Cysteinyl-containing Leukotrienes Research Articles

Leukotriene D4 receptor antagonist montelukast alleviates protamine sulphate‐induced changes in rat urinary bladder

What's known on the subject? and What does the study add? The mastocytosis in detrusor muscle and the leaky epithelium in interstitial cystitis were the most studied features. In this study the leaky epithelium was shown using the ruthenium red staining in electron microscopy and uroplakin distribution in light microscopy besides the mast cell concentration in detrusor muscle using tryptase immunohistochemistry. • To study the effects of montelukast (ML), a leukotriene receptor antagonist which has been shown to be effective in inhibiting the action of cysteinyl-containing leukotrienes, on protamine sulphate (PS)-induced changes in rat urinary bladder. • Wistar female rats were catheterized and intravesically infused with PBS (control group) or PS (PS group) dissolved in PBS twice in 24 h. • In the PS-applied and ML-treated group (PS + ML group) after the 10 mg/kg PS instillation, ML was injected i.p. twice daily for 3 days. • The urinary bladder was investigated for general morphology under a light microscope. • Tryptase immunohistochemistry was used to observe mast cell distribution and activation. Uroplakin distribution was also identified with immunohistochemistry. • Alterations of glycosaminoglycan (GAG) and urothelial permeability were seen with ruthenium red (RR) staining techniques under a transmission electron microscope, and topographical changes of luminal urothelial structure were seen with a scanning electron microscope. • Biochemically malondialdehyde (MDA) and gluthatione (GSH) concentrations were analysed. In the PS group, there was degenerated urothelium with irregular uroplakin distribution, increased inflammatory cell infiltration, increased number of both granulated and activated mast cells, irregularity of GAG and penetration of RR into the intercellular spaces and dilated tight junctions. • In PS + ML group, there was relatively regular uroplakin distribution, a decrease in inflammatory cell infiltration, a decreased number of both activated and granulated mast cells in the mucosa, regular GAG and no penetration of RR into the intercellular areas, and regular tight junctions in most regions. • The significant decrease in MDA and the increased GSH concentrations in the PS + ML group was in accordance with the histological findings. • Montelukast appears to have a protective function in the bladder injury model via the anti-inflammatory effects of this leukotriene receptor antagonist.

The ACh-induced contraction in rat aortas is mediated by the Cys Lt1 receptor via intracellular calcium mobilization in smooth muscle cells.

1. Our previously published data indicate that an endogenously produced 5-lipoxygenase metabolite can strongly contract isolated endothelium-preserved rat aortic strips when cyclo-oxygenase isoenzymes are inhibited. Therefore, we decided to investigate if cysteinyl-containing leukotrienes (Cys Lts) are involved in this endothelium-dependent contraction. 2. The isometric contraction of endothelium-preserved rat aortic strips was recorded in preparations preincubated with 5 microM indomethacin and precontracted with phenylephrine, adjusting resting tension at 0.7 g. Acetylcholine (ACh) contracted control strips. Montelukast and MK-571, selective type 1 Cys Lts receptor (Cys Lt(1)) antagonists and the Cys Lt(1)/Cys Lt(2) (type 2 Cys Lts receptor) antagonist BAYu9773 dose-dependently prevented ACh-induced contraction, their IC(50)s being 2.2, 3.1 and 7.9 nM respectively. The leukotriene B4 receptor antagonist U75302 was far less potent (IC(50) 1.5 microM). 3. In rat aorta smooth muscle cells (RASMs), Western blot analysis showed the presence of Cys Lt(1) and Cys Lt(2) receptors, the Cys Lt(1) receptor being predominantly expressed. 4. In fura-2 loaded RASMs, LTD4 (0.01-100 nM) and LTC4 (200-800 nM) dose-dependently increased intracellular calcium concentration ([Ca(2+)](i)). Montelukast (1-100 nM) reduced LTD4-induced [Ca(2+)](i) increase, its IC(50) being approximately 10 nM. BAY u9773 exhibited significantly low effectiveness. 5. LTD4 (10 nM) induced a redistribution of smooth muscle actin fibres throughout the cytoplasm as visualized by confocal microscopy. 6. In conclusion, Cys Lt(1) activation by endogenously produced Cys Lts, can contract rat aortas, while Cys Lt(2) only marginally influences aortic tone. Intracellularly, this effect is mediated by an increase in [Ca(2+)](i). Therefore, Cys Lts, by inducing vascular contraction, can contribute to systemic hypertension.

Treatment of interstitial cystitis with montelukast, a leukotriene D 4 receptor antagonist

Cysteinyl-containing leukotrienes (CysLTs) are lipid mediators known to possess potent proinflammatory action. We have recently shown the presence of CysLT1 receptors in primary cultures of human detrusor muscle cells. In this pilot study we investigated whether the CysLT1 receptor antagonist montelukast (Singulair ), currently used to treat asthma, had any effect as a new therapy in the treatment of interstitial cystitis (IC). Ten women with diagnosed IC according to National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) criteria and detrusor mastocytosis (with a minimum of 28 mast cells/mm2 muscle tissue) were included in the study. The patients were treated with 10 mg montelukast daily for 2 months. The efficacy of the treatment was determined by the following parameters: 24-hour urinary frequency, pain using visual analog scales (VAS), and use of analgesics. After 2 months of treatment, 24-hour urinary frequency decreased significantly from 17.38 to 12.58 times (P = 0.005) and pain from 46.8 mm to 23.36 mm (P = 0.01) according to VAS. We observed no side effects of montelukast during the study. Only 3 patients used analgesics and there was no change in their use during the study. We conclude that treatment of IC patients with montelukast resulted in a significant improvement of urinary frequency and pain. Additional placebo controlled clinical studies will eventually lead to a better understanding of the pathological and physiological role of CysLTs and the role of CysLT1 receptor antagonists in the treatment of IC.

Increased urinary excretion of leukotriene E 4 in patients with interstitial cystitis

The cysteinyl-containing leukotrienes (CysLTs) have been implicated in the pathology of inflammatory disorders, in particular asthma. CysLTs role in patients with interstitial cystitis (IC) is unknown. The aim of the present study was to measure the concentration of leukotriene E4 (LTE4) in patients with IC and healthy subjects.

Evaluation of the pharmacological activity of the pure cysteinyl-leukotriene receptor antagonists CGP 45715A (iralukast) and CGP 57698 in human airways.

Cysteinyl-containing leukotrienes (cysteinyl-LTs) produce bronchoconstriction, mucus hypersecretion, and pro-inflammatory effects (Dahlén et al., 1980; Dahlén et al., 1981; Foster &Chan, 1991) that may contribute to airway hyper-responsiveness, mucus plug formation, epithelial cell damage and other changes in airway morphology. The pivotal role of cysteinyl-LTs in the pathogenesis of asthma has stimulated many pharmaceutical companies to develop programs aimed at the discovery of cysteinyl-LT antagonists, either based on LTD4 structure or unrelated to it, to provide therapeutic agents for use in asthma. Iralukast and CGP 57698 are two recently developed cysteinyl-LT antagonists that have been shown to be potently active in various animal models, both in vitro and in vivo (Bray et al., 1990; von Sprecher et al., 1991; von Sprecher et al., 1996).KeywordsMucus HypersecretionHuman BronchusCysLT ReceptorEpithelial Cell DamageBronchial ToneThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Altered biosynthesis of leukotrienes and lipoxins and host defense disorders in patients with cirrhosis and ascites

Background & Aims: Advanced cirrhosis is associated with impaired leukocyte function, but the mechanism underlying this host defense alteration is unknown. The aim of this study was to investigate the lipoxygenase pathway of arachidonic acid metabolism and its influence in leukocyte trafficking in patients with cirrhosis and ascites. Methods: Neutrophils (polymorphonuclear leukocytes [PMN]) were isolated from patients with cirrhosis and ascites and healthy subjects, and 5-lipoxygenase (5-LO) messenger RNA levels and 5-LO–derived products were measured. The effect of leukotrienes (LT) and lipoxins (LX) on PMN adhesion and migration was also assessed. Results: PMN from patients with cirrhosis showed increased 5-LO messenger RNA expression. However, in vitro generation of LTB 4, cysteinyl-containing LT and LX was significantly decreased in cirrhotic patients. Interestingly, a close relationship between the activity of the renin-angiotensin system and LXA 4 biosynthesis was observed both in vitro and in vivo. PMN isolated from cirrhotic patients with ascites showed significantly decreased adhesion and migration in response to LTB 4. LXA 4 did not provoke PMN adhesion and migration, but rather abrogated the differences between control and cirrhotic PMN. Cirrhotic monocytes showed marked impairment in adherence to laminin when stimulated with either LTB 4 or LXA 4. Conclusions: These results show the existence of altered biosynthesis of LT and LX and defective response to these lipoxygenase products in leukocytes from patients with cirrhosis and ascites. This abnormality may be relevant to the pathogenesis of host defense disorders in chronic liver disease. GASTROENTEROLOGY 1998;115:147-156

Allergen-induced late-phase airways obstruction in the pig: mediator release and eosinophil recruitment.

The aim of this study was to develop a novel model for studies of mediator mechanisms involved in the late asthmatic reaction in the lower airways, by using the sensitized pig. The release of histamine and cysteinyl-containing leukotrienes (cys-LTs), as well as the levels of inflammatory cells in blood and bronchoalveolar lavage fluid, were determined and their relationship to plasma cortisol levels and pulmonary airways obstruction was noted. Specific-pathogen free pigs were actively sensitized with Ascaris suum allergen, and one group of animals was treated with a cortisol-synthesis inhibitor (metyrapone) by constant intravenous infusion. Ascaris suum allergen was nebulized into the lower airways and total lung resistance, blood leucocyte count and urinary levels of methylhistamine and leukotriene E4 (LTE4) were followed for 8 h, whereafter bronchoalveolar lavage was performed for analysis of leucocytes. An increase in urinary methylhistamine and LTE4 was seen during the acute allergic reaction in both groups of pigs. Metyrapone treatment prolonged the acute release of histamine, and this was seen together with a prolonged acute bronchoconstrictor response. In metyrapone-treated pigs, a continuous release over 8 h was seen for cys-LTs, but not for histamine. A late blood eosinophilia was also seen in metyrapone-treated animals, starting 4-6 h after allergen challenge. Late cys-LT release and eosinophilia were absent in non-metyrapone-treated animals. These results suggest that allergen-induced late release of cys-LTs as well as blood eosinophilia occur simultaneously with late-phase airways obstruction in the pig, and that all these reactions are prevented by high levels of endogenous cortisol.

Taurocholate-stimulated leukotriene C 4 biosynthesis and leukotriene C 4-stimulated choleresis in isolated rat liver

Background/Aims: Cysteinyl-containing leukotrienes seem to exert a cholestatic effect. However, leukotriene inhibitors were found to reduce bile salt efflux in isolated rat hepatocytes, suggesting a role for leukotrienes in bile flow formation. Methods: In the isolated rat liver, the effects of two different concentrations of leukotriene C 4 on bile flow and bile salt excretion are analyzed, as well as the possible effect of taurocholate on the hepatic production of cysteinyl-containing leukotrienes. Results: Leukotriene C 4 (0.25 fmol) increased bile salt excretion (+22.2%; P < 0.05), whereas a much higher dose (0.25 × 10 6 fmol) showed the known cholestatic effect, reducing bile salt excretion (−25.9%; P < 0.01). These dose-dependent biphasic effects were specific because they could be prevented by the simultaneous administration of cysteinyl-containing leukotriene antagonists. On the other hand, taurocholate administration induced a dose-dependent increase in biliary excretion of cysteinyl-containing leukotrienes. Furthermore, taurocholate increased messenger RNA levels of 5-lipoxygenase, a key enzyme in leukotriene biosynthesis. Taurocholate increase of hepatocyte intracellular calcium was not significant, suggesting that taurocholate effects are not mediated by stimulation of calcium metabolism. Conclusions: These results constitute evidence for the existence of a positive feedback mechanism by which bile salts stimulate the synthesis of leukotrienes that, in turn, stimulate bile salt excretion.

Effects of oxygen radicals on cysteinyl leukotriene metabolism and pulmonary circulation in young pigs.

The effects of oxygen radicals, generated by the hypoxanthine-xanthine oxidase (XO) system, on pulmonary circulation and release of cysteinyl-containing leukotrienes (LTs) were studied in pigs after XO infusion into the right atrium. A 2.3-fold increase in pulmonary vascular resistance (PVR) (p < 0.05 vs. baseline) and a 2.1-fold increase in LT release (p < 0.05 vs. baseline) was observed. Pretreatment with indomethacin and allopurinol attenauted the vascular response (p < 0.01 and p < 0.05 vs. XO), and the LT release was inhibited by allopurinol and catalase (p < 0.01 and p < 0.02 vs. XO). We conclude that oxygen radicals stimulate lipoxygenase metabolism. This coincides with the observed increase in PVR, however, no causal relationship can be derived from the data presented.

Somatostatin effectively prevents ethanol- and NSAID-induced gastric mucosal damage in rats.

The interrelationship between somatostatin and its synthetic analog, sandostatin, with neuropeptides and inflammatory mediators, as well as their protection of gastric mucosal damage, were tested in rats. Rats were treated intragastrically with 1.0 ml of 96% ethanol with or without intravenous or intraperitoneal coadministration of somatostatin (1.0 microM/kg). Mucosal damage was also induced by the administration of either indomethacin (30 mg/kg subcutaneously) with or without intravenous sandostatin (10 micrograms/rat), given 30 min prior to damage induction. Somatostatin levels in ethanol-damaged gastric mucosa were significantly lower than in control rats. Substance P and vasoactive intestinal peptide (VIP) levels were significantly higher in the damaged mucosa in rats treated with ethanol, as was the mucosal generation of leukotriene B4 (LTB4) and cysteinyl-containing leukotrienes. The coadministration of somatostatin with ethanol significantly reduced gastric mucosal injury induced by ethanol alone. The protection of the mucosa was accompanied by reduction of mucosal substance P and VIP levels, as well as the generation of leukotrienes, an effect that was reversed by intraperitoneal or intravenous coadministration of somatostatin antagonist, cyclo-(7-aminoheptanoyl-PH-E-D-Trp-Lys-THR), 1.0 microM/100 g, with somatostatin (1.0 microM/kg) and ethanol. When given by itself somatostatin significantly reduced mucosal leukotriene generation compared with their generation in saline-treated rats. Sandostatin completely abolished gastric mucosal damage induced by indomethacin administration. In rats treated with somatostatin and indomethacin, this effect was accompanied by reduction of mucosal leukotriene generation. Administration of sandostatin to pylorus-ligated rats significantly reduced gastric acid output.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary excretion of leukotriene E4 and 11-dehydro-thromboxane B2 in response to bronchial provocations with allergen, aspirin, leukotriene D4, and histamine in asthmatics.

In vivo production of thromboxane (TX) A2 and the cysteinyl-containing leukotrienes (LT) C4, D4, and E4 in correlation to airway responses was studied. Bronchial provocation with specific allergen in atopic asthmatics was followed by a significant increase in urinary concentration of immunoreactive LTE4 (34 +/- 6 before versus 56 +/- 7 ng/mmol creatinine after allergen challenge; n = 5) and 11-dehydro-TXB2 (164 +/- 29 versus 238 +/- 25 ng/mmol creatinine). In the presence of the leukotriene-antagonist ICI-204,219, which significantly increased the PD20 for allergen, the increment in urinary excretion of LTE4 was even higher (60 +/- 8 versus 288 +/- 128 ng/mmol creatinine; n = 5). In contrast, provocation with histamine (n = 5) did not provoke release of leukotrienes or thromboxane, nor was inhalation of LTD4 (n = 7) associated with increased urinary concentration of 11-dehydro-TXB2. Furthermore, bronchoconstriction induced by inhalation of lysine-aspirin in aspirin-sensitive asthmatics (n = 4) was followed by increased levels of LTE4 in the urine, whereas the levels of 11-dehydro-TXB2 remained the same. Finally, the basal levels of LTE4 in the urine of nine aspirin-sensitive asthmatics were elevated as compared with 15 other asthmatics (112 +/- 54 versus 38 +/- 20 ng/mmol creatinine; p less than 0.001). The findings support that the cysteinyl-leukotrienes are potential mediators of allergen-induced asthma and that the release of LTE4 and 11-dehydro-TXB2 into the urine appeared to be a direct and dose-dependent effect of the antigen-antibody reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

On the mechanism of induction of DNA synthesis in cultured arterial smooth muscle cells by leukotrienes. Possible role of prostaglandin endoperoxide synthase products and platelet-derived growth factor.

The induction of DNA replication in rat aortic smooth muscle cells (SMCs) by leukotrienes (LTs) was studied in order to elucidate the mechanisms of action in further detail. The effect of LTB4 was blocked by the prostaglandin (PG) synthesis inhibitor indomethacin and the effects of LTC4 and LTD4 were blocked by the cysteinyl-containing leukotriene receptor antagonists FPL 55712 and ICI 198615. These observations suggest that LTB4 and the cysteinyl-containing leukotrienes act via distinct receptors and point to a role for prostaglandin endoperoxide synthase products in bringing about the effect of LTB4. Radioimmunological determinations and analyses of [3H]arachidonic acid metabolism showed that the SMCs were able to synthesize PGI2 (measured as the stable metabolite 6-keto-PGF1 alpha), PGE2, PGF2 alpha and 15(S)hydroxy-eicosatetraenoic acid (15(S)HETE). Moreover, picomolar concentrations of arachidonic acid, PGI2, PGE2, PGF2 alpha and 15(S)HETE induced DNA replication in the SMCs under serum-free conditions, whereas linoleic acid, 6-keto-PGF1 alpha and 5(S)HETE were inactive in this respect. Analysis of conditioned media for mitogenic activity (with or without antibodies against platelet-derived growth factor, PDGF) and for the presence of material competing with radioiodinated PDGF for binding to specific cell surface receptors indicated that LTB4 stimulated release of PDGF or a PDGF-like molecule from the cells. These findings suggest that the growth-promoting effect of LTB4 is mediated via a prostaglandin endoperoxide synthase product and/or PDGF produced by the cells themselves.

Metabolism of granulocyte-derived leukotriene A4 in human platelets and respiratory tissue: transcellular formation of lipoxins and leukotrienes.

Leukotriene (LT)A4, the unstable intracellular intermediate in leukotriene biosynthesis, may be released to the extracellular space by activated leukocytes (1). As a consequence, the metabolism of LTA4 is not restricted to cells with 5-lipoxygenase activity, but can also be exerted by other cell types equipped with LTA4-metabolizing enzymes. Thus, LTA4, released by 5-lipoxygenase expressing cells, may be converted to LTB4 by surrounding erythrocytes (2), endothelial cells (3) or lymphocytes (4), all possessing LTA4 hydrolase activity. Similarly, mast cells (1), endothelial cells (3, 5) and smooth muscle cells (6) have been demonstrated to convert LTA4 to cysteinyl-containing leukotrienes. The present chapter describes some of our recent data regarding the metabolism of synthetic or granulocyte-derived LTA4 in human platelets and respiratory tissue leading to formation of cysteinyl-containing leukotrienes and lipoxins (LX).

Enzymes involved in the biosynthesis of leukotriene B4

Ten years have elapsed since the discovery and structural elucidation of the leukotriene compounds (1-5). The leukotrienes constitute a group of potent biologically active arachidonic acid-derived metabolites which are implicated as mediators of immediate hypersensitivity reactions and inflammation. They were first discovered in leukocytes and were determined to contain a conjugated triene structure, thus the derivation of their name (6). The cysteinyl-containing leukotrienes (LTC,, LTD4, and LTE,),' which together make up the slow reacting substance of anaphylaxis, are synthesized primarily by eosinophils, mast cells, and macrophages. They stimulate contraction of various smooth muscle cells, promote bronchial mucous secretion, and induce extravasation of plasma proteins by increasing microvascular permeability. Leukotriene B, (LTB,), synthesized predominantly by neutrophils, is capable of stimulating circulating leukocytes to adhere to the vascular endothelium and induces extravasation and chemotaxis of polymorphonuclear leukocytes in nanomolar concentrations. At higher concentrations, LTBI induces polymorphonuclear leukocyte degranulation, superoxide generation, and can augment natural killer cell activity. Several reviews on the structure, biosynthesis, and biological actions of the leukotrienes have been written (7-13). It is the aim of the present review to concentrate on the advances made in recent years, primarily through molecular biology studies, on the reaction pathway leading to production of LTB,. 5-Lipoxygenase

Identification of a novel 7- cis-11- trans-lipoxin A 4 generated by human neutrophils: total synthesis, spasmogenic activities and comparison with other geometric isomers of lipoxins A 4 and B 4

Addition of (15 S)-hydroxy-5,8,11- cis-13- trans-eicosatetraenoic acid (15-HETE) and the ionophore A23187 (2.5 μM) to human neutrophils led to the formation of both lipoxin A 4 and lipoxin B 4 as well as a novel 3,6,15-trihydroxyeicosatetraenoic acid. The new compound was identified using an improved isolation and detection system and its basic structure was determined by physical methods. On the basis of biosynthetic considerations, geometric isomers of lipoxin A 4 and lipoxin B 4 were prepared by total synthesis. Comparison of these synthetic materials with the neutrophil-derived product showed that the new compound is (5 S,6 R,15 S)-trihydroxy-9,11,13- trans-7- cis-eicosatetraenoic acid or the 7- cis-11- trans-isomer of LXA 4 (7- cis-11- trans-LXA 4). LXA 4, 11- trans-LXA 4, 7- cis-LXA 4 and 7- cis-11- trans-LXA 4 all evoked dose-dependent (0.1–10 μM) contractions of the guinea pig lung strip, whereas 6- cis-LXB 4 and 6- cis-8- trans-LXB 4 relaxed this preparation. LXA 4 and 7- cis-LXA 4 were approx. 10-times more potent than the compounds with 11- trans geometry. However, all four double-bond isomers of LXA 4 caused contractions which, based upon pharmacological evidence, appeared to involve specific activation of the same site as cysteinyl-containing leukotrienes. In conclusion, 7- cis-11- trans-LXA 4 was isolated and identified as a novel biologically active eicosanoid formed by human neutrophils.

Novel transcellular interaction: conversion of granulocyte-derived leukotriene A4 to cysteinyl-containing leukotrienes by human platelets.

Human platelets dose-dependently converted exogenous leukotriene A4 to leukotriene C4 and efficiently metabolized this compound to leukotrienes D4 and E4. Neither of these compounds were produced after stimulation of human platelet suspensions with ionophore A23187. After LTA4 incubation of subcellular fractions, formation of leukotriene C4 was exclusively observed in the particulate fraction and was separable from the classical glutathione S-transferase activity. This suggested the presence of a specific leukotriene C4 synthase in human platelets. Addition of physiological amounts of autologous platelets to human granulocyte suspensions significantly increased ionophore A23187-induced formation of leukotriene C4. In contrast, the production of leukotriene B4 was decreased. After preincubation of platelets with [35S]cysteine, 35S-labeled leukotriene C4 was produced by A23187-stimulated platelet-granulocyte suspensions, strongly indicating a transcellular biosynthesis of this compound.

Human endothelial cells stimulate leukotriene synthesis and convert granulocyte released leukotriene A4 into leukotrienes B4, C4, D4 and E4.

Incubation of human endothelial cells with leukotriene A4 resulted in the formation of leukotrienes B4, C4, D4 and E4. Endothelial cells did not produce leukotrienes after stimulation with the ionophore A23187 and/or exogenously added arachidonic acid. However, incubation of polymorphonuclear leukocytes with ionophore A23187 together with endothelial cells led to an increased synthesis of cysteinyl-containing leukotrienes (364%, mean, n = 11) and leukotriene B4 (52%) as compared to leukocytes alone. Thus, the major part of leukotriene C4 recovered in mixed cultures was attributable to the presence of endothelial cells. Similar incubations of leukocytes with fibroblasts or smooth muscle cells did not cause an increased formation of leukotriene C4 or leukotriene B4. The increased biosynthesis of cysteinyl-containing leukotrienes and leukotriene B4 in coincubation of leukocytes and endothelial cells appeared to be caused by two independent mechanisms. First, cell interactions resulted in an increased production of the total amount of leukotrienes, suggesting a stimulation of the leukocyte 5-lipoxygenase pathway, induced by a factor contributed by endothelial cells. Secondly, when endothelial cells prelabeled with [35S]cysteine were incubated with either polymorphonuclear leukocytes and A23187, or synthetic leukotriene A4, the specific activity of the isolated cysteinyl-containing leukotrienes were similar. Thus, transfer of leukotriene A4 from stimulated leukocytes to endothelial cells appeared to be an important mechanism causing an increased formation of cysteinyl-containing leukotrienes in mixed cultures of leukocytes and endothelial cells. In conclusion, the present study indicates that the vascular endothelium, when interacting with activated leukocytes, modulates both the quantity and profile of liberated leukotrienes.

Allergen-specific leukocyte adherence inhibition (LAI) assay: sensitivity, specificity and mechanism

An allergen-specific tube leukocyte adherence inhibition (LAI) assay has been developed in order to study the mechanism by which leukocytes lose their normal property of adherence to glass. Peripheral blood leukocytes (PBL) from 27 individuals allergic to Dermatophagoides farinae (DF), 10 with seasonal rhinitis not induced by DF and 49 non-allergic healthy volunteers were challenged in vitro with DF and a non-relevant allergen, Artemisia vulgaris (AV) and then assayed for the ability to adhere to glass tubes. Challenge by DF, but not by AV, resulted in loss of adherence by PBL from patients allergic to DF, but not in those of normal controls. The specific LAI response was dose-dependent and occurred only when a critical dose of 0.5 × 10 3 was employed. Following in vitro challenge with DF, radioimmunoassay using an antiserum to LTC 4 detected immunoreactive material in supernatants of PBL from DF-allergic individuals. When highly enriched mononuclear cells from non-allergic individuals were armed with cytophilic allergen-specific IgE and challenged with the specific allergen, they lost the property of glass adherence and released a substance that was immunoreactive with LTC 4. The results suggest that the chain of events leading to the LAI response in PBL from allergic individuals involves primary recognition of the allergen by specific IgE antibodies bound to receptors on mononuclear cells. The cells are thus triggered to synthesize cysteinyl-containing leukotrienes which mediate the LAI phenomenon. The results suggest that this assay may be used to study allergen—antibody interaction and the subsequent events leading to the clinical picture of atopic diseases.

The detection of 5-lipoxygenase and cyclo-oxygenase products in sputum of patients with chronic bronchitis and bronchiectasis

Leukotrienes (LTs) and prostanoids (Ps) were detected in sputum of patients with chronic bronchitis and/or bronchiectasis (CB/B) using selective superfusion bioassay and radioimmunoassay (RIA) techniques. Analysis of sputum extracts showed a 4-fold increase in the level of LTB 4 compared to the cysteinyl-containing LTs (LTC 4/LTD 4). The measurement of cyclo-oxygenase products (COPs) indicated relatively greater amounts of the vasodilator prostaglandin E 2 (PGE 2) and prostacyclin (PGI 2) compared to the vasoconstrictor prostaglandin F 2∝ (PGF 2∝) and thromboxane A 2 (TxA 2) agents (70:30% of total COPs respectively). The presence of eicosanoids (LTs and Ps) in sputum of patients with CB/B suggest that these biologically active substances may act as mediators of bronchoconstriction and inflammation in these diseases.

Formation of cysteinyl-containing leukotrienes by human arterial tissues

Human arterial rings incubated in modified Tyrode solution released small amounts of leukotriene (LT) C 4-like material spontaneously and larger amounts upon stimulation with the ionophore A23187 as determined by radioimmunoassay. By reversed phase high pressure liquid chromatography (HPLC) LTC 4-like material was found to comigrate with authentic LTC 4, LTD 4 and LTE 4. Nordihydroguaiaretic acid (NDGA) significantly inhibited the ionophore A23187-induced release of LTC 4-like material, while indomethacin was without effect. Simultaneously the arterial rings released much larger amounts of 6-keto-prostaglandin (PG) F 1α, which were significantly decreased by indomethacin. The results demonstrate that human arterial tissue has the capacity to synthesize cysteinyl-containing LT from endogenous arachidonic acid.