Urinary leukotriene E4 and 11-dehydrothromboxane B2 in patients with aspirin-sensitive asthma

The objective of this study was to define the participation of cysteinyl leukotrienes (LTs) or thromboxane A₂ in the pathogenesis of aspirin-sensitive asthma (ASA). Leukotriene E₄ (LTE₄) and 11-dehydrothromboxane B₂ (11DTXB₂) values in spot urine were measured in 22 asthmatics with a history of aspirin sensitivity and in 17 without such a history of aspirin-sensitive asthma [NASA]) in the outpatient clinic. The urinary LTE₄ value was significantly higher in ASA patients than in NASA (340±47 vs 65±15 pg/mg·cr, P <0.001), but there was no significant difference in urinary 11DTXB2 between the two groups (891±77 vs 657±90 pg/mg·cr). A high value of LTE4 was not associated with type of asthma, severity of disease, oral prednisolone treatment, sex, or age. A higher value of 11DTXB₂ was observed in the atopic type than the nonatopic type in ASA (1086±111 vs 697±147 pg/mg·cr, P<0.05). No correlation was observed between urinary LTE₄ and 11DTXB₂ in either ASA or NASA. In conclusion, LTs may play an important role in the pathogenesis of ASA, and TXA₂ in the pathogenesis of the atopic type in ASA.     

Increased Generation of Leukotriene C4 from Eosinophils in Asthmatic Patients

Human eosinophils with a density of over 1.095 were isolated from peripheral blood by dextran sedimentation, centrifugation with Lymphoprep and density gradients with Percoll. After the cells (1 X 10(5)/ml) were incubated with 1 microgram/ml calcium ionophore A23187 for 20 min, leukotriene C4 (LTC4) content in the supernatant was measured by radioimmunoassay. The generation of LTC4 was significantly higher in the cells from extrinsic asthmatics (23.5 +/- 14.8 ng/10(6) cells, mean +/- SD, n = 26, P less than 0.01) and intrinsic asthmatics (24.6 +/- 20.6 ng/10(6) cells, n = 27, P less than 0.01 as compared with normal healthy subjects (8.3 +/- 7.7 ng/10(6) cells, n = 10). There was no significant difference in the generation of LTC4 between intrinsic and extrinsic asthmatics. These observations indicate that eosinophils from asthmatic patients have increased ability to release LTC4.     

Increase in urinary leukotriene B4 glucuronide concentration in patients with aspirin-intolerant asthma after intravenous aspirin challenge

BACKGROUND: Aspirin challenge of aspirin-intolerant asthma (AIA) patients causes a significant increase in leukotriene E4 (LTE4) concentration in urine. However, knowledge on leukotriene B4 (LTB4) generation in patients with AIA is insufficient. Recent research has demonstrated that exogenously administered LTB4 is excreted as glucuronide into the urine in human healthy subjects. OBJECTIVE: The purpose of this study is to estimate urinary LTB4 glucuronide (LTBG) concentration in the clinically stable condition in healthy subjects and asthmatic patients and to investigate changes in urinary LTBG concentration in patients with AIA after aspirin challenge. METHODS: A provocation test was performed by intravenous aspirin challenge. After urine was hydrolysed by beta-glucuronidase, the fraction containing LTB4 was purified by high-performance liquid chromatography and LTB4 concentration was quantified by enzyme immunoassay. Urinary LTBG concentration was calculated as the difference between the concentration obtained with hydrolysis and that without hydrolysis. RESULTS: (1) After hydrolysis, the presence of urinary LTB4 was verified by gas chromatography-mass spectrometry-selected ion monitoring. (2) The urinary LTBG concentration was significantly higher in the asthmatic patients than in the healthy subjects (median, 5.37 pg/mg creatinine [range 1.2-13] vs. 3.32 pg/mg creatinine [range, 0.14-10.5], P = 0.0159). (3) The patients with AIA (n = 7), but not those with aspirin-tolerant asthma (n = 6), showed significant increases in LTBG and LTE4 excretions after aspirin challenge. (4) When the concentrations after aspirin challenge were analysed simultaneously, a significant linear correlation was observed between urinary LTBG concentration and urinary LTE4 concentration in patients with AIA (Spearman's rank correlation test, r = 0.817, P = 0.0003). CONCLUSION: LTBG is present in human urine, albeit at a concentration lower than urinary LTE4. In addition to a marked increase in cysteinyl-leukotriene production, aspirin challenge induced LTB4 production in AIA patients.     

The challenge procedure influences the extent of allergen-induced urinary excretion of leukotriene E4

BACKGROUND: Cysteinyl-leukotrienes are central mediators in asthma and urinary leukotriene E4 (LTE4) is a reliable marker of their endogenous formation. OBJECTIVE: This study tested the hypothesis that the procedure used for allergen bronchoprovocation influences the bronchoconstrictor response and the amount of LTE4 excreted following allergen challenge. METHODS: Seven atopic asthmatic men underwent two allergen bronchoprovocations 4 weeks apart. The same total dose of allergen was given at both sessions, cumulatively on one occasion and as a single dose at the other session. Urine was collected in hourly samples before and after challenge and LTE4 was measured with previously validated methodology. RESULTS: The mean (+/- SE) drop in FEV1 was not significantly different between the cumulative (29 +/- 2.4%) and the single dose challenge (25 +/- 2.8%). There was a significant increase in post-challenge levels of urinary LTE4 after both sessions. The peak excretion of LTE4 occurred 1 h following the maximal drop in FEV1 for both challenges. However, the post-challenge increase in urinary LTE4 was significantly larger at the cumulative session. In fact, the net increase (post-challenge minus prechallenge) of urinary LTE4 was more than twofold higher after the cumulative session (AUC 0-3 h post-challenge: 46.7 +/- 8.2 vs 22.1 +/- 9.8, P < 0.05). CONCLUSION: The peak excretion of urinary LTE4 occurred within 2 h after the termination of either challenge but the magnitude of urinary excretion of LTE4 was larger when cumulative challenge was performed. The findings are important to consider when designing studies where allergen-induced urinary excretion of LTE4 is an outcome variable.     

Lipoxin A4 levels in asthma: relation with disease severity and aspirin sensitivity

BACKGROUND: Lipoxin (LX) A4, an endogenous anti-inflammatory eicosanoid, has been found to be low in patients with severe asthma. However, few studies also suggested more diminished LX A4 levels in aspirin-exacerbated respiratory disease (AERD) when compared with aspirin-tolerant asthma (ATA). It is, therefore, currently not clear whether the asthma severity or the presence of AERD has a primary role in the disturbed LX metabolism. OBJECTIVE: To detect LX A4 and 15-epi-LX A4 levels in asthma patients with and without AERD of comparable severity. METHODS: The study groups consisted of 22 subjects with AERD, 22 subjects with ATA and 10 volunteers without asthma and aspirin sensitivity. Whole-blood samples were stimulated with calcium ionophore, A23187 (5 x 10(-5) m) and A23187 (5 x 10(-5) m)+aspirin (10(-4) m). LX A4 and 15-epi-LX A4 levels were analysed by the enzyme immune assay method. RESULTS: Severe asthma patients in both AERD [0.5 (0.8)] ng/mL and ATA [0.5 (0.45) ng/mL] groups showed diminished generation for LX A4 to stimulation with A23187 in comparison with other severity degrees in their groups (P=0.02 and 0.046, respectively). LX A4 generation in both severe groups was comparable with each other (P>0.05). Although severe cases with AERD showed a diminished capacity to generate 15-epi-LX A4, this did not reach statistical significance. CONCLUSION: This study indicated that diminished LX A4 generation was unique to severe asthma phenotype regardless of comorbid aspirin sensitivity. Clinical Implications Lower LX A4 levels in severe asthma would suggest a possibility for LX analogues as future treatment options in these patients.     

Correlation among urinary eosinophil protein X, leukotriene E4, and 11-dehydrothromboxane B2 in patients with spontaneous asthmatic attack

Various kinds of cells and their mediators are thought to be involved in the pathogenesis of bronchial asthma. However, changes in each mediator or relationship among mediators during an asthmatic attack have not been well documented. In this study, to clarify whether eosinophil protein X (EPX) is a marker which is distinct from leukotriene E₄ (LTE₄), or 11-dehydrothromboxane B₂ (11DTXB₂), we measured the urinary excretion of EPX, LTE₄, and 11DTXB₂ in 14 asthmatics who were admitted to the hospital with either an acute asthmatic attack or status asthmaticus. These patients included eight atopic and six non-atopic types of bronchial asthma, with a median age of 34.0 years. Urinary excretion of EPX was significantly high on admission with the asthmatic attack, and returned to control levels 175 [122 –384] μg/day when the patients were in the improved state (1036–317 μg/day, P  < 0.01). Similar findings were observed in LTE₄ (155–59 ng/day, P  < 0.01) and 11DTXB₂ (991–442 ng/day, P  < 0.01). No significant differences in values were observed between atopic and non-atopic types of asthma in all three substances. When the individual data during the attack state were analysed, a significant correlation was observed between changes (%) in urinary EPX and those in urinary LTE₄, but no such relationship was observed between changes (%) in urinary EPX and those in urinary 11DTXB₂. These results suggest that measuring urinary EPX levels may be a useful marker for the understanding and management of the disease.     

Leukotriene (LT)‐receptor antagonist is more effective in asthmatic patients with a low baseline ratio of urinary LTE4 to 2,3‐dinor‐6‐keto‐prostaglandin (PG)F1α

BACKGROUND: To test the hypothesis that urinary levels of arachidonic acid metabolites may be a predicting factor of the effects of pranlukast, a selective leukotriene (LT) antagonist, on chronic adult asthma, we investigated the relationship between its clinical efficacy and urinary eicosanoid levels. METHODS: An open, multicenter trial was conducted involving 38 stable moderate and severe asthmatic patients (mean percent predicted FEV1 was 71%). All patients received pranlukast (225 mg twice daily) for 4 weeks after a 2-week run-in period. Urinary levels of LTE4, 11-dehydro-thromboxane (TX) B2, 2,3-dinor-6-keto-prostaglandin (PG) F1alpha, and creatinine were measured in 3-h urine collected on day 1 of the treatment. The responder was defined by an improvement of asthma symptom scores and peak expiratory flow rate (PEFR). RESULTS: One patient was excluded because of an adverse effect, nausea. Thirteen out of 37 subjects were responders and 24 were nonresponders. There were no significant differences in patients' backgrounds and urinary arachidonate levels between the two groups. The urinary LTE4 to 2,3-dinor-6-keto-PGF1alpha ratio in the responder was significantly lower (P=0.01) than that in the nonresponder. In all patients, a significant inverse correlation was revealed between the baseline urinary LTE4/2,3-dinor-6-keto-PGF1alpha ratio and the improvement of PEFR in the morning (r=-0.43, P=0.007). CONCLUSIONS: These data suggested that the urinary ratio of LTE4 to 2,3-dinor-6-keto-PGF1alpha might be one of the predictive markers of the clinical efficacy of this LT-receptor antagonist in asthmatic subjects.     

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)E₂, 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 PGE₂, cysLT, 8‐isoprostane and nitrate concentrations, as well as urinary leukotriene (LT)E₄ and 9α,11β‐prostaglandin (PG)F₂ 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 PGE₂, cysLT and 8‐isoprostane, and urinary LTE₄ 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 PGE₂ 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 PGE₂ 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 LTE₄ 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 PGE₂ are consistent with the hypothesis that PGE₂ has a bronchoprotective and anti‐inflammatory role in patients with more severe asthma. A reduced PGE₂ 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. Cite this as: S. Aggarwal, Y. P. Moodley, P. J. Thompson and N. L. Misso, Clinical & Experimental Allergy, 2010 (40) 85–93.     

Reproducibility of leukotriene D4 inhalation challenge in asthmatics

We have studied the reproducibility of a bronchial leukotriene (LT) provocation test in asthmatics, and the effect of prior treatment with an oral leukotriene D4/E4 antagonist (SR 2640) on LTD4-induced bronchoconstriction in nine asthmatics in a double-blind placebo-controlled randomized cross-over trial. The reproducibility of the bronchial leukotriene provocation test was high. For a specific patient, the replication variance is 0.2303, and the standard deviation is thus 0.4799, corresponding to 48%, i.e. one halving of the dose or half doubling of the dose. SR 2640 antagonised LTD4 induced bronchoconstriction causing a mean shift of 48% to the right of the dose-response curve as compared with placebo (95% confidence interval being 11-137%). This study demonstrates that bronchial LTD4 provocation test is a safe and reproducible method in asthmatics, and that the method can be used to detect LT-antagonism; furthermore that SR 2640 is a weak LTD4-antagonist in asthmatics.     

Leukotriene B4 in atopic dermatitis: increased skin levels and altered sensitivity of peripheral blood T-cells

Employing a radioimmunoassay, de-proteinated suction blister fluid from 12 patients with active atopic dermatitis appeared to contain higher levels of the pro-inflammatory and immunomodulatory mediator leukotriene B4 (LTB4) than suction blister fluid from 12 non-atopic individuals. Indirect support for the identity of the mediator was obtained by HPLC of pooled samples. Nylon wool enriched T cells from six patients with atopic dermatitis and six non-atopic people preincubated with LTB4 (10(-10) M - 10(-8) M) expressed no statistically significant suppression in co-culture with mitogen stimulated autologous mononuclear cells, and there was no difference between atopic and non-atopic T cells in this respect. In contrast, LTB4 induced a dose-dependent reduction in the percentage of phenotypic Leu 2a (suppressor) cells leading to an increased helper/suppressor ratio in five atopic patients that was not observed in five non-atopics. Elevated skin levels of LTB4 may initiate or amplify dermal inflammation, and abnormal T cell response to the mediator may account for the increased helper/suppressor ratio characteristic of patients with atopic dermatitis.     

Human bronchial epithelial cells express an active and inducible biosynthetic pathway for leukotrienes B4 and C4

BACKGROUND: Human bronchial epithelial cells synthesize cyclooxygenase and 15-lipoxygenase products, but the 5-lipoxygenase (5-LO) pathway that generates the leukotriene (LT) family of bronchoconstrictor and pro-inflammatory mediators is thought to be restricted to leucocytes. OBJECTIVE: We hypothesized that human bronchial epithelial cells (HBECs) express a complete and active 5-LO pathway for the synthesis of LTB4 and LTC4, either constitutively or after stimulation. METHODS: Flow cytometry, RT-PCR, Western blotting, enzyme immunoassays and reverse-phase high-performance liquid chromatography were used to investigate constitutive and stimulated expression of 5-LO pathway enzymes and the synthesis of LTs B4 and C4 in primary HBECs and in the 16-HBE 14o- cell line. RESULTS: Constitutive mRNA and protein expression for 5-LO, 5-LO-activating protein (FLAP), LTA4 hydrolase and LTC4 synthase were demonstrated in primary HBECs and in the 16-HBE 14o- cell line. In 16-HBE 14o- cells, treatment with calcium ionophore A23187, bradykinin or LPS up-regulated the expression of these enzymes. The up-regulation of 5-LO was blocked by the anti-inflammatory glucocorticoid dexamethasone. Human bronchial epithelial cells were shown to generate bioactive LTs, with primary HBECs generating 11-fold more LTC4 and five-fold more LTB4 than 16-HBE 14o- cells. LT production was enhanced by ionophore treatment and blocked by the FLAP inhibitor MK-886. CONCLUSIONS: Expression of an active and inducible 5-LO pathway in HBEC suggests that damaged or inflamed bronchial epithelium may synthesize LTs that contribute directly to bronchoconstriction and leucocytosis in airway inflammation.     

Urinary leukotriene E4 in Henoch–Schonlein purpura

BACKGROUND: Children with Henoch-Schonlein purpura (HSP) occasionally have allergic disease. We have previously shown that pranlukast hydrate was effective for purpura in HSP. Pranlukast hydrate is a leukotriene (LT) receptor antagonist; therefore, it is likely that LTs take part in the cause of HSP. Urinary leukotriene E4 (LTE4u) levels are a useful index of whole-body cysteinyl LT production in vivo. In this study, LTE4u was examined in children with HSP. OBJECTIVE: The purpose of this study was to examine the relation between the level of LTE4u and the cause of HSP. METHODS: Eighteen HSP children (six boys and 12 girls) and six healthy children were enrolled. RESULTS: LTE4u levels in patients with HSP were significantly higher (P< 0.05) at the onset than those in healthy children. Four weeks therapy with pranlukast hydrate lowers LTE4u levels in patients with HSP (P< 0.05). There were no differences in LTE4u between the group of HSP patients with purpura nephritis and the group of HSP patients without purpura nephritis. CONCLUSION: These results indicate that csyteinyl LTs may play a role in the pathophysiology of purpura in HSP.     

Urinary LTE4 excretion in antigen-provoked asthmatic patients treated with the inhaled LTD4 antagonist, L-648,051

Leukotriene (LT) E4 represents the major LT metabolite in man, and its urinary excretion can be used as an indirect marker of systemic LTC4 and/or LTD4 synthesis and release. In the present study LTE4 excretion was monitored for 24 h in 12 atopic patients with mild asthma undergoing antigen bronchoprovocation as part of a double-blind, placebo-controlled, two-period cross-over study of the aerosol-delivered LTD4 antagonist, L-648,051. Urinary LTE4 excretion was also studied separately in six of the patients after inhaling only diluent. Urine was sampled before, and serially after antigen challenge, at intervals corresponding to the immediate (0-3 h postchallenge) and late (3-6, 6-12, 12-24 h postchallenge) asthmatic reactions. LTE4 was determined by reversed-phase HPLC and radioimmunoassay. Forced expiratory volume in 1 s (FEV1) was recorded serially through 8 h after inhalation of antigen and diluent. Compared to base-line measurements, antigen bronchoprovocation induced significant increases in mean LTE4 excretion rates 0-3 h postchallenge (i.e. during the immediate asthmatic response) after treatment with both placebo (P < 0.01) and L-648,051 (P < 0.05). These mean LTE4 excretion rates in the immediate phase were also significantly higher than the mean rates in the late phase (3-6 h and beyond); the excretion rates of LTE4 at these later time intervals were similar to base-line values. After inhalation of diluent, the LTE4 excretion rates in the intervals 0-3, 3-6, 6-12 and 12-24 h were unchanged from base-line values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urinary leukotriene E4 and 9α, 11β-prostaglandin F2 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.     

Leukotriene B4 production by blood neutrophils in allergic rhinitis—effects of cetirizine

Background: Mucosal inflammatory processes in late phase of allergic diseases involve cytokine production, cell adhesion molecule overexpression and release of inflammatory mediators with chemotactic activity, such as leukolriene B₄ (LTB₄), We had previously observed increased production of LTB₄ by neutrophils in patients with allergic rhinitis and discussed the role of granulocyte macrophage-colony stimulating factor (GM-CSF) priming. Some antihislaminic compounds were shown to diminish the production of leukotrienes by neutrophils. Objective: In a first step, we evaluated in ex vivo and in vitro studies, the effects of cetirizine on LTB₄ production by blood neutrophils from allergic and healthy subjects. In a second step, we studied the in vitro effect of cetirizine on LTB₄ production by neutrophils from healthy subjects during GM-CSF priming of these cells. Methods: Neutrophils from both populations were purified from venous blood and LTB₄ production was measured using high performance liquid cromatography (HPLC) method. Results: In ex vivo studies, cetirizine treatment induced a decreased LTB₄ production by neutrophils in allergic rhinitis. This effect of decreased LTB₄ production was reproduced in vitro with 10^?8–10^-6 cetirizine. Nevertheless, this anti-Hl compound had no effect on neutrophil priming with GM-CSF. Conclusion: As LTB₄ is an important chemotactic factor, Cetirizine could act on inflammatory cell recruitment by inhibiting LTB₄ production by neutrophils.     

Uncoupled regulation of leukotriene C4 synthase in platelets from aspirin-intolerant asthmatics and healthy volunteers after aspirin treatment

BACKGROUND: We have reported that thromboxane A2 induces suppression of leukotriene (LT) C4 synthase activity in human platelets. AIM: In the present study, we describe a mechanism whereby aspirin treatment can lead to increased formation of LTC4, which is a potent bronchoconstrictor and inflammatory mediator. This mechanism is also demonstrated to be present in platelets from aspirin-intolerant asthmatics (AIA). METHODS: The effect of arachidonic acid or platelet agonists on LTC4 synthase activity was investigated in platelets obtained from healthy volunteers, aspirin-intolerant asthmatics or aspirin-tolerant asthmatics after in vivo treatment or in vitro pre-incubation with aspirin. RESULTS: Incubation of normal platelets with arachidonic acid or collagen provoked approximately 50% reduction of platelet LTC4 synthase activity, as determined by the conversion of LTA4 to LTC4. However, the inhibitory effect of arachidonic acid or collagen was not observed after oral administration of aspirin prior to collection of the platelets. Arachidonic acid-induced inhibition of LTC4 synthase activity was totally abolished in platelets collected from peripheral blood already 30 min after aspirin ingestion but was fully restored in platelets collected 3 to 7 days after the administration of aspirin. Treatment of platelet suspensions with aspirin in vitro dose-dependently counteracted the suppressive effect of arachidonic acid on LTC4 formation, with total reversal at approximately 40 microm. In contrast, the major aspirin metabolite, salicylic acid did not alter arachidonic acid-induced reduction of LTC4 synthase activity. Similarly, LTC4 synthase activity in platelets from AIA and aspirin-tolerant asthmatics (ATA) was reduced by approximately 50% after pre-treatment with arachidonic acid in vitro. Again the inhibitory effect was abolished when platelets were pre-incubated in the presence of aspirin. CONCLUSION: The results indicate that oral aspirin administration can lead to uncoupling of thromboxane A2-dependent negative feedback mechanisms, which may normally restrict the production of cysteinyl leukotrienes. This mechanism can be of potential interest in aspirin-induced asthma.     

Leukotriene B4 enhances tumour necrosis factor-α-induced CCL27 production in human keratinocytes

BACKGROUND: A chemokine CCL27 recruits skin-homing T cells. CCL27 production by epidermal keratinocytes is dependent on nuclear factor-kappaB (NF-kappaB) activity and is enhanced in lesions with atopic dermatitis or allergic contact dermatitis. A lipid mediator leukotriene B(4) (LTB(4)) may be involved in the development of these allergic dermatoses. LTB(4) acts on cell surface G-protein-coupled receptors, BLT1 and BLT2. OBJECTIVE: The aim of this study was to investigate the in vitro effects of LTB(4) on CCL27 production in human keratinocytes. METHODS: Keratinocytes were incubated with TNF-alpha and LTB(4). CCL27 secretion and mRNA levels were analysed by ELISA and RT-PCR, respectively. NF-kappaB activities were analysed by luciferase assays. Protein levels or phosphorylation status were analysed by cell-based ELISA. RESULTS: LTB(4) alone did not enhance CCL27 production and modestly enhanced NF-kappaB activity in human keratinocytes. However, LTB(4) potently enhanced TNF-alpha-induced CCL27 secretion and mRNA expression and NF-kappaB activity. LTB(4) alone or together with TNF-alpha, induced phosphorylation and degradation of inhibitory NF-kappaB alpha (IkappaBalpha) and phosphorylation of NF-kappaB p65. These effects of LTB(4) were suppressed by BLT1 antagonist U75302, pertussis toxin, phosphoinositide-3 kinase (PI3K) inhibitor LY294002 and extracellular signal-regulated kinase (ERK) kinase inhibitor U0126, but not by BLT2 antagonist LY255283. LTB(4) induced phosphorylation of ERK and Akt, downstream kinase of PI3K; LY294002 suppressed phosphorylation of both kinases while U0126 suppressed only the former. CONCLUSION: These results suggest that LTB(4) may enhance TNF-alpha-induced CCL27 production by activating NF-kappaB via the BLT1/G(i/o)/PI3K/ERK pathway in human keratinocytes. LTB(4) may contribute to the enhanced CCL27 production of keratinocytes in lesions with atopic dermatitis or allergic contact dermatitis.