Degranulation Of Primary Granules Research Articles

Polycarbonate and polystyrene nanoplastic particles act as stressors to the innate immune system of fathead minnow (Pimephales promelas).

Water pollution with large-scale and small-scale plastic litter is an area of growing concern. Macro-plastic litter is a well-known threat to aquatic wildlife; however, the effects of micro-sized and nano-sized plastic particles on the health of organisms are not well understood. Small-scale plastic particles can easily be ingested by various aquatic organisms and potentially interfere with their immune system; therefore, the authors used a freshwater fish species as a model organism for nanoplastic exposure. Characterization of polystyrene (41.0 nm) and polycarbonate (158.7 nm) nanoplastic particles (PSNPs and PCNPs, respectively) in plasma was performed, and the effects of PSNPs and PCNPs on the innate immune system of fathead minnow were investigated. In vitro effects of PSNPs and PCNPs on neutrophil function were determined using a battery of neutrophil function assays. Exposure of neutrophils to PSNPs or PCNPs caused significant increases in degranulation of primary granules and neutrophil extracellular trap release compared to a nontreated control, whereas oxidative burst was less affected. The present study outlines the stress response of the cellular component of fish innate immune system to polystyrene and polycarbonate nanoparticles/aggregates and indicates their potential to interfere with disease resistance in fish populations. Environ Toxicol Chem 2016;35:3093-3100. © 2016 SETAC.

Immunological stimuli change expression of genes and neutrophil function in fathead minnow Pimephales promelas Rafinesque

Fathead minnows Pimephales promelas were exposed to lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid [poly(I:C)] to observe immunological responses during simulated bacterial and viral challenge at the level of gene expression and granulocyte function. Complementary DNA libraries were created from LPS- and poly(I:C)-treated fish and c. 5000 expressed sequence tags (ESTs) were sequenced. The ESTs were subjected to BLASTx analysis and 1500 genes were annotated, grouped by function and 20 immune genes were selected for expression studies by real-time PCR. Lipopolysaccharide treatment significantly downregulated expression of interferon regulatory factor 2 binding protein 1 (nine-fold), Chemokine (C-X-C motif) ligand 12a (three-fold) and TNF-related apoptosis-inducing ligand, TRAIL (two-fold). In poly(I:C)-treated fish, a significant upregulation was observed for IFN-inducible and antiviral proteins belonging to the family of Mx proteins (73-fold) and chemokine CCL-C5a (28-fold). Blood neutrophil count was significantly increased in poly(I:C)-treated fish at 24 and 48 h post-injection. Neutrophil extracellular trap release and respiratory burst of kidney granulocytes were suppressed in poly(I:C)-treated fish, while degranulation of primary granules was not affected significantly by the treatment. The changes in gene expression and neutrophil function in P. promelas exposed to LPS and poly(I:C) support the use of this species as an alternative model for studies of pathogen effects on the innate immune system of fishes.

Hydroxylated fullerenes inhibit neutrophil function in fathead minnow ( Pimephales promelas Rafinesque, 1820)

Hydroxylated fullerenes act as potent inhibitors of cytochrome P450-dependent monooxygenases, and are reported to be very strong antioxidants quenching reactive oxygen species (ROS) production. Effects of nanosized hydroxylated fullerenes on fish neutrophil function and immune gene transcription was investigated using fathead minnow ( Pimephales pr omelas). Neutrophil function assays were used to determine the effects of fullerene exposure in vitro and in vivo on oxidative burst, degranulation and extracellular trap (NETs) release, and the innate immune gene trascription was determined with quantitative PCR (qPCR). Application of fullerenes (0.2–200 μg mL −1 in vitro) caused concentration dependent inhibition of oxidative burst and suppressed the release of NETs and degranulation of primary granules (up to 70, 40, and 50% reduction in activity compared to non-treated control, respectively). Transcription of interleukin 11 and myeloperoxidase genes was significantly increased and transcription of elastase 2 gene was significantly decreased in fish exposed to hydroxylated fullerenes for 48 h in vivo (12 and 3 fold increase, and 5 fold decrease, respectively). Observed changes in gene transcription and neutrophil function indicate potential for hydroxylated fullerenes to interfere with the evolutionary conserved innate immune system responses and encourages the use of fish models in studies of nanoparticle immunotoxicity.

Effects of nanosized titanium dioxide on innate immune system of fathead minnow ( Pimephales promelas Rafinesque, 1820)

Effects of nanosized (<100 nm) titanium dioxide (TiO 2) particles on fish neutrophils and immune gene expression was investigated using the fathead minnow ( Pimpehales promelas). Expanded use of TiO 2 in the cosmetic industry has increased the potential exposure risk to aquatic ecosystems and human health. Effects of nano-TiO 2 on neutrophil function of the fathead minnow was investigated using oxidative burst, neutrophil extracellular traps (NETs) release and degranulation of primary granules. The innate immune gene expression was determined with quantitative PCR (qPCR). Application of 0.1 μg mL −1 of nano-TiO 2 in vitro stimulated oxidative burst and NET release. Intraperitoneal injection of 10 μg g −1 of nano-TiO 2 caused a significant decrease in oxidative burst, NETs release and degranulation (21%; 11%; and 30%, decrease, respectively). Fish exposed to nano-TiO 2 for 48 h in vivo had significantly increased expression of interleukin 11, macrophage stimulating factor 1, and neutrophil cytosolic factor 2 (4; 2.5; and 2 fold increase, respectively). Nano-TiO 2 has potential to interfere with the evolutionary conserved innate immune system responses, as evidenced with observed changes in gene expression and neutrophil function. This finding encourages the use of fish models in the studies of nanoparticle immunotoxicity. The lowest significant response concentration studied in vitro is four times greater than the estimated environmental concentration for TiO 2 (0.025 μg mL −1) causing concern about potential impact of nano-TiO 2 on aquatic animals and ecosystems.

Zebrafish ( Danio rerio) whole kidney assays to measure neutrophil extracellular trap release and degranulation of primary granules

The zebrafish ( Danio rerio) is an excellent model system for studies in developmental biology, genetics, and toxicology, and is increasingly gaining importance in the field of immunology. The use of whole zebrafish kidneys as source of neutrophils for degranulation assays and detection of neutrophil extracellular traps is described for the first time. Neutrophils from zebrafish kidneys released neutrophil extracellular traps (NETs) and myeloperoxidase (MPO) upon stimulation with calcium ionophore, phorbol myristate acetate, and β-glucan. Immunocytochemical study of zebrafish kidney cells revealed that NETs are made of DNA fibers associated with neutrophil granular proteins, but not with cytoskeleton. Rapid, direct MPO and extracellular DNA detection assays were developed to quantify NET release and degranulation of neutrophil primary granules from whole zebrafish kidneys. The assays were used to measure the effects of acute crowding and handling stress on neutrophils, and to examine the potential for use of zebrafish whole kidney assays in evaluation of neutrophil function under different conditions in vivo. The whole kidney NET release and degranulation assays are quantitative, can rapidly measure a large number of samples, and are capable of detecting inhibition of neutrophil activity in stressed fish, overcoming the limitations that prevented use of zebrafish in the investigations of cellular innate immune function. The assays can be used as a new research model to study effects of stress, immunomodulators, toxicants, and diseases on fish neutrophil biology.

Immunomodulatory effects of β-glucan on neutrophil function in fathead minnows ( Pimephales promelas Rafinesque, 1820)

Stimulatory effects of yeast β-1,3–1,6-glucans on neutrophils have long been recognized, but effects of glucans on degranulation of primary granules in fish neutrophils have not been previously reported. Neutrophil function was monitored during in vitro and in vivo application of glucans to non- (NS), acute- (AS) and chronically stressed (CS) fish. β-Glucan proved to be a strong and quick (80%, 2 min) stimulant of degranulation. Dietary glucan increased degranulation in NS fish, and prevented a decrease in AS fish. Degranulation in CS fish returned to NS levels 3 days after the glucan diet was fed. Fathead minnows appear to be a useful model to investigate neutrophil degranulation in fish exposed to different environmental conditions and immunomodulators. Use of β-glucans in fish diets prior to AS and during chronic stress can enhance neutrophil function, potentially increasing disease resistance and survival rates after transportation or exposure to poor water quality.

Anesthetic efficacy of tricaine methanesulfonate, metomidate and eugenol: Effects on plasma cortisol concentration and neutrophil function in fathead minnows ( Pimephales promelas Rafinesque, 1820)

Anesthetic efficacy, plasma cortisol concentration, and two parameters of neutrophil function (oxidative burst and degranulation of primary granules) were compared among three anesthetics in the fathead minnow: tricaine methanesulfonate (MS 222), metomidate hydrochloride (MTMD), and eugenol (EUG). The optimum anesthetic concentration was determined as: MS 222 75 mg L − 1 , EUG 30 mg L − 1 and MTMD 4 mg L − 1 . Handling and crowding stress was induced in fish with (SA) and without (S) anesthetic. Plasma cortisol concentration was measured at 0, 30, 90, and 240 min after stress and found to increase at 30 min post-stress in S and SA MS 222 groups, but not in SA MTMD and SA EUG groups. To test the effects of different anesthetics on neutrophil function, fish were divided into a baseline control group, a group exposed to handling and crowding stress (S) and a stressed anesthetized group (SA). Fish were assayed for neutrophil function before and after stress (24 h, 72 h and 7 days). The degranulation of neutrophil primary granules was measured as exocytosis of myeloperoxidase (MPO) using 3, 3′, 5, 5′-tetramethylbenzidine as a substrate. Degranulation of primary granules was decreased to 60–75% of non-stressed control in stressed and fish treated with MS 222, and was not affected when MTMD and EUG were used. The degranulation of primary granules proved to be a useful assay for measuring the effects of stress on neutrophil function in fish. Eugenol and metomidate prevented stress-induced decrease of neutrophil function while MS 222 did not.

Differential Regulation of Neutrophil Phospholipase D Activity and Degranulation

One of the proposed functions of phosphatidic acid (PA) formation from phospholipase D (PLD) activation in neutrophils is to promote degranulation induced by receptor agonists. The present study shows that the time course and dose response of PA formation and degranulation induced by N-formyl–methionyl–leucyl-phenylalanine (fMLP) differed. PLD activation and degranulation also exhibited different dose response to genistein and epigallocatechin gallate (EGCG), inhibitors of protein tyrosine kinases. Genistein inhibited PLD activity with an IC50 value of 12.2 μM in fMLP- and 107 μM in phorbol myristate acetate (PMA)-stimulated cells. It required higher concentrations of genistein to inhibit degranulation than to inhibit PLD activity induced by fMLP. EGCG in the range of 40–400 μM had no effect on PLD activity but it inhibited the release of β-glucuronidase and elastase by fMLP-stimulated cells. These results demonstrate differential regulation of PLD activity and degranulation of primary granules by genistein and EGCG in fMLP-stimulated neutrophils.

Priming response to inflammatory mediators in hyperreactive peripheral neutrophils from adult periodontitis.

To study the response to in vitro priming of peripheral neutrophils from patients with periodontitis compared to healthy controls. Peripheral neutrophils from these patients had shown increased production of oxygen radicals after activation with opsonized bacteria and a difference in priming response could suggest an explanation for this hyperreactivity. Peripheral neutrophils from a group of patients with periodontitis and from age- and sex-matched controls were preincubated with tumor necrosis factor alpha, lipopolysaccharide (LPS), formyl-methionyl-leucyl-phenylalanine and the tetra peptide arginyl-glycyl-aspartate-serine. After preincubation, the cells were activated with gammaglobulin opsonized-bacteria, i.e., a Fc gamma R-stimulation. The priming effect was assessed as the production of oxygen radicals and as the degranulation or primary granules. Showed that the patients had a slightly lower responsiveness to priming than had the controls. This difference in priming response was most pronounced when measured as degranulation of primary granules after preincubation with LPS and 20 min of activation. This study shows no difference in response to priming, with optimal concentrations of inflammatory mediators, between peripheral neutrophils from patients with adult periodontitis and healthy controls.

Partial Myeloperoxidase Deficiency in a Case of Preleukaemia

A patient with a refractory anaemia preceding acute myeloblastic leukaemia had an increased susceptibility to infection due to Staphylococcus aureus. 36% of neutrophils lacked myeloperoxidase (MPO) activity and, in vitro, these polymorphonuclear neutrophils (PMN) had a defect of bactericidal activity against Staphylococcus aureus. Cytochemical studies of phagocytosis with the electron miscroscope have shown that the degranulation of primary granules (MPO+ or MPO-) was normal after phagocytosis of Escherichia coli which were normally lysed. A defective destruction of Staphylococcus aureus and Candida albicans was observed in some PMN with or without MPO activity, suggesting that MPO deficiency itself was not the only cause of this defect. In PMN which appeared normal, most MPO(+) granules were unable to fuse with the phagocytic vacuole containing intact germs even after 90 min of contact. There was, therefore, in addition to a partial MPO deficiency, a defect in cellular degranulation. This defect, the mechanism of which is unknown, may be in part responsible for the defective bacterial degradation.