N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils

N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca²⁺ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca²⁺]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 [Ca²⁺]ᵢ in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca²⁺]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 [Ca²⁺]ᵢ in human neutrophils was observed. In Ca²⁺-free buffer, NAC- and cysteine-induced [Ca²⁺]ᵢ increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca²⁺]ᵢ in human neutrophils occur through Ca²⁺ influx. NAC- and cysteine-induced [Ca²⁺]ᵢ increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na⁺-free HEPES, both NAC and cysteine induced a marked increase in [Ca²⁺]ᵢ in human neutrophils, arguing against the possibility that Na⁺-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca²⁺]ᵢ increasing activity. Our results show that NAC and cysteine induce [Ca²⁺]ᵢ increase through Ca²⁺ influx in human neutrophils via SKF96365- and ruthenium red-dependent way.

Identification of Lys49-PLA2 from crude venom of Crotalus atrox as a human neutrophil-calcium modulating protein

We fortuitously observed a human neutrophil intracellular free-calcium concentration ([Ca2+]i) increasing activity in the commercially available phosphodiesterase I (PDE I), which is actually dried crude venom of Crotalus atrox. As this activity was not observed with another commercially available pure PDE I, we tried to find out the causative molecule(s) present in 'crude' PDE, and identified Lys49-phospholipase A2 (Lys49-PLA2 or K49-PLA2), a catalytically inactive protein which belongs to the phospholipase A2 family, by activity-driven three HPLC (reverse phase, size exclusion, reverse phase) steps followed by SDS-PAGE and LC-MS/MS. K49-PLA2 induced Ca2+ infl ux in human neutrophils without any cytotoxic eff ect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 microM) and SKF-96365 (20 microM) signifi cantly inhibited K49-PLA2-induced [Ca2+]i increase. These results suggest that K49-PLA2 modulates [Ca2+]i in human neutrophils via 2-APB- and SKF-96365-sensitive calcium channels without causing membrane disruption.

Anti-inflammatory sesquiterpene lactones from Tithonia diversifolia trigger different effects on human neutrophils

Abstract The tagitinins isolated of Tithonia diversifolia (Hemsl.) A. Gray, Asteraceae, are the most studied sesquiterpene lactones due to their wide spectrum of pharmacologic activities, especially related with nuclear factor-kappa B inhibition. Nevertheless, detailed studies about the mechanism of action of its active compounds are still lacking. Neutrophils perform a fundamental role in the inflammatory response to several etiologic factors. However, the effect of tagitinins on human neutrophil is not yet clearly known. We investigated the role of tagitinin C (1), tagitinin F (2) and tagitinin A (3) in activation and survival of human neutrophils to establish possible effects in their mechanisms of inflammation. Human neutrophils were purified from the peripheral blood and cultivated with tagitinins C (1), F (2) and A (3) in the presence or not of Escherichia coli lipopolysaccharide. The enzymatic activity, apoptosis and secretion of cytokines rate were determined after 18 h. Lipopolysaccharide-induced myeloperoxidase activity of human neutrophils was significantly inhibited only by tagitinin F (2). Apoptosis of neutrophils was increased in the presence of tagitinin C (1), and it occurred independently of the presence of lipopolysaccharide or dexamethasone. Tagitinins C (1), F (2) and A (3) decrease lipopolysaccharide-induced interleukin-6, interleukin-8 and Tumor necrosis factor alpha production by human neutrophils. Together, these results indicate that tagitinins exhibit anti-inflammatory action on human neutrophils. However, tagitinin F (2) was the only sesquiterpene lactone that decreased secretion of inflammatory products by neutrophils without inducing neutrophil apoptosis.

Scant Extracellular NAD Cleaving Activity of Human Neutrophils is Down-Regulated by fMLP via FPRL1

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,N6-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.

G-test positive serum inhibits ROS-dependent killing of Candida albicans by interfering with internal- ized expression of neutrophil Dectin-1 / 中华微生物学和免疫学杂志

Objective To investigate the mechanism of soluble β-1, 3-D-glucan in G-test positive serum in inhibiting ROS-dependent killing of Candida albicans ( C.albicans ) mediated by neutrophil Dectin-1.Methods The expression and distribution of internalized Dectin-1 and triggered ROS in human neutrophils were detected by using confocal/two-photon laser scanning microscopy upon stimulation with C.albicans (MOI=10) which was pretreated with β-1, 3-D-glucanase (10 U/ml) or not.Abrogation test was used to analyze whether intracellular Dectin-1 was involved in C.albicans-triggered ROS production in human neutrophils.Furthermore, flow cytometry analysis was performed to detect the expression of intracel-lular Dectin-1 and ROS in neutrophils which were pretreated respectively with G-test positive serum at differ-ent dilutions for 60 min and then stimulated with C.albicans for another 60 min at 37℃.Results After stimulated with C.albicans (MOI=10) for 60 min, the expression of Dectin-1 in neutrophils was recruited to the spores of opsonophagocytized C.albicans, and partly co-localized with the triggered ROS production . However, the expression of intracellular Dectin-1 was not observed in neutrophils when stimulated with β-1, 3-D-glucanase pretreated C.albicans for 60 min at 37℃.Abrogation test further showed that C.albicans-trig-gered ROS production in neutrophils was partly and irreversibly inhibited by adding Dectin -1 blocking mAb of 5 μg/ml.In addition , both the triggered expression of intracellular Dectin-1 and ROS production in neu-trophils stimulated with C.albicans ( MOI=10 ) in the presence of G-test positive serum were significantly lower than those of neutrophils stimulated only with C.albicans (LSD-t test, P<0.01).Linear regression a-nalysis suggested that the triggered intracellular Dectin-1 and ROS production in neutrophils upon stimulation with C.albicans were both inhibited by soluble β-1, 3-D-glucan in a dose-dependent manner (Dectin-1,R2=0.702,P<0.01;ROS,R2=0.588,P<0.01 ).Conclusion Taken together, these results demonstrated that the soluble β-1, 3-D-glucan in G-test positive serum may play a role in inhibiting the ROS-dependent killing of C.albicans by interfering with internalized expression of neutrophil Dectin-1.

Antioxidant effects of protocatechuic acid, ferulic acid, and caffeic acid in human neutrophils using a fluorescent substance / Efectos antioxidantes del ácido protocatéquico, ácido ferúlico y el ácido caféico usando una sustancia fluorescente en los neutrófilos humanos

Human neutrophils stimulated by phorbol myristate acetate (PMA), an activator of protein kinase C, produce active oxygen by NADPH oxidase in intracellular structures. We added succinimidyl ester of dichlorodihydrofluorescein diacetate (H2DCFDA), which first emits fluorescence when oxidized with active oxygen species, to neutrophils to produce active oxygen, in order to investigate the antioxidant effects of protocatechuic acid, ferulic acid, and caffeic acid which belong to polyphenols and are widely distributed among plants. Particularly, we focused on examining whether these substances capture and eliminate active oxygen inside or outside the neutrophil cytoplasm and whether these substances inhibit NADPH oxidase. Fluorescence microscopy demonstrated that fluorescence-positive intracellular structures were decreased in neutrophils when stimulated by PMA and exposed to an antioxidant. Quantitative measurement by flow cytometry revealed that the fluorescence intensities in neutrophils, exposed to protocatechuic acid, ferulic acid, or caffeic acid, were decreased by 62.9 percent, 71.4 percent, and 86.1 percent, respectively, as compared with those stimulated by PMA but not exposed to an antioxidant. Judging from fluorescence microscopy and dot blots by flow cytometry, these antioxidants had no effects on neutrophil morphology. On the other hand, the fluorescence intensities of the active oxygen released from neutrophils were decreased by 81.4 percent, 46.7 percent, and 27.4 percent, respectively. Diphenylene iodonium, a specific inhibitor of NADPH oxidase, inhibited the enzyme by 92.1 percent in the PMA-stimulated neutrophils. Protocatechuic acid, ferulic acid, and caffeic acid inhibited the enzyme by 36.5 percent, 54.6 percent, and 27.4 percent, respectively. These results demonstrate that protocatechuic acid, ferulic acid, and caffeic acid capture and eliminate active oxygen, produced by PMA-stimulated neutrophils, intracellularly and extracellularly. Furthe...

Los neutrófilos humanos estimulados por forbol-miristato-acetato (PMA), un activador de la proteína quinasa C, producen oxígeno activo por la NADPH oxidasa en las estructuras intracelulares. Hemos añadido diacetato de 2', 7-dihidro dicloro fluoresceína (H2DCFDA), que emite fluorescencia cuando se oxida con las especies de oxígeno activo, a neutrófilos para producir oxígeno activo, a fin de investigar el efecto antioxidante del ácido protocatéquico, el ácido ferúlico y el ácido cafeico que pertenecen a polifenoles y se distribuyen ampliamente entre las plantas. Particularmente, nos enfocamos en examinar si estas sustancias capturan y eliminan el oxígeno activo dentro o fuera del citoplasma de neutrófilos y si estas sustancias inhiben la NADPH oxidasa. La microscopia de fluorescencia demostró que las estructuras intracelulares positivas a fluorescencia disminuyeron en los neutrófilos mediante la estimulación de la PMA y exposición a un antioxidante. La medición cuantitativa por citometría de flujo reveló que la intensidad de fluorescencia en los neutrófilos, expuestos al ácido protocatéquico, el ácido ferúlico, o el ácido cafeico, se redujo un 62,9 por ciento, 71,4 por ciento y 86,1 por ciento, respectivamente, en comparación con las estimuladas por PMA pero no expuestas a un antioxidante. A juzgar desde la microscopía de fluorescencia y la citometría de flujo, estos antioxidantes no tuvieron efectos sobre la morfología de los neutrófilos. Por otra parte, la intensidad de fluorescencia del oxígeno activo liberado por los neutrófilos se redujeron un 81,4 por ciento, 46,7 por ciento y 27,4 por ciento, respectivamente. El DPI (difenileno-iodonio), un inhibidor específico de la NADPH oxidasa, inhibió a la enzima en el 92,1 por ciento en los neutrófilos estimulados por PMA. El ácido protocatéquico, el ácido ferúlico y el ácido caféico inhiben la enzima en un 36,5 por ciento, 54,6 por ciento y 27,4 por ciento, respectivamente. Estos resultados demuestran...

Tetrachloroauric acide depresses the activation processes of phagocytic cells

Gold compounds depress phagocytic cell responses, including chemotaxis, and respiratory burst. However, the effects of gold compounds on the function of phagocytic cells are variable according to the preparation of medicine. In this study, effect of tetrachloroauric acid on activated neutrophil responses, including respiratory burst, lysosomal enzyme release and change of intracellular Ca2+ level and on the synthesis of interleukin-8 and granulocyte-macrophage colony stimulating factor by macrophages was studied. This study further examines how gold compounds affect the activation processes. The respiratory burst stimulated by complement C5a, degraded IgG and PMA in neutrophils was inhibited by tetrachloroauric acid. In contrast to C5a and degraded IgG, PMA-stimulated superoxide production was weakly inhibited by tetrachloroauric acid. Staurosporine, genistein, EGTA and verapamil inhibited superoxide and H2O2 production caused by C5a and degraded IgG. PMA-stimulated superoxide production was inhibited by staurosporine but was not affected by genistein. Tetrachloroauric acid, genistein, EGTA and verapamil inhibited the release of acid phosphatase and myeloperoxidase, while the effect of staurosporine was not detected. The synthesis of interleukin-8 and granulocyte-macrophage colony stimulating factor by interleukin-1beta in macrophages was inhibited by tetrachloroauric acid. Preincubation with tetrachloroauric acid, genistein, EGTA and verapamil, the elevation of (Ca2+)i evoked by C5a was inhibited. Store-regulated Ca2+ entry in thapsigargin-pretreated neutrophils was decreased by the addition of tetrachloroauric acid and genistein. The effect of staurosporine on intracellular Ca2+ mobilization was not observed. In conclusion, tetrachloroauric acid may suppress neutrophil responses through its inhibitory action on elevation of intracellular Ca2+ level and protein kinase C. It might exhibit an inhibitory effect on the action of protein tyrosine kinase. Tetrachloroauric acid depresses cytokine production by macrophages.

Effect of ketamine on stimulus-induced superoxide generation and intracellular calcium in human neutrophils in vitro / 中国药理学通报

Aim To study the effect of two racemic ketamine, S(+)-ketamine and R(-)-ketamine on stimulus-induced superoxide generation and intracellular calcium in vitro. Methods The stimulus-induced superoxide generation in human neutrophils was determined by using method of cytochrome C reduction. The intracellular calcium in human neutrophils was measured by chemiluminescence with Fura-2 loading. The phosphorylation of p47phox of NADPH oxidase in neutrophils was detected by Western blotting. Results S(+)-Ket and R(-)-Ket inhibited fMLP-induced superoxide generation in neutrophils in a concentration-dependent manner. In the case of PMA, S(+)-Ket inhibited PMA-induced superoxide generation and elevation of intracellular calcium of neutrophils in a concentration-dependent manner, whereas R(-)-Ket slightly increased PMA-induced superoxide generation and elevation of intracellular calcium of neutrophils. On the other hand S(+)-Ket inhibited the phosphorytion of p47phox of NADPH oxidase subunit,which R(-)-Ket was increased. EGTA can abolished the inhibition of S(+)-Ket on PMA-induced phosphorytion of p47phox.Conclusion S(+)-Ket inhibits the phosphorylation of p47phox of NADPH oxidase subunit and the superoxide generation in human neutrophils via PKC-calcium signal pathway.