Reactive oxygen intermediate-dependent NF-kappaB activation by interleukin-1beta requires 5-lipoxygenase or NADPH oxidase activity.
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We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-kappaB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells. Stimulation of lymphoid cells with interleukin-1beta (IL-1beta) led to ROI production and NF-kappaB activation, which could both be blocked by antioxidants or FLAP inhibitors, confirming that 5-LOX was the source of ROIs and was required for NF-kappaB activation in these cells. IL-1beta stimulation of epithelial cells did not generate any ROIs and NF-kappaB induction was not influenced by 5-LOX inhibitors. However, reintroduction of a functional 5-LOX system in these cells allowed ROI production and 5-LOX-dependent NF-kappaB activation. In monocytic cells, IL-1beta treatment led to a production of ROIs which is independent of the 5-LOX enzyme but requires the NADPH oxidase activity. This pathway involves the Rac1 and Cdc42 GTPases, two enzymes which are not required for NF-kappaB activation by IL-1beta in epithelial cells. In conclusion, three different cell-specific pathways lead to NF-kappaB activation by IL-1beta: a pathway dependent on ROI production by 5-LOX in lymphoid cells, an ROI- and 5-LOX-independent pathway in epithelial cells, and a pathway requiring ROI production by NADPH oxidase in monocytic cells. (+info)
Relationship of arachidonic acid metabolizing enzyme expression in epithelial cancer cell lines to the growth effect of selective biochemical inhibitors.
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Arachidonic acid (AA) metabolizing enzymes are emerging as significant mediators of growth stimulation for epithelial cells. The relative contribution of the various family members of AA metabolizing enzymes to epithelial cancer cell growth is not known. To study this question, we first analyzed a series of epithelial cancer cells to establish the relative frequency of expression for the various enzymes. We analyzed the expression of five AA metabolizing enzymes as well as 5-lipoxygenase activating protein (FLAP) in a panel of human epithelial cancer cell lines (n = 20) using reverse transcription-PCR. From this analysis, we found that cyclooxygenase-1 (COX-1), 5-lipoxygenase (5-LOX), and FLAP were universally expressed in all cancer cell lines tested. For the remaining enzymes, the expression of COX-2, 12-LOX, and 15-LOX varied among cell lines, 60, 35, and 90%, respectively. Although the pattern of expression varied among the different cell types, all of the enzymes were expressed in all major cancer histologies. Using a panel of selective biochemical AA metabolizing enzyme inhibitors, we then evaluated the effect of these agents on cell lines with known expression status for the AA metabolizing enzymes. For the enzymes that were not universally expressed, growth inhibition by selective biochemical inhibitors did not closely correlate with the expression status of specific enzymes (P > 0.05). For the universally expressed enzymes, the LOX inhibitors were more potent growth inhibitors than the COX inhibitors. The frequent expression of the AA metabolizing enzymes suggests that AA metabolism pathway may be modulated in response to xenobiotic exposure during carcinogenesis. Although establishing a priori AA metabolizing enzyme status was not consistently informative about what AA metabolizing enzyme inhibition would be most growth inhibitory, the frequent inhibition of many epithelial cancers by these biochemical inhibitors opens a new avenue for cancer therapy and intervention in carcinogenesis. (+info)
The 5-lipoxygenase-activating protein (FLAP) inhibitor, MK886, induces apoptosis independently of FLAP.
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The ability of various inhibitors of lipoxygenase (LOX) enzymes and 5-lipoxygenase-activating protein (FLAP) to induce apoptosis has implicated these pathways in the mechanism(s) of this form of cell death. Although FLAP plays an important role in 5-LOX activity, this protein is found at high levels in some cells lacking LOX, suggesting it might mediate other effects. Furthermore, the concentration of MK886, a FLAP inhibitor, required to induce apoptosis is approximately 100-fold more than that required to inhibit LOX, and this compound remains effective in cells lacking LOX. The present study examines the role of FLAP in MK886-induced apoptosis. MK886 induced apoptosis in WSU cells, a human chronic lymphocytic leukaemia cell line that lacks FLAP protein and mRNA, suggesting that this agent is acting independently of FLAP. This conclusion was further supported by the fact that a more specific FLAP inhibitor, MK591, induced only minimal apoptosis in FL5.12 cells, a murine prolymphoid cell line containing FLAP. The role of FLAP was examined more directly by decreasing its expression by more than 50% in FL5.12 cells treated with 10 microM of an antisense oligonucleotide for 48h. This change in FLAP was not accompanied by any increase in apoptosis. Furthermore, FLAP-depleted cells exhibited the same level of apoptosis 8 h after treatment with 10 microM MK886, as did control cells. The increased fluorescence seen in MK886-treated cells loaded with carboxydichlorofluorescein indicates that oxidative reactions are stimulated by this compound, possibly via the release of fatty acids from fatty acid-binding proteins and their subsequent oxidation. (+info)
Expression of 5-lipoxygenase and 5-lipoxygenase-activating protein in human fetal membranes throughout pregnancy and at term.
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Lipoxygenase metabolites may be involved in human parturition. 5-lipoxygenase (5-LOX) catalyses the first steps in the synthesis of leukotrienes from arachidonic acid, and its activity is dependent on 5-LOX activating protein (FLAP). The expression of 5-LOX and FLAP were investigated in fetal membranes to determine whether there are changes with gestational age or at term with the onset of labour. No significant differences were found in the expression of 5-LOX or FLAP mRNA in the amnion at different gestational ages or at term. In the chorion-decidua, 5-LOX mRNA expression was significantly higher in the first trimester of pregnancy than in the second and third trimesters. At term, there was a significant increase in both 5-LOX mRNA and protein expression in the chorion-decidua in the time after labour, compared with the time before labour. The expression of FLAP mRNA was also significantly higher in the chorion-decidua in the first trimester of pregnancy compared with the third trimester, and at term in the time after labour compared with the time before labour. Expression of FLAP protein was not studied, as an antibody is not currently available. These results are consistent with a role for 5-LOX and FLAP in the control of parturition at term, and also suggest an involvement earlier in pregnancy. (+info)
IL-5 increases expression of 5-lipoxygenase-activating protein and translocates 5-lipoxygenase to the nucleus in human blood eosinophils.
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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. (+info)
Increased cyclooxygenase-2 and 5-lipoxygenase activating protein expression in peritoneal macrophages during ovalbumin immunization of mice and cytosolic phospholipase A(2) activation after antigen challenge.
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The present study investigates phenotypic and functional differentiation of peritoneal macrophages during ovalbumin-induced subcutaneous immunization of mice. For the first time we show that, in mouse peritoneal macrophages, ovalbumin immunization induces an increase in cyclooxygenase-2 (COX-2) and 5-lipoxygenase activating protein (FLAP) expression whereas it inhibits cytosolic phospholipase A(2) (cPLA2) expression. The study of arachidonic acid (AA) metabolism in peritoneal macrophages from control (cPM) and ovalbumin-immunized (iPM) mice shows that the reduced cPLA2 expression is correlated to a reduced basal AA metabolism, but is not a limiting factor for the opsonized zymosan-, PMA-, or A23187-triggered AA metabolism. We also show that in vitro ovalbumin challenge induces, only in iPM, cPLA2 activation through phosphorylation of serine residues, via a mechanism involving MAP kinases, and through increased intracellular calcium concentrations, leading to eicosanoid production. In parallel, we report that, in peritoneal macrophages, ovalbumin immunization induces the expression of CD23, the low affinity receptor for IgEs known for its involvement in allergic diseases. Thus, the modified expression of the enzymes involved in AA metabolism and the difference of response of cPM and iPM toward the antigen are important elements to understand the underlying mechanisms of ovalbumin-induced allergic responses. (+info)
Prolonged exposure to lipopolysaccharide inhibits macrophage 5-lipoxygenase metabolism via induction of nitric oxide synthesis.
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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. (+info)
In situ amplification of 5-lipoxygenase and 5-lipoxygenase-activating protein in allergic airway inflammation and inhibition by leukotriene blockade.
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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. (+info)