Flavonoid impairment of neutrophil response

Flavonoids are a class of phenolic plant pigments which impair the oxidative burst of neutrophils to an extent dependent on their hydrophobicity. The distribution of quercetin and of morin in nitrogen-cavitated neutrophils paralleled their respective hydrophobic characteristics and respiratory burst inhibition. While both flavonoids were localized primarily in the specific granule membrane of neutrophils, the amount of quercetin was considerably greater than that of morin. We here demonstrate inhibition of the initial stimulation response, depolarization of the membrane potential as monitored by fluorescence of the membrane probe diS-C3-(5), and of the respiratory burst, monitored by following the destruction of diS-C3-(5), a reaction mediated by the H2O2 produced in the burst. The flavonoids kaempferol, morin, quercetin, or fisetin were preincubated with human neutrophils at a concentration of 100 microM per 2 X 10(6) cells/ml for 2-3 min and subsequently stimulated with 1 microgram/ml of the tumor promoter phorbol myristate acetate (PMA) or with 60 micrograms/ml of immune complex. The effect of each compound differed, i.e. depolarization was enhanced by some and inhibited by others, while H2O2 generation was inhibited by each, supporting our previous findings that membrane potential depolarization and the respiratory burst are dissociable events. Concentration-response experiments, performed at flavonoid concentrations between 12.5 and 500 microM to determine the IC50 values of these compounds for depolarization and burst activation, indicated that none of the flavonoids affected the resting potential, while all perturbed the stimulus-coupled response, the direction and extent of the perturbation depending upon the stimulus, and the function assessed. These data show that the effects of flavonoids on human neutrophils are complex and suggest several sites of action depending upon the flavonoid's subcellular distribution and pathway of stimulation.     

The inhibitory effect of ambroxol on respiratory burst, degranulation and cytosolic Ca2+ change in degraded immunoglobulin G-activated neutrophils

Superoxide and H2O2 production by neutrophils stimulated by 0.5 mg/ml degraded immunoglobulin G (IgG) and 1 microM N-formyl-methionyl-leucyl-phenylalanine (fMLP) was inhibited by ambroxol in a dose-dependent fashion, and at the concentration of 100 microM, 43.3% to 64.3% of inhibitions were detected. The inhibitory effect of ambroxol on H2O2 production by neutrophils was greater than that on superoxide production. The production of nitrite by lipopolysaccharide-activated murine peritoneal macrophages was significantly attenuated by ambroxol in a dose-dependent fashion and NG-monomethyl-L-arginine (NMMA). Ambroxol decreased the release of myeloperoxidase and lysozyme evoked by 0.5 mg/ml degraded immunoglobulin G and 1 microM fMLP in a dose-dependent fashion, and at the concentration of 100 microM, 37.1% to 64.2% of inhibitions were observed. The stimulatory effect of phorbol 12-myristate 13-acetate (PMA) (0.1 microg/ml) on superoxide production and myeloperoxidase, which is inhibited by 100 nM staurosporine, was not affected by 100 microM ambroxol. Degraded immunoglobulin G (0.5 mg/ml) caused an immediate elevation of [Ca2+]i in fura-2 load neutrophils in 1.23 mM Ca2+-containing medium. Preincubation of neutrophils with 10 microM to 100 microM ambroxol, 5 mM EGTA and 100 microM verapamil depressed the elevation of [Ca2+]i elicited by 0.5 mg/ml degraded immunoglobulin G. In conclusion, the inhibitory action of ambroxol on stimulated neutrophil responses, including respiratory burst and lysosomal enzyme release, appears to be attributed to its depressant action on the activation process, including the change in intracellular Ca2+ level. in which the role of protein kinase C is uncertain.     

Selective inhibition of protein kinase C, mitogen-activated protein kinase, and neutrophil activation in response to calcium pyrophosphate dihydrate crystals, formyl-methionyl-leucyl-phenylalanine, and phorbol ester by O-(chloroacetyl-carbamoyl) fumagillol (AGM-1470; TNP-470)

The effect of O-(chloroacetyl-carbamoyl) fumagillol (AGM-1470; TNP-470) was investigated on protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation in neutrophils stimulated by plasma-opsonized crystals of calcium pyrophosphate dihydrate (triclinic) [CPPD(T)], formyl-Met-Leu-Phe (fMLP), and phorbol 12-myristate 13-acetate (PMA). Neutrophil respiratory burst responses also were determined in AGM-1470-pretreated cells stimulated with the same agonists, using chemiluminescence and superoxide anion generation assays. AGM-1470 (5 microM) effectively inhibited PKC activation in cells treated with CPPD(T) crystals (50 mg/mL, 2 min) and fMLP (1 microM, 1 min), but had no effect on PMA-treated cells (0.5 microM, 5 min). AGM-1470 blocked MAPK activity completely and reduced neutrophil activation induced by fMLP and PMA but not by CPPD(T). The degree of inhibition of the respiratory burst plateaued at approximately 46+/-9 and 54+/-3% in fMLP- and PMA-treated cells, respectively. These data indicate that activation of neutrophil respiratory burst activity may be mediated through the MAPK pathway. AGM-1470 pretreatment did not inhibit CPPD(T) crystal- or fMLP-stimulated phosphatidylinositol 3-kinase (PI 3-kinase) activity. These findings, coupled with further observations that the PI 3-kinase inhibitor wortmannin (10 nM) inhibited fMLP- and CPPD(T) crystal-induced but not PMA-induced chemiluminescence, indicate that at least two distinct signaling pathways (mediated by PI 3-kinase or MAPK) lead to neutrophil respiratory burst responses. PKC may also be required in the MAPK-stimulated pathway. We propose that the inhibitory effect of AGM-1470 on the neutrophil respiratory burst may be due to its ability to inhibit PKC and MAPK activation.     

Inhibition by gomisin C (a lignan from Schizandra chinensis) of the respiratory burst of rat neutrophils.

1. The possible mechanisms of action of the inhibitory effect of gomisin C on the respiratory burst of rat neutrophils in vitro was investigated. 2. The peptide formyl-Met-Leu-Phe (FMLP) induced superoxide anion (O2-) formation and O2 consumption, which was inhibited by gomisin C in a concentration-dependent manner (IC50 21.5 +/- 4.2 micrograms ml-1 for O2- formation). Gomisin C also suppressed O2- formation and consumption at low concentrations of phorbol myristate acetate (PMA) with an IC50 value of 26.9 +/- 2.1 micrograms ml-1 for O2- formation. However, gomisin C did not affect the responses induced by a high concentration of PMA. 3. Gomisin C had no effect on O2- generation and uric acid formation in the xanthine-xanthine oxidase system, and failed to alter O2- generation during dihydroxyfumaric acid (DHF) autoxidation, indicating that it does not scavenge superoxide. 4. Like trifluoperazine (TFP), gomisin C attenuated the activity of PMA-activated neutrophil particulate NADPH oxidase in a concentration-dependent manner. 5. Gomisin C reduced the elevations of cytosolic free Ca2+ in neutrophils stimulated by FMLP in the presence or absence of EDTA. Cyclopiazonic acid (CPA) induced the release of Ca2+ from intracellular stores and this was also reduced by gomisin C. However, the Ca2+ influx pathway activated by CPA was not affected by gomisin C. 6. The cellular cyclic AMP level was markedly increased by forskolin, but not by gomisin C. Moreover, the inositol phosphate levels in FMLP-activated neutrophils were not affected by gomisin C. 7. These results show that the inhibitory action of gomisin C on the respiratory burst is not mediated by changes in cellular cyclic AMP or in inositol phosphates, or by scavenging O2- released from neutrophils, but may be mediated partly by the suppression of NADPH oxidase and partly by the decrease of cytosolic Ca2+ released from an agonist-sensitive intracellular store.     

阿魏酸钠对人类白细胞呼吸爆发氧代谢的影响

目的　研究阿魏酸钠 (SF)对白细胞呼吸爆发期间氧消耗和过氧化物的影响。方法　采用电子自旋共振自旋捕集技术 ,自旋探针氧测定法和化学发光法检测白细胞呼吸爆发期间氧消耗和活性氧自由基。结果　SF( 1mmol·L-1)对白细胞呼吸爆发期间的氧消耗无影响 (P >0 0 5 ) ,但能抑制白细胞产生的化学发光反应 (P <0 0 1) ,清除白细胞释放的O·2 和·OH ,并在黄嘌呤 (Xan) /黄嘌呤氧化酶 (XO)体系和Fentons反应体系中证实 (P <0 0 1)。结论　SF不影响白细胞氧化代谢功能 ,能通过清除氧自由基防止激活白细胞对组织的损伤。     

Activation of cellular functions in macrophages by venom secretory Asp-49 and Lys-49 phospholipases A(2).

The in vitro effects of myotoxin III (MT-III), an Asp-49 catalytically-active phospholipase A(2), and myotoxin II (MT-II), a catalytically-inactive Lys-49 variant, isolated from Bothrops asper snake venom, on phagocytosis and production of hydrogen peroxide (H(2)O(2)) by thioglycollate-elicited macrophages were investigated. MT-II and MT-III were cytotoxic to mouse peritoneal macrophages at concentrations higher than 25 microg/ml. At non-cytotoxic concentrations, MT-II stimulated Fcgamma, complement, mannose and beta-glucan receptors-mediated phagocytosis, whereas MT-III stimulated only the mannose and beta-glucan receptors-mediated phagocytosis. Moreover, both myotoxins induced the release of H(2)O(2) by thioglycollate-elicited macrophages, MT-III being the most potent stimulator. MT-II induced the release of H(2)O(2) only at a concentration of 3.2 microg/ml (130% increment) while MT-III induced this effect at all concentrations tested (0.5-2.5 microg/ml; average of 206% increment). It is concluded that, at non-cytotoxic concentrations, MT-II and MT-III activate defense mechanisms in macrophages up regulating phagocytosis, mainly via mannose and beta-glucan receptors, and the respiratory burst.     

Differential inhibition of human neutrophil functions. Role of cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase.

Multiple molecular forms of cyclic nucleotide phosphodiesterase have been characterized in various tissues and cells according to their substrate specificity, intracellular location, and calmodulin dependence. The purpose of this study was to evaluate the possible involvement of different molecular forms of phosphodiesterase in regulating the respiratory burst and lysosomal enzyme release responses of human neutrophils. Treatment with the selective cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase inhibitors Ro 20-1724 or rolipram, or the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), resulted in inhibition of respiratory burst stimulated by the chemoattractants formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) (IC50 values: 0.71-17 microM) and complement fragment C5a (IC50 values: 61-93 microM), but did not inhibit phagocytosis-stimulated respiratory burst (less than 10% inhibition at 100 microM). Selective inhibitors of calmodulin-dependent phosphodiesterase (ICI 74,917), calmodulin-insensitive, cyclic GMP-specific phosphodiesterase (M & B 22,948), cyclic GMP-stimulated phosphodiesterase (AR-L 57), or cyclic AMP-specific, cyclic GMP-inhibited phosphodiesterase (amrinone and cilostamide) exhibited little or no inhibitory effect on FMLP- or phagocytosis-stimulated respiratory burst (0-42% inhibition at 100 microM). Regulation of neutrophil activation by phosphodiesterase was also response specific, as Ro 20-1724, rolipram and IBMX were less potent inhibitors of FMLP-induced lysosomal enzyme release (0-14% inhibition at 100 microM). Analysis of human neutrophil preparations confirmed the existence of a cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase, which was associated with the particulate fraction of the cell. These results demonstrate a role for the cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase in the regulation of human neutrophil functions, which appears to be both stimulus specific and response specific.     

Diclofenac binding to human polymorphonuclear neutrophils: effect on respiratory burst and N-formylated peptide binding

The respiratory burst of human polymorphonuclear neutrophils (PMN) induced by particle or soluble stimuli was measured in the presence of the nonsteroidal anti-inflammatory drug, diclofenac sodium (Voltaren). Diclofenac (25-100 micrograms/ml) inhibited the oxygen consumption of PMN stimulated by 5 X 10(-7) M of N-formyl-methionyl-leucyl-phenylalanine (FMLP). The inhibition was linearly correlated to diclofenac concentration. By contrast, diclofenac did not affect the rate of heat-killed Klebsiella pneumoniae ingestion of PMN, or the PMN O2-uptake induced by (0.67 microgram/ml) serum-opsonized zymosan or (1 microgram/ml) phorbol myristate acetate (PMA). The PMN production of superoxide anion induced by various FMLP concentrations (10(-7), 10(-6) and 10(-5) M) was also decreased by diclofenac. However, this inhibition declined when the formylated peptide concentration was raised suggesting that diclofenac could alter FMLP binding to the PMN membrane. Binding experiments of tritiated FMLP to intact PMN performed at 22 degrees and 4 degrees showed high- and low-affinity FMLP sites with dissociation constant (Kd) values of approximately 2 X 10(-8) M and 10(-5) M respectively. Diclofenac did not significantly alter the low-affinity component but induced modifications of the high-affinity component which were different at 22 degrees and 4 degrees. At 22 degrees only the dissociation constant value was enhanced by diclofenac (competitive inhibition) whereas at 4 degrees both binding parameters (i.e. dissociation constant and number of available binding sites) were modified (mixed inhibition). Diclofenac was also shown to bind to PMN with a low affinity. This binding was not diminished at 4 degrees by various concentrations of FMLP which even increased the number of diclofenac binding sites on PMN at 22 degrees. These data suggest that diclofenac binding to PMN may decrease FMLP-induced PMN respiratory burst by interfering with the peptide recognition by specific FMLP receptors.     

Effects of acute and long-term diazepam administrations on neutrophil activity: a flow cytometric study

This study analyzed the effects of acute and long-term diazepam treatments on rat peripheral blood neutrophil activity and cortisol serum levels. Rats were acutely and long-term (21 days, once daily) treated with diazepam (10 mg/kg) or its vehicle (1.0 ml/kg). Blood was collected 1 h after treatments for flow cytometric analysis of neutrophil oxidative burst and phagocytosis. Corticosterone and diazepam concentrations were also determined. Results showed that: (1) both diazepam treatments increased lipopolysaccharide (LPS) and phorbol myristate acetate (PMA)-induced neutrophil oxidative burst; (2) the increase in oxidative burst after Staphylococcus aureus induction in acutely treated animals was higher than that observed after long-term treatment; (3) phagocytosis is increased by acute diazepam treatment and decreased by a long-term regimen; (4) acute, but not long-term, diazepam treatment increased corticosterone levels; (5) diazepam plasmatic levels after acute and long-term treatments were not different. These results indicate the development of tolerance to diazepam effects on corticosterone serum levels but not on neutrophil activity.     

虎杖晶4号对人血PMNs呼吸暴发和氧自由基的作用

应用化学发光法测定①人血PMNs受PMA刺激产生的化学发光;②黄嘌呤—黄嘌呤氧化酶体系产生O_2;③VitC—CU~(2+)—酵母多糖产主·OH;④H_2O_2的释放。观察虎杖晶4号对上述体系产生自由基的影响。结果提示.虎杖晶4号对PMN呼吸暴发的早期(1～6 min)有明显的抑制作用,呈剂量依赖性;而在12min时发光强度均高于空白对照.表明因虎杖晶4号而使呼吸暴发滞后。其对O_2.·OH.H_2O_2均有清除作用.且呈剂量依赖性.IC_(50)分别为14.6.29.6和13.0μmol·L~(-1).     

埃必定对人血白细胞呼吸爆发和氧自由基的抑制作用

目的观察埃必定对人血白细胞呼吸爆发和氧自由基的影响，初步探讨埃必定的抗炎作用机制。方法用化学发光法测定人血白细胞受调理的酵母多糖刺激发生呼吸爆发的活性氧、碱性二甲基亚砜－鲁米诺体系产生的超氧阴离子自由基（Ｏ·２）、ＶｉｔＣ－Ｃｕ２＋酵母多糖产生的羟自由基（·ＯＨ）及过氧化氢（Ｈ２Ｏ２）的释放，观察了埃必定对上述体系产生的自由基的影响。结果埃必定对白细胞的呼吸爆发有显著的抑制作用；对Ｏ·２、·ＯＨ有清除作用，呈剂量依赖性，对Ｈ２Ｏ２无清除作用。结论埃必定对人血白细胞呼吸爆发具有抑制作用，对氧自由基具有清除作用     

Inhibition by magnolol of formylmethionyl-leucyl-phenyl alanine-induced respiratory burst in rat neutrophils.

The influence of the plant product magnolol on neutrophil superoxide anion (O2-*) generation has been investigated in the rat. Intraperitoneal injection of magnolol (30mg kg(-1)) significantly inhibited the formylmethionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst in rat whole blood ex-vivo. Magnolol also inhibited the 02-* generation with an IC50 (concentration resulting in 50% inhibition) of 15.4+/-1.6 microM and O2 consumption in rat neutrophils in-vitro. Magnolol weakly inhibited the O2-* generation in the xanthine-xanthine oxidase system, decreased cellular cyclic AMP level and had no effect on cyclic GMP levels. It weakly inhibited neutrophil cytosolic protein kinase C activity but did not alter porcine heart protein kinase A activity. Magnolol attenuated fMLP-induced protein tyrosine phosphorylation with an IC50 of 24.0+/-1.9 microM and the phosphorylation of mitogen-activated protein kinase p42/44 with an IC50 of 28.5+/-4.5 microM. However, magnolol alone activated neutrophil phospholipase D activity as determined by the formation of phosphatidic acid and phosphatidyl-ethanol in the presence of ethanol. In the presence of NADPH, the arachidonate-activated NADPH oxidase activity in a cell-free system was weakly suppressed by magnolol. These results suggest that the inhibition of respiratory burst in fMLP-activated neutrophils by magnolol is probably attributable mainly to the attenuation of protein tyrosine phosphorylation and p42/44 mitogen-activated protein kinase activation, and partly to the suppression of protein kinase C and NADPH oxidase activities.     

The effect of putative protein kinase C inhibitors, K252a and staurosporine, on the human neutrophil respiratory burst activated by both receptor stimulation and post-receptor mechanisms.

1. Two compounds, reported to be potent inhibitors of protein kinase C (PKC), K252a and staurosporine, have been examined in order to gain further information as to the possible role played by PKC in the signal transduction sequence of the neutrophil respiratory burst as determined by superoxide (O2-) production. 2. A number of stimuli were used in the study, some acting at receptors i.e. fMet-Leu-Phe (fMLP), opsonized zymosan and heat-aggregated IgG (HAGG), one acting on a G-protein, fluoride, and two direct PKC activators, dioctanoylglycerol (diC8) and phorbol myristate acetate (PMA). 3. K252a and staurosporine inhibited the respiratory burst with all the stimuli but the order of agonist sensitivity was very different with the two inhibitors. 4. For K252a-induced inhibition of O2- release, the order of potency was fluoride greater than fMLP, HAGG greater than opsonized zymosan greater than PMA, DiC8. For staurosporine-induced inhibition of O2- release, the order of potency changed to fluoride greater than DiC8, PMA greater than HAGG, fMLP greater than opsonized zymosan. The significance of this unexpected difference in relative rank order of potency is discussed with reference to the reported mechanism of action of the two inhibitors and the events involved in the oxidative burst. 5. Staurosporine at low concentrations increased the fMLP-stimulated O2- response by 100%, the maximum effect occurring at 35 nM. 6. To the extent that the compounds used are specific inhibitors of PKC, these findings support a role for the enzyme PKC in stimulus-activation coupling in O2- generation with all the stimuli used in this study.     

维拉帕米对人血PMN细胞呼吸暴发和氧自由基的抑制作用

用化学发光法测定：人血ＰＭＮ细胞受ＰＭＡ刺激发生呼吸暴发产生的活性氧；黄嘌呤－黄嘌呤氧化酶体系产生的Ｏ；Ｖｉｔｃ－ＣＵ２＋－酵母多糖产生的·ＯＨ及Ｈ２Ｏ２的释放，同时也观察了维拉帕米对上述体系产生的自由基的影响。结果提示，维拉帕米对ＰＭＮ细胞的呼吸暴发有显著的抑制作用；对Ｏ、Ｈ２Ｏ２有清除作用，呈剂量依赖性，其ＩＣ５０分别为２８．１，２２．３ｎｍｏｌ·Ｌ－１；对·ＯＨ则无作用。     

In vitro activity of aminoglycosides on the respiratory burst response in human polymorphonuclear neutrophils

In response to phagocytosis of microbes or chemical stimuli, neutrophils generate reactive oxygen species which represent the major bactericidal mechanism of these cells. We have investigated the influence of aminoglycosides (amikacin, gentamicin, netilmicin and tobramycin), antibiotics with a marked concentration-dependent killing effect, on the human neutrophil oxidative burst represented by the release of hydrogen peroxide. This study was performed using time-dependent cellular experiments and a cell-free system. In the cellular model, 2-h incubation with amikacin (ranging from 10 mg/l to 1 g/l), enhanced hydrogen peroxide release by stimulated human neutrophils. At higher concentrations (1 to 5 g/l), hydrogen peroxide production was decreased. No significant effects were observed with the other aminoglycosides at concentrations ranging from 10 mg/l to 2.5 g/l. The pro-oxidant activity of amikacin may be due to a cellular mechanism through oxidative metabolism of human neutrophils as demonstrated in the cell washing experiment, whereas the antioxidant activity observed for higher concentrations may be a result of a scavenging effect as demonstrated in the cell-free system. The enhanced of hydrogen peroxide production observed for therapeutic concentrations of amikacin could be a beneficial effect for neutrophil bactericidal functions.     

Differential regulation of the activation of human eosinophils, macrophages, and neutrophils: effect of the allergic mediator release inhibitor CI-949

The allergic mediator release inhibitor CI-949 [5-methoxy-3-(1- methylethoxy)-1-phenyl-N-1H-tetrazol-5-yl-1H-indole-2-carbox amide L-arginine salt] was evaluated for its effect on the activation of human eosinophils, macrophages, and neutrophils by the phagocytic stimulus serum-opsonized zymosan (SOZ). CI-949 inhibited the SOZ- stimulated respiratory burst of eosinophils, measured as the generation of superoxide anion, with an IC50 of 22.8 microM. At concentrations of 100 microM, CI-949 had no inhibitory effect against lysosomal enzyme release by these cells. At 100 microM, CI-949 had no inhibitory effect against release of eosinophil peroxidase while inhibiting release of the macrophage lysosomal enzyme N-acetyl-beta-D- glucosaminidase by only 11.7 percent. In contrast, CI-949 inhibited the release of the neutrophil primary granule enzyme myeloperoxidase inhibiting of 21.4 microM, while inhibiting release of lysozyme from lysosomal enzyme release from secondary granules with an IC50 of 99.3 microM. These results demonstrate that oxygen radical generation and lysosomal enzyme release by human eosinophils, macrophages and neutrophils are differentially regulated by CI-949. These results suggest that these inflammatory cells may have distinct stimulus-related coupling mechanisms.     

Budesonide reduces superoxide and peroxynitrite anion chemiluminescence during human neutrophil bursts

Many lung disorders are characterized by airway inflammation involving the recruitment of inflammatory cells, and leading to the release of oxidant and inflammatory mediators. The overproduction of superoxide anion (O(2)(*-)) and nitric oxide (NO) during the respiratory bursts of neutrophils leads to production of peroxynitrite, a highly damaging oxidant with an important role in the inflammatory loop causing airway hyper-reactivity in respiratory diseases like asthma. The aim of this study was to investigate in vitro the effects of a 1-hour incubation with budesonide at 2.5 x 10(-7), 5 x 10(-7), 1 x 10(-6), 2 x 10(-6) and 4 x 10(-6) mol/l on O(2)(*-), NO, and peroxynitrite production during the respiratory burst of human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 x 10(-7) mol/l) or phorbol 12-myristate 13-acetate (PMA, 2 x 10(-6) mol/l), as documented by luminol-amplified chemiluminescence (LACL). In absence of L-arginine, budesonide (5 x 10(-7) to 4 x 10(-6) mol/l) dose-dependently reduced both fMLP- and PMA-induced LACL (18.3-50.6%). In the presence of L-arginine (100 microg/ml), a NO donor increasing peroxynitrite production, LACL increased 3-5 times compared with baseline, but budesonide dose-dependently reduced LACL (25.5-59.6%). Mifepristone (4 x 10(-6) mol/l), a glucocorticoid receptor antagonist, inhibited the effect of budesonide on LACL, thus confirming that budesonide reacts with glucocorticoid receptors to exert an antioxidant activity. These results suggest that budesonide target rapidly human neutrophils leading to a fast reduction in both NO and peroxynitrite production, and are consistent with decrease in exhaled NO levels after treatment with budesonide in patients with asthma.     

Protective effect of indomethacin against chemotactic deactivation of human neutrophils induced by formylated peptide

The effects of the nonsteroidal antiinflammatory drug indomethacin on the parameters relating to the migration and respiratory burst of human polymorphonuclear leukocytes (PMN) were studied in an attempt to clarify the mechanism of this drug's action on PMN. At various concentrations below 200 micrograms/ml, indomethacin partially inhibited the spontaneous migration of PMN but did not alter the directional migration induced by C5a-activated serum. In the presence of N-formyl-methionyl-leucyl-phenylalanine (FMLP) as chemoattractant, directed PMN migration was either inhibited or stimulated by indomethacin, depending on FMLP concentration. When PMN migration was induced by the optimal and suboptimal FMLP concentrations of 10(-7) and 10(-8) M, indomethacin inhibited this migration, but when the high FMLP concentration of 10(-6) M depressed this migration by chemotactic deactivation, indomethacin restored it to its maximum. Both the inhibitory and stimulatory effects of indomethacin on FMLP-induced PMN migration were due to changes in the migration speed. Indomethacin also inhibited FMLP-induced changes in the shape of floating PMN, and in respiratory burst, as well as specific FMLP binding to PMN. In contrast, indomethacin did not alter the PMN respiratory burst induced by phorbol myristate acetate or C5a-activated serum. These data show that indomethacin is able to prevent the loss of PMN chemokinetic activity induced by formylated peptides and suggest that it might be useful for investigating the mechanism of peptide-induced chemotactic deactivation.     

Effect of decaglycerol monooleate on phagocytosis and respiratory burst activity of human neutrophils: an in vitro study.

Decaglycerol monooleate (DGMO), a type of polyglycerol esters of fatty acids (PGEF), was evaluated for its in vitro effect on phagocytosis and respiratory burst activity of isolated human neutrophils using flow cytometric assay. Opsonized zymosan particles labelled with FITC (FITC-OZ) were employed as an indicator of phagocytosis. Fluorescence of FITC-OZ attached on to the surface of neutrophils was quenched by addition of trypan blue solution. After 10 minutes of incubation with DGMO up to a concentration of 10 mg/ml, neutrophil phagocytosis was not affected markedly. At the same time, the DGMO emulsion left little influence on complement receptor type three (CR3) that is associated with phagocytosis. On the other hand, oxidation of hydroethidine, which was used as an indicator of intracellular generation of reactive oxygen species (mainly for superoxide anion), was significantly inhibited by DGMO over 1 mg/ml. However, this phenomenon was not seen in DGMO-treated neutrophils when DGMO was removed after incubation. The present data suggest that DGMO does not affect phagocytosis of human neutrophils but down-regulates respiratory burst activity.     

Effect of low-dose prednisone in vivo on the ability of complement receptor to mediate an oxidative burst in rat neutrophils

Glucocorticoids have been used in the treatment of a variety of inflammatory processes including autoimmune diseases. However, the influence of low-dose glucocorticoids on the respiratory burst activity of neutrophils has not been studied. The aim of this work was to study the effect of treatment with low-dose prednisone on the oxidative burst of rat peripheral blood neutrophils. Wistar male rats were treated with prednisone by gavage (28, 87 or 257 microg/animal/day) for 7 or 15 days. These doses are equivalent to 10, 30 or 90 mg/adult human ( approximately 70 kg)/day, respectively. Sera from normal rats were used to opsonize zymosan (opZy). Neutrophils (1x10(5)) were stimulated by opZy and the oxidative burst of control or treated rat cells was measured by luminol-dependent chemiluminescence (CL). Prednisone did not affect the CL of rat neutrophils for either period of treatment, or any studied doses, when compared with controls. These results suggest that the low-dose prednisone has no effect on the oxidative burst mediated by complement receptors during the rat neutrophil phagocytosis of complement-opZy.