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Rationale: Obstructive sleep apnea, which is characterized by intermittent hypoxia (IH) during sleep, has emerged as an independent risk factor for cardiovascular disease, including atherosclerosis. Leukotriene B4 (LTB4) production is increased in patients with obstructive sleep apnea and negatively correlates to hypoxic levels during sleep, with continuous positive airway pressure therapy decreasing LTB4 production. Objectives: DeterminethepotentialroleofLTB4inIH-inducedatherosclerosis in a monocyte cellular model and a murine model. Methods: THP-1 cells were exposed to IH for 3, 6, 24, and 48 hours. Macrophage transformation and foam cell formation were assessed after IH exposures. Apolipopotein E (ApoE) 2/2 or BLT1 2/2 /ApoE 2/2 micewerefedanatherogenicdietandexposedtoIH(alternating21% and 5.7%O2from7 AM to 7 PMeachday)for10weeks.Atherosclerotic lesion formation in en face aorta was examined by oil red O staining. Measurements and Main Results: IH increased production of LTB4 and theexpressionof5-lipoxygenaseandleukotrieneA4hydrolase,thekey enzymes for producing LTB4. IH was associated with transformation of monocytes to activated macrophages, as evidenced by increased expression of CD14 and CD68. In addition, IH exposures promoted increased cellular cholesterol accumulation and foam cell formation. The LTB4 receptor 1 (BLT1) antagonist U-75302 markedly attenuated IH-induced changes. Furthermore, IH promoted atherosclerotic lesion formation in ApoE 2/2 mice. IH-induced lesion formation was markedly attenuated in BLT1 2/2 /ApoE 2/2 mice. Conclusions: BLT1-dependent pathways underlie IH-induced atherogenesis, and may become a potential novel therapeutic target for obstructive sleep apnea–associated cardiovascular disease.

Rationale: Mechanical ventilation with high VT (HVT) progressively leads to lung injury and decreased efficiency of gas exchange. Hypoxic pulmonary vasoconstriction (HPV) directs blood flow to well-ventilated lung regions, preserving systemic oxygenation during pulmonary injury. Recent experimental studies have revealed an important role for leukotriene (LT) biosynthesis by 5-lipoxygenase (5LO) in the impairment of HPV by endotoxin. Objectives: To investigate whether or not impairment of HPV contributes to the hypoxemia associated with HVT and to evaluate the role of LTs in ventilator-induced lung injury. Methods: We studied wild-type and 5LO-deficient mice ventilated for up to 10 hours with low VT (LVT) or HVT. Results: In wild-type mice, HVT, but not LVT, increased pulmonary vascular permeability and edema formation, impaired systemic oxygenation, and reduced survival. HPV, as reflected by the increase in left pulmonary vascular resistance induced by left mainstem bronchus occlusion, was markedly impaired in animals ventilated with HVT. HVT ventilation increased bronchoalveolar lavage levels of LTs and neutrophils. In 5LO-deficient mice, the HVT-induced increase of pulmonary vascular permeability and worsening of respiratory mechanics were markedly attenuated, systemic oxygenation was preserved, and survival increased. Moreover, in 5LO-deficient mice, HVT ventilation did not impair the ability of left mainstem bronchus occlusion to increase left pulmonary vascular resistance. Administration of MK886, a 5LO-activity inhibitor, or MK571, a selective cysteinyl-LT1 receptor antagonist, largely prevented ventilator-induced lung injury. Conclusions: These results indicate that LTs play a central role in the lung injury and impaired oxygenation induced by HVT ventilation.

The objective of this study was to determine the effects of allergen exposure on leukotriene generation and inflammation within the airways of allergic asthmatics and evaluate the effects of the 5-lipoxygenase inhibitor zileuton on these responses. We measured leukotriene-B(4) (LTB(4)) and LTC(4)/D(4)/E(4), inflammatory cytokine mediators, and cellular responses in bronchoalveolar lavage fluid (BALF) before and 24 h after segmental ragweed antigen challenge in 18 asthmatic subjects at baseline. Before initiating therapy with the 5-lipoxygenase inhibitor or placebo, only nine of 18 asthmatic subjects had a significant increase (234 +/- 102-fold, mean +/- SE) in BALF LTC(4)/D(4)/E(4) levels 24 h after segmental antigen challenge, whereas leukotriene levels were essentially unchanged (1.14 +/- 0.22-fold) in the other nine subjects. The high LT producers also had higher postantigen BALF levels of LTB(4), total protein, IL-5, IL-6, TNF-alpha, and recovery of more eosinophils than the low LT producers. Treatment with the 5-lipoxygenase inhibitor zileuton reduced postantigen BALF eosinophil count by 68% in the high LT producers, but had no detectable effect on BALF composition in the low LT producers. These data suggest that leukotriene inhibition may be more effective in a subset of asthmatics in whom leukotrienes are a major contributory factor in causing allergic inflammation.

Leukotrienes (LTs) are potent bioactive lipids derived from the 5-lipoxygenase (5-LO) pathway of arachidonic acid (AA) metabolism. Although they have been conventionally viewed as paracrine mediators of inflammatory disease processes such as asthma (1), more recent information suggests that they are also important participants in disease processes characterized by cellular proliferation and fibrogenesis (2-4), and that they subserve a homeostatic role in antimicrobial host defense (5). In view of the actions and importance of LTs, substantial effort has been directed at increasing our understanding of the regulation of their synthesis. An extensive body of research, reviewed elsewhere in this symposium, has identified three key factors that regulate LT synthesis: (1) the steady state levels of key proteins necessary for AA release and 5-LO metabolism; (2) posttranslational modifications (such as phosphorylation) that alter the catalytic activities of these proteins; and (3) the concentrations of small molecules (e.g., Ca 2+ , ATP, and glutathione) that serve as cofactors for certain of these proteins. One additional determinant of LT biosynthesis that has been recognized is the intracellular compartmentalization of these LT-forming proteins. This topic has become a focus of investigation in our own and other laboratories, and this article reviews the current state of knowledge in this area. Indeed, a number of unexpected findings that have emerged from these studies underscore the appropriateness of a perspective on LT biology that transcends conventional notions.

Blinded, randomized, and placebo-controlled clinical trials have established that cysteinyl leukotriene (cysLT) receptor antagonists and 5-lipoxygenase (5-LO) inhibitors are safe and effective asthma treatments. Trials of 13- to 26-wks' duration demonstrate that both the cysLT1 receptor antagonist, zafirlukast, and the 5-LO inhibitor, zileuton, improve pulmonary function and decrease daytime and nocturnal symptoms. Concomitant rescue β -agonist inhaler use and the need for corticosteroid rescue are also reduced. Preliminary studies suggest that antileukotriene agents may also reduce indices of airway inflammation, including inflammatory cell counts and airway hyperresponsiveness. Both cysLT1 antagonists and 5-LO inhibitors offer a new approach to asthma management. Drazen J. Clinical pharmacology of leukotriene receptor antagonists and 5-lipoxygenase inhibitors.

Inflammatory infiltrates and endothelial cell proliferation have been appreciated in plexiform and concentric lesions, which characterize the vascular remodeling in primary pulmonary hypertension (PPH). Leukotriene production by perivascular and alveolar macrophages relies on activation of 5-lipoxygenase (5-LO), with translocation of the enzyme to the nuclear membrane, and association with the 5-LO activating protein (FLAP). Using immunohistochemical staining, we localized and semi-quantitatively estimated the abundance of 5-LO and FLAP in lungs obtained from patients with PPH, patients with interstitial lung disease (ILD), and normal control subjects. Expression of 5-LO and FLAP was prominent in alveolar macrophages in both the normal and PPH lungs; however, alveolar macrophages were more frequently clustered in the vicinity of remodeled blood vessel in PPH. Medium- and small-size pulmonary arteries in PPH showed more abundant FLAP expression than in control and ILD lungs. 5-LO expression in small arteries in PPH was more intense than in control and ILD patients. Endothelial cells in plexiform and concentric lesions in PPH expressed both 5-LO and FLAP. In situ hybridization confirmed the presence of 5-LO transcripts in macrophages and endothelial cells of the remodeled vessels in PPH. We propose that the overexpression of 5-LO and FLAP represents evidence for the participation of inflammation in the process of PPH vasculopathy or, alternatively, that the overabundance of the enzymes involved in generation of inflammatory mediators may themselves be related to vascular cell proliferation and cell growth.

We studied the effects of the 5-lipoxygenase inhibition and sulfidopeptidyl leukotriene receptor antagonism on lumenal chemotaxis of eosinophils in 124 guinea pig tracheal explant preparations from 62 animals. Cell migration was assessed histologically and by differential cell count, and airway narrowing was measured by calibrated micrometry. Intralumenal instillation of the chemotaxin, formyl-met-leu-phe (FMLP) caused migration of 163,509 +/- 18,103 eosinophils/cm segment (eos/cm) versus 15,443 +/- 3,557 eos/cm for segments receiving vehicle only (p0.001). Coincubation of FMLP with zileuton, a selective inhibitor of 5-lipoxygenase, caused a concentration-related inhibition of eosinophil migration. At 10(-10) M zileuton, cell migration caused by FMLP was decreased by 57% and nearly complete reduction to 17,200 +/- 3,620 eos/cm resulted after 10(-6) M zileuton (p0.001 versus FMLP). Lumenal narrowing caused by FMLP (15.3 +/- 3.4%) was attenuated maximally to 1.15 +/- 2.51% after 10(-8) M zileuton (p0.02). In 36 preparations, concentration of leukotriene B4 (LTB4) was measured in treated tracheal perfusate. LTB4 secretion caused by FMLP was 6.4 +/- 0.48 pg/ml versus 3.32 +/- 0.89 pg/ml for buffer control at 5 min (p0.02) and was undetectable 120 min after activation with FMLP. Blockade of LTB4-receptor with the selective antagonist, LTB4 dimethyl amide, caused90% inhibition of eosinophil migration (p0.001). Comparable results were obtained with zafirlukast, an LTD4-receptor antagonist. Our data demonstrate that both LTB4 and LTD4 facilitate eosinophil migration from lamina propria to lumen caused by the chemotaxin, FMLP, and that LTB4-induced eosinophil migration is accompanied by initial lumenal secretion of LTB4.

We have shown that inhalation of lysine aspirin enhances leukotriene production in the lungs of patients with aspirin-induced asthma (AIA). To assess the specificity of this reaction, we compared two well-matched groups of patients: eleven with AIA versus 14 asthmatics tolerant to aspirin (ATA). All subjects underwent bronchoalveolar lavage (BAL) with saline followed immediately by instillation of 10 mg of lysine aspirin, into a right middle lobe segmental bronchus, which was lavaged 15 min later. At baseline the two groups did not differ with respect to BAL fluid concentrations of cyclooxygenase products, peptido-leukotrienes, histamine, tryptase, interleukin-5 (IL-5), eosinophil cationic protein (ECP), or eosinophil number. Fifteen minutes after aspirin instillation, there was a statistically significant rise in peptido-leukotrienes, IL-5, and eosinophil number in AIA, but not in ATA, but not in ATA patients. In the former, but not in the latter group, mean histamine concentrations rose in response to aspirin, approaching the level of statistical significance. Tryptase and ECP levels showed no significant change. Aspirin significantly depressed PGE2 and thromboxane B2 (TXB2) in both groups, however PGD2, PGF2 alpha, and 9 alpha, 11 beta-PGF2 decreased only in ATA patients. A characteristic disturbance in eicosanoid balance, produced by aspirin in patients intolerant to this drug, might explain precipitation of asthma attacks.

We studied the urinary excretion of the isoprostane 8-iso-prostaglandin F(2alpha) as an index of in vivo oxidant stress, and the production of leukotriene (LT) B(4) (LTB(4)) by neutrophils in subjects with chronic obstructive pulmonary disease (COPD) and normal subjects. Overnight urinary excretion of the isoprostane was significantly higher in patients with COPD than in control subjects, and LTB(4) production by challenge of neutrophils obtained from patients with COPD was also significantly higher than that observed in control neutrophils. Treatment with a standardized polyphenol extract caused a significant decrease in isoprostane excretion, accompanied by a statistically significant increase of Pa(O(2)). Furthermore, changes in FEV(1) significantly correlated with the changes in isoprostane urinary excretion observed from enrollment to the end of treatment. The results of this study suggest that enhanced oxidative stress in subjects with COPD is paralleled by the increased ability of neutrophils to synthesize the chemotactic factor LTB(4), and may ultimately contribute to the infiltration/activation of neutrophils into the airways of subjects with COPD. Antioxidant treatment in subjects with COPD is effective in reducing oxidant stress as shown by the decrease of urinary isoprostane, a reduction that correlates with the severity of the disease, as indicated by changes in Pa(O(2)) and FEV(1).

Although overproduction of proinflammatory 5-lipoxygenase (5-LO)-derived leukotrienes (LTs) has been demonstrated in the lungs of patients with pulmonary fibrosis, their causal involvement in this condition has not been established. Bleomycin-induced pulmonary fibrosis was studied in mice rendered LT deficient by knockout of the 5-LO gene (KO) and in wild-type (WT) control mice. Following administration of bleomycin, lung lavage fluid of WT mice demonstrated an approximately 5-fold increase in levels of cysteinyl-LTs over baseline levels at Day 1, with persistent elevation up to Day 21. As compared with WT mice, 5-LO KO mice demonstrated reduced amounts of histologically evident collagen as well as an approximately 60% reduction in lung hydroxyproline levels postbleomycin. Unlike WT mice, KO mice showed no increases in the numbers of lung inflammatory cells postbleomycin. Furthermore, in situ expression and stimulated production by mixed lung leukocytes of the antifibrotic cytokine interferon-gamma were significantly greater in cells from the 5-LO KO mice. Finally, lavage levels of the antiinflammatory and antifibrotic molecule, prostaglandin E(2), were significantly greater in the KO animals. These results provide strong evidence that LTs may participate in the pathogenesis of pulmonary fibrosis, and they may do so by direct effects as well as indirect effects occurring via their modulation of the synthesis of other inflammatory mediators.

Cysteinyl-leukotrienes and prostaglandin D2 generated by the 5-lipoxygenase (5-LO) and cyclooxygenase (COX) pathways, respectively, cause bronchoconstriction, leukocyte recruitment, and bronchial hyperresponsiveness in asthma. We characterized the cellular expression of 5-LO and COX enzymes using immunohistochemistry on bronchial biopsies from 12 allergic asthmatic patients before and during seasonal exposure to birch pollen. Bronchial responsiveness (p = 0.004) and symptoms (p0.005) increased and peak expiratory flow (PEF; por = 0.02) decreased in the pollen season. In-season biopsies had 2-fold more cells immunostaining for 5-LO (p = 0.02), 5-LO-activating protein (FLAP; p = 0.04), and leukotriene (LT)A4 hydrolase (p = 0.05), and 4-fold more for the terminal enzyme for cysteinyl-leukotriene synthesis, LTC4 synthase (p = 0.02). Immunostaining for COX-1, COX-2, and PGD2 synthase was unchanged. Increased staining for LTC4 synthase was due to increased eosinophils (p = 0.035) and an increased proportion of eosinophils expressing the enzyme (p = 0.047). Macrophages also increased (p = 0.019), but mast cells and T-lymphocyte subsets were unchanged. Inverse correlations between PEF and 5-LO(+) cell counts link increased expression of 5-LO pathway enzymes in eosinophils and macrophages within the bronchial mucosa to deterioration of lung function during seasonal allergen exposure.

5-Lipoxygenase (5LO) catalyzes the conversion of arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE), and further to the allylic epoxide 5(S)-trans-7,9-trans-11,14-cis-eicosatetrenoic acid (leukotriene A 4 , LTA 4 ). Thus, 5LO has a central role in leukotriene biosynthesis. This article covers some basic properties of 5LO. Studies regarding the 5LO gene and targeted gene disruption, and 5LO intracellular compartmentalization, are described in other parts of this supplement.

A decade has passed since the molecular cloning of the 5-lipoxygenase gene and nearly twenty years since the discovery of leukotrienes. In our laboratory, we have strived to make advances in the realm of basic biology and understanding of the 5-lipoxygenase/leukotriene pathway by the use of knockout mice and other cell biology techniques. The aim of this article is to survey the studies conducted with these mice and to present novel information about the nuclear localization of 5-lipoxygenase.

In a study of the transformation of polyunsaturated fatty acids in rabbit polymorphonuclear leukocytes in 1976 it was discovered that arachidonic acid is oxygenated at C-5 (1). Subsequently a number of derivatives, including leukotriene B 4 (LTB 4 ), were identified (2-4). Extension of these studies led in 1979 to the discovery of the pivot epoxide intermediate, LTA 4 (5), and the elucidation of the structure of SRS-A (slow-reacting substance of anaphylaxis) as a group of cysteinyl-leukotrienes, namely LTC 4 , LTD 4 , and LTE 4 (6-10). Earlier work had shown that the prostaglandins are formed by oxygenation and further transformation of arachidonic acid and other polyunsaturated fatty acids (11). The first intermediate in the formation of prostaglandins, the endoperoxide PGG 2 , was isolated and identified in 1974 (12). At the same time we introduced the name cyclooxygenase for the enzyme catalyzing the transformation of arachidonic acid into PGG 2 (12). However, the endoperoxides, PGG 2 and PGH 2 , had some biological effects that could not be explained by their conversion to the known prostaglandins (12). This finding eventually led to the discovery of thromboxane A 2 , an unstable platelet-aggregating and vasoconstrictor substance (13). Subsequent work showed that the endoperoxide can also be converted into a derivative, prostacyclin (PGI 2 ), with opposite biological effects (14). Prostaglandins E 2 and I 2 have strong proinflammatory effects. Aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) inhibit the enzyme (cyclooxygenase) responsible for conversion of arachidonic acid into prostaglandins and thromboxanes (15). An induced form of the cyclooxygenase (COX-2) seems to play an important role in inflammation, thus opening the possibility of developing antiinflammatory NSAIDs that lack the side effects of the previous generation of such drugs (16, 17). In 1975 antiinflammatory steroids were proposed to inhibit prostaglandin formation by blocking of the release of the precursor acid from phospholipid stores (18). Since steroids and NSAIDs have significantly different antiinflammatory effects it seemed conceivable that some of these differences might be explained by the formation of proinflammatory derivatives of arachidonic acid by cyclooxygenase-independent reactions. Our studies of the metabolism of arachidonic acid in leukocytes unraveled a new metabolic pathway and led to the discovery of the leukotrienes (8). These compounds play a role in allergy and asthma and have pronounced proinflammatory effects (19, 20).

Leukotrienes are important chemical mediators in a variety of inflammatory and allergic conditions, including those affecting the respiratory system. These signaling molecules can be divided into two groups: the proinflammatory leukotriene B 4 (LTB 4 ) and the spasmogenic leukotrienes C 4 . D 4 , and E 4 , also termed cysteinyl-leukotrienes (1). LTB 4 is one of the most powerful chemotactic agents known to date and acts via specific seven-transmembrane, G protein-coupled surface receptors on the target cells (2). The profile of the biological properties of LTB 4 makes it a key component in the complex network of soluble and cell-bound factors that govern the development and maintenance of inflammation. Hence, LTB 4 has been proposed to play a role in a variety of acute and chronic inflammatory diseases such as arthritis, dermatoses, inflammatory bowel disease (IBD), and chronic obstructive pulmonary disease (COPD). In particular, LTB 4 seems to play a role in the recruitment of inflammatory cells to the site of tissue injury. This article gives a brief overview of the biochemistry and molecular biology of LTA 4 hydrolase, the enzyme catalyzing the final step in the biosynthesis of LTB 4 .

Persistent neutrophilia is a feature of chronic obstructive pulmonary disease (COPD). Leukotriene synthesis inhibitors and cysteinyl leukotriene receptor antagonists have shown efficacy in the treatment of asthma. Antagonism of leukotriene (LT)B(4) receptors is being considered as a mode of treating COPD. We examined the capacity for inhibition of leukotriene synthesis and LTB(4) receptor antagonism to reduce survival of neutrophils from patients with COPD and those from normal subjects. The basal apoptosis level of these cells was 55.4 +/- 2.4% (mean +/- SEM) of total cells. Separate exposure to lipopolysaccharide (LPS), granulocyte-macrophage colony-stimulating factor (GM-CSF), dexamethasone (DEX), and LTB(4) increased neutrophil survival (p0. 001). The LTB(4) receptor antagonist SB201146 abolished LPS-induced survival in a concentration-dependent manner (10 pmol to 0.1 microM), with an IC(50) of 1.9 nM. Combined exposure to SB201146 and to the cysteinyl leukotriene antagonist SKF104353 did not have a greater effect on survival than did exposure to SB201146 alone. Inhibition of 5-lipoxygenase (5-LO) with BWA4C and of 5-LO-activating protein (FLAP) with MK886 abolished GM-CSF- and DEX-induced neutrophil survival. BWA4C and MK886 abolished GM-CSF- induced neotrophil survival in a concentration-dependent manner (1 nM to 10 microM), with IC(50) values of 182.0 nM and 63.1 nM, respectively. These findings demonstrate reversal of LPS-, GM-CSF-, and DEX-induced neutrophil survival by LTB(4) receptor antagonism and inhibitors of 5-LO and FLAP. They also suggest a potential additional antiinflammatory mode of action of these compounds through reduction of cell survival.

The initial enzymatic steps in leukotriene synthesis occur at the nuclear envelope. Cytosolic phospholipase A2 translocates from the cytoplasm to selectively hydrolyze nuclear envelope phospholipids, releasing free arachidonate. 5-Lipoxygenase-activating protein, an arachidonate transfer protein, then binds arachidonate and presents it to 5-lipoxygenase (5-LO), which catalyzes a two-step reaction to produce leukotriene A4. In resting human and rat peripheral blood neutrophils, 5-LO is localized to the cytoplasm; in rat basophilic leukemia cells and human alveolar macrophages, however, it is found predominantly in the nucleus. Immunofluorescence microscopy studies demonstrate that both cytoplasmic and nuclear 5-LO move to the nuclear envelope following cell activation. Many questions remain unanswered regarding the significance of nuclear 5-LO, potential autocrine actions of leukotrienes, and intracellular trafficking of these enzymes and their products. Peters-Golden M. Cell biology of the 5-lipoxygenase pathway.

Recent evidence suggests that leukotrienes may have a causative role in exercise-induced asthma. Twenty-four subjects with exercise-induced asthma received either 600 mg zileuton, a 5-lipoxygenase inhibitor, or a placebo four times daily for 2 d prior to exercise challenge (a total of nine doses). The last dose was administered in the laboratory 2 h before the exercise challenge. There was no bronchodilation after nine doses of the 5-lipoxygenase inhibitor (p=0.95). The administration of zileuton inhibited bronchospasm after exercise challenge by 40.75% as compared with placebo. Five minutes after the completion of exercise, the zileuton group's FEV1 was 85.76% of the preexercise value, compared with 73.92% of the preexercise value in the placebo group (p0.01). The maximum percent change in baseline FEV1 after zileuton was a 15.58% decrement from the preexercise level, as compared with a 28.1% decrease after placebo (p0.001). Five minutes after exercise, the FVC after zileuton was 92.76% of the preexercise value, as compared with 86.26% after placebo (p0.05). This is the first study in which a 5-lipoxygenase inhibitor has been shown to attenuate exercise-induced asthma. These results suggest that leukotrienes are important biochemical mediators in the development of exercise-induced bronchospasm, and that leukotriene inhibit may have a role in the treatment of this disorder.

Each of these effector systems has the capacity to initiate airway obstruction or alter airway responsiveness in asthma. It is likely that they act in concert in certain asthmatic settings. Further basic and applied research will define their relative roles in asthma.