Induced sputum eicosanoid concentrations in asthma

Further definition of the role of leukotrienes (LT) and prostaglandins (PG) in asthma would be helped by a noninvasive method for assessing airway production. The supernatant from sputum induced with hypertonic saline and dispersed using dithiotrietol has been successfully used to measure other molecular markers of airway inflammation and might be a useful method. We have measured induced sputum supernatant LTC(4)/D(4)/E(4) concentrations using enzyme immunoassay and PGE(2), PGD(2), TXB(2), and PGF(2alpha) using gas chromatography-negative ion chemical ionization-mass spectroscopy in 10 normal subjects and in 26 subjects with asthma of variable severity. Sputum cysteinyl-leukotrienes concentrations were significantly greater in subjects with asthma (median, 9.5 ng/ml) than in normal control subjects (6.4 ng/ml; p < 0.02) and greater in subjects with persistent asthma requiring inhaled corticosteroids (median, 11.4 ng/ml) or studied within 48 h of an acute severe exacerbation of asthma (13 ng/ml) than in subjects with episodic asthma treated with inhaled beta(2)-agonists only (7.2 ng/ml). There were no significant differences in the concentrations of other eicosanoids between groups, although there was a negative correlation between the percentage sputum eosinophil count and sputum PGE(2) concentration (r = -0.48; p < 0.01) in subjects with asthma. We conclude that induced sputum contains high concentrations of eicosanoids and that sputum LTC(4)/D(4)/E(4) concentrations are significantly greater in subjects with asthma than in normal subjects. The inverse relationship between eosinophilic airway inflammation and sputum PGE(2) concentration would be consistent, with the latter having an anti-inflammatory role.     

Phospholipases,eicosanoid production and inflammation

Conclusions The release of AA from its phospholipids pool, which is a basic initial stage in the biosynthesis of eicosanoids, is a complex mechanism. To understand it is very important for a better comprehension of the inflammatory process and its treatment. In the near future drugs such as synthetic macrocortin, lipomodulin or others which could act on these very early stages of inflammation might be produced by the drug industry for the benefit of alle rheumatic patients.     

Vascular responsiveness and eicosanoid production in diabetic rats

Vascular responsiveness to vasoactive eicosanoids as well as vascular prostacyclin and thromboxane production was investigated in 7-10 weeks alloxan-diabetic rats. Aortic rings from diabetic rats exhibited increased responsiveness to carbocyclic thromboxane A2, a thromboxane analogue, when compared to control rat aortae. Isolated perfused hearts of diabetic rats showed increased vascular responsiveness to 9,11-methanoepoxy PGH2 (U-46619), an endoperoxide analogue. Diabetes resulted in a reduction in prostacyclin generation by isolated incubated aortae which was overcome by the addition of arachidonic acid but not by homogenization of incubated aortic tissue. In contrast, prostacyclin, but not thromboxane, generation was elevated in isolated perfused hearts of diabetic animals in response to moderate doses of arachidonic acid, but at high doses of arachidonate, more thromboxane was formed by perfused hearts of diabetic rats. These results suggest that different vessels can either increase or decrease their prostaglandin production in response to diabetes. The alterations in prostanoid production may be due to differential changes in prostacyclin and thromboxane synthesis in vessels which, in turn, may be related to the changes in vascular responsiveness.     

Candida albicans stimulates endothelial cell eicosanoid production

The response of human umbilical vein endothelial cells to invasion by Candida species was examined in vitro. Live Candida albicans caused significant endothelial release of eicosanoids, mainly prostaglandins. Since prostaglandin I2 (PGI2) is an important prostaglandin produced by endothelial cells, factors influencing its release were studied. The ability of different strains and species of Candida to induce endothelial PGI2 release was closely related to their capacity to injure the endothelium (r = .99). C. albicans was the only species tested that either stimulated PGI2 release or damaged the endothelial cells; only this organism possessed detectable phospholipase activity. Candida glabrata and Candida tropicalis had no phospholipase activity and neither increased PGI2 release nor caused significant endothelial damage. Close proximity with germinated C. albicans was required for endothelial injury and PGI2 release. The ability of C. albicans to stimulate endothelial cells may have important implications in regulating neutrophil activities against organisms that interact with endothelial cells.     

Regulation of eicosanoid production in rabbit colon by interleukin-1

Prostaglandins and thromboxanes are increased in human and experimental colitis, but the factors that regulate this enhanced production are unclear. The present studies evaluate the effects of the monokines, interleukin-1 alpha and beta on eicosanoid production in rabbit colon. In tissue incubations the peak dose response of eicosanoid release to human recombinant interleukin-1 is 50 ng/ml. Interleukin-1 alpha increases prostaglandin E2 (PGE2) by 4.5 +/- 1.9 ng/g tissue, 6-keto PGF1 alpha by 6.2 +/- 2.7 ng/g, and thromboxane B2 by 2.1 +/- 0.3 ng/g compared to placebo. In isolated rabbit colons perfused with Krebs' solution, 10-h infusion of interleukin-1 alpha (50 ng/ml) progressively increases production of PGE2, 6-keto PGF1 alpha, and thromboxane B2. Bolus injections of bradykinin increase production of PGE2, but not 6-keto PGF1 alpha and thromboxane B2, and these responses are markedly augmented by interleukin-1 alpha: at 10 h bradykinin-stimulated PGE2 production is 518 +/- 104 vs. 95 +/- 18 ng/5 min (p less than 0.005), 6-keto PGF1 alpha is 172 +/- 88 vs. 8 +/- 2 ng/5 min (p less than 0.02), and thromboxane B2 is 60 +/- 14 vs. 13 +/- 4 ng/5 min (p less than 0.02) for interleukin-treated colons vs. placebo-treated colons, respectively. The response is greater with interleukin-1 alpha than interleukin-1 beta. This study demonstrates that interleukin-1 stimulates prostaglandin and thromboxane production in normal colon tissue. These data are consistent with the concept that interleukin-1 production by inflammatory cells may augment prostaglandin and thromboxane production in colitis.     

上皮源性细胞因子与支气管哮喘

支气管哮喘(简称哮喘)是全球最常见的慢性气道炎症性疾病之一.慢性气道炎症是哮喘的内在发病机制,炎症细胞在气道的局部聚集、炎症介质和细胞因子的释放促使气道炎症发生.上皮源性细胞因子是免疫活性细胞的效应因子,其免疫调节功能在哮喘发病机制中处于中心地位.在人类肺组织及免疫细胞中,细胞因子的表达增高,从而导致肺部变态反应性疾病的发生.近年来利用哮喘发病机制中细胞因子作为靶点进行靶向治疗哮喘已成为未来哮喘治疗研究的主要热点.本文主要就上皮源性细胞因子中IL-25、IL-33及胸腺基质淋巴细胞生成素的结构和功能进行介绍,并对其与哮喘的关系作一简单综述.     

Platelet-activating factor stimulates eicosanoid production in cultured feline tracheal explants.

Eicosanoids have been shown to be major mediators of airway inflammation. Platelet-activating factor (PAF), a potent bronchoconstrictor and stimulator of respiratory mucous secretion, may mediate some of its effects via eicosanoid production. To explore eicosanoid generation by cultured feline tracheal explants, eicosanoids were measured following PAF stimulation. After labeling the explants with [3H]arachidonic acid, supernatant from control and PAF treated explants was fractionated by reverse phase high-performance liquid chromatography (HPLC). The resulting elution pattern suggested the release of arachidonic acid (AA), 15-hydroxyeicosatetraenoic acid (HETE), leukotriene (LT)B4, C4, prostaglandin (PG) D2/E2/F2 alpha, and 6-keto-PGF1 alpha. Radioimmunoassay (RIA) following HPLC resolution confirmed that PAF induced a significantly increased release of peptido-leukotrienes, PGD2, PGE2, PGF2 alpha, LTB4, and 5-HETE, as well as thromboxane (TX) B2. The most remarkable increase was LTC4/D4/E4 (15 x control) and PGD2 (4 x control). The PAF antagonist Ro 19-3704 had an inhibitory effect on the PAF-stimulated release of peptido-leukotrienes. We conclude that PAF stimulates the production of a variety of lipoxygenase and cyclooxygenase pathway metabolites in feline airways.     

Platelet-activating factor stimulates eicosanoid production in cultured feline tracheal epithelial cells

The effect of platelet-activating factor (PAF) on eicosanoid generation and release in cultured feline tracheal epithelial cells was investigated by measuring a wide range of lipoxygenase and cyclooxygenase pathway products. Subconfluent epithelial cell cultures were stimulated by PAF and eicosanoid production was determined by high performance liquid chromatography (HPLC) of [³H]-labeled arachidonic acid (AA) metabolites and by radioimmunoassay (RIA) following HPLC separation. The HPLC chromatograms revealed that PAF augmented the release of prostaglandin (PG)E₂, PGF_2α, 12-hydroxyeicosatetraenoic acid (HETE), and AA. Among these eicosanoids, PGE₂ predominated under baseline conditions and following PAF exposure. RIAs of the nonradiolabeled HPLC elution corresponding to various eicosanoid standards demonstrated that PAF increased the production of 6-keto-PGF_1α, thromboxane B₂ (TXB₂), PGD₂, 5-HETE, and 15-HETE, as well as PGE₂, PGF_2α, and 12-HETE. The PAF-induced eicosanoid augmentation was dose-dependent and occurred within 1 hour with a prompt decline following termination of PAF exposure. This stimulating effect of PAF on eicosanoid release was blocked by two PAF receptor antagonists, Ro 19-3704 and WEB 2086. The PAF-induced increase in eicosanoid release was similar in magnitude to the increase caused by calcium ionophore (Ca-ionophore) A23187, a potent known stimulus for eicosanoid release. Cells of different culture durations (3 and 6 days) showed similar capacity for eicosanoid production. We conclude that PAF stimulates the production of cyclooxygenase and lipoxygenase pathway products from airway epithelial cells via PAF receptors, and that these epithelium-derived eicosanoids may be responsible for some of the PAF-induced respiratory physiological and pathophysiological effects.     

Leukocyte and platelet function and eicosanoid production in subjects with hypercholesterolaemia

A group of 22 subjects with type IIA hypercholesterolaemia (mean serum cholesterol = 8.3 +/- 0.3 mmol/l) were sex, age and weight matched with 22 control subjects (mean serum cholesterol = 4.5 +/- 0.1 mmol/l). Diastolic blood pressure was significantly higher in hypercholesterolaemic subjects (79.2 +/- 1.4 mm Hg) than in control subjects (71.9 +/- 1.4 mm Hg). While the high cholesterol group had 52% greater thromboxane production in clotted whole blood than controls this difference was not significant, and the platelet aggregation and serotonin secretion response to doses of collagen, ADP and arachidonic acid were similar between the 2 groups. Polymorphonuclear leukocyte (PMN) chemiluminescence (used as a measure of reactive oxygen species production) in response to low doses of the chemotactic-peptide FMLP and opsonized zymosan was significantly greater in high cholesterol subjects compared to their matched controls. The production of platelet activating factor (PAF) by calcium ionophore (2.5 micrograms) stimulated PMN isolated from hypercholesterolaemic subjects (11.5 +/- 1.4 ng/10(6) cells) was significantly greater than PAF production by cells from the control group (8.3 +/- 1.0 ng/10(6) cells). Leukotriene B4 release by PMN in response to calcium ionophore did not differ between the 2 groups. These data suggest a degree of leukocyte activation in hypercholesterolaemic subjects compared to controls with normal cholesterol. In addition, plasma levels of lyso-PAF were higher in high cholesterol subjects (317 +/- 21 ng/ml) compared to their matched controls (271 +/- 18 ng/ml) perhaps indicating increased plasma acetylhydrolase activity in subjects with raised cholesterol levels. Recently described biological activity for lyso PAF suggests a possible role for this substance in atherogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endotoxin-induced eicosanoid production by equine vascular endothelial cells and neutrophils

Dispersed equine vascular endothelial cells grown in tissue culture, and freshly isolated neutrophils were used to determine direct effects of endotoxin on cyclooxygenase and lipoxygenase products. Endothelial cells (10(7)/ml) or neutrophils (2 X 10(6)/ml) were incubated with (a) buffer, (b) endotoxin (10 micrograms/ml), (c) endotoxin + flunixin meglumine (10 micrograms/ml), or (d) calcium ionophore, A23187 (10 micrograms/ml). Thromboxane (TxB2), prostacyclin (6-keto-PGF1 alpha), and leukotriene C4 (LTC4) were determined in the incubation fluid by radioimmunoassay. Thromboxane and prostacyclin levels increased in endothelial cells incubated with endotoxin. Treatment with flunixin meglumine prevented the endotoxin-induced release of these cyclooxygenase products to levels below those observed in control cells. Leukotriene production was increased in endothelial cells incubated with endotoxin plus flunixin meglumine. Endotoxin as well as endotoxin plus flunixin meglumine increased the production of prostacyclin and LTC4 by freshly isolated neutrophils. Cells exposed to endotoxin plus flunixin meglumine produced more LTC4 than cells exposed to endotoxin. The data revealed that endotoxin has a direct effect on arachidonic acid metabolism in endothelial cells and neutrophils. Flunixin meglumine reduced the level of cyclooxygenase products but increased the level of lipoxygenase products. Therefore, the well-established beneficial effects of cyclooxygenase inhibitors during endotoxemia may be improved even more if they are used in conjunction with lipoxygenase inhibitors or a combined cyclooxygenase-lipoxygenase inhibitor.     

Epithelial cell proliferation contributes to airway remodeling in severe asthma

RATIONALE: Despite long-term therapy with corticosteroids, patients with severe asthma develop irreversible airway obstruction. OBJECTIVES: To evaluate if there are structural and functional differences in the airway epithelium in severe asthma associated with airway remodeling. METHODS: In bronchial biopsies from 21 normal subjects, 11 subjects with chronic bronchitis, 9 subjects with mild asthma, and 31 subjects with severe asthma, we evaluated epithelial cell morphology: epithelial thickness, lamina reticularis (LR) thickness, and epithelial desquamation. Levels of retinoblastoma protein (Rb), Ki67, and Bcl-2 were measured, reflecting cellular proliferation and death. Terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL) was used to study cellular apoptosis. MEASUREMENTS AND MAIN RESULTS: Airway epithelial and LR thickness was greater in subjects with severe asthma compared with those with mild asthma, normal subjects, and diseased control subjects (p=0.009 and 0.033, respectively). There was no significant difference in epithelial desquamation between groups. Active, hypophosphorylated Rb expression was decreased (p=0.002) and Ki67 was increased (p<0.01) in the epithelium of subjects with severe asthma as compared with normal subjects, indicating increased cellular proliferation. Bcl-2 expression was decreased (p<0.001), indicating decreased cell death suppression. There was a greater level of apoptotic activity in the airway biopsy in subjects with severe asthma as compared with the normal subjects using the TUNEL assay (p=0.002), suggesting increased cell death. CONCLUSIONS: In subjects with severe asthma, as compared with subjects with mild asthma, normal subjects, and diseased control subjects, we found novel evidence of increased cellular proliferation in the airway contributing to a thickened epithelium and LR. These changes may contribute to the progressive decline in lung function and airway remodeling in patients with severe asthma.     

Role of gastric mucosal eicosanoid production in the cytoprotection induced by nitecapone.

Nitecapone (3-100 mg/kg orally) dose-dependently (40-97%) decreased the macroscopic gastric lesions induced by ethanol, NaOH, or HCl in the rat. The duration of protection was long, being still 70% at 6 h after dosing. Nitecapone (10-100 mg/kg orally) induced at 1 h after dosing a significant and dose-dependent increase in gastric mucosal prostaglandin E2 release. After the dose of 30 mg/kg the release was sixfold at 2 h and threefold at 12 h. Colloidal bismuth subcitrate (30 mg/kg) stimulated the prostaglandin E2 release only transiently, and sucralfate (400 mg/kg) showed only a tendency to stimulate the release. Indomethacin prevented the nitecapone-induced stimulation of prostaglandin E2 but was unable to counteract the cytoprotective activity of the compound. Nitecapone (30 mg/kg) also caused transient increase (twofold) in the release of 6-keto-prostaglandin F1 alpha and thromboxane B2 and a decrease in both basal and ethanol-induced release of leukotriene C4.     

Epithelial damage and angiogenesis in the airways of children with asthma

RATIONALE: Airway remodeling and inflammation are characteristic features of adult asthma that are still poorly investigated in childhood asthma. OBJECTIVES: To examine epithelial and vascular changes as well as the inflammatory response in airways of children with asthma. METHODS: We analyzed bronchial biopsies obtained from 44 children undergoing bronchoscopy for appropriate clinical indications other than asthma: 17 with mild/moderate asthma (aged 2-15 yr), 12 with atopy without asthma (1-11 yr), and 15 control children without atopy or asthma (1-14 yr). By histochemistry and immunohistochemistry, we quantified epithelial loss, basement membrane thickness, number of vessels, and inflammatory cells in subepithelium. RESULTS: Epithelial loss and basement membrane thickness were increased in children with asthma compared with control subjects (p = 0.005 and p = 0.0002, respectively) and atopic children (p = 0.002 and p = 0.005, respectively). The number of vessels and eosinophils was increased not only in asthmatic children (p = 0.03 and p = 0.0002, respectively) but also in atopic children without asthma (p = 0.03 and p = 0.008, respectively) compared with control subjects. When we stratified the analysis according to age, we observed that children with asthma younger than 6 yr had increased epithelial loss, basement membrane thickening, and eosinophilia compared with control subjects of the same age. CONCLUSIONS: Epithelial damage and basement membrane thickening, which are pathologic features characteristic of adult asthma, are present even in childhood asthma. Other changes, such as airway eosinophilia and angiogenesis, were also observed in atopic children without asthma. These observations suggest that pathologic changes occur early in the natural history of asthma and emphasize the concept that some of these lesions may characterize atopy even in the absence of asthmatic symptoms.     

The regulation of fibrosis in airway remodeling in asthma

Fibrosis is one of the key pathological features of airway remodeling in asthma. In the normal airway the amount of collagen and other extracellular matrix components is kept in equilibrium by regulation of synthesis and degradation. In asthma this homeostasis is disrupted due to genetic and environmental factors. In the airways of patients with the disease there is increased extracellular matrix deposition, particularly in the reticular basement membrane region, lamina propria and submucosa. Fibrosis is important as it can occur early in the pathogenesis of asthma, be associated with severity and resistant to therapy. In this review we will discuss current knowledge of relaxin and other key regulators of fibrosis in the airway including TGFβ, Smad2/3 and matrix metalloproteinases. As fibrosis is not directly targeted or effectively treated by current asthma drugs including corticosteroids, characterization of airway fibrosis and how it is regulated will be essential for the development of novel therapies for asthma.     

Measurement of in vivo rectal mucosal cytokine and eicosanoid production in ulcerative colitis using filter paper

BACKGROUND: Excessive mucosal generation of cytokines and eicosanoids has been reported in vitro in ulcerative colitis (UC) using traumatising biopsy techniques, and in vivo using time consuming rectal dialysis. AIMS: To validate a simple filter paper technique to profile rectal mucosal production of cytokines and eicosanoids in vivo in patients with UC compared with controls. PATIENTS: Forty one patients with UC (21 with active disease) and 16 controls were studied. METHODS: In vitro, recovery of known concentrations of cytokine or mediator applied to filter papers was measured by ELISA following incubation in buffer. In vivo, patients and controls had filter papers apposed to the rectal mucosa briefly through a rigid sigmoidoscope. Filter papers were then incubated prior to assay by ELISA. RESULTS: In vitro validation studies showed that the filter paper technique could be used to measure mucosal release of interleukin-1beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha), thromboxane B(2) (TXB(2)), and prostaglandin E(2) (PGE(2)), but not interferon gamma (IFN-gamma). Mucosal release of IL-1beta, TNF-alpha, TXB(2) and PGE(2) were significantly increased in active UC (p=0.001) and correlated directly with disease activity (p=0.02). CONCLUSIONS: The filter paper technique confirmed increased rectal mucosal release of cytokines and eicosanoids in UC, in proportion to disease activity. The simplicity, safety and speed of the technique make it a practicable option for use in the outpatient clinic to study the pathogenesis of inflammatory bowel disease, and potentially its response to treatment.