Galina F. Sud’ina

Microbial alkaloid staurosporine induces formation of nanometer-wide membrane tubular extensions (cytonemes, membrane tethers) in human neutrophils

In the present work, we demonstrate that microbial alkaloid staurosporine (STS) and Ro 31-8220, structurally related to STS protein kinase C inhibitor, caused development of membrane tubular extensions in human neutrophils upon adhesion to fibronectin-coated substrata. STS-induced tubular extensions interconnected neutrophils in a network and bound serum-opsonized bacteria Salmonella enterica serovar Typhimurium. The diameter of STS-induced extensions varied in the range 160-200 nm. The extensions were filled with cytoplasm and covered with membrane, as they included fluorescent cytoplasmic and lipid dyes. Neither protein kinase C inhibitors H-7 and bisindolylmaleimide VII, nor tyrosine protein kinase inhibitors tyrphostin AG 82 and genistein caused such extensions formation. Supposedly, STS induces membrane tubular extension formation promoting actin cytoskeleton depolymerization or affecting NO synthesis.

Nitric oxide mediates distinct effects of various LPS chemotypes on phagocytosis and leukotriene synthesis in human neutrophils

We investigated the effect of lipopolysaccharide (LPS) chemotypes differing in their carbohydrate chain length on phagocytosis of serum-opsonized zymosan (OZ) particles and related functions of human polymorphonuclear leukocyte (PMNL, neutrophils). LPS from deep core mutant (Re), complete core (Ra) and smooth (S) phenotypes of Salmonella typhimurium was studied. Priming of neutrophils with various LPSs caused prominent enhancement of OZ phagocytosis, superoxide production and leukotriene (LT) synthesis in neutrophils, with LPS effects increasing as Re<S<Ra. The LPS forms were less potent to activate OZ uptake in the presence of MK-886, 5-lipoxygenase activating protein inhibitor, suggesting the regulatory function of 5-lipoxygenase (5-LO)-derived LTs. Direct measurement of nitrite release from OZ-stimulated neutrophils revealed that the effects of LPS on NO synthesis increased in the range of Ra<S<Re. Nitric oxide synthase (NOS) inhibitor l-NAME increased phagocytosis, LT and superoxide formation by neutrophils, and abolished the difference in the action of the LPSs forms. Further mechanistic studies revealed that NO modulates cellular 5-LO activity in a guanylyl cyclase and protein kinase G dependent manner, as well as interplay between NO and superoxide, and peroxynitrite generation contribute to distinct effects of LPS chemotypes on phagocytosis and LT synthesis in human neutrophils. Our investigation of the three LPS species demonstrates that the LPS polysaccharide core is mostly essential for the PMNL activation and is able to suppress lipid A-induced increase in NOS activity in phagocyting cells by triggering specific signaling cascades.

Mitochondrial reactive oxygen species are involved in chemoattractant-induced oxidative burst and degranulation of human neutrophils in vitro

Activation of neutrophils is accompanied by the oxidative burst, exocytosis of various granule types (degranulation) and a delay in spontaneous apoptosis. The major source of reactive oxygen species (ROS) in human neutrophils is NADPH oxidase (NOX2), however, other sources of ROS also exist. Although the function of ROS is mainly defensive, they can also play a regulatory role in cell signaling. However, the contribution of various sources of ROS in these processes is not clear. We investigated a possible role of mitochondria-derived ROS (mtROS) in the regulation of neutrophil activation induced by chemoattractant fMLP in vitro. Using the mitochondria-targeted antioxidant SkQ1, we demonstrated that mtROS are implicated in the oxidative burst caused by NOX2 activation as well as in the exocytosis of primary (azurophil) and secondary (specific) granules. Scavenging of mtROS with SkQ1 slightly accelerated spontaneous apoptosis and significantly stimulated apoptosis of fMLP-activated neutrophils. These data indicate that mtROS play a critical role in signal transduction that mediates the major neutrophil functional responses in the process of activation.

Membrane tubulovesicular extensions (cytonemes): secretory and adhesive cellular organelles.

In this review, we summarized data on the formation and structure of the long and highly adhesive membrane tubulovesicular extensions (TVEs, membrane tethers or cytonemes) observed in human neutrophils and other mammalian cells, protozoan parasites and bacteria. We determined that TVEs are membrane protrusions characterized by a uniform diameter (130–250 nm for eukaryotic cells and 60–90 nm for bacteria) along the entire length, an outstanding length and high rate of development and a high degree of flexibility and capacity for shedding from the cells. This review represents TVEs as protrusions of the cellular secretory process, serving as intercellular adhesive organelles in eukaryotic cells and bacteria. An analysis of the physical and chemical approaches to induce TVEs formation revealed that disrupting the actin cytoskeleton and inhibiting glucose metabolism or vacuolar-type ATPase induces TVE formation in eukaryotic cells. Nitric oxide is represented as a physiological regulator of TVE formation.

Involvement of red blood cells in the regulation of leukotriene synthesis in polymorphonuclear leucocytes upon interaction with Salmonella Typhimurium.

Golenkina EA, Galkina SI, Romanova JM, Lazarenko MI, Sud’ina GF. Involvement of red blood cells in the regulation of leukotriene synthesis in polymorphonuclear leukocytes upon interaction with Salmonella Typhimurium. APMIS 2011.

Ceruloplasmin-derived peptide is the strongest regulator of oxidative stress and leukotriene synthesis in neutrophils

Ceruloplasmin, an acute-phase protein, can affect the activity of leukocytes through its various enzymatic activities and protein-protein interactions (with lactoferrin, myeloperoxidase, eosinophil peroxidase, serprocidins, and 5-lipoxygenase (5-LOX), among others). However, the molecular mechanisms of ceruloplasmin activity are not clearly understood. In this study, we tested the ability of two synthetic peptides, RPYLKVFNPR (883-892) (P1) and RRPYLKVFNPRR (882-893) (P2), corresponding to the indicated fragments of the ceruloplasmin sequence, to affect neutrophil activation. Leukotriene (LT) B4 is the primary eicosanoid product of polymorphonuclear leukocytes (PMNLs, neutrophils). We studied leukotriene synthesis in PMNLs upon interaction with Salmonella enterica serovar Typhimurium. Priming of neutrophils with phorbol 12-myristate 13-acetate (PMA) elicited the strong regulatory function of P2 peptide as a superoxide formation inducer and leukotriene synthesis inhibitor. Ceruloplasmin-derived P2 peptide appeared to be a strong inhibitor of 5-LOX product synthesis under conditions of oxidative stress.

Neutrophils Release Metalloproteinases during Adhesion in the Presence of Insulin, but Cathepsin G in the Presence of Glucagon

In patients with reperfusion after ischemia and early development of diabetes, neutrophils can attach to blood vessel walls and release their aggressive bactericide agents, which damage the vascular walls. Insulin and 17β-estradiol (E2) relieve the vascular complications observed in metabolic disorders. In contrast, glucagon plays an essential role in the pathophysiology of diabetes. We studied the effect of hormones on neutrophil secretion during adhesion to fibronectin. Amino acid analysis revealed that proteins secreted by neutrophils are characterized by a stable amino acid profile enriched with glutamate, leucine, lysine, and arginine. The total amount of secreted proteins defined as the sum of detected amino acids was increased in the presence of insulin and reduced in the presence of glucagon. E2 did not affect the amount of protein secretion. Proteome analysis showed that in the presence of insulin and E2, neutrophils secreted metalloproteinases MMP-9 and MMP-8 playing a key role in modulation of the extracellular matrix. In contrast, glucagon induced the secretion of cathepsin G, a key bactericide protease of neutrophils. Cathepsin G can promote the development of vascular complications because of its proinflammatory activity and ability to stimulate neutrophil adhesion via the proteolysis of surface receptors.

Fine Regulation of Neutrophil Oxidative Status and Apoptosis by Ceruloplasmin and Its Derivatives

Timely neutrophil apoptosis is an essential part of the resolution phase of acute inflammation. Ceruloplasmin, an acute-phase protein, which is the predominant copper-carrying protein in the blood, has been suggested to have a marked effect on neutrophil life span. The present work is a comparative study on the effects of intact holo-ceruloplasmin, its copper-free (apo-) and partially proteolyzed forms, and synthetic free peptides RPYLKVFNPR (883–892) and RRPYLKVFNPRR (882–893) on polymorphonuclear leukocyte (PMNL, neutrophil) oxidant status and apoptosis. The most pronounced effect on both investigated parameters was found with copper-containing samples, namely, intact and proteolyzed proteins. Both effectively reduced spontaneous and tumor necrosis factor-α (TNF-α)-induced extracellular and intracellular accumulation of superoxide radicals, but induced a sharp increase in the oxidation of intracellular 2′,7′-dichlorofluorescein upon short exposure. Therefore, intact and proteolyzed ceruloplasmin have both anti- and pro-oxidant effects on PMNLs wherein the latter effect is diminished by TNF-α and lactoferrin. Additionally, all compounds investigated were determined to be inhibitors of delayed spontaneous apoptosis. Intact enzyme retained its pro-survival activity, whereas proteolytic degradation converts ceruloplasmin from a mild inhibitor to a potent activator of TNF-α-induced neutrophil apoptosis.

Erratum to “Mold Alkaloid Cytochalasin D Modifies the Morphology and Secretion of fMLP-, LPS-, or PMA-Stimulated Neutrophils upon Adhesion to Fibronectin”

Neutrophils play an essential role in innate immunity due to their ability to migrate into infected tissues and kill microbes with bactericides located in their secretory granules. Neutrophil transmigration and degranulation are tightly regulated by actin cytoskeleton. Invading pathogens produce alkaloids that cause the depolymerization of actin, such as the mold alkaloid cytochalasin D. We studied the effect of cytochalasin D on the morphology and secretion of fMLP-, LPS-, or PMA-stimulated human neutrophils upon adhesion to fibronectin. Electron microscopy showed that the morphology of the neutrophils adherent to fibronectin in the presence of various stimuli differed. But in the presence of cytochalasin D, all stimulated neutrophils exhibited a uniform nonspread shape and developed thread-like membrane tubulovesicular extensions (cytonemes) measuring 200 nm in diameter. Simultaneous detection of neutrophil secretory products by mass spectrometry showed that all tested stimuli caused the secretion of MMP-9, a key enzyme in the neutrophil migration. Cytochalasin D impaired the MMP-9 secretion but initiated the release of cathepsin G and other granular bactericides, proinflammatory agents. The release of bactericides apparently occurs through the formation, shedding, and lysis of cytonemes. The production of alkaloids which modify neutrophil responses to stimulation via actin depolymerization may be part of the strategy of pathogen invasion.

G-quadruplex-forming oligodeoxyribonucleotides activate leukotriene synthesis in human neutrophils

Abstract Human polymorphonuclear leukocytes (PMNLs, neutrophils) play a major role in the immune response to bacterial and fungal infections and eliminate pathogens through phagocytosis. During phagocytosis of microorganisms, the 5-lipoxygenase (5-LOX) pathway is activated resulting in generation of leukotrienes, which mediate host defense. In this study, a library of oligodeoxyribonucleotides (ODNs) with varying numbers of human telomeric repeats (d(TTAGGG)n) and their analogues with phosphorothioate internucleotide linkages and single-nucleotide substitutions was designed. These ODNs with the potential to fold into G-quadruplex structures were studied from structural and functional perspectives. We showed that exogenous G-quadruplex-forming ODNs significantly enhanced 5-LOX metabolite formation in human neutrophils exposed to Salmonella Typhimurium bacteria. However, the activation of leukotriene synthesis was completely lost when G-quadruplex formation was prevented by substitution of guanosine with 7-deazaguanosine or adenosine residues at several positions. To our knowledge, this study is the first to demonstrate that G-quadruplex structures are potent regulators of 5-LOX product synthesis in human neutrophils in the presence of targets of phagocytosis. Communicated by Ramaswamy H. Sarma