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. The addition of Pseudomonas aeruginosa lipopolysaccharides (LPS) with lipid vesicles containing phosphatidylcholine or phosphatidylcholine/cholesterol (70:30) showed that omitting cholesterol abolished the effect of LPS on LT synthesis. We show here the capacity of cholesterol sulfate, the most abundant sulfated sterol in human blood, to suppress LT production in human neutrophils and to neutralize the effect of P. aeruginosa LPS on LT synthesis. We suggest that sulfated lipids serve as specific endogenous regulators of LT synthesis in neutrophils, and anti-inflammatory therapy may be based on modification of cholesterol level and its conversion to anionic derivatives.

Cell-to-cell cross-talk between mesenchymal stem cells and cardiomyocytes in co-culture.

The goals of the study were: (1) to explore the communication between human mesenchymal stem cells (MSC) and rat cardiac myocytes resulting in differentiation of the stem cells and, (2) to evaluate the role of mitochondria in it. Light and fluorescence microscopy as well as scanning electron microscopy revealed that after co-cultivation, cells formed intercellular contacts and transient exchange with cytosolic elements could be observed. The transport of cytosolic entity had no specific direction. Noticeably, mitochondria also could be transferred to the recipient cells in a unidirectional fashion (towards cardiomyocytes only). Transmission electron microscopy revealed significant variability in both the diameter of intercellular contacting tubes and their shape. Inside of these nanotubes mitochondria-resembling structures were identified. Moreover, after co-cultivation with cardiomyocytes, expression of human-specific myosin was revealed in MSC. Thus, we speculate that: (1) transport of intracellular elements to MSC possibly can determine the direction of their differentiation and, (2) mitochondria may be involved in the mechanism of the stem cell differentiation. It looks plausible that mitochondrial transfer to recipient cardiomyocytes may be involved in the mechanism of failed myocardium repair after stem cells transplantation.

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. Studies with blocking antibodies indicated that the increased attachment was mediated by CD11b/CD18 (Mac-1) beta 2 integrin. Scanning electron microscopy indicated that sulphatides also greatly enhanced the degree of cell spreading. In PMNs treated in suspension, sulphatides had no effect on the ionophore A23187-stimulated release of arachidonic acid and the synthesis of 5-LO metabolites. In contrast, in PMNs attached to collagen, the enzymic conversion of arachidonic acid by 5-LO was inhibited by sulphatides. Inhibition of 5-LO metabolism by sulphatides was observed even in the presence of exogenous substrate, suggesting that sulphatides directly inhibited 5-LO action. Consistent with this, sulphatides interfered with ionophore-induced translocation of the 5-LO to the nuclear envelope. Substances competing with sulphatide binding to cells, like dextran sulphate, or a strong inhibitor of cell spreading, like the actin-polymerizing agent jasplakinolide, prevented the effects of sulphatides on PMN attachment and spreading and leukotriene synthesis. We conclude that shape changes occurring in response to sulphatides specifically impair PMN leukotriene synthesis by inhibiting translocation of 5-LO.

Inhibition of neutrophil spreading during adhesion to fibronectin reveals formation of long tubulovesicular cell extensions (cytonemes).

Human neutrophils developed long thin tubulovesicular extensions (cytonemes) upon adhesion to fibronectin-coated substrata, when spreading was blocked. We observed extension formation when neutrophils were plated to fibronectin-coated substrata in Na(+)-free extracellular medium or in the presence of drugs capable of inhibiting spreading: 4-bromophenacyl bromide, N-ethylmaleimide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and cytochalasin D. Addition of Na(+) ions or washing of inhibitors restored neutrophil spreading. Phase-contrast and scanning electron microscopy revealed two types of extensions: (1) highly dynamic, flexible tubulovesicular extensions with unattached tips 0.2-0.4 microm in diameter, which can achieve 70-80 microm in length during 20 min, and (2) thinner straight extensions with flattened tips, which were formed in the presence of phorbol 12-myristate 13-acetate and connected cells to substratum or to the neighboring cells several cell diameters away. The latter may have derived from the former through tension after attachment of the tips. Spreading and extension formation may represent two states of the cell adhesive and communicative mechanism.

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. Suramin decreased the release of radiolabeled arachidonic acid (AA) and 5-lipoxygenase (5-LO) metabolites by prelabeled PMN stimulated with A23187. Using agents releasing the suramin-stimulated adhesion namely jasplakonolide and dextran sulfate, we observed a reversal of the suramin effect on leukotriene synthesis. Jasplakonolide released the adhesion of PMN on endothelial and collagen-coated surfaces and restored 5-LO activity. Dextran-sulfate released adhesion on collagen-coated surfaces and abolished suramin inhibition. Arachidonate could also overcome adhesion and inhibition of 5-LO. We conclude that suramin-induced tight attachment of PMN on to solid surfaces lead to decreased leukotriene synthesis during subsequent A23187 stimulation in the absence of exogenous substrates.

Dependence of neutrophil activation on cell density and adhesion.

Upon an increasing cell density human neutrophils develop more cell-to-cell contacts in conjunction with an increase in the pHi. These changes are accompanied by decreased superoxide formation after adherence, and a decrease in the total amount of 5-lipoxygenase products after various stimuli. Among the various arachidonate metabolites, leukotriene formation remained almost constant but the yield in 5-HETE decreased. This drop in could account for the decrease in total 5-lipoxygenase products observed when the cell density increased. We conclude that cellular signalling can be affected by an increase of cell-cell interactions. Whether the increase in cellular pH is a cause or consequence of such contact inhibition has yet be answered.

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. The degree of ATP hydrolysis during PMN adherence depended on solid substrata and decreased in the order: non-stimulated endothelial cells, TNF-stimulated endothelial cells, collagen-coated surface, Fn-coated surface. In the same order adherence increased. The endogenous level of extracellular ATP in the PMN-endothelial coculture was around 25 nM. We conclude that PMN-endothelial adhesion is counteracted by an ecto-ATPase or by ATP receptors with ATPase activity. Such interactions may play a role in PMN rolling and diapedesis as well as in the pathophysiology of PMN activation by an anergic endothelium.

Regulation of intracellular pH by phospholipase A2 and protein kinase C upon neutrophil adhesion to solid substrata.

Adhesion to solid substrata has been shown to increase intracellular pH (pH(i)) of fibroblasts and of other cells (FEBS Lett. (1988) 234, 449-450; Proc. Natl. Acad. Sci. USA (1989) 86, 4525-4529; J. Biol. Chem. (1990) 265, 1327-1332; Exp. Cell Res. (1992) 200, 211-214; FEBS Lett. (1995) 374, 17-20). We have found that the inhibitors of PLA2, 4-bromophenacyl bromide and manoalide, completely blocked the increase of pH(i) and spreading of neutrophils upon adhesion to solid substrata. Inhibition of phospholipase C with neomycin or removal of extracellular Ca2+ affects neither neutrophil spreading nor their pH(i). Inhibition of PKC with H-7 or staurosporin increased pH(i). PMA, an activator of PKC, dramatically decreased pH(i) but did not impair the spreading of neutrophils. The effect of arachidonic acid, a product of PLA2 activity, on neutrophil pH(i) and spreading was similar to that of PMA. H-7, an inhibitor of PKC, partially blocked the effect of arachidonic acid (AA) on pH(i). BW755C, an inhibitor of AA metabolism by cyclooxygenase or lipoxygenase, affected neither the pH(i) nor cell spreading. We propose that the increase of pH(i) upon neutrophil adhesion is mediated by PLA2 activity, while PKC decreased pH(i). AA produced by PLA2 activates PKC, thus forming a feedback regulation of pH(i).

Regulation of intracellular pH by cell-cell adhesive interactions.

As was shown in our previous work, the intracellular pH (pHi) of cultured human fibroblasts depends on cell density. The pHi is low in single cells, higher in cells, forming small groups and maximal in a sparse monolayer. On the other hand, the pHi is low in areas of confluent monolayers. In the present work, we show that the effects of inhibitors of various pH-controlling mechanisms as well as inhibitors of key enzymes in signal transduction pathways depend on the local cell density. We have found that N-ethylmaleimide and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, known as inhibitors of V-type H+ ATPase, inhibit the elevation of pHi induced by cell-cell contact interactions; meanwhile Cd2+ ions, which inhibit H+ conductive pathway, cause an increase of pHi in a confluent monolayer. Our data revealed also that the Na+/H+ antiporter does not play an essential role in the pHi regulation by intercellular contacts. Inhibitors of phospholipase A2 (4-bromophenacyl-bromide), phospholipase C (neomycin) and protein kinase C (H-7) dramatically change the way the pHi is modulated by local cell density. It is suggested that cell-cell interactions regulate cell activities via modulation of pHi, which is under positive control from phospholipase A2 and under negative control from protein kinase C.

Adhesive interactions of neutrophils and leukotriene synthesis.

Cell-substrate and cell-cell adhesion of neutrophils has been found to slow down the calcium ionophore A23187-induced synthesis of 5-lipoxygenase (5-LO) metabolites of arachidonic acid. Addition of the exogenous substrate, arachidonic acid (AA), together with A23187, resulted in the enhanced production of leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) by adherent neutrophils in comparison with those by the cells in suspension. We observed also the enhanced production of 5-LO metabolites in attached cells when we stimulated the cells by the combined action of phorbol 12-myristate 13-acetate (PMA) and A23187. Thus, the adhesion to solid substrate and to other cells, an important regulatory factor for the activity of many cells, is a powerful regulator of leukotriene production by neutrophils.

Cell-cell contacts alter intracellular pH.

Intracellular pH, an important regulatory factor for many cellular activities, was shown to be modulated by cell adhesion to the solid substratum. In the present work we have shown that cell-cell contacts also affect intracellular pH. pH(i) depends on how many contacts the cell has established with the substratum and the neighboring cells. pH(i) is low in single cells, not contacting each other. It increased with the increase of cell density. pH(i) is again decreased in confluent (topoinhibited) monolayers. pH(i)-shifts triggered by cell-cell contacts seem to be mediated by Na+/H(+)-antiporter. Dependence of pH(i) on cell density could be simulated by different concentration of Arg-Gly-Asp--which is part of the site of extracellular matrix proteins involved in integrin binding. The dependence of pH(i) on cell-cell contacts is discussed in relation to the phenomena of topoinhibition.

Low-density lipoproteins interact with liposome-binding sites on the cell surface.

Under physiological conditions significant amounts of low-density lipoprotein LDL particles ar taken up by cells independently of specific high-affinity LDL receptors (apo-B receptors). Previously it was established that some cells contain surface sites capable of binding liposomes. We proposed that liposome-binding sites could contribute to LDL interaction with the cell surface via phospholipid molecules of LDL particles. To check this hypothesis we studied the competitive interaction of human LDL and DPPC liposomes with mouse embryo fibroblasts depleted of apo-B receptors by preliminary incubation with LDL. We have found that after removal of the liposome-binding sites from cell lamellae these areas of the cell surface lose their ability to bind LDL.

[The effect of aspirin and indomethacin on intracellular pH and on the activation of the Na+/H+ antiporter in neutrophils]. / Vliianie aspirina i indometatsina na vnutrikletochnyi pH i aktivatsiiu Na+/H+ antiportera neitrofilov.

Using microfluorometry, aspirin and indomethacine effects on intracellular pH and Na+/H+ neutrophilic antiporter activation were studied. Aspirin and indomethacine were shown to decrease intracellular pH of quiescent neutrophil cells. Aspirin completely blocked basifying of cytoplasm, which is resulted from Na+/H+ neutrophilic antiporter activation.

[Competition of solid and fluid liposomes for binding and metabolism with lipids from the cell surface]. / Konkurentsiia "tverdykh" i "zhidkikh" liposom za sviazyvanie i obmen lipidami s kletochnoi poverkhnost'iu.

The competitive behavior of solid vs. fluid liposomes in liposome-cell adsorption and cell-to-liposome lipid transfer processes was investigated with L cells and FBT epithelial sheets. Binding and transfer experiments have demonstrated that: solid liposomes adhere to the cell surface as integral vesicles retaining the entrapped substance; fluid liposomes are partly disintegrated at the cell surface with concomitant entry of entrapped substances into the cytoplasm, while their lipids remain on the cell surface; fluid liposomes that escape lysis dissociate from the cell taking away cell lipid molecules. No lipid transfer occurs between the plasma membrane and solid liposomes. Cell-bound solid liposomes interfere with the transfer of cell lipids to fluid liposomes, while these in turn inhibit the binding of solid liposomes to the cell surface.

[Effect of different forms of vitamin A and its combination with vitamin E on the peroxidation of lipids]. / Vliianie razlichnykh form vitamina A i ego sochetaniia s vitaminom E na perekisnoe okislenie lipidov.

Effect of vitamins A and E derivatives on Fe-ascorbate dependent peroxidation was studied by means of thiobarbituric acid test in liposomes prepared from egg lecithin. Retinol, incorporated into liposomes at concentration less than 0.1 mol%, increases the lecithin oxidation, retinyl palmitate was less active and alpha-tocopherol inhibited the reaction. Synergistic effect of alpha-tocopherol and retinol was observed. After simultaneous administration of retinol and alpha-tocopherol into liposomes, vitamin A did not accelerate the lecithin oxidation but increased distinctly the inhibitory effect of alpha-tocopherol. This interaction did not occur between retinol and alpha-tocopherol acetate as well as between m-retinyl acetate and alpha-tocopherol. The data obtained suggest that vitamins A and E affect the lipid peroxidation not only via direct participation in free radical oxidation but also due to transformations of biological membranes structure.

Lipid-cell interactions. Liposome adsorption and cell-to-liposome lipid transfer are mediated by the same cell-surface sites.

The competitive behavior of solid vs. fluid liposomes in liposome-to-cell adsorption and cell-to-liposome lipid transfer processes was investigated with L cells and FBT epithelial sheets. Binding, transfer and 31P-NMR experiments have demonstrated that: (i) solid liposomes adhere to the cell surface as integral vesicles retaining the entrapped substances; (ii) fluid liposomes are partly disintegrated at the cell surface with concomitant entry of entrapped substances into the cytoplasm, while their lipids remain on the cell surface; (iii) fluid liposomes that escape lysis dissociate from the cell, taking away cell lipid molecules. The latter process underlies the mechanism of cell-to-fluid liposome lipid transfer. In contrast, no lipid transfer occurs between the plasma membrane and solid liposomes. Cell-bound solid liposomes interfere with the transfer of cell lipids to fluid liposomes, while these in turn inhibit the binding of solid liposomes to the cell surface. Moreover, cell-induced aggregation of both fluid and solid freshly added liposomes is also inhibited by preincubation of the cells with either solid or fluid liposomes. Thus, different types of interaction of both fluid and solid liposomes with the cell are mediated by the same (or closely related) sites on the cell surface.