Sulfatides inhibit leukotriene synthesis in human polymorphonuclear granulocytes by a mechanism involving lipid rearrangement in intracellular membranes.

Sulfatides - sulfated derivatives of galactocerebroside - are endogenous ligands for P- and L-selectins and are able to induce intracellular signaling in neutrophils through a L-selectin dependent pathway. Sulfatides are implicated in a variety of physiological functions and have been found to suppress the synthesis of 5-lipoxygenase (5-LO) metabolites and impede 5-LO translocation to the nuclear envelope in adherent human polymorphonuclear leukocytes (PMNs) [Sud'ina, G. F., Brock, T. G., Pushkareva, M. A., Galkina, S. I., Turutin, D. V., Peters-Golden, M., et al. (2001). Sulphatides trigger polymorphonuclear granulocyte spreading on collagen-coated surfaces and inhibit subsequent activation of 5-lipoxygenase. The Biochemical Journal, 359, 621-629]. In this study we investigated the mechanism of the leukotriene (LT) synthesis inhibition by sulfatides. Sulfatides neither attenuated the ionophore-induced rise in [Ca(2+)](i) nor promoted PKA activation. We demonstrated that sulfatides directly inhibited 5-LO enzyme activity in a cell-free assay. BODIPY-labeled sulfatides were able to rapidly penetrate into the cells. Sulfatides induced rearrangement and redistribution of cytoskeletal components in adherent PMNs. The lipid incorporation as well as sulfatide-induced inhibition of LT synthesis were abolished by cytochalasin D, an inhibitor of actin polymerization and endocytosis. Importantly, sulfatides caused a prominent intracellular cholesterol redistribution, increasing its abundance at the uropod region. On the basis of these data, we suggest that increased cholesterol accumulation in cell compartments represents a novel mechanism by which sulfatides abrogate 5-LO translocation and activation.

Cholesterol and its anionic derivatives inhibit 5-lipoxygenase activation in polymorphonuclear leukocytes and MonoMac6 cells.

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes (LTs), biological mediators of host defense reactions and of inflammatory diseases. While the role of membrane binding in the regulation of 5-LO activity is well established, the effects of lipids on cellular activity when added to the medium has not been characterized. Here, we show such a novel function of the most abundant sulfated sterol in human blood, cholesterol sulfate (CS), to suppress LT production in human polymorphonuclear leukocytes (PMNL) and Mono Mac6 cells. We synthesized another anionic lipid, cholesterol phosphate, which demonstrated a similar capacity in suppression of LT synthesis in PMNL. Cholesteryl acetate was without effect. Cholesterol increased the effect of CS on 5-LO product synthesis. CS and cholesterol also inhibited arachidonic acid (AA) release from PMNL. Addition of exogenous AA increased the threshold concentration of CS required to inhibit LT synthesis. The effect of cholesterol and its anionic derivatives can arise from remodeling of the cell membrane, which interferes with 5-LO activation. The fact that cellular LT production is regulated by sulfated cholesterol highlights a possible regulatory role of sulfotransferases/sulfatases in 5-LO product synthesis.

Endothelium-leukocyte interactions under the influence of the superoxide-nitrogen monoxide system.

BACKGROUND: The production of reactive oxygen and nitrogen species contributes to the development of vascular injury and inflammation. The present study was focused on neutrophil adhesion to monolayers of primary endothelial cells in the presence of NO donors, a superoxide anion producing system (hypoxanthine-xanthine oxidase, HX-XO) and peroxynitrite under static conditions. MATERIAL/METHODS: Phase contrast and scanning electron microscopy was used to study endothelial monolayer integrity. Neutrophil attachment to surfaces was quantified by myeloperoxidase assay in parallel with microscopic assessment of cell count. RESULTS: In the presence of HX-XO, the endothelial monolayer was destroyed and neutrophil adhesion to the endothelium and exposed subendothelial matrix was drastically increased. Neutrophil attachment was mainly CD18 integrins-mediated and depended on P-selectin, but not on the endothelial adhesion molecules E-selectin, ICAM-1 or PECAM-1. The endothelial monolayer damage caused by HX-XO was a result of superoxide-induced oxidative destruction, since tocopherol and superoxide dismutase protected the monolayer and reduced the number of attached PMNs. Together with the superoxide-producing system, nitric oxide donor diethylamine NONOate also protected the endothelium monolayer from disruption and reduced the number of PMNs attached. Additional exogenous peroxynitrite slightly enhanced neutrophil adhesion to endothelial cells, without monolayer injury. CONCLUSIONS: Superoxide anions induced endothelium injury and neutrophil attachment, but nitric oxide played a protective role.

Activation of NF-kappa B transcription factor in human neutrophils by sulphatides and L-selectin cross-linking.

Sulphated galactocerebroside (sulphatide) has been established as a ligand for L-selectin and shown to trigger intracellular signals in human neutrophils. We have found that sulphatide activated transcription factor NF-kappa B in human neutrophils in a concentration-dependent manner whereas non-sulphated galactocerebroside did not demonstrate such an effect. The activation was inhibitable by pretreatment with primary monoclonal anti-L-selectin antibody (clone LAM1-116). Binding of the primary antibody to L-selectin was insufficient to induce NF-kappa B activation but cross-linking of L-selectin with a secondary antibody was effective. alpha-Chymotrypsin, the agent known to shed L-selectin, activated NF-kappa B by itself. The response to sulphatides was inhibited by jasplakinolide, an actin-polymerising agent known to downregulate surface expression of L-selectin, Fc gamma RIIIb, CD43 and CD44. Recently we have reported that sulphatide stimulated the attachment of human neutrophils to collagen via Mac1 (CD11b/CD18) integrin [Sud'ina et al., Biochem. J. 359 (2001) 621-629]. We now show signalling from sulphatide to NF-kappa B activation and discuss its involvement in neutrophil adhesion.

Regulation of leukotriene synthesis by arachidonic acid in human polymorphonuclear leukocyte adhesive interactions is dependent on the presence of albumin.

We have previously demonstrated that the pretreatment of polymorphonuclear leukocytes (PMNs) with the chemotherapeutic drug, Suramin, increases both cell attachment and inhibits calcium ionophore A23187-stimulated leukotriene (LT) synthesis. Here, we examined the effects of extracellular arachidonic acid (AA) and albumin on attachment and LT synthesis in the interaction of PMNs with both collagen-coated surfaces and human umbilical vein endothelial cell (HUVEC) monolayers. Suramin decreased the release of radiolabelled AA and 5-lipoxygenase metabolites by [(14)C-AA]-prelabelled PMNs stimulated with A23187, with and without human serum albumin (HSA) in the culture medium. Addition of 1 microM AA together with calcium ionophore stimulated the release of endogenous AA to the same level as control and Suramin-pretreated cells, but attachment was unaffected and LT synthesis was still inhibited with Suramin treatment. Using 24 microM AA, regulation of LT synthesis was dependent on the presence of HSA in the medium. Without HSA, 24 microM AA induced detachment of PMNs and increased LT synthesis in Suramin-treated cells above the control level. In the presence of HSA, 24 microM AA did not influence PMN attachment or abolish Suramin-induced inhibition of LT synthesis. These results suggest that tight attachment of PMNs to a solid surface leads to decreased LT synthesis during subsequent stimulation of the cells by A23187 in the presence or absence of exogenous substrate.