Effect of Dexamethasone on Adhesion of Human Neutrophils and Concomitant Secretion.

Neutrophils play a dual role in protecting the body. They are able to penetrate infected tissues and destroy pathogens there by releasing aggressive bactericidal substances. While into the surrounding tissues, the aggressive products secreted by neutrophils initiate development of inflammatory processes. Invasion of neutrophils into tissues is observed during the development of pneumonia in the patients with lung diseases of various etiologies, including acute respiratory distress syndrome caused by coronavirus disease. Synthetic corticosteroid hormone dexamethasone has a therapeutic effect in treatment of lung diseases, including reducing mortality in the patients with severe COVID-19. The acute (short-term) effect of dexamethasone on neutrophil adhesion to fibrinogen and concomitant secretion was studied. Dexamethasone did not affect either attachment of neutrophils to the substrate or their morphology. Production of reactive oxygen species (ROS) and nitric oxide (NO) by neutrophils during adhesion also did not change in the presence of dexamethasone. Dexamethasone stimulated release of metalloproteinases in addition to the proteins secreted by neutrophils during adhesion under control conditions, and selectively stimulated release of free amino acid hydroxylysine, a product of lysyl hydroxylase. Metalloproteinases play a key role and closely interact with lysyl hydroxylase in the processes of modification of the extracellular matrix. Therapeutic effect of dexamethasone could be associated with its ability to reorganize extracellular matrix in the tissues by changing composition of the neutrophil secretions, which could result in the improved gas exchange in the patients with severe lung diseases.

Ivermectin Affects Neutrophil-Induced Inflammation through Inhibition of Hydroxylysine but Stimulation of Cathepsin G and Phenylalanine Secretion.

The invasion and integrin-dependent adhesion of neutrophils to lung tissues and their secretion lead to the development of pneumonia in various pulmonary pathologies, including acute respiratory distress syndrome in coronavirus disease. We studied the effect of ivermectin, a possible therapeutic agent for inflammation and cancer, on integrin-dependent neutrophil adhesion to fibronectin and the concomitant secretion. Ivermectin did not affect the attachment of neutrophils to the substrate and the reactive oxygen species production but sharply inhibited the adhesion-induced release of hydroxylysine and stimulated the release of phenylalanine and cathepsin G. Hydroxylysine is a product of lysyl hydroxylase, which is overexpressed in tumor cells with an increased ability to invade and metastasize. The inhibition of hydroxylysine release by ivermectin, by analogy, may indicate the suppression of neutrophil invasion into tissue. The increase in the release of phenylalanine in our experiments coincided with the secretion of cathepsin G, which indicates the possible role of this enzyme in the cleavage of phenylalanine. What is the substrate in such a reaction is unknown. We demonstrated that exogenously added angiotensin II (1-8) can serve as a substrate for phenylalanine cleavage. Mass spectrometry revealed the formation of angiotensin II (1-7) in the secretion of neutrophils, which attached to fibronectin in the presence of ivermectin and exogenous angiotensin II (1-8), indicating a possible involvement of ivermectin in the inactivation of angiotensin II.

Inhibitor of Hyaluronic Acid Synthesis 4-Methylumbelliferone Suppresses the Secretory Processes That Ensure the Invasion of Neutrophils into Tissues and Induce Inflammation.

Integrin-dependent adhesion of neutrophils to tissue, accompanied by the development of neutrophil-induced inflammation, occurs both in the focus of infection and in the absence of infection in metabolic disorders such as reperfusion after ischemia, diabetes mellitus, or the development of pneumonia in patients with cystic fibrosis or viral diseases. Hyaluronic acid (HA) plays an important role in the recruitment of neutrophils to tissues. 4-methylumbilliferon (4-MU), an inhibitor of HA synthesis, is used to treat inflammation, but its mechanism of action is unknown. We studied the effect of 4-MU on neutrophil adhesion and concomitant secretion using adhesion to fibronectin as a model for integrin-dependent adhesion. 4-MU reduced the spreading of neutrophils on the substrate and the concomitant secretion of granule proteins, including pro-inflammatory components. 4-MU also selectively blocked adhesion-induced release of the free amino acid hydroxylysine, a product of lysyl hydroxylase, which can influence cell invasion by modifying the extracellular matrix. Finally, 4-MU inhibited the formation of cytonemes, the extracellular membrane secretory structures containing the pro-inflammatory bactericides of the primary granules. The anti-inflammatory effect of 4-MU may be associated with the suppression of secretory processes that ensure the neutrophil invasion and initiate inflammation. We suggest that HA, due to the peculiarities of its synthesis, can promote the release of secretory carriers from the cell and 4-MU can block this process.

Inhibition of Neutrophil Secretion Upon Adhesion as a Basis for the Anti-Inflammatory Effect of the Tricyclic Antidepressant Imipramine.

Recent studies demonstrate the involvement of inflammatory processes in the development of depression and the anti-inflammatory effects of antidepressants. Infiltration and adhesion of neutrophils to nerve tissues and their aggressive secretion are considered as possible causes of inflammatory processes in depression. We studied the effect of the antidepressant imipramine on the adhesion and accompanied secretion of neutrophils under control conditions and in the presence of lipopolysaccharides (LPS). As a model of integrin-dependent neutrophil infiltration into tissues, we used integrin-dependent adhesion of neutrophils to the fibronectin-coated substrate. Imipramine inhibited neutrophil adhesion and concomitant secretion of proteins, including matrix metalloproteinase 9 (MMP-9) and neutrophil gelatinase-associated lipocalin (NGAL), which modify the extracellular matrix and basement membranes required for cell migration. Imipramine also significantly and selectively blocked the release of the free amino acid hydroxylysine, a product of lysyl hydroxylase, an enzyme that affects the organization of the extracellular matrix by modifying collagen lysine residues. In contrast, imipramine enhanced the release of ROS by neutrophils during adhesion to fibronectin and stimulated apoptosis. The anti-inflammatory effect of imipramine may be associated with the suppression of neutrophil infiltration and their adhesion to nerve tissues by inhibiting the secretion of neutrophils, which provides these processes.

Neutrophil Adhesion and the Release of the Free Amino Acid Hydroxylysine.

During infection or certain metabolic disorders, neutrophils can escape from blood vessels, invade and attach to other tissues. The invasion and adhesion of neutrophils is accompanied and maintained by their own secretion. We have previously found that adhesion of neutrophils to fibronectin dramatically and selectively stimulates the release of the free amino acid hydroxylysine. The role of hydroxylysine and lysyl hydroxylase in neutrophil adhesion has not been studied, nor have the processes that control them. Using amino acid analysis, mass spectrometry and electron microscopy, we found that the lysyl hydroxylase inhibitor minoxidil, the matrix metalloproteinase inhibitor doxycycline, the PI3K/Akt pathway inhibitors wortmannin and the Akt1/2 inhibitor and drugs that affect the actin cytoskeleton significantly and selectively block the release of hydroxylysine and partially or completely suppress spreading of neutrophils. The actin cytoskeleton effectors and the Akt 1/2 inhibitor also increase the phenylalanine release. We hypothesize that hydroxylysine release upon adhesion is the result of the activation of lysyl hydroxylase in interaction with matrix metalloproteinase, the PI3K/Akt pathway and intact actin cytoskeleton, which play important roles in the recruitment of neutrophils into tissue through extracellular matrix remodeling.

Cytonemes Versus Neutrophil Extracellular Traps in the Fight of Neutrophils with Microbes.

Neutrophils can phagocytose microorganisms and destroy them intracellularly using special bactericides located in intracellular granules. Recent evidence suggests that neutrophils can catch and kill pathogens extracellularly using the same bactericidal agents. For this, live neutrophils create a cytoneme network, and dead neutrophils provide chromatin and proteins to form neutrophil extracellular traps (NETs). Cytonemes are filamentous tubulovesicular secretory protrusions of living neutrophils with intact nuclei. Granular bactericides are localized in membrane vesicles and tubules of which cytonemes are composed. NETs are strands of decondensed DNA associated with histones released by died neutrophils. In NETs, bactericidal neutrophilic agents are adsorbed onto DNA strands and are not covered with a membrane. Cytonemes and NETs occupy different places in protecting the body against infections. Cytonemes can develop within a few minutes at the site of infection through the action of nitric oxide or actin-depolymerizing alkaloids of invading microbes. The formation of NET in vitro occurs due to chromatin decondensation resulting from prolonged activation of neutrophils with PMA (phorbol 12-myristate 13-acetate) or other stimuli, or in vivo due to citrullination of histones with peptidylarginine deiminase 4. In addition to antibacterial activity, cytonemes are involved in cell adhesion and communications. NETs play a role in autoimmunity and thrombosis.

Neutrophils as a source of branched-chain, aromatic and positively charged free amino acids.

Neutrophils release branched-chain (valine, isoleucine, leucine), aromatic (tyrosine, phenylalanine) and positively charged free amino acids (arginine, ornithine, lysine, hydroxylysine, histidine) when adhere and spread onto fibronectin. In the presence of agents that impair cell spreading or adhesion (cytochalasin D, fMLP, nonadhesive substrate), neutrophils release the same amino acids, except for a sharp decrease in hydroxylysine and an increase in phenylalanine, indicating their special connection with cell adhesion. Plasma of patients with diabetes is characterized by an increased content of branched-chain and aromatic amino acids and a reduced ratio of arginine/ornithine compared to healthy human plasma. Our data showed that the secretion of neutrophils, regardless of their adhesion state, can contribute to this shift in the amino acid content. Abbreviations: BCAAs: branched-chain amino acids; Е2: 17ß-estradiol; LPS: lipopolysaccharide from Salmonella enterica serovar Typhimurium; fMLP: N-formylmethionyl-leucyl-phenylalanine.

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

[This corrects the article DOI: 10.1155/2017/4308684.].

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 17ß-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.

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.

Inhibition of the GTPase dynamin or actin depolymerisation initiates outward plasma membrane tubulation/vesiculation (cytoneme formation) in neutrophils.

BACKGROUND INFORMATION: In a previous study, we demonstrated that human neutrophils can develop membrane tubulovesicular extensions (TVEs) that are 160-250 nm in width and several micrometres long. These extensions, or cytonemes, are capable of establishing long-range contacts with other cells or bacteria. Cytonemes consist of membrane tubules and vesicles of a uniform diameter aligned in a row. The mechanism of membrane tubulation/vesiculation to form cytonemes remains unknown. Upon endocytosis, the GTPase dynamin and an intact actin cytoskeleton are required for endocytic vesicles scission from the plasma membrane. RESULTS: We examined the effects of dynasore (a dynamin specific inhibitor), and of cytochalasin D and latrunculin A (actin cytoskeleton disruption agents), on cytoneme formation in neutrophils. Scanning and transmission electron microscopy were used to observe cytoneme formation. High-performance chromatography and mass spectrometry were used to estimate the protein composition of the cytonemes. In neutrophils, dynasore and cytochalasin D or latrunculin A initiated the formation of tubular cytonemes that were similar in diameter and composition. The formation of cytonemes in cells treated with cytochalasin D was accompanied by the appearance of tubular invaginations of the same diameter on the plasma membrane of neutrophils. The formation of dynasore- or cytochalasin D-induced cytonemes, however, was blocked by the nitric oxide (NO) synthases inhibitor l-NAME, indicating that NO is involved in cytoneme development. Proteome analysis indicated that dynasore- or cytochalasin D-induced cytonemes are secretory protrusions that contain neutrophil bactericides along with cytoplasmic proteins, such as glycolytic enzymes and actin cytoskeleton components. CONCLUSIONS: Inhibition of dynamin with dynasore or actin depolymerisation with cytochalasin D or latrunculin A might impair the membrane fusion/fission events that are required for the separation of secretory vesicles from the plasma membrane and from each other. As a result, the secretory process extends from the cells as membrane TVEs or cytonemes. Modification of secretion gives neutrophils the possibility to communicate with other cells over distance via highly adhesive cellular secretory protrusions (cytonemes). Cytonemes deliver their membrane-packed content exactly to the destination without dilution and without harm to the surrounding tissues.

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.

Proteome analysis identified human neutrophil membrane tubulovesicular extensions (cytonemes, membrane tethers) as bactericide trafficking.

BACKGROUND: Following adhesion to fibronectin neutrophils can develop membrane tubulovesicular extensions (TVEs) that can be 200nm wide and several cell diameters long. TVEs attach neutrophils to the other cells, substrata or bacteria over distance. To understand the physiological significance of TVEs we performed proteome analysis of TVE content in neutrophils plated to fibronectin in the presence of compounds known to induce TVE formation (nitric oxide donor diethylamine NONOate, 4-bromophenacyl bromide, cytochalasin D). METHODS: Development of TVEs was confirmed by scanning electron microscopy. TVEs were disrupted following removal of inductors and biochemical, high-performance liquid chromatography and mass spectrometry investigations were employed to characterize the proteins within the incubation media. RESULTS: TVE disruption released (a) the granular bactericides lactoferrin, lipocalin, myeloperoxidase, cathepsin G and defensins; (b) energy metabolism enzymes; (c) actin cytoskeleton proteins; (d) S100 proteins; and (e) annexin 1. CONCLUSIONS: The data confirm that TVEs represent a means of secretory bactericide trafficking, where the protrusions fuse with the plasma membrane upon neutrophil adhesion or extend from the cell surface when fusion is impaired. It is proposed that proteins abundantly presented in TVE (energy metabolism enzymes, actin cytoskeleton and S100 proteins, annexin 1) play an important role in fusion of TVE with the plasma membrane. GENERAL SIGNIFICANCE: Our study confirms TVEs as neutrophil secretory protrusions that make direct contacts with cells and bacteria over distance. The membrane-packed content and outstanding length of TVEs might allow targeted neutrophil secretion of aggressive bactericides over a long distance without dilution or injury to surrounding tissues.

Membrane tubules attach Salmonella Typhimurium to eukaryotic cells and bacteria.

Using scanning electron microscopy techniques we measured the diameter of adhesive tubular appendages of Salmonella enterica serovar S. Typhimurium. The appendages interconnected bacteria in biofilms grown on gallstones or coverslips, or attached bacteria to host cells (human neutrophils). The tubular appendage diameter of bacteria of virulent flagellated C53 strain varied between 60 and 70 nm, thus considerably exceeding in size of flagella or pili. Nonflagellated bacteria of mutant SJW 880 strain in biofilms grown on gallstones or coverslips were also interconnected by 60-90-nm tubular appendages. Transmission electron microscopy studies of thin sections of S. Typhimurium biofilms grown on agar or coverslips revealed numerous fragments of membrane tubular and vesicular structures between bacteria of both flagellated and nonflagellated strains. The membrane structures had the same diameter as tubular appendages observed by scanning electron microscopy, indicating that tubular appendages might represent membrane tubules (tethers). Previously, we have shown that neutrophils can contact cells and bacteria over distance via membrane tubulovesicular extensions (TVE) (cytonemes). The present electron microscopy study revealed the similarities in size and behavior of bacterial tubular appendages and neutrophil TVE. Our data support the hypothesis that bacteria establish long-range adhesive interactions via membrane tubules.

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-induced membrane tubulovesicular extensions (cytonemes) of human neutrophils catch and hold Salmonella enterica serovar Typhimurium at a distance from the cell surface.

Nitric oxide (NO) plays an important role in host defense against bacterial infections such as salmonellosis. NO and 4-bromophenacyl bromide (BPB) induce the formation of long tubulovesicular extensions (TVE, cytonemes, membrane tethers) from human neutrophils. These TVE serve as cellular sensory and adhesive organelles. In the present study, we demonstrated that in the presence of the NO donor, diethylamine NONOate or BPB human neutrophils bound and aggregated Salmonella enterica serovar Typhimurium bacteria extracellularly by TVE. In contrast, inhibition of NO-synthase activity by N(omega)-nitro-L-arginine methyl ester stimulated neutrophil phagocytosis (ingestion) of bacteria. Neutrophil TVE consisted of membrane-covered cytoplasm as was shown by the fluorescent cytoplasmic dye 2',7'-bis(2carboxyethyl)-5,(6)-carboxyfluorescein, and the fluorescent lipid, BODIPY-labeled sulfatide. Disruption and shedding of TVE were accompanied by the appearance of specific invaginations (porosomes) on neutrophil cell bodies. These invaginations corresponded to the variations in diameter of TVE (160-240 nm). We hypothesized that TVE represented protrusions of neutrophil exocytotic trafficking through special structures on the neutrophil surface. In conclusion, we propose a novel mechanism by which NO-induced TVE formation enables neutrophils to bind and aggregate bacteria at a distance.