Fetal hepatic expression of 5-lipoxygenase activating protein is confined to colonizing hematopoietic cells

Leukotriene C₄ is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C₄ synthase (LTC₄S) participate in its biosynthesis. We report evidence from insitu hybridization experiments that FLAP mRNA is abundantly expressed in fetal mouse liver from e11.5 until delivery. In contrast very little or no FLAP mRNA was detected in adult liver. The fetal expression in liver was not uniform but occurred in patches. Cells from e15.5 livers were fractionated by fluorescence activated cell sorting into hepatocytes and other CD45⁻ cells and CD45⁺ hematopoietic cells. The latter were further separated into immature (Lin⁻) and mature (Lin⁺) cells and analyzed for FLAP mRNA content by quantitative RT-PCR. FLAP mRNA expression was confined to CD45⁺ cells and the mature cells had approximately 4-fold higher FLAP mRNA levels compared to the immature cells.     

An experimental cell-based model for studying the cell biology and molecular pharmacology of 5-lipoxygenase-activating protein in leukotriene biosynthesis

BackgroundSubcellular distribution of 5-lipoxygenase (5-LO) to the perinuclear region and interaction with the 5-LO-activating protein (FLAP) are assumed as key steps in leukotriene biosynthesis and are prone to FLAP antagonists.MethodsFLAP and/or 5-LO were stably expressed in HEK293 cells, 5-LO products were analyzed by HPLC, and 5-LO and FLAP subcellular localization was visualized by immunofluorescence microscopy.Results5-LO and FLAP were stably expressed in HEK293 cells, and upon Ca^2 +-ionophore A23187 stimulation exogenous AA was efficiently transformed into the 5-LO products 5-hydro(pero)xyeicosatetraenoic acid (5-H(p)ETE) and the trans-isomers of LTB₄. A23187 stimulation caused 5-LO accumulation at the nuclear membrane only when FLAP was co-expressed. Unexpectedly, A23187 stimulation of HEK cells expressing 5-LO and FLAP without exogenous AA failed in 5-LO product synthesis. HEK cells liberated AA in response to A23187, and transfected HEK cells expressing 12-LO generated 12-HETE after A23187 challenge from endogenous AA. FLAP co-expression increased 5-LO product formation in A23187-stimulated cells at low AA concentrations. Only in cells expressing FLAP and 5-LO, the FLAP antagonist MK886 blocked FLAP-mediated increase in 5-LO product formation, and prevented 5-LO nuclear membrane translocation and co-localization with FLAP.ConclusionThe cellular biosynthesis of 5-LO products from endogenously derived substrate requires not only functional 5-LO/FLAP co-localization but also additional prerequisites which are dispensable when exogenous AA is supplied; identification of these determinants is challenging.General significanceWe present a cell model to study the role of FLAP as 5-LO interacting protein in LT biosynthesis in intact cells and for characterization of putative FLAP antagonists.     

IL-5 increases expression of 5-lipoxygenase-activating protein and translocates 5-lipoxygenase to the nucleus in human blood eosinophils.

Cysteinyl-leukotrienes are potent bronchoconstrictor mediators synthesized by the 5-lipoxygenase (5-LO) pathway. Eosinophilopoietic cytokines such as IL-5 enhance cysteinyl-leukotriene synthesis in eosinophils in vitro, mimicking changes in eosinophils from asthmatic patients, but the mechanism is unknown. We hypothesized that IL-5 induces the expression of 5-LO and/or its activating protein FLAP in eosinophils, and that this might be modulated by anti-inflammatory corticosteroids. Compared with control cultures, IL-5 increased the proportion of normal blood eosinophils immunostaining for FLAP (65 +/- 4 vs 34 +/- 4%; p < 0.0001), enhanced immunoblot levels of FLAP by 51 +/- 14% (p = 0.03), and quadrupled ionophore-stimulated leukotriene C4 synthesis from 5.7 to 20.8 ng/106 cells (p < 0.02). IL-5 effects persisted for 24 h and were abolished by cycloheximide and actinomycin D. The proportion of FLAP+ eosinophils was also increased by dexamethasone (p < 0.0001). Neither IL-5 nor dexamethasone altered 5-LO expression, but IL-5 significantly increased 5-LO immunofluorescence localizing to eosinophil nuclei. Compared with normal subjects, allergic asthmatic patients had a greater proportion of circulating FLAP+ eosinophils (46 +/- 6 vs 27 +/- 3%; p < 0.03) and a smaller IL-5-induced increase in FLAP immunoreactivity (p < 0.05). Thus, IL-5 increases FLAP expression and translocates 5-LO to the nucleus in normal blood eosinophils in vitro. This is associated with an enhanced capacity for cysteinyl-leukotriene synthesis and mimics in vivo increases in FLAP expression in eosinophils from allergic asthmatics.     

A23187-induced translocation of 5-lipoxygenase in osteosarcoma cells.

In a previous study, osteosarcoma cells expressing both 5-lipoxygenase (5-LO) and 5 lipoxygenase-activating protein (FLAP) synthesized leukotrienes upon A23187 stimulation (Dixon, R. A. F., R. E. Diehl, E. Opas, E. Rands, P. J. Vickers, J. F. Evans, J. W. Gillard, and D. K. Miller. 1990. Nature (Lond.). 343:282-284). Osteosarcoma cells expressing 5-LO but not expressing FLAP were unable to synthesize leukotrienes. Thus, it was determined that FLAP was required for the cellular synthesis of leukotrienes. To examine the role of FLAP in A23187-induced translocation of 5-LO to a membrane fraction, we have studied the A23187-stimulated translocation of 5-LO in osteosarcoma cells expressing both 5-LO and FLAP, and in osteosarcoma cells expressing 5-LO only. We demonstrate that in cells expressing both 5-LO and FLAP, 5-LO translocates to membranes in response to A23187 stimulation. This 5-LO translocation is inhibited when cells are stimulated in the presence of MK-886. In osteosarcoma cells expressing 5-LO but not expressing FLAP, 5-LO is able to associate with membranes following A23187 stimulation. In contrast to the cells containing both 5-LO and FLAP, MK-886 is unable to prevent 5-LO membrane association in cells transfected with 5-LO alone. Therefore, we have demonstrated that in this cell system, 5-LO membrane association and activation can be separated into at least two distinct steps: (1) calcium-dependent movement of 5-LO to membranes without product formation, which can occur in the absence of FLAP (membrane association), and (2) activation of 5-LO with product formation, which is FLAP dependent and inhibited by MK-886 (enzyme activation).     

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.     

In situ amplification of 5-lipoxygenase and 5-lipoxygenase-activating protein in allergic airway inflammation and inhibition by leukotriene blockade

Leukotrienes are important mediators of the eosinophilic influx and mucus hypersecretion in the lungs in a murine model of asthma. We used in situ PCR in this model of human asthma to detect lung mRNA for 5-lipoxygenase (5-LO) and 5-LO-activating protein (FLAP), key proteins necessary for leukotriene synthesis. Lung tissue was obtained on day 28 from mice treated with i.p. (days 0 and 14) and intranasal (days 14, 25, 26, and 27) OVA or saline. After fixation, the tissue sections underwent protease- and RNase-free DNase digestion, before in situ RT-PCR using target-specific cDNA amplification. 5-LO and FLAP-specific mRNA was visualized by a digoxigenin detection system, and positive cells were analyzed by morphometry. 5-LO and FLAP-specific mRNA and protein were associated primarily with eosinophils and alveolar macrophages in the airways and pulmonary blood vessels in OVA-sensitized/challenged mice. 5-LO and FLAP protein expression increased on a per-cell basis in alveolar macrophages of OVA-treated mice compared with saline controls. Pulmonary blood vessel endothelial cells were also positive for 5-LO, FLAP mRNA, and protein. 5-LO inhibition significantly decreased 5-LO and FLAP-specific mRNA and protein expression in the lung inflammatory cells and endothelial cells. These studies demonstrate a marked increase in key 5-LO pathway proteins in the allergic lung inflammatory response and an important immunomodulatory effect of leukotriene blockade to decrease 5-LO and FLAP gene expression.     

Molecular basis of the specific subcellular localization of the C2-like domain of 5-lipoxygenase.

The activation of 5-lipoxygenase (5-LO) involves its calcium-dependent translocation to the nuclear envelope, where it catalyzes the two-step transformation of arachidonic acid into leukotriene A(4), leading to the synthesis of various leukotrienes. To understand the mechanism by which 5-LO is specifically targeted to the nuclear envelope, we studied the membrane binding properties of the amino-terminal domain of 5-LO, which has been proposed to have a C2 domain-like structure. The model building, electrostatic potential calculation, and in vitro membrane binding studies of the isolated C2-like domain of 5-LO and selected mutants show that this Ca(2+)-dependent domain selectively binds zwitterionic phosphatidylcholine, which is conferred by tryptophan residues (Trp(13), Trp(75), and Trp(102)) located in the putative Ca(2+)-binding loops. The spatiotemporal dynamics of the enhanced green fluorescence protein-tagged C2-like domain of 5-LO and mutants in living cells also show that the phosphatidylcholine selectivity of the C2-like domain accounts for the specific targeting of 5-LO to the nuclear envelope. Together, these results show that the C2-like domain of 5-LO is a genuine Ca(2+)-dependent membrane-targeting domain and that the subcellular localization of the domain is governed in large part by its membrane binding properties.     

Development of smart cell-free and cell-based assay systems for investigation of leukotriene C4 synthase activity and evaluation of inhibitors

Cysteinyl leukotrienes (cys-LTs) cause bronchoconstriction in anaphylaxis and asthma. They are formed by 5-lipoxygenase (5-LOX) from arachidonic acid (AA) yielding the unstable leukotriene A₄ (LTA₄) that is subsequently conjugated with glutathione (GSH) by LTC₄ synthase (LTC₄S). Cys-LT receptor antagonists and LTC₄S inhibitors have been developed, but only the former have reached the market. High structural homology to related enzymes and lack of convenient test systems due to instability of added LTA₄ have hampered the development of LTC₄S inhibitors. We present smart cell-free and cell-based assay systems based on in situ-generated LTA₄ that allow studying LTC₄S activity and investigating LTC₄S inhibitors. Co-incubations of microsomes from HEK293 cells expressing LTC₄S with isolated 5-LOX efficiently converted exogenous AA to LTC₄ (~ 1.3 μg/200 μg protein). Stimulation of HEK293 cells co-expressing 5-LOX and LTC₄S with Ca^2 +-ionophore A23187 and 20 μM AA resulted in strong LTC₄ formation (~ 250 ng/10⁶ cells). MK-886, a well-known 5-LOX activating protein (FLAP) inhibitor that also acts on LTC₄S, consistently inhibited LTC₄ formation in all assay types (IC₅₀ = 3.1–3.5 μM) and we successfully confirmed TK04a as potent LTC₄S inhibitor in these assay systems (IC₅₀ = 17 and 300 nM, respectively). We demonstrated transcellular LTC₄ biosynthesis between neutrophils or 5-LOX-expressing HEK293 cells that produce LTA₄ from AA and HEK293 cells expressing LTC₄S that transform LTA₄ to LTC₄. In conclusion, our assay approaches are advantageous as the substrate LTA₄ is generated in situ and are suitable for studying enzymatic functionality of LTC₄S including site-directed mutations and evaluation of LTC₄S inhibitors.     

Location,Location, Location: Compartmentalization of Early Events in Leukotriene Biosynthesis

Leukotrienes (LTs), derived from arachidonic acid (AA) released from the membrane by the action of phospholipase A₂, are potent lipid mediators of the inflammatory response. In 1983, Dahlén et al. demonstrated that LTC₄, LTD₄, and LTE₄ mediate antigen-induced constriction of bronchi in tissue obtained from subjects with asthma (Dahlén, S. E., Hansson, G., Hedqvist, P., Björck, T., Granström, E., and Dahlén, B. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1712–1716). Over the last 25+ years, substantial progress has been made in understanding how LTs exert their effects, and a broader appreciation for the numerous biological processes they mediate has emerged. LT biosynthesis is initiated by the action of 5-lipoxygenase (5-LOX), which catalyzes the transformation of AA to LTA₄ in a two-step reaction. Ca²⁺ targets 5-LOX to the nuclear membrane, where it co-localizes with the 5-LOX-activating protein FLAP and, when present, the downstream enzyme LTC₄ synthase, both transmembrane proteins. Crystal structures of the AA-metabolizing LOXs, LTC₄ synthase, and FLAP combined with biochemical data provide a framework for understanding how subcellular organizations optimize the biosynthesis of these labile hydrophobic signaling compounds, which must navigate pathways that include both membrane and soluble enzymes. The insights these structures afford and the questions they engender are discussed in this minireview.     

The nuclear membrane leukotriene synthetic complex is a signal integrator and transducer

Leukotrienes (LTs) are lipid-signaling molecules derived from arachidonic acid (AA) that initiate and amplify inflammation. To initiate LT formation, the 5-lipoxygenase (5-LO) enzyme translocates to nuclear membranes, where it associates with its scaffold protein, 5-lipoxygenase–activating protein (FLAP), to form the core of the multiprotein LT synthetic complex. FLAP is considered to function by binding free AA and facilitating its use as a substrate by 5-LO to form the initial LT, LTA₄. We used a combination of fluorescence lifetime imaging microscopy, cell biology, and biochemistry to identify discrete AA-dependent and AA-independent steps that occur on nuclear membranes to control the assembly of the LT synthetic complex in polymorphonuclear leukocytes. The association of AA with FLAP changes the configuration of the scaffold protein, enhances recruitment of membrane-associated 5-LO to form complexes with FLAP, and controls the closeness of this association. Granulocyte monocyte colony–stimulating factor provides a second AA-independent signal that controls the closeness of 5-LO and FLAP within complexes but not the number of complexes that are assembled. Our results demonstrate that the LT synthetic complex is a signal integrator that transduces extracellular signals to modulate the interaction of 5-LO and FLAP.     

4-Hydroxynonenal enhances CD36 expression on murine macrophages via p38 MAPK-mediated activation of 5-lipoxygenase

Increased levels of 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LO) coexist in atherosclerotic lesions but their relationship in atherogenesis is unclear. This study investigated the role of 5-LO in HNE-induced CD36 expression and macrophage foam cell formation, and the link between HNE and 5-LO. In J774A.1 murine macrophages, HNE (10 μM) enhanced CD36 expression in association with an increased uptake of oxLDL, which was blunted by inhibition of 5-LO with MK886, a 5-LO inhibitor, or with 5-LO siRNA. In peritoneal macrophages from 5-LO-deficient mice, HNE-induced CD36 expression was markedly attenuated, confirming a pivotal role of 5-LO in HNE-induced CD36 expression. In an assay for 5-LO activity, stimulation of macrophages with HNE led to increased leukotriene B₄ production in the presence of exogenous arachidonic acid in association with an increased association of 5-LO to the nuclear membrane. Among the mitogen-activated protein kinase (MAPK) pathways involved in 5-LO phosphorylation, HNE predominantly activated p38 MAPK in macrophages, and the p38 MAPK inhibitor SB203580, but not an extracellular signal-regulated kinase inhibitor, suppressed HNE-induced LTB₄ production. Collectively, these data suggest that p38 MAPK-mediated activation of 5-LO by HNE might enhance CD36 expression, consequently leading to the formation of macrophage foam cells.     

Trout thrombocytes contain 12- but not 5-lipoxygenase activity

Fish thrombocytes are thought to be the evolutionary forerunners of mammalian platelets. Thrombocyte preparations made by conventional methods, such as density gradient centrifugation, contain other cell types such as neutrophilic granulocytes and lymphocytes that could interfere with subsequent experiments. In this study, rainbow trout thrombocytes were separated by density gradient centrifugation and further purified by magnetic cell sorting (MACS) using the thrombocyte specific monoclonal antibody, 30D8. Thrombocyte purity was assessed by reactivity to 30D8 using flow cytometry and immunocytochemistry. Following purification by density gradient centrifugation, thrombocytes were 66.9±9.2% (mean value±S.E.M., n=3) pure. Further purification by MACS significantly increased thrombocyte purity to 97.3±0.6%, whereas only 1.4% of the MACS ?ve fraction were identified as these cells. Incubation of thrombocytes isolated by density gradient alone with calcium ionophore, A23187, generated a range of eicosanoids derived from arachidonic or eicosapentaenoic acids, namely, leukotriene (LT)B₄, LTB₅, lipoxin (LX)A₄, LXA₅, 12-hydroxyeicosatetraenoic acid (12-HETE) and 12-hydroxyeicosapentaenoic acid (12-HEPE). A similar eicosanoid generation profile was observed for cells in the MACS ?ve fraction; however, MACS +ve cells (thrombocytes) generated no 4 or 5 series LT or LX but did generate significant amounts of the 12-lipoxygenase (LO) products, 12-HETE and 12-HEPE. These results indicate that trout thrombocytes contain no demonstrable 5-LO activity and like their mammalian counterparts possess 12-LO activity.     

Interplay of cysteinyl leukotrienes and TGF-β in the activation of hepatic stellate cells from Schistosoma mansoni granulomas

Hepatic stellate cells (HSCs) have a critical role in liver physiology, and in the pathogenesis of liver inflammation and fibrosis. Here, we investigated the interplay between leukotrienes (LT) and TGF-β in the activation mechanisms of HSCs from schistosomal granulomas (GR-HSCs). First, we demonstrated that GR-HSCs express 5-lipoxygenase (5-LO), as detected by immunolocalization in whole cells and confirmed in cell lysates through western blotting and by mRNA expression through RT-PCR. Moreover, mRNA expression of 5-LO activating protein (FLAP) and LTC₄-synthase was also documented, indicating that GR-HSCs have the molecular machinery required for LT synthesis. Morphological analysis of osmium and Oil-Red O-stained HSC revealed large numbers of small lipid droplets (also known as lipid bodies). We observed co-localization of lipid droplet protein marker (ADRP) and 5-LO by immunofluorescence microscopy. We demonstrated that GR-HSCs were able to spontaneously release cysteinyl-LTs (CysLTs), but not LTB_4, into culture supernatants. CysLT production was highly enhanced after TGF-β-stimulation. Moreover, the 5-LO inhibitor zileuton and 5-LO gene deletion were able to inhibit the TGF-β-stimulated proliferation of GR-HSCs, suggesting a role for LTs in HSC activation. Here, we extend the immunoregulatory function of HSC by demonstrating that HSC from liver granulomas of schistosome-infected mouse are able to release Cys-LTs in a TGF-β-regulated manner, potentially impacting pathogenesis and liver fibrosis in schistosomiasis.     

Leukotriene-C4 Synthase,a Critical Enzyme in the Activation of Store-independent Orai1/Orai3 Channels,Is Required for Neointimal Hyperplasia

Leukotriene-C₄ synthase (LTC₄S) generates LTC₄ from arachidonic acid metabolism. LTC₄ is a proinflammatory factor that acts on plasma membrane cysteinyl leukotriene receptors. Recently, however, we showed that LTC₄ was also a cytosolic second messenger that activated store-independent LTC₄-regulated Ca²⁺ (LRC) channels encoded by Orai1/Orai3 heteromultimers in vascular smooth muscle cells (VSMCs). We showed that Orai3 and LRC currents were up-regulated in medial and neointimal VSMCs after vascular injury and that Orai3 knockdown inhibited LRC currents and neointimal hyperplasia. However, the role of LTC₄S in neointima formation remains unknown. Here we show that LTC₄S knockdown inhibited LRC currents in VSMCs. We performed in vivo experiments where rat left carotid arteries were injured using balloon angioplasty to cause neointimal hyperplasia. Neointima formation was associated with up-regulation of LTC₄S protein expression in VSMCs. Inhibition of LTC₄S expression in injured carotids by lentiviral particles encoding shRNA inhibited neointima formation and inward and outward vessel remodeling. LRC current activation did not cause nuclear factor for activated T cells (NFAT) nuclear translocation in VSMCs. Surprisingly, knockdown of either LTC₄S or Orai3 yielded more robust and sustained Akt1 and Akt2 phosphorylation on Ser-473/Ser-474 upon serum stimulation. LTC₄S and Orai3 knockdown inhibited VSMC migration in vitro with no effect on proliferation. Akt activity was suppressed in neointimal and medial VSMCs from injured vessels at 2 weeks postinjury but was restored when the up-regulation of either LTC₄S or Orai3 was prevented by shRNA. We conclude that LTC₄S and Orai3 altered Akt signaling to promote VSMC migration and neointima formation.     

Roles of 5-lipoxygenase activating protein in cell proliferation and apoptosis

5-Lipoxygenase activating protein (FLAP) functions as a facilitator of 5-lipoxygenase (5-LOX) activity. However, on the basis of the induction of apoptosis by the FLAP inhibitor MK886 in cells lacking 5-LOX, it is possible that this fatty acid-binding protein has other activities. This study was designed to examine potential roles of FLAP in apoptosis and cell proliferation. Overexpression of FLAP protein (2.2-fold) was achieved by stable transfection of IL-3-dependent murine prolymphoid progenitor cells (FL5.12) with a construct expressing the cDNA under a CMV promoter. The overexpressed protein was localized to nuclear membranes as with endogenous FLAP. The initial growth rate of FLAP-transfected cells was greater than that of control cells. After 48 h, when cell density had increased, the growth rate of FLAP-transfected cells declined substantially and there and there was a decrease in viability relative to control transfected cells. The FLAP-transfected cells were also more susceptible to withdrawal of IL-3 than were control cells. There was, however, no difference between FLAP and control cells in their susceptibility to MK886, NDGA, or etoposide during the log growth phase. Overexpression of FLAP did not alter Bcl-x_L protein expression, but did decrease Bax protein and somewhat increased COX-1 and COX-2 mRNA levels. The failure of increased FLAP to alter susceptibility to MK886 provides further support to the concept that this agent induces apoptosis by mechanisms unrelated to FLAP. The data also suggest that FLAP can affect cell proliferation.     

Differential localization of 5- and 15-lipoxygenases to the nuclear envelope in RAW macrophages.

Leukotriene formation is initiated in myeloid cells by an increase in intracellular calcium and translocation of 5-lipoxygenase from the cytoplasm to the nuclear envelope where it can utilize arachidonic acid. Monocyte- macrophages and eosinophils also express 15-lipoxygenase, which converts arachidonic acid to 15(S)-hydroxyeicosatetraenoic acid. Enhanced green fluorescent 5-lipoxygenase (5-LO) and 15-lipoxygenase (15-LO) fusion proteins were expressed in the cytoplasm of RAW 264.7 macrophages. Only 5-lipoxygenase translocated to the nuclear envelope after cell stimulation, suggesting that differential subcellular compartmentalization can regulate the generation of leukotrienes versus 15(S)-hydroxyeicosatetraenoic acid in cells that possess both lipoxygenases. A series of truncation mutants of 5-LO were created to identify putative targeting domains; none of these mutants localized to the nuclear envelope. The lack of targeting of 15-LO was then exploited to search for specific targeting motifs in 5-LO, by creating 5-LO/15-LO chimeric molecules. The only chimera that could sustain nuclear envelope translocation was one which involved replacement of the N-terminal 237 amino acids with the corresponding segment of 15-LO. Significantly, no discrete targeting domain could be identified in 5-LO, suggesting that sequences throughout the molecule are required for nuclear envelope localization.     

The shunt from the cyclooxygenase to lipoxygenase pathway in human osteoarthritic subchondral osteoblasts is linked with a variable expression of the 5-lipoxygenase-activating protein

Osteoarthritis (OA) is characterized by articular cartilage degradation and hypertrophic bone changes with osteophyte formation and abnormal bone remodeling. Two groups of OA patients were identified via the production of variable and opposite levels of prostaglandin E₂ (PGE₂) or leukotriene B₄ (LTB₄) by subchondral osteoblasts, PGE₂ levels discriminating between low and high subgroups. We studied whether the expression of 5-lipoxygenase (5-LO) or 5-LO-activating protein (FLAP) is responsible for the shunt from prostaglandins to leukotrienes. FLAP mRNA levels varied in low and high OA groups compared with normal, whereas mRNA levels of 5-LO were similar in all osteoblasts. Selective inhibition of cyclooxygenase-2 (COX-2) with NS-398-stimulated FLAP expression in the high OA osteoblasts subgroup, whereas it was without effect in the low OA osteoblasts subgroup. The addition of PGE₂ to the low OA osteoblasts subgroup decreased FLAP expression but failed to affect it in the high OA osteoblasts subgroup. LTB₄ levels in OA osteoblasts were stimulated about twofold by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) plus transforming growth factor-β (TGF-β), a situation corresponding to their effect on FLAP mRNA levels. Treatments with 1,25(OH)₂D₃ and TGF-β also modulated PGE₂ production. TGF-β stimulated PGE₂ production in both OA osteoblast groups, whereas 1,25(OH)₂D₃ alone had a limited effect but decreased the effect of TGF-β in the low OA osteoblasts subgroup. This modulation of PGE₂ production was mirrored by the synthesis of COX-2. IL-18 levels were only slightly increased in a subgroup of OA osteoblasts compared with normal; however, no relationship was observed overall between IL-18 and PGE₂ levels in normal and OA osteoblasts. These results suggest that the shunt from the production of PGE₂ to LTB₄ is through regulation of the expression of FLAP, not 5-LO, in OA osteoblasts. The expression of FLAP in OA osteoblasts is also modulated differently by 1,25(OH)₂D₃ and TGF-β depending on their endogenous low and high PGE₂ levels.     

Prolonged exposure to lipopolysaccharide inhibits macrophage 5-lipoxygenase metabolism via induction of nitric oxide synthesis

LPS from bacteria can result in the development of sepsis syndrome and acute lung injury. Although acute exposure to endotoxin primes leukocytes for enhanced synthesis of leukotrienes (LT), little is known about the effect of chronic exposure. Therefore, we determined the effect of prolonged LPS treatment on 5-lipoxygenase (5-LO) metabolism of arachidonic acid in alveolar macrophages (AM) and in peripheral blood monocytes. Pretreatment of AM with LPS caused time- and dose-dependent suppression of LT synthetic capacity. LPS pretreatment failed to inhibit arachidonic acid (AA) release. The fact that LPS inhibited LT synthesis from endogenous AA more than from exogenous AA suggested an effect on 5-LO-activating protein (FLAP). In addition, an inhibitory effect of LPS treatment on AM 5-LO activity was suggested by cell-free 5-LO enzyme assay. No effect on the expression of either 5-LO or FLAP proteins was observed. New protein synthesis was necessary for LPS-induced reduction of 5-LO metabolism in AM, and immunoblotting demonstrated marked induction of NO synthase (NOS). Inhibition by LPS was reproduced by an NO donor and was abrogated by inhibitors of constitutive and inducible NOS. Compared with AM, peripheral blood monocytes exhibited no suppression by LPS of 5-LO metabolism and no induction of inducible NOS. We conclude that prolonged exposure to LPS impairs AM 5-LO metabolism by NO-mediated suppression of both 5-LO and FLAP function. Because LT contribute to antimicrobial defense, this down-regulation of 5-LO metabolism may contribute to the increased susceptibility to pneumonia in patients following sepsis.     

Contrasting effect of interleukin-4 on the expression of cytosolic phospholipase A2, 5-lipoxygenase and 5-lipoxygenase-activating protein in peritoneal macrophages from control and ovalbumin-sensitized rats

Interleukin-4 (IL-4), which has been widely described as an anti-inflammatory cytokine, can also exert proinflammatory effects. In this study, we extend these findings to demonstrate, in an allergic model, the dual effect of IL-4 on arachidonic acid (AA) metabolism in macrophages. In peritoneal macrophages from control rats (cPM), IL-4 had no effect on cPLA2 and 5-LO expression, but increased FLAP expression without affecting basal and A23187- or PMA-challenged arachidonic acid (AA) metabolism. In contrast, in peritoneal macrophages from ovalbumin-sensitized rats (sPM), IL-4 decreased cPLA2, 5-LO and FLAP expression and PMA-challenged eicosanoid production. A23187-challenged AA metabolism of sPM was not affected by IL-4 pretreatment. Thus, IL-4 acts differently on cPLA2, 5-LO and FLAP expression and AA metabolism in peritoneal macrophages depending on their resident or sensitization-induced differentiated status.