Leukotrienes: mediators that have been typecast as villains.

As befalls many mediators that act upon the human stage, leukotrienes have become identified with their most powerful roles as villains of the immune system. They are well known for their leading roles in allergic diseases, including asthma. They also have gained recognition for their dramatic role as promoters of inflammation. As new roles for these lipid messengers are sought, it is becoming apparent that the leukotrienes have been typecast as bad guys of the immune system. As examples, their more recent roles have been in atherosclerosis, pulmonary fibrosis and cancer. However, upon further evaluation, we can begin to see their versatility. Thus, leukotrienes stimulate innate immunity against pathogens. In addition, they promote the expression of mediators, receptors and other molecules that are important for immune defense. In these lesser known roles, they lead the fight against bacterial, fungal and viral infection. This review is intended to shed light on the leukotrienes, where they come from and what we really know about them.

5-lipoxygenase and FLAP.

The initial steps in the biosynthesis of leukotrienes from arachidonic acid are carried out by the enzyme 5-lipoxygenase (5-LO). In intact cells, the helper protein 5-LO activating protein (FLAP) is necessary for efficient enzyme utilization of endogenous substrate. The last decade has witnessed remarkable progress in our understanding of these two proteins. Here we review the molecular and cellular aspects of the expression, function, and regulation of 5-LO and FLAP.

Leukotriene synthesis by epithelial cells.

Leukotrienes (LTs) are intercellular signaling molecules that evoke a variety of responses. They are best known as potent promoters of inflammation. Normally, LTs are produced primarily by leukocytes. As a result, current models regarding the production of LTs in the context of disease focus on the leukocytes as the site of production. Structural cells, including epithelial cells, are typically relegated to supportive roles. It is recognized that epithelial cells normally contain all the components necessary for LT synthesis except the enzyme 5-lipoxygenase (5-LO). There is accumulating evidence that some populations of epithelial cells normally express low levels of 5-LO and can synthesize LTs autonomously. Moreover, certain factors, including bacterial and viral infection, can promote the expression of 5-LO in airway, gastrointestinal and skin epithelial cells. The appearance of active 5-LO enzyme in epithelial cells at these sites may contribute to diseases like cancer, colitis and psoriasis. This paper reviews the state of our knowledge regarding the expression of 5-LO in epithelial cells, the factors that modify that expression, and the implications regarding pathogenesis.

Sulphatides trigger polymorphonuclear granulocyte spreading on collagen-coated surfaces and inhibit subsequent activation of 5-lipoxygenase.

Sulphatides are sulphate esters of galactocerebrosides that are present on the surfaces of many cell types and act as specific ligands to selectins. The present study was undertaken to investigate the effect of sulphatides on polymorphonuclear granulocyte (PMN) attachment, spreading and 5-lipoxygenase (5-LO) metabolism. Sulphatides, but not non-sulphated galactocerebrosides, dose-dependently enhanced attachment to collagen, as measured by the myeloperoxidase assay. Studies with blocking antibodies indicated that the increased attachment was mediated by CD11b/CD18 (Mac-1) beta 2 integrin. Scanning electron microscopy indicated that sulphatides also greatly enhanced the degree of cell spreading. In PMNs treated in suspension, sulphatides had no effect on the ionophore A23187-stimulated release of arachidonic acid and the synthesis of 5-LO metabolites. In contrast, in PMNs attached to collagen, the enzymic conversion of arachidonic acid by 5-LO was inhibited by sulphatides. Inhibition of 5-LO metabolism by sulphatides was observed even in the presence of exogenous substrate, suggesting that sulphatides directly inhibited 5-LO action. Consistent with this, sulphatides interfered with ionophore-induced translocation of the 5-LO to the nuclear envelope. Substances competing with sulphatide binding to cells, like dextran sulphate, or a strong inhibitor of cell spreading, like the actin-polymerizing agent jasplakinolide, prevented the effects of sulphatides on PMN attachment and spreading and leukotriene synthesis. We conclude that shape changes occurring in response to sulphatides specifically impair PMN leukotriene synthesis by inhibiting translocation of 5-LO.

Evaluation of phagocytosis and arachidonate metabolism by alveolar macrophages and recruited neutrophils from F344xBN rats of different ages.

The incidence of infectious respiratory diseases increases with aging. Resident alveolar macrophages (AMs) and recruited leukocytes (PMNL) mediate cellular defense against bacterial infections in the lung, and phagocytosis and lipid mediator synthesis are important components of their antimicrobial capacity. The objective of this study was to determine if either phagocytic capacity or lipid mediator generation declines with normal aging, in either AMs or PMNL recruited to a site of inflammation. The F344xBN rat hybrid has a lower incidence of pathologies associated with aging, particularly up to 20 months; animals aged 6,12 and 18 months were chosen to evaluate changes associated with normal aging. As previously reported for peripheral blood leukocytes, phagocytosis by recruited PMNL declined with aging: recruited PMNL from 18 months rats showed a significantly decreased capacity to phagocytose live Klebsiella pneumoniae bacteria, compared to PMNL from 6 months rats. Surprisingly, however, the phagocytic capacity of AMs increased with aging: the phagocytic index of AMs from 18 months rats was more than three times that of AMs from 6 months rats. The capacity of AMs and recruited PMNL to release arachidonic acid or synthesize leukotrienes or prostaglandins did not change with aging. This study demonstrates that, although phagocytosis by recruited PMNL declines with aging, other aspects of immune function do not decline, and may actually increase, with normal aging. These results suggest that impaired phagocytosis by recruited PMNL may be an important component of the increased susceptibility to infectious respiratory diseases during normal aging.

Co-localization of leukotriene a4 hydrolase with 5-lipoxygenase in nuclei of alveolar macrophages and rat basophilic leukemia cells but not neutrophils.

The synthesis of leukotriene B(4) from arachidonic acid requires the sequential action of two enzymes: 5-lipoxygenase and leukotriene A(4) hydrolase. 5-Lipoxygenase is known to be present in the cytoplasm of some leukocytes and able to accumulate in the nucleoplasm of others. In this study, we asked if leukotriene A(4) hydrolase co-localizes with 5-lipoxygenase in different types of leukocytes. Examination of rat basophilic leukemia cells by both immunocytochemistry and immunofluorescence revealed that leukotriene A(4) hydrolase, like 5-lipoxygenase, was most abundant in the nucleus, with only minor occurrences in the cytoplasm. The finding of abundant leukotriene A(4) hydrolase in the soluble nuclear fraction was substantiated by two different cell fractionation techniques. Leukotriene A(4) hydrolase was also found to accumulate together with 5-lipoxygenase in the nucleus of alveolar macrophages. This result was obtained using both in situ and ex vivo techniques. In contrast to these results, peripheral blood neutrophils contained both leukotriene A(4) hydrolase and 5-lipoxygenase exclusively in the cytoplasm. After adherence of neutrophils, 5-lipoxygenase was rapidly imported into the nucleus, whereas leukotriene A(4) hydrolase remained cytosolic. Similarly, 5-lipoxygenase was localized in the nucleus of neutrophils recruited into inflamed appendix tissue, whereas leukotriene A(4) hydrolase remained cytosolic. These results demonstrate for the first time that leukotriene A(4) hydrolase can be accumulated in the nucleus, where it co-localizes with 5-lipoxygenase. As with 5-lipoxygenase, the subcellular distribution of leukotriene A(4) hydrolase is cell-specific and dynamic, but differences in the mechanisms regulating nuclear import must exist. The degree to which these two enzymes are co-localized may influence their metabolic coupling in the conversion of arachidonic acid to leukotriene B(4).

Intracellular compartmentalization of leukotriene synthesis: unexpected nuclear secrets.

Leukotrienes are important lipid mediators implicated in the regulation of various cellular processes and in disease states as well as homeostasis. Regulation of leukotriene biosynthesis is therefore of considerable interest. Although the levels of expression and catalytic activity of leukotriene-forming proteins have long been recognized as important determinants of leukotriene biosynthesis, it has recently become apparent that their intracellular compartmentalization also affects the integrated output of this biosynthetic pathway. In this minireview, we focus on the unexpected discovery that the nucleus is the key intracellular site for leukotriene biosynthesis and discuss the mechanisms that regulate protein localization and the potential implications of these findings.

Nuclear import of arachidonate 5-lipoxygenase.

Leukotrienes are lipid messenger molecules that are secreted by leukocytes to orchestrate a rapid and prolonged immune response. The enzyme 5-lipoxygenase catalyzes the rate-limiting first two steps in the synthesis of leukotrienes from arachidonic acid. Although it has long been known that 5-lipoxygenase moves from the cytoplasm to a membrane following activation, it has only recently been recognized that the enzyme may shuttle into and out of the nucleus before activation. The regulation of this movement of soluble 5-lipoxygenase between the cytoplasm and the nucleoplasm, as well as its impact on 5-lipoxygenase action, leukotriene synthesis and cell function, is only now being elucidated. This review details the state of our understanding of the nuclear import of 5-lipoxygenase and its potential importance in immunity.

Identification of a bipartite nuclear localization sequence necessary for nuclear import of 5-lipoxygenase.

5-Lipoxygenase catalyzes the synthesis of leukotrienes from arachidonic acid. This enzyme can reside either in the cytoplasm or the nucleus; its subcellular distribution is influenced by extracellular factors, and its nuclear import correlates with changes in leukotriene synthetic capacity. To identify sequences responsible for the nuclear import of 5-lipoxygenase, we transfected NIH 3T3 cells and RAW 264.7 macrophages with expression vectors encoding various 5-lipoxygenase constructs fused to green fluorescent protein. Overexpression of wild type 5-lipoxygenase with or without fusion to green fluorescent protein resulted in a predominantly intranuclear pattern of fluorescence, similar to the distribution of native 5-lipoxygenase in primary alveolar macrophages. Within the 5-lipoxygenase protein is a sequence (Arg(638)-Lys(655)) that closely resembles a bipartite nuclear localization signal. Studies using deletion mutants indicated that this region was necessary for nuclear import of 5-lipoxygenase. Analysis of mutants containing specific amino acid substitutions within this sequence confirmed that it was this sequence that was necessary for nuclear import of 5-lipoxygenase and that a specific arginine residue was critical for this function. As nuclear import of 5-lipoxygenase may regulate leukotriene production, natural or induced mutations in this bipartite nuclear localization sequence may also be important in affecting leukotriene synthesis.

Induction of ICAM-1 expression on alveolar epithelial cells during lung development in rats and humans.

Intercellular adhesion molecule-1 (ICAM-1) is an adhesion protein involved in immune and inflammatory cell recruitment and activation. In normal, uninflamed adult rat lung, ICAM-1 is expressed at high levels on type I alveolar epithelial cells and is minimally expressed on type II cells. ICAM-1 expression by alveolar epithelial cells in vitro is a function of the state of cellular differentiation, and is regulated by factors influencing cell shape. Based upon this observation, we hypothesized that ICAM-1 expression by fetal lung epithelial cells is developmentally regulated. To investigate this hypothesis, rat and human lung tissues were obtained at time points that represent the canalicular, saccular, and alveolar stages of development. The relative expression of ICAM-1 protein and mRNA were determined in rat lungs from gestational days 18 and 21 (term = 22 days), from day 8 neonatal rats, and from adult rats. ICAM-1 protein was detectable at low level on day 18 and increased progressively during development. Relative expression of ICAM-1 protein was maximal in adult lung. Expression of ICAM-1 mRNA paralleled that of ICAM-1 protein. By immunohistochemical methods in rat and human lung, ICAM-1 was expressed at low level on cuboidal and flattening epithelial cells in the developing alveolar space at the canalicular and saccular stages; however, ICAM-1 expression was increased as epithelial cells spread and flattened during alveolarization. ICAM-1 was predominantly expressed on type I cells rather than type II cells at the alveolar stage in both the rat and human lungs. Thus, relative ICAM-1 expression progressively increased during lung development. ICAM-1 expression is correlated with the increase in surface area as alveolar structures develop and type I cell differentiation takes place. These data indicate that alveolar epithelial cell ICAM-1 expression is developmentally regulated.

Arachidonic acid is preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglandin E2.

The two cyclooxygenase isoforms, cyclooxygenase-1 and cyclooxygenase-2, both metabolize arachidonic acid to prostaglandin H2, which is subsequently processed by downstream enzymes to the various prostanoids. In the present study, we asked if the two isoforms differ in the profile of prostanoids that ultimately arise from their action on arachidonic acid. Resident peritoneal macrophages contained only cyclooxygenase-1 and synthesized (from either endogenous or exogenous arachidonic acid) a balance of four major prostanoids: prostacyclin, thromboxane A2, prostaglandin D2, and 12-hydroxyheptadecatrienoic acid. Prostaglandin E2 was a minor fifth product, although these cells efficiently converted exogenous prostaglandin H2 to prostaglandin E2. By contrast, induction of cyclooxygenase-2 with lipopol- ysaccharide resulted in the preferential production of prostacyclin and prostaglandin E2. This shift in product profile was accentuated if cyclooxygenase-1 was permanently inactivated with aspirin before cyclooxygenase-2 induction. The conversion of exogenous prostaglandin H2 to prostaglandin E2 was only modestly increased by lipopolysaccharide treatment. Thus, cyclooxygenase-2 induction leads to a shift in arachidonic acid metabolism from the production of several prostanoids with diverse effects as mediated by cyclooxygenase-1 to the preferential synthesis of two prostanoids, prostacyclin and prostaglandin E2, which evoke common effects at the cellular level.

Decreased leukotriene C4 synthesis accompanies adherence-dependent nuclear import of 5-lipoxygenase in human blood eosinophils.

The enzyme 5-lipoxygenase (5-LO) catalyzes the synthesis of leukotrienes (LTs) from arachidonic acid (AA). Adherence or recruitment of polymorphonuclear neutrophils (PMN) induces nuclear import of 5-LO from the cytosol, which is associated with enhanced LTB4 synthesis upon subsequent cell stimulation. In this study, we asked whether adherence of human eosinophils (EOS) causes a similar redistribution of 5-LO and an increase in LTC4 synthesis. Purified blood EOS examined either in suspension or after adherence to fibronectin for 5 min contained only cytosolic 5-LO. Cell stimulation resulted in activation of 5-LO, as evidenced by its translocation to membranes and LTC4 synthesis. As with PMN, adherence of EOS to fibronectin for 120 min caused nuclear import of 5-LO. Unexpectedly, however, adherence also caused a time-dependent decrease in LTC4 synthesis: EOS adhered for 120 min produced 90% less LTC4 than did cells adhered for 5 min. Adherence did not diminish the release of [3H]AA from prelabeled EOS or reduce the synthesis of the prostanoids thromboxane and PGE2. Also, inhibition of LTC4 production caused by adherence could not be overcome by the addition of exogenous AA. Adherence increased, rather than decreased, LTC4 synthase activity. However, the stimulation of adherent EOS failed to induce translocation of 5-LO from the nucleoplasm to the nuclear envelope. This resistance to activation of the nuclear pool of 5-LO with diminished LT production represents a novel mode of regulation of the enzyme, distinct from the paradigm of up-regulated LT synthesis associated with intranuclear localization of 5-LO observed in PMN and other cell types.

Altered expression and localization of 5-lipoxygenase accompany macrophage differentiation in the lung.

The alveolar macrophage (AM) exhibits a greater capacity to synthesize bioactive leukotrienes from arachidonic acid than does its circulating precursor the peripheral blood monocyte. Macrophage differentiation in the lung entails cellular residence within both the pulmonary interstitial and alveolar compartments. In the present study, we sought to determine 1) whether this enhanced metabolic activity was acquired during maturation within the alveolar space and 2) the underlying mechanisms responsible for this upregulation. Rat AMs were separated by Percoll gradient centrifugation into four density-defined subpopulations thought to reflect their degree of maturation. On stimulation with a calcium ionophore, synthesis of leukotriene B4 increased with the degree of maturation, although it was diminished in the oldest subpopulation. This maturation-dependent upregulation was not explained by increases in arachidonic acid release but was associated with increased expression of 5-lipoxygenase (5-LO) protein as determined by immunoblot analysis. Whereas 5-LO is primarily cytosolic in monocytes, it is known to be primarily intranuclear in unfractionated AMs. Here, the localization of 5-LO was investigated by immunofluorescence microscopy and was found to be predominantly nuclear in all AM subpopulations; by contrast, the protein was cytosolic in interstitial macrophages isolated by mechanical and enzymatic lung digestion. These divergent localization patterns in AMs and interstitial macrophages were verified in situ by immunohistochemical staining of sections of normal rat lung. When unfractionated AMs were isolated and maintained in culture for 3 days, a shift in 5-LO distribution from nucleus to cytosol was observed. We conclude that 1) nuclear import of 5-LO occurs within the alveolar space and is reversible on removal from the alveolar milieu and 2) leukotriene synthetic capacity increases further during AM residence within the alveolar space as a result of a progressive increase in the amount of 5-LO protein.

Identification of glyceraldehyde-3-phosphate dehydrogenase as a Ca2+-dependent fusogen in human neutrophil cytosol.

The membrane fusion events observed during neutrophil degranulation are important aspects of the immunoregulatory system. In an attempt to understand the regulation of granule-plasma membrane fusion, we have begun characterizing human neutrophil cytosol for fusion activity, finding that 50% of the fusogenic activity could be attributed to members of the annexin family of proteins. The major non-annexin fusion activity (25% of the total cytosolic activity) was enriched by ion exchange chromatography after depletion of annexins by Ca2+-dependent phospholipid affinity chromatography. The fusion activity co-purified with a 10,14-kDa dimer identified as leukocyte L1 (which was non-fusogenic), along with an approximately 36-kDa protein. This protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by amino-terminal sequencing, and the fusion activity was verified using commercially available GAPDH. GAPDH may play an important role in degranulation because it is as potent as annexin I on a mass basis and may constitute up to 25% of the total cytosolic fusion activity of the neutrophil.

Capacity for repeatable leukotriene generation after transient stimulation of mast cells and macrophages.

Leukotriene (LT) synthesis is initiated by the enzyme 5-lipoxygenase (5-LO). Prolonged cell stimulation causes the translocation of 5-LO to the nuclear envelope and the synthesis of LT, with subsequent inactivation and persistent membrane association of 5-LO. In this study, we examined whether persistent membrane association of 5-LO, as well as the inactivation of 5-LO, could be prevented by shortening the length of cell stimulation or by blocking LT synthesis. As expected, stimulation of rat basophilic leukaemia (RBL) cells, a mast cell model, or alveolar macrophages (AMs) with calcium ionophore for 15 min caused 5-LO translocation, LT generation and the inactivation and persistent membrane association of 5-LO. When RBL cells or AMs instead were stimulated for 0.5-5 min, translocation of 5-LO and synthesis of LT still occurred. However, after washing and resting, the 5-LO enzyme returned to its original intracellular distribution. Furthermore these cells showed a retained capacity for LT synthesis on subsequent re-stimulation. Similar results were obtained when cells were stimulated with either formyl peptide or zymosan, instead of ionophore. In contrast, blockade of LT synthesis during the initial stimulation, with the selective inhibitors zileuton or MK-886, did not inhibit 5-LO translocation, inactivation or persistent membrane association resulting from prolonged cell stimulation. We conclude that, in long-lived immune cells, 5-LO translocation is reversible when cell stimulation is short, but persistent after prolonged stimulation. In addition 5-LO remains active and LT synthetic capacity is retained after transient stimulation, whereas significant inactivation of 5-LO occurs after prolonged stimulation. Finally, results with LT synthesis inhibitors indicate that inactivation and persistent membrane association of 5-LO can result independently of 5-LO activation.

Rapid import of cytosolic 5-lipoxygenase into the nucleus of neutrophils after in vivo recruitment and in vitro adherence.

5-Lipoxygenase catalyzes the synthesis of leukotrienes from arachidonic acid. The subcellular distribution of 5-lipoxygenase is known to be cell type-dependent and is cytosolic in blood neutrophils. In this study, we asked whether neutrophil recruitment into sites of inflammation can alter the subcellular compartmentation of 5-lipoxygenase. In peripheral blood neutrophils from rats, 5-lipoxygenase was exclusively cytosolic, as expected. However, in glycogen-elicited peritoneal neutrophils, abundant soluble 5-lipoxygenase was in the nucleus. Upon activation with calcium ionophore A23187, intranuclear 5-lipoxygenase translocated to the nuclear envelope. Elicited neutrophils required a greater concentration of A23187 for activation than did blood neutrophils (half-maximal response, 160 versus 52 nM, respectively) but generated greater amounts of leukotriene B4 upon maximal stimulation (26.6 versus 7.68 ng/10(6) cells, respectively). Intranuclear 5-lipoxygenase was also evident in human blood neutrophils after adherence to a variety of surfaces, suggesting that adherence alone is sufficient to drive 5-lipoxygenase redistribution. These results demonstrate a physiologically relevant circumstance in which the subcellular distribution of 5-lipoxygenase can be rapidly altered in resting cells, independent of 5-lipoxygenase activation. Nuclear import of 5-lipoxygenase may be a universal accompaniment of neutrophil recruitment into sites of inflammation, and this may be associated with alterations in enzymatic function.

Colchicine inhibits arachidonate release and 5-lipoxygenase action in alveolar macrophages.

Although colchicine is known to inhibit leukotriene synthesis in neutrophils, its effect on other aspects of arachidonic acid (AA) metabolism as well as its mechanism of action are unknown. To address these questions, we investigated the effects of colchicine on resident rat alveolar macrophages (AM), cells that generate a variety of lipoxygenase and cyclooxygenase products after stimulation. Pretreatment of AM with 10 microM colchicine for 1 h dramatically inhibited the synthesis of all 5-lipoxygenase (5-LO) metabolites from endogenous AA in ionophore A-23187-stimulated cells. In addition, colchicine inhibited the total release of AA as well as prostanoids to a lesser extent. Similar effects were observed with the other microtubule-disruptive agents nocodazole and vinblastine, and 5-LO product formation stimulated by the particulate agonist zymosan was inhibited as well. A selective inhibitory effect of colchicine on the 5-LO pathway was demonstrated by monitoring the synthesis of 5-LO products from exogenously supplied AA. Cell-free enzyme assays showed that this effect was not through a direct inhibition of the 5-LO enzyme. Moreover, colchicine did not affect the translocation of 5-LO to the nuclear envelope. We next evaluated the effect of colchicine on the levels of the two 5-LO cofactors, ATP and Ca2+. Although colchicine did not affect ATP levels, it did abrogate the ionophore-induced increase in intracellular Ca2+ concentration; the inhibitory effect of colchicine on 5-LO metabolism in AM was partially overcome by stimulation with higher doses of A-23187. We conclude that microtubular disruption inhibits agonist-induced increase in intracellular Ca2+ concentration, with multiple consequences for AA metabolism. These include a reduction in the liberation of AA from membrane phospholipids as well as the selective inhibition of processing of AA by 5-LO.

Effects of granulocyte-macrophage colony-stimulating factor on eicosanoid production by mononuclear phagocytes.

Granulocyte-macrophage CSF (GM-CSF) primes granulocytes for leukotriene (LT) synthesis. Here, we examined the effects of GM-CSF on arachidonic acid (AA) metabolism in rat alveolar macrophages (AM), peritoneal macrophages, and human peripheral blood monocytes. Pretreatment of AMs with GM-CSF for 24 h significantly increased the synthesis of immunoreactive LTB4 upon subsequent stimulation with calcium ionophore. Enhanced LT synthesis required a minimum of 6 h of GM-CSF pretreatment, suggesting that protein synthesis was required for enhanced LT production; indeed, cycloheximide completely abolished the GM-CSF effect on LT synthesis. HPLC analysis confirmed that GM-CSF primed AMs for enhanced generation of LTB4, as well as the 5-lipoxygenase products LTC, and 5-HETE. Moreover, parallel increases in other AA metabolites and free AA were observed following GM-CSF pretreatment. The enhanced production of all AA metabolites indicated that GM-CSF up-regulated AA release. Consistent with this, whole cell lysates from GM-CSF-treated AMs demonstrated increased phospholipase A2 (PLA2) activity. The increased activity was resistant to DTT, indicating the involvement of a PLA2 other than the 14-kDa PLA2s. By immunoblot analysis, GM-CSF treatment caused an increase in the 85-kDa PLA2 protein comparable to the observed increase in PLA2 activity. Unlike AMs, neither peritoneal macrophages nor peripheral blood monocytes showed increased eicosanoid generation or increased expression of the 85-kDa PLA2 protein following GM-CSF pretreatment. These results indicate that GM-CSF increases the capacity of AMs, but not peritoneal macrophages or peripheral blood monocytes, to generate eicosanoids. This effect results from increased PLA2 activity, due at least in part to increased levels of the 85-kDa PLA2 protein.

Translocation and leukotriene synthetic capacity of nuclear 5-lipoxygenase in rat basophilic leukemia cells and alveolar macrophages.

Leukotriene (LT) synthesis involves the translocation of enzymatically active 5-lipoxygenase (5-LO) from a soluble site to a bound site, where it interacts with 5-lipoxygenase-activating protein (FLAP). In human polymorphonuclear leukocytes (PMNs), 5-LO moves from the cytosol to the nuclear envelope (NE) to interact with FLAP. However, 5-LO has recently been found within the nucleus, as well as the cytosol, of rat basophilic leukemia (RBL) cells and alveolar macrophages (AMs). To assess whether nuclear 5-LO can contribute to LT synthesis in these cells, we investigated whether this enzyme pool 1) translocates upon cell activation, 2) colocalizes with FLAP, and 3) is enzymatically active. By cell fractionation followed by immunoblotting, both cytosolic and nuclear soluble 5-LO decreased dramatically in RBL cells following activation with the calcium ionophore A23187. Concurrently, 5-LO increased in the pelletable nuclear pool, where FLAP was also detected. The loss of both cytosolic and nuclear soluble 5-LO, with concomitant increase exclusively at the NE, as well as co-localization with FLAP, were confirmed by indirect immunofluorescent and confocal microscopy. In AMs, the nuclear soluble pool of 5-LO moved to the NE, where FLAP was also found; however, the cytosolic 5-LO pool did not translocate. Application of these methods to PMNs confirmed that cytosolic 5-LO moved to the nuclear envelope and co-localized with FLAP. By cell-free assay, nuclear soluble proteins from both RBL cells and AMs, but not PMNs, were able to generate 5-LO products from arachidonate, and this was inhibited by the direct 5-LO inhibitor zileuton. Cytosolic proteins from all cell types also showed cell-free 5-LO activity. These results demonstrate three distinct patterns of 5-LO translocation that are specific for each cell type: translocation of only a cytosolic pool in PMNs, of only a nuclear pool in AMs, and of both cytosolic and nuclear pools in RBL cells. By virtue of its enzymatic activity and ability to translocate, nuclear 5-LO has the potential to contribute to LT synthesis in RBL cells and AMs. Finally, these results provide a foundation for considering the individual functions of discrete pools of 5-LO in future studies.