• The Korean Journal of Physiology and Pharmacology
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• v.20 no.5
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• pp.449-457
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• 2016

N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though $Ca^{2+}$ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ($[Ca^{2+}]_i$) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on $[Ca^{2+}]_i$ in human neutrophils. We observed that NAC ($1{\mu}M{\sim}1mM$) and cysteine ($10{\mu}M{\sim}1mM$) increased $[Ca^{2+}]_i$ in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in $[Ca^{2+}]_i$ in human neutrophils was observed. In $Ca^{2+}$-free buffer, NAC- and cysteine-induced $[Ca^{2+}]_i$ increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in $[Ca^{2+}]_i$ in human neutrophils occur through $Ca^{2+}$ influx. NAC- and cysteine-induced $[Ca^{2+}]_i$ increase was effectively inhibited by calcium channel inhibitors SKF96365 ($10{\mu}m$) and ruthenium red ($20{\mu}m$). In $Na^+$-free HEPES, both NAC and cysteine induced a marked increase in $[Ca^{2+}]_i$ in human neutrophils, arguing against the possibility that $Na^+$-dependent intracellular uptake of NAC and cysteine is necessary for their $[Ca^{2+}]_i$ increasing activity. Our results show that NAC and cysteine induce $[Ca^{2+}]_i$ increase through $Ca^{2+}$ influx in human neutrophils via SKF96365- and ruthenium red-dependent way.

• Biomedical Science Letters
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• v.16 no.2
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• pp.123-126
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• 2010

Volatile organic compounds are commonly off gassed from various building materials and can induce sick building syndrome. Volatile organic compounds such as formaldehyde, xylene and toluene are known as toxic agents in immune cells. Human leukocytes, particularly, neutrophils and eosinophils play important roles in the regulation of immune responses. In this study, we investigated the toxic effects of formaldehyde, ortho-xylene (o-xylene), para-xylene (p-xylene) and toluene on the apoptosis of neutrophils and eosinophils isolated from the blood of healthy donors. Formaldehyde increased the constitutive apoptosis of neutrophils and eosinophils. o-xylene, p-xylene and toluene increased the spontaneous apoptosis of eosinophils, but not that of neutrophils. Formaldehyde increased the protein level of IL-8 in neutrophils and eosinophils, and suppressed the MCP-1 expression in neutrophils. The release of IL-6 from neutrophils was diminished by volatile organic compounds used in this study. In conclusion, formaldehyde, xylene and toluene elevate the apoptosis of neutrophils and eosinophils, and regulate the release of cytokine and chemokine in neutrophils and eosinophils. These results indicate that formaldehyde, xylene and toluene have a cytotoxicity in human neutrophils and eosinophils and may damage the modulation of immune responses.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.6
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• pp.497-502
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• 2014

Extracellular nicotinamide adenine dinucleotide (NAD) cleaving activity of a particular cell type determines the rate of the degradation of extracellular NAD with formation of metabolites in the vicinity of the plasma membrane, which has important physiological consequences. It is yet to be elucidated whether intact human neutrophils have any extracellular NAD cleaving activity. In this study, with a simple fluorometric assay utilizing $1,N^6$-ethenoadenine dinucleotide (etheno-NAD) as the substrate, we have shown that intact peripheral human neutrophils have scant extracellular etheno-NAD cleaving activity, which is much less than that of mouse bone marrow neutrophils, mouse peripheral neutrophils, human monocytes and lymphocytes. With high performance liquid chromatography (HPLC), we have identified that ADP-ribose (ADPR) is the major extracellular metabolite of NAD degradation by intact human neutrophils. The scant extracellular etheno-NAD cleaving activity is decreased further by N-formyl-methionine-leucine-phenylalanine (fMLP), a chemoattractant for neutrophils. The fMLP-mediated decrease in the extracellular etheno-NAD cleaving activity is reversed by WRW4, a potent FPRL1 antagonist. These findings show that a much less extracellular etheno-NAD cleaving activity of intact human neutrophils compared to other immune cell types is down-regulated by fMLP via a low affinity fMLP receptor FPRL1.

• The Korean Journal of Physiology and Pharmacology
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• v.11 no.6
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• pp.259-262
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• 2007

Diffuse panbronchiolitis (DPB) is a pulmonary disease characterized by chronic inflammation of the bronchioles and chronic infiltration of inflammatory cells in the lungs. Macrolides are effective therapeutic agents for chronic respiratory tract diseases, such as DPB. However, the mechanisms by which macrolides modulate the immune responses in patients with DPB remain unclear. To understand clinical efficacy for the treatment of DPB by macrolides, the effects of erythromycin (EM) on the expression of pro-inflammatory cytokines such as interleukin-6 (IL-6) and interleukin-8 (IL-8) by human neutrophils were examined. Pre-treatment with EM significantly decreased the expression of IL-6 and IL-8 transcripts by lipopolysaccharide (LPS)-stimulated human neutrophils. EM also reversed the enhanced survival of human neutrophils by LPS. These data indicate that EM has achieved therapeutic effect for patients with DPB, in part, through decreasing the expression of pro-inflammatory cytokines and the survival of neutrophils.

• Parasites, Hosts and Diseases
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• v.51 no.4
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• pp.479-484
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• 2013

Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion ($O^{\cdot}_{2^-}$) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.

• Parasites, Hosts and Diseases
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• v.49 no.3
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• pp.291-294
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• 2011

Trichomonas vaginalis is a flagellated lumen-dwelling extracellular protozoan parasite that causes human trichomoniasis via sexual intercourse. Human neutrophils play a crucial role in acute tissue inflammatory responses in T. vaginalis infection. In this study, we investigated the signaling mechanism of neutrophil responses when stimulated with T. vaginalis-derived secretory products (TvSP), which were collected from $1{\times}10^7$ live trichomonads. Incubation of human neutrophils isolated from peripheral blood with TvSP induced up-regulation of IL-8 protein secretion. In addition, stimulation with TvSP induced phosphorylation of NF-${\kappa}B$ and CREB in neutrophils. Moreover, TvSP-induced IL-8 production was also significantly inhibited by pretreatment of neutrophils with $i{\kappa}B$ inhibitor or CREB inhibitor. These results suggest that transcription factors NF-${\kappa}B$ and CREB are involved in IL-8 production in human neutrophils induced by stimulation with T. vaginalis infection.

• Parasites, Hosts and Diseases
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• v.48 no.1
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• pp.1-7
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• 2010

Neutrophils play an important role in the human immune system for protection against such microorganisms as a protozoan parasite, Trichomonas vaginalis; however, the precise role of neutrophils in the pathogenesis of trichomoniasis is still unknown. Moreover, it is thought that trichomonal lysates and excretory-secretory products (ESP), as well as live T. vaginalis, could possibly interact with neutrophils in local tissues, including areas of inflammation induced by T. vaginalis in humans. The aim of this study was to investigate the influence of T. vaginalis lysate on the fate of neutrophils. We found that T. vaginalis lysate inhibits apoptosis of human neutrophils as revealed by Giemsa stain. Less altered mitochondrial membrane potential (MMP) and surface CD16 receptor expression also supported the idea that neutrophil apoptosis is delayed after T. vaginalis lysate stimulation. In contrast, ESP stimulated-neutrophils were similar in apoptotic features of untreated neutrophils. Maintained caspase-3 and myeloid cell leukemia-1 (Mcl-1) in neutrophils co-cultured with trichomonad lysate suggest that an intrinsic mitochondrial pathway of apoptosis was involved in T. vaginalis lysate-induced delayed neutrophil apoptosis; this phenomenon may contribute to local inflammation in trichomoniasis.

• Journal of Pharmacopuncture
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• v.20 no.2
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• pp.127-131
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• 2017

Objectives: Polymorphonuclear neutrophils (PMNs) constitute the first line of defense against invading microbial pathogens. Early events in inflammation involve the recruitment of neutrophils to the site of injury or damage where changes in intracellular calcium can cause the activation of pro-inflammatory mediators from neutrophils including superoxide generation, degranulation and release of myeloperoxidase (MPO), productions of interleukin (IL)-8 and tumor necrosis factor ${\alpha}$ ($TNF-{\alpha}$), and adhesion to the vascular endothelium. To address the anti-inflammatory role of flavonoids, in the present study, we investigated the effects of the flavonoids quercetin and vitexin on the stimulus-induced nitric oxide (NO), $TNF-{\alpha}$, and MPO productions in human neutrophils. Methods: Human peripheral blood neutrophils were isolated, and their viabilities were determined by using the Trypan Blue exclusion test. The polymorphonuclear leukocyte (PMNL) preparations contained more than 98% neutrophils as determined by morphological examination with Giemsa staining. The viabilities of cultured neutrophils with various concentrations of quercetin and vitexin ($1-100{\mu}M$) were studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. Neutrophils were cultured in complete Roswell Park Memorial Institute (RPMI) medium, pre-incubated with or without quercetin and vitexin ($25{\mu}M$) for 45 min, and stimulated with phorbol 12-myristate 13-acetate (PMA) ($10^{-7}M$). NO production was carried out through nitrite determination by using the Griess method. Also, the $TNF-{\alpha}$ and the MPO productions were measured using enzyme-linked immunosorbent assay (ELISA) kits and MPO assay kits. Results: Neutrophil viability was not affected up to a concentration of $100{\mu}M$ of quercetin or vitexin. Both quercetin and vitexin significantly inhibited $TNF-{\alpha}$, NO, and MPO productions in human neutrophils (P < 0.001). Conclusion:The present study showed that both quercetin and vitexin had significant anti-inflammatory effects. Thus, treatment with either quercetin or vitexin may be considered as a therapeutic strategy for treating patients with neutrophil-mediated inflammatory diseases.

• Journal of Pharmacopuncture
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• v.20 no.1
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• pp.52-56
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• 2017

Objectives: Neutrophils represent the front line of human defense against infections. Immediately after stimulation, neutrophilic enzymes are activated and produce toxic mediators such as pro-inflammatory cytokines, nitric oxide (NO) and myeloperoxidase (MPO). These mediators can be toxic not only to infectious agents but also to host tissues. Because flavonoids exhibit antioxidant and anti-inflammatory effects, they are subjects of interest for pharmacological modulation of inflammation. In the present study, the effects of rutin on stimulus-induced NO and tumor necrosis factor $(TNF)-{\alpha}$ productions and MPO activity in human neutrophils were investigated. Methods: Human peripheral blood neutrophils were isolated using Ficoll-Hypaque density gradient centrifugation coupled with dextran T500 sedimentation. The cell preparations containing > 98% granulocytes were determined by morphological examination through Giemsa staining. Neutrophils were cultured in complete Roswell Park Memorial Institute (RPMI) medium, pre-incubated with or without rutin ($25{\mu}M$) for 45 minutes, and stimulated with phorbol 12-myristate 13-acetate (PMA). Then, the $TNF-{\alpha}$, NO and MPO productions were analyzed using enzyme-linked immunosorbent assay (ELISA), Griess Reagent, and MPO assay kits, respectively. Also, the viability of human neutrophils was assessed using tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and neutrophils were treated with various concentrations of rutin ($1-100{\mu}M$), after which MTT was appended and incubated at $37^{\circ}C$ for 4 hour. Results: Rutin at concentrations up to $100{\mu}M$ did not affect neutrophil viability during the 4-hour incubation period. Rutin significantly decreased the NO and $TNF-{\alpha}$ productions in human peripheral blood neutrophils compared to PMA-control cells (P < 0.001). Also, MPO activity was significantly reduced by rutin (P < 0.001). Conclusion: In this in vitro study, rutin had an anti-inflammatory effect due to its inhibiting NO and $TNF-{\alpha}$ productions, as well as MPO activity, in activated human neutrophils. Treatment with rutin may be considered as a therapeutic strategy for neutrophil-mediated inflammatory/autoimmune diseases.

• BMB Reports
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• v.36 no.6
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• pp.565-571
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• 2003

The locomotor responses of human peripheral blood neutrophils and lymphocytes were measured by the change from spherical to polarized shapes in the presence of endotoxins (lipopolysaccharide, LPS) of enteric pathogens: S. dysenteriae type 1, V. cholerae Inaba 569B, S. typhimurium, and K. pneumoniae. We reported earlier that these endotoxins are chemotactic factors for the neutrophils since they stimulated cell polarization within a few minutes of incubation. Endotoxins had an inhibitory effect upon neutrophil phagocytosis of opsonized yeast and the cells engulfed fewer yeasts. Interestingly, endotoxins increased neutrophil adhesion to clean glass surfaces, but stimulated the cells to exhibit increased random locomotion (chemokinesis) through cellulose nitrate filters and show an enhanced ability to reduce nitroblue tetrazolium (NBT) dye. Unlike neutrophils, lymphocytes direct from blood do not show polarized morphology towards chemotactic factors but the cells acquire locomotor capacity during 24-72 h culture with mitogens such as phytohemagglutinin (PHA), phorbol myristate acetate or concanavalin A. Stimulation of blood lymphocytes with endotoxins did not induce cell polarization in short-term but long-term culture resulted in an increase in the proportion of polarized cells that acquired locomotor morphologies. The majority of these cells were identified as esterase negative B-lymphocytes that migrated through filters. Despite the optimum time of incubation for each of these cell types being different, we found that lymphocytes respond to much lower concentrations of endotoxins than the neutrophils. These findings suggest that endotoxins of enteric pathogens modulate the functions of human blood neutrophils and lymphocytes.