Biosynthesis of leukotriene B4 in human polymorphonuclear leukocytes: regulation by cholesterol and other lipids.

Leukotriene B4 (LTB4) is one of the most potent chemotactic compounds produced in macrophages and neutrophils. LTB4 is a product of arachidonic acid oxygenation by 5-lipoxygenase pathway. We present here the data on regulation of LT synthesis in human polymorphonuclear leukocytes by cholesterol, cholesterol sulfate and cholesterol phosphate.

Cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) selectivity of COX inhibitors.

In vitro evaluations of the selectivity of COX inhibitors are based on a great variety of experimental protocols. As a result, data available on cyclooxygenase (COX)-1/COX-2/5- lipoxygenase (LOX) selectivity of COX inhibitors lack consistency. We, therefore, performed a systematic analysis of the COX-1/COX-2/5-LOX selectivity of 14 compounds with selective COX inhibitory activity (Coxibs).

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.

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.

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.

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).

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.

Sulphatides trigger polymorphonuclear granulocyte spreading on collagen-coated surfaces and inhibit subsequent activation of 5-lipoxygenase.

Sulphatides are sulphate esters of galactocerebrosides that are present on the surfaces of many cell types and act as specific ligands to selectins. The present study was undertaken to investigate the effect of sulphatides on polymorphonuclear granulocyte (PMN) attachment, spreading and 5-lipoxygenase (5-LO) metabolism. Sulphatides, but not non-sulphated galactocerebrosides, dose-dependently enhanced attachment to collagen, as measured by the myeloperoxidase assay.

Effects of suramin on PMN interactions with different surfaces.

Human polymorphonuclear leukocytes (PMN) were found to tightly adhere on endothelial (lines EAhy926 and ECV304) and collagen surfaces under the influence of the chemotherapeutic drug suramin. This was observed by scanning electron microscopy and quantitated by myeloperoxidase assays. Suramin also inhibited Ca2+ ionophore A23187-stimulated leukotriene (LT) synthesis in PMN interaction with endothelial cells or with collagen surface.

Inhibition of cell division but not nuclear division by 1-O- octadecyl-2-O-methyl-Sn-glycero-3-phosphocholine.

1-O-Octadecyl-2-O-methyl-glycero-3-phosphocholine (ET-18-OCH(3)) selectively inhibits the growth of cancer cells. Here we show that in some cell types ET-18-OCH(3)and liposome-associated ET-18-OCH(3)inhibit cell division without concurrent inhibition of nuclear division, leading to multinucleate cell formation, and cell death through apoptosis. Cell cycle analysis revealed that ET-18-OCH(3)-treated U-937 cells continued to move through the cell cycle, but many cells were not able to divide and instead accumulated as tetraploid cells or octaploid cells in the G0/G1 phase of the cell cycle.

Increased cell-surface receptor expression on U-937 cells induced by 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine.

Association of the ether lipid, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3) with liposomes (ELL-12) reduces acute toxicity while maintaining or enhancing anticancer activity in experimental tumor models. ELL-12 has been shown to induce apoptosis by a cytochrome-c-dependent caspase-mediated pathway, which results in proteolytic cleavage of poly(ADP-ribose) polymerase and lamins, but the antitumor effects of ET-18-OCH3 or ELL-12 could result from tumor cell differentiation or activation. Here we compared the effects of ET-18-OCH3 and ELL-12 on the expression of cell-surface proteins associated with cell differentiation and/or activation in U-937 cells.

p53-dependent ceramide response to genotoxic stress.

Both p53 and ceramide have been implicated in the regulation of growth suppression. p53 has been proposed as the "guardian of the genome" and ceramide has been suggested as a "tumor suppressor lipid. " Both molecules appear to regulate cell cycle arrest, senescence, and apoptosis.

Involvement of ecto-ATPase and extracellular ATP in polymorphonuclear granulocyte-endothelial interactions.

The adhesion of human polymorphonuclear granulocytes (PMN) with confluent human endothelial cells (line EAhy926) and with solid substrate coated by collagen and fibronectin (Fn) was studied by phase contrast microscopy and by the measurement of myeloperoxidase activity. The ecto-ATPase inhibitors suramin and Reactive Blue 2 (RB2) more than doubled the adhesion of PMN to endothelial cells. The cells hydrolyzed added ATP and this reaction was inhibited by suramin and RB2.

Growth inhibitory effects of liposome-associated 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine.

The growth inhibitory effects of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3) and various liposome compositions of ET-18-OCH3 were compared in a standardized growth inhibition assay utilizing a diverse tumor cell line panel including cell lines expressing multidrug resistance. ET-18-OCH3 and ELL-12 (4:3:1:2, dioleoylphosphatidylcholine/ cholesterol/dioleoylphosphatidylethanolamine-glutaric acid/ET-18-OCH3), an optimal liposomal ET-18-OCH3 formulation, inhibited growth in the micromolar range in drug-sensitive and -resistant cells. In general, ET-18-OCH3-liposomes were about twofold less growth inhibitory than ET-18-OCH3.

Competitive inhibition of the 5-lipoxygenase-catalysed linoleate oxidation by arachidonic and 5-hydroperoxy-eicosatetraenoic acids.

Linoleic and arachidonic acids are competing substrates for 5-lipoxygenase from barley. When these two substrates are added simultaneously, arachidonic acid acts as a competitive inhibitor of linoleic acid oxidation with Ki of 20 microM, the same value as the Michaelis constant for arachidonate oxygenation by this enzyme (22 +/- 3 microM). Linoleic acid hydroperoxide accumulated in the reaction mixture does not inhibit the enzymatic process, while arachidonic acid hydroperoxy product (5-hydroperoxy-6,8,11,14-eicosatetraenoic acid) inhibits it with very low Ki equal to 0.

Ceramide: an endogenous regulator of apoptosis and growth suppression.

The action of several cytokines is mediated via activation of the sphingomyelin cycle of signal transduction. Ceramide, the product of this pathway, is emerging as an intracellular messenger that mediates effects on terminal differentiation, apoptosis and cell-cycle arrest. Here, Marina Pushkareva, Lina Obeid and Yusuf Hannun discuss the regulation and mechanism of action of this stress-activated pathway.

Retinoblastoma gene product as a downstream target for a ceramide-dependent pathway of growth arrest.

Ceramide, a lipid mediator, has been most closely associated with antiproliferative activities. In this study, we examine the mechanism by which ceramide induces growth suppression and the role of the retinoblastoma gene product (Rb) in this process. Withdrawal of serum from the serum-dependent MOLT-4 cells resulted in significant dephosphorylation of Rb, correlating with the induction of G0/G1 cell cycle arrest.

Stereoselectivity of induction of the retinoblastoma gene product (pRb) dephosphorylation by D-erythro-sphingosine supports a role for pRb in growth suppression by sphingosine.

Sphingosine has been shown to inhibit cell growth in many cell lines although the mechanism of this effect remains obscure. More recently, D-erythro-sphingosine has been shown to act as an early inducer of dephosphorylation of the retinoblastoma gene product (pRb) in the lymphoblastic leukemia cell line MOLT-4 [Chao, R., Khan, W.

[Lipophilic derivatives of caffeic acid as lipoxygenase inhibitors with antioxidant properties].

We have prepared two lipophilic derivatives of caffeic acid at the carboxylic function--caffeic acid phenethyl ester, an active component of propolis, and N,N'-dicyclohexyl-O-(3,4-dihydroxycinnamoyl)-isourea. Both substances inhibit barley 5-lipoxygenase and soybean 15-lipoxygenase at micromolar concentrations. The inhibition is uncompetitive, dose-dependent and reversible.

Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties.

Caffeic acid phenethyl ester, an active component of propolis extract, inhibits 5-lipoxygenase in the micromolar concentration range. The inhibition is of an uncompetitive type, i.e.

[The effect of tumor necrosis factor on the free sphingosine level and sphingomyelinase in murine liver cells and nuclei].

The effects of the human recombinant tumour necrosis factor (TNF) (10 and 40 mg/kg of body mass) on sphingomyelinase activity and sphingosine content in mouse (C57bl) liver cells and nuclei have been studied. Whereas sphingomyelinase is known to be a key enzyme of sphingomyelin metabolism, sphingosine, being a product of deep enzymatic hydrolysis of sphingomyelin, controls the activity of various phosphokinases. The primary response of liver cell to TNF consists in the inhibition of sphingomyelinase; its activation occurs at later periods: after 2 hours at 10 mg/kg TNF and after 4 hours at 40 mg/kg TNF.

[Change in the level of endogenous sphingosine in cell nuclei from regenerating rat liver].

High-performance liquid chromatography was used to study the changes in the sphingosine content in regenerating rat liver cell nuclei during RNA and DNA synthesis. It was found that activation of nucleic acid synthesis was accompanied by sphingosine accumulation in cell nuclei in parallel with the induction of the sphingomyelin cycle consisting in the increasing activity of sphingomyelinase and alteration of the sphingomyelin and ceramide content. To clarify the mechanism of sphingosine involvement in replication and transcription, the ability of this product to interact with DNA and modify the activity of RNA-polymerase in vitro was studied.

[Effect of sphingomyelin and its enzymatic hydrolysis products on heterologous methylation of calf thymus DNA by cytosine-DNA-methyltransferase EcoRII].

It was found that sphingomyelin and its enzymatic hydrolysis products, choline and sphingosine, influence the degree of DNA methylation in the reaction of heterologous methylation by methylase EcoRII in vitro. Sphingomyelin was found to be able to was activate (by 20%), sphingosine and choline inhibit methylation. Phosphatidylcholine had no effect on DNA methylation in an in vitro system.

[A comparative evaluation of the level of sphingomyelinase activity in liver cell nuclei and in the brain].

It was discovered that there is sphingomyelinase activity in the rat liver nuclei. The maximum of enzyme activity is at pH 7.1.