Effect of Arachidonic Acid Reacylation on Leukotriene Biosynthesis in Human Neutrophils Stimulated with Granulocyte-macrophage Colony-stimulating Factor and Formyl-methionyl-leucyl-phenylalanine

Abstract

Priming of human neutrophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) followed by treatment with formyl-methionyl-leucyl-phenylalanine (fMLP) stimulates cells in a physiologically relevant manner with modest 5-lipoxygenase activation and formation of leukotrienes. However, pretreatment of neutrophils with thimerosal, an organomercury thiosalicylic acid derivative, led to a dramatic increase (>50-fold) in the production of leukotriene B 4 and 5-hydroxyeicosatetraenoic acid, significantly higher than that observed after stimulation with calcium ionophore A23187. Little or no effect was observed with thimerosal alone or in combination with either GM-CSF or fMLP. Elevation of [Ca 2؉ ] i induced by thimerosal in neutrophils stimulated with GM-CSF/ fMLP was similar but more sustained compared with samples where thimerosal was absent. However, [Ca 2؉ ] i was significantly lower compared with calcium ionophore-treated cells, suggesting that a sustained calcium rise was necessary but not sufficient to explain the effects of this compound on the GM-CSF/fMLP-stimulated neutrophil. Thimerosal was found to directly inhibit neutrophil lysophospholipid:acyl-CoA acyltransferase activity at the doses that stimulate leukotriene production, and analysis of lysates from neutrophil preparations stimulated in the presence of thimerosal showed a marked increase in free arachidonic acid, supporting the inhibition of the reincorporation of this fatty acid into the membrane phospholipids as a mechanism of action for this compound. The dramatic increase in production of leukotrienes by neutrophils when a physiological stimulus such as GM-CSF/fMLP is employed in the presence of thimerosal suggests a critical regulatory role of arachidonate reacylation that limits leukotriene biosynthesis in concert with 5-lipoxygenase and cytosolic phospholipase A 2 ␣ activation.Neutrophils play an important role in natural immunity and function to eliminate microbes and dead tissues. They are also a major cell involved in the acute inflammatory response, since they respond rapidly to chemotactic stimuli and can be activated by cytokines produced primarily by macrophages to perform degradative and phagocytic functions. Upon activation at the site of inflammation, they release lysosomal enzymes and neutral proteinases, reactive oxygen species, including H 2 O 2 and O 2 .(1, 2), and metabolites of arachidonic acid (AA), 2 in particular leukotriene B 4 (LTB 4 ) (3). Biosynthesis of leukotrienes involves the release of AA from membrane phospholipids by Ca 2ϩ -dependent cytosolic phospholipase A 2 ␣ (cPLA 2 ␣) (4) and its conversion into 5-hydroperoxyeicosatetraenoic acid and subsequently leukotriene A 4 (LTA 4 ), catalyzed by 5-lipoxygenase (5-LO) (5). LTA 4 is then enzymatically converted to LTB 4 by LTA 4 -hydrolase (6) or to leukotriene C 4 (LTC 4 ) by the addition of a molecule of glutathione through the action of LTC 4 -synthase (7). The site for most of these biochemical events is the perinuclear region (8).Regulation of leukotrie...

Biosynthesis of eicosanoids and transcellular metabolism of leukotrienes in murine bone marrow cells

“…hPMNs synthesized much lower amounts of TXB 2 and PGE 2 than mBMCs. As reported previously (22), hPMNs did produce detectable levels of LTC 4 , possibly because of contamination with eosinophils, and significant quantities of 20-OH-LTB 4 (Fig. 1E), an v-oxidation derivative of LTB 4 mediated by CYP4F4 (26).…”

“…Eicosanoid production by mBMCs could also be initiated by the formylated peptide fMLP and the phagocytic stimulus zymosan, coated (opsonized) or uncoated with complement fragments (Table 1). The concentration of fMLP necessary to trigger a robust synthesis of LTs was 10 mM, significantly higher than that observed for hPMNs (0.1 mM) (22), suggesting lower expression of the formylated peptide receptor in less mature PMNs. This was consistent with recent findings that a concentration as high as 10 mM fMLP is necessary for full superoxide production and primary granule release by mouse bone marrow PMNs (20).…”

“…Human PMNs (hPMNs) were isolated from peripheral blood of healthy volunteers as described previously (22). Mouse bone marrow cells (mBMCs) were obtained from the femurs and tibias of mice as described previously (19).…”

Thyroid hormone responsive protein Spot14 enhances catalysis of fatty acid synthase in lactating mammary epithelium

“…Coexpression of Spot14 with Spot14-R was determined to be inhibitory, as Spot14 heterodimerizes with Spot14-R to prevent Spot14-R association with ACC and decrease activation of the hepatic de novo fatty acid synthesis pathway ( 43 ( 29,41 ). In mammary cancer cells, antisense Spot14 knock down decreased in acetonitrile to initiate derivatization and incubated at room temperature for 30 min ( 50 ). Samples were dried under N 2 , resulting pellets were resuspended in 1 ml of isooctane, vortexed, and 50 l was transferred into vials for GC-MS.…”

Dietary Intake, Neutrophil Fatty Acid Profile, Serum Antioxidant Vitamins and Oxygen Radical Absorbance Capacity in Patients with Ulcerative Colitis

“…An increase in the arachidonic acid concentration of phospholipids may be accompanied by a rise in phospholipase A2 activity within the intestinal mucosa (23). A significant increase of arachidonic acid reacylation in the stimulated neutrophils induced a dramatic increase of production of LTs (24). LTB4 is a chemotactic factor that facilitates the accumulation of neutrophils with the consequent production of reactive oxygen species causing damage from oxidative stress (25).…”