were significantly reduced compared with mild asthmatic patients (P<0.05 and <0.001, respectively). Urinary 9 alpha, 11 beta-PGF(2), but not LTE(4) was lower in asthmatic patients using inhaled corticosteroids (P<0.02). Multiple regression analysis indicated that urinary 9 alpha, 11 beta-PGF(2) concentration was negatively correlated with asthma severity (P=0.003) and also with % predicted FEV(1) (forced expiratory volume in 1 s) (P=0.005). CONCLUSIONS: Baseline urinary LTE(4) and 9 alpha, 11 beta-PGF(2) concentrations are of limited value in discriminating between patients with different severities of asthma. Reduced urinary LTE(4) and 9 alpha, 11 beta-PGF(2) in patients with severe asthma suggest that direct or indirect effects of high-dose corticosteroid therapy combined with other factors associated with severe asthma may influence eicosanoid production. However, the negative association of urinary 9 alpha, 11 beta-PGF(2) with lung function suggests an adverse effect of chronic PGD(2) production on lung function in asthma, irrespective of severity.

Oxidant stress induces gamma-glutamylcysteine synthetase and glutathione synthesis in human bronchial epithelial NCI-H292 cells.

BACKGROUND: The bronchial epithelium is exposed to reactive oxygen species (ROS) derived from cigarette smoke, air pollutants and activated leucocytes. Glutathione (GSH) prevents ROS-mediated loss of cell function, tissue injury and inflammation, and its synthesis is regulated by gamma-glutamylcysteine synthetase (gamma-GCS). However, the capacity of bronchial epithelial cells to adapt to oxidative stress and the mechanisms involved are not known. OBJECTIVE: To investigate the effects of oxidative stress on the regulation of GSH synthesis in human bronchial epithelial (NCI-H292) cells. METHODS: NCI-H292 cells were exposed to menadione and intracellular GSH concentrations were measured by spectrophotometry. gamma-GCS activity was measured by HPLC assay and changes in gamma-GCS mRNA by Northern blotting. RESULTS: Exposure to menadione (MQ, 10-200 microm, 30-120 min) decreased total cellular GSH content, measured immediately after exposure to MQ. However, GSH content measured 6-12 h after withdrawal of the oxidant stress (MQ, 50 microm, 30 min), increased c. two fold over baseline levels (P < 0.001). gamma-GCS activity measured 6 h (21.7 +/- 3.4 nmol/min/mg, SD, n = 5, P < 0.01) or 12 h (23.2 +/- 4.6, P < 0.001) after MQ treatment was also significantly increased compared with untreated cells (12.8 +/- 1.0). Similarly, gamma-GCS mRNA expression increased 1.3-1.6-fold relative to GAPDH mRNA, 3-6 h after MQ treatment. The MQ-induced increase in gamma-GCS mRNA expression was completely inhibited by actinomycin D. CONCLUSIONS: Bronchial epithelial (NCI-H292) cells respond rapidly and sensitively to oxidant stress, and this adaptive response is mediated by increased gamma-GCS mRNA transcription and enzyme activity.

A simple assay for a human serum phospholipase A2 that is associated with high-density lipoproteins.

Phospholipase A2 (PLA2) activity is usually assayed with expensive radioactive or chromogenic substrates unsuitable for performing large numbers of assays. We have designed a simple microplate assay for human serum PLA2 using the chromogenic substrate 4-nitro-3-octanoyloxy-benzoic acid. Using this substrate, serum PLA2 activity was similar to that measured with the previously characterized chromogenic phospholipid substrate 1,2-bis-heptanoylthio-glycerophosphocholine. However, the assay described here appears to be more sensitive. The mean PLA2 activity in serum from healthy volunteers (n = 30) measured by this assay was 10.4 +/- 1.6 micromol x h(-1) x ml(-1). The assay is reproducible and is suitable for the analysis of large numbers of samples in a clinical setting. We have also demonstrated that 94% of the PLA2 activity in normal human serum is associated with high-density lipoproteins and that serum PLA2 activity is positively correlated with the lipoprotein parameters total triglyceride (P < 0.0001), total cholesterol (P < 0.0001), and atherogenic index (P = 0.008). The serum PLA2 activity was calcium dependent and was inhibited by the serine protease inhibitor 3,4-dichloroisocoumarin (EC(50) = 0.4 mM). The PLA2 activity characterized here is unlikely to be due to plasma platelet-activating factor acetylhydrolase or low molecular weight His-Asp sPLA2, and may represent a new sPLA2 type.

Oncostatin M synergises with house dust mite proteases to induce the production of PGE(2) from cultured lung epithelial cells.

The release of PGE(2) and nitric oxide (NO) from the respiratory epithelium may act to dampen inflammation. In other tissues, oncostatin M (OSM), a potent inducer of epithelial antiproteases, has also been shown to interact with IL-1beta to stimulate PGE(2) release. However, whether OSM interacts with pro-inflammatory cytokines and proteases in the production of anti-inflammatory eicosanoids and NO from airway epithelium is unknown. The effect of OSM and the related cytokine leukaemia inhibitory factor (LIF) on PGE(2) and NO production by the respiratory epithelial cell line, A549 in response to pro-inflammatory cytokines as well as protease-rich house dust mite (HDM) fractions and a protease-deficient rye grass pollen extract was examined by immunohistochemistry, cell culture, ELISA and enzyme-immunoassay. Cells treated with a mixture of IL-1beta, IFNgamma and LPS for 48 h produced a 9 fold increase in PGE(2) and a 3 fold increase in NO levels (both P<0.05). Both OSM and LIF were without effect. However, OSM added together with the cytokine mixture synergistically enhanced PGE(2) production (22 fold, P<0.05). OSM also synergistically enhanced PGE(2) production in response to a cysteine protease-enriched, but not serine protease-enriched HDM fraction (P<0.05). Rye grass extract, neither alone nor in combination with OSM, induced PGE(2) or NO production, although it did induce the release of GM-CSF. These observations suggest that OSM is an important co-factor in the release of PGE(2) and NO from respiratory epithelial cells and may play a role in defense against exogenous proteases such as those derived from HDM.

Nitrite generation and antioxidant effects during neutrophil apoptosis.

Neutrophil apoptosis is important for the resolution of airway inflammation in a number of lung diseases. Inflammatory mediators, endogenous reactive oxygen and nitrogen species, and intracellular and extracellular antioxidants may all influence neutrophil apoptosis. This study investigated the involvement of these factors during apoptosis of neutrophils cultured in vitro. Neutrophils undergoing spontaneous apoptosis in culture as assessed by annexin V binding generated significant amounts of nitrite. Incubation with agonistic anti-Fas monoclonal antibody or tumor necrosis factor-alpha (TNF-alpha) enhanced neutrophil apoptosis at 6 h, although it decreased nitrite accumulation. Although granulocyte-macrophage colony-stimulating factor significantly reduced neutrophil apoptosis, this was also associated with decreased nitrite accumulation. In contrast, inhibition of apoptosis at 16 h by dibutyryl cyclic adenosine monophosphate was associated with increased nitrite accumulation. Exogenous glutathione (GSH) or N-acetylcysteine significantly enhanced neutrophil apoptosis at 6 h and stimulated the production of H(2)O(2), which may mediate apoptosis through intracellular hydroxyl radical production. Intracellular GSH concentrations decreased in neutrophils undergoing apoptosis, and this was more marked in neutrophils treated with anti-Fas or TNF-alpha. These results suggest a causal association between reduced endogenous nitric oxide production, reduced intracellular GSH, and Fas- and TNF-alpha-mediated neutrophil apoptosis, whereas enhanced neutrophil survival mediated by dibutyryl cyclic adenosine monophosphate is associated with increased nitrite generation and maintenance of intracellular GSH. The interaction of endogenous reactive oxygen species with extracellular antioxidants such as GSH could also contribute to the complex processes regulating neutrophil apoptosis and hence the resolution of inflammation in the lung.

Differential expression of neutrophil adhesion molecules during coronary artery surgery with cardiopulmonary bypass.

BACKGROUND: Activation of neutrophil adhesion molecules and subsequent neutrophil adhesion to vascular endothelium are key events initiating inflammatory organ dysfunction after cardiopulmonary bypass and ischemic reperfusion. OBJECTIVES: We sought to characterize neutrophil integrin CD11b and L-selectin activation associated with coronary artery bypass graft surgery and to determine whether neutrophil activation contributes to their sequestration on postbypass reperfusion. METHODS: Twenty patients undergoing routine coronary artery bypass were studied. Heparinized whole blood was simultaneously sampled from a central venous line, aorta, coronary sinus, and right and left atrium before, during, and up to 20 minutes after cardiopulmonary bypass. Neutrophil counts were obtained, and neutrophil CD11b and L-selectin expression was determined by flow cytometric analysis in whole blood. RESULTS: CD11b expression on circulating neutrophils increased during cardiopulmonary bypass, peaking at 145% of baseline level after release of the aortic clamp and then declined by 20 minutes after bypass (analysis of variance, P =.003). No change in neutrophil L-selectin expression was observed during cardiopulmonary bypass. Neutrophils responded to ex vivo stimulation by C5a and leukotriene B(4) during cardiopulmonary bypass but not at 24 hours after the operation. After reperfusion, neutrophil loss, but not local activation, was demonstrated in the coronary and pulmonary circulations. CONCLUSIONS: Upregulated CD11b expression on neutrophils is likely to contribute to neutrophil sequestration in the heart and lungs after bypass, but neutrophil activation may be limited by their reduced responsiveness to agonist stimulation. CD11b represents a potential therapeutic target for diminishing inflammation after cardiac operations.

PGE 2 and dibutyryl cyclic adenosine monophosphate prolong eosinophil survival in vitro.

BACKGROUND: Apoptosis represents a mechanism by which the accumulation and inflammatory potential of eosinophils in asthma might be limited. Mediators derived from the airway epithelium may influence the rate of eosinophil apoptosis. OBJECTIVE: We have investigated the effects on eosinophil apoptosis of 3 mediators that are likely to be produced by the airway epithelium, namely PGE2, TNF-alpha, and nitric oxide. METHODS: Peripheral blood eosinophils from healthy adult volunteers were purified by density gradient centrifugation and negative immunomagnetic selection. Eosinophils were cultured for 16 or 40 hours with PGE2 (10 nmol/L), dibutyryl cyclic adenosine monophosphate (AMP; 100 micromol/L), TNF-alpha (500 U/mL), the nitric oxide donors, S-nitroso-N-acetylpenicillamine (100 micromol/L), and 2,2;-(hydroxynitrosohydrazono)bis-ethanamine (1 mmol/L), or dibutyryl cyclic guanosine monophosphate (100 micromol/L). Control cultures consisted of untreated, IL5-treated (100 U/mL), and anti-Fas-treated (400 ng/mL) cells. Eosinophil apoptosis was assessed by flow cytometric analysis of annexin V-FITC binding to externalized phosphatidylserine, by electrophoresis of phosphorus 32 end-labeled DNA fragments, and by flow cytometric assessment of hypodiploid DNA with propidium iodide. RESULTS: PGE2 and cyclic AMP inhibited spontaneous eosinophil apoptosis at both 16 and 40 hours as did the PGEP2 receptor agonist, 11-deoxy PGE1, at 40 hours, but these effects were not inhibited by a protein kinase A antagonist. TNF-alpha delayed apoptosis in eosinophil cultures at 16 hours, whereas S-nitroso- N-acetylpenicillamine, 2, 2;-(hydroxynitrosohydrazono)bis-ethanamine, and cyclic guanosine monophosphate had little effect. Anti-Fas had little effect on spontaneous eosinophil apoptosis but significantly reduced the inhibitory effects of PGE2, cyclic AMP, and TNF-alpha. Assessments of apoptosis by DNA fragmentation gave similar but quantitatively less sensitive results. CONCLUSION: Inhibition of spontaneous eosinophil apoptosis by PGE2 appears to be mediated by EP2 receptors but is not protein kinase A dependent. By enhancing eosinophil survival, PGE2 may increase the proinflammatory potential of these cells in chronic asthma.

Gamma-glutamylcysteine synthetase activity in human lung epithelial (A549) cells: factors influencing its measurement.

Despite the central role of gamma-glutamylcysteine synthetase (gammaGCS) in lung antioxidant defenses, the limited studies of the activity of this enzyme in respiratory cells have produced variable results. This study has examined the factors, which may influence the measurement of gammaGCS activity in cultured human lung epithelial cells (A549). Although a source of potential error, gammaGCS activity in A549 cell extracts did not vary significantly when appropriately assayed by three different methods or after removal of the endogenous inhibitor, glutathione (GSH). However, gammaGCS activity did increase significantly during the early stages of cell proliferation (3.50 +/- 0.31 vs. 2.35 +/- 0.16 nmol/min/10(6) cells for baseline, p < .001) and thereafter returned to baseline levels during the later stages of cell growth. Variations in initial plating density also significantly altered gammaGCS activity (3.11 +/- 0.14 vs. 4.04 +/- 0.50 nmol/min/10(6) cells, at 0.25 x 10(5) and 0.58 x 10(5) cells/cm2, respectively, p < .001) and GSH content (45.43 +/- 4.43 vs. 63.64 +/- 3.28 nmol/10(6) cells at 0.25 x 10(5) and 0.58 x 10(5) cells/cm2, respectively, p < .001) during the early stages of cell proliferation. In addition, gammaGCS activity and GSH content were highest in A549 cells grown in medium containing cystine as the predominant sulfur-containing amino acid. These results suggest that gammaGCS activity of A549 cells is strongly dependent on initial plating density, stage of cell growth and sulfur amino acid content of the medium and may account for some of the variation in values reported by different investigators. Whether gammaGCS has an important role in the early phase of cell proliferation needs further investigation.

The effect of aspirin on thrombin stimulated platelet adhesion receptor expression and the role of neutrophils.

AIMS: Aspirin has proven clinical efficacy in limiting the thrombotic complications of atherosclerotic vascular disease but its mechanism of action remains unclear. Recent evidence suggests the anti-platelet action of aspirin may be partly mediated by neutrophil derived nitric oxide (NO). The aim of the study was to determine the effects of aspirin on thrombin-induced platelet expression of the alpha-granule membrane protein, P-selectin, and the platelet surface glycoprotein required for aggregation, GPIIb-IIIa, and to assess whether this was enhanced by the presence of neutrophils. METHODS: Platelet P-selectin and GPIIb-IIIa receptor expression were assessed by flow cytometric analysis of washed platelets stimulated with thrombin (0.025 iu ml(-1), sub aggregatory concentration) alone or after pre-incubation with aspirin (0.05, 0.1, 0.5, 1.0 mg m1(-1) either in the presence or absence of neutrophils (100 platelets per neutrophil). NO release was determined by assay of nitrite in the supernatants from parallel samples. RESULTS: In preliminary aggregation studies, aspirin at all concentrations inhibited arachidonic acid but not thrombin-induced platelet aggregation. Similarly, aspirin at all concentrations failed to inhibit thrombin-induced platelet P-selectin or GPIIb-IIIa expression and this was not influenced by the presence of neutrophils. A reduction in P-selectin and GPIIb-IIIa receptor density on non-activated platelets co-incubated with unstimulated neutrophils was associated with NO release from neutrophils, but this was not enhanced by the addition of aspirin. CONCLUSIONS: These results confirm that thrombin-induced platelet alpha-granule release, with consequent P-selectin expression, and platelet GPIIb-IIIa expression, are not affected by aspirin inhibition of cyclo-oxygenase and suggest that the anti-thrombotic efficacy of aspirin in vivo may partly depend on other mechanisms. This study did not demonstrate an effect of neutrophils or neutrophil derived NO on aspirin inhibition of platelet adhesion receptor expression.

Neutrophil cathepsin G modulates platelet P-selectin expression and inhibits P-selectin-mediated platelet-neutrophil adhesion.

1. Close contact between platelets and neutrophils modulates their cellular interactions in thrombotic and inflammatory states, with stimulation of P-selectin expression on platelets by agonists such as thrombin and neutrophil-derived cathepsin G being critical in mediating platelet-neutrophil adhesion. This study compared the effects of thrombin and cathepsin G on platelet P-selectin expression and on P-selectin-mediated platelet-neutrophil adhesion. 2. Washed platelets and platelet-neutrophil mixed cell suspensions (platelet/neutrophil ratio, 10:1) were incubated with either the supernatant of activated neutrophils, purified cathepsin G or thrombin. Platelet P-selectin expression and platelet adhesion to neutrophils was quantified by flow fluorocytometric analysis. 3. The supernatant from activated neutrophils stimulated platelet P-selectin expression comparable to that produced by purified cathepsin G or thrombin. P-selectin expression induced by both activated neutrophil supernatant and purified cathepsin G was completely inhibited by alpha 1-antichymotrypsin, a specific inhibitor of cathepsin G. Unlike thrombin, which induced maximum platelet P-selectin expression by 10 min, sustained to 120 min, cathepsin G induced an initial large increase in platelet P-selectin expression, followed by a progressive reduction over 30-60 min to baseline levels. 4. Co-incubation of neutrophils with thrombin-stimulated platelets resulted in a significant increase in P-selectin-mediated platelet-neutrophil adhesion, which was completely inhibited by preincubation of neutrophils with anti-sialyl Lewis(x) monoclonal antibody. Thrombin produced maximum platelet-neutrophil adhesion by 10 min which remained stable over 120 min. In contrast, cathepsin G-stimulated platelets did not adhere to neutrophils over 120 min of co-incubation. Addition of cathepsin G to thrombin-stimulated platelets caused a progressive reduction over 30-60 min to baseline levels of platelet-neutrophil adhesion. 5. Neutrophil-derived cathepsin G is a potent platelet activator, but unlike thrombin it causes a time-dependent loss of platelet P-selectin expression and inhibits P-selectin-mediated platelet-neutrophil adhesion. Therefore, cathepsin G may modulate thrombin-mediated platelet-neutrophil adhesive interactions in inflammation and thrombosis.

Glutathione peroxidase activity and mRNA expression in eosinophils and neutrophils of asthmatic and non-asthmatic subjects.

Asthma has been reported to be associated with a reduction in the activity of glutathione peroxidase (GSH-Px), an important antioxidant enzyme. However, the expression of GSH-Px enzyme activity has not previously been investigated in human eosinophils, which are important inflammatory cells involved in asthma. Reverse transcriptase-polymerase chain reaction and Southern blotting demonstrated that eosinophils express GSH-Px mRNA and the relative expression of GSH-Px was greater in eosinophils than in neutrophils for both asthmatic and non-asthmatic subjects. The presence of GSH-Px protein in eosinophil and neutrophil lysates was confirmed by size exclusion chromatography and by Western blotting. GSH-Px enzyme activity as measured by a spectrophotometric assay was greater in eosinophil (48.4+/-1.6 micromol NADPH oxidized x min(-1) x g(-1) protein) than in neutrophil lysates (18.1+/-0.4, n = 24, P < 0.0001). GSH-Px activities of eosinophils and neutrophils from asthmatic subjects did not differ from those of non-asthmatic subjects. Eosinophil GSH-Px activity was correlated with peripheral blood eosinophil count only in asthmatic subjects (rs = 0.59, n = 12, P = 0.04). Increased GSH-Px expression in eosinophils compared with neutrophils of asthmatic patients may provide antioxidant protection against the greater amounts of reactive oxygen species generated by these cells and may enhance the survival of eosinophils at sites of inflammation in asthma.