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)alpha activation.
Highlights
Marily by macrophages to perform degradative and phagocytic functions
Ca2ϩ binds to a calciumdependent phospholipid binding domain (C2) on cPLA2␣ that triggers its translocation from the cytosol to the membrane of the Golgi apparatus, nuclear envelope, and endoplasmic reticulum, where it can access its phospholipid substrate [9]. 5-LO activity is enhanced by ATP-dependent phosphorylation, and micromolar concentrations of Ca2ϩ are required for its catalytic activity and translocation from the cytosol to the nuclear membrane, where it interacts with the 5-LO-activating protein, which facilitates the conversion of arachidonic acid (AA) into leukotrienes by “presenting” the substrate to 5-LO [10]
The stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) and fMLP resulted in a modest production of total leukotriene B4 (LTB4) (0.24 Ϯ 0.09 ng/106 cells) and 5-HETE (0.22 Ϯ 0.08 ng/106 cells) (Fig. 1A, inset), with total LTB4 corresponding to the sum of LTB4 and the -oxidized metabolite 20-OH-LTB4
Summary
Materials—Salts and solvents were purchased from Fisher. Percoll was obtained from Amersham Biosciences. Neutrophils were centrifuged and resuspended in calcium/magnesium-free Hanks’ balanced salt solution (Invitrogen) at a concentration of 10 ϫ 106 cells/ml and primed with either GM-CSF (1 nM), tumor necrosis factor ␣ (10 ng/ml), or Kdo2-lipid A (100 ng/ml) for 30 min at 37 °C. After GM-CSF/fMLP stimulation, cells (e.g. 5 ϫ 106 neutrophils in 0.5 ml) were not lysed but centrifuged (1000 ϫ g, 5 min), and [15N5]adenosine (25 ng; Spectra Stable Isotopes, Columbia, MD) was added to the supernatant. Gas Chromatography (GC)/MS and Quantitation of Free Fatty Acids— One aliquot (corresponding to 1 ϫ 106 cells) of neutrophil lysate (1:2; H2O/ MeOH) was acidified with HCl (70 mM final concentration), and stable isotope-labeled fatty acid standards [13C4]palmitic, [d3]stearic, [d2]oleic, and [d8]arachidonic acid were added. The rate of conversion of the lysophosphatidylcholines into the corresponding phosphatidylcholines was expressed as a ratio between the peak areas of the product diacylphospholipid to the lysophospholipid substrate
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